Partial Characterisation of 4 (DP 4) for the Treatment of Type 2 Diabetes and its Identification as a novel Pharmaceutical Target for Stress-related Indications

Beiträge zur Charakterisierung der Dipeptidylpeptidase 4 (DP 4) für die Behandlung von Typ-2-Diabetes und deren Identifizierung als ein neuartiges pharmazeutisches Ziel für stressvermittelte Indikationen

Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg

zur Erlangung des Doktorgrades Dr. rer. nat.

vorgelegt von M. Sc. (Biochemie) Leona Wagner aus Rheinfelden (Baden)

Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg

Tag der mündlichen Prüfung: 23.10.2012

Vorsitzender der Promotionskommission: Prof. Dr. Rainer Fink

Erstberichterstatter: Prof. Dr. med. Stephan von Hörsten

Zweitberichterstatter: Prof. Dr. Johann Helmut Brandstätter

Publications, Presentations and Posters

Part of the present study have been published or submitted in the following journals or presented on the following conferences:

Publications:

1. Hinke, S.A., McIntosh, C.H., Hoffmann, T., Kuhn-Wache, K., Wagner, L., Bar, J., Manhart, S., Wermann, M., Pederson, R.A., and Demuth, H.U. (2002) On combination therapy of diabetes with metformin and dipeptidyl peptidase IV inhibitors. Diabetes Care 25, 1490- 1491. 2. Bär, J., Weber, A., Hoffmann, T., Stork, J., Wermann, M., Wagner, L., Aust, S., Gerhartz, B., and Demuth, H.U. (2003) Characterisation of human dipeptidyl peptidase IV expressed in Pichia pastoris. A structural and mechanistic comparison between the recombinant human and the purified porcine . Biol. Chem. 384, 1553-1563. 3. Engel, M., Hoffmann, T., Wagner, L., Wermann, M., Heiser, U., Kiefersauer, R., Huber, R., Bode, W., Demuth, H.U., and Brandstetter, H. (2003). The crystal structure of dipeptidyl peptidase IV (CD26) reveals its functional regulation and enzymatic mechanism. Proc. Natl. Acad. Sci. U. S. A. 100, 5063-5068. 4. Wagner, L., Hoffmann, T., Rahfeld, J.U., and Demuth, H.U. (2006) Distribution of dipeptidyl peptidase IV-like activity in canine and porcine tissue sections by RT-PCR. Adv. Exp. Med. Biol. 575, 109-116. 5. Frerker, N*., Wagner, L*., Wolf, R., Heiser, U., Hoffmann, T., Rahfeld, J.U., Schade, J., Karl, T., Naim, H.Y., Alfalah, M., Demuth, H.U., and von Hörsten, S. (2007) Neuropeptide Y (NPY) cleaving enzymes: structural and functional homologues of dipeptidyl peptidase 4. Peptides 28, 257-268. 6. Forssmann, U., Stoetzer, C., Stephan, M., Kruschinski, C., Skripuletz, T., Schade, J., Schmiedl, A., Pabst, R., Wagner, L., Hoffmann, T., Kehlen, A., Escher, S.E., Forssmann, W.G., Elsner, J., and von Hörsten, S. (2008) Inhibition of CD26/dipeptidyl peptidase IV enhances CCL11/eotaxin-mediated recruitment of eosinophils in vivo. J Immunol. 181, 1120-1127. 7. Schade, J., Stephan, M., Schmiedl, A., Wagner, L., Niestroj, A.J., Demuth, H.U., Frerker, N., Klemann, C., Raber, K.A., Pabst, R., and von Hörsten, S. (2008) Regulation of expression and function of dipeptidyl peptidase 4 (DP4), DP8/9, and DP10 in allergic responses of the lung in rats. J. Histochem. Cytochem. 56, 147-155. 8. Frerker, N., Raber, K., Bode, F., Skripuletz, T., Nave, H., Klemann, C., Pabst, R., Stephan, M., Schade, J., Brabant, G., Wedekind, D., Jacobs, R., Jorns, A., Forssmann, U., Straub, R.H., Johannes, S., Hoffmann, T., Wagner, L., Demuth, H.U., and von Hörsten, S. (2009) Phenotyping of congenic dipeptidyl peptidase 4 (DP4) deficient Dark Agouti (DA) rats suggests involvement of DP4 in neuro-, endocrine, and immune functions. Clin. Chem. Lab Med. 47, 275-287. 9. Wagner, L., Wermann, M., Rosche, F., Rahfeld, J.-U., Hoffmann, T., and Demuth, H.-U. (2011) Isolation of dipeptidyl peptidase IV isoforms from porcine kidney by preparative isoelectric focusing. Biol. Chem. 392, 665-677. 10. Alcázar, M.A., Boehler, E., Klaffenbach, D., Hartner, A., Allabauer, I., Wagner, L., von Hörsten, S., Plank, C., and Doetsch, J. (2011) Persistent changes within the intrinsic kidney- associated NPY system and tubular function by litter size reduction. Nephrol.Dial.Transplant. 26, 2453-2465.

Publications, Presentations and Posters

11. Wagner, L., Wolf, R., Zeitschel, U., Rossner, S., Pedersen, A., Leavitt, B., Haydn, M., Kästner, F., Rothermundt, M., Gärtner, U.-T, Weber, A., Schlenzig, D., Frerker, N., Schade, J., Manhart, S., Rahfeld, J.U., Hoffmann, T., Demuth, H.-U., and von Hörsten, S. (2012a) of neuropeptide Y (NPY) from head to toe identified novel NPY-cleaving enzymes and revealed potential drug interactions. (in preparation). 12. Ruiz-Carrillo, D*.Wagner, L*., Jaenckel, N., Börgel, A., Parthier, C., Böhme M., Rosche, F., von Hörsten, S. Demuth H.-U., Rahfeld J.-U., and Stubbs, M. T. (2012) Structural characterization of the recombinant human dipeptidyl peptidase 2. (in preparation). 13. Wagner, L., Kästner, F., Wolf, R., Stiller, H., Heiser, U., Gärtner, U.-T., Manhart, S., Klemm, K., Hoffmann, T., Rahfeld, J., Demuth, H.-U., von Hörsten, S., and Rothermundt, M. (2012b) Identifying neuropeptide Y (NPY) as the main stress-related substrate of dipeptidyl peptidase 4 in blood. (in preparation). 14. Wagner, L., Hult, S., Wolf, R., Börgel, A., Schlenzig, D., Manhart, S., Rahfeld, J.-U., Leavitt, B., Haydn, M., Demuth, H.-U., Petersén, A., von Hörsten, S. (2012c) Verification of CSF NPY levels in Huntington patients revealed differential degradation of NPY1-30 metabolite. (in preparation). 15. Wagner, L., Kästner, F., Wolf, R., Stiller, H., Gans, K., Ruiz-Carrillo, D., Niestroj, A.J., Manhart, S., Hoffmann, T., Rahfeld, J.-U., Demuth, H.-U., von Hörsten, S., and Rothermundt, M. (2012d) Galanin (GAL) is a substrate of prolyl (PEP) in the human blood. (in preparation).

Oral Presentations:

Wagner,L., Gündel,D., Zeitschel,U., Rossner,S.,Frerker,N., Schade,J.Stiller,J., Niestroj, A.J., Thümmler,A., Wolf,R., Gärtner, U.-T., Manhart,S., Kehlen,A., Rahfeld, J.-U., Hoffmann,T., von Hörsten,S., and Demuth, H.-U. “Tissue-distribution of dipeptidyl peptidase 4-like enzymes in distinct brain areas and neuronal tissues” 3rd International Conference on Dipeptidyl Peptidases and Related . 23 - 25 April 2008, Antwerp, Belgium. Wagner, L., Wolf R., Zeitschel U., Rossner S., Petersen A., Leavitt, B., Haydn M., Kästner M., Rothermundt M., Gärtner U., Weber A., Schlenzig S., Frerker N., Schade J., Manhart S., Rahfeld J-U., von Hörsten S., Demuth H.-U. Metabolomic investigations of neuropeptide Y (NPY) identified seven novel NPY-cleaving enzymes and revealed potential drug interactions. 27th Winter School. 24 - 28 February 2010, Tiers, Italy. Wagner, L., Wolf R., Zeitschel U., Rossner S., Gündel D., Stiller H, Petersen A, Leavitt B., Haydn M. , Kästner F., Rothermundt M., Gärtner U., Weber A., Schlenzig D., Frerker N, Schade J., Manhart S, Rahfeld J-U, Demuth H-U., von Hörsten, S. “Dipeptidyl Peptidase 4 (DP 4) and Neuropeptide Y (NPY): A novel pharmaceutical target against stress?” International Meeting on Proline-Specific cleavage and Oxoprolyl Formation. 26 - 29 May 2010, Halle, Germany. Wagner, L., Wolf R., Zeitschel U., Rossner S., Gündel D., Stiller H, Petersen A, Leavitt B., Haydn M. , Kästner F., Rothermundt M., Gärtner U., Weber A., Schlenzig D., Frerker N, Schade J., Manhart S, Rahfeld J-U, Demuth H-U., von Hörsten S. “Dipeptidyl Peptidase 4 (DP 4) and Neuropeptide Y (NPY): A novel pharmaceutical target against stress?” Neuropeptides 2010 (The 7th Joint Meeting of the European Neuropeptide Club and the American Summer Neuropeptide Conference) 21 - 24 June 2010, Pécs, Hungary.

Publications, Presentations and Posters

Posters:

Wagner, L., Wermann, M., Hoffmann, T., and Demuth, H.-U. “Purification of porcine dipeptidyl peptidase IV from the kidney and isolation of its isoforms by preparative isoelectric focusing.” International Symposium (IPS), 1 - 5 November 2001, Freising, Germany. Wagner, L., Hoffmann, T., Gerhartz, B., and Demuth, H.-U. “Distribution of dipeptidyl peptidase IV activity-like enzymes in canine and porcine tissue sections by nested RT-PCR” Dipeptidyl-: Basic Science and Clinical Applications. 26 - 29 September 2002, Berlin, Germany. Friedrich,D., Wermann, M., Wagner,L., Kühn-Wache, K., Hoffmann,T., and Demuth,H.-U. “Isolation and Characterization of Attractin-2” Dipeptidyl-Aminopeptidases: Basic Science and Clinical Applications. 26 - 29 September 2002, Berlin, Germany. Engel, M., Hoffmann, T., Wagner, L., Wermann, M., Heiser, U., Kiefersauer, R., Huber, R., Bode, W., Demuth, H.U., and Brandstetter, H. “1.8 Å Crystal structure of native porcine Dipeptidyl peptidase IV (CD26).” ScreenTech World Summit Protease Inhibitors. 23 - 27 March 2003 San Diego, USA. Wagner L., Rahfeld, J.-U., Niestroj,A.J., Heiser,U., and Demuth,H.-U. “Distribution of dipeptidyl peptidase IV activity-like enzymes in canine and porcine tissue sections by nested RT- PCR” 2nd International Conference of Dipeptidyl Aminopeptidases: Basic Science and Clinical Applications. 13 - 15 April 2005, Magdeburg, Germany. Wagner, L., Hoffmann,T., Rahfeld,J.-U., Niestroj,A.J., Heiser,U., and Demuth, H.-U. “Characterisation of Dipeptidyl peptidase IV-like activity enzymes in porcine pancreas and isolated islets of Langerhans” 7th Young Scientists Meeting of DGZ. 22 - 24 September 2005, Jena, Germany. Wagner,L., Frerker,N., Gündel,G., Wolf,R., Kehlen,A., Thümmler,A., Niestroj,A.J., Heiser,U., Rahfeld,J.-U., Hoffmann,T., von Hörsten,S., and Demuth, H.-U. “Characterization of DP 4-like Activity Enzymes and NP Y in Neuronal Tissue” 8th NPY Conference. 22 - 26 April 2006, St. Petersberg, Florida, USA. Wagner, L., Wolf, R., Zeitschel, U., Rossner, S., Petersen, A., Leavitt, B., Haydn, M., Kästner, M., Rothermundt, M., Gärtner U.-T., Weber, A., Schlenzig, D., Frerker, N., Schade, J., Manhart, S., Rahfeld, J.-U., von Hörsten S., and Demuth, H.-U. “Metabolomic investigations of neuropeptide Y (NPY) identified seven novel NPY-cleaving enzymes and revealed potential drug interactions.” Proteolytic Enzymes & Their Inhibitors (GRC). 2 - 7 May 2010, Lucca, Italy. Wagner,L., Wolf,R., Zeitschel, U., Rossner, S., Petersen, A., Leavitt, B., Haydn, M., Kästner, M., Rothermundt, M., Gärtner, U.-T., Weber, A., Schlenzig, D., Frerker N., Schade J., Manhart S., Rahfeld, J.-U., von Hörsten S., and Demuth, H.-U. “Metabolomic investigations of neuropeptide Y (NPY) identified seven novel NPY-cleaving enzymes and revealed potential drug interactions.” Neuropeptides 2010 (The 7th Joint Meeting of the European Neuropeptide Club and the American Summer Neuropeptide Conference). 21 - 24 June 2010, Pécs, Hungary. Wagner,L., Wolf,R., Zeitschel, U., Rossner, S., Petersen, A., Leavitt, B., Haydn, M., Kästner, M., Rothermundt, M., Gärtner, U.-T., Weber, A., Schlenzig, D., Frerker N., Schade J., Manhart S., Rahfeld, J.-U., von Hörsten S., and Demuth, H.-U. “Metabolomic investigations of neuropeptide Y (NPY) identified seven novel NPY-cleaving enzymes and revealed potential drug interactions.”6th Summer School of Mass Spectrometry for Biotechnology and Medicine” 3-9 July 2011, Dubrovnik, Croatia.

Content

Content

Abbreviations ______vi 1 Introduction______1 1.1 Dipeptidyl peptidase 4 ______1 1.1.1 General Properties ______1 1.1.2 Distribution ______3 1.1.3 Gene and Expression ______4 1.1.4 Glycosylation-based Heterogeneity ______4 1.1.5 DP 4-like Enzymes ______5 1.1.5.1 DP 4 Gene Family ______5 1.1.5.2 Non-related DP 4-like Enzymes ______9 1.1.6 Physiological Role of DP 4 ______11 1.1.6.1 Natural Substrates of DP 4 ______12 1.1.6.2 Binding Partners______13 1.1.6.3 Knock-out, Deficient and Transgenic DP 4-like Animal Models ______15 1.1.7 Involvement of DP 4 in Pathology ______17 1.1.7.1 Involvement of DP 4 in Diabetes Type 2 and Psychiatric Diseases ______20 1.1.7.1.1 Diabetes Type 2 ______20 1.1.7.1.2 Role of DP 4 Substrates in Behaviour ______22

1.2 Objectives and Introduction to the Present Study ______25 1.2.1 Characterisation of DP 4-like Enzymes in Diabetes Type 2 ______25 1.2.1.1 Crystallisation of DP 4 ______25 1.2.1.2 Distribution of DP 4-like Enzymes ______25 1.2.1.3 Analysis of DP 4- like Enzymes in Pancreas and Islets of Langerhans ______26 1.2.2 Identification of DP 4 as a Pharmaceutical Target for Psychiatric Disorders ______26 1.2.2.1 Analysis of the DP 4-like Enzymes in Neuronal Rat Tissue ______27 1.2.2.2 Analysis of Neuropeptide Hydrolysis by DP 4 in the Periphery ______27 1.2.2.3 Analysis of DP 4-like Enzymes in Sera from Depressed Patience ______27

2 Materials and Methods ______28 2.1 Materials ______28 2.1.1 Equipment ______28 2.1.2 Consumables______28 2.1.3 Chemicals ______28

2.2 Methods ______28 2.2.1 DP 4 Purification and Isolation of its Isoforms ______28 2.2.1.1 DP 4 Purification ______28 2.2.1.2 Isolation of purified DP 4 into its Isoforms ______29 2.2.1.2.1 Initial Parallel Rotofor Runs ______30 2.2.1.2.2 First Refractionation ______30 2.2.1.2.3 Second Refractionation ______30 2.2.1.3 Characterisation of Purified DP 4 and its Isolated Isoforms ______31 2.2.1.3.1 Enzyme Activity ______31 i Content

2.2.1.3.2 Determination ______31 2.2.1.3.3 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis ______31 2.2.1.3.4 Native Polyacrylamide Gel Electrophoresis (Native PAGE) ______32 2.2.1.3.4.1 Detection of Proteins after SDS-PAGE or Native PAGE______32 2.2.1.3.5 MALDI-TOF Mass Spectrometry ______32 2.2.1.3.6 Analytical Isoelectrofocusing Electrophoresis ______32 2.2.1.3.6.1 Protein and Activity Staining of Analytical IEF Agarose Gels ______33 2.2.2 Distribution Analysis of DP 4-like Enzymes ______33 2.2.2.1 Design of Primers ______33 2.2.2.2 Tissue IsIolation ______33 2.2.2.3 RNA Isolation ______34 2.2.2.4 Transcription ______34 2.2.2.5 PCR ______34 2.2.2.6 Nested PCR______34 2.2.2.7 Analysis of PCR Products ______34 2.2.2.7.1 Submarine Agarose Gel Electrophoresis ______34 2.2.2.7.2 Determination of DNA and RNA Concentrations ______35 2.2.2.7.3 Preparation of PCR Products for Sequencing ______35 2.2.2.8 Activity Analysis of DP 4-like Enzymes in Tissue Extracts ______35 2.2.2.8.1 Inhibition Studies with selective DP 4-like Inhibitors ______35 2.2.2.8.2 Protein Determination ______35 2.2.3 Analysis of DP 4-like Enzymes in Pancreas and Islets of Langerhans ______36 2.2.3.1 Isolation of Islets of Langerhans from Porcine Pancreas______36 2.2.3.2 Isolation of Islets of Langerhans from Canine Pancreas ______37 2.2.3.3 Isolation of Secretory Granules from Isolated Islets of Langerhans ______37 2.2.3.4 Characterisation of Porcine Islets of Langerhans and Secretory Granules ______38 2.2.3.4.1 Islet Viability ______38 2.2.3.4.2 Enzymatic Analysis ______38 2.2.3.4.2.1 Solubilisation ______38 2.2.3.4.2.2 Activity of DP 4-like Enzymes ______38 2.2.3.4.2.3 Inhibition Studies ______38 2.2.3.4.2.4 Protein Determination ______39 2.2.3.4.3 Activity Assay for Organelle Enzymes ______39 2.2.3.4.3.1 Alkaline Phosphodiesterase I: Marker of Plasma Membrane ______39 2.2.3.4.3.2 Lactate Dehydrogenase: Marker of Cytosol ______39 2.2.3.4.4 Analysis of DP 4-like Enzymes in Isolated Islets by RT-PCR ______39 2.2.3.5 Histochemical Analysis of Porcine Pancreas ______39 2.2.3.5.1 Immunhistochemical Analysis ______39 2.2.3.5.1.1 Production of Rabbit Anti-Porcine DP 4 Polyclonal Antibody ______39 2.2.3.5.1.2 Purification of Guinea Pig Anti-Glucagon Antiserum ______39 2.2.3.5.1.3 Sample Collecting ______40 2.2.3.5.2 Histochemical Activity Staining ______40 2.2.3.5.2.1 Staining of DP 4 with Gly-Pro-1-hydroxy-4-naphthylamide ______40 2.2.3.5.2.2 Staining of DP 2 with Lys-Ala-5-chloro-1-anthraquinonyl hydrazide ______41 2.2.3.6 Characterisation of Islets from Langerhans from Rats ______41 ii Content

2.2.4 Identification of DP 4 as a Pharmaceutical Target for Psychiatric Disorders ______42 2.2.4.1 Analysis of DP 4-like Enzymes in Neuronal Tissues ______42 2.2.4.1.1 Animals, Brain, Neuronal and Glial Tissues ______42 2.2.4.1.2 Tissue Extraction______42 2.2.4.1.3 Protein, Activity and Inhibition Studies ______42 2.2.4.1.4 MALDI-TOF-MS Studies ______42 2.2.4.1.4.1 Neuropeptide Hydrolysis by Neuronal, Glial and Brain Tissue Extracts ______43 2.2.4.1.5 Histochemical Analysis ______43 2.2.4.1.6 Capillary Depletion ______43 2.2.4.1.6.1 Identification of NPY Degrading Enzymes______44 2.2.4.2 Analysis of Neuropeptide Hydrolysis by DP 4 in the Periphery ______44 2.2.4.2.1 Human and Rat Serum ______44 2.2.4.2.1.1 Activity and Inhibition Studies ______44 2.2.4.2.1.2 Influence of Quenching Effects by Haemolysis on DP 4 Activity ______45 2.2.4.2.1.3 MALDI-TOF-MS ______45 2.2.4.2.2 Neuropeptide Hydrolysis ex vivo in Human Blood ______45 2.2.4.2.3 Human CSF ______45 2.2.4.2.3.1 Activity, Inhibition Studies and Protein Determination ______45 2.2.4.2.3.2 MALDI-TOF-MS ______45 2.2.4.2.3.3 Western Blot ______45 2.2.4.3 Characterisation of DP 4-like Enzymes in Sera from Depressed Patients ______46 2.2.4.3.1 Serum Samples from Depressed Patients ______46 2.2.4.3.1.1 Activity and Inhibition Studies ______46 2.2.4.3.1.2 Analysis of NPY and SP Hydrolysis by MALDI-TOF-MS ______46 2.2.4.3.1.3 Analysis of DP 4-like Activity by Analytical Agarose IEF ______46 2.2.4.3.1.4 Determination of DP 4 Concentration by sCD26 ELISA Kit ______46

3 Results and Discussion ______47 3.1 Purification of DP 4 and Isolation of its Isoforms ______47 3.1.1 Purification of DP 4 ______47 3.1.2 Isolation of DP 4 Isoforms______48 3.1.2.1 Parallel Rotofor Runs ______48 3.1.2.2 First Refractionation ______50 3.1.2.3 Second Refractionation ______51

3.2 Distribution of DP 4-like Enzymes in Porcine and Canine Tissues ______58 3.2.1 RT-PCR______58 3.2.1.1 Nested RT-PCR ______58 3.2.2 DP 4-like Activity in Porcine and Canine Tissues ______59 3.2.2.1 Specific Activities ______59 3.2.2.2 Inhibition Studies ______60

3.3 Analysis of DP 4-like Enzymes in Porcine Pancreas ______63 3.3.1 Isolation of Porcine Islets of Langerhans ______63 3.3.2 Activity Analysis of Isolated Islets ______63 3.3.3 Expression of DP 4-like Enzymes by RT-PCR ______65 iii Content

3.3.3.1 Nested RT-PCR ______65 3.3.4 Histochemical Analysis ______66 3.3.4.1 Activity Staining ______66 3.3.4.1.1 DP 4 ______66 3.3.4.1.2 DP 2 ______68 3.3.4.2 Immunohistochemical Staining ______68 3.3.5 Perfusion, Perifusion and Influence of Media Components on DP 4 Activity ______69

3.4 Identification of DP 4 as a Target for Psychiatric Disorders ______70 3.4.1 Analysis of DP 4-like Enzymes in Neuronal Rat Tissues ______70 3.4.1.1 Specific Activities of DP 4-like Enzymes in Neuronal Rat Tissues ______70 3.4.1.1.1 DP 4-like Acitivity in Wildtype and DP 4-deficient Fischer Rats ______70 3.4.1.1.2 DP 4-like Activity in Neuronal Primary Cell Cultures from Rats ______71 3.4.1.2 Histochemical Analysis______71 3.4.1.3 Specific Activity of DP 4 in Circumventricular Organs ______73 3.4.1.4 Analysis of Neuropeptide Hydrolysis ______74 3.4.1.4.1 No Hydrolysis of Neuropeptides by DP 2 ______74 3.4.1.4.2 Hydrolysis of Neuropeptides by Neuronal Tissues______75 3.4.2 Analysis of Neuropeptide Hydrolysis by DP 4 in the Periphery ______76 3.4.2.1 Capillary Depletion______76 3.4.2.1.1 Identification of NPY-Metabolising Enzymes ______77 3.4.2.2 Hydrolysis of NPY in Human CSF ______81 3.4.2.3 Processing of Neuropeptides in the Blood Circulation ______81 3.4.2.3.1 In vitro Metabolism of NPY in Human Serum ______81 3.4.2.3.2 Metabolism of Stress-related Neuropeptides in Human and Rat Sera ______82 3.4.2.3.3 Ex vivo Metabolism of Stress-related Neuropeptides in Human Blood ______84 3.4.3 Analysis of DP 4-like Enzymes in Sera from Depressed Patients ______85 3.4.3.1 DP 4-like Activity in Sera from Depressed Patients ______85 3.4.3.2 Hydrolysis of NPY in Sera from Depressed Patients ______86

4 Conclusion ______87 4.1 Crystal Structure ______87

4.2 Distribution of DP 4-like Enzymes in Porcine and Canine Tissue Sections ______92

4.3 Analysis of DP 4-like Enzymes in Porcine Pancreas ______94

4.4 Identification of DP 4 as Pharmaceutical Target for Psychiatric Diseases ______97

5 Summary ______104 6 Zusammenfassung ______106 7 References ______109 8 Appendix ______142 8.1 Introduction ______142 8.1.1 DP 4-like Enzymes ______142

8.2 Material and Methods ______144 8.2.1 Materials ______144 iv Content

8.2.2 Methods ______149 8.2.2.1 Isolation of DP 4 Isoforms from Porcine Kidney ______149 8.2.2.2 Distribution of DP 4 – like Enzymes in Porcine and Canine Tissues ______150 8.2.2.3 Specific Activities of DP 4-like Enzymes in Porcine and Canine Tissues ______152 8.2.2.4 DP 4-like enzymes in pancreas and islets of Langerhans ______153 8.2.2.5 Identification of DP 4 as a Pharmaceutical Target for Stress Disorders ______155

8.3 Results ______159 8.3.1 Isolation of DP 4 Isoforms from Porcine Kidneys for Crystallisation ______159 8.3.2 Results of RT-PCR from Porcine and Canine Tissues ______160 8.3.3 DP 4-like Activities in various Porcine and Caninine Tissues ______162 8.3.4 DP 4 and NPY as a Pharmaceutical Target for Psychiatric Diseases ______164 8.3.5 Characterisation of NPY Hydrolysis in Sera from Depressed Patients ______168

8.4 Conclusion ______169 8.4.1 DP 4- and DP 4-like Inhibitors ______169 8.4.2 Characterisation of DP 4-like Enzymes in Porcine Pancreas ______174 8.4.3 Identification of DP 4 as a Target for Psychiatric Disorders ______176

9 List of Figures ______181 10 List of Tables ______189 11 Acknowledgement ______191

v Abbreviations

Abbreviations 3V/4V 3rd/4th ventricle aa Ab/pAb/mAb Antibody/polyclonal antibody/monoclonal antibody Ac Nucleus accumbens ACE Angiotensin-converting enzyme ACN Acetonitrile ACTH Adreno-corticotropic hormone ADA Adenosine deaminase ADAbp Adenosine deaminase binding protein ADHS Attention deficit hyperactivity syndrome AIDS Acquired immune deficiency syndrome Ala Alanine AMC /-AMC 7-Amino-4-methylcoumarin/7-Amido-4-methylcoumarin amu Atomic mass unit Amy Amygdala AP Area postrema APC Antigen presenting cell AP N N Arc Nucleus Arcuate Att Attractin AVP Arg-vasopressin BCA Bicinchoninic acid BSA Bovine Serum Albumin CAH 5-chloro-1-anthraquinonylhydrazide CD Cluster of differentiation cDNA Copy DNA CLIP Corticotropin-like intermediate lobe peptide CN-Pyr Cyano-pyrrolidine CNS Central nervous system CRF Corticotropin-releasing factor CSF Cerebrospinal fluids CTSB Cathepsin B CTSD Cathepsin D CTSG CTSL Cathepsin L CTSS Cathepsin S CVO Circumventricular Organ Da Dalton DAB/Dab-Pip Diaminobutyryl piperidinamide DAHC Diammonium hydrogen citrate DCI 3,4-dichloroisocoumarin DEAE Diethylaminoethyl DEPC Diethylpyrocarbonate DHAP 2´, 6´-dihydroxyacetonephenone DMF Dimethylformamide DNA Desoxyribonucleic acid DP 2 Dipeptidyl peptidase 2 DP 4 Dipeptidyl peptidase 4 DP 6 Dipeptidyl peptidase 6 vi Abbreviations

DP 7 Dipeptidyl peptidase 7 DP 8 Dipeptidyl peptidase 8 DP 9 Dipeptidyl peptidase 9 DP 10 Dipeptidyl peptidase 10 DPL - 1/2 Dipeptidyl peptidase like 1/2 DSM IV Diagnostic and Statistical Manual of Mental Disorders IV EAE Experimental autoimmune encephalomyelitis EC Endothelial cells ECM Extracellular matrix EGF Epidermal-growth factor EPM Elevated plus maize EtOH Ethanol FAP Fibroblast activation protein  FBS Fetal bovine serum FDA Fluorescein diacetate FITC Fluorescein isothiocyanate FN Fibronectin Fuc Fucose GABA -butyric acid Gal Galanin GALP Galanin-like peptide GCP-2 Granulocyte chemotactic protein - 2 GHRH Growth hormone releasing hormone GIP Glucose-dependent insulinotropic peptide GlcNAc Glucosamine GLP Glucagon-like-peptide Gly Glycine GRF Growth hormone releasing factor GRP Gastrin-releasing peptide HCCA -cyano-4-hydroxy-cinnamic acid HEPES N-2-Hydroxyethylpiperazine-N’-2-ethane sulfonic acid HIV Human immunodeficiency virus HIV-Tat protein Human immunodeficiency virus trans activatior protein hK1 Human kallikrein 1 / tissue kallikrein HPA Hypothalamic-pituitary adrenal HUVEC Human umbilical vascular endothelial cells

IC50 Inhibition constant ICD-10 International Classification of Disease and Related Health Problems icv Intracerebroventricular IEF Isoelectric focusing IGF-1 Insulin-like growth factor – 1 IGFIIR Insulin-like growth factor II receptor IgG Immunoglobulin G Ile Isoleucine IP - 10 Interferon –  inducible protein Kal Plasma Kallikrein

KD Diassociation equilibrium constant Ki Inhibitory constant

Km Michaelis Menten constant k.o. Knock-out LAP-1 Los Angeles preservation solution vii Abbreviations

LC Locus coerulus LD Laser desorption LS Lateral septum Lys Lysine M6P Mannose-6-phosphate M6PR/IGFRII Mannose-6-phosphate-receptor/insulin-like-growth factor receptor II mAb Monoclonal antibody MALDI-TOF MS Matrix assisted laser desorption time of flight mass spectrometry MADRS Montgomery and Asberg Depression Scale MCP 1/2/3 Monocyte chemotactic protein – 1/2/3 ME Median eminence 4-Me-2-NA 4-methoxy-2-naphtylamide Mep-A Meprin-A Mep-B Meprin-B Mig Monokine induced by interferon- min Minutes MS Mass spectrometry MS* Multiple Sclerosis NAALADASE N-acetylated α-linked acidic NBT Nitro Blue Tetrazolium NCS Newborn calf serum nd Not determined NDRIs Norepinephrine-dopamine-reuptake inhibitors NE Norepinephrine NEP Neprilysin NK-cells Natural killer cells NPY Neuropeptide Y NRIs Norepinephrine reuptake inhibitors NSA Neuro-sympathico-axis NT Neurotensin NTS Nucleus tractus solitary 1-OH-4-NA 1-hydroxy-4-naphtylamide OP Open field test ORF Open reading frame OrxA Orexin A OrxB Orexin B OVLT Organum vasculosum of lamina termianlis Oxy Oxytocin pAb Polyclonal antibody PACAP Pituitary adenylate-cyclase activating peptide PAG Periaqueductal gray Pb Parabracheal nucleus Pbd Probiodrug AG PBMC Peripheral blood mononucleated cells PBS Phosphate balanced solution PCR Polymerase chain reaction PCT Procalcitonin PEG Polyethylene glycol PEP/POP Prolyl endopeptidase/Prolyl Pg 2 Plasminogen type 2 PHM Peptide histidine-methionine viii Abbreviations

PK Plasma Prekallikrein PLP-IEF Preparative liquid phase isoelectric focusing Pl Plasmin pNA/-pNA para-nitroaniline/-para-nitroanilide pNBA para-nitrobenzaldehyde POP/PEP Prolyl oligopeptidase/Prolyl endopeptidase PP Pancreatic polypeptide PTP Protein tyrosine phosphotase Pro Proline PVN Paraventricular nucleus PVS Perivascular space PYY Peptide YY QPP Quiescent proline cell dipeptidase r Correlation coefficient RA Retinoic acid RACE Rapid amplification of cDNA ends RANTES Regulated upon activation, normal T-cell expressed and secreted Ref Reference RNA Ribonucleic acid RT-PCR Reverse transcriptase polymerase chain reaction SA Sinapinic acid SbTI Soybean trypsin Inhibitor SDF-/ Stromal derived factor / SDM Standard deviation of mean SDS Sodium dodecylsulfate SDS-PAGE Sodium dodecyl-polyacrylamide gel electrophoresis sec seconds SEM Standard error of mean SFO Subfornical organ SHR Spontaneously hypertensive rat SI Social interaction test Sia Sialic acid SK Streptokinase SNP Sodium nitroprusside SNRIs Serotonine-norepinephrine reuptake inhibitors SP Substance P SSRIs Selective serotonine reuptake inhibitors TAC1 Tachykinin 1 gene TCA Trichlor acetic acid TEMED N,N,N’,N’-Tetramethylethylenediamine TFA Trifluor acetic acid Tris Tris(hydoxymethyl)aminomethane TRITC Tetramethyl rhodamine isothiocyanate U Units uPA Urokinase/urine plasmin activator UV Ultraviolet VIP Vasoactive intestinal peptide VPACR VIP/PACAP receptor WKY Wistar Kyoto rat (normotensive)

Xaa Amino acid Y1 – 5-R’s Receptors of neuropeptide Y, peptide YY or/and pancreatic polypeptide ix Introduction

1 Introduction

Nature has evolved a number of regulatory, neuronal and immune peptides with a proline residue at the penultimate position determining their structural conformation and biological activity. The proline peptide bond has shown to be resistant to proteolytic cleavage and therefore an exclusive number of post-proline specific peptidases have emerged to regulate these peptides (1). The best-characterised one so far is dipeptidyl peptidase 4 (DP 4) (2). However, only recently, five additional post-proline dipeptidyl aminopeptidases, i.e. functional homologues, have been discovered, some structurally related, others without any structural homology to DP 4 (3). Since DP 4 is involved in glucose homeostasis and immune response, it is of medical and pharmaceutical interest to pursue structural and distribution analysis of these enzymes.

1.1 Dipeptidyl peptidase 4 1.1.1 General Properties DP 4 [EC 3.4.14.5] was first discovered in 1966 by Hopsu-Havu and Glenner, and denoted with glycyl-prolyl--naphtylamidase (4). Other names include post-proline dipeptidyl aminopeptidase IV, glycoprotein gp 110 (5), peptide Hep 105, glycoprotein gp 108 (6), adenosine deaminase binding protein ADabp (7) and T cell activation antigen CD26 (8). The enzyme is a that hydrolyses proline, hydroxyproline, dehydroproline, to a lesser extent alanine, and at slow rates serine, glycine, threonine, valine and leucine at the penultimate position (9). To allow , the N-terminus must be protonated, P1 and P2 in trans configuration and the P’1 position must not be occupied by proline or hydroxyproline (Figure 1) (10).

DP 4

P2 - P1 - P1’ - P2’ - P2’

+ - H3N - - Xaa Pro Yaa

Hyp Pro Ala HYP Ser Gly Val Thr Leu

Figure 1: Substrate specificity of DP 4. Xaa and Yaa indicate any amino acid. Decreasing font of amino acid at P1 position represents declining rate of hydrolysis. Amino acids crossed out must not occupy P1’. Arrow indicates site of cleavage (2;10).

DP 4 is a homodimer and an integral type II glycoprotein anchored to the membrane by its signal peptide. The primary structure consists of a short six amino acid cytoplasmic tail, a 22 amino acid transmembrane, a 738 amino acid extracellular portion comprised of a flexible stalk, glycosylation rich region, cysteine rich region and catalytic region with the

1 Introduction

Ser630, Asp708 and His740 (Figure 2). Recently, the crystal structures of human and porcine DP 4 have been elucidated revealing two domains, an 8 bladed propeller and a /- domain. The propeller is open and consists of two subdomains made up of blades II-V and VI-VIII, I, respectively. Each blade has four anti-parallel -sheets, except for blade IV that has an additional -helix and two -sheets forming an extended arm. Dimerisation occurs hydrophilically at the extendend arm and hydrophobically at the /-hydrolase domain as well as at the transmembrane domain (11). There are two openings, a side opening and a propeller tunnel (Figure 2) (12;13). In a recent crystal structure, the substrate NPY suggested an entry at the side opening (14).

IVb C A B 90° V COOH IV VI 766 III 740 H 708 D Propeller Catalytic /-Hydrolase 630 S opening domain domain VII II 506 I VIII

Cysteine-rich Propeller region 351 domain Glu206 D Asn710 Glycosylation- Glu205 Arg125 rich region Side Asp708 100 Val-Pyr 51 C C opening His740 /-Hydrolase domain N Tyr666 Flexible stalk 39 N Transmembrane Ser 6 630 Intracellular tail Tyr547 1 NH2

Figure 2: Primary and quaternary structure of human DP 4, based on pdb: 1N1M. A, primary structure of DP 4 subunit, consisting of an intracellular tail (aa 1 - 6), transmembrane region (aa 7 - 28), flexible stalk (aa 29 - 39), glycosylated-rich region (aa 101 - 350), cysteine-rich region (aa 55 - 100, 351 - 497) and catalytic region (aa 506 - 766). , N-glycosylation; , potential unoccupied N-glycosylation; , cysteine residues involved in S-bridges; red numbers and letters indicate the catalytic triad. B, quaternary structure of homodimeric human recombinant DP 4 as determined by Rassmussen et al., 2003, (11) showing the /- hydrolase domain (aa 39 - 51 and 506 - 766) in green and propeller domain (aa 55 - 497) with the glycosylation-rich subdomain (red) and the cystein-rich subdomain (blue). Inhibitor Val-Pyr is presented in spheres, indicating that catalysis occurs at the interface of the two domains. C, Propeller domain viewed from the top, illustrating the 8 propeller blades designated with roman numbers and two subdomains. D, zoomed in, depicting the residues involved in catalysis, catalytic triad Ser630, Asp708, His740 are shown in red, Tyr547 responsible for in pale green, Tyr666 forming the hydrophobic pocket in beige, Arg125 and Asn710, contributing to an electrostatic sink in yellow and orange, respectively, and Glu205 and Glu206 ensuring N-terminal anchoring in pink; inhibitor Val-Pyr is presented in cyano. S-S bridges are illustrated in yellow and carbohydrates in orange. The broken line separates propeller from /-hydrolase domain. Structures were drawn with pymol, using pdb: 1N1M.

2 Introduction

1.1.2 Distribution DP 4 is distributed ubiquitously, with the highest expression in the kidney, liver and small intestine (2). It is predominantly found on endothelial and epithelial cells throughout the body (15). Table 1 summarises the distribution of DP 4 in various tissues. Table 1: Tissue distribution of DP 4 in various species as detected by different methods. Relative Method§ Species Organ Tissue abundance* Reference I, H, A Rat (16-19) Brush border membrane of + + + + + I Rabbit (20) proximal tubule cells I Human (21) Kidney Capillary endothelium of I,H Rat (16;18;19) + + + glomeri H Guinea pig, rat (17) Epithelium of Bownman's I,H Rat (16;18;19) + + + capsule H Guinea pig, rat (17) H,I Guinea pig, rat (18;19;21) Hepatocytes + + + + Liver I, P, A Rat (22) Bile canaculi duct + + + + I, P, A Rat (5) Microvilli zone of intestinal I,H Rat (16;18) + + + + crypts and villi H Guinea pig (17) Small Brush border membrane of I,H Rat (16;18) intestinal enterocytes and + + + H Guinea pig (17) Intestines lysosome I Human (21) Golgi apparatus of some + + I Rat (16) intestinal enterocytes H Guinea pig, rat (17) Bile canaculi + + + P, A Pig (23) Pancreas H Rat (18) Excretory duct + + + P,A Pig (24) A Human (25) Alveoli + + + + P, A Pig (26) Lung A, H Rat (27;28) Bronchial epithel +++ H Guinea pig, rat (17) Secretory vesicles, acinar I,H Guinea pig, rat, (16;17) Salivary gland + + + + cells of parotid gland A Rat (27;29) Placenta + + + A Human (30;31) Reproductive Uterus + + H Guinea pig, rat (17) organs, Fallopian tube + + H Guinea pig, rat (17) female Luteal cells + + + I,A Human (32) Reproductive Epididymis + H Guinea pig, rat (17;18) organs, male Seminal fluid + + P, A Human (33;34) Epithelial cells + + + A Guinea pig (35) A Guinea pig (35) Ubiquitous Fibroblasts + + A Human (36) tissue Vascular endothelial cells + + + A, R Human (33;37) Lymphatic endothelial cells + + + A Human (38) Soluble shedded DP 4, A Rat (27) Serum/Blood + + + + Lymphocytes A Human (33;34) R Rat (39) Spleen + + + A Human (37) Thymus Lymphocytes + + + A Human (40) Lymph nodes + + + A Human (40) Urine + + A Human (41) H Guinea pig, rat (17) Brain + A Rat (27) H Guinea pig (42) Spinal cord Meningeal lamellae + + A Human (43) A Rat (27) H Guinea pig, rat (17) Heart + R Rat (39) H Guinea pig, rat (17) Stomach + R Rat (39) Muscle + R Rat (39) * Different methods have been applied in the various references, thus distribution is described only qualitatively. Level of activity/expression: + + + + + = very high ; + + + + = high; + + + = medium ; + + = low, and + = very low . § Abbreviation of methods: I, immunostaining; H, histochemical staining with substrate; A, activity analysis; P, purification; R, RNA analysis by Northern blot. 3 Introduction

1.1.3 Gene and Expression The gene structures of human and mouse DP 4 have been studied in detail and found to be almost identical, mainly varying in size of the genes as well as exons (44;45). In human, the gene is located on chromosome 2q24.3, spans 81.8 kb and contains 26 exons. The nucleotides encoding the sequence around the active site serine (Gly-X-Ser-X-Gly) are split between exons 21 and 22. Likewise, the exons of the catalytic triad are 22 for Ser, 24 for Asp and 26 for His (44). DP 4 contains neither TATAA nor CCAAT box as a promoter, but has a C and G rich region containing several consensus binding sites for transcriptional factors (46;47). Expression is regulated at RNA level and is organ specific (39;48-50). Within an organ it is dependent on cell type, differentiation state and activation state (2). Several cytokines are known to regulate DP 4 expression in a cell-type specific manner (48;51-54). In some pathogenic tissues the binding of the transcription factors is enhanced by certain cytokines (55).

1.1.4 Glycosylation-based Heterogeneity Carbohydrates contribute about 18 - 25 % of the total molecular weight, and mammalian DP 4 has been found to contain 8 - 11 potential N-glycosylation sites (2;13;56-60). Structural analysis of oligosaccharides from rat kidney revealed extensive hetereogeneity composed of one high mannose type and several mono-, bi-, tri and tetraantennary complex type of N- glycans (61). Detailed studies of sialylation of N-glycans from rat liver DP 4 showed predominantly bisialylation, followed by mono- and trisialylation, to a lesser extent tetrasialylation and only small traces of pentasialylation (62). Predominantly sialylation based microheterogeneity is observed when purified DP 4 is transferred onto an analytical IEF, resulting in a pattern of several bands between pH 3.5 - 6.0. The number of bands and pH of such IEF pattern is species-, tissue- and epitope-specific as well as dependent on the differentiation state of cells and age (63-66). While co-translational core N-glycosylation is responsible for folding and stability of DP 4 (67-69), N-terminal sialylation appears to play a more physiological and pathological role as summarised in Figure 3.

Properties and Functions of DP 4 Sialylation  Bisialylation > monosialylation = trisialylation > tetrasialylation HO >> pentasialylation  Species specific HO H -  Tissue specific O O HO H  Epitope specific  Dependent on cell differentiation state O  Age dependent OH  Dynamic: De- and resialylation OH  Facilitates trafficking to apical membrane

Increased Sialylation: Decreased Sialylation: HN CH3  HIV infection  Lung cancer  Rheumatoid arthritis  Activated T-cells O  Systemic Lupus erythematosus  Young age Sialic acid  Old age  Resting T-cells Figure 3: Structure, properties and functions of DP 4 sialylation (25;62-65;70-78). 4 Introduction

Resting T-cells were determined to be more sialylated than activated ones (64). Hypersialylation has been associated with HIV-infection, rheumatoid arthritis, systemic lupus erythematosus and aging (64;78), whereas decreased sialylation was found in lung cancer (25). The process of sialylation seems to be dynamic, as de- and resialylation was detected in rat hepatocytes (70-74;79). Furthermore, trafficking of soluble transfected mouse DP 4 to the apical surfaces of Caco-2 and HT-29 cells was greatly influenced by terminal sialylation (80;81). In addition, DP 4 has also been reported to be O-glycosylated (76;77). Recent crystal structure of bovine ADA bound to human DP 4 showed the interaction of carbohydrates at N-229 of DP 4 with ADA (82).

1.1.5 DP 4-like Enzymes Lately, a number of enzymes have been discovered also to display DP 4 activity. These enzymes can be grouped into the DP 4 gene family and non-related DP 4-like enzymes such as dipeptidyl peptidase 2. 1.1.5.1 DP 4 Gene Family DP 4 belongs to the serine peptidase clan SC, subfamily 9b. Peptidases of the SC clan have a unique catalytic triad in the order of Ser, Asp and His located in a /-hydrolase fold compared to the chymotrypsin catalytic triad of His, Asp, Ser. The SC9 family, also referred to as prolyl oligopeptidase family, consists of four subfamilies represented by their respective enzymes prolyl endopeptidase (9a), dipeptidyl peptidase 4 (9b), acyl aminoacyl peptidase (9c) and only a recent member glutamyl endopeptidase (9d) [http://merops.sanger.ac.uk/famcards/s9.htm] (83). Currently, six members have been identified belonging to the dipeptidyl peptidase subfamily 9b, including dipeptidyl peptidase 4, fibroblast activation protein alpha (FAP) (84), dipeptidyl peptidase 8 (DP 8) (85), dipeptidyl peptidase 9 (DP 9) (86), dipeptidyl peptidase 4-like protein 1 (DPL 1, DP 6, DPPX L, DPPX S) (87) and dipeptidyl peptidase 4-like protein 2 (DPL 2, DP 10) (88). Their amino acid identities and similarities, phylogenetic relationship, based on cDNA, as well as their arrangement of structural domains are outlined in Figures 4 and 5, respectively, whereas Table 2 summarises the order of the catalytic serine and arrangement at the active site. A comparison of their properties, in turn, is given in Table 3. Except for DPL 1 and DPL 2 that contain each a mutation at the active serine, all members display dipeptidyl peptidase 4-like activity with neutral to basic pH optima and similar inhibition profiles (85;89-91).

Table 2: Order of catalytic triad and arrangement around serine recognition sites of DP 4-like enzymes (92;93). Clan SC Clan SC Classification Family S9b Family S28 DP 4, FAP, Enzyme DP 2 DP 8, DP 9 Order of catalytic residues Ser-Asp-His Ser-Asp-His Arrangement around serine GWSYG GGSYG GxSxG

5 Introduction

FAP DPL 1 DPL 2 DP 8 DP 9 A 760aa 865aa 789aa 882aa 969aa B FAP DP 4 52 % 33 % 36 % 26 % 25 % 766 aa 71 % 55 % 57 % 49 % 47 % DP 4

FAP 32 % 34 % 26% 23 % DP 9 760aa 54 % 55 % 49 % 48 % DPL 1 53 % 27 % 25 % DP 8

865 aa 70 % 52 % 47 % DPL 1- long DPL 2 24 % 26 % 789 aa 48 % 49 % DPL 1- short DP 8 58 % DPL-2 882 aa 72 %

Figure 4: Phylogenetic relationship between the members of the S 9b subfamily (DP 4-gene family). A, percent of amino acid identity (bold) and similarity (92); B, dentogram showing the phylogenetic relationship of human members of the S9b subfamily. The analysis was based on the cDNA sequences of DP 4, FAP, DP 8, DP 9, DPL 1-long, DPL 1-short and DPL 2, using standard parameters in Vector NTI 6.

Figure 5: Schematic presentation of the proteins belonging to the DP 4-gene family of S9b. The arrangement of structural domains is depicted. Approximate positions of N-glycosylation sites, cysteine residues and some residues required for enzyme activity and ADA or antibody sites are shown. Not to scale. [Taken and adapted from Gorrell, 2005 (94)].

FAP, also referred to as seprase, has the highest sequence identity to DP 4 and is believed to arise from gene duplication due to its gene proximity (84). FAP has recently been shown to readily hydrolyse NPY, BNP, substance P and PYY as well as to a lower rate GLP-1 and GIP, whereas chemokines were not truncated by FAP (95). In addition to DP 4 activity, FAP also exhibits gelatinase and collagenase activity, which is collagen type I specific. Its endopeptidase activity requires the sequence Xaa-Gly-Pro-Yaa… as shown in Appendix 1 (96;97). Recently, the crystal structure of FAP has been elucidated and comparison with the crystal structure of DP 4 points to a lower anchoring of substrates by Glu203-Glu204 due to shielding effects of surrounding hydrophobic residues and lack of Asp663. This in turn, results in a lower activity and enables its endopeptidase activity as confirmed by site-directed mutagenesis with subsequent kinetic studies (98). Although FAP expression is restricted to reactive stromal fibroblasts of epithelial cancers, subsets of bone and soft tissue sarcomas, activated stellate cells, arthritic chondrocytes as well as granulation tissue of healing wounds (92;94;99;100), co-expression of DP 4 and FAP in these cells results in the formation of a heteromeric complex (101). The DP 4 activity is maintained by both enzymes in the complex 6 Introduction as well as gelatine degradation by FAP (102). In tumourgenic cells and wounds, this heteromeric complex is localised on the advancing portion of invadopodia that is believed to play an important role in tumour invasion, spreading of metastasis, angiogenesis (103-107) and wound healing, respectively (102). Thus, as to identify it as a pharmaceutical target, the expression of FAP has been investigated in several types of cancers and been associated with epithelial colon-, gastric-, intestinal-, esophageal-, undifferentiated thyroid-, lung-, breast-, ovarian- and cervical-cancers, cutaneous melanoma as well as aggressive fibromatosus (108- 120). Interestingly, it was shown that stromal FAP is more prominent in early-stage of colorectal cancer and smaller tumour xenografts, with increased expression of FAP being an adverse prognostic indicator in patients with advanced metastatic disease. In contrast, prolonged survival of patients with breast cancer was associated with high FAP expression, whereas in cervical cancer, FAP was correlated with increased dysplasia and carcinoma development, suggesting FAP being an invasion marker (109;110;113). Co-localisation of FAP and urokinase-type plasminogen activator receptor Annexin2 (AnxA2) was detected in malignant melanoma by FRET and the complex appeared to be dependent on both cytoskeleton and integrins (121). Knock-out mice of FAP confirmed its role in wound-healing, however no change of phenotype was observed with regards to cancer (122). Expression of FAP was found to be up-regulated by IL-1 and oncostatin M in arthritic chondrocytes from patients with osteoarthritis, while it was down-regulated in patients with systemic lupus erythematosus (100). Recently, a soluble form has been found in serum where it was shown to hydrolyse 2-antiplasmin to promote fibrosis (123;124). Thus, as opposed to DP 4 that is involved fibrinolysis and scar resolution, FAP promotes fibrosis and scar formation (92). A possible role of FAP in neutropenia and anemia has only been recently elucidated (125;126). Dipeptidyl peptidase 8 (DP 8) consists of 882 amino acids and has a molecular weight of 100 kDa (84). Although DP 8 has previously been reported to be monomeric, current data gave strong evidence for a dimeric structure with a suggested molecular weight above 200 kDa (127). So far, it has been proposed to be located in the cytoplasm as a soluble protein and up to now, there has been no evidence for any secretion (85;128). Recent proteomic screening has revealed phosphorylation of Tyr331 and Thr334 (129). Using several chromogenic substrates, DP 8 was shown to display post-proline dipeptidyl aminopeptidase activity similar to that of DP 4 as depicted in Appendices 1 and 2 (85;127). Hydrolysis of NPY, GLP-1, GLP-2, PYY, ITAC, IP-10, SDF-1 and SDF-1, but not of MIG, Gro and Eotaxin could be demonstrated in vitro, though the rate of cleavage was slower compared to DP 4, in particular for PYY as illustrated in Appendix 3 (127;130). DP 8 mRNA is ubiquitously distributed with its highest expression in testis, prostates, ovaries, placenta and brain (Table 4) (85;88;131). Furthermore, it is up-regulated in activated lymphocytes (84). However, its physiological function is presently unknown and still awaits further studies. The human gene localisation is 15q22. Dipeptidyl peptidase 9 (DP 9) has previously been reported to be active as a cytosolic monomer comprised of 863 amino acids with a molecular weight of approximately 100 kDa (90). It lacks a transmembrane domain and is found intracellular near the Golgi apparatus, though secretion from transfected cells has not been observed yet (90). Further ORFs of 2913 bp (127) and 3006 bp (90) have been described. Recently, a new DP 9 variant with another start site in a prolonged ORF has been found, leading to an enzymatically active protein of 892 amino acids. This variant was shown to be active as homodimer with an estimated molecular weight above 200 kDa, whereas no activity could be detected for the 7 Introduction shorter variant comprised of 863 amino acids (127). Using several chromogenic substrates, DP 9 exhibited post-proline dipeptidyl aminopeptidase activity similar to that of DP 4 and it was shown to truncate NPY, GLP-1, GLP-2 and to a far lesser extent for PYY in vitro as illustrated in Appendices 1 - 3 (90;127). However, recently the cytoplasmic proteasome-derived antigenic peptide RU134-42 could be identified as the first natural substrate of DP 9, suggesting DP 9 to play an important role in peptide turnover and antigen presentation (132). Furthermore, DP 9 was also shown to enzymatically inactivate Akt, thereby interfering with EGF-signaling (133). DP 9 contains an Arg-Gly-Asp cell attachment motif and 2 potential glycosylation sites, though SDS-analysis of expressed DP 9 revealed no mass differences based on deglycosylation (86;88). Like DP 4 and DP 8, DP 9 mRNA is ubiquitously distributed, with its highest expression in liver, heart and skeletal muscle (Table 4) (86;88;90). Its physiological function has not been elucidated yet, though an up-regulation of DP 9 mRNA was detected in human testicular tumour (134). The gene is located on chromosome 19p13.3. So far, one can not differentiate between DP 8 and DP 9 enzymatic activity due to lack of selective inhibitors, however DP 8/DP 9 activity could be detected in human leukocytes, rat brain, lung and testis, bovine testis, murine brain, organs of the immune system such as thymus, spleen, lymph nodes and PBMC, testis, skeletal and uterine muscles as well as colon (28;131;134-140). Nevertheless, brain and testis have been the only organs in which DP 8/DP 9 activities precede over DP 4 activity (134-139). Association of DP 8 and DP 9 with H-Ras suggests a functional role in signal transduction (133). Furthermore, DP 8/DP 9 appear to be involved in T- cell proliferation thereby releasing IL-2 as well as macrophage activation causing activation of caspase 1 and induction of IL-1 (141-144). Yet, the suggested cytotoxicity of DP 8/DP 9 inhibition is currently controversially discussed (145-148). Recently, an increase of DP 8/DP 9 activity has been associated with asthma (28). Extra-enzymatic functions of DP 8/DP 9 include cell adhesion, migration and apoptosis (149). Interestingly, similar to PEP, but unlike DP 4 and

FAP, DP 8 and DP 9 are reversibly inactivated by H2O2 oxidation involving two cysteins in each monomer (150). Up to now, there are not any crystal structures available, neither of DP 8 nor of DP 9, although molecular modelling based on DP 4 and FAP crystal structures indicate similar overall structures comprised of a -propeller and a -hydrolase domain, respectively, with the active site being located at the interphase of the two domains. However, two loops and one helix of the propeller domain extending to the interphase cavity appear to play a role at the active site. While the R125-loop and the E205E206-helix have been described in the crystal structures of DP 4, the P2-loop, containing F357 and R358, seems to be unique to DP 8 and

DP 9 and is suggested to influence substrate and inhibitor binding to the P2-pocket (12;13;151- 154). Due to the shortest gene sizes, lowest numbers of exons, the active site being located on one exon in comparison to DP 4 and FAPand their closest phylogenetic relationship with respect to prokaryotic members of the family, DP 8 and DP 9 are believed to be the ancestral genes of the DP 4 gene family (92). Dipeptidyl peptidase-like proteins 1 (DPL-1) and 2 (DPL-2) lack DP 4-like activity due mutations at the active site. Both of them are type II membrane-bound glycoproteins, suggested to interact with the voltage-gated potassium channel Kv4 (87;88;92;128;155-159). While DPL-1 is exclusively expressed in the brain as two variants, i.e. a short and a long form, DPL-2 is found in brain, pancreas and adrenal gland (88;158). The crystal structure of DPL-1 resembles that of DP 4 (151). DPL-2 has additionally been associated with asthma (28;155;160- 162). Table 3 summarises the properties of all the members of the DP 4 gene family. 8 Introduction

1.1.5.2 Non-related DP 4-like Enzymes In addition, enzymes structurally unrelated to the DP 4 gene family have also been reported to display DP 4 activity (3). These include Dipeptidyl peptidase 2 (DP 2, EC3.4.14.2) of SC clan 28 (163), attractin (164) and N-acetyl alpha-linked acidic (NAALADases I, NAALADases II and NAALADases L) from the metalloprotease clan MH, family M28B (165). However, elevated DP 4-like activity of the NAALADases has previously been detected only in crude extracts after cloning and expression (165), whereas detailed kinetic analysis of expressed and purified NAALADase I did not reveal any DP 4-like activity (166). Likewise, the DP 4-like activity of attractin in serum has been controversially discussed for several years (164;167-169) and only recently been disproved (170). DP 2 has been identified with quiescent proline cell dipeptidase (QPP) based on genetic homology and kinetic parameters (171-173). The soluble serine protease contains a proform and has a length of 492 amino acids with a molecular weight of 58 kDa (174-176). Glycosylation and dimerisation are required for the catalytic activity and latter occurs via a leucine zipper motif, which is novel for (175;177). The homodimer is located in cellular vesicles that are distinct from lysosomes and secretion is regulated by an increased Ca2+ flux (175). Using chromogenic substrates, DP 2 displays post-proline dipeptidyl aminopeptidase activity similar to DP 4, however over a broad pH range with an acidic to neutral pH optimum as shown in Appendices 1 and 3 (172-174;176;178). While DP 2 readily hydrolyses tripeptides, its activity decreases rapidly with increasing chain length of peptide.

Thus, it was shown to cleave only fragments of substance P1-4, bradykinin1-3 or bradykinin1-5 (135;163;179). DP 2 has been reported to be involved in apoptosis as a decrease of DP 2 activity caused cells to exit their G0-phase in quiescent lymphocytes and fibroblasts, resulting in an induction of apoptosis by up-regulation of p53 and c-Myc as well as a down-regulation of Blc-2 (180-182). Furthermore, DP 2 was found to be essential for maintaining cell quiescence of lymphocytes, in which the transcription factors KLF2 and TOB1 regulate the expression of DP 2 (183). Nevertheless, another study report participation in necrosis rather than apoptosis (184). DP 2 is ubiquitously distributed with high expression in kidney, brain, testis, heart, resting lymphocytes and differentiated macrophages (Table 4) (171;174;176;181;184;185). Since it was previously thought to be a lysosomal enzyme, its physiological function to date is unknown. However, altered serum activities of DP 2 have been associated with various pathogenic conditions, such as Sjörgen Syndrome, rheumathoid arthritis, Lupus erythematosus, various cancers and Parkinson disease (168). Lately, neurogenin 3-specific DP 2-/- deficiency revealed a phenotype with impaired glucose tolerance, insulin resistance and visceral obesity (186). Interestingly, ADA was recently discovered to also bind to DP 2 though with an order of magnitude lower compared to DP 4 (187). The human gene localization is 9q34.3. Since DP 2 also belongs to the SC clan, its order of catalytic residues is Ser, Asp and His, located in an / hydrolase fold as summarised in Table 2. Recently, the crystal structure of DP 2 was deposit in the as pdb 3JYH, revealing a /-hydrolase domain as well as a novel SKS domain, comprised of 5 -helices arranged in a helix bundle fold, capping the active site. An insertion from the SKS domain to the acive site results in steric hinderance of larger substrates and contains Asp334 for anchoring N-terminus of the peptide substrate. 9 Introduction

Table 3: Summary of the DP 4-like enzymes comprised of the human DP 4-gene family (S9b) and DP 2 (90). Clan SC Family S9b (DP4-gene family) S28 DP 4 FAP DPL-1 DPL-2 DP 8 DP 9 DP 2 DPIV, DPPIV, Seprase, DP 6, DPP 6, DAP IV, CD26, antiplasmin DP 10, DPP 10, DP II, Synonyms DPPX-S, DPPX-L, DPP 8 DPP 9 ADAbp, Gp 108, cleaving DPP Y DPP 7,QPP DPPX Gp 110, Hep 105 enzyme, APCE EC Number 3.4.14.5 - - - - - 3.4.14.2 Merops S09.003 S09.007 S09.973 S09.974 S09.018 S09.019 S28.002 M96859 Accession M80536 U09278 AY172659 AF221634 AF452102 AF154502 M96860 Human 2q24.3 2q23 7q36.2-3 2q12.3-2q14.2 15q22 19p13.3 9q34.3 Gene Dog 36 36 16 19 30 20 9 Location Pig 15q21 1q 1q Rat 3q21 3q21 4q11 13q11 8q24 9q12 3p13 Gene size (kb) 81.8 72.8 659 535 71 48.7 2,850 # Exons 26 26 26 26 20 22 13 Promoter TATA-less, GC rich ? ? ? TATA-less, GC rich ? ? Tissue-specific Regulation of HNF/ ? ? ? ? ? KLF2, TOB1 expression Interferons // RA, Cytokines # Amino-acids 766 760 803/859 796 882 863/892 492 Mr (kDa) 110 97 115/120 91 100 98 58 Proform ------yes (SP) Transmembrane     - - - Cysteines 12 13 11 9 12 17 8 # Gylcosylation 9 (2 - 9)* 5 7 10 - (2) 6 Glycosylation N/O N N N - N N Catalytic triad   - -    Intron at catalytic S   - - - -  Activity DP 4 DP 4 + gelatinase unknown unknown DP 4 DP 4 DP 4 (acidic pH) Quaternary structure dimer/tetramer dimer dimer dimer dimer dimer dimer Activity needs dimer   - - - -  Crystal structure     - -  Membrane Secretory Subcellular Membrane Membrane Cytosolic/ type II / Membrane type II Cytosolic vesicles / type II / soluble type II secretory? location soluble Lysosomes ADA, CD45, Cav-1   Integrins   A-type K(+) Binding Collagen, FAP, PgR   Integrins A-type K(+)   channel H-Ras H-Ras ADA CARMA-1, HIV-TAT channel (Kv4.2) partners Annexin 2 (Kv4.2) CXCR-4, M6P/IGFII uPA-R, DP 4 Ubiquitous Activated- Ubiquitous Ubiquitous Ubiquitous Fibroblasts Brain, Pancreas, Distribution Brain Kidney > small Activated- Adrenal gland Reproductive Heart, Liver Skeletal , Kidney, Testis, Intestine > Lung Stellate cells Organs, Brain Brain, Muscle,Testis Brain Knock-out    - - -  Transgenic  ------ in activated T-cells,  peripheral  peripheral  Macrophages B-cells Leucocytes Leucocytes Leucocytes - - - Resting NK-cells Lymphocytes  Lymphocytes  Lymphocytes Macrophages Monocytes Monocytes T-cell Glucose-metabolism, Cell growth Embryonic Signal Proliferation? Antigen presenting Neuro- Cell adhesion, regulation, development, transduction, Known Signal Signal Transduction protection? Immune response, Tumour Signal transduction, Regulation of Physiological Transduction Cell-survival Maintaining Tumour progression, progression, Regulation of synaptic Cell-survival Cell-proliferation lymphocyte Functions Psychoneuro- Wound healing synaptic plasticity, plasticity, + + Cell-proliferation quiescence endocrine function, Fibrogenesis K channel K channel

Cancer, Diabetes Asthma Necrosis of Liver Cirrhosis Neurodegeneration Pathological Cancer Amytrophic leucocytes Arthritis Progressive spinal Prostate Cancer ? MS, AIDS lateral sclerosis Neuroprotection Role Pulmory muscular atrophy Anxiety (ALS) Diabetes fibrosis (92;94;98;100) (88;92;94;128) (85;92;94;128) (90;92;94;128); (2;12;13;92;94;128;188) (92;94;128;157) (92;94;128;171) References (119;122;128) (155;160;191) (130;131;134;135) (131-140;143) (189;190) (159;189-192) (172-186); (123;124;189) (193-195) (140;143;189;190)) (148;189;190) *Number of glycosylation sites being occupied in the crystal structures.

10 Introduction

Table 4: Relative abundace of RNA-expression of the DP 4-like enzymes by Northern blot analysis.

ef.

rain eart iver

ung

estis

uscle

R

idney

pleen

L

L

pecies

B H

rostate

lacenta T

tomach

ancreas

S

K

M

ntestine

S

P

I P

S P DP 4 H, R, +++ +++ ++ + +++ - ++ ++ nd + + (39;44) DP 8 H + + + nd - - - ++ - + +++ + - (85;88) DP 9 H ++ + +++ ++ ++ + + + + ++ ++ +++ +++ (86;88;90) DP 2 R +++ + - nd nd nd - ++ nd nd ++ ++ - (171) +++ = very strong expression, ++ = strong expression, + = weak expression,- = faint expression; nd = not determined, H = human, R = rat

Although all of these enzymes described above display DP 4-like activity (Table 3), they exhibit distinct features with respect to cellular compartmentation and glycosylation as illustrated in Figure 6. In addition, DP 4 has also been reported to be internalised upon association with binding partners such as CXCR 4 and M6P/IGFII (196-198), recycling of terminal carbohydrates (70;75) and assembled to lipid rafts (199).

Figure 6: Cellular compartmentation of DP 4-like enzymes. DP 4 and FAP are located either as homodimers or heteromeric complex on the plasma membrane or shedded into the serum. DP 2, a homodimer, is distributed as a zymogen in secretory vesicles or lysosome. DP 8 and DP 9 are also homodimers and located cytosolically. , glycosylation.

1.1.6 Physiological Role of DP 4

DP 4 has recently been described as a moonlightening protein due to its multiple functions. However, the physiological roles of DP 4 are not only based on its enzymatic activity, resulting in the regulation of many peptides, but also on its interactions with a variety of binding partners (200). Thus, it is involved in processes such as nutrition, nociception, cell- adhesion, psychoneuro-endocrinology, immune response and cardiovascular system as recently reviewed (2;94;189;200-204) and summarised in Figure 7. Nevertheless, it should be born in mind that some functions may also be accounted to either DP 8, DP 9 or FAP, respectively and await further investigations in future. Results obtained from research on knock-out models such as knock-out mice and DP 4 deficient Fischer rats as well as transgenic mice could give further clues on the physiological involvement of the other DP 4- like enzymes as outlined in Table 7. Interestingly, DP 4 and FAP appear to play opposing roles in fibrinology. While DP 4 enhances activation of plasminogen 2 and therefore assists in fibrinolysis and scar resolution (94;205), FAP converts 2-antiplasmin into a more active form, thereby promoting fibrinogenesis and scar formation (94;123;124). 11 Introduction

Figure 7: Physiological processes influenced by DP 4 or/and DP 4-like enzymes (2;206).

1.1.6.1 Natural Substrates of DP 4 Hydrolysis of natural substrates by DP 4 results either in inactivation of regulatory peptides or changes in receptor specificity or binding (2;207;208). Three peptide families, including the chemokine family, pancreatic polypeptide family and PACAP/glucagon superfamily, have been shown to contain a great number of DP 4 substrates and are therefore highlighted in Table 5 (2;201;207;208). Since the physiological concentrations of these substrates range between nanolar and picomolar in vivo, the overall reaction rate is given by v0 = kcat/KM [E] [S], with the selectivity constant kcat/KM being a good indicator for the likelihood of the reaction to occur in vivo (2). Table 5 summarises the substrates known to be hydrolysed by DP 4 in vitro and in vivo. Interestingly, some substrates with alanine or glycine at the penultimate position are more rapidly cleaved than the ones with proline, indicating that stereochemistry and a possible second may also contribute to the overall reaction (209). Upon investigation of natural substrates, GLP-1, GLP-2, NPY, PYY, SDF-, SDF-, IP-10 and ITAC have been demonstrated to be cleaved by DP 8 and/or DP 9, however with lower efficiencies compared to DP 4. In fact, NPY was demonstrated to be the best substrate for DP 8 and DP 9, whereas PYY was cleaved at slow rates and GLP-1 and GLP-2 at intermediate rates without producing a second dipeptide like DP 4 (127;130;210). This would imply that the specificity is extended to

P´1, which differ in serine and isoleucine for NPY and PYY, respectively. Furthermore, unlike

DP 4, DP 8 and DP 9 are only able to cleave substrates with Pro and Ala at P1 position. Intriguingly, a great number cytokines contain proline at the penultimate position, yet only their N-terminal fragments were shown to be susceptible to DP 4 hydrolysis (211). Likewise, FAP is also not able to truncate any chemokines, but has recently been found to hydrolyse NPY, BNP, substance P, PYY as well as to a lesser extent GLP-1 and GIP (95). 12 Introduction

Table 5: Summary of natural substrates known to be hydrolysed by DP 4.

# Amino Peptide N-terminus Selectivity* Assay system Reference acids Pancreatic Polypeptide Peptide YY YPIKPE... 36 +(++)a in vitro/in vivo (2;212;213) Neuropeptide Y YPSKPD... 36 +++ in vitro/in vivo (2;212;214)a Chemokines

SDF-1 KPVSLS... 68 ++++ in vitro/in vivo (2;215;216) MDC GPYGAN… 69 ++++ (++)b in vitro/in vivo (2;216;217) I-TAC FPMFKR... 73 +++ in vitro/in vivo (2;216;218) IP-10 VPLSRT... 77 ++ in vitro/in vivo (2;216;219)

specificity Mig TPVVRK... 10 ++ in vitro/in vivo (2;216;219) RANTES SPYSSD... 68 + in vitro/in vivo (2;216;220)

Alteration of receptor of receptor Alteration Eotaxin GPASVP... 74 + in vitro/in vivo (2;216;217) LD78 APLAAD... 70 + in vitro/in vivo (2;216;221) SDF-1 KPVSLS... 72 nd in vitro (2;222) PACAP/Glucagon family GHRH44/GHRH29-NH2 YADAIF... 44/29 +++ in vitro (223;224) a a GLP-1 HAEGTF... 30 ++(+++) in vitro/in vivo (224-226) GIP YAEGTF... 42 ++ in vitro/in vivo (224;226;227) PACAP38/PACAP27 HSEGIF... 38/27 ++(+)b/+(+)b in vitro/in vivo (2;214;224) Glucagon HSQGTF... 29 ++ in vitro/in vivo (224;228;229) Oxyntomodulin HSQGTF... 37 ++ in vitro (224) PHM HADGVF... 27 ++ in vitro (224) GLP-2 HADGSF... 33 + in vitro/in vivo (224;225;227) Secretin HSDGTF... 27 + in vitro (224) VIP HSDAVF... 59 + (+)b in vitro (214) Neuropeptides/Peptides Substance P RPKPQ... 11 ++(++)b in vitro/in vivo (230;231)

Inactivation BNP-32 SPKMVQG… 32 ++ in vitro (179;232) IGF-I GPETLCGA… 105 + in vitro (233) Hemorphin-7 LVVYPW… 10 ++ in vitro (179;234) -casomorphin YPFVEPI 7 ++ in vitro (235) Endomorphin-1 YPFF-NH2 4 + in vitro (236) Endomorphin-2 YPWF-NH2 4 + in vitro/in vivo (237;238) Enterostatin VPDPR 5 + in vitro (239) Tyr-MIF-1 YPLG-NH2 4 + in vitro (235) Morphiceptin YPFP-NH2 4 nd in vitro (240) Kentsin TPRK 4 nd in vitro (235) Chemokine GCP-2 GPVS... 75 nd in vitro (220) Others GRP VPLPAG... 27 +++(+++)b in vitro/in vivo (214)

Vasostatin-1 (Chromogranin A1-76)) LPVNSPM... 76 +++ in vitro (241) SR-17 (Chromogranin B586-602)) SAEFPDFY… 17 + in vitro (242) Pro-colipase VPDPR... 101 + in vitro (24) CLIP RPV... 22 + in vitro (235) Trypsinogen pro-peptide FPT... 8 + in vitro (235) Trypsinogen (pig) FPT... 231 + in vitro (24) Prolactin (sheep) TPV… 198 + in vitro (235) Aprotinin RPD... 58 + in vitro (235)

Biological effectunknown Biological Chorionic gonadotropin APD... 243 + in vitro (235) Promellitin APEPEP 50 nd in vitro (243) Procalcitonin APFRSA... 116 nd in vitro/ in vivo (244;245) a, different values obtained by various laboratories; b, selectivity of second cleavage of the same substrate; *,in vitro values 5 -1 -1 5 -1 -1 5 -1 -1 5 -1 -1 kcat/KM values: + = 0 – 1 x 10 M s , ++ = 1-10 x 10 M s , +++ = 10-30 x 10 M s , ++++ = 30 –50 x 10 M s

1.1.6.2 Binding Partners Several molecules have been shown to bind to DP 4, thereby causing various physiological and immunological responses as summarised in Table 6. These can be subdivided into three categories: Immunology, cell adhesion or cell-cell communication and peptide transport.

13 Introduction

Table 6: Summary of molecules known to associate with DP 4. Binding Binding site Function Refs partner α1 and α2 of ADA bind to DP 4 via loop A between blades IV and V, and loop B between (246) Binding of extracellular ADA to A Receptor on dentritic β 3 and β 4 of blade V, respectively. B2 (247) APC cells and CD26 on T-cells to form a ternary Glycosylation of DP 4-Asn-229 involved, as (7;82) complex, resulting in: observed in crystal structure. (248) ADA Ternary complex between A R-ADA of (249) 2B Co-stimulation of T-cells dentritic APC to lymphocytic CD26. ADA (250) T-cell proliferation, binding only in higher mammalian and species (251) T-cell protection dependent: (252) Human  porcine  rat  mouse Binding of DP 4 at the intracellular PTP2 (253) Signal transduction resulting in phosphorylation of CD45 domain of CD45 causes recruitment of both (199) Erk1/2TCR-zeta, ZAP70 by p56lck enzymes on lipid rafts (254) M6P Carbohydrate moiety of DP 4 Induces association of M6P/IGFRII and DP 4 (197) Binding of activated caveolin-1 on APC cells to extra-cellular/soluble CD26 at aa 201-210 (255) and Ser630 leading to: ((256) T-cell proliferation +  CD26 on T-cells  (257) binding of CARMA-1 on cytoplasmic tail of Causes upregulation of CD86 on TT-loaded dentritic (258) Caveolin-1 monocytes, thus leading to the association of APC with CD26 + association to lipid rafts  (259) CD28 on T-cells and subsequently to T-cell activation. phosphorylation of caveolin-1 on APC  (260) dissociation of Tollip and IRAK-1  (261) phosphorylation of IRAK-1  activation of (262) NF-B   CD86 Immunology binding of CARMA-1 on cytoplasmic tail of (260) CD26  recruitment of CARMA-1, CD26, Leading to activation of ZAP70, PLC, MAPK, phospatyl CARMA-1 (261) Bcl10 and IkappaB kinase complex to lipid inositol and  IL-2 (262) rafts  signal transduction T-cell activation, internalisation of DP 4, transendothelial (198) M6P/IGFRII Needs M6P bound on DP 4 migration by binding of lymphocytes to endothelial DP 4 (197) (196) Reduction of chemoattraction, co-internalisation in presence CXCR4 (215) ? of SDF-, formation of invadopodia in presence of SDF- receptor (263) and gp120, (264) Thromboxane Natural DP 4 Inhibitor, (265) ? A2 receptor T-cell suppression (266) 2 binding sites, sialic acid moiety and active site of DP 4. Crystal structures shows P and HIV-entry, inhibitor of DP 4 due to reverse binding at the HIV-TAT 2 P1 of Tat1-9 bind to S1 and S2 of DP 4, active site (64;248) respectively HIV-entry and subsequent apoptosis; inhibits ADA binding Cysteine rich region, HIV-gp 120 interacts (267-271) HIV-gp120 to DP 4 in presence of CXCR4 , though binding site distinct via its C3 region with DP 4 on lymphocytes (196) to ADA Cysteine rich region between aa 238 and (272) Collagen Extracellular adhesion? Cancer? Metastasis? 495 (103) Fibronectin Cysteine rich region of DP 4 between Fibronectin mediated spreading of fibroblasts, lung (273-275) (FN) aa 469 – 479 via aa LTSRPA motif (FN) metastasis, dissociates in presence of soluble DP 4 Cysteine rich region of DP 4 close to ADA binding site, sialic acid carbohydrate moieties of 2+ Plasminogen  Ca response in synovial fibroblasts, activation of receptor plasminogen binds to Pg-receptor/DP 4 synovial fibroblasts signal transduction in prostate cancer (276-280) (PgR) complex (aa 313 – 319)/IIb3 and urinary/tissue

cells resulting in  MMP 9. Quintary complex of ADA, Pg 2, (205) Plasminogen/ plasminogen activator uPA/tPA. Activated DP 4 and urinary plasminogen activator (uPA/tPA) and PgR (94) Plasmin plasmin (Pl) changes conformation and binds to (Pg/Pl) DP 4. Quintary complex abolished by   Pg 2 to plasmin angiostatin binding to DP 4. Cysteine rich region of DP 4 only from Streptokinase Ca2+ response in synovial fibroblasts,  DP 4 (279) rheumatoid synovial fibroblasts via aa LTSRPA (SK) autoantibodies, SK bound to DP 4 hydrolysis FN (276)

motif (SK) Cell adhesion/ adhesion/ Cell

cellcommunication Extracellular adhesion? Metastasis? Complement

- Vitronectin Sialic acid moiety of DP 4 (64) system? Coagulation?

cell Both glycosylated and unglycosylated Natural DP 4 Inhibitor. Binding of soluble glypican 3 to Glypican 3 (281) glypican 3 bind to DP 4 CD26  cell-proliferation and induces apoptosis. Heteromeric complex on invadopodia causing metastasis, (105) FAP Heteromeric complex tumor invasion, angiogenesis, wound healing and fibroblast (107) migration Blades IV of each subunit align to form an Tetramerisation, cell-adhesion, cell-cell communication (?), DP 4 eight-stranded antiparallel sheet, possibly (13) chemotaxis(?) Asn229 involved

Peptide transporter on microvilli membrane of renal Na+/H+ proximal tubule, reabsorption of dipeptides with proline. (282) exchanger ? In prostate cancer, association of DP 4, Pg 2 and NH3 (283)

isoform NH3 results in Ca2+ signal cascade and in intra-cellular pH ((284) Peptide Peptide

Transport  tumour cell proliferation + invasivness

14 Introduction

1.1.6.3 Knock-out, Deficient and Transgenic DP 4-like Animal Models DP 4-knock-out, -deficient and-transgenic animal models have been useful to elucidate the physiological role of DP 4-like enzymes and in vivo substrates. The phenotypes of such animal models are summarised in Table 7. Thus, DP 4(-/-) mice gave evidence on the important role of DP 4 in the incretin metabolism of the insulinotropic peptides GLP-1 and GIP. Additional increased energy expenditure and decreased food intake make DP 4 inhibitors a favourite pharmaceutical target compared to other known anti-diabetic drugs (285;286). Furthermore, behavioural studies point to a possible negative involvement of DP 4 in stress-related behaviour probably due to the modulation and/or inactivation of neuropeptide substrates, therefore identifying DP 4 as a potential pharmaceutical target in stress-related diseases (287). Intriguingly, DP 4(-/-) seemed to be vital with normal immune response, though their serum showed altered cytokine secretion and antibody production upon mitogen stimulation, a down-regulation of CD4+ T-cells as well as up-regulation of NK cells in the spleen and a marked decrease of peripheral blood CD4+ NKT (288). Two substrains of Fischer 344 rats, the F344/Crl(Ger/DP4-) and F344/CuCrj(Jpn/DP4-) lack endogenous DP 4 at protein levels, while F344/Crl (USA/DP4+) represents the wildtype (289). However, genetic analysis indicated the mRNA synthesis of both mutant substrains, with the F344/CuCrj(Jpn/DP4-) having a mutation of Gly633 to Arg633 at the active site followed by rapid degradation in the endoplasmatic reticulum (289-291). Studies on these animals confirm the in vivo role of DP 4 in diabetes, intestinotrophic peptide GLP-2 and stress-related behaviour, whereas isolated PMBC from mutant rats could be activated with mitogens similarly to the wildtype (292-299). DP 4 deficient Fischer rats are commonly used as host animals for the transplantation of hepatocytes or stem cells obtained from wilde-type animals, as DP 4 is a hepatic differentiation marker and as to distinguish between transplanted cells from the host cells (300-302). Interestingly, mutant and wildtype F344 displayed the same phenotype with regards to arthritis and non-selective DP 4-like inhibitor were able to suppress induced arthritis in both subspecies, implying the involvement of a DP 4-like enzyme other than DP 4, possible FAP (94;122;303). Similarly, the stimulation of neutrophils and erythrocytes from haematopoietic prognitor cells by a non-selective DP 4-like inhibitor was observed in both the mutant and wild type F344 subspecies, again suggesting an involvement of another DP 4-like enzyme such as FAP, DP 8 or DP 9 (125). A novel congenic DP 4 deficient DA.F344-Dpp4m/SvH rat model confirmed the physiological role of DP 4 in glucose-metabolism and stress-related diseases (304). Like the DP 4 knock-out and deficient animal models, transgenic mice with human CD26 displayed normal immunological phenotype, though thymocyte proliferation as well as CD4+ and CD8+ T-cell differentiation and viability was impaired, suggesting an important role of DP 4 in the T-lymphocyte homeostasis in peripheral blood (305). FAP(-/-) in turn, indicated the contribution of FAP in wound-healing, while no general susceptibly in cancer was observed, suggesting a more advanced mechanism (122). DPL 1(-/-) and DP 2 have been the only knockout of the DP 4- like enzymes found to be lethal, so far (306). Only recently the phenotype of neurogenin 3- (-/-) (-/-) specific DP 2 mice has been reported, which appears to be opposite to DP 4 , resulting in increased hyperinsulaemia, glucose intolerance, insulin resistance, visceral obesity and liver steatosis (186). 15 Introduction

Table 7: Summary of phenotypes obtained from DP 4-knock-out (k.o.), DP 4-deficient and DP4- transgenic animal models as well as FAP-/- k.o.-, DPL1-/- k.o.- and DP 2-/- k.o.-mice. Model Investigation Phenotype Ref Protection from  Energy expenditure,  Serum GLP-1, leptin, insulin, (285) obesity and  Glucose tolerance  food intake (286) insulin resistance: DP 4(-/-)  DP 4-inhibition  DP 4(+/+)  DP 4 = target enzyme Spleen:  CD4+ T-cells,  NK cells, Stimulation (PWM):  IL-4, IL-10, IFN- Peripheral blood:  CD4+ NKT-lymphocytes (143;288; Immunology: After immunization (PWM):  IgG, IgG1, IgG2a, IgE,  IL-4, IL-2 + delayed IFN- (-/-) (+/+) 307-309) (-/-) DP 4  DP 4  non-selective + DP 8/DP 9 inhibitors  T-cell proliferation  DP 8/DP 9 DP4 mice involved

Nociception:  plasma Substance P, delayed pain response (310) Cancer : DP 4(-/-)  DP 4(+/+)  DP 4-like inhibitor  tumour cells (311)  Depression-like behaviour according to tail suspension and forced swim test Behaviour : (287) DP 4Mut  DP 4WT  DP 4 = target enzyme Disease Experimental colitis: DP 4(-/-)  DP 4(+/+)  DP 4-like inhibitor intestinal adaptation (312-314) MS:  TGF-1,  TH -immunity  clinical experimental autoimmune encephalomyelitis Autoimmunity: 1 (315;316) Arthritis:  Serum SDF-  arthritic inflammation

Protection from Serum:  GLP-1 GIP  glucose tolerance  insulin after high fat diet or gucose  GLP-13-36 (294;295; obesity and  insulin reistance  food intake after high fat diet  weight gain 299;317- insulin resistance: DP 4Mut  DP 4-inhibition  DP 4WT  DP 4 = target enzyme 319) Mut DP 4 :  food intake + weight gain,  postprandial [PYY] after 24 h fast due to PYY1-36 Satiety DP 4WT: PYY  food intake but not in DP 4Mut (320) Short term DP 4-like inhibition: no anorectic effect of PYY1-36 Asthma: Peritracheal oedema:  oedema due to ACE inhibitors Glomerulonephritis: DP 4Mut = resistant to experimental induced glomerulonephritis Cholestasis:  serum DP4-activity after induction of cholestasis in DP 4WT rats, no serum Mut (303;321- Diseases: DP 4 activity in DP4 326) Cancer:  DP 4Mut expression   metastasis + cell adhesion Arthritis: DP 4Mut  DP 4WT  arthritic inflammation with DP 4 -like inhibitors  other DP 4-like enzymes involved in arthritis Isolated PMBC from DP 4WT and DP 4Mut are able to be activated after mitogen activation  F344/Crl DP4 may be involved but not necessary for T-cell activation in rats (Ger/DP4-) rats Isolated splenic leucocytes from DP 4WT and DP 4Mut have the same proliferative response of

in vitro stimulation by T-cells (Con A), B-cells (LPS) and T- + B-cell (PWM) mitogens  DP4 or may be involved but not necessary for lymphocyte proliferation in rats. Altered age Mut dependent leucocyte subset + thymic emigration pattern in DP 4 (109;125; F344/DuCrji Immunology: WT + + + 322;327- Asthma: DP 4 :  CD4 /CD26 /CD25 -T-cells recruitment in asthma induced lungs of rats (DP4-) rats 333)  CD26+ : CD26- TCR-cells   IgE DP 4Mut:  CD4+ T-cells  IgE,  recruitment of Eosinphils,  recruitment of T-cells Mut Cancer:  NK cytotoxicity in DP 4 rats  DP 4 activity sustains NK cytotoxicity Haematopoiesis: DP 4Mut  DP 4WT DP4-like inhibitor  neutrophils + erythrocytes from prognitor stem cells via G-CSFDP 4-like enzyme involved in neutropenia and acute anemia Nociception:  sensitivity to pain (310)  stress response in OF, SI, passive avoidance + EPM  motor activity Behaviour : (297;334) DP 4Mut  DP 4WT  DP 4 = target enzyme Small Intestine DP 4Mut  DP4-inhibition  GLP-2 +  bowel weight + resistance to gastrointestinal damage (293;335) Assimilation of Mut  excretion proline containing peptides in urine;  weight in DP 4 after fed with gliadin (240;336; Pro in kidney Isolated brush border membranes from small intestines and kidney unable to hydrolyse 337) and s. intestine proline containing peptides Used as a model of stem cells/hepatocyte transplantation to distinguish between donor and (300;302) Transplantation recipient, DP4 = HAM.4 = differentiation marker of hepatocytes (338-343) Protection from Serum:  GLP-1  Glucose tolerance  insulin after OGT  weight gain after high-calorie obesity and diet  islet size insulin resistance DP 4Mut  DP 4WT  DP 4 = target enzyme  Leptin signaling  bound leptin in plasma  free leptin in liver  triglycerides  alkaline Lipid metabolism phosphatase  aminotransferases Da.F344- Assimilation of m (304) Dpp4 /SvH Pro in kidney  weight in DP 4Mut after fed with gliadin and s. intestine Immunology  Lymphocytes  Eosinophils  NK-cells  B-cells  NK cytoxicity  T-cell prolifaration  IL-6  ACTH  Corticosterone  stress-induced hyperthermia  stress response in hole board Behaviour test, SI + EPM DP 4Mut  DP 4WT  DP 4 = target enzyme  age-related thymus cellularity with impaired thymocyte proliferation huCD26tg mice Immunology (305)  peripheral T-cell pool with  apoptosis in CD4+ and CD8+ subpopulations FAP (-/-) mice General Delayed wound-healing (122) DPL 1 (-/-) mice General DPL 1 (-/-)  Embryonic lethal, DPL 1(+/-)  pigmentation defect (306) NGN3-specific  Hyperinsulaemia,  glucose intolerance,  insulin resistance,  liver steatosis,  adipocytes (-/-) Metabolism (186) DP 2 mice resulting in visceral obesity. 16 Introduction

1.1.7 Involvement of DP 4 in Pathology Due to its ubiquitous distribution and involvement in various physiological processes, a great number of pathological conditions are associated with either altered DP 4 expression and/or activity correlating to the severity of the respective condition. These can be subdivided into five categories as illustrated in Figure 8: psychoneuroendocrine disorders, autoimmune and inflammatory diseases, infectious diseases, haematological malignancies as well as solid tumours (2). Thus, DP 4 expression and activity is often determined for clinical diagnostic. In particular altered CD26 (DP 4) activities and/or expressions in serum have been associated with various pathogenic conditions involving psychological-, autoimmune-, inflammatory-, infectious-, metabolic-, cardiovascular-disorders as well as tumour and cancer as summarised in Table 8 (2;94;189;190;202;204;344;345). Although, previously several DP 4-like enzymes were described to contribute to the overall DP 4-like activity in serum such as attractin and - DPP IV, it is now generally accepted that CD26 (DP 4) constitute to more than 90 % of the overall DP 4-like activity in serum and plasma (164;167;169;170;346-349). Furthermore, the increased activity of DP 2 in serum and synovial fluid, resulting in an altered ratio with respect to DP 4 activity has been determined in the case of rheumatoid arthritis and lupus erythematosus, whereas elevated DP 2 activity was found in leucocytes of patients with Sjörgen Syndrom (350-352). Changes in activity of DP 2 have also been associated in neurodegenerative disorders and myopathies such as Alzheimer, Huntington’s and Parkinson disease as well as muscular dystrophy and polymyosites (353). As already mentioned above, the expression of FAP is analysed as a marker of several solid tumours, such as epithelial colon-, gastric-, intestinal-, esophageal-, undifferentiated thyroid-, lung-, breast-, ovarian- and cervical-cancers, cutaneous melanoma as well as aggressive fibromatosus (108-120;189). The physiological and pathophysiological of DP 9 has only recently been elucidated, being involved in antigen presenting and EGF signaling pathway (132;133). This suggest DP 9 playing a role in inflammation as well as cell proliferation and apoptosis (132;133;190;309). Furthermore, an up-regulation of DP 9 has been detected in testicular tumours (134).

Figure 8: Pathophysiological role of DP 4 with either altered expression and/or activity (2;204). 17 Introduction

Table 8: Summary of altered CD26 (DP 4) activity and expression in human sera (2;94;202;204;344;345) Serum Serum Disease CD26 Remarks Reference [CD26] Activity Male  (2;202;204;344;345) Healthy Female  (2;202;204;344;345) Age  (2;202;204;344;345) Major depression   ADA activity (354-356) Schizophrenia  (357) Anxiety  (358) Psychological Stress (359) Diseases  Anorexia nervosa   T+CD26/CD25-cells (360;361) Bulimia   T+CD26/CD25-cells (360;361) Alcoholism  (362)  sDP 2 Rheumatoid arthritis   activity,  sCD30 (78;363-365)  SynoviocytesCD26 Lupus Erythematosus    DP 2  PBMCCD26 (78;364;366;367) Sjögren Syndrome    DP 2 in leucocytes (78) Autoimmune Psoriaris   ADA (368;369) Diseases Skleroderma  (370) Anca-associated vasculitis   sCD30  IL-10 (371) Celiac Disease   intestinal CD26 (372)  TCD26/CD4-cell,  iNK Allergic Asthmatics   (373)  Eosinophils Diabetes type 1    TCD26/CD4  TCD26/CD8 (374;375) Pancreatitis  (376) Gastric ulcer  (376) Inflammatory / Acute hepatitis   (325) Infectious Chronic hepatitis   (377;378) Diseases Crohn’s disease  (379-381) HIV    ADA,  TCD26-cell (382) Sepsis  (383) Diabetes type 2  (374) Metabolic/ Hypertension  (384) Cardiovascular Cirrhosis  (385) Osteoporosis  (386) Gastric cancer   (376;387) Bile duct cancer  (376) Colorectum   (388-394) Pancreatic cancer  (376) Cancer/Tumour Oral sqamous cell carcinoma   (395;396) Hepatocellular carcinoma  (397) Multiple myeloma  (397) Hodgkin’s Disease  (398) Lymphosarcoma  (387)

In addition, administration of DP 4 inhibitors or anti-DP 4-mAb have been demonstrated to improve disease conditions in various animal models, cell cultures and in case of type 2 diabetes also in human, by altering the natural substrate concentrations (399) or interfering with the interaction of binding partners. It should be mentioned that side effects obtained during the course of toxicological studies of a non-selective inhibitor were proposed to be due the inhibition of DP 8 and/or DP 9 (142). Symptoms include alopecia, thrombocytopenia, anemia, spenomegaly and mortality in rats and bloody diarrhea in dogs. However, this issue is currently controversially discussed (141;142;145-147). Nevertheless, a non-selective DP 4-like inhibitor PT-100 had been in clinical trials II-III for various types of cancers based on its dual inhibitory action against FAP as well as DP 8 and/or DP 9, though it was discontinued in 2007 (125;126;144;311;400). Radioactive labeled anti-FAP-mAb has been applied for targeting tumour cells (401-405). 18 Introduction

Table 9: Summary of disease conditions, being potential targets for DP 4 and/or FAP inhibition or modulated by anti-DP 4-mAb (399). Development Target disease Effect of Inhibitors Comments Inhibitor*/mAb Ref. stage FDA approved: Sitagliptin (Januvia) (399) Inhibition of incretin deactivation, Sitagliptin, Saxagliptin, Many pharmaceutical Saxagliptin (Onglyza) (406) Type 2 improvement of postprandially Linagliptin companies are targeting Linagliptin (Ondero) (407) stimulated insulin secretion and EMEA: Vildagliptin diabetes DP 4 inhibition Vildagliptin (Galvus) (408) glucose tolerance Japan: Alogliptin Alogliptin (Nesina) (409) Korea:Gemigliptin Prevents spreading of metastases, (311) inhibits tumour migration and Phase III: Disapproved Talabostat (144) angiogenesis by inhibiting FAP. FDA put on hold treatment on lung- (PT-100) (400) Inhibition of DP 8 + DP 9:  IL- Phase II studies: and pancreas cancer (Val-boroPro) (126)  cytokines +  chemokines  Melanomas Clinical trials for Disconinued (125) melanomas  neutrophils,  T-lymphocytes,  macrophages Tumours (401- Cell-specific suppression of Human Clinical F19-Antibody FAP-mAbF19 405) stromal tumours expression high Phase I radio-labeled Oral DNA vaccine (410) levels of FAP Animal model chemotherapy against FAP (411) (104) Prevents spreading of Cell culture, Hetero-complex with DP 4-mAb 1F7 (203) metastases, inhibition of tumour animal models FAP involved Pro-boroPro (275) migration and angiogenesis (274) Multiple Animal experiments, (412) Suppression of EAE human primary cell Autoimmune disease Lys[Z(NO2)]-Pyr (413) sclerosis culture (414) Cell culture, animal Lys[Z(NO )]-Thia (415) Reduction of keratinocyte 2 Psoriasis models Lys[Z(NO2)]-Pyr (416) hyperproliferation Preclinical studies P32/98 (417) DP 4 inhibition leads to Rheumatoid TMC-2A (418) Suppression of symptoms Animal models inhibition of CD45 PT, TSL-225 (323) arthritis may involve DP 2 + FAP Suppression of NPY cleavage Effective in animal Suggested to mainly Anxiety/stress P32/98 (334) decreases anxiety models (rats) involve NPY DP 4 inhibitor blocks mescaline- Psychosis/ Effective in animal induced scratching and AMAC (419) models (mice) schizophrenia amphetamine- induced hyperactivity Pro-Pro diphenyl- (420- Graft rejection Suppression of graft rejection Animal experiments phosphonate ester 424) (prodipine) High doses Lys[Z(NO )]-Thia 2 (425) necessary Lys[Z(NO )]-Pyr Autoimmune General immunosuppressive Cell culture, animal 2 (426) Dual inhibition of Ala-boroPro effects models (427- disease DP 4 and AP N Pro-boroPro 432) (PETIR-001) PETIR-001 Enhanced cell cycle arrest at the Influences survival DP 4-mAb 1F7 T-cell lymphoid Cell culture, G -S checkpoint, resulting in and de novo tumour Diprotin A (433) 1 animal models malignancies increased p21 expression growth Val-Pyr Suppression of SDF- cleavage, (222) Mechanism Phe-Pyrr-2-CN AIDS suppression of gp 120 and DP 4 Cell culture (434- not fully understood Arg(PMC)- Pyrr-2-CN interaction 436) Heteromeric complex DP 4-mAbE26 (103) Wound healing Promotion of wound healing Cell culture of DP 4 with FAP DP 4-mAbE19 (437) involved DP 4-mAbE3 *, a detailed list of DP 4 inhibitors is given in Appendices 77 + 82. A, PT = phosphotase activity

Furthermore, Pentostatin, an ADA inhibitor in clinical phase II, was shown to preferentially reduce CD26+ T-lymphocytes (438).

19 Introduction

1.1.7.1 Involvement of DP 4 in Diabetes Type 2 and Psychiatric Diseases

1.1.7.1.1 Diabetes Type 2 The development of DP 4 inhibitors for the treatment of diabetes type 2 has previously been the main focus of the company where the present study was performed. The principles of this new approach are outlined in Figure 9. Upon intake of food, the resulting glucose stimulates the secretion of gastric hormones such as GLP-1 and GIP from the intestinal epithelial cells into the blood circulation. Once arrived at the -cells of the pancreas, they bind to their respective receptors, which in turn transduce a signal cascade to induce insulin secretion. However, the half-lives of GLP-1 and GIP in the blood circulation are only 1½ and 7 minutes, respectively, due to their inactivation by DP 4, that cleaves off dipeptides from their N-termini. Thus, application of DP 4 inhibitors prevents such degradation of GLP-1 and GIP, thereby extending their half-lives by a multitude (407;408;439-446). As a consequence, there is an increased and prolonged stimulation of the GLP-1 and GIP receptors at pancreatic -cells, resulting in a boost of insulin secretion.

DP 4 DP 4 + (inactive) 2 x Inhibitor Glucose 

Pancreas (active GIP or GLP- + H3N 1)- COOH Small Incretin Insulin release by (active) Intestine islets of Langerhans

DP 4 + H3N - COOH DP IV Incretin Decreased blood (active) - (inactive) glucose level Figure 9: Schematic presentation of the enteroinsular axis, illustrating the release and insulinotropic action of GLP-1 and GIP as well as the influence of DP 4 and the effect of DP 4 inhibition (401).

The pancreas is situated inferior to the stomach between the curvatures of the duodenum. It can be divided into three portions, i.e. the head at the duodenum, the body and the tail near the spleen. The organ is comprised of two types of glandular tissues, called exocrine and endocrine tissue, respectively. The exocrine tissue is made up of a cluster acinar cells that secrete gastric juice including several digestive enzymes, into the small intestine via a network of ducts. The endocrine tissue, also referred to as islets of Langerhans, in turn, is a cluster of highly specific cells that secrete hormones and neuropeptides to maintain glucose homeostasis. The islets of Langerhans are highly vascularised and innervated. The main hormones are glucagon and insulin, secreted by the - and -cells, respectively as illustrated in Figure 10. Glucagon increases glucogenolysis, gluconeogenesis and the release of glucose by the liver, whereas insulin promotes uptake of glucose by the cells, glucogenesis, lipogenesis and opposes glucogenolysis and gluconeogenesis. According to several histochemical and immunohistochemical analyses, DP 4 is claimed to be present in various pancreatic tissues as summarised in Table 10. These include ductal epithelial cells of intra and interlobular ducts, capillaries and surprisingly in a soluble state of the -cell granules of the islets of Langerhans, where it is thought to colocalise with 20 Introduction glucagon. However, since no alternative splicing of DP 4 has been reported so far, a soluble DP 4 within granules is unlikely due to its signal peptide properties of the transmembrane. Therefore, either DP 2 or even DP 9 might be a DP 4-like enzyme located in the granules. DP 9 is highly expressed in the pancreas, though it is currently described as cytosolic enzyme (86;88;90;94;134;189;447;448). DP 2 has been proven to be located in secretory vesicle (167;410). Furthermore, polyclonal antibodies specific against DP 2 as well as histochemical analysis identified the presence of DP 2 in the Langerhans islets (171;185). However, due to size-restrictions regarding peptide length, none of the known DP 4 substrate is cleaved by DP 2 (135;179). Intriguingly, FAP has been described to be expressed in a subset of islet cells, presumably α–cells and a soluble alternative spliced isoform has been reported (449;450).

Figure 10: Anatomical presentation of the pancreas, illustrating the exocrine and endocine tissue. Islets of Langerhans consist predominantly of 3 different cells: blue, -cells secreting glucagon; yellow, -cells secreting insulin; green, -cells secreting somatostatin and VIP.

Table 10: Distribution DP 4 (-like enzymes) in the pancreas. Tissue Cells Organelle Enzyme Reference Exocrine intralobular and interlobular ducts Epithelial Membrane bound DP 4 (17;18;451) Exocrine acinar gland Acinar cells Cytosole DP8/DP9 (134) -cells Soluble in granules DP 4 (447;448) Islets of Langerhans -cells Secretory vesicles DP 2 (171;185) Capillaries Endothelial Membrane bound DP 4 (17;33;452;453)

The presence of a DP 4-like enzyme co-localising with glucagon in the alpha cell granules, is of great significance, since most of the insulinotropic receptors for natural DP 4 substrates are found on the beta cells as summarised in Table 11. Except for GRP-R, Y1-R and PRL-R, all receptors belong to the Family-B of the G-coupled protein receptors (GCPR), also referred to as secretin receptors. These receptors have been proven to regulate intracellular concentrations of cyclic AMP, resulting in an altered Ca2+ concentration (454). The concurrent release of a DP 4-like enzyme with glucagon would result in a switch of glucose homostasis as glucagon counteracts the effect of insulin, whereas deactivation of insulinotropic peptide hormones by a DP 4-like enzyme would stop their signal transduction capacity to stimulate insulin secretion. In addition to the abundance of secretin receptors, a number of natural DP 4 substrates are secreted by the islets of Langerhans or the surrounding tissues such as vagus neurons as outline in Table 12. The primary function of most of these substrates is the paracrine control of glucose homeostasis.

21 Introduction

Table 11: Summary of insulinotropic receptors for natural DP 4 substrates found in the islets of Langerhans. Receptor Cell Ligand Function Ref Glucagon >> GLP-1 >> Stimulate insulin secretion in a glucose dependent manner to (2;454) Glucagon-R  ()a oxytonmodulin maintain glucose homostasis (455) Stimulate insulin secretion in a glucose dependent manner, (2;454) GIP-R  (?)b GIP  uptake of glucose and triglycerides (455) Stimulate insulin secretion in a glucose dependent manner, induces (2;454) GLP-1R  (?)b GLP-1 >> Glucagon insulin biosynthesis,   cell mass,  insulin granules,  islet (455) neogenesis Stimulate insulin or glucagon secretion in a glucose dependent (2;454) GRF-R  ()a GHRH manner (455) Stimulate insulin or glucagon secretion in a glucose dependent VPAC a VIP, PACAP > PHM (2;455) 2  () manner

a Stimulate insulin or glucagon secretion in a glucose dependent (2;454) PAC1  () PACAP manner by PACAP (455) Secretin-R  Secretin Stimulate insulin secretion in a glucose dependent manner (454;455) Vagus Stimulate the secretion of VIP and PACAP from Vagus neurons, GRP-R neurons GRP together they stimulate insulin secretion, involved in islet cell (2;456) , ()a differentiation Regulation of energy homostasis such as increased glucose uptake, Y -R NPY and PYY (2;456) 1  NPY inhibits insulin secretion Elevate insulin secretion during pregnancy, PRL release is stimulated PRL  Prolactin (454;455) by GRP, PACAP. VIP and PHM a) only small number present, b) controversal results obtained from literature

Table 12: Natural DP 4 substrates in pancreas with neuronal, paracrine or endocrine functions. Substrate Cell Function Ref  Glucogenolysis,  Gluconeogenesis ,  glucose release by the liver, Glucagon  (455)  Glucogenesis,  insulin secretion in a glucose dependent manner GLP-1  Only small amounts, paracrine insulinotropic function (455) GLP-2  Only small amounts, alternative spliced  no secretion (455) Oxytonmodulin  Only small amounts, paracrine insulinotropic function (455) IGF-1  Co-secreted with glucagon, paracrine action,  insulin secretion (219) (457- PYY , pp,  Paracrine action,  insulin secretion in a glucose dependent manner 461) Released by GRP, together they stimulate insulin or glucagon secretion in a glucose VIP , vagus (455) neurons dependent manner, vasorelexant of smooth muscles vagus Released by GRP, together they stimulate insulin or glucagon secretion in a glucose PACAP (455) neurons dependent manner, vasorelexant of smooth muscles, vasodilation vagus Released by GRP, together they stimulate insulin or glucagon secretion in a glucose PHM (455) neurons dependent manner, vasorelexant of smooth muscles, vasodilation , ductal Only present in -cells during development,  insulin or glucagon secretion in a glucose Secretin - (455) cells dependent manner, releases of HCO3 from the ductal cells Stimulates insulin or glucagon secretion in a glucose dependent manner, GHRH , vagus (455) neurons  -cell division and growth vagus Stimulates the secretion of VIP and PACAP from vagus neurons, GRP (455) neurons together they stimulate insulin secretion, involved in islet cell differentiation splachnic NPY Co-secreted with norepinephrine  insulin secretion (462) neurons

1.1.7.1.2 Role of DP 4 Substrates in Behaviour Although very little is known about DP 4 and DP 4-like enzymes within the central nervous system (CNS), DP 4-deficient rats, DP 4-/- k.o. mice and in vivo inhibition have resulted in altered stress-related behaviour as summarised in Table 13 (135;287;296;297;304;419;463). A reason for this phenomenon could be the great number of neuropeptides that are metabolised by DP 4 in vivo as shown in Table 5. Their distribution in the (CNS) and neurological functions are outlined in Table 14. All of these substrates are found in the hypothalamus, pointing to neuroendocrinological functions, which can be subdivided into feeding, behaviour, circardian rhythm, nociception, cardiovascular and neurogenesis. Altered expression of several DP 4 substrates have been associated with mood disorders like anxiety, post-traumatic stress disorder (PTSD) and depression as well as schizophrenia, autism and several neurodegeneration diseases such as Alzheimer, Huntington and 22 Introduction

Parkison disease as confirmed by knock-out studies of NPY, Y2-R, Y1-R, TAC1, NK1-R, PACAP, VIP, secretin and GRP-R in mice, respectively (464-475). A link between NPY hydrolysis by DP 4 and stress has already been confirmed in in vivo studies, as truncated

NPY3-36 loses its selectivity towards the anxiolytic Y1-receptor, but is still a good ligand for anxiogenic Y2- receptor as illustrated in Figure 11 (296). Successive cleavage of SP1-11 to

SP5-11 in turn, results in loss of selectivity, whereas PACAP1-27 and PACAP1-38 are inactivated by DP 4, repectively (Figure 11).

Table 13: Various animal models used for investigations of DP 4 as a psychopharmaceutical target. Animal Model  DP 4 Results Tests Ref.

-/-  Depression Swim test, tail suspension test, hole DP 4 mice DP 4 knock-out (287)  Exploration board test, locomotor activity test F344/Crl(Ger/DP 4-) rats Elevated plus maze, open field, social (296) DP 4-deficient F344/DuCrj(DP 4-) rats  Anxiety interaction test, passive avoidance (297) Social interaction, open field, F344/Crl(Por) rats (wt) wt + P32/98 icv  Anxiety (463) ultrasonic vocalisation test  Anxiety Elevated plus maze, hole board test, DP 4-deficient DA/F344  Exploration social-interaction test, body temperature DP 4-deficient (304) rats  Social interacion before/after transport stress,  stress-induced hyperthermia  ACTH + glucocorticoids AMAC, oral Mescaline-induced scratching, Spargue Dawley rats  Psychosis (419) 6-60 mg/kg amphetamine-induced hyperactivity  Psychosis P32/98, PD83  Depression  Autism Wistar rats (SHR) Tetradic encounter test pdb oral, 1 mg/kg  Social interacion  Social cognitive behaviour

Figure 11: Influence of N-terminal truncation of neuropeptides by dipeptidyl peptidase 4 (DP 4) on receptor binding. A, truncation of NPY to NPY3-36 alters receptor selectivity; B, successive cleavage of SP to SP5-11 causes loss of NK1-R selectivity; C, DP 4 inactivates PACAP1-27/ PACAP1-38 (472;476-484). 23 Introduction

Table 14: Summary of neuropeptides/peptides hydrolysed by DP 4 and found in the brain. Peptide crossing BBB Neuropeptides/P Distribution in the Brain Function Brain  Blood Ref. eptide Blood  Brain Food intake, stress,  HPA, NPY  anxiety, 1-36  NS-D* Neuropeptide Y ArcN, Amy, Hip, LC, LatSep NPY  anxiety, schizophrenia, epilepsy, (212;213;485;486) 3-36 BloodBrain substance dependence, circardian, Peptide YY Hypothalamus (ArcN)  Food intake  energy expenditure  NS-D* BloodBrain (212-214;487-489)

Enterostatin PVN, Amy, dorTha  Food intake  energy expenditure  ? (239;490-492) Hypothalamus  Food intake  drinking  memory (Alzheimer)  NS-D* GLP-1 (214;455;493-499) (PVN, SON, ArcN, NST, AP)  neuronal survival  HPA  nausea/vomiting BloodBrain GLP-2 Hypothalamus ? ? (292;455;494;500) Hip, OlfB, cerebellum, Amy, SN,  Exploration,  locomotion,  prognitor cell GIP ? (2;494;501-504) LatSept, ThaNu, Hyth, brainstem proliferation  neurogenesis? Glucagon Hypotalamus, brainstem  Hyperglycaemia  ? BloodBrain (228;455;494;500) Cortex, limbic system, cerebellum, activity + curiosity HPA catecholamine     NS-D* Secretin midbrain, hindbrain, pineal gland, synthesis, involved in social and cognitive (224;455;494;500)  S (Choroid Plexus) Hip, Hyth ArcN, dHyth, brainstem behaviour, autism

PACAP38 >>> Hypothalamus (ME, PVN, Schizophrenia, circardian,  neurotrophic  NS-D* PACAP27 (224;455;494;500) PACAP27 ArcN) Amygdala, Astrocytes  Food intake; Pit:  GH,  PRL,  LH,  ACTH  S(PTS6) PACAP38 (465-467;477;505) Circardian rhythm, sleep (2;214;224;227) VIP Hypothalamus (SCN, SON)  NS-D* BloodBrain Pituitary gland:  PRL  GH  LH (455;494) GHRH44/GHR Hyth (ArcN), ME >> Cortex  Pit:  GH  ? BloodBrain (455;494) H29-NH2 Cerebellum  Brainstem Other brain areas:  neurotrophic PHM Hypothalamus Pit:  PRL  GH  LH  D BloodBrain (455;494) CLIP Ant + intermed Pit, ArcN, dor.RaNu = ACTH(18-39), involved in stress and sleep ? (235;506;507) AV3V, Ba-Me Hyth, CVO’s, brain Regulation of water + salt intake, blood pressure, BNP-32 ? (2;232;508;509) stem  vasopressin  exploration  anxiety  HPA # Olfactory tubercle, Amy, CaNu, ADHS, depression, aggression, anxiety, stress,  S /C-M (NK1) GP, Hyth (PVN, ArcN), ME, schizophrenia, neurogenensis (Parkinson, SP  BBB (2;230;231;483) Substance P 2-11 habenula, post. Pit, ThaNu, SN, Huntington, Alzheimer), respiratory rhythm, SP1-9  BBB (510-517) PAG, LC, PBrNu, NST, AP neurotoxicity, nausea/emesis, nociception (pain) BloodBrain  Nociception,  learning/memory by binding Hemorphin-7 Hyth, LC, CSF to AT4-R similar as AngIV (Alzheimer), ? (234;518) regulates blood pressure,  Nociception,  motivation via reward, Hyth (PVN, ArcN., VmNu + DmNu,  feeding response ,  oxygen consumption, SON), NTS, LC, PBrNu, PAG,  S /C-M Endomorphin-1  blood pressure regulation, (236;519-525) NuAmb, MidTh, Amy, LSepNu,  BloodBrain BNST, NuAcc, OVLT, Cortex, antidepressive,  anxiety (anxiolytic),  vassopressin + oxytocin Caudal nucleus of spinal trigeminal  Nociception,  motivation via reward, tract, superficial laminae of the #  feeding response ,  oxygen consumption,  S /C-M (235;237;238) Endomorphin-2 spinal dorsal horn, NTS,  BloodBrain (519-524) parasolitary nucleus, lateral  blood pressure regulation, reticular nucleus, NuAmb, antidepressive action,  anxiety (anxiolytic),  MSH, anxiolytic, antidepressive,  nociception  S /C-M# (PTS-1), Hypothalamus, cortex, striatum, Tyr-MIF-1 under stress,  stress-induced analgesia,  body- BrainBlood (235;526-533) amygdala, pons-medulla weight during/after chronic stress,  nociception  NS-D#, BloodBrain Circadian rhythm,  sympathetic nervous system Hyth (SCN), Hip, NST, PAG, AP, modulating stress, fear and anxiety response GRP SN, Amy, LC, dNuRa, ant.+ (anxiety, schizophrenia, depression, dementia, ? (235;534-539) intermed.Pit autism) GRP-R receptor at hippocampus and amygdala,  PRL and GH secretion Chromogranin B: Involved in the regulated secretory pathway by assembling, aggregating, + transporting of NP in SV within in the TGN. SR-17 Hyth, ant.Pit, Hip, temporal lobe, mRNA expression altered in epilepsy (Fragment of NTS, LC, OlfB, sympathetic ? (242;540-549) (hippocampus + temporal lobe), schizophrenia Chromogranin B) ganglions (hippocampus) and Alzheimer (hippocampus), SR-17: co-expressed + released with NE in sympahec nerves at the spleen Vasostatin-1 Ant. and post. Pit, CVO’s, Hyth, Chromogranin A: Involved in the regulated (Fragment of Hip, brainstem, cerebellum, secretory pathway,  vasopressin + oxytocin ? (550-554) Chromogranin A) neurons + astrocytes vasostatin-1: cardiovascular function Prolactin Pituitary gland  Milk production ? (2;235;555;556) (sheep) Amy, amygdala; ArcNu, Nucleus Arcuate; AP, Area Postrema; AV3V, Anteroventral Third Ventricle; Ba-MeHyth, Basomedial Hypothalamus; BNST, Bed Nucleus of Stria Terminalis; CaNu, Caudate Nucleus; CVO, Circumventricular Organs; DmNu, Dorsomedial Nucleus of Hypothalamus; dNuRa, dorsal Nucleus Raphe; GP, Globus Pallidus; Hip, Hippocampus, Hyth, Hypothalamus; LatSep; Lateral Septum; LatSepNu, Lateral Septum Nucleus; LC, Locus Coerulus; ME, Eminentia Mediana; MidTh, Midline Thalamic Nuclei; NuAcc, Nucleus Accumbens; NuAmb, Nucleus Ambiguus; NST, Nucleus of the Solitary Tract, OlfB, Olfactory Bulb; OVLT, Organum Vasculosum of the Lamina Terminalis; PAG, periaqueductal gray; PbrNu, Parabrachial Nucleus; Pit, pituitary gland; PVN, Paraventricular Nucleus; SCN, Suprachiastic Nucleus, SON, Supraorbital Nucleus; SN, Substantia Nigra; ThaNu, Thalamic Nuclei; VmNu, Venteromedial Nucleus of Hypothalamus; * NS/D, non-saturable/diffusion transport; #, S/CM, saturable/carrier- mediated transport; PTS-1, BBB transporter PTS-1. 24 Objectives of the Present Study

1.2 Objectives and Introduction to the Present Study 1.2.1 Characterisation of DP 4-like Enzymes in Diabetes Type 2 Although the mechanism of incretins and DP 4 of the enteroinsular axis in diabetes Type 2 is well understood, the following aspects still had to be investigated: 1. A high-resolution crystal structure of DP 4 for the design of inhibitors, and elucidation of substrate and inhibition mechanisms. 2. The tissue distribution of DP 4-like enzymes in various tissue sections to investigate where possible inhibition cross-reactivity might occur. Contribution DP 4-like enzymes towards the overall DP 4-like activity in various tissues. 3. Analysis of pancreatic islets with respect to DP 4-like enzymes due to the presence of a great number of insulinotropic receptors for natural DP 4 substrates.

1.2.1.1 Crystallisation of DP 4 For a successful crystallisation of a membrane protein, the following prerequisites are required:  Large quantities of protein  Soluble state  High purity  Homogeneous with respect to carbohydrate content Although purification of DP 4 from porcine kidney had been well established and applied for many years, its glycosylation-based microheterogeneity, resulting in 17 isoform on an analytical IEF, had to be resolved for efficient crystallisation (89;557). Therefore, on the basis of charge differences of the various DP 4 isoforms, a preparative liquid-phase isoelectric focusing has been implemented to separate them, using the Rotofor system (558). The crystallisation of porcine DP 4 was performed in collaboration with Prof. Bode’s group of the Max-Planck Institute for Structural Analysis in Martinsried, Germany and was comprised of the following steps (13): i. Purification of porcine DP 4 from the kidney cortex Present study at ii. Isolation of DP 4 isoforms by preparative isoelectric focusing probiodrug AG iii. Crystallisation iv. Cell units and space co-ordinates- - Prof. Bode’s group v. Native dataset MPI, Martinsried vi. Phase-solving vii. Structure solving

1.2.1.2 Distribution of DP 4-like Enzymes Based on cytotoxic effects of anti-diabetic DP 4-inhibitors with cross-reactivity to DP 8 and/or DP 9, the tissue distribution of DP 4-like enzymes DP 4, FAP, DP 8, DP 9 and DP 2 was investigated in Sus scrofa and Canis lupus familaris (131;141;142). Several sections obtained from kidney, lung, small intestine and pancreas were screened for mRNA expression by nested RT-PCR and the contributions of DP 4, DP 8/DP 9 and DP 2 towards the overall DP 4-like activity were evaluated, using selective inhibitors as well as a non- selective DP 4-like inhibitor P32/98 and an inhibitor mix as a control (131). A general scheme of the method implemented is given in Figure 12. However, it should be mentioned that two filed patents concerning DP 8 and DP 9, respectively, restricted the scope of research during the course of study, so that mRNA expression could not be quantified (559-561).

25 Objectives of the Present Study

Figure 12: Scheme of methods employed to analyse DP 4-like enzymes in porcine and canine tissues.

1.2.1.3 Analysis of DP 4- like Enzymes in Pancreas and Islets of Langerhans While the endocrinological function of DP 4 in glucose homeostasis has been confirmed by DP 4 deficient rats and DP 4-/- k.o. mice models, a possible paracrine or neuronal role in the regulation of insulin secretion by DP 4 and/or DP 4-like enzymes expressed in pancreatic islets of Langerhans still had to be investigated: i. Isolation of islets of Langerhans from the pig. ii. Isolation of secretory granules from isolated porcine islets. iii. Activity and inhibition studies of islets and granules with respect to DP 4-like enzymes. iv. Nested RT-PCR. v. Enzymatic histochemical analysis with respect to DP 4-like activity and DP 2. vi. Immunological analysis with respect to DP 4, insulin and glucagon in collaboration with Dr. Hause, Biocentre, Martin-Luther University, Halle. vii. Preliminary analysis of concurrent DP 4 and glucagon secretion after stimulation of perfused pancreas in collaboration with Prof. R. Petersen from University of Britsh Columbia (UBC), Vancouver, Canada.

1.2.2 Identification of DP 4 as a Pharmaceutical Target for Psychiatric Disorders Based on altered stress-related behaviour in DP 4-deficient rats, DP 4-/- k.o. mice and upon in vivo inhibition of DP 4 in two rat models as outlined in Table 13, several basic biochemical investigations were performed to identify DP 4 as a possible pharmaceutical target for stress- related disorders such as depression, anxiety and schizophrenia. Thus, the present study was designed to address the following objectives:

1. Target enzyme: Involvement of additional DP 4-like enzymes? 2. Target substrate: NPY, SP, PACAP? 3. Target organ: CNS versus periphery? 4. Inhibitor profile: Selectivity? BBB? Long-term versus short-term? 5. Target disease: Anxiety, depression, schizophrenia?

For this reason, the study was divided into three sections, i.e. analysis of DP 4-like enzymes in rat neuronal tissues, investigation of DP 4-like enzymes and its neuropeptide substrates in rat and human peripheral body fluids as well as the analysis of DP 4-like enzymes in sera obtained from depressed patients.

26 Objectives of the Present Study

1.2.2.1 Analysis of the DP 4-like Enzymes in Neuronal Rat Tissue As little is known about the distribution and functions of DP 4-like enzymes in the brain, the following investigations were performed: i. Activity and inhibition studies of DP 4-like enzymes in brain extracts obtained from F344/ZTM and F344/Crl(Wiga)SvH-Dpp4 rats. ii. Activity and inhibition studies of DP 4-like enzymes in primary cell cultures of neurons, astrocytes and microglia. iii. Hydrolysis of NPY, Substance P and PACAP by DP 4-like enzymes in brain extracts obtained from F344/ZTM and F344/Crl(Wiga)SvH-Dpp4 rats using MALDI-TOF-MS. iv. Hydrolysis of NPY, Substance P and PACAP by DP 4-like enzymes in primary cell cultures of neurons, astrocytes and microglia using MALDI-TOF-MS. Identification of NPY as the major DP 4 substrate in the brain. v. Histochemical analysis of DP 4-like enzymes in F344/ZTM and F344/Crl(Wiga)SvH-Dpp4 rats. vi. Activity and inhibition studies of DP 4-like enzymes in various brain areas. vii. Establishmnet of capillary depletion.

1.2.2.2 Analysis of Neuropeptide Hydrolysis by DP 4 in the Periphery As already indicated in Table 13, most of the DP 4 substrates in the brain are also able to cross the blood brain barrier, thus being able to communicate from the periphery to the CNS and vice versa (511;517;562-564). In particular the hypothalamus and the nucleus tractus solitary are linked with the neuroendocrine and neuro-sympathicus system via the median eminence and area postrema in the stress response, respectively (565-570). Furthermore, radiolabelled PYY3-36 was peripherally injected and found to bind at the Y2-R at the median eminence and area postrema (571). Interestingly, NPY and its DP 4-truncated metabolites have different physiological functions in the periphery and CNS. Therefore, DP 4-like enzymes and hydrolysis of stress-related neuropeptides were analysed in peripheral body fluid as as followed: i. Activity and inhibition studies of DP 4-like enzymes in human serum. ii. Hydrolysis of NPY, SP, rat Orexin B, GALP in human/rat serum by MALDI-TOF-MS. iii. Establishment of ex vivo hydrolysis of NPY, substance P and GALP in whole blood by MALDI-TOF-MS and identification of NPY as the main stress-related substrate for DP 4. iv. Activity studies of DP 4-like enzymes and hydrolysis of NPY in human CSF.

1.2.2.3 Analysis of DP 4-like Enzymes in Sera from Depressed Patience As DP 4-like activity in human serum has been proposed as a biomarker for clinical depression (Table 8), activity of DP 4, its concentration of DP 4, its post-translational modification and neuropeptide hydrolysis were re-evaluated in sera from depressed patients, thereby also considering the additional DP 4-like enzymes (354-357;359). i. Activity and inhibition studies of DP 4-like enzymes in human sera obtained from patients diagnosed with clinical depression. ii. Determination of DP 4 concentration by commercial ELISA in human sera obtained from patients diagnosed with clinical depression. iii. Analysis of post-translational modification by analytical IEF in human sera obtained from patients diagnosed with depression. iv. Hydrolysis of NPY and Substance P in human sera obtained from patients diagnosed with clinical depression by MALDI-TOF-MS.

27 Materials and Methods

2 Materials and Methods 2.1 Materials 2.1.1 Equipment All equipment used in the present study are summarised in Appendix 4. 2.1.2 Consumables Unless otherwise stated in Appendix 5 all consumables were purchased from Roth, Karlsruhe, Germany. 2.1.3 Chemicals Unless otherwise stated in Appendices 6-13, all chemicals were of high analytical grade and either purchased from Roth, Karsruhe (Germany) or Sigma-Aldrich, Deisenhofen (Germany). All buffers were prepared with triple distilled water. 2.2 Methods 2.2.1 DP 4 Purification and Isolation of its Isoforms 2.2.1.1 DP 4 Purification Fresh porcine kidney was obtained from the abattoir in Köthen (Germany), transported on ice to the laboratory (± 30 - 60 min), where the cortex was immediately removed, portioned and stored at -20° C until required. Purification of DP 4 was performed as follows (89;558): Homogenisation 1,200 g of porcine kidney cortex was soaked for ± 3 h in 2 l saline solution at room temperature to drain the blood and to thaw. It was subsequently homogenised with a 1000 ml

0.02 M saccharose solution, containing 0.2% Triton X-100 by an Ultra Torrax T25 (Janke and Kunkel, Staufen). Autolysis The homogenate was incubated overnight, whilst stirring at 30 - 32° C. The solid particles were removed by centrifugation at 12,200 g at 22° C for 20 minutes. The precipitate was discarded. 60 % Ammonium Sulphate Fractionation

507 g of (NH4)2SO4 was slowly added to the 1,300 ml supernatant, whilst stirring at room temperature. After six hours of stirring, the solution was centrifuged at 12,200 g at 22° C for 20 min. The precipitate was discarded and the supernatant (1,370 ml) pooled. 85 % Ammonium Sulphate Fractionation

250.71 g of (NH4)2SO4 was slowly added to the supernatant, whilst stirring. The solution was left stirring overnight and afterwards centrifuged at 12,200 g at 22° C for 20 min. The supernatant was decanted and the precipitate resuspended in 130 ml of 20 mM NaH2PO4 buffer, pH 6.8. The ammonium sulphate was removed by means of a 20 h dialysis, increasing the final volume to 191 ml. Ultrafiltration The dialysed solution was concentrated by ultrafiltration to 20 ml with an YM 50 membrane for 3 h at 3 bar and 4° C. Sepharose 6 B 19 ml of sample was loaded onto a 2.6 x 100 cm Sepharose 6B (Amersham Pharmacia Biotech, GE Healthcare, Uppsala) column, and the proteins were eluted with a 20 mM

28 Materials and Methods

NaH2PO4 buffer, pH 6.8 at a flow-rate of 0.25 ml/min. The DP 4 active fractions were divided into two pools of 78 ml (fractions 70-88) and 71 ml (fractions 89-102) based on SDS-PAGE and specific activities, and designated with Sepharose 6B Pool 1 and Sepharose 6B Pool 2, respectively. DEAE-Sephacel Each pool was loaded onto a 2.5 x 22 cm DEAE-Sephacel (Amersham Pharmacia Biotech, GE Healthcare, Uppsala) column, employing the BioCad FPLC system (Perspective Biosystems, Wiesbaden). The column was washed with two column volumes of 20 mM

NaH2PO4 buffer, pH 6.8 at a flow-rate of 2.0 ml/min. Proteins were initially eluted with half a column volume of a linear 0 - 40 mM NaCl gradient in 20 mM NaH2PO4 buffer, pH 6.8, which was subsequently increased to a linear 40 - 150 mM NaCl gradient within five column volumes. The DP 4 active fractions originating from Sepharose Pool 1 were again divided into two pooled fractions referred to as DEAE Sephacel Pool 1 and DEAE-Sephacel Pool 2 respectively, due to their different intensities of isoform bands in an analytical IEF, pH 4 - 6. DP 4 active fractions derived from Sepharose 6B Pool 2 were pooled and denoted with DEAE-Sephacel Pool 3. All pooled fractions were free of any AP N activity. Uno Q In some purification batches, DEAE-Sephacel Pool 2 required further purification with Uno Q according to SDS-PAGE analysis

2.2.1.2 Isolation of purified DP 4 into its Isoforms Purified porcine DP 4 was further separated into its isoforms by means of preparative liquid phase isoelectric focusing using the Rotofor system from Bio-Rad, a device invented by Egen et al., 1988 (572). In principal, the Rotofor system is a preparative IEF in free solution. The method consists of three steps i.e. loading of sample, focusing and elution as illustrated in Figure 13 (573).

Figure 13: Scheme of Rotofor cell device, involving loading, focusing and elution under vacuum.

After a rotofor run, the collected samples can be further separated by refractionation, which entails pooling selected samples, diluting them to a desired volume and rerunning them

29 Materials and Methods

(574;575). In the present study, the refraction method was elaborated by introducing of a second refractionation step as illustrated in Figure 14 (558).

Figure 14: Schematic overview of the isolation of DP 4 isoforms comprised of parallel Rotofor separation runs, 1st refractionation and 2nd refractionation steps (558). 2.2.1.2.1 Initial Parallel Rotofor Runs Each DEAE-Sephacel Pool was diluted with distilled water to a final volume of 56 ml, also containing 2 % (v/v) Servalyt, pH 5 - 6, and loaded into the focusing chamber of a Rotofor cell from Bio-Rad (Hercules, USA). Focusing was performed at constant power of 15 W at 0° C for 5 hours, using 0.5 M acetic acid and 0.5 M ethanolamine as anode and cathode buffers, respectively. 20 fractions of about 2.5 ml were rapidly harvested at 1 barr and the fractions were characterised with respect to their pH, analytical IEF profiles and specific activities. The three parallel Rotofor runs derived from DEAE-Sephacel Pools 1 - 3 were named Rotofor 1 - 3, respectively. 2.2.1.2.2 First Refractionation According to similar pH, analytical IEF isoform pattern and specific activities, Rotofor 1 - 3 could be divided between fraction of most acidic isoform clusters (closely spaced isoform pattern at pI 5.2), acidic (broad isoform pattern between pI 5.2 and 5.85) and slightly acidic (closely spaced isoform pattern around pI 5.85) isoform clusters. The most acidic and slightly acidic isoform clusters from Rotofor 1 - 3 were each pooled respectively, diluted to 56 ml with water and loaded into the Rotofor chamber to be further refractionated under the same running conditions described above. After harvesting, the fractions were immediately analysed with regards to pH, analytical IEF profiles and specific activities. The first refractionation resulted in two Rotofor runs referred to as Rotofor 4 Pool 1 (originating from most acidic isoform clusters of Rotofor 1 - 3) and Rotofor 5 Pool 2 (derived from acidic clusters of Rotofor 1-3), respectively. Rotofor 4 Pool 1 resulted in most acidic isoform clusters (closly spaced isoform pattern at pI 5.2) and acidic (broad isoform pattern between pI 5.2 and 5.5), whereas Rotofor 5 Pool 2 in most acidic, acidic (isoform pattern between pI 5.5 and 5.85) and slightly acidic (closely spaced isoform pattern around pI 5.85) clusters of DP 4 isoform. 2.2.1.2.3 Second Refractionation Most acidic and acidic isoform clusters from the first refractionation step were each pooled together, respectively, diluted to 56 ml and refractionated for a second time employing the same running conditions described above. Second refractionation of the most acidic isoform

30 Materials and Methods cluster was performed in Rotofor 6 Pool 3, whereas Rotofor 7 Pool 4 further refractionated the acidic one. Due to the low specific activities and protein contents as well as the presence of similar, but few isoform bands on analytical agarose IEF, pH 4 - 6, slightly acidic isoform cluster from Rotofor 1 - 3 and Rotofor 5 Pool 2 were pooled together, diluted to 56 ml and rerun in Rotofor 8 Pool 5. Thus, the second refractionation step consisted of three Rotofor runs named Rotofor 6 Pool 3 to Rotofor 8 Pool 5. After characterisation of fractions from the second refractionation step, identical isolated DP 4 isoforms were pooled together and the ones showing the highest analytical IEF homogeneity as well as specific activity were further processed for crystallisation (13). Table 15 outlines the compositions of the various Rotofor pools prior to their run. Table 15: Composition of Pools 1 - 5, consisting of fractions from Rotofor 1 - 5. F r a c t i o n s Volume Rotofor 1 Rotofor 2 Rotofor 3 Rotofor 4 Rotofor 5 (ml) Pool 1 Pool 2 First Rotofor 4 Pool 1 30 3 - 8 3 - 8 3 - 8 Refractionation Rotofor 5 Pool 2 24 9 - 11 9 - 12 9 - 12 Rotofor 6 Pool 3 28 13 - 17 1- 6 Second Rotofor 7 Pool 4 24 18 - 20 7 - 13 Refractionation Rotofor 8 Pool 5 28 12 - 15 13 - 15 13 - 15 14 - 20

2.2.1.3 Characterisation of Purified DP 4 and its Isolated Isoforms 2.2.1.3.1 Enzyme Activity DP 4 activity was measured with a spectrophotometer U2000 at 30° C, using 0.4 mM Gly- Pro-pNA·HCl as substrate, buffered in 40 mM Hepes with an ionic strength of 0.125 at pH 7.4. The release of para-nitroaniline (pNA) could be determined at =390 nm, and the resulting E/min be converted into enzymatic units by means of the Lambert- Beer’s law: E =  · C · d Since the extinction coefficient for pNA is given with 11.5 mM-1cm-1 and the pathlength of absorbing beam is 1 cm due to the thickness of the cuvette, the concentration of product formation per time interval is directly proportional to the difference of absorption per time. As for DP 4, the product formation has a stoichiometry with respect to substrate hydrolysis of 1:1. Therefore, the computed substrate hydrolysis can be converted into enzyme units. One enzyme unit was defined as the amount of enzyme necessary to hydrolyse one µmol substrate per minute. In order to test for aminopeptidase N activity, 0.4 mM Ala-pNA· HCl in presence of 1 mM

MgCl2 was applied under the same assay conditions described above. 2.2.1.3.2 Protein Determination Protein was determined according to the method of Bradford (576), using BSA for a standards curve ranging from 25 – 1000 µg/ml. Absorption was measured at  = 595 nm at room temperature (Appendix 14). 2.2.1.3.3 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the instruction manual of the Bio-Rad Mini- Protean II Dual Slab cell system, based on the Laemmli SDS system (577). A 7.5 % gel was prepared and samples (5 - 30 µg) were run at 200 V and 64 mA for approximately 45 minutes. The electrophoretic run was carried out under reducing conditions i.e. in presence of a 1:1 sample buffer containing - mercaptoethanol and all samples were incubated for 5 min in a boiling waterbath prior to loading. 31 Materials and Methods

2.2.1.3.4 Native Polyacrylamide Gel Electrophoresis (Native PAGE) A commercially ready made native gel of 4 - 12 % from Novex (Invitrogen, Franfurt/M) was applied. Samples (5 - 30 µg) were diluted with a Tris-Glycine native sample buffer from Novex at a ratio of 1:1 according to the instruction manual and run with a commercially available Tris-Glycine running buffer (Novex) at constant 125 V for about 2 h. 2.2.1.3.4.1 Detection of Proteins after SDS-PAGE or Native PAGE The protocol for protein staining of SDS-PAGE and native PAGE gels with Serva-Blue is given in Appendix 15. 2.2.1.3.5 MALDI-TOF Mass Spectrometry Mass spectrometry of purified DP 4 was performed with a LD-TOF system G2025 provided by Hewlett Packard (Waldbronn, Germany). Prior to mixing with the matrix, the samples were desalted by dialysis against water and diluted to a 150 µg/ml protein concentration. A 1:1 mixture of analyte and matrix solution, composed of 30 mg DHAP, 44 mg DAHC dissolved in 1 ml ACN/0.1 % (v/v) TFA, were combined and 1 µl was transferred to the sample tip. The analyte/matrix complex was immediately evaporated in a vacuum chamber of sample preparation accessory HP G2024A from Helwett Packard (Waldbronn, Germany) in order to obtain a homogeneous crystallization. Calibration of the mass spectrometer was carried out with bovine serum albumin. The mass spectrum represents the cumulative m/z signal for 184 out of 185 single laser shots with a laser power of 1.0 - 2.5 µJ. 2.2.1.3.6 Analytical Isoelectrofocusing Electrophoresis Isoelectric focusing (IEF) is a procedure used to determine the pI of a protein. A continuous pH gradient is established by a mixture of low molecular weight polyamino, polysulfonic and polycarboxylic acids also referred to as ampholytes that distribute themselves in an electric field. Due to the various differently charged side chains of amino acids, proteins differ in their overall net charge, depending on their primary structure, pH of surrounding medium and to a lesser extent their three dimensional structure. Furthermore, glycosylation with sialic acid and phosphorylation can cause microheterogeneity of some proteins in isoelectrofocusing. Therefore, when a protein mixture is applied on a gel with a pH gradient, each protein migrates until it reaches the pH that matches its pI at which it exists as a zwitterion with zero net charge. Proteins with different isoelectric points are thus distributed throughout the gel (575). In the present study analytical isoelectrofocusing was used to investigate the micro- heterogeneity of purified DP 4. As DP 4 was analysed by means of activity staining, only 0.8 % agarose gels were casted according to an adapted method of Kähne et al., 1996 (63). In order to decrease the viscosity of migrating proteins and increase the resolution, 12 % (w/v) sorbitol, and 2.6 % serine (pKa = 5.5) as a separator were added to 0.8 % (w/v) agarose, respectively. The mixture was dissolved by heating and subsequently cooled down to 65 - 70° C for the addition of ampholytes consisting of 7 % (v/v) Servalyt® pH 5 - 6 and 3 % Servalyt® pH 4 - 6 (Serva, Invitrogen, Heidelberg). The gels (0.5 mm thick) were casted by means of the capillary method onto a polyester film (gel bond) and were stored at 8° C overnight. The gel bond was transferred onto horizontal electrophoresis apparatus (Multiphore II, Amersham Pharmacia Biotech, GE, Healthcare, Uppsala) with the ceramic plate being previously covered with a thin film of kerosene to ensure equal heat distribution during the run. Cooling was performed by a cryostat at 0° C and the electrical field was applied by a Power Pac 3000 (Bio-Rad, Munich) and transferred onto the gel via paper electrodes previously soaked in 0.05 M H2SO4 and 0.25 M NaOH buffers for anode and 32 Materials and Methods cathode, respectively. The focusing was accomplished under the following conditions:

Prefocusing: 30 min at constant 1400 V, 30 mA (limit) and 8 W (limit). Loading: Application of samples and markers. Focusing: a) 1 h at 1400 V (constant), 35 mA (limit) and 8 W (limit). b) 30 min at 2000 V (constant), 30 mA (limit) and 35 W (limit).

2.2.1.3.6.1 Protein and Activity Staining of Analytical IEF Agarose Gels Protein Staining The protocol for protein staining with Serva Blue on analytical IEF agarose gels is outlined in Appendix 16. Activity Staining Activity staining consisted of the following two steps: A, capillary blotting; B, activity staining as summarised in Appendix 17, using Gly-Pro-4-methoxy--naphtylamide (Sigma-Aldrich, Steinheim) as DP 4 substrate. The hydrolysed β-methoxy-naphtylamide forms an insoluble red-violet Azo-product with Fast Blue, which acts as an azo-coupler.

2.2.2 Distribution Analysis of DP 4-like Enzymes 2.2.2.1 Design of Primers The following primers were designed with the aid of Vector NTI and ordered from Metabion (Munich) as shown in Appendices 18 - 20, respectively (578). The accession numbers of the templates are outlined in Appendix 21. All primers had the following characteristics:  100 % similarity  no palidrome  no dimers  no hairpin loops  40-60 % GC percentage  last five nucleotides from the 3’ ends  3 deoxyadenylate or deoxythymidylate  all PCR fragments covered  3 exon-intron junction of the gene template As controls, porcine and canine actin primers were designed as shown in Appendix 22. 2.2.2.2 Tissue IsIolation Porcine tissue was obtained from the abattoir at the Institute for Veterinary Physiology of the University of Leipzig. A ±14 month old pig was bolted and exsanguated. Tissue was removed immediately, cut into ±1 - 2 cm3 sections, shock-frozen in liquid nitrogen and stored at -80° C until required. Canine tissue was obtained from LPT in Hamburg. Beagles were anaethesised, tissue was removed, cut into its sections, shock-frozen, sent via courier on dry ice within 3 hours and stored at -80° C until required. The following sections were analysed as summarised in Table 16: Table 16: Tissue sections, used to investigate the distribution of DP 4-like enzymes. Organ Kidney Small Intestine Lung Pancreas

Kidney Cortex Duodenum Bronchial Epithelium Head

Outer medulla, outer stripe Jejunum Alveoli Body Tissue Tissue Section Outer medulla inner stripe Ileum Tail

33 Materials and Methods

2.2.2.3 RNA Isolation The tissues were taken out of -80° C freezer, kept on dry ice and weighed off. RNA was isolated using TRIzol reagent from Invitrogen, Life Technology according to manufacturer instructions. All glasswares and equipment’s were treated and incubated overnight at 37° C with 0.1 % DEPC water, followed by autoclavation at 121° C and 1 bar for 20 min. 2.2.2.4 Transcription Transcription kit from Invitrogen, Life technologies was employed, using Superscript II transcriptase. Except for random hexamer primer from Promega, all reagents were part of a 3’ RACE kit. The protocol was performed according to the description of the 3’ RACE kit, using 10 µg total RNA. 2.2.2.5 PCR For the first PCR, the cDNA template was used and the Herculase polymerase system from Stratagene was implemented. The various primers are described in Appendices 19 and 22, respectively. Briefly, 1 µl cDNA was added to a final 25 µl solution composed of 100 nM of each Primer, 200 µM NTP mix (Promega), 10 x Herculase buffer and 1 Unit of enhanced Herculase polymerase (Stratagene, Agilent Technology, Waldbronn). PCR was performed with an Eppendorf PCR mastercycler, using the following PCR program as outlined in Appendix 23. The temperature gradient and distribution of PCR tubes can be depicted in Appendix 24. The PCR fragments were analysed in a 1 % agarose gel and PCR products with the right size were further used for nested PCR. 2.2.2.6 Nested PCR In order to further verify the PCR products; a nested PCR was performed, applying Taq polymerase from Promega. The primers are summarised in Appendices 20 and 22, respectively. For the nested PCR, 1 µl of PCR product was added to a final 100 µl solution composed of 100 nM of each Primer, 200 µM NTP mix, 20 mM Taq polymerase buffer and 2 Units of Taq polymerase. PCR was performed with an Eppendorf PCR mastercycler, using the following PCR program as outlined in Appendix 25. The temperature gradient and distribution of PCR tubes can be depicted in Appendix 26. The PCR results were analysed on a 1.5 % agarose gel with positive PCR bands either being gel extracted or PCR purified and sent to SeqLab (Göttingen) to verify the sequence. 2.2.2.7 Analysis of PCR Products 2.2.2.7.1 Submarine Agarose Gel Electrophoresis All PCR reactions were analysed on either 1 % or 1.5 % agarose gels, using the horizontal DNA/RNA Minigel System GE-B1A by Hyabaid AGS (Heidelberg) and fragment sizes were determined by comparing them with a 100 bp ladder marker from PeqLab (Heidelberg). 1.0 g or 1.5 g agarose from PeqLab (Heidelberg) were dissolved in 100 ml TBE buffer (579) and 1 µg/ml Ethidium bromide dye by boiling, and a 10 cm gel was poured with 2 rows of 50 µl wells. 10 µl of sample was diluted with 2 µl of sample buffer from PeqLab (Heidelberg), loaded into the wells and the gel was run for about 30 min at 9 V/cm. The results were documented with UV-System from iNTAS (Göttingen).

34 Materials and Methods

2.2.2.7.2 Determination of DNA and RNA Concentrations The concentrations of purified DNA and isolated RNA were determined at 260 nm by Spectro-photometer SmartSpec from BIO-RAD, using the conversion factors of 50 µg/µl and 40 µg/µl per absorption unit for DNA and RNA, respectively.

2.2.2.7.3 Preparation of PCR Products for Sequencing Depending on the quality of PCR fragments, samples were either PCR purified with a JET quick PCR purification kit or in case of alternative splicing, gel extracted with a JET quick gel extraction spin kit. The purified PCR fragment was sent to SeqLab (Göttingen) for sequencing. 2.2.2.8 Activity Analysis of DP 4-like Enzymes in Tissue Extracts

300 mg to 1 g of tissue was homogenised with Ultra Torrex in 1 ml/500 mgtissue of 20 mM Tris- HCl, pH 7.6, containing 0.1 % octyl- glucopyranoside. Solubilisation was achieved by a sonicator (Sonicator Sonoplus SH70G, Bandelin, Berlin), applying 5 x 10 strokes at 50 % power with 20 seconds intervals, whilst keeping the sample on ice. Cell debris was removed by centrifugation at 13,000 g for 15 minutes at 4° C. Activity was determined as outlined in Appendices 27 and 28, using Ala-Pro-AMC and adapting the assay to the microplate reader SUNRISE TECAN (Crailsheim, Germany).

2.2.2.8.1 Inhibition Studies with selective DP 4-like Inhibitors The following selective inhibitors have been applied on the tissue extracts, as summarised in Appendix 29. DP 4-, DP 8- and DP 9- inhibitors were each analysed with 0.125 mM Ala-Pro- AMC, in 40 mM Hepes, pH 7.6, whereas DP 2 was analysed with 0.225 mM Ala-Pro-AMC in 80 mM NaAcetate, pH 5.5. IC50 of Sitagliptin (DP 4-inhibitor) and UG93 (DP 8/9-inhibitor) had previously been determined (135;170). Additionally, an inhibitor-mix composed of the three inhibitors was applied to the tissue extracts and body fluids. In order to convert fluorescence units into enzyme activity units, AMC standard curves were established ranging between 0.018 - 10 µM AMC (Appendix 30) for each assay condition. One enzymatic unit was defined as the amount of enzyme necessary to convert 1 µmol substrate in 1 min. Since substrate hydrolysis and product yield had a stoichometry of 1:1, the decline of substrate equals the product synthesis, and can therefore be deduced from the product (AMC) concentration from the following equation:

Δ Fluorescence x vtotal

Units/l = Δ time x venzyme x Fluorescent Unit / µM AMC

2.2.2.8.2 Protein Determination BCA protein determination assay (Sigma-Aldrich, Steinheim) was implemented, using the microtiter plate procedure described by Redinbaugh and Turley (580). 200 µl BCA assay solution was added to 25 µl of sample, subsequently the microtiter plate was shaken for 10 sec and incubated for 30 min at 30° C. Absorption was taken at 535 nm and a standard curve with BSA (Sigma, Steinheim) was performed, ranging between 0.05 - 1.0 mg/ml with a correlation coefficient of 0.996 as shown in Appendix 31.

35 Materials and Methods

2.2.3 Analysis of DP 4-like Enzymes in Pancreas and Islets of Langerhans 2.2.3.1 Isolation of Islets of Langerhans from Porcine Pancreas Porcine islets of Langerhans were isolated according to the method of Brandhorst et al., 1995 (581). Prior to isolation, all equipment and solutions were autoclaved except for the HBSS and CMRL 1066 solutions that were sterile filtered with a top bottle unit containing 2 µm pores (Appendix 32). Fresh pancreas with portions of duodenum was obtained directly after bolting and exsanguating of a ± 14 month old pig at the Institute for Veterinary Physiology of the University of Leipzig. The pancreas was placed on ice, washed shortly with cold PBS and 200 ml of cold HBSS comprised of 1.0 mg/ml collagenase (Serva, Invitrogen), 10 % FBS (Gibco, Invitrogen) and 0.1 g/ml Trypsin inhibitor (Fluka, Sigma-Aldrich) was injected at the papilla duodeni minor (Appendix 32). Sometimes the papilla duodeni minor could not be found, in such cases the HBSS/collagenase was injected into the lobuli of the pancreas. Once inflated, the pancreas was put on ice and transported to the laboratory. The pancreas was incubated at 32° C for 30 - 40 min. After 15 minutes, aliquots were taken in 5 minute intervals and investigated under a fluorescence microscope Axiovert S100 (Zeiss, Jena) using the phase contrast mode. The incubation was stopped immediately, when the vast majority of islets of Langerhans were solubilised from acinar tissue by the addition of about 100 ml ice cold HBSS with 10 % FBS and by tranferring the extract and remaining pancreas into a kidney bowl, placed on ice. The pancreas was chopped into small pieces, dissociated by a meat grinder, washed with 300 ml of ice cold HBSS + 10 % FBS solution and sieved through a wire mesh with 1000 µm pores. While the filtrate was collected into a 1 l centrifuge flask and kept on ice, the remaining pieces of pancreas were resuspended in 200 ml HBSS with 10% FBS and incubated at 32° C for approximately 20 minutes, taking aliquots in 2 minutes intervals to analyse them under the phase contrast microscope. Incubation was stopped by the addition of 100 ml ice cold HBSS + 10 % FBS and placing the solution on ice. It was then filtered through the meshed wire, washed with 100 ml HBSS + 10 % FBS and collected into a 1 l centrifuge flask. The extract was centrifuged at 200 g for 10 minutes at 4° C. The supernatant was discarded, whereas the pellet resuspended in 300 ml ice cold HBSS with 10 % FBS and centrifuged at 200 g for 10 min at 4° C. This last step was repeated and the resulting pellet was resuspended in 6 ml of HBSS solution containing 25 % Ficoll (w/v). Three layers of 4 ml HBSS solutions, containing 23 %, 20.5 % and 11 % Ficoll (w/v) respectively (Appendix 32), were carefully added onto the suspension and the density gradient was centrifuged at 800 g for 10 min at 4° C. Each layer was removed and twice washed with PBS by resuspension and centrifugation at 500 g for 15 min at 4° C. A schematic illustration of the isolation of porcine islets of Langerhans is given in Figure 15. Finally, isolated islets were resuspended in 5 ml CMRL-11066 supplemented with 10 % NCS, 10 mM Hepes, 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin and 60 µg/ml gentamycin (Appendix 32) and incubated in

25 cm² suspension flasks overnight at 37° C and 5 % CO2.

Figure 15: Schematic overview of the islet isolation from porcine pancreas. 36 Materials and Methods

2.2.3.2 Isolation of Islets of Langerhans from Canine Pancreas Isoloation of canine islets of Langerhans (582) was similar to the method described in the previous section, except that the beagles were anaethesised at LPT, Hamburg, the pancreas was removed, cut into pieces, preserved in LAP-1 solution (Appendix 32), comprised of 3 30 mM D-mannitol, 100 mM K-lactobionate, 15 mM KH2PO4, 5 mM MgSO4, 30 x 10 U/l superoxide dismutase, 10 mM nicotinamide at pH 7.3 according (583) and transported on ice to our laboratory by courier after approximately 2½ hours. Collagenase incubation with HBSS containing 1 mg/ml collagenase, 0.1 mg/ml trypsin inhibitor and 10 % FBS (Appendix 32) was only 15 minutes with an analysis of aliquots after 5 min in 2-5 minutes intervals under a phase contrast microsope. 2.2.3.3 Isolation of Secretory Granules from Isolated Islets of Langerhans Secretory granules from isolated islets of Langerhans were isolated according to the method of Howell et al., 1969 (584). Isolated islets from porcine pancreas were homogenised in 15 ml of

0.3 M sucrose, supplemented with 5 mM Na2HPO4, pH 6.0 at 4° C by applying 20 strokes with a glass teflon homogeniser. After leaving the homogenate to settle for two minutes, the supernatant was removed, the pellet resuspended in 5 ml buffer and rehomogenised with 20 strokes. Both homogenates were combined and centrifuged at 600 g at 4° C for 5 min. The supernatant was removed, the pellet resuspended in 5 ml buffer and centrifuged again at 600 g for 5 min at 4° C. Since the resulting pellets represented nuclei and cell debris, they were discarded, whereas the supernatants were combined and centrifuged at 5,500 g at 4° C for 5 min. After removing the supernatant, the pellet was resuspended in 5 ml buffer and centrifuged at 5,500 g at 4° C for 5 min. The resulting pellets, mainly consisting of mitochondria, were discarded, the supernatants combined and centrifuged at 24,000 g at 4° C for 10 min to obtain a pellet comprised of secretory granules, lysosomes and endosomes. The granules were further purified with a sucrose density gradient, prepared two hours prior to centrifugation and comprised of 1.7 M (0.4 ml), 1.6 M (0.6 ml), 1.55 M (0.6 ml) and 1.4 M (0.4 ml) sucrose supplemented with 5 mM Na2HPO4, pH 6.0. The pellet containing the secretotry granules was resuspended in 2 ml of buffer and 0.6 ml of the suspension was carefully layered on top of the gradient. Sucrose density gradient was performed at 105,000 g at 4° C for 1 h with an ultracentrifuge (Optima Max, Beckmann Coulter). Figure 16 gives a schematic outline of the isolation of secretory granules.

Figure 16: Schematic outline of the differential fractionation to isolate secretory granules from porcine islets of Langerhans. Buffer: 5 mM Na2HPO4/NaH2PO4, pH 6.0, 300 mM sucrose. 37 Materials and Methods

2.2.3.4 Characterisation of Porcine Islets of Langerhans and Secretory Granules 2.2.3.4.1 Islet Viability The membrane integrity of freshly isolated or cultured porcine islets was assessed by a fluorometric viability assay, using fluorescein diacetate and propidium iodide for staining of viable and dead islet cells, respectively (585;586).

2.2.3.4.2 Enzymatic Analysis 2.2.3.4.2.1 Solubilisation Islets of Langerhans After isolation of islets and washing them with PBS, each pellet was resuspended in 5 mM

Na2HPO4 pH 6.0, containing 25 mM sucrose and 1 % octyl- glucopyranoside. Solubilisation was achieved by a sonicator (Sonicator Sonoplus SH70G, Bandelin, Berlin), applying 5 x 10 strokes at 50 % power with 20 seconds intervals, whilst keeping the sample on ice. Cell debris was removed by centrifugation at 13,000 g. For the separation of cytosol from the plasma membrane, isolated islets were resuspended in 5 ml 5 mM Na2HPO4 pH 6.0, containing 25 mM sucrose. After solubilisation and centrifugation as described above, the supernatant was further centrifuged at 105,000 g at 4° C for 1 h by an ultracentrifuge (Optima Max, Beckmann-Coulter). The resulting supernatant was removed, representing the cytosole fraction, whereas the pellet, containing the plasma membrane, was resuspended in equal volume as the cytosole fraction with 5 mM Na2HPO4 pH 6.0, containing 25 mM sucrose and 1 % octyl-ß glucopyranoside. Secretory Granules After the sucrose density gradient, each layer was carefully removed, diluted to 0.8 M sucrose with 5 mM Na2HPO4 and lysed by five cycles of freezing (dry ice) and thawing (37° C). Subsequently, the samples were centrifuged at 105,000 g at 4° C for 1 h by an ultracentrifuge (Optima Max, Beckmann-Coulter). Afterwards, the supernatant was removed, representing the intra-granular fraction, whereas pellet was resuspended in equal volumes of

0.8 M sucrose with 5 mM Na2HPO4 containing 1 % octyl- glucopyranoside (granular membrane). 2.2.3.4.2.2 Activity of DP 4-like Enzymes The activity of isolated islets was determined as described in section 2.2.1.3.1, with the exception that spectrophotometer Lamda 20 (Applied Biosystems, Weiterstadt) was applied and the assay temperature was 37° C. For the analysis of DP 2 activity, 1 mM Lys-Ala-pNA HCl as substrate was implemented, buffered in 80 mM sodium acetate at pH 5.5. Due to its higher sensitivity, 200 µM Gly-Pro-AMCHBr was used to assess the DP 4 activity in isolated secretory granules, buffered in 40 mM Hepes pH 7.6 and 0.7 M KCl. Likewise, 1 mM Lys- Ala-AMCHBr was applied to determine the DP 2 activity, buffered in 80 mM NaAcetate, pH 5.5. The fluorometric assay was performed in microplates with an excitation of 380 nm and an emission of 465 nm, at a gain of 100 and an incubation temperature of 37° C, using microplate reader SUNRISE (TECAN, Crailshaim). In order to convert fluorescence units into enzyme activity units, AMC standard curves were established ranging between 0.018–10 µM AMC (Appendix 30) as described in section 2.2.2.10. 2.2.3.4.2.3 Inhibition Studies The selective DP 4-like inhibitors as summarised in Appendix 29, have been applied, using the assay described 2.2.2.11.

38 Materials and Methods

2.2.3.4.2.4 Protein Determination BCA protein determination assay (Sigma-Aldrich, Steinheim) was implemented, using the microtiter plate procedure descibed by Redinbaugh and Turley (580), outlined in section 2.2.2.11 and shown in Appendix 31.

2.2.3.4.3 Activity Assay for Organelle Enzymes 2.2.3.4.3.1 Alkaline Phosphodiesterase I: Marker of Plasma Membrane Alkaline phosphodiesterase was determined according to Storrie and Madden, 1990 (587), using 0.4 mM sodium thymidine 5’-monophosphate, p-nitrophenylester in 40 mM Tris-HCl, pH 9.0 and adapting the method for a microplate reader. The reaction was stopped with

0.13 M glycine/0.13 M Na2CO3 after 2 hours and adsorption was read at 405 nm. To convert adsorption into enzymatic units per hour, a standard curve was performed with p-nitrophenol, ranging between 312.5 nM - 2.44 µM with a correlation coefficient of 0.999. 2.2.3.4.3.2 Lactate Dehydrogenase: Marker of Cytosol Lactate dehydrogenase (LDH) was determined, using CytoTox 96 non-radioactive cytotoxicity assay (Promega, Darmstadt). The assay utilises the resulting NADH from the conversion of lactate to pyruvate by LDH to reconvert it back to NAD+ in presence of diaphorase, thereby forming the chromophore formazan. The assay was incubated for half an hour and adsorption was read at 490 nm. 2.2.3.4.4 Analysis of DP 4-like Enzymes in Isolated Islets by RT-PCR The same methods were applied as described in sections 2.2.2.1 - 2.2.2.8 for the analysis of DP 4-like enzymes in isolated islets of Langerhans by nested RT-PCR, using the primers summarised in Appendices 19, 20 and 22, respectively. However, to identify any contaminating tissue, the following primers were designed with the aid of Vector NTI software and synthesised by Metabion (Munich) as summarised in Appendix 33. Due to the high GC content of the proinsulin template, the PCR mixture also contained 5 % DMSO (Stratagene, Amsterdam) and the denaturing temperature was increased to 98° C. Kidney cortex and pancreas were used as tissue controls for the primers CD31, carbonic anhydrase and amylase, preproglucagon and proinsulin, respectively.

2.2.3.5 Histochemical Analysis of Porcine Pancreas 2.2.3.5.1 Immunhistochemical Analysis 2.2.3.5.1.1 Production of Rabbit Anti-Porcine DP 4 Polyclonal Antibody 2 mg of purified DP 4 as described in section 2.2.1.1, was injected into a rabbit at Biogene (Berlin) and the antiserum was purified using a 5 ml HiTrap Protein A HP column (Amersham Pharmacia Biotech, GE Healthcare,Uppsala) with a BioCad 700E FPLC system (Perspective Biosystems, Wiesbaden), following the instruction manual of Amersham Biosciences, (Uppsala). Protein concentration was determined according to the Bradford method as described in section 2.2.1.3.2. 2.2.3.5.1.2 Purification of Guinea Pig Anti-Glucagon Antiserum The antiserum was purified, by manually applying 5 ml HiTrap Protein A HP column (Amersham Pharmacia Biotech, GE Healthcare, Uppsala) according to the instruction

39 Materials and Methods manual of Amersham Biosciences, (Uppsala). Protein concentration was determined according to the Bradford method as described in section 2.2.1.3.2. 2.2.3.5.1.3 Sample Collecting Fresh pancreas was obtained from the abattoir at the Institute of Veterinary Physiology of the University of Leipzig. Small squared sections of 2-5 mm3 were cut and immediately fixed in 0.25 M paraformaldehyde and transported to the laboratory. Embedding and immuno- staining was performed by Dr. Gerd Hause from Bioservice (Halle, Germany) according to the method of Hause et al., 1992 (588). Briefly, after fixation, the samples were rinsed, dehydrated and embedded in PEG, sectioned in 2 µm and mounted onto poly-L-lysine coated slides. The slides were pretreated for 10 minutes with PBS, 5 minutes with 0.1 M

NH4Cl dissolved in PBS, followed by 30 minutes with PBS containing 1 % BSA. Incubation with the respective primary antibody/antibodies took place overnight at 4° C, diluted with the appropriate dilution as summarised in Appendix 34 and dissolved in PBS containing 1 % BSA. The slides were subsequently washed four times with PBS containing 0.1 % BSA. The secondary antibodies were incubated at 37° C for 2 hours; their dilutions are indicated in Appendix 34. Finally the slides were twice washed for 10 minutes with PBS, followed by incubation with DABI for 20 minute and twice a wash with PBS for 5 minutes. Afterwards, 60 µl of Citifluor-glycerol was layered and the cover slide was sealed with nail polish.

2.2.3.5.2 Histochemical Activity Staining

2.2.3.5.2.1 Staining of DP 4 with Gly-Pro-1-hydroxy-4-naphthylamide DP 4 histochmical analysis was performed according to the method of Dikov et al, 1999 (18). The substrate was synthesised according to an adapted method by Dr. A. Niestroj from the Department of Medical Chemistry of probiodrug AG (28). Porcine samples of pancreas and kidney were obtained from the abattoir at the Institute for Veterinary Physiology of the University of Leipzig, cut into 1 cm3 sections and immediately shock-frozen in liquid nitrogen. They were transported to the laboratory, where they were stored in -80 °C until required. 10 µm sections were cut on a cryotome at -25° C, mounted onto a non-precooled slide and fixed in acetone at -25° C for five minutes. The slides were incubated in 0.1 M NaH2PO4, pH 7.8 buffer for about 5 min, followed by an incubation in 0.375 mM Gly-Pro-1-hydroxy-4- naphthylamide (structure, Appendix 35) in 0.1 M NaH2PO4, pH 7.8, containing 1 mg/ml NBT, previously dissolved in 200 µl DMF, at 37° C in darkness for 10, 20 or 30 min. After the assay, the slides were twice washed with 0.1 M NaH2PO4, pH 7.0 for 5 minutes, post-fixed with 4 % paraformaldehyde, dissolved in 0.1 M NaH2PO4, pH 7.0 for 10 minutes and washed with 0.1 M NaH2PO4, pH 7.0 for 5 minutes. The slides were embedded in glycerol-gelatine and the cover slide was sealed with nail polish. Three controls were performed, one with an irreversible serine protease inhibitor of 4 mM Pefabloc (La Rosche, Mannheim) dissolved in

0.375 mM Gly-Pro-1-hydroxy-4-naphtylamide, containing 1 mg/ml NBT, in 0.1 M NaH2PO4, pH 7.8 for 3 hours at 37° C in darkness. The second one was the competitive reversible DP 4-like inhibitor of 1 mM P32/98 dissolved 0.375 mM Gly-Pro-1-hydroxy-4-naphthylamide, containing 1 mg/ml NBT, in 0.1 M NaH2PO4, pH 7.8 for 10, 20 or 30 min at 37° C in darkness. The third one was an incubation of buffer containing 1 mg/ml NBT for 10, 20 or 30 min at 37° C in darkness.

40 Materials and Methods

2.2.3.5.2.2 Staining of DP 2 with Lys-Ala-5-chloro-1-anthraquinonyl hydrazide DP 2 histochmical analysis was performed according to an adapted method of Dikov et al, 2000 (589). The substrate was synthesised by Dr. A. Niestroj from the Department of Medical Chemistry (probiodrug AG). Porcine samples of pancreas and kidney were obtained from the abattoir at the Institute for Veterinary Physiology of the University of Leipzig, cut into 1 cm² sections and immediately shock-frozen in liquid nitrogen. They were transported to the laboratory, where they were stored in -80° C until required. 10 µm sections were cut on a cryotome at -25° C, mounted onto a poly-L-lysine coated slide and fixed in 2 % paraformaldehyde at -25° C for five minutes. The slides were incubated in 1 mM Lys-Ala-

CAHHBr (structure Appendix 36) in 0.1 M NaH2PO4, pH 5.5, containing 1 mg/ml pNBA, previously dissolved in 200 µl DMF at 37° C in darkness for 30, 60 or 90 min. After the assay,

the slides were twice washed with 0.1 M NaH2PO4, pH 7.0 for 5 minutes, post-fixed with 4 %

paraformaldehyde, incubated in 0.1 M NaH2PO4, pH 7.0 for 10 minutes and washed with

0.1 M NaH2PO4, pH 7.0 for 5 minutes. The slides were embedded in glycerol-gelatine and the cover slide was sealed with nail polish. Three controls were performed, first with a strong reversible DP 4-specific inhibitor, using 2.3 µM Ile-CN-Pyr (Dr. U. Heiser, Department of

Medical Chemistry, probiodrug AG, Halle) dissolved in 0.1 M NaH2PO4, pH 5.5, containing 1 mM Lys-Ala-CAHHBr and 1 mg/ml pNBA, and an incubation at 37° C in darkness for 30, 60 or 90 minutes (590). Second, was the competitive reversible DP 2-specific inhibitor Dab-

Pip, of which 20 µM Dab-Pip was applied, dissolved in 0.1 M NaH2PO4, pH 5.5, containing 1.0 M Lys-Ala-CAHHBr and 1 mg/ml pNBA; and the samples were incubated at 37° C in darkness for 30, 60 or 90 min (591). Third, sample was incubated with buffer containing 1 mg/ml pNBA for 30, 60 or 90 min at 37° C in darkness.

2.2.3.6 Characterisation of Islets from Langerhans from Rats DP 4 and/or DP 4-like enzymes were assessed at the Department of Physiology of the University of British Columbia (Vancouver, Canada) with respect to activities, perfusion, perifusion and granules, using 70 µM Gly-Pro-AMCHBr as substrate. During the investigations of islets in cell culture, perfusion and perifusion, a number of media components and stimulants were found to interfere with DP 4 activity as summarised in Appendices 37 - 39. Pancreata were perfused as previously described (592), using Somnotol as anesthesia and a modified Krebs Ringer perfusion buffer as summarised in Appendix 40. Glucagon secretion was stimulated with sodium nitroprusside, norepinephrine or splanchic nerve stimulation as outlined in Appendix 39 and analysed by RIA. Samples were immediately stored at -20° C until required. Prior to activity analysis of DP 4 and glucagon, the samples were ultracentrifuged at 105,000 g for 1 h at 4° C to remove endothelial DP 4 derived from haemolysis due to the high pressure of fast flow rate.

41 Materials and Methods

2.2.4 Identification of DP 4 as a Pharmaceutical Target for Psychiatric Disorders 2.2.4.1 Analysis of DP 4-like Enzymes in Neuronal Tissues 2.2.4.1.1 Animals, Brain, Neuronal and Glial Tissues F344/Ztm and F344/Crl(Wiga)SvH-Dpp4 rat substrains were originally obtained from Charles River in 1998 as F344/Crl(Por/98) rat and further inbred at the Central Animal Facility of the Hannover Medical School. While the F344/Ztm rat substrain exhibit a DP4 wildtype-like phenotype, the substrain F344/Crl(Wiga)SvH-Dpp4 is deficient for DP 4 activity due to a DP 4 mutation (275). Brains, and other organs were removed and further dissected into their brain areas by Prof. S. von Hörsten, Dr. N. Frerker and Dr. J. Schade from the Department of Functional and Applied Anatomy at the Hannover Medical School or by Prof. S. von Hörsten and Jenny Stiller from the Department of Experimental Therapy, Franz-Penzoldt-Center, Friedrich-Alexander-University Erlangen-Nürnberg. Primary cell cultures of neurons, astrocytes and microglia were acquired from neonatal brains of Spargue Dawley rats isolated by Dr. Ulrike Zeitschel and Prof. Steffen Rossner from the Paul-Flechsig Institute for Brain Research at the University of Leipzig.

2.2.4.1.2 Tissue Extraction Brains, obtained from F344/Ztm and F344/Crl(Wiga)SvH-Dpp4 rats, were thawed and washed in PBS to remove any remaining blood, and extracted with 20 mM Tris/HCl, pH 7.6, 0.1% - octylglucopyranoside by homogenisation, sonification and subsequent centrifugation at 13,000 g for 15 min at 4° C. Similarly, the isolated and cultivated neuronal primary cell cultures were suspended in 20 mM Tris/HCl, pH 7.6, for 10 min on ice, sonificated and centrifugated at 13,000 g for 15 min at 4° C. Additional brain extract from F344/Ztm rats devoid of any detergents, was further fractionated into cytosolic and membrane by ultra- centrifugation at 105,000 g for 1 h. The resulting pellet was resupended with equal volumes as the supernatant with 20 mM Tris-HCl, pH 7.6, containing 0.1% -octylglucopyranoside.

2.2.4.1.3 Protein, Activity and Inhibition Studies Protein concentration was determined as described in section 2.2.1.3.2 (Appendix 14), activity assay as outlined in section 2.2.2.9 and actvitiy/inhibition studies as summarised in section 2.2.2.9 and 2.2.2.10 (Appendices 27-29), respectively.

2.2.4.1.4 MALDI-TOF-MS Studies In general, a defined amount of tissue extract or enzyme was incubated with an optimised concentration of NPY or neuropeptide (NP) (Dr. S. Manhart, probiodrug AG) ranging between 12.5 - 50 µM in 20 mM Tris-HCl, pH 7.6 at 37° C, and several aliquots were taken either ranging between 0 - 24 h or 0 - 3 h depending on how rapid the NP’s were degraded. The reaction was stopped with 0.1 % TFA at a ratio of 1:1. For the zero time intervals the tissue extract or enzyme was inactivated with 0.1 % TFA prior to the addition of neuropeptide (Appendix 42). Selective inhibitors summarised in Appendix 46 were used to identify enzymes. Afterwards, the samples were either mixed with the matrix sinapinic acid or -cyano-4-hydroxy-cinnamic acid at a ratio 1:1 and analysed with MALDI-TOF mass spectrometry (Voyager-DE Pro Biospectrometry workstation from Applied Biosystem) as described in (135) or illustrated in Figure 17.

42 Materials and Methods

Figure 17: Principles of sample preparation and detection by MALDI-TOF-MS using Voyager-DE Pro Biospectrometry workstation from Applied Biosystem (insert photo). [Taken and adapted from http://www.ncbi.nlm.nih.gov/books/NBK22410/figure/A481/, Berg JM, Tymoczko JL, Stryer L. (2002) Biochemistry. 5th edition. New York: W H Freeman.]

2.2.4.1.4.1 Neuropeptide Hydrolysis by Neuronal, Glial and Brain Tissue Extracts Neuronal and glial tissue extracts were each diluted to a final protein concentration of 40 µg/ml with 20 mM Tris-HCl, pH 7.6 and incubated with 25 µM of either NPY, Substance P or PACAP, taking several aliquots between 2, 10, 30 min, 1, 2, 3, 6 and 24 h at 37° C (Appendix 42). Each tissue extract was analysed with 1 µM DP 4-inhibitor Sitagliptin, 1 mM P32/98, 200 µM phosphoramidon and 2 mM EDTA, respectively. In addition, cleavage of NPY, Substance P and PACAP were also investigated in neuronal tissue extracts with a protein concentration of 400 µg/ml. The cytosolic and membrane fractions of brain extracts obtained from F344/Ztm rats were evaluated at a protein concentration of 200 µg/ml and applying 25 µM NPY. Furthermore, utilising 200 µg/ml brain extract, the influence of 200 µM actinonin, Pepstatin A, Cpp-AAF-pAb (Bachem, Bubendorf) and Pro-Ile was examined on the degradation of 25 µM

NPY1-36, NPY1-30, NPY1-24, NPY1-25 and NPY1-20, respectively. 2.2.4.1.5 Histochemical Analysis Histochemical analysis was performed with rat brain and kidneys sections as outlined in section 2.2.3.5.2.1, however, implementing some adaptations regarding thickness of sections, substrate concentrations and incubation times as summarised in Appendix 41. 2.2.4.1.6 Capillary Depletion Preliminary capillary depletion was carried out according to Triguero et al., 1990 (593), in order to analyse the crossing of peripherally injected NPY1-36-Biotin (50 µM) and its resulting metabolites over the blood brain barrier (BBB) as it has been previously described (562). The buffers employed for brain perfusion and capillary depletion are outlined in Appendix 43. A 10 x conservation solution was added to the perfusate, brain parenchyma and capillary pellet, respectively (Appendix 44). Dormitor/Ketamie was used as anaesthetics and surgery was carried out by Prof. S. von Hörsten as illustrated in Figure 18. Chlorpromazine was implemented as a positive control for BBB, Fluorescein as a negative control of BBB, F344/Crl(Wiga)SvH-Dpp4 rat substrains and Sitagliptin as negative control for DP 4-activity and alkaline phosphatase as a marker for capillaries (Appendix 45). 43 Materials and Methods

Figure 18: Scheme illustrating the method of capillary depletion, comprised of 1st surgery, 2nd perfusion and 3rd capillary depletion (537).

2.2.4.1.6.1 Identification of NPY Degrading Enzymes Due to proteolytical degradation of NPY during the course of capillary depletion, selective inhibitors were implemented against neuropeptidases described in the databases of merops- sanger, pub-med as well as online-handbook of Sigma-Aldrich-homepage on neuopeptidases (594-596) (http://www.merops.sanger.ac.uk, http://www.ncbi.nlm.nih.gov/ sites/entrez?cmd=search&db=pubmed,http://www.sigmaaldrich.com/etc/medialib/docs/Sigm a/RBIHandbook/rbibook5neuropep.Par.0001.File.tmp/rbibook5neuropep.pdf). The list of inhibitors as well as the respective commercial recombinant or purified enzymes are outlined in Appendix 46. MALDI-TOF-MS analysis was performed as described in section 2.2.4.1.4, using 25 µM NPY and extracts of brain parenchyma. Appendix 47 summarises the activation of Mep-A and Mep-B. As to identify smaller NPY fragments, aliquots were additionally analysed with the matrix -cyano-4-hydroxy-cinnamic acid such as for meprin A, meprin B, trypsin, thrombin, plasmin, urokinase, plasma kallikrein and tissue kallikrein. Furthermore, degradation of NPY by cathepsin D was investigated both at pH 5.5 as well as pH 7.6. The composition of improved 10 x conservation solution is outlined in Appendix 48. 2.2.4.2 Analysis of Neuropeptide Hydrolysis by DP 4 in the Periphery 2.2.4.2.1 Human and Rat Serum Human sera were obtained from 18 volunteers and either immediatedly analysed or 1 x frozen. Sera from 6 Wistar rats were immediately frozen and stored at –80° C until required. 2.2.4.2.1.1 Activity and Inhibition Studies Human serum was diluted 1:9 prior to measurements and DP 4-like activity in was analysed similar to section 2.2.2.9 and 2.2.2.10, however, using 200 µM Gly-Pro-AMC and 1 mM Gly- Pro-AMC as substrates for DP 4-like enzymes and DP 2, respectively. The assay was adapted for Costar 96-well microplates half-area to allow reduction of volumes of all assay reagents by half (Appendices 27 and 29) and DP 4-like activity was determined with microplate reader Fluostar (BMG Laboratories) at a gain of 1200. Inhibitors Sitagliptin, P32/98 and Dab-Pip were implemented as outlined in Appendix 30. 44 Materials and Methods

2.2.4.2.1.2 Influence of Quenching Effects by Haemolysis on DP 4 Activity Since fluorogenic AMC detection is quenched by haemolytic serum, a cruor was diluted 200 - 1,000,000-fold with PBS and for each dilution an AMC standard curve, ranging between 9.5 nM - 10 µM was performed (Appendix 49). The degree of haemolysis was measuered by the adsorption at 405 nm and 690 nm, taking the difference of these two readings (Appendix 50). An arbitary cut-off limit of 0.1 was set for the difference 405 nm-690 nm. 2.2.4.2.1.3 MALDI-TOF-MS MALDI-TOF-MS analysis was implemented as described section 2.2.4.1.4 and Appendix 42, using 50 µM of the stress-related neuropeptides NPY, SP, GALP, Gal, -Endo, rat and human OrxB, OrxA, Oxy, AVP, NT, ACTH and CRF as summarised in Appendix 51. Neuropeptide stability was determined either in 1 x frozen or fresh serum, using 5-fold or 25- fold dilutions. 5-fold diluted sera were desalted by C18-ZipTips (Millipore) prior to the addition of matrix as outlined in Appendix 52. 2.2.4.2.2 Neuropeptide Hydrolysis ex vivo in Human Blood Heparin-blood was either diluted 5-fold or 10-fold with 20 mM Tris-HCl, pH 7.6 containing sucrose to obtain a final isotonic concentration of 300 mM (Appendix 53). As a control haemolysed blood diluted with hypotonic 20 mM Tris-HCl, pH 7.6 was also implemented. The hydrolysis of 25 µM NPY, GALP and SP were analysed as described in Appendix 53 and the reaction was stopped by C18-ZipTips (Appendix 52) and matrix instead of 0.1 % TFA. 2.2.4.2.3 Human CSF Frozen samples of human cerebrospinal fluid from eight healthy volunteers were kindly supplied Prof. Dr. B. Leavitt from the Centre for Molecular Medicine and Therapeutics at the Child and Family Research Institute of the University of British Columbia, Vancouver, Canada. The samples were never thawn before and of good quality as summarised in Table 17.

Table 17: Properties of human CSF, used to analyse DP 4-like activity and hydrolysis of 12.5 µM NPY. n male/female Average Age Range of Age RBC Count (x 106/L) WBC COUNT (x 106/L) 8 4/4 51.1 ± 15.5 27 - 66 < 8 (2.5 ± 3.3) < 2 (1.4 ± 0.74)

2.2.4.2.3.1 Activity, Inhibition Studies and Protein Determination DP 4-like activity in human CSF was analysed as described in section 2.2.4.2.1.1 and protein was determined according to the Bradford method as outlined in section 2.2.1.3.2 (576). 2.2.4.2.3.2 MALDI-TOF-MS NPY hydrolysis by human CSF was analysed by MALDI-TOF-MS as explained in section 2.2.4.1.4 and Appendix 42, however doubling the volume of CSF samples to obtain a 2.5-fold final dilution and applying 12.5 µM NPY. 2.2.4.2.3.3 Western Blot Medium of hCTSD-HEK-cells (Dr. D. Schlenzig) was concentrated 5-fold with vivaspin 500 (Satorius). 25 µg of the concentrated medium, 1 µg purified CTSD and 20 µl human CSF were loaded and separated by 12 % SDS/PAGE under reducing conditions and then transferred to a nitrocellulose membrane (Millipore). Immunodetection was performed with rabbit anti-human-CTSD-pAb (Cell Signaling Technology, Inc) according to the manufacturer’s instructions and antibody complexes were detected by SuperSignal West Femto substrate (Perbio, Thermo Scientific, Erembodemgem-Aalst). 45 Materials and Methods

2.2.4.3 Characterisation of DP 4-like Enzymes in Sera from Depressed Patients 2.2.4.3.1 Serum Samples from Depressed Patients Frozen serum samples from 32 depressed patients and 30 healthy controls were kindly provided by PD. Dr. M. Rothermundt (head of department) and Dr. F. Kästner from the Department of Psychiatry at the University of Münster. Patients were diagnosed for a clinical major depressive episode according to DMS IV, 296.1 and 296.2, (Diagnostic and Statistical Manual of Mental Disorders) as well as ICD 10, F32 and F33 (International Classification of Disease and Related Health Problems) and further subdiagnosed for melancholic ad non- melancholic depression as summarised in Table 18 and Appendix 54 (597;598). Healthy controls were age and sex matched and free of any psychiatric history as well as medication. All subjects underwent medical examinations for past and chronic illnesses as well as acute infections. Furthermore, serum was drawn from depressed patients prior to treatment (t1) and after complete clinical remission (t2), having a MADRS (Montgomery and Asberg Depression Scale)  10 (597-599). Treatment consisted of antidepressants, predominantly comprised of re- uptake inhibitors (± 80 %) and to a lesser extent of tricyclics (± 10 %) and monoamine oxidase inhibtors (MAOI’s) (± 10 %) (597;598). All sera were stored upon arrival at - 80° C until required, slowly thawn on ice and analysed for DP 4-like activity, stored again overnight at 4° C and analysed for NPY and SP hydrolysis as well as post-translational modifications, using MALDI-TOF-MS and analytical agarose IEF, respectively. Afterwards they were frozen again at -80° C until the CD26 (DP 4) concentration was determined by ELISA. Table 18: Summary of depressed patients and the respective control subjects for serum analysis. Average Age (Range) Gender (Females/Males) Total 44.91 ± 14.28 23/9 Melancholic 51.63 ± 11.12 11/5 Non-melancholic 38.00 ± 14.06 12/4 Healthy Subjects 42.33 ± 12.72 21/9 2.2.4.3.1.1 Activity and Inhibition Studies Serum samples were diluted 10-fold and activity was determined as described in section 2.2.4.2.1.1, using inhibitors Sitagliptin, P32/98 and Dab-Pip (Appendix 30). Each sample was evaluated for haemolysis as explained in section 2.2.4.2.1.2 by taking the difference 405 nm - 690 nm.

2.2.4.3.1.2 Analysis of NPY and SP Hydrolysis by MALDI-TOF-MS Serum samples were diluted 5-fold and the hydrolysis of 50 µM NPY and SP was determined as described in section 2.2.4.2.1.3 by MALDI-TOF-MS (Appendix 42).

2.2.4.3.1.3 Analysis of DP 4-like Activity by Analytical Agarose IEF 20 µl of serum was loaded onto a 0.8 mm agorose gel, pH 3 - 7 with IEF electrophoresis and activity staining being carried out as in section 2.2.1.3.6 and 2.2.1.3.7.2, respectively.

2.2.4.3.1.4 Determination of DP 4 Concentration by sCD26 ELISA Kit DP 4 concentrations were determined, using sCD26 ELISA kit (Bender Medsystem GmbH, eBioscience, Vienna) according to the instruction manual and deriving DP 4 concentration from a standard curve ranging between 15.6 - 500 ng/ml as depicted in Appendix 55. Samples were measured in duplicates and differed  20 % from the mean. Specific Activity of DP 4 in serum was computed as mU/µg. 46 Results and Discussion

3 Results and Discussion 3.1 Purification of DP 4 and Isolation of its Isoforms 3.1.1 Purification of DP 4 The results of the DP 4 purification are summarised in Table 19. The final pooled fractions of purified DP 4 obtained from DEAE-Sephacel P1 - 3 have specific activities and purification factors ranging between 31.26 to 37.16 and 200.9 to 238.7, respectively. In total, more than 110 mg of DP 4 protein was purified from 1 kg porcine kidney cortex. No AP N activity could be detected in any of the DEAE-Sephacel Pools. SDS-PAGE analysis displayed broad bands of 100 - 115 kDa, typical for DP 4 and partially indicating post-translational microheterogeneity due to glycosylation (Fig 19 B). In order to ensure homogeneity of the protein, native gradient gel and MALDI-TOF mass spectrometry was performed. Native gradient PAGE gel showed a single broad band at about 200 kDa (Figure 19 A) and MALDI-TOF-MS resulted in single, but broad peaks of 203, 102, 68, 51, 34 kDa, representing dimer, monomer, 3-, 4- and 6-fold charged molecules, respectively (Figure 19D). However, analytical agarose IEF in Figure 19C revealed a high degree of microheterogeneity, resulting in at least 17 isoforms that range between pH 4.8 and 6.0. These isoforms appeared to occur in clusters with different concentrations. Upon activity staining, bands around pI 5.2 - 5.3 became first visible, followed by pI 5.4 - 5.5 and 4.8 - 5.0. Bands around pI 5.8 - 6.0 only showed up when over-staining were performed. Figure 19C also demonstrates the higher sensitivity of activity staining compared to Serva blue staining. Separator molecule serine refined the resolution around pI 5.5.

A B C D a b c d e f kDa pI

205 - 6.15 - 5.85

116 97 84 66 - 5.2 - 4.15 55 - 3.75

Figure 19: Analysis of purified porcine DP 4 from kidney cortex. A, Native PAGE with a 4 -12 % gradient; a, purified porcine DP 4. B, 7.5 % SDS-PAGE of purified DP 4, b, marker; c, purified porcine DP 4; C, analytical 0.8 % agarose IEF, pH 4 - 6; d, DP 4 activity stained with Gly-Pro-4-methoxy--naphtylamide; e, DP 4, Serva Blue stained, f, IEF standards. D, MALDI-TOF mass spectra of purified porcine DP 4 from kidney cortex. Peak 1 - 5 correspond to the mass of 6-, 4-, 3-, 2- and 1- fold charged molecule, respectively. The mass spectrum represents the cumulative m/z signal for 184 out of 185 single laser shots with a laser power of 1.0 - 2.5 µJ (558).

47 Results and Discussion

Table 19: Purification table of porcine DP 4, isolated from 1,200 g kidney cortex. D P 4 AP N Volume Total Protein Total Specific Purification Specific % Purification step Units/ml Yield (ml) Units mg/ml protein Activity factor Activity AP N Autolysis 1,300 4.67 6,071 30.01 39,013 0.16 100 1 0.001 0.63

60 % (NH4)2SO4 1,370 3.99 5,468 6.59 9,034 0.61 90.1 3.89 0.002 0.33 85 % (NH4)2SO4 132 33.7 4,449 47.63 6,287 0.71 73.3 4.55 0.01 1.45 Sepharose 6B P1a 78 41.07 3,203 22.31 1,740 1.84 52.8 11.83 0.02 1.14 Sepharose 6B P2a 71 15.52 1,102 15.44 1,096 1.00 18.2 6.45 n.d.d n.d.d DEAE-Sephacel P1b 30 82.85 2,485 2.28 68.4 36.34 41.0 233.5 n.d.d n.d.d DEAE-Sephacel P2b 12 84.84 1,018 2.28 27.4 37.16 16.8 238.7 n.d.d n.d.d DEAE-Sephacel P3c 18 26.57 478 0.85 15.3 31.26 7.9 200.9 n.d.d n.d.d a, DP 4 active fractions were divided into two pools designated with Sepharose 6B P1 and Sepharose 6B P2; b, DEAE-Sephacel P1 and P2 derived from Sepharose 6B P1; c, DEAE-Sephacel P3 originated from Sepharose 6B P2; d, n.d. not detectable

3.1.2 Isolation of DP 4 Isoforms 3.1.2.1 Parallel Rotofor Runs A general overview of the purified DP 4 pools applied for the three parallel Rotofor runs and the resulting IEF patterns is given in Figure 20. All three parallel Rotofor runs resulted in similar IEF patterns that could be divided into most acidic- (closly spaced isoform pattern at pI 5.2), acidic- (broad isoform pattern between pI 5.2 and 5.85) and slightly acidic clusters (closly spaced isoform pattern at pI 5.85). An example of these IEF clusters is illustrated Figure 21, representing the protein-stained IEF profile of Rotofor 1 (Figure 21A) and activity stained IEF profiles of Rotofor 1 - 3, respectively (Figure 21C - E). The corresponding elution profile of Rotofor 1 is depicted in Figure 21B, showing a single broad peak of DP 4-specific activities, with a maximum between fractions 6 and 12 and a narrow protein peak ranging between 10 and 13.

Figure 20: Scheme of initial parallel separation runs of Rotofor 1 - 3. Sample composition of purified DP 4, 2 % Servalytes pH 5 - 6 and triple distilled water to a volume of 56 ml. Running conditions: 15 W for 5 h at 0° C.

Gradual separation between the acidic and less acidic isoforms could already be achieved during the parallel Rotofor runs and activity staining of analytic IEF showed highly similar pI profiles for Rotofor 1 - 3 as demonstrated in Figure 21C - E. The pH gradient in Figure 21F represents the mean of all three parallel Rotofor runs with standard errors of means and indicates that the conditions chosen for the preparative IEF were highly reproducible. This is further supported by comparing the mean pH gradient profile of two independent DP 4

48 Results and Discussion preparations consisting of 3 and 4 independent parallel runs as shown in Figure 22. The statistical analysis with respect to pH gradient is summarised in Table 20. The linear portion of the mean pH gradient from all three Rotofor runs ranged between 4.3 - 5.5 (fractions 5 - 16), had a slope of 0.099 pH units per fraction and a correlation coefficient of 0.9921. Furthermore, a standard mean deviation of 0.047 was obtained for Rotofor 1 - 3. Due to the high reproducibility of the parallel Rotofor runs, fractions were pooled together according to their pH, IEF-pattern and specific activities for further refractionations as indicated in Figure 21.

F

Figure 21: Analysis of fractions obtained from initial parallel separation runs of Rotofor 1 - 3, using purified DP 4. A + B, Analysis of the Rotofor 1, using purified porcine DP 4 from DEAE- Sephacel-Pool 1. A, analytical agarose IEF, pH 4 - 6, Serva Blue stained of Rotofor 1; B, elution profile representing Rotofor 1; solid line, protein concentration; ▲,specific activity;■, pH profile of Rotofor fractions. C - E, represent analytical IEF profiles, pH 4 - 6 from Rotofor’s 1 - 3 respectively, activity stained with Gly-Pro 4-methoxy--naphthylamide; dotted lines indicate most acidic-, acidic- and slightly acidic clusters pooled together for subsequent refractionation. F, mean pH profile with standard error of mean from Rotofor’s 1 - 3 (558).

Table 20: Statistical analysis of parallel Rotofor runs of 2 independent DP 4 preparations with respect to pH-gradient.

Linear range pH –range of pH gradient Standard mean Batch # n R (# fraction) linear portion (pH/fraction) deviation 1 3 5 - 16 4.3 - 5.5 0.099 0.992 0.047 2 4 6 - 16 4.9 - 6.6 0.139 0.960 0.142

49 Results and Discussion

Figure 22: pH-gradient of parallel Rotofor separation runs, using 2 independent DP 4 preparations. ▲, first DP 4 preparation for preparative IEF, n = 3; , second DP 4 preparation for preparative IEF, n = 4.

3.1.2.2 First Refractionation The composition and properties of the pooled fractions used for the first refractionation are described in Tables 21 and 22, respectively. The gels and the corresponding elution profiles are depicted in Figure 23. Enhanced separation of the isoforms could be seen on both analytical agarose IEF’s gels (Figure 23 A and C) compared to previous Rotofor separation runs (Figure 21). However, the gels also indicated that an additional step would be required for further separation. Analytical IEF of Rotofor 4 Pool 1 resulted in two distinct isoform patterns of most acidic- (closely spaced bands at pI 5.2) and acidic clusters (broadly spaced bands between pI 5.2 and 5.5) as illustrated in Figure 23A. Rotofor 5 Pool 2 in turn, revealed three different isoform patterns of most acidic- (closely spaced bands at pI 5.2), acidic- (broadly spaced bands between pI 5.2 and 5.5) and slightly acidic clusters (isoforms between pI 5.5 and 5.85) as depicted in Figure 23C. Furthermore, isoforms below pI 5.2 and above pI 5.85 became more apparent in most acidic and slightly acidic clusters, respectively. The linear pH range of Rotofor 4 Pool 1 was 4.5 to 5.35 with a slope of 0.67 pH units per fraction (Figure 23B) in contrast to 5.15 - 5.75 for Rotofor 5 Pool 2 with a gradient of 0.033 pH units per fraction (Figure 23D). Interestingly, according to the pH gradients of the Rotofor fractions from the first refractionation, no isoforms were observed below pH 5.0 in Rotofor 4 Pool 1 (Figure 23B), although it arose from the most acidic clusters of Rotofor 1 - 3; whereas Rotofor 5 Pool 2 showed isoforms ranging between pH 4 and 7 (Figure 23 D). This implies that the DP 4 isoforms may have aggregated in liquid solution to have an altered overall surface net charge, whereas migration in an agarose IEF gel appeared to resolve the aggregation due to viscosity. In such case, the deciding factor for pooling was the IEF pattern (Table 21 and Figure 23). Furthermore, distribution of Servalytes was shown to differ in liquid-phase IEF, implementing the Rotofor cell (545-457).

Table 21: Composition of pooled fractions derived from the three parallel Rotofor runs and used for the first refraction. Vol. (ml) Rotofor 1 Rotofor 2 Rotofor 3 Rotofor 4 Pool 1 30 3 - 8 3 - 8 3 - 8 Rotofor 5 Pool 2 24 9 - 12 9 - 12 9 - 11

Table 22: Properties of pooled fractions derived from parallel Rotofor runs 1 - 3, diluted to 56 ml and employed for the first refractionation. Rotofor # Vol. [Ampholyte] Total [Protein] Total Protein Spec. Act. pH Units/ml Pool # (ml) % Units (mg/ml) (mg) (Units/mg) Rotofor 4 P1 5.11 56 1.6 14.06 489.11 0.60 33.66 23.39 Rotofor 5 P2 5.61 56 1.1 16.45 659.01 0.65 36.52 25.22 50 Results and Discussion

pI 2 4 6 8 10 12 14 16 18 20

5.85 A

5.2 most acidic 3.75 acidic

2 4 6 8 10 12 14 16 18 20 5.85 C

5.2 most slightly acidic 3.75 acidic acidic

Figure 23: Characterisation of first refractionation step. A, Analyical agarose IEF, pH 4 - 6 of Rotofor 4 Pool 1. DP 4 isoforms are stained with Gly-Pro 4-methoxy--naphthylamide. B, elution profile of Rotofor 4 Pool 1; solid line, protein concentration; ▲, specific activity;, pH profile of Rotofor fractions. C, Analyical agarose IEF, pH 4 - 6 of Rotofor 5 Pool 2. DP 4 isoforms are stained with Gly-Pro 4-methoxy--naphthylamide. D, elution profile of Rotofor 5 Pool 2; solid line, protein concentration; ▲, specific activity; , pH profile of Rotofor fractions (558).

3.1.2.3 Second Refractionation Excessive dilution of ampholyte concentrations with similar pH was avoided by pooling fractions from parallel Rotofor separation runs. Thus, a second refractionation step could be introduced to further separate the isoforms of DP 4. The composition and properties of the pooled fractions employed for the second refractionation are summarised in Tables 23 and 24. In the second refractionation, a pH gradient of less than 0.028 pH units per fraction could be obtained as illustrated in Figures 24B, D, and F. Hence, the linear pH region ranged from 5.35 - 5.60 with a slope of 0.028 for Rotofor 6 Pool 3, 5.40 - 5.75 with a gradient of 0.025 for Rotofor 7 Pool 4 and 5.60 - 5.90 with a slope of 0.022 pH units per fraction for Rotofor 8 Pool 5. A drop of pH in the gradient was observed in Rotofor 7 Pool 4. This phenomenon has been repetitively observed and can be explained by ampholytes (Servalytes) binding to these isoforms once there is no high electrical field. Latter explanation is further substantiated by the distortion of isoform bands in the analytical IEF in this particular pH region (Figures 22C and D). Interestingly, isoform bands in Rotofor 6 Pool 3 appear in the acidic region on the analytical IEF, but have an overall pH of 5.4 - 8.55 (Figures 22A and B). A possible reason could be the aggregation of the isoforms in absence of an electrical field with an altered surface net charge, explaining the presence of two or more isoforms per fraction. Fractions with only one isoform were obtained in Rotofor 8 Pool 5, however, these fractions displayed the least specific activity ranging between 1 - 3 Units/mg (Figures 22E and F) compared to 15

51 Results and Discussion

- 50 Units/mg in Rotofor 6 Pool 3 and Rotofor 7 Pool 4 (Figures 22A - D). Rotofor 6 Pool 3 attained the highest purity with only 2 isoforms per fractions. Isoforms framed in Figure 24 were pooled, characterised as outlined in Table 25 and sent to Munich for crystallisation. Figure 25 represents the crystals and spacer group data obtained from the isoforms, respectively.

Table 23: Composition of pooled fractions used for the second refractionation. Fractions were either derived from the first refractionation or parallel Rotofor runs as indicated.

Parallel Rotofor runs 1st refractionation Vol. (ml) Rotofor 1 Rotofor 2 Rotofor 3 Rotofor 4 Pool 1 Rotofor 5 Pool 2 Rotofor 6 Pool 3 28 13 - 17 1 - 6 Rotofor 7 Pool 4 24 18 - 20 7 - 13 Rotofor 8 Pool 5 28 13 - 14 13 - 14 12 - 15 14 - 20

pI 2 4 6 8 10 12 14 16 18 20

5.85 A

2 5.2 1

pI 2 4 6 8 10 12 14 16 18 20 C 5.85

4 5.2 3

pI 2 4 6 8 10 12 14 16 18 20 E 5.85

5.2

Figure 24: Analysis of fractions obtained from Rotofor separation runs of the second refractionation step. A, analytical IEF, pH 4-6 of Rotofor 6 Pool 3 representing fractions 1 - 20, activity stained with Gly-Pro 4-methoxy--naphthylamide. B, elution profile of Rotofor 6 Pool 3, solid line, protein; ▲, specific activity (Units/mg); , pH; frame indicate pooled isolated isoforms used for crystallisation. C, analytical IEF, pH 4 - 6 of Rotofor 7 Pool 4 representing fractions 1 - 20, activity stained with Gly-Pro 4-methoxy-- naphthylamide. D, elution profile of Rotofor 7 Pool 4, solid line, protein; ▲, specific activity (Units/mg); , pH; frame indicate pooled isolated isoforms used for crystallisation. E, analytical IEF, pH 4 - 6 of Rotofor 8 Pool 5 representing fractions 1 - 20, activity stained with Gly-Pro 4-methoxy--naphthylamide. F, elution profile of Rotofor 8 Pool 5, solid line, protein; ▲, specific activity (Units/mg); , pH. Frame indicates pooled isoform fractions sent to Martinsried, Germany for crystallisation (558). 52 Results and Discussion

Table 24: Properties of pooled fractions derived either from first refractionation step or parallel Rotofor 1 - 3, diluted to 56 ml and applied for the second refractionation.

Total Specific Rotofor # Volume [Ampholyte] Total [Protein] pH Units/ml Protein Activity Pool # (ml) % Units (mg/ml) (mg) (Units/mg) Rotofor 6 P3 5.80 56 0.6 3.18 178.32 0.26 14.62 12.2 Rotofor 7 P4 6.21 56 0.5 8.32 466.17 0.48 27.03 17.25 Rotofor 8 P5 6.14 56 1.1 3.85 215.58 0.28 15.81 13.64

Table 25: Properties of purified and pooled DP 4-isoforms containing only 2 - 3 isoforms per pool, which were sent to the working group led by Prof. Bode, Martinsried, Germany for further processing of crystallisation. All pools of purified DP 4-isoforms were obtained from the second refractionation.

Rotofor # # pooled Volume Total [Protein] Total Protein Specific Activity Fractions pH Units/ml Pool # isoforms (ml) Units (mg/ml) (mg) (Units/mg) Rotofor 6 P3 1 8 - 17 5.23 19 6.44 122.44 0.25 4.80 25.53 Rotofor 6 P3 2 18 - 20 6.66 6 24.16 144.97 0.62 3.72 39.00 Rotofor 7 P4 3 1 - 7 4.31 13.8 11.07 152.76 0.44 6.06 25.20 Rotofor 7 P4 4 8 - 10 4.82 5.2 21.74 115.22 0.84 4.34 26.03

Space group P1

Space co-ordinates a 61.973 Å b 118.069 Å c 133.506 Å  112.788 Å  94.972 Å  91.175 Å Dataset Best resolution of complete native 1.8 Å Dataset Figure 25: DP 4 crystals obtained from the isoforms by the collaboration partners of the working group led by Prof. Bode, MPI, Martinsried, Germany. Crystallography data acquired from DP 4 isoforms by the collaboration partners of the working group led by Prof. Bode, MPI, Martinsried, Germany, resulting in the first native DP 4 crystal pdb: 1ORV (13).

This is the first time that DP 4 isoforms have been isolated on a preparative scale. Only gradual separation from purified rat kidney had been accomplished with crown ether silica chromatography, yielding clusters of 6 - 18 DP 4 isoforms between pI 5.5 and 6.8 (65). Unlike human DP 4, deglycosylation of porcine DP 4 resulted in a reduced enzymatic activity as demonstrated in Figure 28. Since these isoforms were shown to be closely spaced together in several reports (63-65) and the present study, preparative isoelectric focusing appeared to be the only option to separate them. In an initial attempt to separate the DP 4 isoforms over a narrow pH range with non-ampholytic Biolytes (Bio-Rad) covering pH range of 4.9 to 6.2, the obtained fractions resulted in precipitation and denaturation of DP 4 due to uneven distribution of Biolytes (600-602). Gradual separation with an ampholytic pH gradient of 5 to 6, using Servalytes, maintained DP 4 activity, and therefore appeared to be a more gentle method (600;602). However, investigating the results of the Rotofor fractions from an initial Rotofor and subsequent refractionation run on an analytical IEF did not give the satisfactory homogeneity required for crystallisation as seen in Figures 21 and 23. Thus, the method of refractionation had to be elaborated. This could be achieved by initially running three parallel Rotofor separation runs under the same conditions in presence of 2 % ampholytes, having a protein concentration between 0.27 - 1.22 mg/ml. Since the analytical IEF profiles were highly reproducible, fractions 53 Results and Discussion with similar IEF pattern could be pooled together so that the ampholyte concentrations of the pooled fractions for the first refractionation were 1.6 and 1.1 % for Rotofor 4 Pool 1 and Rotofor 5 Pool 2, respectively (Table 22). This was sufficient for a protein concentration of 0.6 and 0.65 mg/ml (Table 22) according to the instruction manual for the Rotofor by Bio-Rad. The high reproducibility of the pH gradients obtained from the separation runs of Rotofor 1 - 3 indicated that the apparent heterogeneity of DP 4 in Figures 21 and 22 was not a result of remixing upon harvesting. Furthermore, comparing the parallel Rotofor separation runs between two batches of purification and isoform isolation even using a different Rotofor apparatus showed that the method is of general applicability as seen in Figure 22. The main achievement of the first refractionation was the separation of the most acidic isoforms around pI 5.2 from the acidic ones with pI 5.5 - 5.85, (Figure 23) compared to 5.2 - 5.85 in the initial parallel Rotofor runs (Figure 21). By pooling similar fractions from Rotofor 4 Pool 1 and Rotofor 5 Pool 2, sufficient ampholyte concentrations of 0.6, 0.5 and 1.1 % were attained for Rotofor 6 Pool 3, Rotofor 7 Pool 4 and Rotofor 8 Pool 5, respectively (Table 24). Thus, a second refractionation step could be introduced, eventually giving satisfactory results with regards to the homogeneity of the isoforms (Figure 24). Hence, porcine DP 4 was finally separated into most acidic (Rotofor 6 Pool 3), acidic (Rotofor 7 Pool 4) and slightly acidic (Rotofor 8 Pool 5) isoforms (Figure 22). Figures 24A and C showed fractions with only two to three closely spaced isoforms per fraction, having sufficient protein yields and specific activities to be further processed for crystallisation (Table 25) (13). Separation of isoforms could also be observed in the elution profiles of Figures 21, 23 and 24, in which the specific activities changed from a single broad peak (Figure 19) to several distinct ones (Figures 21 and 22). Fractions with only one isoform were obtained in Rotofor 8 Pool 5, however, these fractions displayed the least specific activities ranging between 1 - 3 Units/mg compared to 15 - 50 Units/mg in Rotofor 6 Pool 3 and Rotofor 7 Pool 4 (Figure 24). In our present study we found instability of the more cationic DP 4 isoforms considering the low specific activity in Rotofor 8 Pool 5 and fragmentation observed in SDS-PAGE (data not shown) and MALDI-TOF analysis after several weeks of storage (558). Such degradation may have resulted from the autolysis step during the DP 4 purification, in which lysosomal glycosidases including sialidase were released. Therefore, they can be considered as possible artefacts. Thus, by developing this method of preparative isoform isolation, new insight might be obtained by physical and biochemical analysis of DP 4 microheterogeneity. Agarose IEF has proven to be a powerful tool to analyse this microheterogeneity of DP 4 due to the ease and gentleness of blotting, ensuring that no activity or isoform of DP 4 was lost. Apparent lower resolution compared to polyacrylamide IEF was compensated by adding the separator molecule serine. This enhanced the resolution around pH 5.5, though it abolished the linearity of pH gradient. Furthermore, the agarose IEF has also the potential to be used for the investigation of DP 4 isoforms from cell lysates by activity staining because of its large pores and lack of any toxic additives (63). The analytical IEF showed a great reduction of DP 4 heterogeneity from the initial parallel Rotofor run to the final Rotofors of the second refractionation. This was in agreement with the gradual decrease of pH gradients from 0.099 pH units per fraction for the initial parallel Rotofor runs to 0.067 and 0.033 in the first refractionation to 0.022 - 0.028 pH units per fraction in the second refractionation (Figure 26). Intriguingly, the pH gradient of the Rotofor fraction covers a different pH range than the corresponding isoform

54 Results and Discussion bands observed in the analytical IEF. Rotofor 6 Pool 3 in particular, had isoform bands in the acidic region of the analytical IEF, but an overall pH range between 5.4 - 8.55 (Fig. 22A and B). A possible reason could be the aggregation of the isoforms in solution, having a different overall surface net charge. While the pH gradient of the Rotofor was distributed only over 20 fractions, analytical IEF appeared to have more pH increments, explaining the presence of two or more isoforms per fraction (600;602). Furthermore, higher viscosity due to presence of sorbitol may have also resolve isoform aggregation.

Figure 26: Example of pH refinement of Rotofor runs comprised of initial parallel Rotofors, first refractionation and second refractionation. , mean pH of Rotofor 1 - 3 with SEM; , Rotofor 4 Pool 1; , Rotofor 5 Pool 2; , Rotofor 6 Pool 3; , Rotofor 7 Pool 4; , Rotofor 8 Pool 5.

Aggregation of isoforms was also substantiated by the crystal structure, which revealed to be a tetramer (Figure 27). A native dataset of 1.8 Å was obtained with the isoforms and the elucidated crystal structure has already been published (13). Glycosylation site Asn-279 on each subunit appeared to play a crucial role in the tetramerisation process, while Asn-229 was reported to be involved to a lesser extent (Figure 27 C). This was the first crystal structure of a native DP 4, also exhibiting the second highest resolution compared to human recombinant DP 4 with resolutions ranging from 1.62 Å to 3.64 Å (12-14;153;154;188;603-607). Only very recently, a crystal structure was reported having a resolution of 1.62 Å. Up to now, 93 crystal structures have currently been solved, most of them from human recombinant, one bacterial, eight of native porcine DP 4 and six of native rat (Appendix 57). The human recombinant DP 4’s were either expressed in insect cells with immature glycans (12;14;606) or in Pichia pastoris and deglycosylated prior to crystallisation (153;154;608). All of the crystals revealed the same structure topology of a DP 4 subunit, consisting of a catalytic /-hydrolase domain and an eight-bladed--propeller domain with a bottom opening of the propeller funnel and a side opening at the interface of the two domains as shown in Figure 27. However, they exhibited different numbers and sites of glycosylation, ranging between 2 - 9 glycosylation sites per subunit as summarised in Table 26. One structure of the human recombinant DP 4, expressed in Pichia pastoris differed even in the sites of glycosylation per subunit (154). Interestingly, four chains per asymmetric unit have been described in several crystal structures (pdb’s: 3CCB, 3CCC, 2QKY, 2ONC, 2I78, 2OAG, 2OQI, 2IO3, 2GG3, 2AJ8, 2AJB, 2AJC, 2AJD, 2BUA, 2BUC, 1R9N, 1R9M, 2BGN, 1W1J, 1ORV, 1ORW), however, only in 1ORV and 1ORW tetrameric protein interaction could be observed (13;14;82;603;604;609-617).

55 Results and Discussion

A B

-Hydrolase domain Side opening

Propeller domain

Tetramer Propeller opening interphase C

Asn279 Asn229 Asn229

Plasma Membrane

Figure 27: Schematic presentation of crystal structure of native porcine DP 4 from kidney cortex (pdb 1orv). A, soluble DP 4 forms a 222 symmetric assembly as a dimer of dimers. Arrows indicate respective propeller and side opening. Each subunit consists of a catalytic /- hydrolase (green shades) and -propeller domain as indicated. The -propeller is again subdivided into a carbohydrate rich sub-domain (red shades) and cystein-rich sub-domain (blue shades). Carbohydrates and disulfite bonds at the -propeller tunnel are indicated in orange and yellow, respectively. B, tetramerisation interphase of -propepller domains of sub- unit A and sub-unit C as viewed from the top, illustrating opposite facing of the sub-units. C, tetramerisation cavity suggesting that glycosylation at Asn-279 and Asn-229 appears to play a crucial role in the tetramerisation of DP 4. The transmembrane helices and their orientation to the membrane were drawn in to illustrate how tetramerisation of DP 4 may mediate cell–cell contacts (11;618). Structures were drawn with Pymol, using pdb 1orv.

Table 26: Comparison of glycosylation sites occupied in various DP 4 crystal structures. # Sites of Glycosylation as seen Res. Species Source Assemly pdb Ref. Glycosylation in the crystal Å

Isoform from kidney 6 mature a Native porcine Tetramer 85 92 229 279 321 685 1.8 1ORV (13) cortex complex type 6 mature Native porcine kidney cortex Tetramer 85 92 229 279 321 685 2.56 2BUA (604) complex type 5 mature Native rat kidney cortex Dimer 83 90 227 319 582 2.8 2GBC (188) complex type Human Insect cell Hi5 7 immature Dimer 85 150 219 229 281 321 520 2.1 1N1M (12) recombinant Human (605) Insect cell SF29 5 immature Dimer 85 219 229 281 321 2.6 1J2E recombinant (606) Human Insect cell SF29 5 immature Dimer 85 92 150 219 229 281 321 520 684 2.1 1R9M (14) recombinant Human Pichia pastoris 2 single Dimer 85 229 1.9 1PFQ (153) recombinant deglycosylated with Endo F GlcNAC Human Pichia pastoris 4 single GlcNAC, b c Dimer 85 92 150 229 281 2.1 1NUE (154) recombinant deglycosylated with Endo F subunits differ a, only described in pdb-file: http://www.biochem.ucl.ac.uk/bsm/pdbsum/1orv/main.html; b, only found in subunit B; c, only found in subunit A. Res. = resolution; Ref = reference

According to our knowledge, this is the first time that the Rotofor apparatus has been implemented to separate glycosylated isoforms of a purified enzyme as to improve crystallisation conditions. A high resolution was achieved with the isoforms despite of complex glycosylation

56 Results and Discussion and tetramerization as a result of cell unit compactness of the crystal (13;153). Glycosylation is often necessary to maintain enzymatic and biological activity and therefore yeast, plant and insect expression systems are employed to produce active glycosylated proteins for crystallisation (619). Deglycolysation of porcine DP 4 reduced the number of isoforms, but also drastically diminished its activity as depicted in Figure 28A. Human recombinant DP 4 from Pichia pastoris produced in our laboratory showed even a higher microheterogeneity when applied in an analytical agarose IEF, pH 3 - 7, counting more than 30 isoforms due to phosphorylation of the mannose residues as seen in Figure 28 B (620;621).

pI A B C D

1 2 3 4 5 6 1 2 3 4 5 Total DP4-like Activity 6.15 6 35 5.85 30 25 20 15 10

Activ(mU/mgl) ity 5 5.2 0 4.55 4.15 3.75

Figure 28: Analytical IEF, pH 3 - 7 of purified porcine and human recombinant DP 4 activity stained with Gly-Pro 4-methoxy--naphthylamide. A, porcine DP 4 (pDP 4) purified from kidney cortex; 1, pDP 4 + pNGNASE; 2, pDP 4 + Endo H; 3, pDP 4 + Neuramidase; 4, pDP 4 dephosphorylated; 5, native pDP 4. B, human recombinant DP 4 expressed in Pichia pastoris (lane 6); C, Serum DP 4-like activity, 1, without inhibitor; 2, + DP 4-selective inhibitor Sitagliptin; 3, + DP 8/9-selective inhibitor UG93; 4, + Dab-Pip; 5, + P32/98; 6, + Inhibitor mix. D, diagram of DP 4-like activity in serum. Sitagliptin, DP 4 selective inhibitor; UG93, DP 8/9 selective inhibitor; Dap-Pip, DP 2 selective inhibitor; Mix, Sitagliptin + UG93 + Dab-Pip; P32/98, non-selective DP 4-like inhibitor (135;558;622).

Thus, this newly developed technique is a powerful tool to separate post-translational heterogeneity of purified native and recombinant proteins for further analysis such as glycoproteomics (623-626). Since RNA analysis of DP 4 expression revealed no alternative splicing (39), only post- translational modifications could account for the isoforms. Microheterogeneity of DP 4 in an analytical IEF has often been reported and sialylation has mainly been accounted for these isoforms due to the cationic shift of the acidic isoforms and reduction of their numbers in presence of neuramidase and also seen in Figure 28A (63-65). Tyrosine-phosphorylation of DP 4 has only recently been demonstrated to occur in Golgi/endosome fractions in association with cellular c-scr and appeared to be insulin dependent (248;627). Furthermore, it was shown that dephosphorylation of precipitated DP 4 interfered with mannose 6-phosphate binding (198). Dephosphorylation of porcine DP 4 resulted in loss of activity as demonstrated in Figure 28A. Sulfation or/and acetylation have been speculated, but not proven so far (63). As already discussed in section 1.1.4 and summarised in Figure 3, terminal sialylation appears to play an important physiological role, as it is dependent on species (Appendix 57), tissues, age of organism and differentiation status of the cell as well as epitope specific (63-65). Moreover, it is associated with several diseases such as AIDS, rheumatoid arthritis, lupus erythematodus and lung cancer (25;64;78). For example the degree of hetereogeneity was shown to differ from the kidney of various species, as distinct isoform patterns were observed in human with 57 Results and Discussion pI 4.4 - 5.3 (63), rat with pI 5.0 - 7.0 (65) and pig with pI 4.8 - 6.0 in the present study. Furthermore, analysis of various tissues from human (64) and rat (63;65) with analytical IEF indicated tissue specific isoform patterns, which was also confirmed by investigating the carbohydrate composition of isolated porcine liver and kidney DP 4 (66). Such tissue-specific sialylation was shown to arise from tissue-specific expression of sialyltransferases (628). The immunological importance of the microheterogeneity of DP 4 isoforms in human PBMC has been studied in great details by analytical IEF and revealed different staining with various monoclonal antibodies (63;629). Moreover, an increase in sialylation was associated with age and HIV infection, with the latter resulting in a stronger inhibition by cationic HIV-tat protein (64). Contrary, activation of resting T-lymphocytes led to a decrease in sialylation (64). Furthermore, lysosomal sialidase was reported to migrate to the plasma membrane upon T- cell activation (630) and DP 4 in turn, requires M-6P at its carbohydrate moiety to associate with M-6P/IGFII-R and subsequently internalise (198). Generally, sialylation has been reported to be involved in cell-cell interactions, T and B cell activation, haematopoietic cell differentiation and tumorgenicity (631), processes that are also linked to DP 4 (200). Interestingly, asialoglycoproteins from the serum were uptaken by the asialoglycoprotein receptor systems via clathrin coated pits. Thus, it appears that sialylation has a protective role and hyper- or desialylation results in degradation. Interestingly, human serum DP 4 has highly acidic isoforms with pI’s ranging from 3.75 - 4.5 and with lower activity from 5.5 - 5.7 as illustrated in Figure 28C. Applying selective inhibitors against the various DP 4-like enzymes we could demonstrate that virtually all isoforms exhibiting DP 4-like activity were derived from DP 4 as also shown in activity studies of Figure 28C and D, respectively (135;558;622).

3.2 Distribution of DP 4-like Enzymes in Porcine and Canine Tissues 3.2.1 RT-PCR 3.2.1.1 Nested RT-PCR The results obtained from mRNA screening by nested RT-PCR are given in Appendices 58 - 60 and have already been published (131;578). Generally, the expression of several DP 4-like enzymes arising from the same tissue section could be detected. Overall, DP 4 and DP 9 were most frequently expressed, followed by FAP and DP 2, whereas DP 8 was expressed in the least number of tissues. Except for FAP, this is in strong agreement with the data of Northern blot analysis, summarised in Table 4 (39;44;49;85;86;88;90;171). A more significant finding is the expression of FAP in almost all tissues except for canine ileum. FAP is reported to be a tumour marker and not associated with healthy tissue. However, in situ hybridisation revealed expression of FAP at mRNA level, but not at protein level in many healthy liver cells. Nevertheless, the expression of FAP mRNA was up-regulated upon activation of liver stellate cells and the detection of protein FAP became detectable in liver cirrhosis by immuno-staining (92;632;633). Likewise, in silico analysis of FAP mRNA expression revealed very low levels of FAP mRNA in normal tissues, which was up-regulated in tumours (634). Therefore Appendix 61 gives the alignment of the porcine and canine nested RT-PCR products in comparison with the cDNA of human FAP. Appendix 62, in turn, summarises the percentage of homology of the nested RT-PCR products of the DP 4-like enzymes with their respective templates.

58 Results and Discussion

Kidney In kidney expression of DP 4-like enzymes decreases from cortex to outer and inner stripe of outer medulla, with all DP 4-like enzymes being expressed in the cortex, whereas only DP 4, DP 8 and FAP could be detected in the medulla (Appendix 58). Lung All DP 4-like enzymes are present in bronchial epithelium and alveolar tissue from dog and pig as illustrated in Appendix 59, thus implying the presence of DP 4-like enzymes in various sub- cellular compartments of lung cells. In situ hybridisation studies on human and baboon lung tissues revealed staining of DP 8 and DP 9 in bronchiolar lymphocytes and endothelial vessels (134). Interestingly, up-regulation of DP 8 and DP 9 mRNA’s were found upon induction of asthma, whereas mRNA levels of DP 4 remained unchanged (28). Small Intestine Similar to the kidney, the number of DP 4-like enzymes decreases from duodenum, jejunum to ileum as depicted in Appendix 60. DP 4, DP 8 and DP 9 are present in all three tissues from both pig and dog, whereas FAP only in porcine jejunum and DP 2 in canine jejunum. Interestingly, the pH of the small instestine is the lowest at the atrium and duodenum due to the acidic chyme from the stomach. It is therefore not surprising to find DP 2 only in the duodenum. In situ hybridisation of DP 8 and DP 9 in small intestine tissues from human and baboon showed staining of DP 8 and DP 9 in goblet cells, enterocytes, lymphocytes and endothelial capillaries. No distinction was made between duodenum, jejunum and ileum, yet colon showed higher staining (134). Lower mRNA expression of DP 4 was observed during intestinal adaptation in rats (635).

3.2.2 DP 4-like Activity in Porcine and Canine Tissues 3.2.2.1 Specific Activities The specific activities of the various porcine and canine tissues are outlined in Table 27 and represent the overall DP 4-activity at pH 7.6. As already described in literature, the highest DP 4- activity is found in the kidney decreasing from kidney cortex to outer medulla outer stripe to outer medulla inner stripe. In contrast, specific activities increase from duodenum to jejunum to ileum in the small intestine, with canine tissues having higher specific activities. Low specific activities between 9.3 - 1.7 mU/mg were obtained in the various parts of the pancreas, with highest of 9.3 mU/mg being found in porcine pancreatic tail, whereas for canine the highest specific activity was found in the pancreatic head with 3.4 mU/mg. Generally higher levels of specific activities were found in bronchial epithelium compared to alveolar lung, particularly in canine bronchial epithelium with 39.4 mU/mg compared to 9.7 mU/mg in porcine.

Table 27: Specific activities of various porcine and canine tissues, using 0.1 mM Ala-Pro-AMC, pH 7.6 (131). Specific Activity (mUnits/ mg) Organ Tissue section Porcine Canine Kidney Cortex 195.8 ± 27.2 150.0 ± 17.3 Kidney Outer Medulla, outer stripe 141.8 ± 3.0 110.3 ± 0.1 Outer Medulla, inner stripe 107.3 ± 4.0 98.4 ± 2.4 Bronchial Epithelium 9.7 ± 0.9 39.4 ± 2.6 Lung Alveolar Lung 7.9 ± 5.6 4.9 ± 2.8 Duodenum 8.2 ± 1.4 27.6 ± 0.9 Small Intestine Jejunum 23.9 ± 0.3 40.8 ± 0.8 Ileum 27.6 ± 0.4 42.8 ± 1.4 Pancreatic Head 5.2 ± 0.3 3.4 ± 0.7 Pancreas Pancreatic Body 3.2 ± 0.6 1.7 ± 0.1 Pancreatic Tail 9.3 ± 2.5 1.7 ± 0.0

59 Results and Discussion

3.2.2.2 Inhibition Studies The contribution of the various DP 4-like enzymes to the overall specific activities is illustrated in Figure 29, representing the remaining activities after applying selective inhibitors against DP 4, DP 8/9 and DP 2, respectively (131). Generally, the results revealed that DP 4 is the most dominant enzyme in all tissue extracts, except for canine pancreas, corresponding to recent findings (134). Kidney Specific activity of DP 4 is particularly high in kidney tissues, where the highest overall specific activity has been detected and over 75 % of DP 4 inhibition was achieved. Histochemical activity studies by others confirmed association of DP 4 (-like) activity to microstructures of the kidney, since DP 4 (-like) activity was found predominantly on proximal tubules and Bowmann’s capsule (17;18;451), while DP 2 mainly stained distal tubules (171;589). No information is yet available regarding DP 8/9 activities in the kidney and only small levels could be detected in the present study in porcine kidney cortex. The main function of DP 4 in the kidney known so far, is the reabsorption of proline containing dipeptides by its enzymatic activity and association with the co-transporter Na+/H+ exchanger isoform NHE3 (240;283;284;337;636-639). In addition, DP 4 activity also appears to be involved in the clearance of DP 4 substrates such as GLP-1 (640-642). The gene expression of DP 4 was found to be regulated at mRNA levels (39;49) and it is interesting to note, that glomerular DP 4 expression is up-regulated by glucose (643). Furthermore, DP 4 is associated with a number of kidney diseases such as lupus nephritis, Heymann nephritis, renal cell injury and various glomerular diseases (6;644-647). Lung DP 4 is also the most dominant enzyme in lung tissues at neutral pH compared to DP 8/9 and DP 2, with selective DP 4 inhibitors resulting in about 40 - 75 % inhibition of the overall DP 4- like activity. Unlike the results obtained by nested RT-PCR, hardly any activity is contributed by DP 8/9 in porcine and canine alveolar lung as well as bronchial epithelium, where only 0 - 20 % reduction of the overall DP 4-like activity was obtained. DP 2 appears to be present more in the bronchial epithelium than alveolar lung tissue inhibited by 25 - 50 % and 0 - 10 %, respectively. However, at acidic pH, the inhibition profile of DP 2 in lung tissue was similar to that of DP 4 at neutral pH with about 50 % and 70 - 75 % inhibition in alveolar lung and bronchial epithelium, respectively (Appendix 63). Histochemical analysis located DP 4-like activity to bronchial epithelial cells (648), T-lymphocytes, serosal gland cells (649), endothelial capillaries (17) and lung parenchyma (28), whereas histochemical and immunological investigations identified moderate DP 2 levels in bronchial epithelium, alveolar cells and pleura (171) and high levels in lung macrophages (185;650;651). Distinction between DP 4 and DP 8/9 activity was made by histochemistry and confirmed by immuno-staining, using lung tissue sections from wildtype and DP 4-deficient F344 rats, thereby revealing strong staining of DP 4 in lung parenchyma and moderate staining of DP 8/9 in bronchi, particularily containing bronchoalveolar fluids (28). Nothing specific is known about the physiological function of DP 4 and the lung. However, respiration is highly subjected to the autonomal nervous system, with the sympathetic nervous system secreting NPY and SP, which in turn influence the cardiovascular and respiratory systems that are regulated by natural DP 4 substrates such as NPY, PACAP, SP, VIP, vasostatin I and II, procalcitonin. Thus, degradation of DP 4 substrates may result in an autocrine, paracrine, endocrine and/or neuronal control by membrane bound and soluble DP 4-like enzymes, respectively. In addition, DP 4 involvement in chemotaxis by 60 Results and Discussion

the degradation of chemokines and NPY may also contribute to the immunology of pulmorary infections and allergies (649). The involvement of DP 4 and CD26+ T-cells in the pathogenesis of asthma has recently only been discovered (28;258;321;329;330). Furthermore, the activities of DP 4 as well as DP 8/9 were up-regulated upon induction of asthma (28). DP 4 and FAP appear to play an important role in lung cancer and metastasis (25;84;273;274). In addition, high DP 2 expression has been associated with pulmonary granulomatosis and fibrosis (652). Small Intestine Similarily to the specific activities in Table 27, the contribution of DP 4 increases from duodenum to jejunum to ileum and this trend is more pronounced in the canine small intestine compared to porcine. On the contrary, at pH 5.5, DP 2 activity substantially decreases from duodenum to ileum (Appendix 63) and this trend is more pronounced in pig than dog and far more subtle at neutral pH 7.6 (Figure 29). Generally, activity of DP 8/9 could be detected in the all tissues of small intestine, although the inhibitory effects are stronger in dog. Interestingly, activity studies in DP 4-/- mice showed hardly any activity of DP 8/9 in small intestine, despite strong staining of in situ hybridisation, thus concluding the presence of inactive alternative spliced variants (134). The importance of DP 4-like enzymes in the small intestine is also given by the expression of a great number of natural substrates in this organ, which are summarised in Table 28. Detailed analysis of porcine ileum revealed degradation of GLP-1 (7-36) by DP 4 in the capillaries close to ileal L-cells (653). Similar to the kidney, DP 4 is believed to assist in the assimilation proline containing tri- and dipeptides, since DP 4-deficient F344 rats as well as congenic Da.F344-Dpp4m/SvH rats were found to excrete abnormal high levels of proline containing peptides (240;304). The proline specific H+/peptide symporter has been identified as PEPT-1 (654). Changes of DP 4 activity and / or DP 4-like activity have been associated with coeliac disease and colon cancer of differentiated tumours and the involvement of several DP 4-like enzymes has been proposed in the development of inflammatory bowel diseases (IBD) such as Chrohn’s disease and ulcerative colitis (190;312-314;401;635;655-658). Furthermore, during intestinal adaptation, expression of DP 4 was found to be down-regulated, whereas the DP 4 substrate, GLP-2, was up-regulated (635). FAP, in turn, is more up- regulated in undifferentiated gastric and intestinal cancer (116). Table 28: Summary of natural DP 4 substrates in small intestine. Family Substrate Function Location Ref. releases of HCO - to neutralise chyme from stomach, S-cell of duodenum, Secretin 3 (455) regulates gut motility;  gastric pepsin secretion,  gastric acid secretion proximal jejunum GRF  release of growth hormones;  secretion of insulin intrinsic neurons of GI tract (455) vasorelexant of vascular and non-vascular smooth muscles; nerve ends + PACAP (455)  insulin or glucagon secretion depending on plasma glucose level endocrine cells of the gut vasorelexant of vascular and non-vascular smooth muscles; nerve ends + VIP (455)  insulin / glucagon secretion depending on plasma glucose level endocrine cells of the gut vasorelexant of vascular and non-vascular smooth muscles, nerve ends + PHM (455) PACAP/  insulin / glucagon secretion depending on plasma glucose level endocrine cells of the gut Glucagon Insulinotropic;  synthesis of proinsulin DNA promotes proliferation and L-cells of distal jejunum (208) GLP-1 differentiation of pancreatic -cell + ileum (640)  intestinal growth;regenerates intestinal mucosa and permeability, promotes L-cells of distal jejunum (208) GLP-2 differentiation of enterocytes + ileum (640) Insulinotropic;  secretion of gastric acid  gastric motility; K-cells of duodenum + (208) GIP  Inhibits stomach emptying proximal jejunum (659) Glucagon only small amounts present in the small intestine L-cells of distal jejunum + ileum (455) Oxyntomodulin  gastric acid secretion; regulates gut motility  sensation of hunger L-cells of distal jejunum + ileum (660) Glicentin  insulin secretion; gastric acid secretion  gut growth; regulates gut motility L-cells of distal jejunum + ileum (660) Tachykinin Substance P  gut motility;  vascular permeability endocrine + nerve cells of GI tract (2;483) Pancreatic NPY Intestinal vasoconstriction, Sympathetic neurons (2;208) polypeptide intestinal vasoconstriction;  intestinal fluid secretion,  gut motility; (2) PYY L-cells of distal jejunum + ileum family  exocrine pancreas secretion,  proliferation of gut epithelial cells (208)  secretion of gastric hormones; trophic effect on gut, regulates cell differentiation; (2) Bombesin GRP intrinsic neurons of the gut vasodilator effects on intestinal circulation (208)

61 Results and Discussion

Figure 29: Influence of selective DP 4-like inhibitors on the activity of porcine and canine tissue extracts, at pH 7.6. A, remainng DP 4-like activity of porcine tissue extracts after incubation with selective inhibitors against DP 4, DP 8/DP 9 and DP 2 as well as inhibitor mix and P32/98. B, remaining DP 4-like activity of canine tissue extracts after incubation with selective inhibitors against DP 4, DP 8/DP 9 and DP 2 as well as inhibitor mix and P32/98. , DP 4 inhibitor Sitagliptin; , DP 8/DP 9 inhibitor UG93; , DP 2 inhibitor Dab-Pip; , inhibitor mix (Sitagliptin, UG93, Dab-Pip; , P32/98. Concentrations and IC50 of the selective DP 4-like inhibitors are summarised in Appendix 29. Overall DP 4 activity, Sitagliptin, UG93, Dab-Pip, inhibitor mix and P32/98 were analysed with 0.1 mM Ala-Pro-AMC, in 40 mM Hepes, pH 7.6; n = 6; error bars = SEM (131). Pancreas Substantial differences of the various DP 4-like enzymes could be observed between porcine and canine tissues of the pancreas. While about 60 - 75 % of inhibition of DP 4 and DP 2 was found in pancreatic body and tail as well as 25 - 30 % of inhibition of DP 8/9 in head and tail, only about 10 - 20 % of inhibition of each DP 4-like enzyme could be detected in canine pancreatic tissue. Furthermore, merely 25 - 50 % of inhibition was observed with the inhibitor mix, thus pointing to another unknown enzyme with DP 4-like activity as it has recently been suggested from selective activity studies in pancreatic tissue of DP 4-/- mice (134). Results from nested RT-PCR generally revealed the co-localisation of all DP 4-like enzymes in the pancreatic tissues from pig and dog (Appendix 64), whereas in situ hybridisation showed staining of DP 8 and DP 9 in acinar cells of baboon pancreas (131;134;661). Histochemical enzymatic studies and immuno-staining in turn, confirmed strong epithelial staining of DP 4 (- like) activity in the pancreatic ducts and weak acinar staining, whereas DP 2 was found in the islets (17;18;171;185;451;589;662). Interestingly, soluble DP 4 was also detected in granules of -cells from porcine islets by electron microscopy (447;448). Pancreas, particularly the islets of Langerhans contain a great number of DP 4 substrates and their respective receptors as already summarised in Tables 11 and 12, respectively. 62 Results and Discussion

3.3 Analysis of DP 4-like Enzymes in Porcine Pancreas 3.3.1 Isolation of Porcine Islets of Langerhans Five different fractions were obtained after the centrifugation with a Ficoll density gradient, including top-phase, 1st-phase, 1st-interphase, 2nd-interphase and bottom-phase. In general, the top-phase and 1st-phase contained most of the islets with the least acinar contamination as seen in Figure 30. In contrast, the 1st-interphase had a number of large islets, some fragmented, but was also contaminated with a substantial amount of acinar tissue, whereas the 2nd-interphase consisted mainly of acinar tissue, dispersed with a few islets, most of them being fragmented. The bottom phase mainly consisted of cell debris. Viability test of the top phase and 1st phase resulted in a bright green fluorescent colour of isolated viable islets as seen in Figure 30C and D with a small number of single dead acinar cells in bright red (data not shown) (661). The number of isolated porcine islets varied greatly, which is general phenomenon (663-665). Figure 30B illustrates the irregular shapes and different sizes of porcine islets, ranging between 50 µm to 350 µm (665;666).

A B C D

Figure 30: Purity, morphology and viability of isolated islets of Langerhans from porcine pancreas. A, single islet under phase contrast microscopy; B, islets obtained from the top–phase of Ficoll density gradient and observed under phase contrast microscopy. C, viable single islet stained for fluorescein diacetate; D, viable islets obtained from the top–phase of Ficoll density gradient and stained for fluorescein diacetate (661).

3.3.2 Activity Analysis of Isolated Islets Generally, the highest DP 4 activity was acquired in fractions containing predominantly acinar tissue such as the first and second interphases, followed by top-phase and first phase. Although great variation could be observed between the overall specific activities of the DP 4- like enzymes in the various fractions obtained from different batches of islet isolation, the percentage of total activity of the pure islet and acinar fractions remained fairly consistent as seen in Figure 31. Thus, one can conclude that most of the DP 4-like activity (85 - 90 %) originated from acinar tissue.

Figure 31: DP 4-like activity in fractions obtained from the Ficoll density gradient after islet isolation. A, Specific activity of DP 4-like enzymes in the various fractions. B, Percentage of total DP 4-like activity in the various fractions (661). n = 6; error bars = SEM. 63 Results and Discussion

This was further investigated by analysis the remaining activities of the DP 4-like enzymes in the various fractions from the Ficoll gradient, using selective inhibitors against DP 4, DP 8/9 and DP 2, respectively, as seen in Figure 32. The results confirmed that DP 4 exhibited the highest activity in all fractions, followed by DP 2 and only low percentage of remaining activity was contributed by DP 8/9 in the different fractions as well as the pancreatic tissue extract.

120

100

80

60

40

20 % Remaining% Activity

0 Top 1.Phase 1.Interphase 2.Interphase Pancreas Figure 32: Analysis of DP 4-like enzymes in fractions obtained after centrifugation with Ficoll density gradient as a final step of islet isolation, using selective inhibitors against DP 4, DP 8/DP 9 and DP 2, respectively. , selective DP 4 inhibitor Sitagliptin; , selective DP 8/DP 9 inhibitor UG93; , selective DP 2 inhibitor Dab-Pip; , inhibitor mix. % remaining activity in presence of DP 4 inhibitor, DP 8/ DP 9 inhibitor and inhibitor mix was determined with respect to overall DP 4- like activity at pH 7.6, whereas remaining activity in presence of DP 2 inhibitor was determined with respect to overall DP 4-like activity at pH 5.5 (131;661). n = 3; error bars = SEM.

Furthermore, sub-cellular fractionation indicated the presence of DP 4-like activity in both membrane and cytosolic fractions as illustrated in Figure 33. Surprisingly, the inhibitory effect of DP 4 with less than 10 % remaining activity was stronger in the cytosole of islets than in pancreas with only 70 % remaining activity. As shedded DP 4 would be washed away during the process of islet isolation and DP 4 would be more exposed to proteases in the process of extracting pancreatic or acinar tissue, rather than in the islets, one can assume that the soluble DP 4 is cell specific (414;415). Isolating granules by a sucrose density gradient also suggest the presence of one or several vesicular DP 4-like enzymes, most likely DP 2. As to distinguish between DP 4-like enzymes and DP 2, all the fractions were assayed with Gly-Pro- AMC at pH 7.6 and Lys-Ala-AMC, at pH 5.5, respectively. The results of the sub-cellular fractionation are summarised in Table 29. Since the specific activities of both DP 4-like enzymes and DP 2 were increased in enriched vesicular fractions comprised of granules, lysosomes and endosomes in comparison to the membrane and cytosolic fractions without any vesicles, one can assume the activity to be specific to these organelles. While DP 2 was found in the intra-granular fractions of the sucrose gradient, DP 4 activity could be detected in both granular membrane and intra-granular. However, only membrane DP 4-like activity was determined in the lighter granules at 1.5 M sucrose, whereas the heavy granules at 1.6 M sucrose contained both membrane bound as well as intra-granular DP 4-like activities.

64 Results and Discussion

Table 29: Specific activities of DP 4 (GP-AMC, pH 7.6) and DP 2 (KA-AMC, pH 5.5) after granule isolation. Specific Activity (mUnits/mg) Fraction DP 4-like DP 2 DP 4-like/DP 2 Cytosol without granules, lysosomes, endosomes 3.76 ± 1.76 8.40 ± 7.39 0.45 Membrane without granules, lysosomes, endosomes 5.91 ± 3.31 3.85 ± 0.84 1.54 Granules, lysosomes, endosomes, intra-granular 3.82 ± 0.56 16.98 ± 1.73 0.23 Granules, lysosomes, endosomes, membrane 66.92 ± 1.98 1.26 ± 0.04 52.97 1.5 M sucrose, intra-granular 2.36 ± 1.26 12.27 ± 1.10 0.19 1.5 M sucrose, membrane 26.75 ± 1.28 1.91 ± 0.44 14.01 1.6 M sucrose, intra-granular 22.28 ± 0.72 19.80 ± 0.45 1.126 1.6 M sucrose, membrane 24.29 ± 0.08 5.41 ± 0.63 4.49

4,5 A B C

4

3,5

Specific Activity Specific Activity Specific 3

2,5

2

1,5

1

0,5 Specific Activity (mUnits/mg) SpecificActivity 0 Islet Cytosole Islet Membrane Pancreatic Pancreatic Islets Pancreatic Head Cytosolic Head Head Membrane

Figure 33: Analysis of DP 4-like enzymes by selective DP 4-like inhibitors in cytosolic and membrane fractions of porcine isolated islets, obtained after ultracentriugation at 105,000 g for 1 h at 4° C. A, specific activities in presence and absence of selective inhibitors specific for DP 4, DP 8/DP 9 and DP 2, respectively as well as non-selective DP 4-like inhibitor P32/98. , overall DP 4-like activity; , selective DP 4 inhibitor Sitagliptin; , selective DP 8/9 inhibitor UG93; , DP 4-like activity at pH 5.5; , selective DP 2 inhibitor Dab-Pip; , inhibitor mix; , P32/98. DP 4, Sitagliptin, UG93, inhibitor mix and P32/98 were determined with 0.1 mM Ala-Pro-AMC, pH 7.6, whereas DP 2 and Dab-Pip were analysed with 0.2 mM Ala-Pro-AMC, pH 5.5. n = 6; error bars = SEM. B, Specific activity of lactate dehydrogenase (A/h/mg), a cytosolic marker. C, specific activity of alkaline phosphodiesterase I (mUnits/h/mg), a plasma membrane marker. Cyt, cytosol; mem, membrane, PH, pancreatic head (131;661).

3.3.3 Expression of DP 4-like Enzymes by RT-PCR 3.3.3.1 Nested RT-PCR In order to identify any contaminating tissue in the purified islets, the following control primers for exocrine acinar and ductal tissues, endocrine islet tissues and capillaries were applied as summarised in Table 30. The primers for carbonic anhydrase II (CAII) and CD31, specific for ductal and capillary tissue respectively, were also tested on sections of kidney cortex. As expected, sections from pancreatic tissue had positive nested RT-PCR signals of amylase for acinar tissue, carbonic anhydrase for ductal tissue, insulin for -cells and glucagon for -cells (Figure 34). CD31 could only be identified in the kidney cortex (Figure 34). The presence of all DP 4-like enzymes could be determined in pancreatic tissue as depicted in Figure 34. The results of nested RT-PCR in isolated islets revealed the absence of ductal and vascular tissue, however, acinar contamination was detected by the presence of amylase signals. Therefore, the ubiquitous distribution of DP 4-like enzymes as seen in Figure 34, may also originate from contaminating acinar tissue (661). 65 Results and Discussion

Table 30: Summary of tissue controls used for nested RT-PCR in pancreas and isolated islets. Tissue Cells Tissue controls Reference

Exocrine (667) Amylase Acinar (668) (Am) (669) (667) Carbonic Anhydrase II Epithelial (670) (CAII) (671) Endocrine Insulin (668) -cells (Islet of Langerhans) (Ins) (669;672) -cells Glucagon (668) ---ccceeelllllsss -cells (Glu) (669;672) Capillaries Platelet endothelial cell (673) Endothelial adhesion molecule (674) (PECAM, CD31) (675)

Figure 34: Results of nested RT-PCR of DP 4-like enzymes from porcine and canine pancreas as well as isolated islets (131;661).

3.3.4 Histochemical Analysis 3.3.4.1 Activity Staining 3.3.4.1.1 DP 4 The chemical structures of GP-1-OH-4-NA and P32/98 are given in Appendix 35A+B, respectively. Figure 35A-D illustrates the time-dependent staining of the histochemical assay for DP 4-like activity as well as complete inhibition with P32/98 and 4 mM Pefabloc in porcine kidney, respectively. DP 4-like activity was found in interlobular-, intralobular- and intracalated- ducts of the exocrine tissue as well as endothelial cells of capillaries surrounding islets (Figure 35E-G), which correspond to data attained by others (17;18;451). In addition, we could also detect DP 4-like activity in some pancreatic islets (Figure 35H). However, higher magnification of isolated islets as depicted in Figure 36 suggests the presence of DP 4-like enzymes at different sub-cellular localisations. Larger islets had either the central cells stained for DP 4-

66 Results and Discussion like activity at the membrane or intra-cellularly in peripheral cells as indicated by arrow and arrow-head in Figure 36A, respectively. Smaller islets in turn, revealed only intra-cellular staining of cells at the periphery (Figure 36B). This would imply cell specific protein expression of various DP 4-like enzymes in islets of Langerhans. Soluble granular DP 4 activity has already been described to co-localise with glucagon (447;448). However, inhibition of isolated islets with 1 mM P32/98 showed reduced staining of membrane DP 4-like activity, but lacks any inhibition of the intra-cellular staining of peripheral cells (Figure 36C). Likewise, incubating sections of isolated islets with 4 mM Pefabloc drastically reduced positve intra-cellular staining of peripheral cells, thus pointing to a serine protease other than DP 4, DP 8/ 9 or DP 2. Yet, a few cells were also stained positively at the membrane, thereby hinting to another enzyme of a different class such as consecutive cleavage by the metalloproteases aminopeptidase P and aminopeptidase N (Figure 36D) (676). This result would also explain the lower inhibition of islets and pancreatic tissue by inhibitor mix and P32/98 compared to other tissues as illustrated in Figures 29, 32 and 33, respectively and also recently observed by others (134;190).

Figure 35: Results obtained from the histochemical staining for DP 4-like activity in porcine kidney and pancreas, using Gly-Pro-1-OH-4-naphthylamide. A, staining of porcine kidney after 10 min; B, staining of porcine kidney after 30 min, showing strong DP 4 activity at the brush border of proximal tubules; C, staining of kidney in presence of 1 mM P32/98. D, staining of kidney in presence of 4 mM Pefabloc; E, interlobular duct of porcine pancreas; F, intercalated ducts linking to intralobular duct of pancreas; G, endothelial DP 4 surrounding a small islet in pancreas. H, islets in pancreas, showing differential staining of DP 4-like activity; I, staining of pancreas in presence of 1 mM P32/98.

Figure 36: Histochemical staining for DP 4-like activity in isolated porcine islets. A, staining of porcine islet after 30 min, showing DP 4-like activity at surface of the central islet cells as indicated by arrows and intra- cellular staining of peripheral islet cells marked with arrow head; B, positive staining of DP 4-like activity in peripheral islet cells after 30 min; C, staining of DP 4-like activity in presence of 1 mM P32/98 after 30 min; D, staining of DP 4-like activity in presence of 4 mM Pefabloc after 30 min (661).

67 Results and Discussion

3.3.4.1.2 DP 2 Since NBT inhibits DP 2, the substrate KA-CAH (Appendix 36) and inhibitor Dab-Pip were applied in the histochemical assay for DP 2 activity at pH 5.5 (589). Figure 37A-D illustrate the time-dependent activity staining of DP 2, showing initial cell-specific staining of islets corresponding to the number of -cells after 30 minutes as already previously described (171;185). However, after 60 and 90 minutes small fibres suggesting additionally staining of surrounding network of a neuronal plexus (Figure 37B-D). Likewise, staining of isolated islets showed cell-specific DP 2 activity (Figure 37E). Strong DP 2 activity was also found in the kidney, mainly staining the distal tubules, but not glomerulus as depicted in Figure 37F and reported by others (171;185;589).

Figure 37: Histochemical staining for DP 2 activity in porcine kidney, pancreas and isolated islets, using KA-CAH, pH 5.5. A, staining of porcine pancreas after 30 min, implying specific cell-type staining of possible -cells in islets; B, staining of porcine pancreas after 60 min, indicating additional peripheral staining of islets; C+D, islet in porcine pancreas after 90 min, showing staining of islet cells and most likely its surrounding neuronal network; E, staining of isolatetd islets after 90 min; F, staining of porcine kidney after 30 min, showing strong DP 2 activity at the brush border of distal tubules.

3.3.4.2 Immunohistochemical Staining Immunohisochemical analysis of porcine pancreas with polyclonal pig anti-DP 4 antibody could detect strong DP 4 expression in capillaries surrounding the islet of Langerhans (Figure 38F+J), in epithelial exocrine tissue comprised of interlobular- and intralobular-ducts (Figure 38H) as well as intercalated-ducts (Figure 38G, arrow) and faint staining in acinar cells (Figure 38G+I). The results were in agreement with the immunological analysis by Hartel-Schenk et al., 1990 and Hartel et al., 1988, who investigated the tissue distribution of DP 4 in rat with four monoclonal and one polyclonal antibodies (451;648). However, co-localisation of DP 4 with glucagon in -cells of an islet as described by Grondin et al., 1999 and Poulson et al., 1993 was not observed in the present study as seen in Figure 38J, where DP 4 staining represents capillaries surrounding an islet (447;448). Double-staining of islets of Langerhans with monoclonal anti-insulin and polyclonal anti-glucagon antibodies could identify the three types of islets found in porcine pancreas, i.e. small islets with low number of -cell (Figure 38I), large islets with –cells in the core of islets (Figure 38C) and large islets with -cells at the periphery of the islet (data not shown) as well as single -cells found in the pancreatic head (data not shown) (672).

68 Results and Discussion

Figure 38: Immunohistochemical analysis of porcine pancreas, staining various tissue sections either with anti-DP 4 pAb, anti-insulin mAb or anti-glucagon pAb or in combination of two. A, staining of a large islet for insulin (red). B, staining of the islet for glucagon (green). C, super-imposing the islet, stained for insulin (red) and glucagon (green). D, staining of an islet for insulin (red). E, staining of the islet for DP 4 (green). F, super-imposing the islet, stained for insulin (red) and surrounded by capillaries expressing DP 4 (green). G, staining of exocrine tissue, locating DP 4 mainly to intercalated- (arrow) and intralobular-ducts and more faintly to acinar cells. H, DP 4 staining of an interlobular- (arrow head) and intralobular- (arrow) duct. I, double-staining of a small islet for insulin (red) surrounded by exocrine tissue expressing DP 4 (green). J, double-staining of an islet for glucagon (red) surrounded by capillaries expressing DP 4 (green) (661). In co-laboration with Dr. Hause, Biocentre, Halle.

3.3.5 Perfusion, Perifusion and Influence of Media Components on DP 4 Activity Upon investigating secretion of DP 4-like enzymes by cell culture, perfusion and perifusion experiments, several media components and stimulants such as BSA, and nitroprusside were found to interfere with DP 4 activity (Appendices 65 and 66). Preliminary perfusion studies of rat pancreas revealed secretion of glucagon and a DP 4-like enzyme after stimulation of splanchic nerve (Figure 39). Delayed detection of DP 4 activity compared to

glucagon may be due to higher viscosity of DP 4 based on its molecular size.

Units/l)

(

DP 4 Activity 4 DP

Figure 39: Perfusion studies of rat pancreas, showing secretion of DP 4-like enzymes and glucagon after stimulation of splanchic nerve in Wistar rats. ▲, DP 4 activity, measured with 0,1 M Gly-Pro-AMC after ultracentrifugating samples at 105,000 g for 1 h and deducting residual DP 4 activity in BSA (RIA grade); -, glucagon concentration; black arrow, splanchic nerve stimulation; blue arrow, 10 µM sodium nitroprusside (SNP) stimulation. n = 3; error bars = SEM. 69 Results and Discussion

3.4 Identification of DP 4 as a Target for Psychiatric Disorders 3.4.1 Analysis of DP 4-like Enzymes in Neuronal Rat Tissues 3.4.1.1 Specific Activities of DP 4-like Enzymes in Neuronal Rat Tissues

3.4.1.1.1 DP 4-like Acitivity in Wildtype and DP 4-deficient Fischer Rats Analysing the activity of the DP 4-like enzymes in brain extracts of DP 4-deficient Fischer rats F344/Crl(Wiga)SvH-Dpp4 and the corresponding wildtype F344/Ztm, revealed at neutral pH equal contribution by DP 8/DP 9 and DP 2, respectively, whereas the activity of DP 4 was comprised of only a tenth of the total activity as depicted in Figure 40A (135;622;677). However, at acidic pH the overall DP 4-like activity was almost doubled due to increased activity of DP 2. Interestinly, hardly any differences were observed between the total DP 4-like activity obtained from the F344/Ztm and F344/Crl(Wiga)SvH-Dpp4, respectively. While this confirms the low contribution DP 4 to the overall DP 4-like activity in the brain on the one hand, it also implies on the other hand, that there is no compensatory mechanism of any other DP 4- like enzyme if DP 4 is lacking. These results correspond to findings recently obtained by activity studies with DP 4-/- mice (134;143).

Figure 40: Analysis of specific activities, contributed by the various DP 4-like enzymes in brain, neuronal and glial tissues, using selective inhibitors against the DP 4-like enzymes. A, Analysis of the specific activities of the DP 4-like enzymes in brain extracts obtained from wildtype Fischer rats (F344/Ztm) and DP 4-deficient Fischer rats (F344/Crl(Wiga)SvH-Dpp4) as well as cell extracts from primary rat neurons. B, Analysis of the specific activities of the DP 4-like enzymes in brain extracts as well as cell extracts from primary rat neurons, astrocytes and microglia. Specific activities were determined using 0.1 mM Ala-Pro-AMC, in 40 mM Hepes, pH 7.6 for DP 4 and DP 8/9 as well as 0.2 mM Ala-Pro-AMC in 80 mM NaAcetate, pH 5.5 for DP 2, respectively. Except for the inhibition by non-selective DP 4-like inhibitor P32/98, the specific activities of the DP 4-like enzymes are presented as the difference of total specific activity - selectively inhibited specific activity. , brain extract F344/Ztm; , brain extract F344/Crl(Wiga)SvH-Dpp4; , rat neuronal cell extract; , rat microglial cell extract; , rat astrocytic cell extract (135;622;677). n = 6; error bars = SEM. 70 Results and Discussion

3.4.1.1.2 DP 4-like Activity in Neuronal Primary Cell Cultures from Rats Further analysis of specific activities of the DP 4-like enzymes in primary cell extracts from rat neurons, rat microglia and rat astrocytes revealed substantial increases in specific activities as seen in Figure 40A+B (677). Particularly at acidic pH, the specific activities of microglial and astrocytic cell extracts rose 20 - 30-fold and 40 - 50-fold compared to neuronal cell and brain extracts, respectively. Applying selective inhibitor against DP 2 confirmed that the specific activity is comprised of more than 90 % by DP 2. High levels of DP 2 have already been reported in astrocytes (43). Interestingly, the contribution of DP 8/9 to the overall specific activity ranged only 10 - 15 % in astrocytes and microglia, whereas in neurons and brain extracts it was between 40 - 50 %. Hardly any specific activity of DP 4 could be detected in astrocytes, while in neurons, microglia and brain at least 10 - 15 % of the total specific activity was owed to DP 4, with microglia exhibiting the highest DP 4 activity of 1.4 mU/mg. It is noteworthy to mention, that of all the tissues investigated in the present study (Figures 29 and 33), brain and neuronal tissue is the only one, where the activity of DP 8/9 exceeds the one of DP 4. This is in agreement with recent data obtained from activity studies in various murine tissues and rat brain (134-136).

3.4.1.2 Histochemical Analysis

As to elucidate whether the DP 4-like activity was specific to a certain brain area, histochemical analysis were performed, using Gly-Pro-4-OH-1-NA and NBT. In order to distinguish between DP 4 and DP 8/9, selective inhibitors and F344/Crl(Wiga)SvH-Dpp4 versus F344/Ztm rats were implemented, which were first tested on kidney sections as illustrated in Figure 41. The results confirmed that in rat kidney, most of the DP 4-like activity is derived from DP 4, staining the brush border membranes of the proximal tubules as well as the glomerulus, whereas weak staining of DP 8/9 could be observed in tubular cells, which appears to be intra-cellular (18).

Neg (NBT) Pos Sitagliptin UG93 Inh. Mix P32/92 (GP-4-OH-1-NA) (1mM ) A 10 x F344/Ztm (pos. Con.) 40 x

B 10 x F344/Crl(Wiga)SvH-Dpp4 (neg. Con.) 40 x

Figure 41: Histochemical analysis of DP 4-like enzymes in 5 µm kidney sections from F344/Ztm and F344/Crl(Wiga)SvH-Dpp4 Fischer rats after 30 minutes, using Gly-Pro-4-OH-1-NA and NBT as substrates, and selective inhibitors against DP 4 (Sitagliptin) and DP 8/9 (UG93) as well as non-selective DP 4-like inhibitor P32/98. A, staining of kidney section from wildtype Fischer rats F344/Ztm in absence or presence of Sitagliptin or UG93 or non-selective P32/98 inhibitors, showing predominantly staining of brushborders of proximal tubules and glomerulus by DP 4 and only weak intra-cellular staining of tubular cells by DP 8/9. B, staining of kidney sections from DP 4- deficient F344/Crl(Wiga)SvH-Dpp4 Fischer rats in absence or presence Sitagliptin or UG93 or non- selective P32/98 inhibitors, resulting in no or hardly any staining due to lack of DP 4 (677). 71 Results and Discussion

However, given that the DP 4-like activity in brain is much lower compared to kidney, the thickness of the sections had to be increased to 50 µm and the incubation times were extended between 1 - 20 hours as outlined in Appendix 41. Figure 42 illustrates positive and ubiquitous staining of both wildtype F344/Ztm and mutant F344/Crl(Wiga)SvH-Dpp4 brain sections after 12 h. Since NBT is inhibiting DP 2, staining of both F344/Ztm as well as F344/Crl(Wiga)SvH-Dpp4 points to ubiquitous activity of DP 8/9 in brain. Exclusively staining of the meninges in only wildtype F344/Ztm brain sections after one hour in turn, implies specific expression and activity of DP 4 in the meninges (Figure 42B). In addition, tissue sections were also investigated using Fast Blue B and Gly-Pro-4-Met-2-NA to include DP 2 activity. Figure 43 illustrates that DP 2 co-localises with DP 4 in the meninges after 1 hour. However, while strong DP 4 activity could be detected in the arachnoidal membrane and pia mater of wilde-typ F344/Ztm rats in presence of DP 2-selective inhibitor Dab-Pip (Figure 43E), positive staining of wildtype F344/Ztm rats in presence of Sitagliptin as well as DP 4-deficient F344/Crl(Wiga)SvH-Dpp4 points to DP 2 activity in the arachnoidal membrane and subarachnoidal space (Figure 43B+C). Furthermore, moderate ubiquitous staining could be observed in brain parenchyma of wildtype F344/Ztm rats after one hour, which was ablated in presence of DP 2-selective inhibitor Dab-Pip as depicted Figure 43A, D+E, respectively. These results also point to moderate ubiquitous distribution of DP 2. No activity was detected in DP 4-deficient F344/Crl(Wiga)SvH-Dpp4 in presence of Dab-Pip, thus indicating that in this assay, activity of only DP 2 and DP 4 were measured after one hour of staining. A possible explanation of the low activity of DP 8/9 in both assay systems using Gly-Pro-4-OH-1-NA and Gly-Pro-4-Met-2-NA, respectively, could be the fact, that some activity of DP 8/9 was lost upon freezing as in case of cryosections. This phenomenon was also observed by others, in particular regarding DP 8 (127;134;138). Thus, one can conclude that DP 8/9 and DP 2 are ubiquitously distributed with DP 2 having additionally elevated activity in the meninges, whereas DP 4 is exclusively expressed in certain areas such as the meninges.

WT WT Mut Mut (GP-4-OH-1-NA + NBT) (NBT) (GP-4-OH-1-NA + NBT) (NBT)

A Brain 50 µm 12 h 10 x

B Meninges 50 µm Meninges 1 h 100 x

Figure 42: Histochemical analysis of DP 4-like enzymes in 50 µm brain sections from F344/Ztm and F344/Crl(Wiga)SvH-Dpp4 Fischer rats after 12 and 1 h, using Gly-Pro-4-OH-1-NA and NBT as substrates. A, staining of brain sections from F344/Ztm and F344/Crl(Wiga)SvH-Dpp4 Fischer rats, respectively, indicating ubiquitous activity of DP 8/9 in brain. B, staining of meninges in F344/Ztm and F344/Crl(Wiga)SvH-Dpp4 Fischer rats after 1 hour, results in exclusively staining of the meninges in only wildtype. WT, wildtype F344/Ztm; Mut, DP 4- deficient F344/Crl(Wiga)SvH-Dpp4 (677).

72 Results and Discussion

Figure 43: Histochemical analysis of DP 4-like enzymes in brain sections from F344/Ztm and F344/Crl(Wiga)SvH-Dpp4 Fischer rats after 1 h, using Gly-Pro-4-Met-2-NA and Fast Blue B as substrate. A, staining of meninges from wildtype F344/Ztm rat at 10 x magnification, showing strong DP 4-like activity in the arachoidal membrane of meninges. B, staining of meninges of wildtype F344/Ztm rat in presence of DP 4-selective inhibitor, indicating DP 2 activity in subarachnoidal space and arachnoidal membrane. C, staining of meninges from DP 4-deficient F344/Crl(Wiga)SvH-Dpp4 rat, pointing to DP 2 activity in subarachnoidal space. D, staining of meninges in wildtype F344/Ztm rats at 60 x magnification. E, staining of meninges from wildtype F344/Ztm rat in presence of DP 2-selective inhibitor Dab-Pip, showing strong activity of DP 4 in arachnoidal membrane and pia mater. F, no staining of meninges from DP 4-deficient F344/Crl(Wiga)SvH-Dpp4 rat in presence of Dab-Pip. DM, dura mater; AM, arachnoidal membrane; SAS, subarachnoidal space; PM, pia mater.

3.4.1.3 Specific Activity of DP 4 in Circumventricular Organs In addition to the histochemical activity assay, extracts from brain areas corresponding to psychological stress and neuronal circuits of NPY were analysed with regards to specific activities of DP 4-like enzymes and the results are summarised in Appendix 67. These include the brain areas cortex, amygdala, hypothalamus, circumventricular organs (CVO’s), hippocampus, striatum, meninges, subventricular zone (SVZ), cerebellum and spinal cord. Like the data obtained from crude brain extracts in Figure 40A, almost all areas exhibit similar levels of specific activities, having the order DP 2  DP 8/9 >> DP 4. However, two exceptions could be identified, where specific activities of DP 4 was exceeding to ones of DP 8/9, i.e. meninges and the CVO’s. Particularly in the CVO’s, the overall specific activity of the DP 4-like enzymes was exceptionally high compared to brain extract and at neutral pH, DP 4 contributed to more than 60 % as seen in Figure 44A. The CVO’s are unique structures of the brain as they lack the blood-brain-barrier and thus represent an interphase between the CNS and the periphery. Altogether, there are nine CVO’s and one distinguishes between sensory and neurosecretory CVO’s as illustrated in Figure 44B. While the sensory CVO’s are comprised of subfornical organ (SFO), organ vasculosum of lamina terminalis (OVLT) and area postrema (AP); the neurosecretory CVO’s include anterior pituitary (Ant.Pit.), median eminence (EM), intermediate lobe of pituitary and pineal gland (PiG). Choroid plexus (Ch.P) and subcommissural organ (SCO) are exceptions due partial BBB (565;567). Apart from being involved in cardiovascular regulation, body-fluid balance, food-intake, immunology, regulation of temperature and reproduction, they are associated with behaviour particularly regarding

73 Results and Discussion stress, as the median eminence is part of the hypothalamic-pituitary-adrenal axis (HPA-axis), the so-called neuro-endocrinological stress axis, whereas the area postrema is linked to the neuro-autonomal-sympathetic stress axis (NAS), also referred to as cardiovascular stress axis (566). Expression of DP 4 in the median eminence has also been demonstrated by immunohistochemical staining (678).

A B 5 DP 4-like activity 4 Sitagliptin

3 (mU/mg)

SpecificActiv ity 2

1

0 Brain CVO's Figure 44: Specific activities of DP 4 in brain and circumventricular organs (CVO’s). A, specific activities of DP 4 in extracts from brain and CVO’s as determined with 0.1 mM Ala-Pro-AMC in 40 mM Hepes pH 7.6 in presence or absence of DP 4 selective inhibitor Sitagliptin. n = 4, error bars = SEM. B, anatomical presentation of CVO’s in a sagittal section of rat brain. Ant.Pit., anterior pituitary; EM, median eminence; OVLT, organ vasculosum of lamina terminalis; SCO, subcommissural organ; SFO, subfornical organ; Ch.P, choroids plexus; PiG, pineal gland; AP, area postrema (679).

3.4.1.4 Analysis of Neuropeptide Hydrolysis

3.4.1.4.1 No Hydrolysis of Neuropeptides by DP 2 Since DP 2 is the most abundant DP 4-like enzyme in the brain, hydrolysis of NPY and SP by human recombinant DP 2 was evaluated. Yet, as one can see from Figure 45, unlike DP 4, human recombinant DP 2 is unable to truncate NPY or SP as already reported (135;163;179;212).

Figure 45: Proteolytic studies of NPY and SP by human recombinant DP 2 and DP 4, using MALDI- TOF-MS. A, 25 µM NPY in presence of human recombinant DP 2 results in no truncation after 6 h. B, 25 µM SP in presence of human recombinant DP 2 results in no truncation after 6 h. C, 25 µM NPY is readily truncated to NPY3-36 by human recombinant DP 4 after 30 min. D, 25 µM SP is truncated to SP3-11 by human recombinant DP 4 after 60 min. Matrices: NPY, sinapinic acid; SP, - cyano-4-hydroxycinnamic acid (135). 74 Results and Discussion

3.4.1.4.2 Hydrolysis of Neuropeptides by Neuronal Tissues Hydrolysis of the neuropeptides NPY, SP and PACAP were studied using tissue extracts obtained from wildtype and DP 4-deficient Fischer rats as well as cell extracts from rat primary neurons, astrocytes and microglia. Applying 40 µg/ml of tissue or cell extract respectively, Figure 46 clearly illustrates that NPY is relatively stable in brain and neuronal cell extracts, whereas in astrocytes and microglia it is readily metabolised to NPY2-36, NPY1-30, NPY3-30 and

NPY1-30, NPY3-36, NPY2-36, respectively. Interestingly, NPY1-30 has been reported to derive from cleavage by neprilysin (NEP), yet applying the NEP-selective inhibitor phosphoramidon at

50 µM to 1 mM did not suppress the formation of this metabolite (485). NPY2-36 is produced by soluble aminopeptidase P (sAmpP) and its presence in astrocytes has already been reported (43;135;485;678;680). Implementing DP 4-selective inhibitor as well as DP 4-like inhibitor

P32/98, NPY3-36 was equally produced by DP 4 and DP 8/9 in astrocytes, whereas in microglia it was mainly derived from DP 4 (data not shown).

Figure 46: Hydrolysis of 25 µM NPY by 40 µg/ml tissue extracts from either rat brain, neurons, astrocytes or microglia after 6 h and 24 h using MALDI-TOF-MS. Matrix, sinapinic acid.

Increasing the protein concentration of the crude extracts from rat brain and neurons 10-fold to 400 µg/ml, respectively, various metabolites of NPY could be detected by these tissues as demonstrated in Figure 47. In brain tissue the main metabolite is initially NPY1-30, followed by

75 Results and Discussion

NPY3-36 and NPY3-30. After 2 h, NPY is additionally C-terminally truncated to NPY1-34 as well as degraded to NPY1-24 and NPY1-23. Interestingly, while after 30 min the NPY3-36 metabolite is equally produced by DP 4 and DP 8/9 as seen in Figure 47A, it is mainly derived from DP8/9 after 2 h as DP 4-selective inhibitor Sitagliptin hardly suppressed NPY3-36 truncation in contrast to DP 4-like inhibitor P32/98 (Figure 47B). The reason for this phenomenon is that DP 4 catalyses NPY much more efficiently compared to DP 8/9 (Appendix 2) (124), yet according to the activity studies in Figure 40A, brain tissues exhibit more DP 8/9 activity than DP 4.

Proteolysis of brain extract after 2 h resulted in smaller NPY metabolites of NPY1-30, NPY3-30,

NPY1-24 and NPY1-23, respectively. In neurons in turn, the N-terminal truncation of NPY1-36 to

NPY3-36 is exclusively contributed by cytosolic DP 8/9 as illustrated after 6 h of hydrolysis in Figure 47C. The hydrolysis of SP and PACAP was additionally analysed by this method, yet it yielded only endoproteolytically degraded products. Thus, one can conclude that NPY is the major stress-related substrate of DP 4 within the CNS.

Figure 47: Hydrolysis of 25 µM NPY by 400 µg/ml tissue extracts from either rat brain or rat primary neurons, using MALDI-TOF-MS. A, hydrolysis of 25 µM NPY by 400 µg/ml brain extracts from rat after 30 min. B, hydrolysis of 25 µM NPY by 400 µg/ml brain extracts from rat after 2 h. C, hydrolysis of 25 µM NPY by 400 µg/ml cell extracts from rat primary neurons after 6 h. Sitagliptin, DP 4-selective inhibitor; P32/98, non-selective DP 4-like inhibitor. Matrix, sinapinic acid.

3.4.2 Analysis of Neuropeptide Hydrolysis by DP 4 in the Periphery 3.4.2.1 Capillary Depletion In the present study high levels of DP 4 were found in the CVO’s and these structures present an interphase between the CNS and the periphery. Furthermore, peripheral NPY was reported to cross the BBB in a non-saturable manner and for this reason an experiment was designed to investigate the crossing of peripherally administered NPY to the brain with the main aim to identify the resulting metabolites during this process (562). As to distinguish between endogenous NPY and peripherally administered NPY, NPY was C-terminally labelled to yield NPY-biotin, which could then be concentrated from brain tissue by immune precipitation with a streptavidin-antibody. Finally, the resulting NPY-metabolites could be identified by MALDI- TOF-MS (681;682). Thus, NPY-biotin was administered and perfused through the blood circulation of the brain, which was subsequently removed and fractionated into brain parenchyma and endothelial capillaries by capillary depletion (593). A scheme illustrating the procedure of capillary depletion is given in Figure 18 and the various controls being applied are summarised in Appendix 45. The preliminiray results of capillary depletion can be depicted 76 Results and Discussion in Appendix 68 and Figure 48, respectively, demonstrating that perfusion of rat brain as well as fractionation into brain parenchyma and endothelial capillaries by capillary depletion were successful (Appendix 68A+C and Figure 48A). However, an exess of the BBB control chlorpromazine was applied (Appendix 68B) and no C-terminally labelled NPY-biotin could be detected in brain parenchyma after immune precipitation due to proteolysis, though a conservation solution comprised of aprotinin, PMSF, phosphoramidon, EDTA, Val-Pyr and DTT had previously been added. Figure 48A shows the N-terminal truncation of NPY-biotin to

NPY2-36-biotin and NPY3-36-biotin by endothelial DP 4 and mAmpP, respectively, obtained from a brain-perfusate corresponding to ± 3 min exposure of blood circulation. Figure 48B in turn, indicates complete degradation of NPY-biotin in brain parenchyma as no metabolite could be detected after immune precipitation. Even brain parenchyma spiked with unlabelled NPY1-36 in presence of the commercial protease inhibitor mix complete mini (Roche), resulted in endoproteolytical degradation, yielding the metabolites NPY1- 29 and NPY1-30, already after 30 min (Figure 48D). Interestingly, comparing the hydrolysis of NPY-biotin by endothelial DP 4 and mAmpP with in vitro metabolism of undiluted serum spiked with 50 µM NPY-biotin after 3 min resulted in the same metabolites with similar % intensities (Figure 48C).

Figure 48: Analysis of C-terminally labelled NPY-biotin and its metabolites after perfusion and capillary depletion of rat brain. A, N-terminal truncation of 50 µM NPY-biotin by endothelial DP 4 (NPY3-36- biotin) and mAmpP (NPY2-36-biotin) after brain perfusion corresponding to ± 3 min blood circulation. The MALDI-TOF-MS spectra was performed after immune precipitation of NPY-biotin with streptavidin-Ab and subsequent C18 zip-tip. B, complete degradation of NPY-biotin in brain parenchyma in presence of conservation solution with inhibitors aprotinin, PMSF, Val-Pyr, EDTA, phosphoramidon and DTT, as no NPY-metabolite could be detected after immune precipitation of NPY-biotin with streptavidin-Ab and subsequent C18 zip-tip, using MADLI-TOF-MS. C, N-terminal truncation of 50 µM NPY-biotin by soluble DP 4 (NPY3-36-biotin) and mAmpP (NPY2-36-biotin) in undiluted serum after ± 3 min. D, endoproteolytical degradation of unlabelled NPY spiked in brain parenchyma in presence of commercial protease inhibitor mix complete mini (Roche). 3.4.2.1.1 Identification of NPY-Metabolising Enzymes Originally, NPY was reported to be metabolised in the brain by NEP, AmpP and to a smaller extent by DP 4, with NEP producing NPY1-20 and NPY1-30, respectively (485). However, applying NEP-specific inhibitor phosphoramidon (Ki = 3 nM) at concentrations ranging between

50 µM and 1 mM, the formation of the metabolite NPY1-30 could not be supressed in all tissue extracts described in section 3.4.2. Furthermore, sub-cellular fractionation gave evidence that the unknown proteolytical enzyme(s) is/are cytosolic (Figure 49B). As to identify the unknown 77 Results and Discussion proteolytic enzyme(s), selective inhibitors against various neuropeptidases (Appendix 46) were added to brain parenchyma, already containing conservation solution. Interestingly, NPY1-30 was already present in the the time zero sample t0, in which tissue extract was added to 0.1 % TFA for inactivation prior to the addition of substrate. This observation pointed to a possible lysosomal enzyme and using the search function of substrate specificity of the merops-sanger data base, cathepsin D (CTSD) turned out to be the main candidate. Thus, applying Pepstatin A against CTSD in brain parenchyma, the dominant NPY1-30 metabolite could be suppressed already after 20 minutes (Figure 49A), resulting in smaller fragments similar to the MALDI-TOF-MS spectra of proteolysis after 90 min without any inhibitor. Applying inhibitor actinonin against Meprin A/Meprin B (MepA/MepB), proteolysis into smaller fragments was suppressed after 90 minutes, however, giving rise to NPY1-30 and NPY2-30 generated by CTSD and AmpP, respectively. Since the smaller degradation metabolites were only found in the cytosolic fractions, secreted soluble Mep-A must have produced them and not the membrane bound Mep-B. Both Mep-A and Mep-B were already described to cleave NPY, though the cleavage sites had not been identified for MepA, yet (683). Consequently, adding Pepstatin A and actinonin to the conservation solution as outlined in Appendix 48, caused NPY to be stable for more than 24 h (Figure 50).

Figure 49: NPY-hydrolysis by secreted Mep-A and lysosomal CTSD from rat brain, investigated by MALDI-TOF-MS. A, metabolism of 25 µM NPY by 500 µg/ml rat tissue extract in presence or absence of pepstatin A and actinonin. B, metabolism of 25 µM NPY by cytosolic and membrane fractions from extracts of rat brain.

Figure 50: Improved conservation solution comprised of aprotinin, PMSF, Val-Pyr, EDTA, E64, pepstatin A, actinonin, phosphoramidon and DTT enabled stability of NPY for more than 24 h as determined by MALDI-TOF-MS. Matrix, sinapinic acid. 78 Results and Discussion

As to confirm the novel NPY-cleaving enzymes, human recombinant MepA and human CTSD purified from spleen were analysed for NPY hydrolysis as illustrated in Figure 51.

A B

Sitagliptin

C D

E F

Figure 51: Hydrolysis of 25 µM NPY by human recombinant/purified enzymes, using MALDI-TOF-MS analysis reveals a meprin-A-mediated degradation and an endoproteolytic inactivation of NPY by cathepsin D and Neprilysin. In addition, the cathepsin D mediated effect is highly pH- dependent. A, DP 4 (1 µg/ml, human recombinant; inhibitor: Sitagliptin). B, NEP (1 µg/ml, human recombinant; inhibitor: phosphoramidon). C, CTSD, pH 7.6 (1 µg/ml, purified from human spleen; inhibitor: pepstatin A). D, CTSD, pH 5.5 (1 µg/ml, purified from human spleen; inhibitor: pepstatin A). E, MepA (400 ng/ml, human recombinant, previously activated with 100 ng/ml trypsin for 1 h, activation stopped by AEBSF achieving a 1 mM final concentration; 1 µg/ml activated MepA resulted in rapid degradation of undetectable peaks after 2 min; inhibitor: actinonin). F, MepB (400 ng/ml, human recombinant, previously activated with 100 ng/ml trypsin for 30 min, activation stopped by AEBSF achieving a 1 mM final concentration; inhibitor: actinonin).

Figure 51 clearly illustrates that DP 4, CTSD, MepB and MepA rapidly hydrolyse NPY, whereas NEP only slowely degrades NPY, requiring an enzyme concentration of  1 µg/ml. For comparison, the trypsin-like enzymes trypsin, plasmin, uPA and plasma kallikrein, were also investigated as they were known to cleave NPY (Appendix 69). While plasmin, uPA and 79 Results and Discussion thrombin slowly hydrolyse NPY, trypsin, plasma kallikrein (Kal) as well as novel NPY-cleaving enzyme tissue kallikrein (hK1) rapidly hydrolyse NPY similar to DP 4, CTSD, MepB and MepA. Interestingly, very little is known about MepA and MepB in the brain, other than in craniofacial development, where it was predominantly found in the ependymal layer of brain ventricles and choroid plexus (684). Since secreted MepA and meprin- subunit of membrane-bound MepB require activation and so far only trypsin, plasmin, hK’s 4, 5 and 8 have been reported to activate it, additional trypsin-like enzymes expressed in the brain were also investigated for possible activation of MepA as outlined in Appendix 47 (685-688). Thus, uPA and hK1 were shown for the first time to activate MepA, and hK1 additionally MepB. In fact, hK1 activated MepA and MepB almost with similar efficiency as trypsin as seen in Appendix 70. Recently, activation of MepA and MepB by kallikrein 8, also known as neuropsin, was shown (687;688). Although degradation of NPY by CTSD is far more rapidly at acidic pH corresponding to the lysosomes, it is also feasible at physiological pH 7.6 as shown in Figure 51 C+D, respectively. CSTD is widely distributed in the brain and particular in microglia, where it is active as a lysosomal as well as extracellularly secreted enzyme. It is secreted via cation-dependent mannose-6-phospate receptor (M6PR) and such secretion is associated with diseases like cancer and neurodegneration (689-695). As the Y-receptors have been reported to internalise to stop signalling, additional lysosomal enzymes such as cathepsin B (CTSB), cathepsin L (CTSL), cathepsin G (CTSG) and cathepsin S (CTSS) were also investigated for NPY degradation. Thus, Figure 52 shows that NPY is rapidly degraded by CTSB, CTSG and CTSS, and to a lesser extend by CTSL. This points to a novel degradive pathway of NPY in lysosomes and supports the data that after agonist induced signal transduction, resulting in intracellular phoshorylation of the Y-receptor, the receptor becomes desensitized and -arrestin-2 binds followed by subsequent clathrin-dependent internalisation of the receptor-agonist complex with according to the present data, NPY being degraded in the lysosomes (696-707).

Figure 52: Hydrolysis of 25 µM NPY by 1 µg/ml Cathepsins B, G, L and S as analysed by MALDI-TOF- MS. As control NPY cleavage was inhibited by inhibitor E64 (A-C) or DIC (D). A, CTSB, pH 5.5; B, CTSL, pH 5.5; C, CTSS, pH 6.0; D, GTSG, pH 7.6. Matrix, sinapinic acid. 80 Results and Discussion

3.4.2.2 Hydrolysis of NPY in Human CSF Interestingly, analysing the ex vivo metabolism of NPY in human cerebrospinal fluid (CSF) by MALDI-TOF-MS resulted in a major metabolite of NPY1-30 by CTSD as illustrated in Figure 53. NPY-cleavage by CTSD from CSF was subject to substrate inhibition as the NPY concentration in the assay had to be reduced to 12.5 µM to detect the NPY1-30 metabolite. Western blot analysis revealed that CTSD is present in the CSF as a single chain active enzyme having a molecular weight of 48 kDa (Figure 53A). This form of CTSD is also found to be secreted into the medium after transfection into HEK-293 cells, whereas purified human CTSD prevailed predominantly in the matured double chain active form, with the heavy chain possessing a molecular weight of 23 kDa (708-710). So far, NPY hydrolysis has only been investigated in human CSF from depressed patients, resulting in shredded metabolites possibly due to Mep-A or cathepsins (711;712). Lack of any DP 4-like activity in human CSF confirmed the absence of any metabolites derived from DP 4-like enzymes.

Figure 53: Hydrolysis of NPY in CSF as analysed by MALDI-TOF-MS. A, Western blot of CTSD. M, marker, lane 1, medium of HEK-cells transfected with CTSD, lane 2, human CTSD purified from spleen, lane 3, CTSD from 20 µl CSF. B, Rapid hydrolysis of NPY by human CSF results in CTSD-specific metabolite NPY1-30 after 30 min at physiological pH 7.4, as detected by MALDI- TOF-MS. Samples were kindly supplied by Dr. B. Leavitt and Prof. M. Haydn from University of British Columbia, Vancouver, Canada.

3.4.2.3 Processing of Neuropeptides in the Blood Circulation

3.4.2.3.1 In vitro Metabolism of NPY in Human Serum The time-dependent hydrolysis of NPY in human serum clearly demonstrates that DP 4 is the major NPY-cleaving enzymes in serum as shown in Figure 54A. In absence of any inhibitors,

NPY3-36 was the major metabolite and in presence of selective DP 4 inhibitor only untruncated

NPY1- 36 and NPY1-34 prevailed. In fact, DP 4-selective inhibition by Sitagliptin substantially increased the contribution of the inactive NPY1-34 metabolite compared to the metabolism without inhibitor (Figure 54 B). Suppression of C-terminal truncation in presence of Captopril points to an ACE-like cleavage, whereas combined inhibition of DP 4 and ACE-like enzyme resulted in only vasoconstrictive of NPY1-36 as well as the less potent metabolite NPY2-36 by AmpP. While C- terminal truncation of NPY by ACE to NPY1-34 compensates supposedly hypertensive side-effects of anitdiabetic DP 4 inhibition due to inactivation of hypertensive NPY1-36, a combined administration of anti-diabetic DP 4 and antihypertensive ACE inhibitors would result in the presence of hypertensive NPY1-36 and less potent NPY2-36. C-terminal truncation of NPY could be detected in more than 20 sera analysed so far, though the NPY1-34 metabolite varied greatly in intensity, thus pointing to a possible genetic disposition. In fact, a combined application of P32/98 81 Results and Discussion and Captopril resulted in hypertension in spontaneously hypertensive rats (SHR), whereas normotensive Wistar Kyoto rats (WKY) remained normotensive. Interestingly, administration of P32/98 alone did not alter the blood pressure in both rat species and applying an antagonist against Y1-R prior to P32/98 and Captopril, prevented the development of hypertension in SHR, thereby confirming the involvement NPY or PYY (713;714). Similarly, in a very recent report a combined administration of Enalapril (ACE inhibitor) and Sitagliptin (DP 4 inhibitor) resulted in hypertension in patients suffering from the metabolic syndrome (715;716). Additional C-terminal truncation of NPY has already been described in stability experiments of NPY-analogues in serum, yielding NPY metabolites NPY1-34, NPY1-35, NPY1-32 and NPY1-26 (717). However, no major

NPY3-35 metabolite produced by plasma kallikrein could be detected in the present study as lately reported (718). Such discrepancy could be explained with different experimental conditions such as salt concentrations and/or storage, since the observed enzyme must have been auto- catalysed -kallikrein due to the lack of factor XIIa in serum, where plasma kallikrein is normally present as inactive plasma prekallikrein (719;720). Furthermore, time-dependent hydrolysis of NPY by purified plasma kallikrein resulted in trypsin-like cleavage of NPY yielding the metabolites

NPY1-18, NPY1-19, NPY1-21, NPY1-25, NPY1-33, NPY1-35, NPY26-33, NPY26-35 and NPY26-36 as shown in Appendix 69 (680). Moreover, we could also show that NPY is a good substrate for tissue kallikrein (hK1). Nevertheless, in serum NPY is predominantly metabolised by DP 4 as already confirmed by selective inhibitors against the DP 4-like enzymes (135).

Without inhibitor

Sitagliptin Sitagliptin

Captopril

Sitagliptin+ Captopril

Figure 54:Hydrolysis of NPY by human serum analysed by MALDI-TOF-MS. A, time-dependent hydrolysis of NPY in human serum revealing additional C-terminal truncation by unknown enzyme. B, identification of C-terminal truncation by ACE-like activity. Note the compensatory increase of ACE-like activity after inhibition of DP 4 by Sitagliptin and the increased half-life of vasoconstrictive full-length NPY1-36 after combined inhibition of ACE + Sitagliptin. Data represent MALDI-TOF-MS analysis after 90 min. Matrix, sinapinic acid. 3.4.2.3.2 Metabolism of Stress-related Neuropeptides in Human and Rat Sera A great number of neuropeptides have been described to be involved in stress such as depression, anxiety, and schizophrenia, including NPY, GALP, rOrxB, hOrxB, SP, Gal, PACAP, NT, AVP, ACTH, AngI, Oxy and CRF. Since all of them contain at least one proline as reviewed in Appendix 51, their stability was analysed in serum, implementing MALDI-TOF-MS (721;722). Out of these, NPY, GALP, rOrxB and to a lesser extent SP were N-terminally truncated by DP 4, Gal endoproteolytically hydrolysed by DP 4-related enzyme proline endopeptidase (PEP), hOrxB was N-terminally truncated by an aminopeptidase, -Endo, SP, 82 Results and Discussion

PACAP, NT, GALP and h/rOrxB were endoproteolytically degraded, AVP, ACTH, AngI and NPY were C-terminally truncated and OrxA, Oxy and CRF were found to be stable for more than 24 h as summarised in Appendix 71. Thus, two novel substrates for DP 4 (GALP and rOrxB) and one for PEP (Gal) could be identified. To substantiate the results, these neuropeptides were also analysed with human recombinant DP 4 and PEP as shown in Figure 55 and Appendix 72, respectively. Interestingly, in presence of selective DP 4 inhibitor, rOrxB is N-terminally truncated by serum Amp P.

Figure 55: Time-dependent hydrolysis of 50 µM galanin-like peptide (GALP) by human recombinant DP 4 and human serum as well as hydrolysis of 50 µM rat Orexin B (OrxB) by DP 4 and rat serum, using MALDI-TOF-MS. A, time-dependent hydrolysis of GALP by human recombinant DP 4. B, time-dependent hydrolysis of GALP by human serum. C, time-dependent hydrolysis of rat Orx B by human recombinant DP 4. D, time-dependent hydrolysis of rat OrxB by rat serum. Sitagliptin, DP 4 selective inhibitor; matrix, sinapinic acid for GALP; -cyano-4-hydroxycinnamic acid for rat OrxB. 83 Results and Discussion

3.4.2.3.3 Ex vivo Metabolism of Stress-related Neuropeptides in Human Blood However, as blood cells also contained proteases on the cell membrane such as DP 4 on a sub- bright set of CD26 memory T-cells and NEP as CD10 on B-cells, neuropeptide hydrolysis was also investigated ex vivo in whole blood (207;723). To ensure no interferance of neuropeptide degradation by cytosolic enzymes due to haemolysis, blood was diluted with an isotonic buffer solution containing 300 mM sucrose as shown in Figure 56. In addition, degradation of NPY, GALP, SP and Gal was also performed using hypotonic blood without any sucrose. Figure 56A clearly demonstrates that NPY is the best neuropeptide substrate for DP 4 in the blood circulation, whereas SP is mainly degraded by NEP (Figure 56 B) and GALP was shown for the first time to be physiological substrate of DP 4. As expected, the neuropeptides were much more rapidly degraded in hypotonic blood as in isotonic solution, and with regards to SP, it even resulted in different metabolites (Figure 56 D). In contrast, the metabolic degradation pattern of NPY-metabolites by hypotonic blood was identical to the isotonic one as depicted in Figure 57, yet the rate of hydrolysis was much faster (3 h compared to 12 h) as it also included cleavage by cytosolic DP 8 and/or DP 9 from leucocytes (127;140;210). Interestingly, the final degradation product of NPY ex vivo blood is NPY3-34, caused by an ACE-like enzyme, whereas the NPY3-35 by plasma kallikrein could not be detected (718).

Figure 56: MALDI-TOF-MS spectra, showing ex vivo hydrolysis of neuropeptide Y (NPY), galanin- like peptide (GALP) and substance P (SP) in blood. A, MALDI-TOF-MS spectra, showing ex vivo hydrolysis of 25 µM NPY in blood diluted 1:4 with 20 mM Tris-HCl, pH 7.6 and 0.3 M sucrose after 30 min.. B, MALDI-TOF-MS spectra showing ex vivo hydrolysis of 25 µM GALP in blood diluted 1:4 with 20 mM Tris-HCl, pH 7.6 and 0.3 M sucrose after 6 h. C, MALDI-TOF-MS spectra showing ex vivo hydrolysis of 25 µM SP in blood diluted 1:4 with 20 mM Tris-HCl, pH 7.6 and 0.3 M sucrose after 3 h. D, MALDI-TOF-MS spectra showing in vitro hydrolysis of 25 µM SP in blood diluted 1:4 with hypotonic 20 mM Tris-HCl, pH 7.6 after 5 min. Matrices, sinapinic acid for NPY and GALP; -cyano-4-hydroxycinnamic acid for SP.

84 Results and Discussion

Figure 57: MALDI-TOF-MS spectra comparing the hydrolysis of 25 µM neuropeptide Y (NPY) ex vivo blood versus in vitro haemolytic blood. A, MALDI-TOF-MS spectra showing the time-dependent ex vivo hydrolysis of 25 µM NPY in blood diluted 1:9 with isotonic 20 mM Tris-HCl, pH 7.6 and 0.3 M sucrose. B, MALDI-TOF-MS spectra showing the time-dependent in vitro hydrolysis of 25 µM NPY in haemolytic blood diluted 1:9 with hypotonic 20 mM Tris-HCl, pH 7.6, indicating additional NPY3-36 truncation by cytosolic DP 8 and/or DP 9. Matrix, sinapinic acid.

3.4.3 Analysis of DP 4-like Enzymes in Sera from Depressed Patients 3.4.3.1 DP 4-like Activity in Sera from Depressed Patients DP 4 has already been suggested to be a biomarker for the diagnosis of major depression and other stress-related diseases at a time when DP 8 and DP 9 were still unknown (354-359;724). For this reason, sera obtained from 32 patients suffering from either melancholic or non- melancholic depression were re-evaluated for DP 4 activity, implementing a selective DP 4 inhibitor Sitagliptin and the non-selective DP 4-like inhibitor P32/98 as a control (Figure 58). The results confirmed a decrease of DP 4-like activity in sera from depressed patients prior to treatment, which increased after treatment and it was even close to the activity of their age and sex-matched controls in case of melancholic depressed patients. Furthermore, in all samples investigated, DP 4-like activity was comprised of more than 90 % by DP 4 (Figure 58). As to determine whether the lower serum DP 4 activity in depressed patients was due to less shedding of DP 4 into the blood circulation or based on post-translational modifications of soluble DP 4 as in case of rheumathoid arthritis or lupus erythematosus (78), the protein concentration of DP 4 was determined by ELISA and correlated to DP 4 activity, whereas post- translational modifications were examined by analytical agarose IEF, pH 3 - 7. Figure 59 clearly demonstrates that DP 4 activity very significantly correlated to DP 4 protein concentration (p < 0.0001) as previously reported (725), while the various DP 4 isoforms in serum were virtually identical in all investigated samples. Thus, one can conclude that in major depression, less DP 4 is shedded into the blood circulation, thereby resulting in a lower serum activity of DP 4. Moreover, it also indicates that serum DP 4 is not the pharmaceutical target for the treatment of major depression, but instead its analysis could serve as a biological marker for the diagnosis of clinical depression as previously suggested (354-357;724).

85 Results and Discussion

Figure 58: DP 4-like activity in sera obtained from depressed patients, using 100 µM Gly-Pro-AMC. A, DP 4-like activity of sera obtained from patients diagnosed with clinical depression (n = 32 for depressed patient and n = 30 for control subjects). B, DP 4-like activity of sera obtained from patients diagnosed with non-melancholic depression (n = 16 for depressed patients and n = 15 for control subjects). C, DP 4-like activity of sera obtained from patients diagnosed with melancholic depression (n = 16 for depressed patients and n = 15 for control subjects). Sitagliptin, selective DP 4 inhibitor; P32/98, non-selective DP 4-like inhibitor; error bars = SEM. Samples were kindly supplied by PD Dr. M. Rothermundt and Dr. F. Kästner (597;598).

Figure 59: Characterisation of DP 4 in sera from depressed patients and control subjects. A, correlation of DP 4 activity with CD26 (DP 4) protein concentration as determined by ELISA, p < 0.0001, n = 94. B, analytical agarose IEF pH 3 - 7 stained with Gly-Pro-4-Met-2-NA, displaying DP 4 isoforms due to heterogeneity in sialynation. Lane 1, non-melancholic depressed patient before treatment; lane 2, non-melancholic depressed patient after treatment; lane 3, healthy control subject matching with respect to age and sex; lane 4, melancholic depressed patient before treatment; lane 5, melancholic depressed patient after treatment; lane 6, healthy control subject matching with respect to age and sex.

3.4.3.2 Hydrolysis of NPY in Sera from Depressed Patients Unlike the hydrolysis of NPY by CSF from depressed patients (711), no alterations of metabolites could be observed comparing NPY hydrolysis by sera from depressed patients with their respective controls, except that the rate of hydrolysis was slower as illustrated in

Appendix 73. As already shown in Figure 53A, DP 4-derived metabolite NPY3-36 was the major cleavage product, followed by NPY1-34, NPY3-34 and sometimes NPY1-30. 86 A 1 h 6 h 12 h 15 h 20 h B 1 h C 12 h

F344/Ztm Conclusion

4 Conclusion 4.1 Crystal Structure With the aid of this newly developed method of isoform isolation to solve the microheterogeneity of purified porcine DP 4, the first native crystal structure of DP 4 was obtained (558). A unique feature of the native porcine crystal structure is its tetramerisation, indicating possible cell to cell communication by either two membrane DP 4’s on different cells as represented in Figure 60, or by a soluble serum DP 4 binding to a membrane one such as serum DP 4 on endothelial membrane-bound DP 4. It has been reported that DP 4 assists in transendothelial migration of T-lymphocytes (726;727) and this process may also involve mannose-6-phosphate receptor (197). Furthermore, binding of T-lymphocytes to epithelial cells was found to be mediated and dependent on the interaction between DP 4 and ADA, as only DP 4-rich T-lymphocytes bound to epithelial cells and this adhesion was abolished by soluble ADA and monoclonal anti-DP 4 antibody Ta5.9, specific to the ADA- binding epitope (252). Interestingly, the tetramerisation interface also covers the ADA binding epitope, including Leu294 and Val341 (13). Gln286 of DP 4 appears to play a key role in both tetramerisation and ADA binding, as confirmed by the recent crystal structure of

(hDP 4bADA)2 complex as well as in vivo cryo-electron microscopy (82;248). On the other hand, using various DP 4 inhibitors and several monoclonal antibodies specific to epitopes at the glycosylated region of DP 4, no relationship was found between DP 4-dependent binding of lymphocytes to endothelial cells or fibroblasts (728). Additionally, lung endothelial DP 4 was shown to be an adhesion receptor for metastasing breast cancer cells, and adhesion was blocked by soluble DPIV and monoclonal anti–DP 4 antibody (273;274). Tetramerisation involves in its centre the strands Asn-279–Gln-286 of each DP 4-dimer to form an antiparallel-sheet, thus extending the propeller blade IV to an eight-stranded antiparallel sheet (Figure 60 C). The resulting tetramer contains a large cavity and may explain why a hexamer assembly was proposed by gel filtration experiments (729). In addition, glycosylation at 279 appears to be important for tetramerisation (13). However, the suggested interference of glycosylation at 279 with ADA binding has recently been argued by site-directed mutagenesis studies as well as the (hDP 4bADA)2 structure (82;730). Nonetheless, glycosylation at Asn-229, which is in close vicinity to the tetramerisation interface, was found to directly bind to ADA. This implies that proper glycosylation may be an important factor for DP 4 to function as cell-cell communication molecule (13). Whether such tetramerisation is specific to the particular DP 4 isoform used for the crystal structure is still subject of current research. Comparing the cell units of the various crystal structures, one can observe substantial differences with respect to their volumes, that range between 978,835 - 2,033,934 Å3 (Table 31). Surprisingly, the smallest cell unit was obtained by the isolated acidic isoforms of native porcine DP 4, although the structure was found to be tetrameric and contained mature complex N-glycans (13). The dimeric structures of human recombinant DP 4 expressed in insect cells in turn, consisted only of immature N-glycans, but revealed the largest cell units, which were more than double the size of the porcine cell unit (606). Deglycosylation of human recombinant DP 4 reduced the size of cell units by a tenth to a quarter (154).

87 Conclusion

A B

-Hydrolase domain Side opening

Propeller domain

Tetramer Propeller opening interphase C

Ile287 Gln Tyr Ser Thr Val Ser Ala Asn279

Asn Ala Ser Val Thr Ser Tyr Gln Ile Plasma Membrane 279 287

Figure 60: Schematic presentation of crystal structure of native porcine DP 4 from kidney cortex, depicting the residues important for tetramerisation (pdb: 1ORV). A, Soluble DP 4 forms a 222 symmetric assembly as a dimer of dimers. Arrows indicate respective propeller and side opening. Each subunit consists of a catalytic /-hydrolase (green shades) and -propeller domain as indicated. The -propeller is again subdivided into a carbohydrate rich sub-domain (red shades) and cystein-rich sub-domain (blue shades). Carbohydrates and disulfite bonds at the - propeller tunnel are indicated in orange and yellow, respectively. B, Tetramerisation interphase of -propepller domains of subunit A and subunit C as viewed from the bottom, illustrating the involvement of the strands Asn279-Gln286 to form an antiparallel sheet. C, residues of strands Asn279-Gln286 of subunits A and C forming an antiparallel sheet, responsible for tetramerisation and stabilised by H-bonds (gray dots). The transmembrane helices and their orientation to the membrane were drawn in to illustrate how tetramerization of DP 4 may mediate cell-cell contacts (11). Figures were prepared with Pymol, using pdb: 1ORV (13).

There appeared to be a direct relationship between the size of cell units and resolution due to the compactness of the molecules. Therefore, a 1.8 Å resolution was obtained with porcine (13), 1.9 - 2.1 Å with deglycosylated human recombinant expressed in Pichia pastoris (153;154) and 2.1 - 2.6 Å with the crystal structures of glycosylated human recombinant DP 4 expressed in insect cells (12;14) (Table 31). Thus, it becomes apparent that glycosylation influenced the sizes of cell unit and resolution due to steric hinderance. Although glycosylated DP 4 exhibited more crystal contacts between DP 4 dimers, most of them were due to the oligosaccharides and therefore were drastically reduced upon exclusion of the sugars. Deglycolylated DP 4 in turn, had mainly protein based crystal contacts (153). Due to these differences one can conclude that DP 4 exhibit great structural flexibility on its surface, mainly influenced by its glycosylation, the flexibility of blade I and the extended arm of blade IV (12;153). Having a crystal structure with a high resolution of 1.8 Å, allows insight in the mechanism of substrate hydrolysis. Furthermore, superimposing the active centres of human recombinant DP 4 (12) and native porcine (13) revealed great similarities differing only in position 562

88 Conclusion which is occupied by serine in pig instead of asparagine in human (Figure 61) (620). Thus, the crystal structure obtained from native porcine DP 4 can serve as a good model for inhibitor design and the structure with the bound inhibitor p-Jodo-Phe-Pyr-CN (Ki = 25.1 ± 0.9 nM) can give insight to mechanism of catalysis as seen in Figure 62 (13).

Table 31: Comparison of cell units obtained from crystal structures of native porcine and various human recombinant DP 4’s. Co-ordinates of Unit cell Crystal Structure Space Volume cell R- cell unit (Å) angle 3 Res. Ref. group unit (Å ) factor pdb Feat. # Glyco Assem. a b c α β γ P, nat. 1ORV 6 mat com. Tetramer P 1 62.0 118.2 133.6 113 95 91 978,835 0.22 1.8 (13) iso. H, Pp a 1PFQ 2 s Dimer P 2 2 2 71.0 118.1 184.8 90 90 90 1,547,889 0.24 1.9 (153) Deglyc 1 1 1 H,Pp 1NU6 4 s Dimer P 2 2 2 65.5 68.2 419.3 90 90 90 1,873,055 0.22 2.1 (154) Deglyc 1 1 1 H, Ic 1N1M 7 im Dimer P 2 2 2 119.0 123.2 130.7 90 90 90 1,916,167 0.21 2.5 (12) Hi5 1 1 1 H, Ic (605) 1J2E 5 im Dimer P 2 2 2 118.0 125.9 136.8 90 90 90 2,033,934 0.25 2.6 SF29 1 1 1 (606) H, Ic 1R9M 9 im Dimer P 2 2 2 121.8 124.1 144.5 90 90 90 2,184,172 0.25 2.1 (14) Hi5 1 1 1 a, authors mentioned structural alteration between their crystal and that by Rasmussen et al., due to crystal contacts and compactness within the cell unit. ; Feat. = features; P = porcine; nat. iso. = native isoform; H = human; Pp = Pichia pastoris; Deglyc = deglycosylated; Glyco. = glycosylation; Ic = Insect cell; mat com. = mature complex; s = single sugar; im = imature; Assem. = assembly; Res. = resolution; Ref. = reference

Figure 61: Superimposition of the space around the catalytic active serine 630 of human DP 4 (orange) and porcine DP 4 (green). Amino acids of the active centre were used for the comparison of both structures. For the calculation of the RMS value (root mean square), the amino acids of the human DP 4 (pdb: 1N1M) were used as the target molecule. Afterwards, each atom (without hydrogen atoms) of the porcine DP 4 (pdb: 1ORV) was put on the atom of the target molecule and a good match was found, (RMS-value: 0.34 Å). This value is in the range of the X-ray determination accuracy. The catalytic triad is shown in red. [Taken from Bär et al., 2003, (620)].

The small hydrophobic S1 pocket, formed by Tyr666, Tyr662, Val711, Val656, Trp659 and Tyr631, gives a good fit for proline as well as other small and uncharged amino acids such as alanine, serine and glycine at P1 position. Stabilisation of the pyrrolidine is achieved by ring stacking to Tyr662 in a parallel fashion and orthogonally to Tyr666. The active Ser630 is covalently bound to the inhibitor by its hydroxyl group, which is also hydrogen bonded to the protonated nitrogen of the imidazole of His740. Latter, in turn is stabilised by a hydrogen bond of the carboxylate oxygen of Asp708. Binding is further enhanced by the formation of a hydrogen bond between the nitrogen of the P1 imidate and Oη atom of Tyr547, as well as the

89 Conclusion

peptide nitrogen of Tyr631. The free N-terminus of P2 is anchored by a twin single salt bridge with Glu205-Glu206, thus defining DP 4 as dipeptidyl aminopeptidase. In addition, Glu205 forms a hydrogen and salt bridge with Arg125. Arg125 and Nδ2 of Asp710 in turn, stabilise the P2- carbonyl oxygen atom, respectively (13;731;732). In contrast to the small S1 pocket, the S2 pocket is large, allowing a great variety of amino acids at P2 and inhibitors with large bulky groups. The porcine crystal structure with the inhibitor represents the acylenzyme in the catalytic mechanism, since the inhibitor acts as a dipeptide-mimic and is covalently bound to the enzyme (13;731;732).

Tyr-631 A B CF COO- Tyr-547 3 Tyr- I NH + 666 3 N

Tyr-662 O CN Ser-630 Glu-206 His-740

Glu-205 Asn-710 Arg-125 Asp-708

Figure 62: Binding of reversible tight binding inhibitor p-Jodo-Phe-Pyr-CN trifluoroacetate (Ki = 25.1 ± 0.9 nM) to the active site (Ser-630) of porcine DP 4 (13). A, presentation of active site showing covalently binding of Ser-630 to the inhibitor, anchoring of free N-terminus of the inhibitor by Glu-205 and Glu-206 and the P2 oxyanion trap by Arg-125 and Asn-710. Drawn with Pymol, using pdb: 1ORW. B, chemical structure of inhibitor p-Jodo-Phe-Pyr-CN trifluoroacetate.

A similar structure was obtained with an iodinated derivative of NVP-DPP 728, also a pyrrolidine-2-nitrile with a bulky pyridine side chain. Due to an ethylenediamine spacer, the inhibitor lacks the interaction with Glu206, but the bulky side chain stabilises the inhibitor by a hydrogen bond with the hydroxyl group of Ser209 to the nitrogen of the pyridine and ring stacking between the planar 5-iodo-pyridine and aromatic ring of Phe357 (153). Two additional crystal structures with tetrahedral intermediates have additionally been elucidated, one with the tripeptide Ile-Pro-Ile also called Diprotin A (154), and another one with the decapeptide of NP Y (14). They displayed similar interactions as shown in Figure 62, however Arg125 also formed a hydrogen bond to the carbonyl group of P1’ and the peptides contained an intramolecular hydrogen bond between the backbone NH group of P1’ and the

P2 carbonyl oxygen (14;154;731;732). The NP Y substrate had an additional hydrogen bond between the P2’ side chain of Lys and the hydroxy group of Tyr752. Since the P’ residues point to side opening and only six of the ten amino acids of the substrate are ordered in the crystal structure, one can conclude that the substrate has utilised the side opening (14). In both crystal structures the tetrahedral intermediate was stabilised in the oxyanion hole by the hydroxyl group of Tyr547 and the peptide NH group of Tyr631 (14;154;731;732). The binding of Val-Pyrrolidide to human DP 4 represented the enzyme in the ground state, thus indicating possible substrate binding. The inhibitor in this structure lacked the covalent bond to Ser630 and the hydrogen bonds of Tyr547 and Tyr631 (12). Using site-directed mutagenesis, the residues Ser630, Asp708, His740, Tyr547, Glu205 and Glu206 have shown to be essential for the catalytic activity (44;733-737). Furthermore, studies in our own laboratory by Kathrin Gans also demonstrated the importance of Asn710 for catalysis, whereas Arg125 influenced the hydrolysis of natural substrates, containing alanine or serine at P1 position. The residues

Tyr631 and Trp629 in turn, did not appear to be crucial for enzyme activity (738). Interestingly,

90 Conclusion the crystal structure of bacterial DAP 4 from Stenotrophomonas maltophilia lacked the residues Arg125, Tyr457 and Tyr631 surrounding the active site. In fact, Asn611, corresponding to

Tyr631, resulted in an increase preference towards hydroxyproline compared to proline as proven by site-directed mutagenesis (607). Recently, the crystal structure of FAP has been elucidated, revealing a similar structural topology compared to DP 4 with respect to –hydrolase, propeller, extended arm, dimer interphase, propeller tunnel and side opening (98). However, the most significant difference was an exchange of Ala657 in FAP instead of Asp663, causing the substrate binding site to be less negatively charged as in DP 4. Furthermore, the anchoring of the free N-terminus by

Glu203 and Glu204 in FAP is reduced, since the side chain of Glu204 forms only one salt bridge with the positively charged N-terminus, whereas the other acidic oxygen is buried in a hydrophobic pocket contributed by Trp199, Val200 and Ala657. This in turn, explains why FAP is a better endopeptidase rather than dipeptidyl aminopeptidase (Appendix 1). Mutating Ala657 to Asp657 in FAP and Asp663 to Ala663 in DP 4 converted FAP to a better dipeptidyl aminopeptidase and DP 4 to a endopeptidase, respectively (98). A recent structure of DPL-1 in turn, showed substantial differences of the residues at the active site, explaining its lack of activity (159). Table 32 outlines the important amino acid residues of the active site that are either present or absent in the members of the DP 4-gene family, as determined by sequence alignment with respect to DP 4. It illustrates that except for Arg125 being absent in DP 8 and DP 9 as well as Asp663 missing in FAP, all the other residues of the active site are present in the members with DP 4 activity. DPL-1 and DPL-2 in turn, lack the residues forming an electrostatic sink to stabilise the P2 carbonyl oxygen, the active serine and the most important residue responsible for the oxyanion hole.

Table 32: Important residues of the active site present/absent in the members of the DP 4- gene family as determined by sequence alignment with respect to DP 4. Propeller domain -Hydrolase domain

ES AN AN OH AS AS AS/CT AS/OH AS HP AN HP CT ES CT

Enzyme Accession # R125 E205 E206 Y457 G628 W629 S630 Y631 G632 Y662 D663 Y666 D708 N710 H740 DP 4 M80536                sDAP 4 P95782             FAP U09278               DP 8 AF221634               DP 9 AF452109               DPL-1 M96859         DPL-2 AY172659         Amino acid numbering is based on DP 4 sequence. ES = electrostatic sink, AN = anchoring of N-terminus, OH = oxyanion hole, AS = active site, CT = catalytic triad, HP = hydrophobic pocket; sDAP = bacterial DP 4 from Stenotrophomonas maltophilia.

So far, most of the inhibitors applied in vivo bind at the active site of the DP 4 as summarised in Appendix 74. The majority of them are competitive inhibitors that act as product analogues lacking the carbonyl group of proline. Nonpeptide DP 4 inhibitors include 7-benzyl-1,3- dimethyl-8-(1-piperazinyl)xanthine and 6,8-dimethoxy-1-aminomethyl-4-isoquinoline with a competitive and mixed type of inhibition mechanism, respectively (731;732;739). TMC-2A and TSL-225 from Aspergillus orizae are the only uncompetitive inhibitiors known up to now with a Ki in the low micromolar range (323). Appendix 74 also indicates that diabetes is the target of most DP 4 inhibitor design with the aim of potential drug development (406- 409;439;440;444-446;731;732;740) and five of them (Sitagliptin, Januvia®, Merck and Co In.; Saxagliptin, Onglyza®, Bristol-Myers Squibb, Linagliptin, Ondero®, Böhringer Ingelheim, Vildagliptin Galvus®, Novartis and Takeda Pharmaceuticals Alogliptin,Nesina® ) are already on the market.

91 Conclusion

4.2 Distribution of DP 4-like Enzymes in Porcine and Canine Tissue Sections

So far, it could be demonstrated in the present study and by others that the DP 4-like enzymes co-localise at mRNA levels in most tissues, using RT-PCR (Appendices 58-62 + 64), Northern blot (Table 4) and in situ hybridisation, respectively (39;44;45;49;85;86;88;90;131;134;171). However, the ubiquitous expression of FAP in healthy tissues could be illustrated for the first time (Appendices 58-62 + 64) and correlated to in silico findings (131;634). Most of the mRNA of DP 8 and DP 9 were located to lymphocytes and epithelial cells in liver, gastrointestinal tract, lymph node, spleen and lung as determined by in situ hybridisation (134). High levels of DP 8 and DP 9 mRNA’s were particularly found in baboon and mice testis at various stages of spermatogenesis (134). The expression of DP 4 generally appeared to be regulated at mRNA levels and was highly specific to organ, cell type and differentiation status (2). In lung, DP 4 was reported to be regulated post-transcriptionally due to differences between expression at mRNA and protein levels (39;49) and such post-transcriptional regulation was also described for FAP (92). Yet, upon induction of asthma only DP 8 and DP 9 mRNA’s were found to be increased in rat lungs (28). Furthermore, expression of DP 4 and FAP were shown to be up-regulated by cytokines (51-54;100;741-746). DP 4, DP 8 and DP 9, in turn, were confirmed to be up- regulated in activated lymphocytes and macrophages (2;85;136;140;143;190;200;207;309;744- 748). In contrast, high expression of DP 2 is associated with quiescent lymphocytes, which is regulated by the transcription factors KLF2 and TOB1 and a down-regulation of DP 2 expression causes resting lymphocytes to exit G0 phase (176;180;182-184;749-751). Increased expression of DP 4 and FAP are associated with various types of cancers, whereas DP 9 appears to be up-regulated in human testical tumours (105;108-120;134;189;752-756). In addition, higher levels of DP 8 and DP 9 were found in human meningiomas (757;758). However, to distinguish between the various DP 4-like enzymes at protein levels is more challenging as only few selective antibodies have recently been available on the market and only a small number of selective inhibitors against DP 4, DP 8/9 and DP 2 have been developed up to now (28;139;141;142;146;591;759-764). Thus, the contribution of the various DP 4-like enzymes to the overall DP 4-like activity has only lately started to be unravelled, making use of selective inhibitors, DP 4-/- knock-out mice, DP 4-deficient Fischer rats, siRNA and selective assay conditions (28;132-136;139;140;143;148;452). In the present study we could show that DP 4 contributes to most of the DP 4-like activity in all porcine and canine tissues being investigated except for canine pancreas (Figure 29), and similar results were recently obtained from comprehensive activity studies on several tissues from wildtype and DP 4 knock-out mice as well as histochemical studies in lungs from wildtype and DP 4-deficient F344 rats (28;131;134). Nevertheless, DP 8/9 activity has been readily detected in human leucocytes and it was even exceeding the activity of DP 4 in rat and murine brains as well as bovine and murine testis (134-140). In fact, natural short form of DP 9 could recently be isolated and characterised from bovine testis (137). Although the physiological roles of DP 8 and DP 9 have not been elucidated in brain and testis yet, recent data suggest immunological functions in T-cell proliferation and antigen presentation which require the activities of DP 9 and/or DP 8 (132;140- 143;189;190;309;765). Thus, controversal results regarding DP 4 activity and T-cell activation as well as T-cell proliferation are now attributed to mainly DP 8 and/or DP 9, while the co- stimilatory functions of DP 4 / CD26 appear to involve binding partners such as ADA, CD45, 92 Conclusion

M6P/IGFIIR and Caveolin-1 as summarised and illustrated in Appendices 75 and 76, respectively (8;54;142;143;196;198-200;207;255-257;259-261;309;765-772). The first generation of DP 4 inhibitors were developed prior to the discovery of DP 8 and DP 9 and these include P32/98 (Ile-Thia), Lys[Z(NO2)]-pyrrolidine, Lys[Z(NO2)]-thiazolidide,

Lys[Z(NO2)]-piperidide, LAF-237 (vildagliptin), NVP-DP728, L-Pro-L-boroPro, Pro-Pro-diphenyl phosphonate esters, aminoacyl-pyrrolidine-2-nitriles, aminoacylpyrrolidides and aminoacyl- thiazolidides (2;85;92;739;773). At this stage, data concerning selectivity were only available on DP 4 and DP 2 as outlined in Appendix 77. Yet, toxicological studies of the first generation inhibitor P32/98 resulted in high toxicity with bloody diarrhea, emesis and tenesmus in dogs and alopecia, thrombocytopenia, anemia, enlarged spleen, multiple histological pathologies and mortality in rats. Investigating the cytotoxic effects, by developing selective inhibitors, revealed inhibition of DP 8 and DP 9 to be responsible for the side-effects (141;142;774). Therefore, the second generation of anti-diabetic DP 4 inhibitors focused on the selectivity of the various DP 4- like enzymes as summarised in Appendix 78 (141;142;732;760-765;774-777). On the other hand, the requirement of selectivity is currently controversially debated, as some of the selective inhibitors against DP 8/9 were unable to enter the cell, suggesting that the cytotoxic effects observed were not due to inhibition of these cytosolic enzymes (145-148). Given that DP 4, FAP, DP 8, DP 9 and even DP 2 have their highest sequence and structure similarities at the catalytic domain (92), uncompetitive inhibition at the propeller domain may be more specific for a particular DP 4-like enzyme. A trapped sulfate in the porcine crystal structure points to a oxyanion pocket at the propeller domain, formed by the amide nitrogens of Glu-361 and Ile-407 as well as by N2 of His-363. This site could be a starting point for rational design of uncompetitive DP 4 inhibitors as potential drug candidates to block the propeller opening (13).

Molecular modelling of DP 8 and DP 9 in turn, revealed a P2-loop at the propeller domain, containing F357 and R358, that seems to be unique to DP 8 and DP 9 and is suggested to influence substrate and inhibitor binding to the P2-pocket (152). Recently, the structure of DP 2 was deposited in the protein data bank as pdb 3JYH, revealing a /-hydrolase domain as well as a novel SKS domain, comprised of 5 -helices arranged in a helix bundle fold, capping the active site. An insertion from the SKS domain to the acive site results in steric hinderance of larger substrates and contains Asp334 for anchoring N-terminus of the substrate (778). In addition to selectivity, the second generation of anti-diabetic DP 4 inhibitors also were designed to have a high potency for long-acting, so that only a once a day application is required. The short acting PSN-9301 appeared to be the only exception (779). While a once a daily application is convenient from a patient point of view, long-acting inhibitors against DP 4 are most likely to interfere with other natural substrates of DP 4 and their associated physiological functions, such as SP in rhinosinusitis and angioedema, SDF- in arthritis and NPY/PYY in blood pressure as recently suggested (316;715;716;780-784). In fact, headache and nasopharyngitis are frequently reported side-effect of gliptin treatments (407;785-789). Recently, the FDA revised its prescribing information to include case reports on acute pancreatitis in patients using Sitagliptin (790). Contradicting case reports have been given, regarding Sitagliptin and psoriasis as it was observed to trigger psoriasis on the one hand, whereas on the other hand it was claimed to cure it (791;792). Interestingly, investigating NPY hydrolysis in serum and blood in the present study, a novel C-terminal truncation by an ACE-like 93 Conclusion enzyme could be detected, which increased in intensity upon DP 4 inhibition. The latter finding suggests a potential interaction within current drug treatments using anti-diabetic DP 4 inhibitors and anti-hypertensive ACE inhibitors in combination. A suspected increase of vasocontrictive

NPY1-36 after treatment with anti-diabetic DP4 inhibitor may be compensated by C-terminal inactivation of NPY mediated by ACE, but fails if ACE is also blocked. This hypothesis has been substantiated by physiological animal studies, using SHR and WKY, respectively (713). Intriguingly, treating SHR and WKY rats with either P32/98 alone or in combination with captopril, only SHR developed hypertension after combined therapy, thus suggesting a genetic background. However, Y1-R antagonists ablated the hypertensive effects of combined treatment with DP 4- and ACE-inhibitors, supporting the involvement of either NPY or PYY (713). This is of pharmacological significance as hypertension is a frequent co-morbidity with diabetes and in very recent reports, the development of hypertension was associated with combined application of anti-diabetic Sitagliptin and anti-hypertensive Enalapril in patients suffering from metabolic syndrome (715;716). Since the anti-diabetic effects of DP 4 inhibitors are only required upon a glucose challenge i.e. just before a meal, the present data suggests the need for the development of a third generation of short-acting anti-diabetic DP 4 inhibitors to avoid potential side-effects and keep possible drug interactions to a minimum.

4.3 Analysis of DP 4-like Enzymes in Porcine Pancreas Nonetheless, while the involvement of DP 4 in glucose metabolism and diabetes is now generally acknowledged as a fact with GLP-1 being the major substrate, little is known about the role(s) of the DP 4 (-likes enzymes) in the pancreas, particularily in the islets of Langerhans. Yet, secreted or membrane-bound DP 4 (-like enzyme) from islets of Langerhans would not only influence the receptor binding of endocrinal DP 4 substrates GLP-1, GIP and GRF, but also the paracrine DP 4 substrates glucagon, oxytonmodulin, GLP-1, PYY and IGF-1 as well as the neuropeptide DP 4 substrates VIP, PACAP, GRP, GHRH and NPY, respectively (Tables 5, 11 and 12). While GLP-1, GIP, VIP, PACAP and GRP are insulinotropic, glucagon, NPY, PYY, IGF-1 and PP inhibit insulin secretion by binding to their respective receptors at the -cell as described in Table 12 and Appendix 79 (2). Therefore, secreted or membrane-bound DP 4 (-like enzymes) would be involved in the endocrinal, paracrinal and neuronal regulation of insulin secretion from the -cells of islets of Langerhans as shown in Appendix 79 (2;233;455-462;793- 795). However, as already shown in Figure 6, DP 8 and DP 9 are cytosolic and for this reason they have no physiological access to the substrates mentioned above, though they were shown to hydrolyse GLP-1 and NPY in vitro (92;94;127;189;190;210). DP 2 in turn, is described to be located in secretory vesicles, having a pH optimum of 5.5, yet, it is not able to cleave any of the above DP 4 substrates (135;163;175;179). Thus, DP 4 would be the only DP 4-like enzyme being involved in such endocrine, paracrine or neuronal regulation of insulin secretion. Generally, the data in the current study revealed the presence of all DP 4-like members in the pancreas. Results obtained from activity studies as well as immunological and histochemical analysis give strong evidence that most of the DP 4 (-like enzymes) and its/their expression originate from the exocrine tissue (Figures 31, 35 and 38), followed by endothelial capillaries surrounding islets of Langerhans (Figure 38E, F and J). However, sub-cellular fractionation, activity/inhibition studies, RT-PCR analysis and histochemical studies in turn, suggest the

94 Conclusion presence of more than one DP 4-like enzyme in and on cells of islets of Langerhans (Figures 33 - 36). Furthermore, separation of cytosolic and membrane fractions, enrichment of granules, lysosomes and endosomes as well as histochemical activity staining indicate different sub- cellular distribution of DP 4 (-like activity enzymes) in cells of islets of Langerhans (Table 29 and Figure 36). These results are in agreement with data obtained by histochemical and immunological investigations of DP 4 activity and expression, although co-localisation of DP 4 with glucagon could not be confirmed by immunohistochemical analysis (Figure 37) (17;18;134;447;448;648;662). A possible explanation of latter discrepancy could be different methods of sacrificing the pig. In the present method the pigs were bolted and therefore underwent extreme stress prior to exodus, resulting in the release of glucagon due to stimulation by norepinephrine. Implementing anaesthetics for sacrifying in turn, would inhibit glucagon secretion due to stimulation of the autonomic parasympathetic nervous system (Appendix 79). Unfortunately, both reports on co-localisation of DP 4 with glucagon in granules or -cells did not specify the exact method of sacrification (447;448). This in turn, implies a regulatory secretion of DP 4 and/or glucagon, which is supported by preliminary results of perfusion experiments resulting in the secretion of glucagon and DP 4 into the perfusate after splanchic stimulation (Figure 39). It has been argued that DP 4 was expressed and sorted to the granules of islet -cells, where it was found to either co-localise with glucagon (448) or be present in lucent granules predominantly distinct from glucagon localisation (447) as depicted in Appendices 80 and 81 respectively. Yet, no alternative splicing has been found with DP 4 mRNA, making granular sorting of DP 4 highly unlikely (39). Furthermore, glucagon was proven to be a DP 4 substrate in vitro and in vivo (228), though the low granular pH would slow down granular cleavage of glucagon by DP 4 (448;796). Alternatively, DP 4 could also bind to glucagon and Glu-R and subsequently internalise, with the extra-cellular structure of DP 4 pointing intra-granularly. This would explain the DP 4 activity of the membrane fraction of isolated granules/endosomes as reported in Table 29, whereas soluble DP 4 of isolated granules/endosomes could result from autolysis due to acidic pH (796;797). Internalisation of DP 4 with binding partners has been reported with mannose-6-phosphate/insulin-like growth factor II receptor (M6PR/IGFRII), CXCR4 receptor as well as caveolin-1 and such process in the islet -cells would prevent local excess of glucagon (196;198;255;798). Nevertheless, although DP 4 has been described in various sub-cellular compartments as summarised in Appendix 82, the granules shown in the electron microscopy staining by Poulsen et al., 1993 and Grondin et al., 1999 (Appendix 80) were spherical in shape, ± 250 nm in size, found close to the membrane and in case of Poulsen et al., 1993 had a electron-dense core (447;448). These characteristics point either to electron- dense secretory granules (80-350 nm) or lucent secretory lysosomes (100-1250 nm) rather than internalised flask-shape caveolae (50-100 nm), clathrin-coated endocytosis (100-150 nm), tubulo-vesicular endosomes (500-2000 nm), grape-like heteromorphic multi-caveolar complexes (500-2000 nm) or transcytotic vesicles (50-150 nm) (75;262;767;798-845). However, in order to draw final conclusions, the co-localisation of specific organelle markers together with DP 4 have to be investigated as described in Appendix 82. Furthermore, secretion or internalisation of DP 4 in pancreatic islets could also be examined by in situ RT-PCR and subsequent immunohistochemical staining with the cell-specific markers glucagon, insulin, somatostatin and 95 Conclusion pancreatic polypeptide for , ,  and pp-cells, respectively (Appendices 81 and 83, (846;847)). Positive mRNA staining of DP 4 and islet cell-marker would favour the secretion hypothesis, whereas only immunohistochemical staining would support the internalisation theory. Nonetheless, preliminary results from perfusion studies in rats also substantiate the secretion of DP 4 and glucagon, with the more viscous DP 4 being detected in later fractions (Figure 39). Thus, in addition to its known endocrinal regulatory functions, DP 4 appears to be also involved in the neuronal and paracrinal control of insulin secretion (Appendix 79). Yet, such paracrine regulatory functions would also be dependent on the microcirculation of a pancreatic islet, which has been reported to differ substantially in the various species (675). For example, rat and dog were shown to contain a vascular plexus that connect the various different cell types of an islet. However, there appeared to be a species dependent order of such connection like     - cells in dog and     -cells in rat (675). Human and pigs do not have such vascular plexus, but a vascular corrosive cast (Appendix 83). Since the prolonged half-life of the insulinotropic peptides/neuropeptides due to DP 4 inhibition exert their action via the blood circulation, such species dependent micro-circulation may influence pharmacokinetics and toxicolgy of DP 4 inhibitors. Finally, the above data implies that soluble DP 4 in the blood circulation is derived from secretion via secretory lysosomes as found in haemopoietic leucocytes such as NK- and Tc-cells rather than from shedding off the plasma membrane , as already reported in studies of organelle proteomics (345;447;448;723;845;848-850). Intriguingly, although DP 2 does not cleave any of the DP 4 substrates mentioned above, it is located in secretory vesicles with a similar density to lysosomes and its low pH optimum would favour the intra-granular pH of 5.5 (172;173;175). Furthermore, the presence of DP 2 in -cells of islets of Langerhans has been well documented by histochemical and immunological staining and recently DP 2-/- knock-out mice revealed a phenotype opposite to DP 4, resulting in insulin resistance, hyperinsulaemia, impaired glucose tolerance, increased liver steatosis and visceral obesity due to higher numbers of adipocytes as summarised in Table 7 (17;171;186;589). In the present study we could confirm DP 2 in islets of Langerhans (Figure 37 A), but we also detected positive staining of possible neurons surrounding the islets as illustrated in Figure 37B-D. In addition, differences in specific activities in cytosolic fractions with and devoid of granules, lysosomes and endosomes as well as isolated granules, give strong evidence of DP 2 being found in granules (Table 29). DP 2 is known to hydrolyse predominantly tripeptides and thus, the naturally occurring IGF-1 fragment IGF-1(1-3), comprised of Gly-Pro-Glu (GPE) may be a potential DP 2 substrate in association with the diabetes pathology (851-853). In contrast to IGF-1 and truncated IGF-1 (-3N:IGF-1), GPE does not bind to IGF-IR to inhibit insulin secretion, but instead has been described to stimulate the release of acetylcholine, which in turn promotes the secretion of insulin via parasympathetic neurons (462;795;853-857). Thus, IGF-1 and GPE have opposing functions regarding insulin secretion. However, as the granules of -cells are known to contain high concentrations of zinc (> 1 mM), which inhibits DP 2 activity, degradation of GPE by DP 2 is most likely to occur extra-cellularly after secretion to ablate stimulation of neuronal acetylcholine secretion (851;858-860). This hypothesis would explain the -/- hyperinsulaemia in DP 2 knock-out mice (186). Nevertheless, whether GPE is a substrate of DP 2 and its involvement in glucose homeostasis as well as the pathology of diabetes still needs to be proven. 96 Conclusion

4.4 Identification of DP 4 as Pharmaceutical Target for Psychiatric Diseases In addition to the well-established role of DP 4 in the pathology of diabetes, DP 4-/- k.o. mice, DP 4-deficient rats and inhibition studies have given strong evidence of DP 4 being involved in stress-related behaviour and therefore have been investigated for a pharmaceutical target as summarised in Table 13. Intriguingly, the various animal models and tests outlined in Table 13 resulted in different psychopharmacological targets such as depression, anxiety and psychosis/schizophrenia. A reason for this phenomenon could be the diathesis-stress model, which entails that depending on the genetic disposition and in presence of chronic or acute stress, a person/rat develops depression, anxiety or schizophrenia as illustrated in Figure 63 (861-864). However, in order to fully understand the mechanisms behind DP 4 and stress- related diseases, several basic biochemical investigations had to be performed, being part of the present study and including target substrate, involvement of additional DP 4-like enzymes, target organ, inhibitor profile and target disease.

Figure 63: Schematic diagram of the Diathesis-Stress Model. Under an existing genetic disposition and in combination with chronic/acute stress, a psychiatric disorder of the hypothalamic- pituitary-adrenal axis (HPA) or neuro-sympathico-axis (NSA) will be developed, resulting either in depression, anxiety or psychosis/schizophrenia.

Although DP 2 and DP 8/9 exhibited substantial higher activities in neuronal and brain tissues compared to DP 4 (Figures 40, 42 and 43), they do not have physiological access to vesicular or secreted neuropeptides such as NPY either due to their sub-cellular localisation such as the cytosolic enzymes DP 8/9 or they lack the catalytic efficiency like DP 2 and to a lesser extent FAP of cleaving any of the DP 4 substrates (Figures 6 and 45, Appendix 84) (94;127;134;135;147;175;179;210). Furthermore, even though soluble sDP 4 and sFAP are present in the blood circulation, sDP 4 is the favoured candidate because of its higher catalytic efficiency and activity i.e. concentration, in blood (94-96;123;124;135;678;718;865-867). Bearing this in mind and together with the in vivo animal data in Table 13, DP 4 becomes therefore the primary focus of investigations for a psychiatric target. For this reason the high specific activity in the circumventricular organs shown in Figure 44 is of great significance, particularly regarding the median eminence and area postrema, as these structures lack the BBB and hence present the interphase between the neuro-endocrino-immuno systems mediated by the two stress axis hypothalamic-pituitary-adrenal axis (HPA-axis) and the neuro- sympathico-axis (NSA) as illustrated in Figure 64 and in more details in Appendix 85.

97 Conclusion

Figure 64: Psycho-neuro-endocrino-immunological role of DP 4 at the CVO’s median eminence (EM) and area postrema (AP). EM and AP form the interphase between the neuronal, endocrinal and immunological systems, involving the two stress axis hypothalamic-pituitary-adrenal (HPA) and the neuro-sympathico-axis (NSA). Activation of HPA and NSA results in the release of stress hormones cortisol and epinephrine, cytokines and NPY, which in turn interact with the brain via EM and AP and the lymph organs such as the spleen (562;565-567;569;571;678;868-874).

Moreover, the presence of DP 4 at the median eminence could also be detected by immunohistochemistry, where it was mainly found at the outer zone of the median eminence

(MEE) close to the fenestrated capillaries corresponding to -tanycytes as shown in Figure 64 and Appendices 86 + 87 (565-567;569;869-874). Figure 65D demonstrates the unique features of tanycytes in general, being able to pick up/pass on neuroendocrine signals from either the blood circulation, perivascular space (PVS) or CSF due to the lack of BBB, PVS-BB or CSF-BB, respectively (565-567;569;869-876). -Tanycytes are found at the lateral (1) and medial (2) regions of the outer zone of median eminence, thereby bridging CSF with portal blood (Appendix 86). They are linked to the HPA-axis, joined together via zona adherens involving - catenin and -cadherins, able to transcytose and endocytose via caveolae and clathrin- dependent endocytosis and are innervated by peptidergic and aminergic neurons (869).

However, whether DP 4 is expressed at the terminal ends of 1,2-tanycytes, on the membrane of endothelial cells of the capillaries in contact with 1,2-tanycytes or whether 1,2-tanycytes are able to pick up sDP 4 from the blood circulation by binding to caveolin-1 still remains to be researched (257;262;877). Co-localisation of DP 4 with NPY could be detected at the lining of the third ventricle, corresponding to either ependymal cells or ventricular cell bodies of 1,2- tanycytes that project to the ventromedial nucleus (1) or nucleus arcuate (2) of the hypothalamus (Appendices 86 and 87) (869). Contrary to -tanycytes, 12-tanycytes bridge CSF to the respective hypothalamic nuclei, do not internalise by means of caveolin-1 or clathrin- dependent endocytosis, are not directly linked to the HPA-axis, contain zona adherens only at the ventricular pole and only 1-tanycytes can transcytose, whereas 2-tanycytes have neurogenic properties as summarised in Appendix 87. Less is known about the properties of tanycytes in the area postrema (AP), however, the AP is comprised of a dorsal ventricular mantle zone, a central zone, and a ventral junctional zone, containing the funiculus separans, i.e. a layer of tanycytes that function as a BBB separating AP from NTS (Appendices 88 and 89 A). The central zone in turn, contains the fenestrated capillaries and neurons, expressing a great number of receptors and projecting to various brain 98 Conclusion areas as illustrated in Appendix 89B and summarised in Appendix 88. Tanycytes at the mantle zone link the 4th ventricle with fenestrated capillaries and/or neurons at the central zone (878;879). In contrast to NTS, capillaries of the AP express the membrane transport system caveolin-1, caveolin-2 and P-glycoprotein (880). Thus, although EM and AP lack BBB, they tightly control transport/signals from blood/CSF to CNS and vice versa via tanycytes, membrane transporter systems and receptors (Appendices 86-89 and Figure 65).

Figure 65: Expression of DP 4 and NPY at the median eminence. A, immunohistochemical staining of DP 4 (blue) and NPY (purple) at the median eminence, showing mainly staining of

terminal ends of 1,2-tanycytes at the portal blood and/or endothelial cells of capillaries (blue arrow) and some staining of basal processes of 1,2-tanycytes of the inner (MEI) and outer zone (MEE) of median eminence (black arrow). Co-localisation of DP 4 and NPY is found on

lining of third ventricle corresponding to cell-bodies of 1,2-tanycytes (purple arrow head). B, schematic presentation of the median eminence, showing a -tanycyte bridging the CSF with portal blood. C, schematic presentation of blood brain barrier (BBB), illustrating the tight junction of overlapping endothelial cells, the tight adherens of astrocytes and pericytes to the capillary. D, schematic presentation of interaction between CVO and a capillary, demonstrating the fenestrated endothelial cells, loose astrocytes, large perivascular space and terminal ends of tanycytes picking up/passing on neuroendocrine signals to and from the periphery such as blood and perivascular space. BBB, blood brain barrier; CVO, circumventricular organ; EM, median eminence; MEE, exterior zone of median eminence; MEI, interior zone of median eminence; ArcL, lateral nucleus arcuate; ArcM, medial nucleus arcuate (869;871). Interestingly, these results explain the different in vivo data summarised in Table 13, in which oral application was only effective in treating psychosis in SHR rats, whereas for anxiolytic effects in adult F344/Crl(Por) rats, the inhibitor had to be i.c.v. injected. However, in young post- natal 10-day-old rats, lacking BBB and fully developed tanycytes, anxiolytic effects could already be observed when P32/98 was i.p. injected. Thus, two conclusions can be drawn from the above. First of all, there appears to be different pharmacological mechanisms behind these two disease targets, implying that for targeting psychosis, the DP 4-selective inhibitor does not have to cross the BBB, whereas for anxiety the potential drug candidate requires a high logBB. 99 Conclusion

A reason for this apparent discrepancy could be the different synaptic plasticity and projections involved in the diseases as hypothesised in Appendix 90. Secondly, with respect to CNS, the CVO’s, particular the median eminence and area postrema, are the most likely target tissues for DP 4-selective inhibitors against psychiatric diseases. However, since CVO’s also form a link between the CNS and the periphery, involving the two stress axis HPA and NSA, soluble DP 4 was additionally investigated in the blood circulation as well in CSF. While in human CSF, no DP 4 activity could be detected and NPY was metabolised only by CTSD (Figure 53), the DP 4-truncated metabolite NPY3-36 was the major metabolite found ex vivo in the blood circulation as well as in the serum (Figures 54 and 56). Novel DP 4 substrate GALP was identified, also showing feasible ex vivo DP 4-truncation by blood, whereas hardly any of the DP 4-metabolite was found regarding substance P (Figure 56). While different metabolites were obtained with substance P in hypotonic solution compared to ex vivo isotonic blood solution, the same metabolites were detected with respect to NPY (NPY1-36, NPY3-36,

NPY1-34, NPY3-34) though the rate of hydrolysis was much faster in hypotonic blood solution (5 min to 120 min) compared to the isotonic one (10 min to 12 h) due to the contribution of DP 8 and/or DP 9 by burst leukocytes (Figure 57) (140). Interestingly, when NPY-biotin was perfused through the brain blood circulation, it was truncated by membrane-bound endothelial DP 4 and mAmpP, respectively. Though only small percentage of intensities of these metabolites was obtained, it was comparable with regards to the truncation of NPY-biotin by undiluted serum after 3 minutes. Thus, endothelial DP 4 truncation of NPY can be considered similar to the metabolism by soluble DP 4 as depicted in Figure 48. Furthermore, while in human serum hardly any Amp metabolite is formed, it is a dominant metabolite in rat serum (Figures 54 and 49). Moreover, rat Orexin B was found to be a novel substrate of DP 4 in serum (Figure 55D), whereas human Orexin B was not at all metabolised by DP 4 (data not shown). Hence, there appears to be a different metabolism of stress-related neuropeptides in rat compared to human, which has to be considered when interpreting data from in vivo animal models. Regarding sera obtained from patients suffering from clinical depression, we could confirm a lower DP 4-like activity in the serum as previously reported (354-357;724). Implementing a selective DP 4 inhibitor, Sitagliptin and non-selective DP 4-like inhibitor P32/98, we demonstrated for first time that these effects are due to DP 4 (Figure 58). Furthermore, the lower activity of serum DP 4 in depressed patients significantly correlated to DP 4 protein concentrations as illustrated in Figure 59A, which means that less DP 4 is either shedded or secreted into the blood circulation upon clinical depression. While this result points to serum DP 4 being a potential biomarker for clinical depression, it also entails that soluble serum DP 4 is not a pharmaceutical target for clinical depression. However, as already mentioned above, endothelial DP 4 equally contributes to the overall DP 4 metabolite and therefore may compensate apparent lower serum activity of DP 4, particularly, since capillary endothelial cells are highly innervated by autonomic nerves (881). In fact, high levels of DP 4-truncated metabolites SP3-11 and SP5-11 were obtained from cultivated bovine endothelial cells isolated from brain microvascular, a so-called in vitro BBB model (516;517). Furthermore, truncation of

PACAP38 by endothelial DP 4 may explain the different results obtained from in vivo metabolism of PACAP in the blood circulation, yielding PACAP3-38 versus its in vitro metabolism in plasma

(PACAP1-14, PACAP1-19, PACAP1-37, PACAP3-19, PACAP6-26) (214;224;477;478;882). Likewise to

100 Conclusion clinical depression, the serum activity of DP 4 was also reported to be decreased in stress- induced anxiety and phobia, whereas in schizophrenia it was found to be increased as summarised in Table 8 (357-359). Again these findings suggest different mechanisms of action regarding DP 4 and circulating neuropeptides, with endothelial DP 4 possibly being the pharmacological target in clinical depression and anxiety, whereas soluble DP 4 in schizophrenia/psychosis. A possible reason for such altered DP 4 activity/concentrations could be changes in cytokines associated with stress-related disorders as DP 4 activity was shown to be increased in cultivated HUVEC cells after cytokine stimulation with either LPS, TNF- or INF- (741). A link between altered cytokines and psychiatric stress-related disorders has frequently been reported, caused by strong innervation of sympathetic nerves in the spleen and lymph nodes on the one hand and the immunosuppressive action of cortisol on the other hand as illustrated in Figure 64 and Appendix 85 (597;598;724;874;883-886). Moreover, decreased activity of DP 4 in sera from depressed patients significantly correlated to decreased ADA activity (356). Comparing the neuropeptide substrates NPY, GALP, SP and PACAP with respect to endothelial DP 4 and soluble DP 4, one can conclude from the present study that NPY is metabolised by both endothelial as well as soluble DP 4, GALP by soluble DP 4 and possibly endothelial DP 4, whereas PACAP and SP exclusively by endothelial DP 4 as depicted in Figures 48, 54, 55 and 56 (224;231;477;478;887). Yet, this also implies a general difference between soluble serum DP 4 and membrane bound endothelial DP 4 regarding their activity and above all substrate specificity. A possible explanation for this phenomenon could be conformational changes of the quaternary structure due to the dimeric transmembrane domain that was recently shown to influence enzymatic activity of DP 4 (11). Furthermore, N-terminal truncation of these neuropeptides by DP 4 has different effects regarding their affinity towards receptor binding as shown in Appendix 91 and Figure 11. Cleavage of NPY1-36 to NPY3-36 modulates receptor selectivity as NPY3-36 does not bind to Y1-R any more (481;482). Truncation of SP1-11 to SP5-11 results in loss of selectivity as SP5-11 binds equally to NK1-R, NK2-R and NK3-

R, respectively (483;510). In contrast, hydrolysis of PACAP1-38 to PACAP3-38 and PACAP5-38 by

DP 4 leads to inactivation, whereas the GALP3-32 metabolite was shown to have increased affinity towards Gal1-R, Gal2-R and Gal3-R, respectively (Figure 11 and Appendix 91) (477;479;887-889). While truncation of NPY and PACAP results in a switch from anti-stress to stress and stress-related diseases, cleavage of SP and GALP only influences the degree of stress-related behaviour (Appendix 91). Therefore, NPY and PACAP are the favoured pharmacological targets. As in vitro and ex vivo metabolism of NPY by neuronal tissue and blood yielded DP 4-metabolites with the highest % intensities in the MALDI-TOF-MS spectra compared to the other neuropeptide substrates and is described as one of the best substrate of DP 4, it can be considered as the main pharmacological target substrate of DP 4 concerning the treatment of stress-related diseases (2). Furthermore, a link between DP 4 and NPY in stress- related behaviour has already been established and the involvement of NPY and its respective -/- -/- Y-receptors in stress-related and social disorders has been further confirmed in NPY , Y1-R -/- and Y2-R k.o. mice models (296;469-471;890;891). Truncation of NPY by DP 4 results in conformational changes of NPY by opening its so-called hair-pin structure as illustrated in

Appendix 92. This altered structure in turn, leads to different ligand-receptor interactions, as R35

101 Conclusion

of NPY1-36 binds to E6.59 of Y1-R, whereas Arg33 and Arg25 of NPY3-36 bind to Glu6.59 and Glu5.77 of

Y2-R, respectively, eventually causing a change in physiological response due to a shift in receptor selectivity (892). Thus, taken together, we identified in the present study that DP 4 is the target enzyme, NPY the target substrate and the CVO’s particular EM and AP, soluble and/or endothelial DP 4 the target organs and/or target body fluid. Moreover, there appears to be a different mechanism regarding anxiety and/or depression on the one hand and psychosis/schizophrenia on the other hand due to different synaptic plasticity (Appendix 90) as well as the contribution of soluble versus endothelial DP 4. Hence, while for the treatment of psychosis/schizophrenia the potential drug candidate does not require to cross the BBB, a high logBB is necessary for the treatment of anxiety. Due to the high contribution of DP 8/9 and DP 2 activity in the CNS, the inhibitor should be DP 4-selective. As to avoid potential side effects as well as drug-interaction with ACE- inhibitors, a rather short-term potential drug candidate is recommended in contrast to the currently on the market available anti-diabetic DP 4 inhibitors. The only issue that has not been resolved in the present study is the disease target. Since a potential drug candidate has to be superior to current drug treatments against depression, anxiety and schizophrenia, a summary of their current medication, including their application requirements, side effects and statistical demands, is given in Table 33. The most severe side effects are outlined in bold and special attention should therefore be paid in possible clinical studies with a potential DP 4 inhibitor as a drug candidate. While there is statistically a substantial demand regarding clinical depression and anxiety, the side effects of the anti-psychotic drugs are more severe and less alternative treatments are currently available on the market. As for the treatment of anxiety the potential drug candidate has to cross the BBB to be effective, a nasal spray is consequently suggested. Based on the experience from the anti-diabetic DP 4 inhibitors currently on the market, a weight gain and development of diabetes can be ruled out as potential side-effects. Furthermore, there have been no reports on substance dependence concerning DP 4 inhibitors. Moreover, in a recent case report of a suicide attempt by an overdose of Sitagliptin did not turn out to be lethal (893). However, since increased levels of immunosuppressive cortisol in serum is associated with clinical depression and the only side-effects known of current DP 4 inhibitors are increased inflammatory response such as acute pancreatitis, rhinosinusitis, nasopharyngitis, upper respiratory tract infection and urinary tract infection, special focus should be spent in these matters during clinical trials (781-783;786;788;790;894). Table 33 also indicates that current treatments of stress-related diseases target various neurotransmitter systems such as serotonine, norepinephrine and dopamine against clinical depression, GABA against anxiety and dompamine against psychosis/schizophrenia. Neuropeptides have a broader specificity being involved in several stress-related diseases as illustrated in Appendix 91. Such target overlap may even be of advantage, since co-morbidities like anxiety in clinical depression or post-schizophrenic depression are often observed as summarised in Appendices 54, 93 and 94. Finally, as to overcome proteolysis problems of NPY in the present study, intensive metabolic investigations of NPY were performed, thereby identifying seven novel NPY-cleaving enzymes as illustrated in Figure 66. These enzymes include CTSD, CTSB, CTSL, CTSS, CTSG, hK1 and ACE as well as the cleavage sites of Mep-A (678). Furthermore, the findings point to novel mechanisms of abrogating NPY-induced signal-transduction, involving on the one hand internalisation of the ligand-receptor complex with subsequent lysosomal degradation of NPY by cathepsins, on the other hand extra-cellular endoproteolytical degradation by activated tissue kallikrein as well as C-terminal inactivation by ACE in serum.

102 Conclusion

Table 33: Current medication for the treatment of depression, anxiety and schizophrenia§. Depression Anxiety Schizophrenia Antidepressiva Benzodiazepines Antipsychotics Medication # Re-uptake inhibitors* Tricyclics, MAOI GABAA-receptors D2-receptor antagonists (typical + atypical) Upon requirement  doses chronically Application Over a longer period $ (except GAD + PTSD)  doses acute phase Delayed onset of therapeutic action Substance dependence Motoric Disorders Resistance towards treatment Cognitive impairment (extra pyramidal reaction) Nausea Sedation/Drowsiness  Weight gain Side-effects Diarrhea  Weight gain Diabetes of current Headaches Hypotension Agranulocytosis medication  Lipido Dizziness Seizures  REM sleep Paradoxial reaction: Dementia  Weight gain  Aggression  Anxiety Pancreatitis Market/Statistics 1:3 1:4 1:99 § Taken from (2010): http://en.wikipedia.org/wiki/Antidepressant, http://en.wikipedia.org/wiki/Benzodiazepines, http://en.wikipedia.org/wiki/Antipsychotic; ICD10 codes F32,F40,F20, * SSRI, selective serotonine re-uptake inhibitors; SNRIs, serotonine-norepinephrine re-uptake inhibitors; NRIs, norepinephrine-re-uptake inhibitors; NDRIs, norepinephrine-dopamine-re-uptake inhibitors ; # MAOI, Monamine Oxidase Inhibitors; §GAD, generalised anxiety disorder; PTSD, post-traumatic stress disorder.

Figure 66: Metabolism of NPY before (A) and after the present study (B), showing the sub-cellular and tissue distribution of NPY-cleaving enzymes. A, metabolism of NPY before the present study, illustrating the N-terminal modulating enzymes mAmpP, sAmpP, DP 4, DP 8 and DP 9, that alter receptor selectivity (1) as well as the proteolytical degrading enzymes NEP, Mep-B, Mep-A, uPA, Pl and Kal (2). B, metabolism of NPY including novel NPY-cleaving enzymes identified in the present study. In addition to the modulating enzymes, six additional degrading enzymes and one C-terminal inactivating enzyme were identified, including lysosomal enzymes CTSD, CTSB, CTSL, CTSS and CTSG, trypsin-like peptidase hK1 and ACE. Furthermore cleavage sites of Mep-A and Kal were discovered and novel Mep-A activating enzymes found. Novel NPY-cleaving enzymes are high-lighted in colour, arrows show cleavage sites (127;135;485;678;680;683;718). 103 Summary

5 Summary Dipeptidyl peptidase 4 (DP 4, EC 3.4.14.5) is an unique serine protease that hydrolyses dipeptides from the N-termini of oligopeptides and small polypeptides with either proline, hydroxyproline or to a lesser extent alanine at the penultimate position. DP 4 is a type II integral membrane glycoprotein, anchored to the plasma membrane by its signal peptide sequence and consists of two identical subunits, each with a molecular mass of 105-110 kDa. It is described as a multifunctional protein that is involved in many physiological and immunological processes such as peptide metabolism, cell adhesion and T-cell activation. The enzyme is responsible for the short-lives of the insulinotropic incretines GLP-1 and GIP due to their inactivation by DP 4, and therefore inhibition of DP 4 activity results in enhanced insulin secretion from the β-cells of the islets of Langerhans. Thus, a great number of DP 4 inhibitors have been developed as anti-diabetic drugs and currently five of them (Sitagliptin, Januvia®, Merck and Co In.; Saxagliptin, Onglyza®, Bristol-Myers Squibb, Vildagliptin Galvus®, Novartis, Linagliptin Ondero®, Boehringer Ingelheim and Alogliptin, Nesina®, Takedo Pharmaceuticals ) are available on the market. An apparent criteria for drug development is the selectivity of DP 4 inhibitors with respect to additional DP 4-like enzymes, comprised of dipeptidyl peptidase 8 (DP 8), dipeptidyl peptidase 9 (DP 9) and fibroblast activation protein alpha (FAP) of the sub-family S9b, the so-called DP 4-gene family, as well as dipeptidyl peptidase 2 (DP 2) of the S28 family. All of these enzymes exhibit DP 4 activity in vitro, though they have different sub-cellular localisation and little is known regarding their physiological role. While the endocrinological function of DP 4 in glucose homeostasis has been confirmed by DP 4 deficient rats and DP 4-/- k.o. mice models, a possible paracrine or neuronal role in the regulation of insulin secretion by DP 4 and/or DP 4-like enzymes expressed in pancreatic islets of Langerhans still had to be investigated. Thus, the first part of the present study dealt with characterisation of DP 4 and/or DP 4-like enzymes for the treatment of type 2 diabetes and included isolation of DP 4 isoforms to improve crystallisation conditions of native glycosylated DP 4 as well as the analysis of DP 4-like enzymes in various tissues with a particular focus on pancreatic islets of Langerhans. The second part of the present study involved the identification and characterisation of DP 4 as an additional pharmaceutical target for the indications anxiety, depression or schizophrenia, based on altered stress-related behaviour in DP 4-deficient rats, DP 4-/- k.o. mice and upon inhibition of DP 4 in vivo. The study included detailed characterisation of DP 4 and DP 4-like enzymes in rat neuronal tissues and human body fluids, identification of target substrates and target organs, inhibitor profile as well as target disease. In order to improve crystallisation conditions, a new method had to be developed to solve the glycosylation based microheterogeneity of 17 isoforms of purified porcine DP 4. This could be achieved by elaborating on the refractionation method of preparative liquid-phase IEF to refine the pH gradient to less than 0.03 pH units per fraction, eventually resulting in 2-3 isoforms per fraction that were pure enough to obtain a 1.8 Å crystal structure. So far, it has been the crystal structure with the highest resolution out of 76 known DP 4 structures. A unique feature of the porcine DP 4 crystal structure is its tetramerisation, indicating possible cell to cell communication and pointing to a biological function of DP 4 isoforms. Screening of mRNA expression of DP 4-like enzymes in various tissues by nested RT-PCR, revealed the co-localisation of several DP 4-like enzymes in porcine and canine tissue sections from kidney, lung, small intestine and pancreas. Implementing selective DP 4-like

104 Summary inhibitors to analyse the contribution of the DP 4-like enzymes towards the overall DP 4-like activity, showed that most of the activity is contributed by DP 4. Detailed characterisation of porcine islets of Langerhans and pancreas, using activity studies, enzymatic histochemistry, immuno-histochemistry and RT-PCR analysis, revealed that the majority of DP 4-like activity originate from the exocrine tissue. Immunohistochemical staining with anti-DP 4 pAb demonstrated the expression of DP 4 on capillaries surrounding pancreatic islets. However, sub-cellular fractionation and histochemical analysis also implied the presence of DP 4-like activity at different sub-cellular localisations and specific to distinct cell types of islet of Langerhans. The presence of DP 2 could also be demonstrated in isolated granules and by enzymatic histochemical analysis. As DP 8 and DP 9 are cytosolic enzymes and DP 2 is not able to cleave any of the known DP 4 substrates, the results therefore suggest that in addition to its well described endocrinological functions, DP 4 at the islets of Langerhans also influences paracrine and neuronal regulation of insulin secretion from the -cells. Furthermore, when the hydrolysis of the DP 4 substrate neuropeptide Y (NPY) was analysed in serum by MALDI-TOF-MS, an additional C-terminal truncation of NPY by angiotensin converting enzyme (ACE) could be detected. The latter finding suggests a potential interaction within current drug treatments using anti-diabetic DP 4 inhibitors and anti- hypertensive ACE inhibitors in combination. A suspected increase of vasocontrictive NPY1-36 after treatment with anti-diabetic DP4 inhibitor may therefore be compensated by C-terminal inactivation of NPY via ACE, but fails if ACE is also blocked. In addition, DP 4 was also identified as a potential target for stress-related diseases such as anxiety, depression and schizophrenia. Although in general, DP 2, DP 8 and DP 9 were shown to mostly contribute towards the overall DP 4-like activity in neuronal tissues, high levels of DP 4 could be detected in the circumventricular organs (CVO). As the CVO’s lack the blood brain barrier (BBB), this result implies an important role of DP 4 at the neuroendocrino-immunological interphase, involving both stress axis, the hypothalamic- pituitary-adrenal axis (HPA) as well as the neuro-sympathico axis (NSA). Furthermore, assessing the metabolism of NPY, substance P (SP), pituitary-adenylate activating peptide (PACAP), orexin B (OrxB) and galanin-like peptide (GALP) in rat neuronal tissues and human body fluids, revealed that NPY is the target substrate of DP 4 with respect to stress, though the two novel DP 4 substrates GALP and rat OrxB could also be identified. While no N-terminal truncation of NPY by DP 4 could be detected in human cerebrospinal fluids, DP 4- derived NPY3-36 was the major metabolite in human sera and ex vivo unfractionated blood, followed by ACE-like C-terminal truncation. Moreover, circulating NPY was found to be equally metabolised by endothelial DP 4 as well as soluble DP 4. Finally, metabolomic studies of NPY identified seven novel NPY-cleaving enzymes, including the lysosomal cathepsins D, B, L, S and G, tissue kallikrein and ACE. These findings point to novel mechanisms of abrogating NPY induced signal-transduction, involving on the one hand internalisation of ligand-receptor complex with subsequent lysosomal degradation by cathepsins, on the other hand extra-cellular endoproteolytical degradation by activated tissue kallikrein as well as C-terminal inactivation by ACE in serum.

105 Summary

6 Zusammenfassung Dipeptidylpeptidase 4 (DP 4, EC: 3.4.14.5) ist eine hochspezifische Serinprotease, die Dipeptide von den N-Termini von Oligopeptiden sowie kleineren Polypeptiden mit Prolin, Hydroxyprolin oder - in geringerem Maße - Alanin an vorletzter Stelle spaltet. DP 4 ist ein integrales Membranglycoprotein vom Typ II, das durch seine Signalsequenz in der Membran verankert wird. Sie ist aus zwei identischen Untereinheiten mit einer Molmasse von jeweils 105-110 kDa zusammengesetzt. DP 4 wird als multifunktionales Protein beschrieben, das in viele physiologische und immunologische Prozesse involviert ist, wie zum Beispiel Peptidstoffwechsel, Zelladhäsion und T-Zellaktivierung. Das Enzym ist verantwortlich für die kurze Halbwertzeit der insulinotropischen Inkretine GLP-1 und GIP, welche durch DP 4 inaktiviert werden. Daher ergibt eine Hemmung der DP 4 eine erhöhte Ausschüttung an Insulin aus den β–Zellen der Langerhans‘schen Inseln. Aus diesem Grund wurde eine Reihe von DP 4 Inhibitoren als Medikamente zur Behandlung von Typ-2 Diabetes entwickelt, von denen drei derzeit auf dem Markt erhältlich sind: Januvia® (Merck und Co Inc, Wirkstoff Sitagliptin), Onglyza® (Bristol-Myers Squibb, Saxagliptin), Galvus® (Norvatis, Vildagliptin), Ondero® (Boehringer Ingelheim, Linagliptin) und Nesina® (Takeda Pharmaceuticals, Alogliptin). Für die Entwicklung von DP 4 Inhibitoren ist jedoch deren Selektivität bezüglich weiterer Enzyme mit DP 4-ähnlicher Aktivität zu berücksichtigen. Dies sind Dipeptidylpeptidase 8 (DP 8), Dipeptidylpeptidase 9 (DP 9) und Fibroblast Aktivierungsprotein alpha (FAP) aus der S9b-Unterfamilie (der sogenannten DP 4- Gen-spezifischen Familie) sowie Dipeptidylpeptidase 2 (DP 2) aus der S28-Familie. Die oben genannten Enzyme besitzen vergleichbare Aktivitäten in vitro, allerdings sind sie subzellulär unterschiedlich verteilt. Darüber hinaus ist über ihre physiologische Rolle wenig bekannt. Während die endokrinologische Funktion der DP 4 in der Glukose-homeostase durch DP 4- defizienten Ratten- und DP 4-/- k.o. Mausmodelle bereits bestätigt wurde, galt es eine mögliche parakrine und neuronale Rolle der DP 4 bzw. DP 4-ähnlichen Enzyme in der Regulierung von Insulinausschüttung aus den Langerhans´schen Inseln im Pankreas nachzuweisen. Im ersten Teil der Dissertation sollten folgende Aspekte in Bezug auf Typ-2-Diabetes untersucht werden: Isolierung der glykosilierten Isoformen der gereinigten DP 4 zur Aufklärung der 3D-Kristallstruktur, Analyse der Verteilung der Enzyme mit DP 4-ähnlicher Aktivität in verschiedenen Schweine-und Hundegeweben sowie Charakterisierung der Enzyme mit DP 4-ähnlicher Aktivität in den Langerhans‘schen Inseln. Ein weiterer Teil dieser Dissertation behandelt die Identifizierung und Charakterisierung der DP 4 als ein zusätzliches pharmazeutisches Behandlungsziel psychiatrischer Krankheiten mit den Indikationen Angst, Depression und Schizophrenie, welche sich auf Verhaltensänderungen bezüglich Stress in DP 4-defizienten Ratten-, DP 4-/- k.o. Mäusemodelle sowie Hemmung der DP 4 in vivo beziehen. Die Untersuchungen umfassten eine detaillierte Charakterisierung der DP 4 und Enzyme mit DP 4-ähnlicher Aktivität in neuronalen Geweben und humanen Körperflüssigkeiten, die Identifizierung der Hauptsubstrate und Zielorgane, das Inhibitorprofil sowie deren Bezug zu psychiatrische Indikationen. Um die Bedingungen der Kristallisation zu verbessern, musste eine neue Methode entwickelt werden, mit deren Hilfe die aus 17 unterschiedlich glykosilierten Isoformen bestehende Mikroheterogenität der gereinigten Schweine-DP 4 in einzelne Isofomen getrennt werden sollte. Diese konnten durch Einführung eines zweiten optimierten Refraktionierungsschrittes mittels einer präparativen Flüssigphase-IEF mit dem Apparat Rotoforzelle aufgrund einer

106 Summary hohen pH- Auflösung von weniger als 0,03 pH-Einheiten pro Fraktion, in nur 2 bis 3 Isoformen pro Fraktion isoliert werden. Die Reinheit der Fraktionen war ausreichend, um eine Kristallstruktur mit einer Auflösung von 1,8 Å zu erhalten. Die mit Isoformen erhaltene erste native Kristallstruktur der Schweine-DP 4 besitzt somit die höchste Auflösung aller 76 derzeit in der Proteindatenbank (pdb) eingetragenen Kristallstrukturen. Die Struktur zeigt eine tetramere Anordnung, was auf eine Kommunikation zweier Zellen und eine mögliche biologische Funktion der Isoformen hinweist. Eine Verteilungsanalyse der Enzyme mit DP 4-ähnlicher Aktivität mittels verschachtelter RT- PCR ergab Kolokalisierung von mehreren Enzymen in Schweine- und Hundegewebe aus Niere, Lunge, Dünndarm und Pankreas. Durch Anwendung selektiver Inhibitoren für die Enzyme mit DP 4-ähnlicher Aktivität konnte der Beitrag der einzelnen Enzyme zur allgemeinen DP 4-Aktivität ermittelt werden und zeigte, dass der Hauptanteil der allgemeinen DP 4-Aktivität auf DP 4 zurückzuführen ist. Eine detaillierte Charakterisierung der Langerhans‘schen Inseln und des Schweinepankreas mittels Aktivitätsstudien, histochemischer Aktivitätsfärbung, Immunhistochemie und RT-PCR Analyse offenbarte, dass der größte Anteil der DP 4-Aktivität aus exokrinem Drüsengewebe des Pankreas stammt. Eine Expression der DP 4 in den die Langerhans´schen Inseln umgebenden Blutgefäßen konnte durch eine immunhistochemische Färbung mit einem polyklonalen anti-DP 4- Antikörper nachgewiesen werden. Mittels subzellulärer Fraktionierung und histochemischer Aktivitätsfärbung wurde die Präsenz mehrerer Enzyme mit DP 4-ähnlicher Aktivität, jedoch mit unterschiedlicher subzellulärer Lokalisierung festgestellt. Diese waren wiederum spezifisch bezüglich bestimmter Zelltypen der Langerhans‘schen Inseln. Die Präsenz der DP 2 wurde ferner mittels isolierter Granulae, durch histochemische Aktivitätsfärbung und Aktivitätsstudien bewiesen. Da sowohl die DP 8 als auch die DP 9 zytosolisch vorkommende Enzyme sind und die DP 2 nicht in der Lage ist DP 4-Substrate zu spalten, weisen die vorliegenden Ergebnisse neben der bekannten endokrinologischen Rolle der DP 4 im Blut auf weitere parakrine und neuronale Funktionen in der Regulierung der Insulinausschüttung hin. Des Weiteren wurde zusätzlich zu der bekannten N-terminalen DP 4-Spaltung von Neuropeptid Y (NPY) im Serum eine C-terminale Inaktivierung durch Angiotensin- konvertierendes Enzym (ACE) detektiert. Diese läßt auf eine mögliche Wechselwirkung bei gleichzeitiger Anwendung von DP 4-Hemmern als Antidiabetikum und anihypertensiven

ACE-Hemmern schließen. Eine zu erwartende Anreicherung von vasokonstriktiven NPY1-36 wird durch die von ACE verursachte C-terminale Inaktivierung zu NPY1-34 kompensiert, jedoch wird diese unterbunden, wenn ACE ebenfalls blockiert wird. In dieser Dissertation konnte darüber hinaus DP 4 als mögliches pharmazeutisches Behandlungsziel stress-verwandter Indikationen wie zum Beispiel Depression, Angst und Schizophrenie identifiziert werden. Obwohl in neuronalen Geweben der Anteil der Enzyme DP 2, DP 8 und DP 9 höher bezüglich der allgemeinen DP 4- ähnlichen Aktivität ist als die der DP 4, wurde eine hohe DP 4-Aktivität und Expression in den sogenannten Zirkumventrikulären Organen (ZVOs) gefunden. Da die ZVOs keine Blut-Hirnschranke (BBB) aufweisen, deutet dies auf eine wichtige Rolle der DP 4 in der neuroendokrinologischen- immunologischer Interphase hin, die die beiden Stressachsen, nämlich die Hypothalamus- Hypophysen-Nebennieren (hypothalamic-pituitary-adrenal, HPA) Achse sowie die neurosympathische Achse (NSA) umfasst. Bei einer Untersuchungsreihe wurde die Hydrolyse von NPY, Substanz P (SP), „pituitary adenylate cyclase-activating peptide“ (PACAP), Orexin B (Orx B) und „Galanin-like peptide“ 107 Summary

(GALP) in neuronalen Rattengeweben und humanen Körperflüssigkeiten mittels MALDI- TOF-MS analysiert. Dabei wurde NPY als das Hauptsubstrat der DP 4 bezüglich des stress- vermittelten Behandlungsziels identifiziert, obwohl außerdem GALP und Ratten-Orx B als neue Substrate der DP 4 entdeckt wurden. Während im humanen Liquor cerebrospinalis keine N-terminale DP 4-Trunkierung von NPY nachgewiesen wurde, ist das durch DP 4 gespaltene NPY3-36 der Hauptmetabolit in humanen Serum und Vollblut, gefolgt von der durch ACE verursachten C-terminalen Inaktivierung zu NPY1-34. Ferner konnte gezeigt werden, dass im Blut vorhandenes NPY gleichermaßen von membrangebundener Endothel- DP 4 als auch von löslicher DP 4 gespalten wird. Darüber hinaus wurden durch Untersuchungen zum NPY-Metabolismus sieben neue NPY- spaltende Proteasen identifiziert, nämlich die lysosomalen Kathepsine D, B, L, S und G, das Trypsin-ähnliche Enzym Kallikrein 1 sowie ACE. Die Beteiligung dieser Proteasen lässt auf einen neuen Mechanismus bezüglich der Unterbindung der NPY vermittelten Signal- Transduktion schließen, welcher einerseits aus einer Internalisierung des Liganden- Rezeptorenkomplexes mit anschließenden lysosomalen Abbau durch Kathepsine, andererseits aus einen extrazellulären endoproteolytischen Abbau durch Kallikrein 1 sowie einer durch ACE verursachten C-terminalen Inaktivierung im Serum besteht.

108 Reference

7 References

1. Yaron, A. and Naider, F. (1993) Proline-dependent structural and biological properties of peptides and proteins. Crit.Rev.Biochem.Mol.Biol. 28, 31-81. 2. Lambeir, A. M., Durinx, C., Scharpe, S., and De Meester, I. (2003) Dipeptidyl-peptidase IV from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme DPP IV. Crit Rev.Clin.Lab Sci. 40, 209- 294. 3. Sedo, A. and Malik, R. (2001) Dipeptidyl peptidase IV-like molecules: homologous proteins or homologous activities? Biochim.Biophys.Acta 1550, 107-116. 4. Hopsu-Havu, V. K. and Glenner, G. G. (1966) A new dipeptide naphthylamidase hydrolyzing glycyl-prolyl-beta- naphthylamide. Histochemie. 7, 197-201. 5. McCaughan, G. W., Wickson, J. E., Creswick, P. F., and Gorrell, M. D. (1990) Identification of the bile canalicular cell surface molecule GP110 as the ectopeptidase dipeptidyl peptidase IV: an analysis by tissue distribution, purification and N-terminal amino acid sequence. Hepatology 11, 534-544. 6. Natori, Y., Hayakawa, I., and Shibata, S. (1987) Identification of gp108, a pathogenic antigen of passive Heymann nephritis, as dipeptidyl peptidase IV. Clin.Exp.Immunol. 70, 434-439. 7. Kameoka, J., Tanaka, T., Nojima, Y., Schlossman, S. F., and Morimoto, C. (1993) Direct association of adenosine deaminase with a T cell activation antigen, CD26. Science 261, 466-469. 8. Fleischer, B. () CD26: a surface protease involved in T-cell activation. Immunology today 9. Martin, R. A., Cleary, D. L., Guido, D. M., Zurcher-Neely, H. A., and Kubiak, T. M. (1993) Dipeptidyl peptidase IV (DPP-IV) from pig kidney cleaves analogs of bovine growth hormone-releasing factor (bGRF) modified at position 2 with Ser, Thr or Val. Extended DPP-IV substrate specificity? Biochim.Biophys.Acta 1164, 252-260. 10. Demuth, H.-U. and Heins, J. (1995) On the Catalytic Mechanism of Dipeptidyl Peptidase IV. In Fleischer, B., editor. Dipeptidyl Peptidase IV (CD26) in Metabolism and the Immune Response, R.G. Landes Company, Austin, USA, 1-35. 11. Chung, K. M., Cheng, J. H., Suen, C. S., Huang, C. H., Tsai, C. H., Huang, L. H., Chen, Y. R., Wang, A. H., Hwang, M. J., and Chen, X. (2010) The dimeric transmembrane domain of prolyl dipeptidase DPP-IV contributes to its quaternary structure and enzymatic activities. Protein Sci. 19, 1627-1638. 12. Rasmussen, H. B., Branner, S., Wiberg, F. C., and Wagtmann, N. (2003) Crystal structure of human dipeptidyl peptidase IV/CD26 in complex with a substrate analog. Nat.Struct.Biol. 10, 19-25. 13. Engel, M., Hoffmann, T., Wagner, L., Wermann, M., Heiser, U., Kiefersauer, R., Huber, R., Bode, W., Demuth, H. U., and Brandstetter, H. (2003) The crystal structure of dipeptidyl peptidase IV (CD26) reveals its functional regulation and enzymatic mechanism. Proc.Natl.Acad.Sci.U.S.A. 100, 5063-5068. 14. Aertgeerts, K., Ye, S., Tennant, M. G., Kraus, M. L., Rogers, J., Sang, B. C., Skene, R. J., Webb, D. R., and Prasad, G. S. (2004) Crystal structure of human dipeptidyl peptidase IV in complex with a decapeptide reveals details on substrate specificity and tetrahedral intermediate formation. Protein Sci. 13, 412-421. 15. Lojda, Z. (1979) Studies on dipeptidyl(amino)peptidase IV (glycyl-proline naphthylamidase).II Blood vessels. Histochemistry 59, 153-166. 16. Sahara, N., Fukasawa, K., Harada, M., and Suzuki, K. (1989) Subcellular localisation of dipeptidyl peptidase IV in rat kidney and small intestine. Acta Histochem.Cytochem. 22, 479-485. 17. Gossrau, R. (1979) [Peptidases II. Localization of dipeptidylpeptidase IV (DPP IV). Histochemical and biochemical study]. Histochemistry 60, 231-248. 18. Dikov, A., Dimitrova, M., Stoineva, I., and Halbhuber, K. J. (1999) New tetrazolium method for the histochemical localization of dipeptidyl peptidase IV. Cell Mol.Biol.(Noisy.-le.-grand.) 45, 225-231. 19. Fukasawa, K. M., Fukasawa, K., Sahara, N., Harada, M., Kondo, Y., and Nagatsu, I. (1981) Immunohistochemical localization of dipeptidyl aminopeptidase IV in rat kidney, liver, and salivary glands. J.Histochem.Cytochem. 29, 337- 343. 20. Tauc, M., Chatelet, F., Verroust, P., Vandewalle, A., Poujeol, P., and Ronco, P. (1988) Characterization of monoclonal antibodies specific for rabbit renal brush-border : application to immunohistological localization. J.Histochem.Cytochem. 36, 523-532. 21. Stange, T., Kettmann, U., and Holzhausen, H. J. (1996) Immunoelectron microscopic single and double labelling of aminopeptidase N (CD 13) and dipeptidyl peptidase IV (CD 26). Acta Histochem. 98, 323-331. 22. Walborg, E. F. J., Tsuchida, S., Weeden, D. S., Thomas, M. W., Barrick, A., McEntire, K. D., Allison, J. P., and Hixson, D. C. (1985) Identification of dipeptidyl peptidase IV as a protein shared by the plasma membrane of hepatocytes and liver biomatrix. Exp.Cell Res. 158, 509-518. 23. Ichinose, M., Takayama, H., Fukuda, T., Watanabe, B., Ishimaru, T., Izumi, M., Miyke, S., and Takamori, M. (1982) Partial purification and characterization of glycylprolyl dipeptidyl aminopeptidase in porcine pancreas. Biochim.Biophys.Acta 719, 527-531. 24. Heymann, E., Mentlein, R., Nausch, I., Erlanson-Albertsson, C., Yoshimoto, T., and Feller, A. C. (1986) Processing of pro-colipase and trypsinogen by pancreatic dipeptidyl peptidase IV. Biomed.Biochim.Acta 45, 575-584. 25. Sedo, A., Krepela, E., Kasafirek, E., Kraml, J., and Kadlecova, L. (1991) Dipeptidyl peptidase IV in the human lung and spinocellular lung cancer. Physiol.Res. 40, 359-362. 26. Barnes, K., Murphy, L. J., and Turner, A. J. (1994) Immunoseparation of membrane peptidases from pig lung membranes using magnetic beads. Biochem.Soc.Trans. 22, 451S. 27. Labsch, B., Heins, J., Hilse, H., Barth, A., and Oehme, P. (1981) [The activity of dipeptidyl peptidase IV in various organs and tissues of the rat and its possible relation with substance P metabolism]. Pharmazie. 36, 161-163. 28. Schade, J., Stephan, M., Schmiedl, A., Wagner, L., Niestroj, A. J., Demuth, H. U., Frerker, N., Klemann, C., Raber, K. A., Pabst, R., and Von Horsten, S. (2008) Regulation of expression and function of dipeptidyl peptidase 4 (DP4), DP8/9, and DP10 in allergic responses of the lung in rats. J.Histochem.Cytochem. 56, 147-155. 29. Sahara, N. and Suzuki, K. (1984) Ultrastructural localization of dipeptidyl peptidase IV in rat salivary glands by immunocytochemistry. Cell Tissue Res. 235, 427-432.

109 Reference

30. Puschel, G., Mentlein, R., and Heymann, E. (1982) Isolation and characterization of dipeptidyl peptidase IV from human placenta. Eur.J.Biochem. 126, 359-365. 31. Shimai, K. (1985) [Activities of dipeptidyl peptidase IV and collagenase-like peptidase released from macrophages stimulated by Bacteroides gingivalis]. Nippon.Shishubyo.Gakkai.Kaishi. 27, 297-306. 32. Fujiwara, H., Maeda, M., Imai, K., Fukuoka, M., Yasuda, K., Takakura, K., and Mori, T. (1992) Human luteal cells express dipeptidyl peptidase IV on the cell surface. J.Clin.Endocrinol.Metab. 75, 1352-1357. 33. Vanhoof, G., De Meester, I., van Sande, M., Scharpe, S., and Yaron, A. (1992) Distribution of proline-specific aminopeptidases in human tissues and body fluids. Eur.J.Clin.Chem.Clin.Biochem. 30, 333-338. 34. Kullertz, G., Barth, A., and Fischer, G. (1986) [Determination of dipeptidyl-peptidase IV in biological materials]. J.Clin.Chem.Clin.Biochem. 24, 551-558. 35. Avasarala, J. R., Naoi, M., Sasaki, S., Ueda, M., Saga, S., Suzuki, T., Yanagita, N., and Nagatsu, T. (1991) Distribution of peptidases in cultured cells from middle ear mucosa: prolyl endopeptidase is localized in fibroblasts and dipeptidyl peptidase IV and II in epithelial cells. Biochem.Med.Metab.Biol. 45, 355-358. 36. Saison, M., Verlinden, J., Van Leuven, F., Cassiman, J. J., and Van den, B. H. (1983) Identification of cell surface dipeptidylpeptidase IV in human fibroblasts. Biochem J 216, 177-183. 37. De Meester, I., Vanhoof, G., Hendriks, D., Demuth, H. U., Yaron, A., and Scharpe, S. (1992) Characterization of dipeptidyl peptidase IV (CD26) from human lymphocytes. Clin.Chim.Acta 210, 23-34. 38. Shin, J. W., Jurisic, G., and Detmar, M. (2008) Lymphatic-specific expression of dipeptidyl peptidase IV and its dual role in lymphatic endothelial function. Exp.Cell Res. 314, 3048-3056. 39. Hong, W. J., Petell, J. K., Swank, D., Sanford, J., Hixson, D. C., and Doyle, D. (1989) Expression of dipeptidyl peptidase IV in rat tissues is mainly regulated at the mRNA levels. Exp.Cell Res. 182, 256-266. 40. Ulmer, A. J., Mattern, T., Feller, A. C., Heymann, E., and Flad, H. D. (1990) CD26 antigen is a surface dipeptidyl peptidase IV (DPPIV) as characterized by monoclonal antibodies clone TII-19-4-7 and 4EL1C7. Scand.J.Immunol. 31, 429-435. 41. Chikuma, T., Hama, T., Nagatsu, T., Kumegawa, M., and Kato, T. (1990) Purification and properties of dipeptidyl peptidase IV from human urine. Biol.Chem.Hoppe Seyler 371, 325-330. 42. Haninec, P. and Dubovy, P. (1988) Fine structure histochemical study of the distribution of dipeptidylpeptidase IV (DPP IV) in the meningeal lamellae of the rat. Experientia 44, 708-710. 43. Mentlein, R., von Kolszynski, M., Sprang, R., and Lucius, R. (1990) Proline-specific proteases in cultivated neuronal and glial cells. Brain Res. 527, 159-162. 44. Abbott, C. A., Baker, E., Sutherland, G. R., and McCaughan, G. W. (1994) Genomic organization, exact localization, and tissue expression of the human CD26 (dipeptidyl peptidase IV) gene [published erratum appears in Immunogenetics 1995;42(1):76]. Immunogenetics 40, 331-338. 45. Bernard, A. M., Mattei, M. G., Pierres, M., and Marguet, D. (1994) Structure of the mouse dipeptidyl peptidase IV (CD26) gene. Biochemistry 33, 15204-15214. 46. Qvist, H., Sjostrom, H., and Noren, O. (1998) The TATA-less, GC-rich porcine dipeptidylpeptidase IV (DPPIV) promoter shows bidirectional activity. Biol.Chem. 379, 75-81. 47. Bohm, S. K., Gum, J. R. J., Erickson, R. H., Hicks, J. W., and Kim, Y. S. (1995) Human dipeptidyl peptidase IV gene promoter: tissue-specific regulation from a TATA-less GC-rich sequence characteristic of a housekeeping gene promoter. Biochem.J. 311, 835-843. 48. Erickson, R. H., Gum, J. R., Lotterman, C. D., Hicks, J. W., Lai, R. S., and Kim, Y. S. (1999) Regulation of the gene for human dipeptidyl peptidase IV by hepatocyte nuclear factor 1 alpha. Biochem.J. 338, 91-97. 49. Hildebrandt, M., Reutter, W., and Gitlin, J. D. (1991) Tissue-specific regulation of dipeptidyl peptidase IV expression during development. Biochem.J. 277, 331-334. 50. Darmoul, D., Voisin, T., Couvineau, A., Rouyer-Fessard, C., Salomon, R., Wang, Y., Swallow, D. M., and Laburthe, M. (1994) Regional expression of epithelial dipeptidyl peptidase IV in the human intestines. Biochem.Biophys.Res.Commun. 203, 1224-1229. 51. Riemann, D., Kehlen, A., and Langner, J. (1995) Stimulation of the expression and the enzyme activity of aminopeptidase N/CD13 and dipeptidylpeptidase IV/CD26 on human renal cell carcinoma cells and renal tubular epithelial cells by T cell-derived cytokines, such as IL-4 and IL-13. Clin.Exp.Immunol. 100, 277-283. 52. Fujiwara, H., Fukuoka, M., Yasuda, K., Ueda, M., Imai, K., Goto, Y., Suginami, H., Kanzaki, H., Maeda, M., and Mori, T. (1994) Cytokines stimulate dipeptidyl peptidase-IV expression on human luteinizing granulosa cells. J.Clin.Endocrinol.Metab. 79, 1007-1011. 53. Kehlen, A., Gohring, B., Langner, J., and Riemann, D. (1998) Regulation of the expression of aminopeptidase A, aminopeptidase N/CD13 and dipeptidylpeptidase IV/CD26 in renal carcinoma cells and renal tubular epithelial cells by cytokines and cAMP-increasing mediators. Clin.Exp.Immunol. 111, 435-441. 54. Cordero, O. J., Salgado, F. J., Vinuela, J. E., and Nogueira, M. (1997) Interleukin-12 enhances CD26 expression and dipeptidyl peptidase IV function on human activated lymphocytes. Immunobiol. 197, 522-533. 55. Bauvois, B., Djavaheri-Mergny, M., Rouillard, D., Dumont, J., and Wietzerbin, J. (2000) Regulation of CD26/DPPIV gene expression by interferons and retinoic acid in tumor B cells. Oncogene 19, 265-272. 56. Marguet, D., Bernard, A. M., Vivier, I., Darmoul, D., Naquet, P., and Pierres, M. (1992) cDNA cloning for mouse thymocyte-activating molecule. A multifunctional ecto-dipeptidyl peptidase IV (CD26) included in a subgroup of serine proteases. J.Biol.Chem. 267, 2200-2208. 57. Ogata, S., Misumi, Y., and Ikehara, Y. (1989) Primary structure of rat liver dipeptidyl peptidase IV deduced from its cDNA and identification of the NH2-terminal signal sequence as the membrane-anchoring domain. J.Biol.Chem. 264, 3596-3601. 58. Nishimura, Y., Miyazawa, T., Ikeda, Y., Izumiya, Y., Nakamura, K., Sato, E., Mikami, T., and Takahashi, E. (1999) Molecular cloning and sequencing of a cDNA encoding the feline T-cell activation antigen CD26 homologue. Immunogenetics 50, 366-368. 59. Lee, S. U., Park, Y. H., Davis, W. C., Hamilton, J., Naessens, J., and Bohach, G. A. (2002) Molecular characterization of bovine CD26 upregulated by a staphylococcal superantigen. Immunogenetics 54, 216-220. 60. Misumi, Y., Hayashi, Y., Arakawa, F., and Ikehara, Y. (1992) Molecular cloning and sequence analysis of human dipeptidyl peptidase IV, a serine proteinase on the cell surface. Biochim.Biophys.Acta 1131, 333-336.

110 Reference

61. Yamashita, K., Tachibana, Y., Matsuda, Y., Katunuma, N., Kochibe, N., and Kobata, A. (1988) Comparative studies of the sugar chains of aminopeptidase N and dipeptidylpeptidase IV purified from rat kidney brush-border membrane. Biochemistry 27, 5565-5573. 62. Stehling, P., Grams, S., Nuck, R., Grunow, D., Reutter, W., and Gohlke, M. (1999) In vivo modulation of the acidic N- glycans from rat liver dipeptidyl peptidase IV by N-propanoyl-D-mannosamine. Biochem.Biophys.Res.Commun. 263, 76-80. 63. Kahne, T., Kroning, H., Thiel, U., Ulmer, A. J., Flad, H. D., and Ansorge, S. (1996) Alterations in structure and cellular localization of molecular forms of DP IV/CD26 during T cell activation. Cell Immunol. 170, 63-70. 64. Smith, R. E., Talhouk, J. W., Brown, E. E., and Edgar, S. E. (1998) The significance of hypersialylation of dipeptidyl peptidase IV (CD26) in the inhibition of its activity by Tat and other cationic peptides. CD26: a subverted adhesion molecule for HIV peptide binding. AIDS Res.Hum.Retroviruses 14, 851-868. 65. Schmauser, B., Kilian, C., Reutter, W., and Tauber, R. (1999) Sialoforms of dipeptidylpeptidase IV from rat kidney and liver. Glycobiology. 9, 1295-1305. 66. Fukasawa, K. M., Fukasawa, K., Hiraoka, B. Y., and Harada, M. (1981) Comparison of dipeptidyl peptidase IV prepared from pig liver and kidney. Biochim.Biophys.Acta 657, 179-189. 67. Fan, H., Meng, W., Kilian, C., Grams, S., and Reutter, W. (1997) Domain-specific N-glycosylation of the membrane glycoprotein dipeptidylpeptidase IV (CD26) influences its subcellular trafficking, biological stability, enzyme activity and protein folding. Eur.J.Biochem. 246, 243-251. 68. Loch, N., Tauber, R., Becker, A., Hartel Schenk, S., and Reutter, W. (1992) Biosynthesis and metabolism of dipeptidylpeptidase IV in primary cultured rat hepatocytes and Morris hepatoma 7777 cells. Eur.J.Biochem. 210, 161- 168. 69. Erickson, R. H., Suzuki, Y., Sedlmayer, A., and Kim, Y. S. (1992) Biosynthesis and degradation of altered immature forms of intestinal dipeptidyl peptidase IV in a rat strain lacking the enzyme. J.Biol.Chem. 267, 21623-21629. 70. Kreisel, W., Hildebrandt, H., Mossner, W., Tauber, R., and Reutter, W. (1993) Oligosaccharide reprocessing and recycling of a cell surface glycoprotein in cultured rat hepatocytes. Biol.Chem.Hoppe Seyler 374, 255-263. 71. Kreisel, W., Hanski, C., Tran-Thi, T. A., Katz, N., Decker, K., Reutter, W., and Gerok, W. (1988) Remodeling of a rat hepatocyte plasma membrane glycoprotein. De- and reglycosylation of dipeptidyl peptidase IV. J.Biol.Chem. 263, 11736-11742. 72. Kreisel, W., Reutter, W., and Gerok, W. (1984) Modification of the intramolecular turnover of terminal carbohydrates of dipeptidylaminopeptidase IV isolated from rat-liver plasma membrane during liver regeneration. Eur.J.Biochem. 138, 435-438. 73. Kreisel, W., Volk, B. A., Buchsel, R., and Reutter, W. (1980) Different half-lives of the carbohydrate and protein moieties of a 110,000-dalton glycoprotein isolated from plasma membranes of rat liver. Proc.Natl.Acad.Sci.U.S.A. 77, 1828-1831. 74. Volk, B. A., Kreisel, W., Kottgen, E., Gerok, W., and Reutter, W. (1983) Heterogeneous turnover of terminal and core sugars within the carbohydrate chain of dipeptidylaminopeptidase IV isolated from rat liver plasma membrane. FEBS Lett. 163, 150-152. 75. Reutter, W., Baum, O., Loster, K., Fan, H., Bork, J. P., Bernt, K., Hanski, C., and Tauber, R. (1995) Functional aspects of the three extracellular regions of dipeptidyl peptidase IV:Characterization of glycosylation events, of the collagen binding site and of endopeptidase activity. In Fleischer, B., editor. Dipeptidyl Peptidase IV (CD26) in Metabolism and Immune Response, R.G. Landes Company, Austin, 55-78. 76. Alfalah, M., Jacob, R., and Naim, H. Y. (2002) Intestinal dipeptidyl peptidase IV is efficiently sorted to the apical membrane through the concerted action of N- and O-glycans as well as association with lipid microdomains. J Biol.Chem. 277, 10683-10690. 77. Naim, H. Y., Joberty, G., Alfalah, M., and Jacob, R. (1999) Temporal association of the N- and O-linked glycosylation events and their implication in the polarized sorting of intestinal brush border sucrase-isomaltase, aminopeptidase N, and dipeptidyl peptidase IV. J.Biol.Chem. 274, 17961-17967. 78. Cuchacovich, M., Gatica, H., Pizzo, S. V., and Gonzalez-Gronow, M. (2001) Characterization of human serum dipeptidyl peptidase IV (CD26) and analysis of its autoantibodies in patients with rheumatoid arthritis and other autoimmune diseases. Clin.Exp.Rheumatol. 19, 673-680. 79. Buchsel, R., Kreisel, W., Fringes, B., Hanski, C., Reutter, W., and Gerok, W. (1986) Localization and turnover of dipeptidylpeptidase IV in the domains of rat liver plasma membrane. Eur.J.Cell Biol. 40, 53-57. 80. Slimane, T. A., Lenoir, C., Sapin, C., Maurice, M., and Trugnan, G. (2000) Apical secretion and sialylation of soluble dipeptidyl peptidase IV are two related events. Exp.Cell Res. 258, 184-194. 81. Delacour, D., Gouyer, V., Leteurtre, E., Ait-Slimane, T., Drobecq, H., Lenoir, C., Moreau-Hannedouche, O., Trugnan, G., and Huet, G. (2003) 1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside blocks the apical biosynthetic pathway in polarized HT-29 cells. J Biol.Chem. 278, 37799-37809. 82. Weihofen, W. A., Liu, J., Reutter, W., Saenger, W., and Fan, H. (2004) Crystal structure of CD26/dipeptidyl-peptidase IV in complex with adenosine deaminase reveals a highly amphiphilic interface. J.Biol.Chem. 279, 43330-43335. 83. Polgar, L. (2002) The prolyl oligopeptidase family. Cell Mol.Life Sci. 59, 349-362. 84. Scanlan, M. J., Raj, B. K., Calvo, B., Garin-Chesa, P., Sanz-Moncasi, M. P., Healey, J. H., Old, L. J., and Rettig, W. J. (1994) Molecular cloning of fibroblast activation protein alpha, a member of the serine protease family selectively expressed in stromal fibroblasts of epithelial cancers. Proc.Natl.Acad.Sci.U.S.A. 91, 5657-5661. 85. Abbott, C. A., Yu, D. M., Woollatt, E., Sutherland, G. R., McCaughan, G. W., and Gorrell, M. D. (2000) Cloning, expression and chromosomal localization of a novel human dipeptidyl peptidase (DPP) IV homolog, DPP8. Eur.J Biochem. 267, 6140-6150. 86. Olsen, C. and Wagtmann, N. (2002) Identification and characterization of human DPP9, a novel homologue of dipeptidyl peptidase IV. Gene 299, 185-193. 87. Wada, K., Yokotani, N., Hunter, C., Doi, K., Wenthold, R. J., and Shimasaki, S. (1992) Differential expression of two distinct forms of mRNA encoding members of a dipeptidyl aminopeptidase family. Proc.Natl.Acad.Sci.U.S.A 89, 197- 201. 88. Qi, S. Y., Riviere, P. J., Trojnar, J., Junien, J. L., and Akinsanya, K. O. (2003) Cloning and characterization of dipeptidyl peptidase 10, a new member of an emerging subgroup of serine proteases. Biochem.J. 373, 179-189. 89. Wolf, B., Fischer, G., and Barth, A. (1978) [Kinetics of dipeptidyl-peptidase IV]. Acta Biol.Med.Ger. 37, 409-420. 111 Reference

90. Ajami, K., Abbott, C. A., McCaughan, G. W., and Gorrell, M. D. (2004) Dipeptidyl peptidase 9 has two forms, a broad tissue distribution, cytoplasmic localization and DPIV-like peptidase activity. Biochim.Biophys.Acta 1679, 18-28. 91. Park, J. E., Lenter, M. C., Zimmermann, R. N., Garin-Chesa, P., Old, L. J., and Rettig, W. J. (1999) Fibroblast activation protein, a dual specificity serine protease expressed in reactive human tumor stromal fibroblasts. J.Biol.Chem. 274, 36505-36512. 92. Abbott, C. A. and Gorrell, M. D. (2002) The family of CD26/DP IV and related Ectopeptidases. In Langner, J. and Ansorge, S., editors. Ectopeptidases, Kluwer Academic/Plenum Publishers, New York, 171-195. 93. Abbott, C. A., Yu, D., McCaughan, G. W., and Gorrell, M. D. (2000) Post-proline-cleaving peptidases having DP IV like enzyme activity. Post-proline peptidases. Adv.Exp.Med.Biol. 477, 103-109. 94. Gorrell, M. D. (2005) Dipeptidyl peptidase IV and related enzymes in cell biology and liver disorders. Clin.Sci.(Lond) 108, 277-292. 95. Keane, F. M., Nadvi, N. A., Yao, T. W., and Gorrell, M. D. (2011) Neuropeptide Y, B-type natriuretic peptide, substance P and peptide YY are novel substrates of fibroblast activation protein-alpha. FEBS J 278, 1316-1332. 96. Edosada, C. Y., Quan, C., Tran, T., Pham, V., Wiesmann, C., Fairbrother, W., and Wolf, B. B. (2006) Peptide substrate profiling defines fibroblast activation protein as an endopeptidase of strict Gly(2)-Pro(1)-cleaving specificity. FEBS Lett. 580, 1581-1586. 97. Edosada, C. Y., Quan, C., Wiesmann, C., Tran, T., Sutherlin, D., Reynolds, M., Elliott, J. M., Raab, H., Fairbrother, W., and Wolf, B. B. (2006) Selective inhibition of fibroblast activation protein protease based on dipeptide substrate specificity. J.Biol.Chem. 281, 7437-7444. 98. Aertgeerts, K., Levin, I., Shi, L., Snell, G. P., Jennings, A., Prasad, G. S., Zhang, Y., Kraus, M. L., Salakian, S., Sridhar, V., Wijnands, R., and Tennant, M. G. (2005) Structural and kinetic analysis of the substrate specificity of human fibroblast activation protein alpha. J Biol.Chem. 280, 19441-19444. 99. Rettig, W. J., Su, S. L., Fortunato, S. R., Scanlan, M. J., Raj, B. K., Garin-Chesa, P., Healey, J. H., and Old, L. J. (1994) Fibroblast activation protein: purification, epitope mapping and induction by growth factors. Int.J.Cancer 58, 385-392. 100. Milner, J. M., Kevorkian, L., Young, D. A., Jones, D., Wait, R., Donell, S. T., Barksby, E., Patterson, A. M., Middleton, J., Cravatt, B. F., Clark, I. M., Rowan, A. D., and Cawston, T. E. (2006) Fibroblast activation protein alpha is expressed by chondrocytes following a pro-inflammatory stimulus and is elevated in osteoarthritis. Arthritis Res.Ther. 8, R23. 101. Rettig, W. J., Garin-Chesa, P., Healey, J. H., Su, S. L., Ozer, H. L., Schwab, M., Albino, A. P., and Old, L. J. (1993) Regulation and heteromeric structure of the fibroblast activation protein in normal and transformed cells of mesenchymal and neuroectodermal origin. Cancer Res. 53, 3327-3335. 102. Ghersi, G., Dong, H., Goldstein, L. A., Yeh, Y., Hakkinen, L., Larjava, H. S., and Chen, W. T. (2003) Seprase-dPPIV association and prolyl peptidase and gelatinase activities of the protease complex. Adv.Exp.Med.Biol. 524, 87-94. 103. Ghersi, G., Dong, H., Goldstein, L. A., Yeh, Y., Hakkinen, L., Larjava, H. S., and Chen, W. T. (2002) Regulation of fibroblast migration on collagenous matrix by a cell surface peptidase complex. J Biol.Chem. 277, 29231-29241. 104. Chen, W. T. and Kelly, T. (2003) Seprase complexes in cellular invasiveness. Cancer Metastasis Rev. 22, 259-269. 105. Chen, W. T., Kelly, T., and Ghersi, G. (2003) DPPIV, seprase, and related serine peptidases in multiple cellular functions. Curr.Top.Dev.Biol. 54, 207-232. 106. Chen, W. T. (2003) DPPIV and seprase in cancer invasion and angiogenesis. Adv.Exp.Med.Biol. 524, 197-203. 107. Chen, W. T. (1996) Proteases associated with invadopodia, and their role in degradation of extracellular matrix. Enzyme Protein 49, 59-71. 108. Garin-Chesa, P., Old, L. J., and Rettig, W. J. (1990) Cell surface glycoprotein of reactive stromal fibroblasts as a potential antibody target in human epithelial cancers. Proc.Natl.Acad.Sci.U.S.A 87, 7235-7239. 109. Henry, L. R., Lee, H. O., Lee, J. S., Klein-Szanto, A., Watts, P., Ross, E. A., Chen, W. T., and Cheng, J. D. (2007) Clinical implications of fibroblast activation protein in patients with colon cancer. Clin.Cancer Res 13, 1736-1741. 110. Ariga, N., Sato, E., Ohuchi, N., Nagura, H., and Ohtani, H. (2001) Stromal expression of fibroblast activation protein/seprase, a cell membrane serine proteinase and gelatinase, is associated with longer survival in patients with invasive ductal carcinoma of breast. Int.J.Cancer 95, 67-72. 111. Iwasa, S., Okada, K., Chen, W. T., Jin, X., Yamane, T., Ooi, A., and Mitsumata, M. (2005) 'Increased expression of seprase, a membrane-type serine protease, is associated with lymph node metastasis in human colorectal cancer'. Cancer Lett. 227, 229-236. 112. Iwasa, S., Jin, X., Okada, K., Mitsumata, M., and Ooi, A. (2003) Increased expression of seprase, a membrane-type serine protease, is associated with lymph node metastasis in human colorectal cancer. Cancer Lett. 199, 91-98. 113. Jin, X., Iwasa, S., Okada, K., Mitsumata, M., and Ooi, A. (2003) Expression patterns of seprase, a membrane serine protease, in cervical carcinoma and cervical intraepithelial neoplasm. Anticancer Res. 23, 3195-3198. 114. Huber, M. A., Kraut, N., Park, J. E., Schubert, R. D., Rettig, W. J., Peter, R. U., and Garin-Chesa, P. (2003) Fibroblast activation protein: differential expression and serine protease activity in reactive stromal fibroblasts of melanocytic skin tumors. J.Invest Dermatol. 120, 182-188. 115. Mori, Y., Kono, K., Matsumoto, Y., Fujii, H., Yamane, T., Mitsumata, M., and Chen, W. T. (2004) The expression of a type II transmembrane serine protease (Seprase) in human gastric carcinoma. Oncology 67, 411-419. 116. Okada, K., Chen, W. T., Iwasa, S., Jin, X., Yamane, T., Ooi, A., and Mitsumata, M. (2003) Seprase, a membrane-type serine protease, has different expression patterns in intestinal- and diffuse-type gastric cancer. Oncology 65, 363-370. 117. Goodman, J. D., Rozypal, T. L., and Kelly, T. (2003) Seprase, a membrane-bound protease, alleviates the serum growth requirement of human breast cancer cells. Clin.Exp.Metastasis 20, 459-470. 118. Kelly, T., Kechelava, S., Rozypal, T. L., West, K. W., and Korourian, S. (1998) Seprase, a membrane-bound protease, is overexpressed by invasive ductal carcinoma cells of human breast cancers. Mod.Pathol. 11, 855-863. 119. Skubitz, K. M. and Skubitz, A. P. (2004) Gene expression in aggressive fibromatosis. J Lab Clin.Med. 143, 89-98. 120. Wesley, U. V., Tiwari, S., and Houghton, A. N. (2004) Role for dipeptidyl peptidase IV in tumor suppression of human non small cell lung carcinoma cells. Int.J.Cancer 109, 855-866. 121. Artym, V. V., Kindzelskii, A. L., Chen, W. T., and Petty, H. R. (2002) Molecular proximity of seprase and the urokinase-type plasminogen activator receptor on malignant melanoma cell membranes: dependence on beta1 integrins and the cytoskeleton. Carcinogenesis 23, 1593-1601.

112 Reference

122. Niedermeyer, J., Kriz, M., Hilberg, F., Garin-Chesa, P., Bamberger, U., Lenter, M. C., Park, J., Viertel, B., Puschner, H., Mauz, M., Rettig, W. J., and Schnapp, A. (2000) Targeted disruption of mouse fibroblast activation protein. Mol.Cell Biol. 20, 1089-1094. 123. Lee, K. N., Jackson, K. W., Christiansen, V. J., Chung, K. H., and McKee, P. A. (2004) A Novel Plasma Proteinase Potentiates {alpha}2-Antiplasmin Inhibition of Fibrin . Blood 103, 3783-3788. 124. Lee, K. N., Jackson, K. W., Christiansen, V. J., Chung, K. H., and McKee, P. A. (2004) Alpha2-antiplasmin: potential therapeutic roles in fibrin survival and removal. Curr.Med.Chem.Cardiovasc.Hematol.Agents 2, 303-310. 125. Jones, B., Adams, S., Miller, G. T., Jesson, M. I., Watanabe, T., and Wallner, B. P. (2003) Hematopoietic stimulation by a dipeptidyl peptidase inhibitor reveals a novel regulatory mechanism and therapeutic treatment for blood cell deficiencies. Blood 102, 641-648. 126. Nemunaitis, J., Vukelja, S. J., Richards, D., Cunningham, C., Senzer, N., Nugent, J., Duncan, H., Jones, B., Haltom, E., and Uprichard, M. J. (2006) Phase I trial of PT-100 (PT-100), a cytokine-inducing small molecule, following chemotherapy for solid tumor malignancy. Cancer Invest 24, 553-561. 127. Bjelke, J. R., Christensen, J., Nielsen, P. F., Branner, S., Kanstrup, A. B., Wagtmann, N., and Rasmussen, H. B. (2006) Dipeptidyl peptidase 8 and 9 specificity and molecular characterization compared to dipeptidyl peptidase IV. Biochem.J. 396, 391-396. 128. Chen, T., Ajami, K., McCaughan, G. W., Gorrell, M. D., and Abbott, C. A. (2003) Dipeptidyl peptidase IV gene family. The DPIV family. Adv.Exp.Med.Biol. 524, 79-86. 129. Yu, L. R., Zhu, Z., Chan, K. C., Issaq, H. J., Dimitrov, D. S., and Veenstra, T. D. (2007) Improved titanium dioxide enrichment of phosphopeptides from HeLa cells and high confident phosphopeptide identification by cross-validation of MS/MS and MS/MS/MS spectra. J.Proteome.Res. 6, 4150-4162. 130. Ajami, K., Pitman, M. R., Wilson, C. H., Park, J., Menz, R. I., Starr, A. E., Cox, J. H., Abbott, C. A., Overall, C. M., and Gorrell, M. D. (2008) Stromal cell-derived factors 1alpha and 1beta, inflammatory protein-10 and interferon-inducible T cell chemo-attractant are novel substrates of dipeptidyl peptidase 8. FEBS Lett. 582, 819-825. 131. Wagner, L., Hoffmann, T., Rahfeld, J. U., and Demuth, H. U. (2006) Distribution of dipeptidyl peptidase IV-like activity enzymes in canine and porcine tissue sections by RT-PCR. Adv.Exp.Med.Biol. 575, 109-116. 132. Geiss-Friedlander, R., Parmentier, N., Moller, U., Urlaub, H., Van den Eynde, B. J., and Melchior, F. (2009) The cytoplasmic peptidase DPP9 is rate-limiting for degradation of proline-containing peptides. J Biol.Chem. 284, 27211- 27219. 133. Yao, T. W., Kim, W. S., Yu, D. M., Sharbeen, G., McCaughan, G. W., Choi, K. Y., Xia, P., and Gorrell, M. D. (2011) A novel role of dipeptidyl peptidase 9 in epidermal growth factor signaling. Mol.Cancer Res. 9, 948-959. 134. Yu, D. M., Ajami, K., Gall, M. G., Park, J., Lee, C. S., Evans, K. A., McLaughlin, E. A., Pitman, M. R., Abbott, C. A., McCaughan, G. W., and Gorrell, M. D. (2009) The In Vivo Expression of Dipeptidyl Peptidases 8 and 9. J.Histochem.Cytochem. 57, 1025-1040. 135. Frerker, N., Wagner, L., Wolf, R., Heiser, U., Hoffmann, T., Rahfeld, J. U., Schade, J., Karl, T., Naim, H. Y., Alfalah, M., Demuth, H. U., and Von Horsten, S. (2007) Neuropeptide Y (NPY) cleaving enzymes: structural and functional homologues of dipeptidyl peptidase 4. Peptides 28, 257-268. 136. Rohnert, P., Schmidt, W., Emmerlich, P., Goihl, A., Wrenger, S., Bank, U., Nordhoff, K., Tager, M., Ansorge, S., Reinhold, D., and Striggow, F. (2012) Dipeptidyl peptidase IV, aminopeptidase N and DPIV/APN-like proteases in cerebral ischemia. J Neuroinflammation. 9, 44. 137. Dubois, V., Lambeir, A. M., Vandamme, S., Matheeussen, V., Guisez, Y., Scharpe, S., and De, M., I (2010) Dipeptidyl peptidase 9 (DPP9) from bovine testes: Identification and characterization as the short form by mass spectrometry. Biochim.Biophys.Acta 1804, 781-788. 138. Dubois, V., Lambeir, A. M., Van der Veken, P., Augustyns, K., Creemers, J., Chen, X., Scharpe, S., and De, M., I (2008) Purification and characterization of dipeptidyl peptidase IV-like enzymes from bovine testes. Front Biosci. 13, 3558-3568. 139. Dubois, V., Van Ginneken, C., De Cock, H., Lambeir, A. M., Van der Veken, P., Augustyns, K., Chen, X., Scharpe, S., and De, M., I (2009) Enzyme activity and immunohistochemical localization of dipeptidyl peptidase 8 and 9 in male reproductive tissues. J.Histochem.Cytochem. 57, 531-541. 140. Maes, M. B., Dubois, V., Brandt, I., Lambeir, A. M., Van der Veken, P., Augustyns, K., Cheng, J. D., Chen, X., Scharpe, S., and De Meester, I. (2007) Dipeptidyl peptidase 8/9-like activity in human leukocytes. J Leukoc.Biol. 81, 1252-1257. 141. Lankas, G., Leiting, B., Sinha, R. R., Eiermann, G., Biftu, T., Kim, D., Ok, H., Weber, A. E., and Thornberry, N. (2004) Inhibition of DP8/9 Results in Toxitiy in Preclinical Species: Potential Imortance of Selective Dipeptidyl Peptidase IV Inhibition for Treatment of Type 2 DM. Diabetes 53, A2. 142. Lankas, G. R., Leiting, B., Roy, R. S., Eiermann, G. J., Beconi, M. G., Biftu, T., Chan, C. C., Edmondson, S., Feeney, W. P., He, H., Ippolito, D. E., Kim, D., Lyons, K. A., Ok, H. O., Patel, R. A., Petrov, A. N., Pryor, K. A., Qian, X., Reigle, L., Woods, A., Wu, J. K., Zaller, D., Zhang, X., Zhu, L., Weber, A. E., and Thornberry, N. A. (2005) Dipeptidyl Peptidase IV Inhibition for the Treatment of Type 2 Diabetes: Potential Importance of Selectivity Over Dipeptidyl Peptidases 8 and 9. Diabetes 54, 2988-2994. 143. Reinhold, D., Goihl, A., Wrenger, S., Reinhold, A., Kuhlmann, U. C., Faust, J., Neubert, K., Thielitz, A., Brocke, S., Tager, M., Ansorge, S., and Bank, U. (2009) Role of dipeptidyl peptidase IV (DP IV)-like enzymes in T lymphocyte activation: investigations in DP IV/CD26-knockout mice. Clin.Chem.Lab Med. 47, 268-274. 144. Eager, R. M., Cunningham, C. C., Senzer, N. N., Stephenson, J., Jr., Anthony, S. P., SJ, O. D., Frenette, G., Pavlick, A. C., Jones, B., Uprichard, M., and Nemunaitis, J. (2009) Phase II assessment of talabostat and cisplatin in second- line stage IV melanoma. BMC.Cancer 9, 263. 145. Burkey, B. F., Hoffmann, P. K., Hassiepen, U., Trappe, J., Juedes, M., and Foley, J. E. (2008) Adverse effects of dipeptidyl peptidases 8 and 9 inhibition in rodents revisited. Diabetes Obes.Metab 10, 1057-1061. 146. Wu, J. J., Tang, H. K., Yeh, T. K., Chen, C. M., Shy, H. S., Chu, Y. R., Chien, C. H., Tsai, T. Y., Huang, Y. C., Huang, Y. L., Huang, C. H., Tseng, H. Y., Jiaang, W. T., Chao, Y. S., and Chen, X. (2009) Biochemistry, pharmacokinetics, and toxicology of a potent and selective DPP8/9 inhibitor. Biochem.Pharmacol. 78, 203-210. 147. Kirby, M., Yu, D. M., O'Connor, S., and Gorrell, M. D. (2010) Inhibitor selectivity in the clinical application of dipeptidyl peptidase-4 inhibition. Clin.Sci.(Lond) 118, 31-41.

113 Reference

148. Bank, U., Heimburg, A., Wohlfarth, A., Koch, G., Nordhoff, K., Julius, H., Helmuth, M., Breyer, D., Reinhold, D., Tager, M., and Ansorge, S. (2011) Outside or inside: role of the subcellular localization of DP4-like enzymes for substrate conversion and inhibitor effects. Biol Chem 392, 169-187. 149. Yu, D. M., Wang, X. M., McCaughan, G. W., and Gorrell, M. D. (2006) Extraenzymatic functions of the dipeptidyl peptidase IV-related proteins DP8 and DP9 in cell adhesion, migration and apoptosis. FEBS J 273, 2447-2460. 150. Park, J., Knott, H. T., Navdi, N. A., Collyer, C. A., Wang, X. M., Church, W. B., and Gorrell, M. D. (2008) Reversible Inactivation of Human Dipeptidyl Peptidases 8 and 9 by Oxidation. The Open Enzyme Inhibition Journal 1, 52-60. 151. Rummey, C., Nordhoff, S., Thiemann, M., and Metz, G. (2006) In silico fragment-based discovery of DPP-IV S1 pocket binders. Bioorg.Med.Chem.Lett. 16, 1405-1409. 152. Rummey, C. and Metz, G. (2007) Homology models of dipeptidyl peptidases 8 and 9 with a focus on loop predictions near the active site. Proteins 66, 160-171. 153. Oefner, C., D'Arcy, A., Mac, S. A., Pierau, S., Gardiner, R., and Dale, G. E. (2003) High-resolution structure of human apo dipeptidyl peptidase IV/CD26 and its complex with 1-[({2-[(5-iodopyridin-2-yl)amino]-ethyl}amino)-acetyl]-2-cyano- (S)-pyrrol idine. Acta Crystallogr.D.Biol.Crystallogr. 59, 1206-1212. 154. Thoma, R., Loffler, B., Stihle, M., Huber, W., Ruf, A., and Hennig, M. (2003) Structural Basis of Proline-Specific Exopeptidase Activity as Observed in Human Dipeptidyl Peptidase-IV. Structure.(Camb.) 11, 947-959. 155. Zagha, E., Ozaita, A., Chang, S. Y., Nadal, M. S., Lin, U., Saganich, M. J., McCormack, T., Akinsanya, K. O., Qi, S. Y., and Rudy, B. (2005) Dipeptidyl peptidase 10 modulates Kv4-mediated A-type potassium channels. J Biol.Chem. 280, 18853-18861. 156. Kin, Y., Misumi, Y., and Ikehara, Y. (2001) Biosynthesis and characterization of the brain-specific membrane protein DPPX, a dipeptidyl peptidase IV-related protein. J.Biochem.(Tokyo) 129, 289-295. 157. Nadal, M. S., Ozaita, A., Amarillo, Y., de Miera, E. V., Ma, Y., Mo, W., Goldberg, E. M., Misumi, Y., Ikehara, Y., Neubert, T. A., and Rudy, B. (2003) The CD26-related dipeptidyl aminopeptidase-like protein DPPX is a critical component of neuronal A-type K+ channels. Neuron 37, 449-461. 158. Chen, T., Ajami, K., McCaughan, G. W., Gai, W. P., Gorrell, M. D., and Abbott, C. A. (2006) Molecular characterization of a novel dipeptidyl peptidase like 2-short form (DPL2-s) that is highly expressed in the brain and lacks dipeptidyl peptidase activity. Biochim.Biophys.Acta. 1764, 33-43. 159. Strop, P., Bankovich, A. J., Hansen, K. C., Garcia, K. C., and Brunger, A. T. (2004) Structure of a human A-type potassium channel interacting protein DPPX, a member of the dipeptidyl aminopeptidase family. J.Mol.Biol. 343, 1055-1065. 160. Blumenthal, M. N. (2005) The role of genetics in the development of asthma and atopy. Curr.Opin.Allergy Clin.Immunol. 5, 141-145. 161. Holgate, S. T., Davies, D. E., Powell, R. M., Howarth, P. H., Haitchi, H. M., and Holloway, J. W. (2007) Local genetic and environmental factors in asthma disease pathogenesis: chronicity and persistence mechanisms. Eur Respir.J 29, 793-803. 162. Weiss, S. T. and Raby, B. A. (2004) Asthma genetics 2003. Hum.Mol.Genet. 13, R83-R89. 163. Mentlein, R. and Struckhoff, G. (1989) Purification of two dipeptidyl aminopeptidases II from rat brain and their action on proline-containing neuropeptides. J Neurochem 52, 1284-1293. 164. Duke-Cohan, J. S., Morimoto, C., Rocker, J. A., and Schlossman, S. F. (1996) Serum high molecular weight dipeptidyl peptidase IV (CD26) is similar to a novel antigen DPPT-L released from activated T cells. J.Immunol. 156, 1714-1721. 165. Pangalos, M. N., Neefs, J. M., Somers, M., Verhasselt, P., Bekkers, M., van der Helm, L., Fraiponts, E., Ashton, D., and Gordon, R. D. (1999) Isolation and expression of novel human glutamate with N-acetylated alpha-linked acidic dipeptidase and dipeptidyl peptidase IV activity. J.Biol.Chem. 274, 8470-8483. 166. Barinka, C., Rinnova, M., Sacha, P., Rojas, C., Majer, P., Slusher, B. S., and Konvalinka, J. (2002) Substrate specificity, inhibition and enzymological analysis of recombinant human glutamate II. J Neurochem. 80, 477-487. 167. Duke-Cohan, J. S., Morimoto, C., Rocker, J. A., and Schlossman, S. F. (1995) A novel form of dipeptidylpeptidase IV found in human serum. Isolation, characterization, and comparison with T lymphocyte membrane dipeptidylpeptidase IV (CD26). J.Biol.Chem. 270, 14107-14114. 168. Duke-Cohan, J. S., Kim, J. H., and Azouz, A. (2004) Attractin: cautionary tales for therapeutic intervention in molecules with pleiotropic functionality. J Environ.Pathol.Toxicol.Oncol. 23, 1-11. 169. Durinx, C., Lambeir, A. M., Bosmans, E., Falmagne, J. B., Berghmans, R., Haemers, A., Scharpe, S., and De Meester, I. (2000) Molecular characterization of dipeptidyl peptidase activity in serum: soluble CD26/dipeptidyl peptidase IV is responsible for the release of X-Pro dipeptides. Eur J Biochem 267, 5608-5613. 170. Friedrich, D., Hoffmann, T., Bar, J., Wermann, M., Manhart, S., Heiser, U., and Demuth, H. U. (2007) Does human attractin have DP4 activity? Biol.Chem. 388, 155-162. 171. Araki, H., Li, Y., Yamamoto, Y., Haneda, M., Nishi, K., Kikkawa, R., and Ohkubo, I. (2001) Purification, molecular cloning, and immunohistochemical localization of dipeptidyl peptidase II from the rat kidney and its identity with quiescent cell proline dipeptidase. J.Biochem.(Tokyo) 129, 279-288. 172. Leiting, B., Pryor, K. D., Wu, J. K., Marsilio, F., Patel, R. A., Craik, C. S., Ellman, J. A., Cummings, R. T., and Thornberry, N. A. (2003) Catalytic properties and inhibition of proline-specific dipeptidyl peptidases II, IV and VII. Biochem J 371, 525-532. 173. Maes, M. B., Lambeir, A. M., Gilany, K., Senten, K., Van der Veken, P., Leiting, B., Augustyns, K., Scharpe, S., and De Meester, I. (2005) Kinetic investigation of human dipeptidyl peptidase II (DPPII)-mediated hydrolysis of dipeptide derivatives and its identification as quiescent cell proline dipeptidase (QPP)/dipeptidyl peptidase 7 (DPP7). Biochem J 386, 315-324. 174. Underwood, R., Chiravuri, M., Lee, H., Schmitz, T., Kabcenell, A. K., Yardley, K., and Huber, B. T. (1999) Sequence, purification, and cloning of an intracellular serine protease, quiescent cell proline dipeptidase. J Biol.Chem. 274, 34053-34058. 175. Chiravuri, M., Agarraberes, F., Mathieu, S. L., Lee, H., and Huber, B. T. (2000) Vesicular localization and characterization of a novel post-proline-cleaving aminodipeptidase, quiescent cell proline dipeptidase. J.Immunol. 165, 5695-5702.

114 Reference

176. Maes, M. B., Scharpe, S., and De Meester, I. (2007) Dipeptidyl peptidase II (DPPII), a review. Clin.Chim.Acta 380, 31- 49. 177. Chiravuri, M., Lee, H., Mathieu, S. L., and Huber, B. T. (2000) Homodimerization via a leucine zipper motif is required for enzymatic activity of quiescent cell proline dipeptidase. J Biol.Chem. 275, 26994-26999. 178. Mentlein, R. and Struckhoff, G. (1989) Purification of two dipeptidyl aminopeptidases II from rat brain and their action on proline-containing neuropeptides. J.Neurochem. 52, 1284-1293. 179. Brandt, I., Lambeir, A. M., Maes, M. B., Scharpe, S., and De, M., I (2006) Peptide substrates of dipeptidyl peptidases. Adv.Exp.Med.Biol. 575, 3-18. 180. Chiravuri, M. and Huber, B. T. (2000) Aminodipeptidase inhibitor-induced cell death in quiescent lymphocytes: a review. Apoptosis 5, 319-322. 181. Chiravuri, M., Schmitz, T., Yardley, K., Underwood, R., Dayal, Y., and Huber, B. T. (1999) A novel apoptotic pathway in quiescent lymphocytes identified by inhibition of a post-proline cleaving aminodipeptidase: a candidate target protease, quiescent cell proline dipeptidase. J.Immunol. 163, 3092-3099. 182. Mele, D. A., Bista, P., Baez, D. V., and Huber, B. T. (2009) Dipeptidyl peptidase 2 is an essential survival factor in the regulation of cell quiescence. Cell Cycle 8, 2425-2434. 183. Bista, P., Mele, D. A., Baez, D. V., and Huber, B. T. (2008) Lymphocyte quiescence factor Dpp2 is transcriptionally activated by KLF2 and TOB1. Mol.Immunol. 45, 3618-3623. 184. Maes, M. B., Martinet, W., Schrijvers, D. M., Van der Veken, P., De Meyer, G. R., Augustyns, K., Lambeir, A. M., Scharpe, S., and De Meester, I. (2006) Dipeptidyl peptidase II and leukocyte cell death. Biochem Pharmacol. 72, 70- 79. 185. Gossrau, R. and Lojda, Z. (1980) Study on dipeptidylpeptidase II (DPP II). Histochemistry 70, 53-76. 186. Danilova, O. V., Tai, A. K., Mele, D. A., Beinborn, M., Leiter, A. B., Greenberg, A. S., Perfield, J. W., Defuria, J., Singru, P. S., Lechan, R. M., and Huber, B. T. (2009) Neurogenin 3-Specific Dipeptidyl Peptidase-2 Deficiency Causes Impaired Glucose Tolerance, Insulin Resistance, and Visceral Obesity. Endocrinology 150, 5240-5248. 187. Sharoyan, S. G., Antonyan, A. A., Mardanyan, S. S., Lupidi, G., Cuccioloni, M., Angeletti, M., and Cristalli, G. (2008) Complex of dipeptidyl peptidase II with adenosine deaminase. Biochemistry (Mosc.) 73, 943-949. 188. Longenecker, K. L., Stewart, K. D., Madar, D. J., Jakob, C. G., Fry, E. H., Wilk, S., Lin, C. W., Ballaron, S. J., Stashko, M. A., Lubben, T. H., Yong, H., Pireh, D., Pei, Z., Basha, F., Wiedeman, P. E., von Geldern, T. W., Trevillyan, J. M., and Stoll, V. S. (2006) Crystal structures of DPP-IV (CD26) from rat kidney exhibit flexible accommodation of peptidase-selective inhibitors. Biochemistry 45, 7474-7482. 189. Yu, D. M., Yao, T. W., Chowdhury, S., Nadvi, N. A., Osborne, B., Church, W. B., McCaughan, G. W., and Gorrell, M. D. (2010) The dipeptidyl peptidase IV family in cancer and cell biology. FEBS J 277, 1126-1144. 190. Yazbeck, R., Howarth, G. S., and Abbott, C. A. (2009) Dipeptidyl peptidase inhibitors, an emerging drug class for inflammatory disease? Trends Pharmacol.Sci. 30, 600-607. 191. McNicholas, K., Chen, T., and Abbott, C. A. (2009) Dipeptidyl peptidase (DP) 6 and DP10: novel brain proteins implicated in human health and disease. Clin.Chem.Lab Med. 47, 262-267. 192. van Es, M. A., van Vught, P. W., van Kempen, G., Blauw, H. M., Veldink, J. H., and van den Berg, L. H. (2009) Dpp6 is associated with susceptibility to progressive spinal muscular atrophy. Neurology 72, 1184-1185. 193. Bezerra, G. A., Dobrovetsky, E., Seitova, A., Dhe-Paganon, S., and Gruber, K. (2012) Crystallization and preliminary X-ray diffraction analysis of human dipeptidyl peptidase 10 (DPPY), a component of voltage-gated potassium channels. Acta Crystallogr.Sect.F.Struct.Biol.Cryst.Commun. 68, 214-217. 194. van Es, M. A., van Vught, P. W., Blauw, H. M., Franke, L., Saris, C. G., Van den, B. L., de Jong, S. W., de, J., V, Baas, F., Van't Slot, R., Lemmens, R., Schelhaas, H. J., Birve, A., Sleegers, K., Van Broeckhoven, C., Schymick, J. C., Traynor, B. J., Wokke, J. H., Wijmenga, C., Robberecht, W., Andersen, P. M., Veldink, J. H., Ophoff, R. A., and van den Berg, L. H. (2008) Genetic variation in DPP6 is associated with susceptibility to amyotrophic lateral sclerosis. Nat.Genet. 40, 29-31. 195. Garber, K. (2008) Genetics. The elusive ALS genes. Science 319, 20. 196. Herrera, C., Morimoto, C., Blanco, J., Mallol, J., Arenzana, F., Lluis, C., and Franco, R. (2001) Comodulation of CXCR4 and CD26 in human lymphocytes. J Biol.Chem. 276, 19532-19539. 197. Ikushima, H., Munakata, Y., Iwata, S., Ohnuma, K., Kobayashi, S., Dang, N. H., and Morimoto, C. (2002) Soluble CD26/dipeptidyl peptidase IV enhances transendothelial migration via its interaction with mannose 6- phosphate/insulin-like growth factor II receptor. Cell Immunol. 215, 106-110. 198. Ikushima, H., Munakata, Y., Ishii, T., Iwata, S., Terashima, M., Tanaka, H., Schlossman, S. F., and Morimoto, C. (2000) Internalization of CD26 by mannose 6-phosphate/insulin-like growth factor II receptor contributes to T cell activation. Proc.Natl.Acad.Sci.U.S.A.. 97, 8439-8444. 199. Ishii, T., Ohnuma, K., Murakami, A., Takasawa, N., Kobayashi, S., Dang, N. H., Schlossman, S. F., and Morimoto, C. (2001) CD26-mediated signaling for T cell activation occurs in lipid rafts through its association with CD45RO. Proc.Natl.Acad.Sci.U.S.A. 98, 12138-12143. 200. Boonacker, E. and Van Noorden, C. J. (2003) The multifunctional or moonlighting protein CD26/DPPIV. Eur.J Cell Biol. 82, 53-73. 201. Mentlein, R. (1999) Dipeptidyl-peptidase IV (CD26)--role in the inactivation of regulatory peptides. Regul.Pept. 85, 9- 24. 202. Gorrell, M. D., Gysbers, V., and McCaughan, G. W. (2001) CD26: a multifunctional integral membrane and secreted protein of activated lymphocytes. Scand.J Immunol. 54, 249-264. 203. Dang, N. H. and Morimoto, C. (2002) CD26: an expanding role in immune regulation and cancer. Histol.Histopathol. 17, 1213-1226. 204. Hildebrandt, M., Reutter, W., Arck, P., Rose, M., and Klapp, B. F. (2000) A guardian angel: the involvement of dipeptidyl peptidase IV in psychoneuroendocrine function, nutrition and immune defence. Clin.Sci.(Lond.) 99, 93-104. 205. Gonzalez-Gronow, M., Hershfield, M. S., Arredondo-Vega, F. X., and Pizzo, S. V. (2004) Cell surface adenosine deaminase binds and stimulates plasminogen activation on 1-LN human prostate cancer cells. J Biol.Chem. 279, 20993-20998. 206. Hildebrandt, M., Reutter, W., Arck, P., Rose, M., and Klapp, B. F. (2000) A guardian angel: the involvement of dipeptidyl peptidase IV in psychoneuroendocrine function, nutrition and immune defence. Clin.Sci.(Lond.) 99, 93-104.

115 Reference

207. De Meester, I., Korom, S., Van Damme, J., and Scharpe, S. (1999) CD26, let it cut or cut it down. Immunol.Today 20, 367-375. 208. De Meester, I., Durinx, C., Proost, P., Scharpe, S., and Lambeir, A. M. (2002) DP IV - Natural substrates of medical importance. In Langner, J. and Ansorge, S., editors. Ectopeptidases, Kluwer Academic/Plenum Publishers, New York, USA, 223-257. 209. Kuhn-Wache, K., Bar, J. W., Hoffmann, T., Wolf, R., Rahfeld, J. U., and Demuth, H. U. (2011) Selective inhibition of dipeptidyl peptidase 4 by targeting a substrate-specific secondary binding site. Biol Chem 392, 223-231. 210. Bjelke, J. R., Kanstrup, A. B., and Rasmussen, H. B. (2006) Selectivity among dipeptidyl peptidases of the s9b family. Cell Mol.Biol.(Noisy.-le-grand) 52, 3-7. 211. Hoffmann, T., Faust, J., Neubert, K., and Ansorge, S. (1993) Dipeptidyl peptidase IV (CD 26) and aminopeptidase N (CD 13) catalyzed hydrolysis of cytokines and peptides with N-terminal cytokine sequences. FEBS Lett. 336, 61-64. 212. Mentlein, R., Dahms, P., Grandt, D., and Kruger, R. (1993) Proteolytic processing of neuropeptide Y and peptide YY by dipeptidyl peptidase IV. Regul.Pept. 49, 133-144. 213. Batterham, R. L. and Bloom, S. R. (2003) The gut hormone peptide YY regulates appetite. Ann.N.Y.Acad.Sci. 994, 162-168. 214. Lambeir, A. M., Durinx, C., Proost, P., Van Damme, J., Scharpe, S., and De Meester, I. (2001) Kinetic study of the processing by dipeptidyl-peptidase IV/CD26 of neuropeptides involved in pancreatic insulin secretion. FEBS Lett. 507, 327-330. 215. Proost, P., Struyf, S., Schols, D., Durinx, C., Wuyts, A., Lenaerts, J. P., De Clercq, E., De Meester, I., and Van Damme, J. (1998) Processing by CD26/dipeptidyl-peptidase IV reduces the chemotactic and anti-HIV-1 activity of stromal-cell-derived factor-1alpha. FEBS Lett. 432, 73-76. 216. Lambeir, A. M., Proost, P., Durinx, C., Bal, G., Senten, K., Augustyns, K., Scharpe, S., Van Damme, J., and De Meester, I. (2001) Kinetic investigation of chemokine truncation by CD26/dipeptidyl peptidase IV reveals a striking selectivity within the chemokine family. J Biol.Chem. 276, 29839-29845. 217. Proost, P., Struyf, S., Schols, D., Opdenakker, G., Sozzani, S., Allavena, P., Mantovani, A., Augustyns, K., Bal, G., Haemers, A., Lambeir, A. M., Scharpe, S., Van Damme, J., and De Meester, I. (1999) Truncation of macrophage- derived chemokine by CD26/ dipeptidyl- peptidase IV beyond its predicted cleavage site affects chemotactic activity and CC chemokine receptor 4 interaction. J.Biol.Chem. 274, 3988-3993. 218. Ludwig, A., Schiemann, F., Mentlein, R., Lindner, B., and Brandt, E. (2002) Dipeptidyl peptidase IV (CD26) on T cells cleaves the CXC chemokine CXCL11 (I-TAC) and abolishes the stimulating but not the desensitizing potential of the chemokine. J Leukoc.Biol. 72, 183-191. 219. Proost, P., Schutyser, E., Menten, P., Struyf, S., Wuyts, A., Opdenakker, G., Detheux, M., Parmentier, M., Durinx, C., Lambeir, A. M., Neyts, J., Liekens, S., Maudgal, P. C., Billiau, A., and Van Damme, J. (2001) Amino-terminal truncation of CXCR3 agonists impairs receptor signaling and lymphocyte chemotaxis, while preserving antiangiogenic properties. Blood 98, 3554-3561. 220. Proost, P., De, M., I, Schols, D., Struyf, S., Lambeir, A. M., Wuyts, A., Opdenakker, G., De Clercq, E., Scharpe, S., and Van Damme, J. (1998) Amino-terminal truncation of chemokines by CD26/dipeptidyl-peptidase IV. Conversion of RANTES into a potent inhibitor of monocyte chemotaxis and HIV-1-infection. J.Biol.Chem. 273, 7222-7227. 221. Proost, P., Menten, P., Struyf, S., Schutyser, E., De Meester, I., and Van Damme, J. (2000) Cleavage by CD26/dipeptidyl peptidase IV converts the chemokine LD78beta into a most efficient monocyte attractant and CCR1 agonist. Blood 96, 1674-1680. 222. Shioda, T., Kato, H., Ohnishi, Y., Tashiro, K., Ikegawa, M., Nakayama, E. E., Hu, H., Kato, A., Sakai, Y., Liu, H., Honjo, T., Nomoto, A., Iwamoto, A., Morimoto, C., and Nagai, Y. (1998) Anti-HIV-1 and chemotactic activities of human stromal cell-derived factor 1alpha (SDF-1alpha) and SDF-1beta are abolished by CD26/dipeptidyl peptidase IV-mediated cleavage. Proc.Natl.Acad.Sci.U.S.A. 95, 6331-6336. 223. Bongers, J., Lambros, T., Ahmad, M., and Heimer, E. P. (1992) Kinetics of dipeptidyl peptidase IV proteolysis of growth hormone- releasing factor and analogs. Biochim.Biophys.Acta 1122, 147-153. 224. Zhu, L., Tamvakopoulos, C., Xie, D., Dragovic, J., Shen, X., Fenyk-Melody, J. E., Schmidt, K., Bagchi, A., Griffin, P. R., Thornberry, N. A., and Sinha, R. R. (2003) The role of dipeptidyl peptidase IV in the cleavage of glucagon family peptides: in vivo metabolism of pituitary adenylate cyclase activating polypeptide [1-38]. J Biol.Chem. 278, 22418- 22423. 225. Lambeir, A. M., Proost, P., Scharpe, S., and De Meester, I. (2002) A kinetic study of glucagon-like peptide-1 and glucagon-like peptide-2 truncation by dipeptidyl peptidase IV, in vitro. Biochem Pharmacol. 64, 1753-1756. 226. Pauly, R. P., Rosche, F., Wermann, M., McIntosh, C. H., Pederson, R. A., and Demuth, H. U. (1996) Investigation of glucose-dependent insulinotropic polypeptide-(1-42) and glucagon-like peptide-1-(7-36) degradation in vitro by dipeptidyl peptidase IV using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. A novel kinetic approach. J.Biol.Chem. 271, 23222-23229. 227. Mentlein, R., Gallwitz, B., and Schmidt, W. E. (1993) Dipeptidyl-peptidase IV hydrolyses gastric inhibitory polypeptide, glucagon-like peptide-1(7-36)amide, peptide histidine methionine and is responsible for their degradation in human serum. Eur.J.Biochem. 214, 829-835. 228. Hinke, S. A., Pospisilik, J. A., Demuth, H. U., Mannhart, S., Kuhn-Wache, K., Hoffmann, T., Nishimura, E., Pederson, R. A., and McIntosh, C. H. (2000) Dipeptidyl peptidase IV (DPIV/CD26) degradation of glucagon. Characterization of glucagon degradation products and DPIV-resistant analogs. J Biol.Chem. 275, 3827-3834. 229. Pospisilik, J. A., Hinke, S. A., Pederson, R. A., Hoffmann, T., Rosche, F., Schlenzig, D., Glund, K., Heiser, U., McIntosh, C. H., and Demuth, H. (2001) Metabolism of glucagon by dipeptidyl peptidase IV (CD26). Regul.Pept. 96, 133-141. 230. Nausch, I. and Heymann, E. (1985) Substance P in human plasma is degraded by dipeptidyl peptidase IV, not by cholinesterase. J.Neurochem. 44, 1354-1357. 231. Ahmad, S., Wang, L., and Ward, P. E. (1992) Dipeptidyl(amino)peptidase IV and aminopeptidase M metabolize circulating substance P in vivo. J.Pharmacol.Exp.Ther. 260, 1257-1261. 232. Brandt, I., Lambeir, A. M., Ketelslegers, J. M., Vanderheyden, M., Scharpe, S., and De, M., I (2006) Dipeptidyl- peptidase IV converts intact B-type natriuretic peptide into its des-SerPro form. Clin.Chem. 52, 82-87. 233. Lin, C.T., Chien, C.H., Tseng, B., Juo, C.G., Han, Y.S., and Chen, X. IGF-1 is a Substrate of Prolyl dipeptidase DPP- IV. 2008, Antwerp, Belgium.

116 Reference

234. Cohen, M., Fruitier-Arnaudin, I., and Piot, J. M. (2004) Hemorphins: substrates and/or inhibitors of dipeptidyl peptidase IV; Hemorphins N-terminus sequence influence on the interaction between hemorphins and DPPIV. Biochimie 86, 31-37. 235. Nausch, I., Mentlein, R., and Heymann, E. (1990) The degradation of bioactive peptides and proteins by dipeptidyl peptidase IV from human placenta. Biol.Chem.Hoppe Seyler 371, 1113-1118. 236. Bird, A. P., Faltinek, J. R., and Shojaei, A. H. (2001) Transbuccal peptide delivery: stability and in vitro permeation studies on endomorphin-1. J Control.Release. 73, 31-36. 237. Shane, R., Wilk, S., and Bodnar, R. J. (1999) Modulation of endomorphin-2-induced analgesia by dipeptidyl peptidase IV. Brain Res. 815, 278-286. 238. Sakurada, C., Sakurada, S., Hayashi, T., Katsuyama, S., Tan-No, K., and Sakurada, T. (2003) Degradation of endomorphin-2 at the supraspinal level in mice is initiated by dipeptidyl peptidase IV: an in vitro and in vivo study. Biochem.Pharmacol. 66, 653-661. 239. Bouras, M., Huneau, J. F., Luengo, C., Erlanson-Albertsson, C., and Tome, D. (1995) Metabolism of enterostatin in rat intestine, brain membranes, and serum: differential involvement of proline-specific peptidases. Peptides 16, 399- 405. 240. Tiruppathi, C., Miyamoto, Y., Ganapathy, V., and Leibach, F. H. (1993) Genetic evidence for role of DPP IV in intestinal hydrolysis and assimilation of prolyl peptides. Am.J.Physiol. 265, G81-G89. 241. Zhang, X. Y., De Meester, I., Lambeir, A. M., Dillen, L., Van Dongen, W., Esmans, E. L., Haemers, A., Scharpe, S., and Claeys, M. (1999) Study of the enzymatic degradation of vasostatin I and II and their precursor chromogranin A by dipeptidyl peptidase IV using high- performance liquid chromatography/electrospray mass spectrometry. J.Mass.Spectrom. 34, 255-263. 242. Depreitere, J., Durinx, C., Wang, Z., Coen, E., Lambeir, A. M., Scharpe, S., De Potter, W., and Nouwen, E. J. (2002) Presence and release of SR-17 (chromogranin B(586-602)) in the porcine splenic nerve and its enzymatic degradation by CD26/dipeptidyl peptidase IV. Regul.Pept. 106, 71-79. 243. Kreil, G., Haiml, L., and Suchanek, G. (1980) Stepwise cleavage of the Pro part of promelittin by dipeptidyl peptidase IV. Eur J Biochem 111, 49-58. 244. Wrenger, S., Kahne, T., Bohuon, C., Weglohner, W., Ansorge, S., and Reinhold, D. (2000) Amino-terminal truncation of procalcitonin, a marker for systemic bacterial infections, by dipeptidyl peptidase IV (DP IV). FEBS Lett. 466, 155- 159. 245. Weglohner, W., Struck, J., Fischer-Schulz, C., Morgenthaler, N. G., Otto, A., Bohuon, C., and Bergmann, A. (2001) Isolation and characterization of serum procalcitonin from patients with sepsis. Peptides 22, 2099-2103. 246. Abbott, C. A., McCaughan, G. W., Levy, M. T., Church, W. B., and Gorrell, M. D. (1999) Binding to human dipeptidyl peptidase IV by adenosine deaminase and antibodies that inhibit ligand binding involves overlapping, discontinuous sites on a predicted beta propeller domain. Eur.J Biochem. 266, 798-810. 247. Dong, R. P., Tachibana, K., Hegen, M., Munakata, Y., Cho, D., Schlossman, S. F., and Morimoto, C. (1997) Determination of adenosine deaminase binding domain on CD26 and its immunoregulatory effect on T cell activation. J.Immunol. 159, 6070-6076. 248. Fan, H., Tansi, F. L., Weihofen, W. A., Bottcher, C., Hu, J., Martinez, J., Saenger, W., and Reutter, W. (2011) Molecular mechanism and structural basis of interactions of dipeptidyl peptidase IV with adenosine deaminase and human immunodeficiency virus type-1 transcription transactivator. Eur J Cell Biol. 249. Richard, E., Alam, S. M., Arredondo-Vega, F. X., Patel, D. D., and Hershfield, M. S. (2002) Clustered charged amino acids of human adenosine deaminase comprise a functional epitope for binding the adenosine deaminase complexing protein CD26/dipeptidyl peptidase IV. J Biol.Chem. 277, 19720-19726. 250. Pacheco, R., Martinez-Navio, J. M., Lejeune, M., Climent, N., Oliva, H., Gatell, J. M., Gallart, T., Mallol, J., Lluis, C., and Franco, R. (2005) CD26, adenosine deaminase, and adenosine receptors mediate costimulatory signals in the immunological synapse. Proc.Natl.Acad.Sci.U.S.A 102, 9583-9588. 251. Richard, E., Arredondo-Vega, F. X., Santisteban, I., Kelly, S. J., Patel, D. D., and Hershfield, M. S. (2000) The binding site of human adenosine deaminase for CD26/Dipeptidyl peptidase IV: the Arg142Gln mutation impairs binding to cd26 but does not cause immune deficiency. J Exp.Med. 192, 1223-1236. 252. Gines, S., Marino, M., Mallol, J., Canela, E. I., Morimoto, C., Callebaut, C., Hovanessian, A., Casado, V., Lluis, C., and Franco, R. (2002) Regulation of epithelial and lymphocyte cell adhesion by adenosine deaminase-CD26 interaction. Biochem J 361, 203-209. 253. Hegen, M., Kameoka, J., Dong, R. P., Schlossman, S. F., and Morimoto, C. (1997) Cross-linking of CD26 by antibody induces tyrosine phosphorylation and activation of mitogen-activated protein kinase. Immunology 90, 257-264. 254. Torimoto, Y., Dang, N. H., Vivier, E., Tanaka, T., Schlossman, S. F., and Morimoto, C. (1991) Coassociation of CD26 (dipeptidyl peptidase IV) with CD45 on the surface of human T lymphocytes. J.Immunol. 147, 2514-2517. 255. Ohnuma, K., Yamochi, T., Uchiyama, M., Nishibashi, K., Yoshikawa, N., Shimizu, N., Iwata, S., Tanaka, H., Dang, N. H., and Morimoto, C. (2004) CD26 up-regulates expression of CD86 on antigen-presenting cells by means of caveolin-1. Proc.Natl.Acad.Sci.U.S.A 101, 14186-14191. 256. Ohnuma, K., Munakata, Y., Ishii, T., Iwata, S., Kobayashi, S., Hosono, O., Kawasaki, H., Dang, N. H., and Morimoto, C. (2001) Soluble CD26/dipeptidyl peptidase IV induces T cell proliferation through CD86 up-regulation on APCs. J Immunol. 167, 6745-6755. 257. Ohnuma, K., Yamochi, T., Uchiyama, M., Nishibashi, K., Iwata, S., Hosono, O., Kawasaki, H., Tanaka, H., Dang, N. H., and Morimoto, C. (2005) CD26 mediates dissociation of Tollip and IRAK-1 from caveolin-1 and induces upregulation of CD86 on antigen-presenting cells. Mol.Cell Biol. 25, 7743-7757. 258. Ohnuma, K., Yamochi, T., Hosono, O., and Morimoto, C. (2005) CD26 T cells in the pathogenesis of asthma. Clin.Exp.Immunol. 139, 13-16. 259. Ohnuma, K., Uchiyama, M., Hatano, R., Takasawa, W., Endo, Y., Dang, N. H., and Morimoto, C. (2009) Blockade of CD26-mediated T cell costimulation with soluble caveolin-1-Ig fusion protein induces anergy in CD4+T cells. Biochem Biophys.Res.Commun. 386, 327-332. 260. Ohnuma, K., Dang, N. H., and Morimoto, C. (2008) Revisiting an old acquaintance: CD26 and its molecular mechanisms in T cell function. Trends Immunol. 29, 295-301. 261. Ohnuma, K., Takahashi, N., Yamochi, T., Hosono, O., Dang, N. H., and Morimoto, C. (2008) Role of CD26/dipeptidyl peptidase IV in human T cell activation and function. Front Biosci. 13, 2299-2310. 117 Reference

262. Ohnuma, K., Uchiyama, M., Yamochi, T., Nishibashi, K., Hosono, O., Takahashi, N., Kina, S., Tanaka, H., Lin, X., Dang, N. H., and Morimoto, C. (2007) Caveolin-1 triggers T-cell activation via CD26 in association with CARMA1. J Biol.Chem. 282, 10117-10131. 263. Callebaut, C., Jacotot, E., Blanco, J., Krust, B., and Hovanessian, A. G. (1998) Increased rate of HIV-1 entry and its cytopathic effect in CD4+/CXCR4+ T cells expressing relatively high levels of CD26. Exp.Cell Res. 241, 352-362. 264. Blanco, J., Barretina, J., Henson, G., Bridger, G., De Clercq, E., Clotet, B., and Este, J. A. (2000) The CXCR4 antagonist AMD3100 efficiently inhibits cell-surface-expressed human immunodeficiency virus type 1 envelope- induced apoptosis. Antimicrob.Agents Chemother. 44, 51-56. 265. Wrenger, S., Faust, J., Mrestani-Klaus, C., Fengler, A., Stockel-Maschek, A., Lorey, S., Kahne, T., Brandt, W., Neubert, K., Ansorge, S., and Reinhold, D. (2000) Down-regulation of T cell activation following inhibition of dipeptidyl peptidase IV/CD26 by the N-terminal part of the thromboxane A2 receptor. J Biol.Chem. 275, 22180-22186. 266. Reinhold, D., Kahne, T., Steinbrecher, A., Wrenger, S., Neubert, K., Ansorge, S., and Brocke, S. (2002) The role of dipeptidyl peptidase IV (DP IV) enzymatic activity in T cell activation and autoimmunity. Biol.Chem. 383, 1133-1138. 267. Valenzuela, A., Blanco, J., Callebaut, C., Jacotot, E., Lluis, C., Hovanessian, A. G., and Franco, R. (1997) HIV-1 envelope gp120 and viral particles block adenosine deaminase binding to human CD26. Adv.Exp.Med.Biol. 421, 185- 192. 268. Valenzuela, A., Blanco, J., Callebaut, C., Jacotot, E., Lluis, C., Hovanessian, A. G., and Franco, R. (1997) Adenosine deaminase binding to human CD26 is inhibited by HIV-1 envelope glycoprotein gp120 and viral particles. J.Immunol. 158, 3721-3729. 269. Jacotot, E., Callebaut, C., Blanco, J., Riviere, Y., Krust, B., and Hovanessian, A. G. (1997) CD26 as a positive regulator of HIV envelope-glycoprotein induced apoptosis in CD4+ T cells. Adv.Exp.Med.Biol. 421, 207-216. 270. Jacotot, E., Callebaut, C., Blanco, J., Riviere, Y., Krust, B., and Hovanessian, A. G. (1996) HIV envelope glycoprotein- induced cell killing by apoptosis is enhanced with increased expression of CD26 in CD4+ T cells. Virology 223, 318- 330. 271. Blanco, J., Valenzuela, A., Herrera, C., Lluis, C., Hovanessian, A. G., and Franco, R. (2000) The HIV-1 gp120 inhibits the binding of adenosine deaminase to CD26 by a mechanism modulated by CD4 and CXCR4 expression. FEBS Lett. 477, 123-128. 272. Loster, K., Zeilinger, K., Schuppan, D., and Reutter, W. (1995) The cysteine-rich region of dipeptidyl peptidase IV (CD 26) is the collagen-binding site. Biochem.Biophys.Res.Commun. 217, 341-348. 273. Cheng, H. C., Abdel-Ghany, M., and Pauli, B. U. (2003) A novel consensus motif in fibronectin mediates dipeptidyl peptidase IV adhesion and metastasis. J Biol.Chem. 278, 24600-24607. 274. Cheng, H. C., Abdel-Ghany, M., Elble, R. C., and Pauli, B. U. (1998) Lung endothelial dipeptidyl peptidase IV promotes adhesion and metastasis of rat breast cancer cells via tumor cell surface-associated fibronectin. J.Biol.Chem. 273, 24207-24215. 275. Abdel-Ghany, M., Cheng, H., Levine, R. A., and Pauli, B. U. (1998) Truncated dipeptidyl peptidase IV is a potent anti- adhesion and anti- metastasis peptide for rat breast cancer cells. Invasion Metastasis 18, 35-43. 276. Cuchacovich, M., Gatica, H., Vial, P., Yovanovich, J., Pizzo, S. V., and Gonzalez-Gronow, M. (2002) Streptokinase promotes development of dipeptidyl peptidase IV (CD26) autoantibodies after fibrinolytic therapy in myocardial infarction patients. Clin.Diagn.Lab.Immunol. 9, 1253-1259. 277. Gonzalez-Gronow, M., Gawdi, G., and Pizzo, S. V. (1994) Characterization of the plasminogen receptors of normal and rheumatoid arthritis human synovial fibroblasts. J.Biol.Chem. 269, 4360-4366. 278. Gonzalez-Gronow, M., Grenett, H. E., Weber, M. R., Gawdi, G., and Pizzo, S. V. (2001) Interaction of plasminogen with dipeptidyl peptidase IV initiates a signal transduction mechanism which regulates expression of matrix metalloproteinase-9 by prostate cancer cells. Biochem J 355, 397-407. 279. Gonzalez-Gronow, M., Weber, M. R., Gawdi, G., and Pizzo, S. V. (1998) Dipeptiyl peptidase (CD26) is a receptor for Streptokinase on rheumatoid synoival fibroblasts. Fibrinolysis Proteolysis 12, 129-135. 280. Gonzalez-Gronow, M., Weber, M. R., Shearin, T. V., Gawdi, G., Pirie-Shepherd, R. S., and Pizzo, S. V. (1998) Plasmin(ogen) carbohydrate chains mediate binding to dipeptidyl peptidase IV (CD26) in rheumatoid arrthritis human synoival fibroblasts. Fibrinolysis Proteolysis 12, 366-374. 281. Davoodi, J., Kelly, J., Gendron, N. H., and MacKenzie, A. E. (2007) The Simpson-Golabi-Behmel syndrome causative glypican-3, binds to and inhibits the dipeptidyl peptidase activity of CD26. Proteomics. 7, 2300-2310. 282. Girardi, A. C., Knauf, F., Demuth, H. U., and Aronson, P. S. (2004) Role of dipeptidyl peptidase IV in regulating activity of Na+/H+ exchanger isoform NHE3 in proximal tubule cells. Am.J.Physiol Cell Physiol 287, C1238-C1245. 283. Girardi, A. C., Degray, B. C., Nagy, T., Biemesderfer, D., and Aronson, P. S. (2001) Association of Na(+)-H(+) exchanger isoform NHE3 and dipeptidyl peptidase IV in the renal proximal tubule. J Biol.Chem. 276, 46671-46677. 284. Gonzalez-Gronow, M., Misra, U. K., Gawdi, G., and Pizzo, S. V. (2005) Association of plasminogen with dipeptidyl peptidase IV and Na+-H+ exchanger isoform NHE3 regulates invasion of human 1-LN prostate tumor cells. J Biol.Chem. .. 285. Conarello, S. L., Li, Z., Ronan, J., Roy, R. S., Zhu, L., Jiang, G., Liu, F., Woods, J., Zycband, E., Moller, D. E., Thornberry, N. A., and Zhang, B. B. (2003) Mice lacking dipeptidyl peptidase IV are protected against obesity and insulin resistance. Proc Natl.Acad Sci U.S A 100, 6825-6830. 286. Marguet, D., Baggio, L., Kobayashi, T., Bernard, A. M., Pierres, M., Nielsen, P. F., Ribel, U., Watanabe, T., Drucker, D. J., and Wagtmann, N. (2000) Enhanced insulin secretion and improved glucose tolerance in mice lacking CD26. Proc.Natl.Acad.Sci.U.S.A. 97, 6874-6879. 287. El Yacoubi, M., Vaugeois, J. M., Marguet, D., Sauze, N., Guieu, R., Costentin, J., and Fenouillet, E. (2006) Behavioral characterization of CD26 deficient mice in animal tests of anxiety and antidepressant-like activity. Behav.Brain Res 171, 279-285. 288. Yan, S., Marguet, D., Dobers, J., Reutter, W., and Fan, H. (2003) Deficiency of CD26 results in a change of cytokine and immunoglobulin secretion after stimulation by pokeweed mitogen. Eur.J.Immunol. 33, 1519-1527. 289. Karl, T., Chwalisz, W. T., Wedekind, D., Hedrich, H. J., Hoffmann, T., Jacobs, R., Pabst, R., and Von Horsten, S. (2003) Localization, transmission, spontaneous mutations, and variation of function of the Dpp4 (Dipeptidyl-peptidase IV; CD26) gene in rats. Regul.Pept. 115, 81-90.

118 Reference

290. Tsuji, E., Misumi, Y., Fujiwara, T., Takami, N., Ogata, S., and Ikehara, Y. (1992) An active-site mutation (Gly633-- >Arg) of dipeptidyl peptidase IV causes its retention and rapid degradation in the endoplasmic reticulum. Biochemistry 31, 11921-11927. 291. Thompson, N. L., Hixson, D. C., Callanan, H., Panzica, M., Flanagan, D., Faris, R. A., Hong, W. J., Hartel-Schenk, S., and Doyle, D. (1991) A Fischer rat substrain deficient in dipeptidyl peptidase IV activity makes normal steady-state RNA levels and an altered protein. Use as a liver-cell transplantation model. Biochem.J. 273, 497-502. 292. Drucker, D. J., Boushey, R. P., Wang, F., Hill, M. E., Brubaker, P. L., and Yusta, B. (1999) Biologic properties and therapeutic potential of glucagon-like peptide-2. JPEN.J Parenter.Enteral.Nutr. 23, S98-100. 293. Drucker, D. J., Shi, Q., Crivici, A., Sumner-Smith, M., Tavares, W., Hill, M., DeForest, L., Cooper, S., and Brubaker, P. L. (1997) Regulation of the biological activity of glucagon-like peptide 2 in vivo by dipeptidyl peptidase IV. Nat.Biotechnol. 15, 673-677. 294. Pederson, R. A., Kieffer, T. J., Pauly, R., Kofod, H., Kwong, J., and McIntosh, C. H. (1996) The enteroinsular axis in dipeptidyl peptidase IV-negative rats. Metabolism 45, 1335-1341. 295. Kieffer, T. J., McIntosh, C. H., and Pederson, R. A. (1995) Degradation of glucose-dependent insulinotropic polypeptide and truncated glucagon-like peptide 1 in vitro and in vivo by dipeptidyl peptidase IV. Endocrinology 136, 3585-3596. 296. Karl, T., Hoffmann, T., Pabst, R., and Von Horsten, S. (2003) Behavioral effects of neuropeptide Y in F344 rat substrains with a reduced dipeptidyl-peptidase IV activity. Pharmacol.Biochem.Behav. 75, 869-879. 297. Karl, T., Hoffmann, T., Pabst, R., and Von Horsten, S. (2003) Extreme reduction of dipeptidyl peptidase IV activity in F344 rat substrains is associated with various behavioral differences. Physiol Behav. 80, 123-134. 298. Yasuda, N., Inoue, T., Nagakura, T., Yamazaki, K., Kira, K., Saeki, T., and Tanaka, I. (2002) Enhanced secretion of glucagon-like peptide 1 by biguanide compounds. Biochem Biophys.Res.Commun. 298, 779-784. 299. Nagakura, T., Yasuda, N., Yamazaki, K., Ikuta, H., Yoshikawa, S., Asano, O., and Tanaka, I. (2001) Improved glucose tolerance via enhanced glucose-dependent insulin secretion in dipeptidyl peptidase IV-deficient Fischer rats. Biochem Biophys.Res.Commun. 284, 501-506. 300. Gupta, S., Rajvanshi, P., and Lee, C. D. (1995) Integration of transplanted hepatocytes into host liver plates demonstrated with dipeptidyl peptidase IV-deficient rats. Proc.Natl.Acad.Sci.U.S.A. 92, 5860-5864. 301. Laconi, E., Oren, R., Mukhopadhyay, D. K., Hurston, E., Laconi, S., Pani, P., Dabeva, M. D., and Shafritz, D. A. (1998) Long-term, near-total liver replacement by transplantation of isolated hepatocytes in rats treated with retrorsine. Am.J.Pathol. 153, 319-329. 302. Laconi, S., Pillai, S., Porcu, P. P., Shafritz, D. A., Pani, P., and Laconi, E. (2001) Massive liver replacement by transplanted hepatocytes in the absence of exogenous growth stimuli in rats treated with retrorsine. Am.J Pathol. 158, 771-777. 303. Tanaka, S., Murakami, T., Horikawa, H., Sugiura, M., Kawashima, K., and Sugita, T. (1997) Suppression of arthritis by the inhibitors of dipeptidyl peptidase IV. Int.J.Immunopharmacol. 19, 15-24. 304. Frerker, N., Raber, K., Bode, F., Skripuletz, T., Nave, H., Klemann, C., Pabst, R., Stephan, M., Schade, J., Brabant, G., Wedekind, D., Jacobs, R., Jorns, A., Forssmann, U., Straub, R. H., Johannes, S., Hoffmann, T., Wagner, L., Demuth, H. U., and Von Horsten, S. (2009) Phenotyping of congenic dipeptidyl peptidase 4 (DP4) deficient Dark Agouti (DA) rats suggests involvement of DP4 in neuro-, endocrine, and immune functions. Clin.Chem.Lab Med. 47, 275-287. 305. Simeoni, L., Rufini, A., Moretti, T., Forte, P., Aiuti, A., and Fantoni, A. (2002) Human CD26 expression in transgenic mice affects murine T-cell populations and modifies their subset distribution. Hum.Immunol. 63, 719-730. 306. Hough, R. B., Lengeling, A., Bedian, V., Lo, C., and Bucan, M. (1998) Rump white inversion in the mouse disrupts dipeptidyl aminopeptidase-like protein 6 and causes dysregulation of Kit expression. Proc.Natl.Acad.Sci.U.S.A 95, 13800-13805. 307. Fan, H., Yan, S., Stehling, S., Marguet, D., Schuppaw, D., and Reutter, W. (2003) Dipeptidyl peptidase IV/CD26 in T cell activation, cytokine secretion and immunoglobulin production. Adv.Exp.Med.Biol. 524, 165-174. 308. Wrenger, S., Faust, J., Mrestani-Klaus, C., Brandt, W., Thielitz, A., Neubert, K., and Reinhold, D. (2008) Non- substrate peptides influencing dipeptidyl peptidase IV/CD26 activity and immune cell function. Front Biosci. 13, 3194- 3201. 309. Vora, K. A., Porter, G., Peng, R., Cui, Y., Pryor, K., Eiermann, G., and Zaller, D. M. (2009) Genetic ablation or pharmacological blockade of dipeptidyl peptidase IV does not impact T cell-dependent immune responses. BMC.Immunol. 10, 19. 310. Guieu, R., Fenouillet, E., Devaux, C., Fajloun, Z., Carrega, L., Sabatier, J. M., Sauze, N., and Marguet, D. (2006) CD26 modulates nociception in mice via its dipeptidyl-peptidase IV activity. Behav.Brain Res 166, 279-285. 311. Adams, S., Miller, G. T., Jesson, M. I., Watanabe, T., Jones, B., and Wallner, B. P. (2004) PT-100, a small molecule dipeptidyl peptidase inhibitor, has potent antitumor effects and augments antibody-mediated cytotoxicity via a novel immune mechanism. Cancer Res. 64, 5471-5480. 312. Geier, M. S., Tenikoff, D., Yazbeck, R., McCaughan, G. W., Abbott, C. A., and Howarth, G. S. (2005) Development and resolution of experimental colitis in mice with targeted deletion of dipeptidyl peptidase IV. J Cell Physiol. 204, 687-692. 313. Abbott, C. A., Yazbeck, R., Geier, M. S., Demuth, H. U., and Howarth, G. S. (2006) Dipeptidyl peptidases and inflammatory bowel disease. Adv.Exp.Med.Biol. 575, 155-162. 314. Yazbeck, R., Howarth, G. S., Butler, R. N., Geier, M. S., and Abbott, C. A. (2011) Biochemical and histological changes in the small intestine of mice with dextran sulfate sodium colitis. J Cell Physiol 226, 3219-3224. 315. Preller, V., Gerber, A., Wrenger, S., Togni, M., Marguet, D., Tadje, J., Lendeckel, U., Rocken, C., Faust, J., Neubert, K., Schraven, B., Martin, R., Ansorge, S., Brocke, S., and Reinhold, D. (2007) TGF-beta1-mediated control of central nervous system inflammation and autoimmunity through the inhibitory receptor CD26. J Immunol. 178, 4632-4640. 316. Busso, N., Wagtmann, N., Herling, C., Chobaz-Peclat, V., Bischof-Delaloye, A., So, A., and Grouzmann, E. (2005) Circulating CD26 is negatively associated with inflammation in human and experimental arthritis. Am.J Pathol. 166, 433-442. 317. Yasuda, N., Nagakura, T., Yamazaki, K., Inoue, T., and Tanaka, I. (2002) Improvement of high fat-diet-induced insulin resistance in dipeptidyl peptidase IV-deficient Fischer rats. Life Sci. 71, 227-238.

119 Reference

318. Mitani, H., Takimoto, M., and Kimura, M. (2002) Dipeptidyl peptidase IV inhibitor NVP-DPP728 ameliorates early insulin response and glucose tolerance in aged rats but not in aged Fischer 344 rats lacking its enzyme activity. Jpn.J Pharmacol. 88, 451-458. 319. Mitani, H., Takimoto, M., Hughes, T. E., and Kimura, M. (2002) Dipeptidyl peptidase IV inhibition improves impaired glucose tolerance in high-fat diet-fed rats: study using a Fischer 344 rat substrain deficient in its enzyme activity. Jpn.J Pharmacol. 88, 442-450. 320. Unniappan, S., McIntosh, C. H., Demuth, H. U., Heiser, U., Wolf, R., and Kieffer, T. J. (2006) Effects of dipeptidyl peptidase IV on the satiety actions of peptide YY. Diabetologia 49, 1915-1923. 321. Kruschinski, C., Skripuletz, T., Bedoui, S., Tschernig, T., Pabst, R., Nassenstein, C., Braun, A., and Von Horsten, S. (2005) CD26 (dipeptidyl-peptidase IV)-dependent recruitment of T cells in a rat asthma model. Clin.Exp.Immunol. 139, 17-24. 322. Shingu, K., Helfritz, A., Zielinska-Skowronek, M., Meyer-Olson, D., Jacobs, R., Schmidt, R. E., Mentlein, R., Pabst, R., and Von Horsten, S. (2003) CD26 expression determines lung metastasis in mutant F344 rats: involvement of NK cell function and soluble CD26. Cancer Immunol.Immunother. 52, 546-554. 323. Tanaka, S., Murakami, T., Nonaka, N., Ohnuki, T., Yamada, M., and Sugita, T. (1998) Anti-arthritic effects of the novel dipeptidyl peptidase IV inhibitors TMC-2A and TSL-225. Immunopharmacology 40, 21-26. 324. Cheng, H. C., Abdel-Ghany, M., Zhang, S., and Pauli, B. U. (1999) Is the Fischer 344/CRJ rat a protein-knock-out model for dipeptidyl peptidase IV-mediated lung metastasis of breast cancer? Clin.Exp.Metastasis 17, 609-615. 325. Perner, F., Gyuris, T., Rakoczy, G., Sarvary, E., Gorog, D., Szalay, F., Kunos, I., Szonyi, L., Peterfy, M., and Takacs, L. (1999) Dipeptidyl peptidase activity of CD26 in serum and urine as a marker of cholestasis: experimental and clinical evidence. J.Lab.Clin.Med. 134, 56-67. 326. Shinosaki, T., Kobayashi, T., Kimura, K., and Kurihara, H. (2002) Involvement of dipeptidyl peptidase IV in immune complex-mediated glomerulonephritis. Lab.Invest. 82, 505-513. 327. Klemann, C., Schade, J., Pabst, R., Leitner, S., Stiller, J., Von Horsten, S., and Stephan, M. (2009) CD26/dipeptidyl peptidase 4-deficiency alters thymic emigration patterns and leukcocyte subsets in F344-rats age-dependently. Clin.Exp.Immunol. 155, 357-365. 328. Forssmann, U., Stoetzer, C., Stephan, M., Kruschinski, C., Skripuletz, T., Schade, J., Schmiedl, A., Pabst, R., Wagner, L., Hoffmann, T., Kehlen, A., Escher, S. E., Forssmann, W. G., Elsner, J., and Von Horsten, S. (2008) Inhibition of CD26/dipeptidyl peptidase IV enhances CCL11/eotaxin-mediated recruitment of eosinophils in vivo. J Immunol. 181, 1120-1127. 329. Schade, J., Schmiedl, A., Kehlen, A., Veres, T. Z., Stephan, M., Pabst, R., and Von Horsten, S. (2009) Airway-specific recruitment of T cells is reduced in a CD26-deficient F344 rat substrain. Clin.Exp.Immunol. 158, 133-142. 330. Schade, J., Schmiedl, A., Stephan, M., Pabst, R., and Von Horsten, S. (2010) Transferred T cells preferentially adhere in the BALT of CD26-deficient recipient lungs during asthma. Immunobiol. 215, 321-331. 331. Skripuletz, T., Schmiedl, A., Schade, J., Bedoui, S., Glaab, T., Pabst, R., Von Horsten, S., and Stephan, M. (2007) Dose-dependent recruitment of CD25+ and CD26+ T cells in a novel F344 rat model of asthma. Am.J Physiol Lung Cell Mol.Physiol 292, L1564-L1571. 332. Coburn, M. C., Hixson, D. C., and Reichner, J. S. (1994) In vitro immune responsiveness of rats lacking active dipeptidylpeptidase IV. Cell Immunol. 158, 269-280. 333. Iwaki-Egawa, S., Watanabe, Y., and Fujimoto, Y. (1995) Is CD26/dipeptidyl peptidase IV a really important molecule in T cell activation of a certain rat strain? Immunobiol. 194, 429-442. 334. Karl, T., Hoffmann, T., Pabst, R., and Von Horsten, S. (2003) Behavioral effects of neuropeptide Y in F344 rat substrains with a reduced dipeptidyl-peptidase IV activity. Pharmacol Biochem Behav. 75, 869-879. 335. Tavares, W., Drucker, D. J., and Brubaker, P. L. (2000) Enzymatic- and renal-dependent catabolism of the intestinotropic hormone glucagon-like peptide-2 in rats. Am.J Physiol.Endocrinol.Metab. 278, E134-E139. 336. Watanabe, Y., Kojima-Komatsu, T., Iwaki-Egawa, S., and Fujimoto, Y. (1993) Increased excretion of proline- containing peptides in dipeptidyl peptidase IV-deficient rats. Res.Commun.Chem.Pathol.Pharmacol. 81, 323-330. 337. Tiruppathi, C., Kulanthaivel, P., Ganapathy, V., and Leibach, F. H. (1990) Evidence for tripeptide/H+ co-transport in rabbit renal brush-border membrane vesicles. Biochem.J. 268, 27-33. 338. Laconi, S., Pani, P., Pillai, S., Pasciu, D., Sarma, D. S., and Laconi, E. (2001) A growth-constrained environment drives tumor progression invivo. Proc.Natl.Acad.Sci.U.S.A. 98, 7806-7811. 339. Gupta, S., Rajvanshi, P., Sokhi, R., Slehria, S., Yam, A., Kerr, A., and Novikoff, P. M. (1999) Entry and integration of transplanted hepatocytes in rat liver plates occur by disruption of hepatic sinusoidal endothelium. Hepatology 29, 509- 519. 340. Gupta, S., Rajvanshi, P., Sokhi, R. P., Vaidya, S., Irani, A. N., and Gorla, G. R. (1999) Position-specific gene expression in the liver lobule is directed by the microenvironment and not by the previous cell differentiation state. J.Biol.Chem. 274, 2157-2165. 341. Sigal, S. H., Rajvanshi, P., Reid, L. M., and Gupta, S. (1995) Demonstration of differentiation in hepatocyte progenitor cells using dipeptidyl peptidase IV deficient mutant rats. Cell Mol.Biol.Res. 41, 39-47. 342. Tsugiki, M., Kobayashi, Y., Kawasaki, T., and Yoshimi, T. (1998) Identification of bile canalicular cell surface antigen HAM.4 as dipeptidyl peptidase IV (DPPIV) and characterization of its role in hepatic regeneration after partial hepatectomy in rats. Dig.Dis.Sci. 43, 2591-2600. 343. Petersen, B. E., Bowen, W. C., Patrene, K. D., Mars, W. M., Sullivan, A. K., Murase, N., Boggs, S. S., Greenberger, J. S., and Goff, J. P. (1999) Bone marrow as a potential source of hepatic oval cells. Science 284, 1168-1170. 344. Durinx, C., Neels, H., Van der Auwera, J. C., Naelaerts, K., Scharpe, S., and De Meester, I. (2001) Reference values for plasma dipeptidyl-peptidase IV activity and their association with other laboratory parameters. Clin.Chem.Lab.Med. 39, 155-159. 345. Cordero, O. J., Salgado, F. J., and Nogueira, M. (2009) On the origin of serum CD26 and its altered concentration in cancer patients. Cancer Immunol.Immunother. 58, 1725-1749. 346. Blanco, J., Jacotot, E., Callebaut, C., Krust, B., and Hovanessian, A. G. (1997) Further characterization of DPP IV- beta, a novel cell surface expressed protein with dipeptidyl peptidase activity. Adv.Exp.Med.Biol. 421, 193-199. 347. Blanco, J., Nguyen, C., Callebaut, C., Jacotot, E., Krust, B., Mazaleyrat, J. P., Wakselman, M., and Hovanessian, A. G. (1998) Dipeptidyl-peptidase IV-beta--further characterization and comparison to dipeptidyl-peptidase IV activity of CD26. Eur.J.Biochem. 256, 369-378. 120 Reference

348. Jacotot, E., Callebaut, C., Blanco, J., Krust, B., Neubert, K., Barth, A., and Hovanessian, A. G. (1996) Dipeptidyl- peptidase IV-beta, a novel form of cell-surface-expressed protein with dipeptidyl-peptidase IV activity. Eur.J.Biochem. 239, 248-258. 349. Friedrich, D., Kuhn-Wache, K., Hoffmann, T., and Demuth, H. U. (2003) Isolation and characterization of attractin-2. Adv.Exp.Med.Biol. 524, 109-113. 350. Gotoh, H., Hagihara, M., Nagatsu, T., Iwata, H., and Miura, T. (1989) Activities of dipeptidyl peptidase II and dipeptidyl peptidase IV in synovial fluid from patients with rheumatoid arthritis and osteoarthritis. Clin.Chem. 35, 1016-1018. 351. Kamori, M., Hagihara, M., Nagatsu, T., Iwata, H., and Miura, T. (1991) Activities of dipeptidyl peptidase II, dipeptidyl peptidase IV, prolyl endopeptidase, and collagenase-like peptidase in synovial membrane from patients with rheumatoid arthritis and osteoarthritis. Biochem.Med.Metab.Biol. 45, 154-160. 352. Sohar, N., Hammer, H., and Sohar, I. (2002) Lysosomal peptidases and glycosidases in rheumatoid arthritis. Biol.Chem. 383, 865-869. 353. Kar, N. C. and Pearson, C. M. (1978) Dipeptidyl peptidases in human muscle disease. Clin.Chim.Acta 82, 185-192. 354. Maes, M., De, M., I, Verkerk, R., De Medts, P., Wauters, A., Vanhoof, G., Vandoolaeghe, E., Neels, H., and Scharpe, S. (1997) Lower serum dipeptidyl peptidase IV activity in treatment resistant major depression: relationships with immune-inflammatory markers. Psychoneuroendocrinology. 22, 65-78. 355. Maes, M., De, M., I, Vanhoof, G., Scharpe, S., Bosmans, E., Vandervorst, C., Verkerk, R., Minner, B., Suy, E., and Raus, J. (1991) Decreased serum dipeptidyl peptidase IV activity in major depression. Biol.Psychiatry 30, 577-586. 356. Elgun, S., Keskinege, A., and Kumbasar, H. (1999) Dipeptidyl peptidase IV and adenosine deaminase activity - Decrease in depression. Psychoneuroendocrinology 24, 823-832. 357. Maes, M., De, M., I, Scharpe, S., Desnyder, R., Ranjan, R., and Meltzer, H. Y. (1996) Alterations in plasma dipeptidyl peptidase IV enzyme activity in depression and schizophrenia: effects of antidepressants and antipsychotic drugs. Acta Psychiatr.Scand. 93, 1-8. 358. Emanuele, E., Minoretti, P., Martinelli, V., Pesenti, S., Olivieri, V., Aldeghi, A., and Politi, P. (2006) Circulating levels of soluble CD26 are associated with phobic anxiety in women. Prog Neuropsychopharmacol.Biol.Psychiatry 30, 1334- 1336. 359. Maes, M., Goossens, F., Lin, A., De, M., I, Van Gastel, A., and Scharpe, S. (1998) Effects of psychological stress on serum prolyl endopeptidase and dipeptidyl peptidase IV activity in humans: higher serum prolyl endopeptidase activity is related to stress-induced anxiety. Psychoneuroendocrinology. 23, 485-495. 360. Hildebrandt, M., Rose, M., Mayr, C., Schuler, C., Reutter, W., Salama, A., and Klapp, B. F. (1999) Alterations in expression and in serum activity of dipeptidyl peptidase IV (DPP IV, CD26) in patients with hyporectic eating disorders. Scand.J Immunol. 50, 536-541. 361. Hildebrandt, M., Rose, M., Monnikes, H., Reutter, W., Keller, W., and Klapp, B. F. (2001) Eating disorders: a role for dipeptidyl peptidase IV in nutritional control. Nutrition 17, 451-454. 362. Maes, M., Lin, A., Bonaccorso, S., Vandoolaeghe, E., Song, C., Goossens, F., De, M., I, Degroote, J., Neels, H., Scharpe, S., and Janca, A. (1999) Lower activity of serum peptidases in abstinent alcohol-dependent patients. Alcohol 17, 1-6. 363. Cordero, O. J., Salgado, F. J., Mera-Varela, A., and Nogueira, M. (2001) Serum interleukin-12, interleukin-15, soluble CD26, and adenosine deaminase in patients with rheumatoid arthritis. Rheumatol.Int. 21, 69-74. 364. Hagihara, M., Ohhashi, M., and Nagatsu, T. (1987) Activities of dipeptidyl peptidase II and dipeptidyl peptidase IV in mice with lupus erythematosus-like syndrome and in patients with lupus erythematosus and rheumatoid arthritis. Clin.Chem. 33, 1463-1465. 365. Ulusoy, H., Kamanli, A., Ilhan, N., Kuru, O., Arslan, S., Alkan, G., and Ozgocmen, S. (2011) Serum levels of soluble CD26 and CD30 and their clinical significance in patients with rheumatoid arthritis. Rheumatol.Int. 366. Kobayashi, H., Hosono, O., Mimori, T., Kawasaki, H., Dang, N. H., Tanaka, H., and Morimoto, C. (2002) Reduction of serum soluble CD26/dipeptidyl peptidase IV enzyme activity and its correlation with disease activity in systemic lupus erythematosus. J Rheumatol. 29, 1858-1866. 367. Stancikova, M., Lojda, Z., Lukac, J., and Ruzickova, M. (1992) Dipeptidyl peptidase IV in patients with systemic lupus erythematosus. Clin.Exp.Rheumatol. 10, 381-385. 368. Miyagaki, T., Sugaya, M., Suga, H., Morimura, S., Kamata, M., Ohmatsu, H., Fujita, H., Asano, Y., Tada, Y., Kadono, T., and Sato, S. (2011) Serum soluble CD26 levels: diagnostic efficiency for atopic dermatitis, cutaneous T-cell lymphoma and psoriasis in combination with serum thymus and activation-regulated chemokine levels. J Eur Acad.Dermatol.Venereol. 369. Yildirim, F. E., Karaduman, A., Pinar, A., and Aksoy, Y. (2011) CD26/dipeptidyl-peptidase IV and adenosine deaminase serum levels in psoriatic patients treated with cyclosporine, etanercept, and psoralen plus ultraviolet A phototherapy. Int.J Dermatol. 50, 948-955. 370. Tamaki, Z., Kubo, M., Yazawa, N., Mimura, Y., Ashida, R., Tomita, M., Tada, Y., Kawashima, T., and Tamaki, K. (2008) Serum levels of soluble CD26 in patients with scleroderma. J Dermatol.Sci. 52, 67-69. 371. Schonermarck, U., Csernok, E., Trabandt, A., Hansen, H., and Gross, W. L. (2000) Circulating cytokines and soluble CD23, CD26 and CD30 in ANCA-associated vasculitides. Clin.Exp.Rheumatol. 18, 457-463. 372. Detel, D., Persic, M., and Varljen, J. (2007) Serum and intestinal dipeptidyl peptidase IV (DPP IV/CD26) activity in children with celiac disease. J Pediatr.Gastroenterol.Nutr. 45, 65-70. 373. Lun, S. W., Wong, C. K., Ko, F. W., Hui, D. S., and Lam, C. W. (2007) Increased expression of plasma and CD4+ T lymphocyte costimulatory molecule CD26 in adult patients with allergic asthma. J Clin.Immunol. 27, 430-437. 374. Mannucci, E., Pala, L., Ciani, S., Bardini, G., Pezzatini, A., Sposato, I., Cremasco, F., Ognibene, A., and Rotella, C. M. (2005) Hyperglycaemia increases dipeptidyl peptidase IV activity in diabetes mellitus. Diabetologia 48, 1168-1172. 375. Varga, T., Somogyi, A., Barna, G., Wichmann, B., Nagy, G., Racz, K., Selmeci, L., and Firneisz, G. (2011) Higher serum DPP-4 enzyme activity and decreased lymphocyte CD26 expression in type 1 diabetes. Pathol.Oncol.Res. 17, 925-930. 376. Hino, M., Nagatsu, T., Kakumu, S., Okuyama, S., Yoshii, Y., and Nagatsu, I. (1975) Glycylprolyl beta- naphthylamidase activity in human serum. Clin.Chim.Acta 62, 5-11. 377. Andrieu, T., Thibault, V., Malet, I., Laporte, J., Bauvois, B., Agut, H., and Cahour, A. (2003) Similar increased serum dipeptidyl peptidase IV activity in chronic hepatitis C and other viral infections. J.Clin.Virol. 27, 59-68.

121 Reference

378. Firneisz, G., Lakatos, P. L., and Szalay, F. (2001) Serum dipeptidyl peptidase IV (DPP IV, CD26) activity in chronic hepatitis C. Scand.J Gastroenterol. 36, 877-880. 379. Xiao, Q., Boushey, R. P., Cino, M., Drucker, D. J., and Brubaker, P. L. (2000) Circulating levels of glucagon-like peptide-2 in human subjects with inflammatory bowel disease. Am.J Physiol.Regul.Integr.Comp.Physiol. 278, R1057- R1063. 380. Hildebrandt, M., Rose, M., Ruter, J., Salama, A., Monnikes, H., and Klapp, B. F. (2001) Dipeptidyl peptidase IV (DP IV, CD26) in patients with inflammatory bowel disease. Scand.J Gastroenterol. 36, 1067-1072. 381. Rose, M., Hildebrandt, M., Fliege, H., Seibold, S., Monnikes, H., and Klapp, B. F. (2002) T-cell immune parameters and depression in patients with Crohn's disease. J Clin.Gastroenterol. 34, 40-48. 382. Hosono, O., Homma, T., Kobayashi, H., Munakata, Y., Nojima, Y., Iwamoto, A., and Morimoto, C. (1999) Decreased dipeptidyl peptidase IV enzyme activity of plasma soluble CD26 and its inverse correlation with HIV-1 RNA in HIV-1 infected individuals. Clin.Immunol. 91, 283-295. 383. Bergmann, A. and Bohuon, C. (2002) Decrease of serum dipeptidylpeptidase activity in severe sepsis patients: relationship to procalcitonin. Clin.Chim.Acta 321, 123-126. 384. Fuyamada, H., Hino, M., Nagatsu, T., Ogawa, K., and Sakakibara, S. (1977) Serum glyccylproline p-nitroanilidase activity in human hypertension. Clin.Chim.Acta 74, 177-181. 385. Lakatos, P. L., Firneisz, G., Rakoczy, G., Selmeci, L., and Szalay, F. (1999) Elevated serum dipeptidyl peptidase IV (CD26, EC 3.4.14.5) activity in patients with primary biliary cirrhosis. J.Hepatol. 30, 740. 386. Gotoh, H., Hagihara, M., Nagatsu, T., Iwata, H., and Miura, T. (1988) Activity of dipeptidyl peptidase IV and post- proline cleaving enzyme in sera from osteoporotic patients. Clin.Chem. 34, 2499-2501. 387. Fujita, K., Hirano, M., Tokunaga, K., Nagatsu, I., Nagatsu, T., and Sakakibara, S. (1977) Serum glycylproline p- nitroanilidase activity in blood cancers. Clin.Chim.Acta 81, 215-217. 388. Kojima, K., Mihara, R., Sakai, T., Togari, A., Matsui, T., Shinpo, K., Fujita, K., Fukasawa, K., Harada, M., and Nagatsu, T. (1987) Serum activities of dipeptidyl-aminopeptidase II and dipeptidyl-aminopeptidase IV in tumor-bearing animals and in cancer patients. Biochem Med.Metab Biol. 37, 35-41. 389. Ayude, D., Paez, d. l. C., Cordero, O. J., Nogueira, M., Ayude, J., Fernandez-Briera, A., and Rodriguez-Berrocal, F. J. (2003) Clinical interest of the combined use of serum CD26 and alpha-L-fucosidase in the early diagnosis of colorectal cancer. Dis.Markers 19, 267-272. 390. De Chiara, L., Rodriguez-Pineiro, A. M., Rodriguez-Berrocal, F. J., Cordero, O. J., Martinez-Ares, D., and Paez, d. l. C. (2010) Serum CD26 is related to histopathological polyp traits and behaves as a marker for colorectal cancer and advanced adenomas. BMC.Cancer 10, 333. 391. De Chiara, L., Rodriguez-Pineiro, A. M., Cordero, O. J., Rodriguez-Berrocal, F. J., Ayude, D., Rivas-Hervada And FJ, and de la Cadena, M. P. (2009) Soluble CD26 levels and its association to epidemiologic parameters in a sample population. Dis.Markers 27, 311-316. 392. Cordero, O. J., De Chiara, L., Lemos-Gonzalez, Y., Paez, d. l. C., and Rodriguez-Berrocal, F. J. (2008) How the measurements of a few serum markers can be combined to enhance their clinical values in the management of cancer. Anticancer Res. 28, 2333-2341. 393. Cordero, O. J., Ayude, D., Nogueira, M., Rodriguez-Berrocal, F. J., and de la Cadena, M. P. (2000) Preoperative serum CD26 levels: diagnostic efficiency and predictive value for colorectal cancer. Br.J Cancer 83, 1139-1146. 394. Cordero, O. J., Imbernon, M., Chiara, L. D., Martinez-Zorzano, V. S., Ayude, D., de la Cadena, M. P., and Rodriguez- Berrocal, F. J. (2011) Potential of soluble CD26 as a serum marker for colorectal cancer detection. World J Clin.Oncol. 2, 245-261. 395. Urade, M., Komatsu, M., Yamaoka, M., Fukasawa, K., Harada, M., Mima, T., and Matsuya, T. (1989) Serum dipeptidyl peptidase activities as a possible marker of oral cancer. Cancer 64, 1274-1280. 396. Fukasawa, K., Harada, M., Komatsu, M., Yamaoka, M., Urade, M., Shirasuna, K., and Miyazaki, T. (1982) Serum dipeptidyl peptidase (DPP) IV activities in oral cancer patients. Int.J Oral Surg. 11, 246-250. 397. Kojima, K., Mihara, R., Sakai, T., Togari, A., Matsui, T., Shinpo, K., Fujita, K., Fukasawa, K., Harada, M., and Nagatsu, T. (1987) Serum activities of dipeptidyl-aminopeptidase II and dipeptidyl-aminopeptidase IV in tumor-bearing animals and in cancer patients. Biochem.Med.Metab Biol. 37, 35-41. 398. Gruss, H. J., Pinto, A., Duyster, J., Poppema, S., and Herrmann, F. (1997) Hodgkin's disease: a tumor with disturbed immunological pathways. Immunol.Today 18, 156-163. 399. Hoffmann, T. and Demuth, H.-U. (2002) Therapeutic strategies exploiting DP IV inhibition - Target Disease: Type 2 Diabetes. In Langner, J. and Ansorge, S., editors. Ectopeptidases, Kluwer Academic/Plenum Publishers, New York, USA, 259-278. 400. Eager, R. M., Cunningham, C. C., Senzer, N., Richards, D. A., Raju, R. N., Jones, B., Uprichard, M., and Nemunaitis, J. (2009) Phase II Trial of Talabostat and Docetaxel in Advanced Non-small Cell Lung Cancer. Clin.Oncol.(R.Coll.Radiol.) 21, 464-472. 401. Welt, S., Divgi, C. R., Scott, A. M., Garin-Chesa, P., Finn, R. D., Graham, M., Carswell, E. A., Cohen, A., Larson, S. M., Old, L. J., and . (1994) Antibody targeting in metastatic colon cancer: a phase I study of monoclonal antibody F19 against a cell-surface protein of reactive tumor stromal fibroblasts. J.Clin.Oncol. 12, 1193-1203. 402. Tanswell, P., Garin-Chesa, P., Rettig, W. J., Welt, S., Divgi, C. R., Casper, E. S., Finn, R. D., Larson, S. M., Old, L. J., and Scott, A. M. (2001) Population pharmacokinetics of antifibroblast activation protein monoclonal antibody F19 in cancer patients. Br.J.Clin.Pharmacol. 51, 177-180. 403. Schmidt, A., Muller, D., Mersmann, M., Wuest, T., Gerlach, E., Garin-Chesa, P., Rettig, W. J., Pfizenmaier, K., and Moosmayer, D. (2001) Generation of human high-affinity antibodies specific for the fibroblast activation protein by guided selection. Eur.J.Biochem. 268, 1730-1738. 404. Scott, A. M., Wiseman, G., Welt, S., Adjei, A., Lee, F. T., Hopkins, W., Divgi, C. R., Hanson, L. H., Mitchell, P., Gansen, D. N., Larson, S. M., Ingle, J. N., Hoffman, E. W., Tanswell, P., Ritter, G., Cohen, L. S., Bette, P., Arvay, L., Amelsberg, A., Vlock, D., Rettig, W. J., and Old, L. J. (2003) A Phase I Dose-Escalation Study of Sibrotuzumab in Patients with Advanced or Metastatic Fibroblast Activation Protein-positive Cancer. Clin.Cancer Res. 9, 1639-1647. 405. Wuest, T., Moosmayer, D., and Pfizenmaier, K. (2001) Construction of a bispecific single chain antibody for recruitment of cytotoxic T cells to the tumour stroma associated antigen fibroblast activation protein. J.Biotechnol. 92, 159-168.

122 Reference

406. Drucker, D. J., Sherman, S. I., Bergenstal, R. M., and Buse, J. B. (2011) The safety of incretin-based therapies-- review of the scientific evidence. J Clin.Endocrinol Metab 96, 2027-2031. 407. Baetta, R. and Corsini, A. (2011) Pharmacology of dipeptidyl peptidase-4 inhibitors: similarities and differences. Drugs 71, 1441-1467. 408. Mendieta, L., Tarrago, T., and Giralt, E. (2011) Recent patents of dipeptidyl peptidase IV inhibitors. Expert.Opin.Ther.Pat 21, 1693-1741. 409. Lim, K. S., Cho, J. Y., Kim, B. H., Kim, J. R., Kim, H. S., Kim, D. K., Kim, S. H., Yim, H. J., Lee, S. H., Shin, S. G., Jang, I. J., and Yu, K. S. (2009) Pharmacokinetics and pharmacodynamics of LC15-0444, a novel dipeptidyl peptidase IV inhibitor, after multiple dosing in healthy volunteers. Br.J Clin.Pharmacol. 68, 883-890. 410. Mersmann, M., Schmidt, A., Rippmann, J. F., Wuest, T., Brocks, B., Rettig, W. J., Garin-Chesa, P., Pfizenmaier, K., and Moosmayer, D. (2001) Human antibody derivatives against the fibroblast activation protein for tumor stroma targeting of carcinomas. Int.J.Cancer 92, 240-248. 411. Loeffler, M., Kruger, J. A., Niethammer, A. G., and Reisfeld, R. A. (2006) Targeting tumor-associated fibroblasts improves cancer chemotherapy by increasing intratumoral drug uptake. J Clin.Invest 116, 1955-1962. 412. Steinbrecher, A., Reinhold, D., Quigley, L., Gado, A., Tresser, N., Izikson, L., Born, I., Faust, J., Neubert, K., Martin, R., Ansorge, S., and Brocke, S. (2000) Dipeptidyl peptidase IV in inflammatory CNS disease. Adv.Exp.Med.Biol. 477, 145-153. 413. Steinbrecher, A., Reinhold, D., Quigley, L., Gado, A., Tresser, N., Izikson, L., Born, I., Faust, J., Neubert, K., Martin, R., Ansorge, S., and Brocke, S. (2001) Targeting dipeptidyl peptidase IV (CD26) suppresses autoimmune encephalomyelitis and up-regulates TGF-beta 1 secretion in vivo. J Immunol. 166, 2041-2048. 414. Reinhold, D., Hemmer, B., Gran, B., Born, I., Faust, J., Neubert, K., McFarland, H. F., Martin, R., and Ansorge, S. (1998) Inhibitors of dipeptidyl peptidase IV/CD26 suppress activation of human MBP-specific CD4+ T cell clones. J.Neuroimmunol. 87, 203-209. 415. Reinhold, D., Vetter, R. W., Mnich, K., Buhling, F., Lendeckel, U., Born, I., Faust, J., Neubert, K., Gollnick, H., and Ansorge, S. (1998) Dipeptidyl peptidase IV (DP IV, CD26) is involved in regulation of DNA synthesis in human keratinocytes. FEBS Lett. 428, 100-104. 416. Vetter, R., Reinhold, D., Buhling, F., Lendeckel, U., Born, I., Faust, J., Neubert, K., Ansorge, S., and Gollnick, H. (2000) DNA synthesis in cultured human keratinocytes and HaCaT keratinocytes is reduced by specific inhibition of dipeptidyl peptidase IV (CD26) enzymatic activity. Adv.Exp.Med.Biol. 477, 167-171. 417. Novelli, M., Savoia, P., Fierro, M. T., Verrone, A., Quaglino, P., and Bernengo, M. G. (1996) Keratinocytes express dipeptidyl-peptidase IV (CD26) in benign and malignant skin diseases. Br.J.Dermatol. 134, 1052-1056. 418. Williams, Y. N., Baba, H., Hayashi, S., Ikai, H., Sugita, T., Tanaka, S., Miyasaka, N., and Kubota, T. (2003) Dipeptidyl peptidase IV on activated T cells as a target molecule for therapy of rheumatoid arthritis. Clin.Exp.Immunol. 131, 68- 74. 419. Lautar, S. L., Rojas, C., Slusher, B. S., Wozniak, K. M., Wu, Y., Thomas, A. G., Waldon, D., Li, W., Ferraris, D., and Belyakov, S. (2005) DPP IV inhibitor blocks mescaline-induced scratching and amphetamine-induced hyperactivity in mice. Brain Res 1048, 177-184. 420. Korom, S., De Meester, I., Stadlbauer, T. H., Chandraker, A., Schaub, M., Sayegh, M. H., Belyaev, A., Haemers, A., Scharpe, S., and Kupiec-Weglinski, J. W. (1997) Inhibition of CD26/dipeptidyl peptidase IV activity in vivo prolongs cardiac allograft survival in rat recipients. Transplantation 63, 1495-1500. 421. Korom, S., De Meester, I., Onodera, K., Stadlbauer, T. H., Borloo, M., Lambeir, A. M., and Kupiec-Weglinski, J. W. (1997) The effects of CD26/DPP IV-targeted therapy on acute allograft rejection. Transplant.Proc. 29, 1274-1275. 422. Korom, S., De Meester, I., Coito, A. J., Graser, E., Pratschke, J., Konig, S., Grimm, H., Volk, H. D., Scharpe, S., and Kupiec-Weglinski, J. W. (1999) CD26/DPP IV-mediated modulation of acute rejection. Transplant.Proc. 31, 873. 423. Korom, S., De Meester, I., Schmidbauer, G., Pratschke, J., Brendel, M. D., Durinx, C., Schwemmle, K., Haemers, A., Scharpe, S., and Kupiec-Weglinski, J. W. (1999) Specific inhibition of CD26/DPP IV enzymatic activity in allograft recipients: effects on humoral immunity. Transplant.Proc. 31, 778. 424. Korom, S., De, M., I, Belyaev, A., Schmidbauer, G., and Schwemmle, K. (2003) CD26/DPP IV in experimental and clinical organ transplantation. Adv.Exp.Med.Biol. 524:133-43., 133-143. 425. Reinhold, D., Hemmer, B., Gran, B., Steinbrecher, A., Brocke, S., Kahne, T., Wrenger, S., Born, I., Faust, J., Neubert, K., Martin, R., and Ansorge, S. (2000) Dipeptidyl peptidase IV (CD26): role in T cell activation and autoimmune disease. Adv.Exp.Med.Biol. 477, 155-160. 426. Kubota, T., Flentke, G. R., Bachovchin, W. W., and Stollar, B. D. (1992) Involvement of dipeptidyl peptidase IV in an in vivo immune response. Clin.Exp.Immunol. 89, 192-197. 427. Ansorge, S., Bank, U., Heimburg, A., Helmuth, M., Koch, G., Tadje, J., Lendeckel, U., Wolke, C., Neubert, K., Faust, J., Fuchs, P., Reinhold, D., Thielitz, A., and Tager, M. (2009) Recent insights into the role of dipeptidyl aminopeptidase IV (DPIV) and aminopeptidase N (APN) families in immune functions. Clin.Chem.Lab Med. 47, 253- 261. 428. Bank, U., Tadje, J., Helmuth, M., Stefin, S., Tager, M., Wolke, C., Wischeropp, A., Ittenson, A., Reinhold, D., Ansorge, S., and Lendeckel, U. (2006) Dipeptidylpeptidase IV (DPIV) and alanyl-aminopeptidases (AAPs) as a new target complex for treatment of autoimmune and inflammatory diseases-proof of concept in a mouse model of colitis. Adv.Exp.Med.Biol. 575, 143-153. 429. Reinhold, D., Bank, U., Entz, D., Goihl, A., Stoye, D., Wrenger, S., Brocke, S., Thielitz, A., Stefin, S., Nordhoff, K., Heimburg, A., Tager, M., and Ansorge, S. (2011) PETIR-001, a dual inhibitor of dipeptidyl peptidase IV (DP IV) and aminopeptidase N (APN), ameliorates experimental autoimmune encephalomyelitis in SJL/J mice. Biol Chem 392, 233-237. 430. Biton, A., Bank, U., Tager, M., Ansorge, S., Reinhold, D., Lendeckel, U., and Brocke, S. (2006) Dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) as regulators of T cell function and targets of immunotherapy in CNS inflammation. Adv.Exp.Med.Biol. 575, 177-186. 431. Bank, U., Heimburg, A., Helmuth, M., Stefin, S., Lendeckel, U., Reinhold, D., Faust, J., Fuchs, P., Sens, B., Neubert, K., Tager, M., and Ansorge, S. (2006) Triggering endogenous immunosuppressive mechanisms by combined targeting of Dipeptidyl peptidase IV (DPIV/CD26) and Aminopeptidase N (APN/ CD13)--a novel approach for the treatment of inflammatory bowel disease. Int.Immunopharmacol. 6, 1925-1934.

123 Reference

432. Thielitz, A., Reinhold, D., Vetter, R., Bank, U., Helmuth, M., Hartig, R., Wrenger, S., Wiswedel, I., Lendeckel, U., Kahne, T., Neubert, K., Faust, J., Zouboulis, C. C., Ansorge, S., and Gollnick, H. (2007) Inhibitors of dipeptidyl peptidase IV and aminopeptidase N target major pathogenetic steps in acne initiation. J Invest Dermatol. 127, 1042- 1051. 433. Kubota, T., Iizuka, H., Bachovchin, W. W., and Stollar, B. D. (1994) Dipeptidyl peptidase IV (DP IV) activity in serum and on lymphocytes of MRL/Mp-lpr/lpr mice correlates with disease onset. Clin.Exp.Immunol. 96, 292-296. 434. Jiang, J. D., Wilk, S., Li, J., Zhang, H., and Bekesi, J. G. (1997) Inhibition of human immunodeficiency virus type 1 infection in a T-cell line (CEM) by new dipeptidyl-peptidase IV (CD26) inhibitors. Res.Virol. 148, 255-266. 435. Schols, D., Proost, P., Struyf, S., Wuyts, A., De, M., I, Scharpe, S., Van Damme, J., and De Clercq, E. (1998) CD26- processed RANTES(3-68), but not intact RANTES, has potent anti-HIV-1 activity. Antiviral Res 39, 175-187. 436. Ohtsuki, T., Tsuda, H., and Morimoto, C. (2000) Good or evil: CD26 and HIV infection. J.Dermatol.Sci. 22, 152-160. 437. Ghersi, G., Chen, W., Lee, E. W., and Zukowska, Z. (2001) Critical role of dipeptidyl peptidase IV in neuropeptide Y- mediated endothelial cell migration in response to wounding. Peptides 22, 453-458. 438. Sato, K. and Dang, N. H. (2003) CD26: A novel treatment target for T-cell lymphoid malignancies? (Review). Int.J Oncol. 22, 481-497. 439. Demuth, H. U., McIntosh, C. H., and Pederson, R. A. (2005) Type 2 diabetes-therapy with dipeptidyl peptidase IV inhibitors. Biochim.Biophys.Acta 1751, 33-44. 440. McIntosh, C. H., Demuth, H.-U., Pospisilik, J. A., and Pederson, R. A. (2005) Dipeptidyl peptidase IV inhibitors: How do they work as new antidabetic agents? Regul.Pept. 128, 159-165. 441. McIntosh, C. H., Demuth, H. U., Kim, S. J., Pospisilik, J. A., and Pederson, R. A. (2006) Applications of dipeptidyl peptidase IV inhibitors in diabetes mellitus. Int.J.Biochem.Cell Biol. 38, 860-872. 442. Pederson, R. A., White, H. A., Schlenzig, D., Pauly, R. P., McIntosh, C. H., and Demuth, H. U. (1998) Improved glucose tolerance in Zucker fatty rats by oral administration of the dipeptidyl peptidase IV inhibitor isoleucine thiazolidide. Diabetes 47, 1253-1258. 443. Gallwitz, B. (2006) Therapies for the treatment of type 2 diabetes mellitus based on incretin action. Minerva Endocrinol. 31, 133-147. 444. Gallwitz, B. (2011) Small molecule dipeptidylpeptidase IV inhibitors under investigation for diabetes mellitus therapy. Expert.Opin.Investig.Drugs 20, 723-732. 445. Weber, A. E. and Thornberry, N. A. (2010) Dipeptidyl peptidase 4 inhibitors. In Abraham, D. J. and Rotella, D. P., editors. Burger's Medicinal Chemistry, Drug Discovery, and Development, John Wiley & Sons, Inc., Hoboken, New Jersey, 1-38. 446. Sebokova, E., Christ, A. D., Boehringer, M., and Mizrahi, J. (2007) Dipeptidyl peptidase IV inhibitors: the next generation of new promising therapies for the management of type 2 diabetes. Curr.Top.Med.Chem. 7, 547-555. 447. Grondin, G., Hooper, N. M., and LeBel, D. (1999) Specific localization of and dipeptidyl peptidase IV in secretion granules of two different pancreatic islet cells. J.Histochem.Cytochem. 47, 489-498. 448. Poulsen, M. D., Hansen, G. H., Dabelsteen, E., Hoyer, P. E., Noren, O., and Sjostrom, H. (1993) Dipeptidyl peptidase IV is sorted to the secretory granules in pancreatic islet A-cells. J.Histochem.Cytochem. 41, 81-88. 449. Rettig, W. J., Garin-Chesa, P., Beresford, H. R., Oettgen, H. F., Melamed, M. R., and Old, L. J. (1988) Cell-surface glycoproteins of human sarcomas: differential expression in normal and malignant tissues and cultured cells. Proc.Natl.Acad.Sci.U.S.A 85, 3110-3114. 450. Goldstein, L. A. and Chen, W. T. (2000) Identification of an alternatively spliced seprase mRNA that encodes a novel intracellular isoform. J Biol.Chem. 275, 2554-2559. 451. Hartel, S., Gossrau, R., Hanski, C., and Reutter, W. (1988) Dipeptidyl peptidase (DPP) IV in rat organs. Comparison of immunohistochemistry and activity histochemistry. Histochemistry 89, 151-161. 452. Matheeussen, V., Baerts, L., De Meyer, G., De Keulenaer, G., Van, D., V, Augustyns, K., Dubois, V., Scharpe, S., and De, M., I (2011) Expression and spatial heterogeneity of dipeptidyl peptidases in endothelial cells of conduct vessels and capillaries. Biol Chem 392, 189-198. 453. Mentlein, R. (2005) Dipeptidyl-peptidase IV and aminopeptidase P: molecular switches of NPY/PYY receptor affinities. In Zukowska, Z. and Feuerstein, G. Z., editors. The NPY Family in Peptides in Immune Disorders, Inflammation, Angiogenesis and Cancer, Birkhäuser Verlag, Basel, 75-84. 454. Harmar, A. J. (2001) Family-B G-protein-coupled receptors. Genome Biol. 2, 3001-3013. 455. Sherwood, N. M., Krueckl, S. L., and McRory, J. E. (2000) The origin and function of the pituitary adenylate cyclase- activating polypeptide (PACAP)/glucagon superfamily. Endocr.Rev. 21, 619-670. 456. De Meester, I., Durinx, C., Bal, G., Proost, P., Struyf, S., Goossens, F., Augustyns, K., and Scharpe, S. (2000) Natural substrates of dipeptidyl peptidase IV. Adv.Exp.Med.Biol. 477, 67-87. 457. Boey, D., Sainsbury, A., and Herzog, H. (2007) The role of peptide YY in regulating glucose homeostasis. Peptides 28, 390-395. 458. Bottcher, G., Ahren, B., Lundquist, I., and Sundler, F. (1989) Peptide YY: intrapancreatic localization and effects on insulin and glucagon secretion in the mouse. Pancreas 4, 282-288. 459. Bottcher, G., Sjoberg, J., Ekman, R., Hakanson, R., and Sundler, F. (1993) Peptide YY in the mammalian pancreas: immunocytochemical localization and immunochemical characterization. Regul.Pept. 43, 115-130. 460. Nieuwenhuizen, A. G., Karlsson, S., Fridolf, T., and Ahren, B. (1994) Mechanisms underlying the insulinostatic effect of peptide YY in mouse pancreatic islets. Diabetologia 37, 871-878. 461. Myrsen-Axcrona, U., Ekblad, E., and Sundler, F. (1997) Developmental expression of NPY, PYY and PP in the rat pancreas and their coexistence with islet hormones. Regul.Pept. 68, 165-175. 462. Ahren, B. (2000) Autonomic regulation of islet hormone secretion--implications for health and disease. Diabetologia 43, 393-410. 463. Stephan, M., Karl, T., von Hoersten, S., and Hoffmann, T. Inhibition of dipeptidyl peptidase 4 modulates anxiety-like behaviors in rats. 2007, Prague, Czech Republic. 464. Yamada, K., Wada, E., and Wada, K. (2000) Bombesin-like peptides: studies on food intake and social behaviour with receptor knock-out mice. Ann.Med. 32, 519-529. 465. Hashimoto, R., Hashimoto, H., Shintani, N., Ohi, K., Hori, H., Saitoh, O., Kosuga, A., Tatsumi, M., Iwata, N., Ozaki, N., Kamijima, K., Baba, A., Takeda, M., and Kunugi, H. (2010) Possible association between the pituitary adenylate cyclase-activating polypeptide (PACAP) gene and major depressive disorder. Neurosci.Lett. 468, 300-302. 124 Reference

466. Fujii, H., Ishihama, T., Ago, Y., Shintani, N., Kakuda, M., Hashimoto, H., Baba, A., and Matsuda, T. (2007) Methamphetamine-induced hyperactivity and behavioral sensitization in PACAP deficient mice. Peptides 28, 1674- 1679. 467. Girard, B. A., Lelievre, V., Braas, K. M., Razinia, T., Vizzard, M. A., Ioffe, Y., El Meskini, R., Ronnett, G. V., Waschek, J. A., and May, V. (2006) Noncompensation in peptide/receptor gene expression and distinct behavioral phenotypes in VIP- and PACAP-deficient mice. J Neurochem. 99, 499-513. 468. Tschenett, A., Singewald, N., Carli, M., Balducci, C., Salchner, P., Vezzani, A., Herzog, H., and Sperk, G. (2003) Reduced anxiety and improved stress coping ability in mice lacking NPY-Y2 receptors. Eur J Neurosci. 18, 143-148. 469. Karl, T. and Herzog, H. (2007) Behavioral profiling of NPY in aggression and neuropsychiatric diseases. Peptides 28, 326-333. 470. Karl, T., Chesworth, R., Duffy, L., and Herzog, H. (2010) Schizophrenia-relevant behaviours in a genetic mouse model for Y2 deficiency. Behav.Brain Res. 207, 434-440. 471. Karl, T., Duffy, L., and Herzog, H. (2008) Behavioural profile of a new mouse model for NPY deficiency. Eur J Neurosci. 28, 173-180. 472. Bilkei-Gorzo, A. and Zimmer, A. (2005) Mutagenesis and knockout models: NK1 and substance P. Handb.Exp.Pharmacol. 143-162. 473. Bilkei-Gorzo, A., Racz, I., Michel, K., and Zimmer, A. (2002) Diminished anxiety- and depression-related behaviors in mice with selective deletion of the Tac1 gene. J Neurosci. 22, 10046-10052. 474. Bilkei-Gorzo, A., Berner, J., Zimmermann, J., Wickstrom, R., Racz, I., and Zimmer, A. (2010) Increased morphine analgesia and reduced side effects in mice lacking the tac1 gene. Br.J Pharmacol. 160, 1443-1452. 475. Santarelli, L., Gobbi, G., Blier, P., and Hen, R. (2002) Behavioral and physiologic effects of genetic or pharmacologic inactivation of the substance P receptor (NK1). J Clin.Psychiatry 63 Suppl 11, 11-17. 476. Sah, R., Parker, S. L., Sheriff, S., Eaton, K., Balasubramaniam, A., and Sallee, F. R. (2007) Interaction of NPY compounds with the rat glucocorticoid-induced receptor (GIR) reveals similarity to the NPY-Y2 receptor. Peptides 28, 302-309. 477. Bourgault, S., Vaudry, D., Dejda, A., Doan, N. D., Vaudry, H., and Fournier, A. (2009) Pituitary adenylate cyclase- activating polypeptide: focus on structure-activity relationships of a neuroprotective Peptide. Curr.Med.Chem. 16, 4462-4480. 478. Bourgault, S., Vaudry, D., Botia, B., Couvineau, A., Laburthe, M., Vaudry, H., and Fournier, A. (2008) Novel stable PACAP analogs with potent activity towards the PAC1 receptor. Peptides 29, 919-932. 479. Vaudry, D., Gonzalez, B. J., Basille, M., Yon, L., Fournier, A., and Vaudry, H. (2000) Pituitary adenylate cyclase- activating polypeptide and its receptors: from structure to functions. Pharmacol.Rev. 52, 269-324. 480. Kask, A., Harro, J., Von Horsten, S., Redrobe, J. P., Dumont, Y., and Quirion, R. (2002) The neurocircuitry and receptor subtypes mediating anxiolytic-like effects of neuropeptide Y. Neurosci.Biobehav.Rev. 26, 259-283. 481. Merten, N. and Beck-Sickinger, A. G. (2006) Molecular ligand-receptor interaction of the NPY/PP peptide family. EXS 95, 35-62. 482. Dumont, Y. and Quirion, R. (2006) An overview of neuropeptide Y: pharmacology to molecular biology and receptor localization. EXS 95, 7-33. 483. Severini, C., Improta, G., Falconieri-Erspamer, G., Salvadori, S., and Erspamer, V. (2002) The tachykinin peptide family. Pharmacol.Rev. 54, 285-322. 484. Ebner, K., Sartori, S. B., and Singewald, N. (2009) Tachykinin receptors as therapeutic targets in stress-related disorders. Curr.Pharm.Des 15, 1647-1674. 485. Medeiros, M. d. and Turner, A. J. (1996) Metabolism and functions of neuropeptide Y. Neurochem.Res. 21, 1125- 1132. 486. Kastin, A. J. and Akerstrom, V. (1999) Nonsaturable entry of neuropeptide Y into brain. Am.J Physiol 276, E479- E482. 487. Medeiros, M. d. and Turner, A. J. (1993) Processing and metabolism of peptide YY. Biochem.Soc.Trans. 21, 248S. 488. Medeiros, M. D. and Turner, A. J. (1994) Processing and metabolism of peptide-YY: pivotal roles of dipeptidylpeptidase-IV, aminopeptidase-P, and endopeptidase-24.11. Endocrinology 134, 2088-2094. 489. Nonaka, N., Shioda, S., Niehoff, M. L., and Banks, W. A. (2003) Characterization of blood-brain barrier permeability to PYY3-36 in the mouse. J Pharmacol.Exp.Ther. 306, 948-953. 490. Bouras, M., Huneau, J. F., and Tome, D. (1996) The inhibition of intestinal dipeptidylaminopeptidase-IV promotes the absorption of enterostatin and des-arginine-enterostatin across rat jejunum in vitro. Life Sci. 59, 2147-2155. 491. Koizumi, M., Nakanishi, Y., Sato, H., Morinaga, Y., Ido, T., and Kimura, S. (2002) Uptake across the blood-brain barrier and tissue distribution of enterostatin after peripheral administration in rats. Physiol Behav. 77, 5-10. 492. York, D. A., Lin, L., Thomas, S. R., Braymer, H. D., and Park, M. (2006) Procolipase gene expression in the rat brain: source of endogenous enterostatin production in the brain. Brain Res 1087, 52-59. 493. Kastin, A. J., Akerstrom, V., and Pan, W. (2002) Interactions of glucagon-like peptide-1 (GLP-1) with the blood-brain barrier. J Mol.Neurosci. 18, 7-14. 494. Dogrukol-Ak, D., Tore, F., and Tuncel, N. (2004) Passage of VIP/PACAP/secretin family across the blood-brain barrier: therapeutic effects. Curr.Pharm.Des 10, 1325-1340. 495. Ussher, J. R. and Drucker, D. J. (2012) Cardiovascular Biology of the Incretin System. Endocr.Rev. 496. Drucker, D. J. (2002) Biological actions and therapeutic potential of the glucagon-like peptides. Gastroenterology 122, 531-544. 497. During, M. J., Cao, L., Zuzga, D. S., Francis, J. S., Fitzsimons, H. L., Jiao, X., Bland, R. J., Klugmann, M., Banks, W. A., Drucker, D. J., and Haile, C. N. (2003) Glucagon-like peptide-1 receptor is involved in learning and neuroprotection. Nat.Med. 9, 1173-1179. 498. Knauf, C., Cani, P. D., Perrin, C., Iglesias, M. A., Maury, J. F., Bernard, E., Benhamed, F., Gremeaux, T., Drucker, D. J., Kahn, C. R., Girard, J., Tanti, J. F., Delzenne, N. M., Postic, C., and Burcelin, R. (2005) Brain glucagon-like peptide-1 increases insulin secretion and muscle insulin resistance to favor hepatic glycogen storage. J Clin.Invest 115, 3554-3563. 499. Knauf, C., Cani, P. D., Kim, D. H., Iglesias, M. A., Chabo, C., Waget, A., Colom, A., Rastrelli, S., Delzenne, N. M., Drucker, D. J., Seeley, R. J., and Burcelin, R. (2008) Role of central nervous system glucagon-like Peptide-1 receptors in enteric glucose sensing. Diabetes 57, 2603-2612. 125 Reference

500. Nussdorfer, G. G., Bahcelioglu, M., Neri, G., and Malendowicz, L. K. (2000) Secretin, glucagon, gastric inhibitory polypeptide, parathyroid hormone, and related peptides in the regulation of the hypothalamus- pituitary-adrenal axis. Peptides 21, 309-324. 501. Baggio, L. L. and Drucker, D. J. (2007) Biology of incretins: GLP-1 and GIP. Gastroenterology 132, 2131-2157. 502. Drucker, D. J. (2006) The biology of incretin hormones. Cell Metab 3, 153-165. 503. Nyberg, J., Anderson, M. F., Meister, B., Alborn, A. M., Strom, A. K., Brederlau, A., Illerskog, A. C., Nilsson, O., Kieffer, T. J., Hietala, M. A., Ricksten, A., and Eriksson, P. S. (2005) Glucose-dependent insulinotropic polypeptide is expressed in adult hippocampus and induces progenitor cell proliferation. J Neurosci. 25, 1816-1825. 504. Nyberg, J., Jacobsson, C., Anderson, M. F., and Eriksson, P. S. (2007) Immunohistochemical distribution of glucose- dependent insulinotropic polypeptide in the adult rat brain. J Neurosci.Res 85, 2099-2119. 505. Green, B. D., Irwin, N., and Flatt, P. R. (2006) Pituitary adenylate cyclase-activating peptide (PACAP): Assessment of dipeptidyl peptidase IV degradation, insulin-releasing activity and antidiabetic potential. Peptides. .. 506. Bonnet, C., Leger, L., Baubet, V., Debilly, G., and Cespuglio, R. (1997) Influence of a 1 h immobilization stress on sleep states and corticotropin-like intermediate lobe peptide (CLIP or ACTH18-39, Ph-ACTH18-39) brain contents in the rat. Brain Res 751, 54-63. 507. Vogel, D., Rosche, F, Wetzel, W, Hartung, K, Seidenbecher, T, Balschun, D, and Demuth, H-U. () The Degradation of Clip(Corticotropin-Like Intermediate Lobe Peptide) And Clip Fragments by dipeptidyl Peptidase IV and prolyl Endopeptidase. unknown 508. Vanderheyden, M., Bartunek, J., Goethals, M., Verstreken, S., Lambeir, A. M., De, M., I, and Scharpe, S. (2009) Dipeptidyl-peptidase IV and B-type natriuretic peptide. From bench to bedside. Clin.Chem.Lab Med. 47, 248-252. 509. Griebel, G. (1999) Is there a future for neuropeptide receptor ligands in the treatment of anxiety disorders? Pharmacol.Ther. 82, 1-61. 510. Katsouni, E., Sakkas, P., Zarros, A., Skandali, N., and Liapi, C. (2009) The involvement of substance P in the induction of aggressive behavior. Peptides 30, 1586-1591. 511. Freed, A. L., Audus, K. L., and Lunte, S. M. (2002) Investigation of substance P transport across the blood-brain barrier. Peptides 23, 157-165. 512. Singewald, N., Chicchi, G. G., Thurner, C. C., Tsao, K. L., Spetea, M., Schmidhammer, H., Sreepathi, H. K., Ferraguti, F., Singewald, G. M., and Ebner, K. (2008) Modulation of basal and stress-induced amygdaloid substance P release by the potent and selective NK1 receptor antagonist L-822429. J Neurochem. 106, 2476-2488. 513. Ebner, K. and Singewald, N. (2006) The role of substance P in stress and anxiety responses. Amino.Acids 31, 251- 272. 514. Ebner, K., Muigg, P., Singewald, G., and Singewald, N. (2008) Substance P in stress and anxiety: NK-1 receptor antagonism interacts with key brain areas of the stress circuitry. Ann.N.Y.Acad.Sci. 1144, 61-73. 515. Yan, T. C., Hunt, S. P., and Stanford, S. C. (2009) Behavioural and neurochemical abnormalities in mice lacking functional tachykinin-1 (NK1) receptors: a model of attention deficit hyperactivity disorder. Neuropharmacology 57, 627-635. 516. Chappa, A. K., Cooper, J. D., Audus, K. L., and Lunte, S. M. (2007) Investigation of the metabolism of substance P at the blood-brain barrier using LC-MS/MS. J Pharm.Biomed.Anal. 43, 1409-1415. 517. Chappa, A. K., Audus, K. L., and Lunte, S. M. (2006) Characteristics of substance P transport across the blood-brain barrier. Pharm.Res 23, 1201-1208. 518. Moeller, I., Lew, R. A., Mendelsohn, F. A., Smith, A. I., Brennan, M. E., Tetaz, T. J., and Chai, S. Y. (1997) The globin fragment LVV-hemorphin-7 is an endogenous ligand for the AT4 receptor in the brain. J Neurochem. 68, 2530-2537. 519. Zadina, J. E. (2002) Isolation and distribution of endomorphins in the central nervous system. Jpn.J Pharmacol. 89, 203-208. 520. Champion, H. C., Zadina, J. E., Kastin, A. J., Hackler, L., Ge, L. J., and Kadowitz, P. J. (1997) Endomorphin 1 and 2, endogenous ligands for the mu-opioid receptor, decrease cardiac output, and total peripheral resistance in the rat. Peptides 18, 1393-1397. 521. Wang, Q. P., Zadina, J. E., Guan, J. L., and Shioda, S. (2002) Morphological studies of the endomorphinergic neurons in the central nervous system. Jpn.J Pharmacol. 89, 209-215. 522. Kastin, A. J., Fasold, M. B., Smith, R. R., Horner, K. A., and Zadina, J. E. (2001) Saturable brain-to-blood transport of endomorphins. Exp.Brain Res 139, 70-75. 523. Fichna, J., Janecka, A., Piestrzeniewicz, M., Costentin, J., and Do Rego, J. C. (2007) Antidepressant-like effect of endomorphin-1 and endomorphin-2 in mice. Neuropsychopharmacology 32, 813-821. 524. Fichna, J., Janecka, A., Costentin, J., and Do Rego, J. C. (2007) The endomorphin system and its evolving neurophysiological role. Pharmacol.Rev. 59, 88-123. 525. Fujita, T. and Kumamoto, E. (2006) Inhibition by endomorphin-1 and endomorphin-2 of excitatory transmission in adult rat substantia gelatinosa neurons. Neuroscience. 139, 1095-1105. 526. Banks, W. A., Kastin, A. J., and Michals, E. A. (1987) Tyr-MIF-1 and Met-enkephalin share a saturable blood-brain barrier transport system. Peptides 8, 899-903. 527. Zadina, J. E., Kastin, A. J., Coy, D. H., and Adinoff, B. A. (1985) Developmental, behavioral, and opiate receptor changes after prenatal or postnatal beta-endorphin, CRF, or Tyr-MIF-1. Psychoneuroendocrinology 10, 367-383. 528. Galina, Z. H. and Kastin, A. J. (1988) Differential activity of the endogenous antiopiate Tyr-MIF-1 after various intensities of stress. Neurosci.Lett. 84, 312-316. 529. Galina, Z. H. and Kastin, A. J. (1987) Tyr-MIF-1 attenuates antinociceptive responses induced by three models of stress-analgesia. Br.J Pharmacol. 90, 669-674. 530. Maresh, G. A., Kastin, A. J., Brown, T. T., Zadina, J. E., and Banks, W. A. (1999) Peptide transport system-1 (PTS-1) for Tyr-MIF-1 and Met-enkephalin differs from the receptors for either. Brain Res 839, 336-340. 531. Reed, G. W., Olson, G. A., and Olson, R. D. (1994) The Tyr-MIF-1 family of peptides. Neurosci.Biobehav.Rev. 18, 519-525. 532. Bocheva, A. and Dzambazova-Maximova, E. (2004) Antiopioid properties of the TYR-MIF-1 family. Methods Find.Exp.Clin.Pharmacol. 26, 673-677. 533. Miller, L. G. and Kastin, A. J. (1987) MIF-1 and Tyr-MIF-1 augment GABA-stimulated benzodiazepine receptor binding. Peptides 8, 751-755.

126 Reference

534. Bedard, T., Mountney, C., Kent, P., Anisman, H., and Merali, Z. (2007) Role of gastrin-releasing peptide and neuromedin B in anxiety and fear-related behavior. Behav.Brain Res 179, 133-140. 535. Mountney, C., Sillberg, V., Kent, P., Anisman, H., and Merali, Z. (2006) The role of gastrin-releasing peptide on conditioned fear: differential cortical and amygdaloid responses in the rat. Psychopharmacology (Berl) 189, 287-296. 536. Kentroti, S., Dees, W. L., and McCann, S. M. (1988) Evidence for a physiological role of hypothalamic gastrin- releasing peptide to suppress growth hormone and prolactin release in the rat. Proc.Natl.Acad.Sci.U.S.A 85, 953-957. 537. Presti-Torres, J., de Lima, M. N., Scalco, F. S., Caldana, F., Garcia, V. A., Guimaraes, M. R., Schwartsmann, G., Roesler, R., and Schroder, N. (2007) Impairments of social behavior and memory after neonatal gastrin-releasing peptide receptor blockade in rats: Implications for an animal model of neurodevelopmental disorders. Neuropharmacology 52, 724-732. 538. Roesler, R., Henriques, J. A., and Schwartsmann, G. (2006) Gastrin-releasing peptide receptor as a molecular target for psychiatric and neurological disorders. CNS.Neurol.Disord.Drug Targets. 5, 197-204. 539. Wada, E., Way, J., Lebacq-Verheyden, A. M., and Battey, J. F. (1990) Neuromedin B and gastrin-releasing peptide mRNAs are differentially distributed in the rat nervous system. J Neurosci. 10, 2917-2930. 540. Ozawa, H. and Takata, K. (1995) The granin family--its role in sorting and secretory granule formation. Cell Struct.Funct. 20, 415-420. 541. Nowakowski, C., Kaufmann, W. A., Adlassnig, C., Maier, H., Salimi, K., Jellinger, K. A., and Marksteiner, J. (2002) Reduction of chromogranin B-like immunoreactivity in distinct subregions of the hippocampus from individuals with schizophrenia. Schizophr.Res 58, 43-53. 542. Gee, P., Rhodes, C. H., Fricker, L. D., and Angeletti, R. H. (1993) Expression of neuropeptide processing enzymes and neurosecretory proteins in ependyma and choroid plexus epithelium. Brain Res 617, 238-248. 543. Kandlhofer, S., Hoertnagl, B., Czech, T., Baumgartner, C., Maier, H., Novak, K., and Sperk, G. (2000) Chromogranins in temporal lobe epilepsy. Epilepsia 41, S111-S114. 544. Landen, M., Grenfeldt, B., Davidsson, P., Stridsberg, M., Regland, B., Gottfries, C. G., and Blennow, K. (1999) Reduction of chromogranin A and B but not C in the cerebrospinal fluid in subjects with schizophrenia. Eur Neuropsychopharmacol. 9, 311-315. 545. Lechner, T., Adlassnig, C., Humpel, C., Kaufmann, W. A., Maier, H., Reinstadler-Kramer, K., Hinterholzl, J., Mahata, S. K., Jellinger, K. A., and Marksteiner, J. (2004) Chromogranin peptides in Alzheimer's disease. Exp.Gerontol. 39, 101-113. 546. Mahata, S. K., Mahata, M., Marksteiner, J., Sperk, G., Fischer-Colbrie, R., and Winkler, H. (1991) Distribution of mRNAs for Chromogranins A and B and Secretogranin II in Rat Brain. Eur J Neurosci. 3, 895-904. 547. Marksteiner, J., Kaufmann, W. A., Gurka, P., and Humpel, C. (2002) Synaptic proteins in Alzheimer's disease. J Mol.Neurosci. 18, 53-63. 548. Weiler, R., Marksteiner, J., Bellmann, R., Wohlfarter, T., Schober, M., Fischer-Colbrie, R., Sperk, G., and Winkler, H. (1990) Chromogranins in rat brain: characterization, topographical distribution and regulation of synthesis. Brain Res. 532, 87-94. 549. Salahuddin, M. J., Sekiya, K., Ghatei, M. A., and Bloom, S. R. (1989) Regional distribution of chromogranin B 420- 493-like immunoreactivity in the pituitary gland and central nervous system of man, guinea-pig and rat. Neuroscience 30, 231-240. 550. Fischer-Colbrie, R., Lassmann, H., Hagn, C., and Winkler, H. (1985) Immunological studies on the distribution of chromogranin A and B in endocrine and nervous tissues. Neuroscience 16, 547-555. 551. Weiler, R., Marksteiner, J., Bellmann, R., Wohlfarter, T., Schober, M., Fischer-Colbrie, R., Sperk, G., and Winkler, H. (1990) Chromogranins in rat brain: characterization, topographical distribution and regulation of synthesis. Brain Res 532, 87-94. 552. Lassmann, H., Hagn, C., Fischer-Colbrie, R., and Winkler, H. (1986) Presence of chromogranin A, B and C in bovine endocrine and nervous tissues: a comparative immunohistochemical study. Histochem.J 18, 380-386. 553. Lloyd, R. V., Hawkins, K., Jin, L., Kulig, E., and Fields, K. (1992) Chromogranin A, chromogranin B and secretogranin II mRNAs in the pituitary and adrenal glands of various mammals. Regulation of chromogranin A, chromogranin B and secretogranin II mRNA levels by estrogen. Lab Invest 67, 394-404. 554. Majdoubi, M. E., Metz-Boutigue, M. H., Garcia-Sablone, P., Theodosis, D. T., and Aunis, D. (1996) Immunocytochemical localization of chromogranin A in the normal and stimulated hypothalamo-neurohypophysial system of the rat. J Neurocytol. 25, 405-416. 555. Foitzik, K., Krause, K., Nixon, A. J., Ford, C. A., Ohnemus, U., Pearson, A. J., and Paus, R. (2003) Prolactin and its receptor are expressed in murine hair follicle epithelium, show hair cycle-dependent expression, and induce catagen. Am.J.Pathol. 162, 1611-1621. 556. Tanaka, S., Tatsumi, K., Okubo, K., Itoh, K., Kawamoto, S., Matsubara, K., and Amino, N. (2002) Expression profile of active genes in the human pituitary gland. J Mol.Endocrinol. 28, 33-44. 557. Seidl, R. and Schaefer, W. (1991) Preparative purification of dipeptidyl peptidase IV. Prep.Biochem. 21, 141-150. 558. Wagner, L., Wermann, M., Rosche, F., Rahfeld, J. U., Hoffmann, T., and Demuth, H. U. (2011) Isolation of dipeptidyl peptidase IV (DP 4) isoforms from porcine kidney by preparative isoelectric focusing to improve crystallization. Biol Chem 392, 665-677. 559. Abbott, C.A. and Gorrel, M.D. (2001) Dipeptidyl peptidases. WO 01/19866 A1 Australia. 560. Abbott, C.A. and Gorrel, M.D. (2004) Dipeptidyl peptidases. US 2004/0053369 A1 Australia. 561. Liou, J.R. (2002) Regulation of human dipeptidyl peptidase 8. W0 02/066627 Germany. 562. Kastin, A. J. and Akerstrom, V. (1999) Nonsaturable entry of neuropeptide Y into brain. Am.J.Physiol. 276, E479- E482. 563. Kastin, A. J., Pan, W., Maness, L. M., and Banks, W. A. (1999) Peptides crossing the blood-brain barrier: some unusual observations. Brain Res 848, 96-100. 564. Kastin, A. J. and Akerstrom, V. (1999) Orexin A but not orexin B rapidly enters brain from blood by simple diffusion. J Pharmacol.Exp.Ther. 289, 219-223. 565. Cottrell, G. T. and Ferguson, A. V. (2004) Sensory circumventricular organs: central roles in integrated autonomic regulation. Regul.Pept. 117, 11-23. 566. Buller, K. M. (2001) Role of circumventricular organs in pro-inflammatory cytokine-induced activation of the hypothalamic-pituitary-adrenal axis. Clin.Exp.Pharmacol.Physiol 28, 581-589. 127 Reference

567. Johnson, A. K. and Gross, P. M. (1993) Sensory circumventricular organs and brain homeostatic pathways. FASEB J 7, 678-686. 568. Miller, C. C., Holmes, P. V., and Edwards, G. L. (2002) Area postrema lesions elevate NPY levels and decrease anxiety-related behavior in rats. Physiol Behav. 77, 135-140. 569. De Seranno, S., Estrella, C., Loyens, A., Cornea, A., Ojeda, S. R., Beauvillain, J. C., and Prevot, V. (2004) Vascular endothelial cells promote acute plasticity in ependymoglial cells of the neuroendocrine brain. J Neurosci. 24, 10353- 10363. 570. Schnabel, R., Bernstein, H. G., Luppa, H., Lojda, Z., and Barth, A. (1992) Aminopeptidases in the circumventricular organs of the mouse brain: a histochemical study. Neuroscience 47, 431-438. 571. Dumont, Y., Moyse, E., Fournier, A., and Quirion, R. (2007) Distribution of peripherally injected peptide YY ([125I] PYY (3-36)) and pancreatic polypeptide ([125I] hPP) in the CNS: enrichment in the area postrema. J Mol.Neurosci. 33, 294-304. 572. Egen, N. B., Bliss, M., Mayersohn, M., Owens, S. M., Arnold, L., and Bier, M. (1988) Isolation of monoclonal antibodies to phencyclidine from ascites fluid by preparative isoelectric focusing in the Rotofor. Anal.Biochem. 172, 488-494. 573. Ayala, A., Parrado, J., and Machado, A. (1998) Use of Rotofor preparative isoelectrofocusing cell in protein purification procedure. Appl.Biochem.Biotechnol. 69, 11-16. 574. Bier, M. (1998) Recycling isoelectric focusing and isotachophoresis. Electrophoresis 19, 1057-1063. 575. Garfin, D. E. (1990) Isoelectric focusing. Methods Enzymol. 182, 459-477. 576. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal.Biochem 72, 248-254. 577. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. 578. Wagner, L., Hoffmann, T., Gerhartz, B., and Demuth, H.-U. “Distribution of dipeptidyl peptidase IV activity-like enzymes in canine and porcine tissue sections by nested RT-PCR” Dipeptidyl-Aminopeptidases: Basic Science and Clinical Applications. 26 - 29 September 2002, Berlin, Germany. 579. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A laboratory manual, 2nd ed Ed., Cold Spring Harbor Laboratory Press, New York, USA 580. Redinbaugh, M. G. and Turley, R. B. (1986) Adaptation of the bicinchoninic acid protein assay for the use with microtiter plates and sucrose gradient fractions. Anal.Biochem. 153, 267-271. 581. Brandhorst, D., Brandhorst, H., Hering, B. J., Federlin, K., and Bretzel, R. G. (1995) Islet isolation from the pancreas of large mammals and humans: 10 years of experience. Exp.Clin.Endocrinol.Diabetes 103, 3-14. 582. Une, S., Arita, S., Ohtsuka, S., Kawahara, T., Atiya, A., Shevlin, L., and Mullen, Y. (1997) Canine islet isolation using a two-step digestion method and the influence of stationary collagenase incubation of the pancreas. Transplant.Proc. 29, 1970. 583. Kenmochi, T., Miyamoto, M., Sasaki, H., Une, S., Nakagawa, Y., Moldovan, S., Benhamou, P. Y., Brunicardi, F. C., Tanaka, H., and Mullen, Y. (1998) LAP-1 cold preservation solution for isolation of high-quality human pancreatic islets. Pancreas 17, 367-377. 584. Howell, S. L., Fink, C. J., and Lacy, P. E. (1969) Isolation and properties of secretory granules from rat islets of Langerhans. I. Isolation of a secretory granule fraction. J Cell Biol. 41, 154-161. 585. London, N. J., Contractor, H., Lake, S. P., Aucott, G. C., Bell, P. R., and James, R. F. (1990) A fluorometric viability assay for single human and rat islets. Horm.Metab Res.Suppl 25, 82-87. 586. London, N. J., Contractor, H., Lake, S. P., Aucott, G. C., Bell, P. R., and James, R. F. (1989) A microfluorometric viability assay for isolated human and rat islets of Langerhans. Diabetes Res. 12, 141-149. 587. Storrie, B. and Madden, E. A. (1990) Isolation of subcellular organelles. Methods Enzymol. 182, 203-225. 588. Hause, G., Hause, B., and Van Lammeren, A. A. M. (1992) Microtubular and actin filament configurations during microspore and pollen development in Brassica napus cv. Topas. Can.J.Bot. 70, 1369-1376. 589. Dikov, A., Dimitrova, M., Pajpanova, T., Krieg, R., and Halbhuber, K. J. (2000) Histochemical method for dipeptidyl aminopeptidase II with a new anthraquinonyl hydrazide substrate. Cell Mol.Biol.(Noisy.-le-grand) 46, 1213-1218. 590. Li, J., Wilk, E., and Wilk, S. (1995) Aminoacylpyrrolidine-2-nitriles: potent and stable inhibitors of dipeptidyl-peptidase IV (CD 26). Arch.Biochem.Biophys. 323, 148-154. 591. Senten, K., Van der Veken, P., Bal, G., De Meester, I., Scharpe, S., Bauvois, B., Haemers, A., and Augustyns, K. (2002) Development of potent and selective dipeptidyl peptidase II inhibitors. Bioorg.Med.Chem.Lett. 12, 2825-2828. 592. Pederson, R. A. and Brown, J. C. (1976) The insulinotropic action of gastric inhibitory polypeptide in the perfused isolated rat pancreas. Endocrinology 99, 780-785. 593. Triguero, D., Buciak, J., and Pardridge, W. M. (1990) Capillary depletion method for quantification of blood-brain barrier transport of circulating peptides and plasma proteins. J Neurochem. 54, 1882-1888. 594. Merops-Sanger (2009) http://www.merops.sanger.ac.uk. 595. pub-med (2009) http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed. 596. Handbook Sigma-Aldrich (2009) http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/RBI_Handbook/rbibook5_ neuropep.Par.0001.File.tmp/rbibook5_neuropep.pdf. 597. Kaestner, F., Hettich, M., Peters, M., Sibrowski, W., Hetzel, G., Ponath, G., Arolt, V., Cassens, U., and Rothermundt, M. (2005) Different activation patterns of proinflammatory cytokines in melancholic and non-melancholic major depression are associated with HPA axis activity. J.Affect.Disord. 87, 305-311. 598. Rothermundt, M., Arolt, V., Fenker, J., Gutbrodt, H., Peters, M., and Kirchner, H. (2001) Different immune patterns in melancholic and non-melancholic major depression. Eur Arch.Psychiatry Clin.Neurosci. 251, 90-97. 599. Montgomery, S. A. and Asberg, M. (1979) A new depression scale designed to be sensitive to change. Br.J Psychiatry 134, 382-389. 600. Simo, C., Mendieta, M. E., Antonioli, P., Sebastiano, R., Citterio, A., Cifuentes, A., Peltre, G., and Righetti, P. G. (2006) Mass distribution, polydispersity and focusing properties of carrier ampholytes for IEF II: pH 4-6 intervals. Electrophoresis 27, 4849-4858.

128 Reference

601. Sebastiano, R., Simo, C., Mendieta, M. E., Antonioli, P., Citterio, A., Cifuentes, A., Peltre, G., and Righetti, P. G. (2006) Mass distribution and focusing properties of carrier ampholytes for isoelectric focusing: I. Novel and unexpected results. Electrophoresis 27, 3919-3934. 602. Righetti, P. G., Simo, C., Sebastiano, R., and Citterio, A. (2007) Carrier ampholytes for IEF, on their fortieth anniversary (1967-2007), brought to trial in court: the verdict. Electrophoresis 28, 3799-3810. 603. Weihofen, W. A., Liu, J., Reutter, W., Saenger, W., and Fan, H. (2005) Crystal structures of HIV-1 Tat-derived nonapeptides Tat-(1-9) and Trp2-Tat-(1-9) bound to the active site of dipeptidyl-peptidase IV (CD26). J Biol.Chem. 280, 14911-14917. 604. Nordhoff, S., Cerezo-Galvez, S., Feurer, A., Hill, O., Matassa, V. G., Metz, G., Rummey, C., Thiemann, M., and Edwards, P. J. (2006) The reversed binding of beta-phenethylamine inhibitors of DPP-IV: X-ray structures and properties of novel fragment and elaborated inhibitors. Bioorg.Med.Chem.Lett. 16, 1744-1748. 605. Hiramatsu, H., Kyono, K., Higashiyama, Y., Fukushima, C., Shima, H., Sugiyama, S., Inaka, K., Yamamoto, A., and Shimizu, R. (2003) The structure and function of human dipeptidyl peptidase IV, possessing a unique eight-bladed beta-propeller fold. Biochem Biophys.Res.Commun. 302, 849-854. 606. Hiramatsu, H., Kyono, K., Shima, H., Fukushima, C., Sugiyama, S., Inaka, K., Yamamoto, A., and Shimizu, R. (2003) Crystallization and preliminary X-ray study of human dipeptidyl peptidase IV (DPPIV). Acta Crystallogr.D.Biol.Crystallogr. 59, 595-596. 607. Nakajima, Y., Ito, K., Toshima, T., Egawa, T., Zheng, H., Oyama, H., Wu, Y. F., Takahashi, E., Kyono, K., and Yoshimoto, T. (2008) Dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia exhibits activity against a substrate containing a 4-hydroxyproline residue. J Bacteriol. 190, 7819-7829. 608. Dale, G. E., D'Arcy, B., Yuvaniyama, C., Wipf, B., Oefner, C., and D'Arcy, A. (2000) Purification and crystallization of the extracellular domain of human neutral endopeptidase (neprilysin) expressed in Pichia pastoris. Acta Crystallogr.D.Biol.Crystallogr. 56, 894-897. 609. Backes, B. J., Longenecker, K., Hamilton, G. L., Stewart, K., Lai, C., Kopecka, H., von Geldern, T. W., Madar, D. J., Pei, Z., Lubben, T. H., Zinker, B. A., Tian, Z., Ballaron, S. J., Stashko, M. A., Mika, A. K., Beno, D. W., Kempf-Grote, A. J., Black-Schaefer, C., Sham, H. L., and Trevillyan, J. M. (2007) Pyrrolidine-constrained phenethylamines: The design of potent, selective, and pharmacologically efficacious dipeptidyl peptidase IV (DPP4) inhibitors from a lead- like screening hit. Bioorg.Med.Chem.Lett. 17, 2005-2012. 610. Wallace, M. B., Feng, J., Zhang, Z., Skene, R. J., Shi, L., Caster, C. L., Kassel, D. B., Xu, R., and Gwaltney, S. L. (2008) Structure-based design and synthesis of benzimidazole derivatives as dipeptidyl peptidase IV inhibitors. Bioorg.Med.Chem.Lett. 18, 2362-2367. 611. Koo, K. D., Kim, M. J., Kim, S., Kim, K. H., Hong, S. Y., Hur, G. C., Yim, H. J., Kim, G. T., Han, H. O., Kwon, O. H., Kwon, T. S., Koh, J. S., and Lee, C. S. (2007) Synthesis, SAR, and X-ray structure of novel potent DPPIV inhibitors: oxadiazolyl ketones. Bioorg.Med.Chem.Lett. 17, 4167-4172. 612. Madar, D. J., Kopecka, H., Pireh, D., Yong, H., Pei, Z., Li, X., Wiedeman, P. E., Djuric, S. W., von Geldern, T. W., Fickes, M. G., Bhagavatula, L., McDermott, T., Wittenberger, S., Richards, S. J., Longenecker, K. L., Stewart, K. D., Lubben, T. H., Ballaron, S. J., Stashko, M. A., Long, M. A., Wells, H., Zinker, B. A., Mika, A. K., Beno, D. W., Kempf- Grote, A. J., Polakowski, J., Segreti, J., Reinhart, G. A., Fryer, R. M., Sham, H. L., and Trevillyan, J. M. (2006) Discovery of 2-[4-{{2-(2S,5R)-2-cyano-5-ethynyl-1-pyrrolidinyl]-2-oxoethyl]amino]- 4-methyl-1-piperidinyl]-4- pyridinecarboxylic acid (ABT-279): a very potent, selective, effective, and well-tolerated inhibitor of dipeptidyl peptidase-IV, useful for the treatment of diabetes. J Med.Chem. 49, 6416-6420. 613. Pei, Z., Li, X., Longenecker, K., von Geldern, T. W., Wiedeman, P. E., Lubben, T. H., Zinker, B. A., Stewart, K., Ballaron, S. J., Stashko, M. A., Mika, A. K., Beno, D. W., Long, M., Wells, H., Kempf-Grote, A. J., Madar, D. J., McDermott, T. S., Bhagavatula, L., Fickes, M. G., Pireh, D., Solomon, L. R., Lake, M. R., Edalji, R., Fry, E. H., Sham, H. L., and Trevillyan, J. M. (2006) Discovery, structure-activity relationship, and pharmacological evaluation of (5- substituted-pyrrolidinyl-2-carbonyl)-2-cyanopyrrolidines as potent dipeptidyl peptidase IV inhibitors. J Med.Chem. 49, 3520-3535. 614. Pei, Z., Li, X., von Geldern, T. W., Longenecker, K., Pireh, D., Stewart, K. D., Backes, B. J., Lai, C., Lubben, T. H., Ballaron, S. J., Beno, D. W., Kempf-Grote, A. J., Sham, H. L., and Trevillyan, J. M. (2007) Discovery and structure- activity relationships of piperidinone- and piperidine-constrained phenethylamines as novel, potent, and selective dipeptidyl peptidase IV inhibitors. J Med.Chem. 50, 1983-1987. 615. Pei, Z. (2008) From the bench to the bedside: dipeptidyl peptidase IV inhibitors, a new class of oral antihyperglycemic agents. Curr.Opin.Drug Discov.Devel. 11, 512-532. 616. Engel, M., Hoffmann, T., Manhart, S., Heiser, U., Chambre, S., Huber, R., Demuth, H. U., and Bode, W. (2006) Rigidity and flexibility of dipeptidyl peptidase IV: crystal structures of and docking experiments with DPIV. J.Mol.Biol. 355, 768-783. 617. Feng, J., Zhang, Z., Wallace, M. B., Stafford, J. A., Kaldor, S. W., Kassel, D. B., Navre, M., Shi, L., Skene, R. J., Asakawa, T., Takeuchi, K., Xu, R., Webb, D. R., and Gwaltney, S. L. (2007) Discovery of alogliptin: a potent, selective, bioavailable, and efficacious inhibitor of dipeptidyl peptidase IV. J Med.Chem. 50, 2297-2300. 618. Chung, K. M., Huang, C. H., Cheng, J. H., Tsai, C. H., Suen, C. S., Hwang, M. J., and Chen, X. (2011) Proline in transmembrane domain of type II protein DPP-IV governs its translocation behavior through endoplasmic reticulum. Biochemistry 50, 7909-7918. 619. Elbein A.D. (1991) The role of N-linked oligosaccharides in glycoprotein function. TIPTECH 9, 346-352. 620. Bar, J., Weber, A., Hoffmann, T., Stork, J., Wermann, M., Wagner, L., Aust, S., Gerhartz, B., and Demuth, H. U. (2003) Characterisation of human dipeptidyl peptidase IV expressed in Pichia pastoris. A structural and mechanistic comparison between the recombinant human and the purified porcine enzyme. Biol.Chem. 384, 1553-1563. 621. Bretthauer, R. K. and Castellino, F. J. (1999) Glycosylation of Pichia pastoris-derived proteins. Biotechnol.Appl.Biochem. 30, 193-200. 622. Wagner,L., Frerker,N., Gündel,G., Wolf,R., Kehlen,A., Thümmler,A., Niestroj,A.J., Heiser,U., Rahfeld,J.-U., Hoffmann,T., von Hörsten,S., and Demuth, H.-U. “Characterization of DP 4-like Activity Enzymes and NP Y in Neuronal Tissue” 8th NPY Conference. 22 - 26 April 2006, St. Petersberg, Florida, USA. 623. Kurogochi, M., Matsushita, T., Amano, M., Furukawa, J. I., Shinohara, Y., Aoshima, M., and Nishimura, S. I. (2010) Sialic acid-focused quantitative mouse serum glycoproteomics by multiple reaction monitoring assay. Mol.Cell Proteomics.

129 Reference

624. Tissot, B., North, S. J., Ceroni, A., Pang, P. C., Panico, M., Rosati, F., Capone, A., Haslam, S. M., Dell, A., and Morris, H. R. (2009) Glycoproteomics: past, present and future. FEBS Lett. 583, 1728-1735. 625. Wei, X. and Li, L. (2009) Comparative glycoproteomics: approaches and applications. Brief.Funct.Genomic.Proteomic. 8, 104-113. 626. Wuhrer, M., Catalina, M. I., Deelder, A. M., and Hokke, C. H. (2007) Glycoproteomics based on tandem mass spectrometry of glycopeptides. J Chromatogr.B Analyt.Technol.Biomed.Life Sci. 849, 115-128. 627. Bilodeau, N., Fiset, A., Poirier, G. G., Fortier, S., Gingras, M. C., Lavoie, J. N., and Faure, R. L. (2006) Insulin- dependent phosphorylation of DPP IV in liver. Evidence for a role of compartmentalized c-Src. FEBS J. 273, 992- 1003. 628. Paulson, J. C., Weinstein, J., and Schauer, A. (1989) Tissue-specific expression of sialyltransferases. J.Biol.Chem. 264, 10931-10934. 629. Torimoto, Y., Dang, N. H., Tanaka, T., Prado, C., Schlossman, S. F., and Morimoto, C. (1992) Biochemical characterization of CD26 (dipeptidyl peptidase IV): functional comparison of distinct epitopes recognized by various anti- CD26 monoclonal antibodies. Mol.Immunol. 29, 183-192. 630. Lukong, K. E., Seyrantepe, V., Landry, K., Trudel, S., Ahmad, A., Gahl, W. A., Lefrancois, S., Morales, C. R., and Pshezhetsky, A. V. (2001) Intracellular distribution of lysosomal sialidase is controlled by the internalization signal in its cytoplasmic tail. J.Biol.Chem. 276, 46172-46181. 631. Keppler O.T., Hinderlich S., Langner J, Schwartz-Albiez R., Reutter W., and Pawlita M. (1999) UDP-GlcNac 2- Epimerase: A regulator of cell surface sialylation. Science 284, 1372-1376. 632. Levy, M. T., McCaughan, G. W., Abbott, C. A., Park, J. E., Cunningham, A. M., Muller, E., Rettig, W. J., and Gorrell, M. D. (1999) Fibroblast activation protein: a cell surface dipeptidyl peptidase and gelatinase expressed by stellate cells at the tissue remodelling interface in human cirrhosis. Hepatology 29, 1768-1778. 633. Levy, M. T., McCaughan, G. W., Marinos, G., and Gorrell, M. D. (2002) Intrahepatic expression of the hepatic stellate cell marker fibroblast activation protein correlates with the degree of fibrosis in hepatitis C virus infection. Liver 22, 93- 101. 634. Dolznig, H., Schweifer, N., Puri, C., Rettig, W. J., Kerjaschki, D., and Garin-Chesa, P. (2005) Characterization of cancer stroma markers: In silico analysis of an mRNA expression database for fibroblast activation protein and endosialin. Cancer Immunity 5. 635. Dunphy, J. L., Justice, F. A., Taylor, R. G., and Fuller, P. J. (1999) mRNA Levels of Dipeptidyl Peptidase IV Decrease during Intestinal Adaptation. J.Surg.Res. 87, 130-133. 636. Tiruppathi, C., Ganapathy, V., and Leibach, F. H. (1990) Evidence for tripeptide-proton symport in renal brush border membrane vesicles. Studies in a novel rat strain with a genetic absence of dipeptidyl peptidase IV. J.Biol.Chem. 265, 2048-2053. 637. Tiruppathi, C., Miyamoto, Y., Ganapathy, V., Roesel, R. A., Whitford, G. M., and Leibach, F. H. (1990) Hydrolysis and transport of proline-containing peptides in renal brush- border membrane vesicles from dipeptidyl peptidase IV- positive and dipeptidyl peptidase IV-negative rat strains. J.Biol.Chem. 265, 1476-1483. 638. Miyamoto, Y., Ganapathy, V., Barlas, A., Neubert, K., Barth, A., and Leibach, F. H. (1987) Role of dipeptidyl peptidase IV in uptake of peptide nitrogen from beta- casomorphin in rabbit renal BBMV. Am.J.Physiol. 252, F670- F677. 639. Andersen, K. J. and McDonald, J. K. (1987) Presence and possible role of a renal brush-border Gly-Pro-X-releasing exopeptidase. Am.J Physiol 253, F648-F655. 640. Kieffer, T. J. and Habener, J. F. (1999) The glucagon-like peptides. Endocr.Rev. 20, 876-913. 641. Plamboeck, A., Holst, J. J., Carr, R. D., and Deacon, C. F. (2003) Neutral endopeptidase 24.11 and dipeptidyl peptidase IV are both involved in regulating the metabolic stability of glucagon-like peptide-1 in vivo. Adv.Exp.Med.Biol. 524, 303-312. 642. Plamboeck, A., Holst, J. J., Carr, R. D., and Deacon, C. F. (2005) Neutral endopeptidase 24.11 and dipeptidyl peptidase IV are both mediators of the degradation of glucagon-like peptide 1 in the anaesthetised pig. Diabetologia 48, 1882-1890. 643. Pala, L., Mannucci, E., Pezzatini, A., Ciani, S., Sardi, J., Raimondi, L., Ognibene, A., Cappadona, A., Vannelli, B. G., and Rotella, C. M. (2003) Dipeptidyl peptidase-IV expression and activity in human glomerular endothelial cells. Biochem.Biophys.Res.Commun. 310, 28-31. 644. van Leer, E. H., Bruijn, J. A., Prins, F. A., Hoedemaeker, P. J., and de Heer, E. (1993) Redistribution of glomerular dipeptidyl peptidase type IV in experimental lupus nephritis. Demonstration of decreased enzyme activity at the ultrastructural level. Lab.Invest. 68, 550-556. 645. Scherberich, J. E., Wiemer, J., and Schoeppe, W. (1992) Biochemical and immunological properties of urinary angiotensinase A and dipeptidylaminopeptidase IV. Their use as markers in patients with renal cell injury. Eur.J.Clin.Chem.Clin.Biochem. 30, 663-668. 646. Stiller, D., Bahn, H., and August, C. (1991) Demonstration of glomerular DPP IV activity in kidney diseases. Acta Histochem. 91, 105-109. 647. Mucke, H. A. (2003) Renal therapeutics: patent highlights January to June 2003. Curr.Opin.Investig.Drugs 4, 1048- 1052. 648. Hartel-Schenk, S., Gossrau, R., and Reutter, W. (1990) Comparative immunohistochemistry and histochemistry of dipeptidyl peptidase IV in rat organs during development. Histochem.J. 22, 567-578. 649. Van Der Velden, V., Wierenga-Wolf, A. F., Adriaansen-Soeting, P. W., Overbeek, S. E., Moller, G. M., Hoogsteden, H. C., and Versnel, M. A. (1998) Expression of aminopeptidase N and dipeptidyl peptidase IV in the healthy and asthmatic bronchus. Clin.Exp.Allergy 28, 110-120. 650. Sannes, P. L. (1983) Subcellular localization of dipeptidyl peptidases II and IV in rat and rabbit alveolar macrophages. J.Histochem.Cytochem. 31, 684-690. 651. Randell, S. H. and Sannes, P. L. (1985) Cytochemical localization and biochemical evaluation of a lysosomal serine protease in lung: dipeptidyl peptidase II in the normal rat. J.Histochem.Cytochem. 33, 677-686. 652. Randell, S. H. and Sannes, P. L. (1988) Biochemical quantitation and histochemical localization of cathepsin B, dipeptidyl peptidases I and II, and acid phosphatase in pulmonary granulomatosis and fibrosis in rats. Inflammation 12, 67-86.

130 Reference

653. Hansen, L., Deacon, C. F., Orskov, C., and Holst, J. J. (1999) Glucagon-like peptide-1-(7-36)amide is transformed to glucagon-like peptide-1-(9-36)amide by dipeptidyl peptidase IV in the capillaries supplying the L cells of the porcine intestine. Endocrinology 140, 5356-5363. 654. Brandsch, M., Brandsch, C., Ganapathy, M. E., Chew, C. S., Ganapathy, V., and Leibach, F. H. (1997) Influence of proton and essential histidyl residues on the transport kinetics of the H+/peptide cotransport systems in intestine (PEPT 1) and kidney (PEPT 2). Biochim.Biophys.Acta 1324, 251-262. 655. Sjostrom, H., Noren, O., Krasilnikoff, P. A., and Gudmand-Hoyer, E. (1981) Intestinal peptidases and sucrase in coeliac disease. Clin.Chim.Acta 109, 53-58. 656. Mercer, J., Eagles, M. E., and Talbot, I. C. (1990) Brush border enzymes in coeliac disease: histochemical evaluation. J.Clin.Pathol. 43, 307-312. 657. Kozakova, H., Stepankova, R., Kolinska, J., Farre, M. A., Funda, D. P., Tuckova, L., and Tlaskalova-Hogenova, H. (1998) Brush border enzyme activities in the small intestine after long-term gliadin feeding in animal models of human coeliac disease. Folia Microbiol.(Praha.) 43, 497-500. 658. Zweibaum, A., Hauri, H. P., Sterchi, E., Chantret, I., Haffen, K., Bamat, J., and Sordat, B. (1984) Immunohistological evidence, obtained with monoclonal antibodies, of small intestinal brush border hydrolases in human colon cancers and foetal colons. Int.J.Cancer 34, 591-598. 659. Kieffer, T. J. (2003) GIP or not GIP? That is the question. Trends Pharmacol.Sci. 24, 110-112. 660. Drucker, D.J. (2012 ) http://www.glucagon.com. 661. Wagner, L., Hoffmann,T., Rahfeld,J.-U., Niestroj,A.J., Heiser,U., and Demuth, H.-U. “Characterisation of Dipeptidyl peptidase IV-like activity enzymes in porcine pancreas and isolated islets of Langerhans” 7th Young Scientists Meeting of DGZ. 22 - 24 September 2005, Jena, Germany. 662. Gutschmidt, S. and Gossrau, R. (1981) A quantitative histochemical study of dipeptidylpeptidase IV (DPP IV). Histochemistry 73, 285-304. 663. Vantyghem, M. C., Kerr-Conte, J., Pattou, F., Gevaert, M. H., Hober, C., Defossez, A., Mazzuca, M., and Beauvillain, J. C. (1996) Immunohistochemical and ultrastructural study of adult porcine endocrine pancreas during the different steps of islet isolation. Histochem.Cell Biol. 106, 511-519. 664. Brandhorst, D., Brandhorst, H., Brendel, M., and Bretzel, R. G. (1998) [Isolation of islands of Langerhans from human and porcine pancreas for transplantation to humans]. Zentralbl.Chir. 123, 814-822. 665. Cui, W., Gu, Y., Miyamoto, M., Tanaka, M., Xu, B., Imamura, M., Iwata, H., Ikada, Y., and Inoue, K. (1999) Novel method for isolation of adult porcine pancreatic islets with two-stage digestion procedure. Cell Transplant. 8, 391-398. 666. Takei, S., Teruya, M., Grunewald, A., Garcia, R., Chan, E. K., and Charles, M. A. (1994) Isolation and function of human and pig islets. Pancreas 9, 150-156. 667. Githens, S., Schexnayder, J. A., Moses, R. L., Denning, G. M., Smith, J. J., and Frazier, M. L. (1994) Mouse pancreatic acinar/ductular tissue gives rise to epithelial cultures that are morphologically, biochemically, and functionally indistinguishable from interlobular duct cell cultures. In Vitro Cell Dev.Biol.Anim 30A, 622-635. 668. Teitelman, G., Lee, J. K., and Alpert, S. (1987) Expression of cell type-specific markers during pancreatic development in the mouse: implications for pancreatic cell lineages. Cell Tissue Res. 250, 435-439. 669. Vantyghem, M. C., Kerr-Conte, J., Pattou, F., Gevaert, M. H., Hober, C., Defossez, A., Beauvillain, J. C., and Mazzuca, M. (1995) Immuno-histochemical and ultrastructural study of adult porcine pancreas during the different steps of islet isolation. Transplant.Proc. 27, 3372-3374. 670. Kadison, A. S., Maldonado, T. S., Crisera, C. A., Longaker, M. T., and Gittes, G. K. (2000) In vitro validation of duct differentiation in developing embryonic mouse pancreas. J Surg.Res. 90, 126-130. 671. Kolar, C., Caffrey, T., Hollingsworth, M., Scheetz, M., Sutherlin, M., Weide, L., and Lawson, T. (1997) Duct epithelial cells cultured from human pancreas processed for transplantation retain differentiated ductal characteristics. Pancreas 15, 265-271. 672. Wieczorek, G., Pospischil, A., and Perentes, E. (1998) A comparative immunohistochemical study of pancreatic islets in laboratory animals (rats, dogs, minipigs, nonhuman primates). Exp.Toxicol.Pathol. 50, 151-172. 673. Vecchi, A., Garlanda, C., Lampugnani, M. G., Resnati, M., Matteucci, C., Stoppacciaro, A., Schnurch, H., Risau, W., Ruco, L., Mantovani, A., and . (1994) Monoclonal antibodies specific for endothelial cells of mouse blood vessels. Their application in the identification of adult and embryonic endothelium. Eur.J Cell Biol. 63, 247-254. 674. Garlanda, C. and Dejana, E. (1997) Heterogeneity of endothelial cells. Specific markers. Arterioscler.Thromb.Vasc.Biol. 17, 1193-1202. 675. Petruzzo, P., Cappai, A., Congiu, T., Riva, A., and Brotzu, G. (1997) Pancreatic islet microcirculation: a scanning electron microscopic study of corrosion casts. Transplant.Proc. 29, 2050-2051. 676. Vanhoof, G., Goossens, F., Juliano, M. A., Juliano, L., Hendriks, D., Schatteman, K., Lin, A. H., and Scharpe, S. (1997) Isolation and sequence analysis of a human cDNA clone (XPNPEPL) homologous to X- (aminopeptidase P). Cytogenet.Cell Genet. 78, 275-280. 677. Wagner, L., Gündel, D, Zeitschel, U, Rossner, S, Frerker, N, Schade, J, Stiller, J, Niestroj, A J, Thümmler, A, Wolf, R, Gärtner, U T, Manhart, S, Kehlen, A, Rahfeld, J-U, Hoffmann, T, von Hoersten, S, and Demuth, H-U. (2008) Tissue- distribution of dipeptidyl peptidase 4-like enzymes in distinct brain areas and neuronal tissues. Clin.Chem Lab Med. 46, A1-A43. 678. Wagner, L., Wolf, R., Zeitschel, U., Rossner, S., Pedersen, A., Leavitt, B., Haydn, M., Kaestner, F., Rothermundt, M., Gaertner, U., Weber, A., Schlenzig, D., Frerker, N., Schade, J., Manhart, S., Rahfeld, J.U., Hoffmann, T., Demuth, H.- U., and von Hoersten, S. (2012)Metabolism of neuropeptide Y (NPY) from head to toe identified novel NPY-cleaving enzymes and revealed potential drug interactions. Proc.Natl.Acad.Sci.U.S.A (in preparation). 679. Wagner, L., Wolf, R, Zeitschel, U, Rossner, S, Gündel, D, Stiller, H, Petersen, A, Leavitt, B, Hayden, M, Kaestner, F, Rothermundt, M, Gärtner, U T, Weber, A, Schlenzig, D, Frerker, N, Schade, J, Manhart, S, Rahfeld, J-U, Demuth, H- U, and von Hoersten, S. (2011) Dipeptidyl peptidase 4 (DP 4) and neuropeptide Y (NPY): a novel pharmaceutical target against stress? Neuropeptides 44, 529. 680. Ludwig, R., Feindt, J., Lucius, R., Petersen, A., and Mentlein, R. (1996) Metabolism of neuropeptide Y and calcitonin gene-related peptide by cultivated neurons and glial cells. Brain Res Mol.Brain Res 37, 181-191. 681. Wolf, R., Rosche, F., Hoffmann, T., and Demuth, H. U. (2001) Immunoprecipitation and liquid chromatographic-mass spectrometric determination of the peptide glucose-dependent insulinotropic polypeptides GIP1-42 and GIP3-42 from

131 Reference

human plasma samples. New sensitive method to analyze physiological concentrations of peptide hormones. J Chromatogr.A. 926, 21-27. 682. Wolf, R., Hoffmann, T., Rosche, F., and Demuth, H. U. (2004) Simultaneous determination of incretin hormones and their truncated forms from human plasma by immunoprecipitation and liquid chromatography-mass spectrometry. J.Chromatogr.B Analyt.Technol.Biomed.Life Sci. 803, 91-99. 683. Bertenshaw, G. P., Turk, B. E., Hubbard, S. J., Matters, G. L., Bylander, J. E., Crisman, J. M., Cantley, L. C., and Bond, J. S. (2001) Marked differences between metalloproteases meprin A and B in substrate and peptide bond specificity. J Biol.Chem. 276, 13248-13255. 684. Spencer-Dene, B., Thorogood, P., Nair, S., Kenny, A. J., Harris, M., and Henderson, B. (1994) Distribution of, and a putative role for, the cell-surface neutral metallo- during mammalian craniofacial development. Development 120, 3213-3226. 685. Becker, C., Kruse, M. N., Slotty, K. A., Kohler, D., Harris, J. R., Rosmann, S., Sterchi, E. E., and Stocker, W. (2003) Differences in the activation mechanism between the alpha and beta subunits of human meprin. Biol Chem 384, 825- 831. 686. Rosmann, S., Hahn, D., Lottaz, D., Kruse, M. N., Stocker, W., and Sterchi, E. E. (2002) Activation of human meprin- alpha in a cell culture model of colorectal cancer is triggered by the plasminogen-activating system. J.Biol.Chem. 277, 40650-40658. 687. Wang, Y., Luo, W., and Reiser, G. (2008) Trypsin and trypsin-like proteases in the brain: proteolysis and cellular functions. Cell Mol.Life Sci. 65, 237-252. 688. Ohler, A., Debela, M., Wagner, S., Magdolen, V., and Becker-Pauly, C. (2010) Analyzing the protease web in skin: meprin metalloproteases are activated specifically by KLK4, 5 and 8 vice versa leading to processing of proKLK7 thereby triggering its activation. Biol.Chem. 391, 455-460. 689. Benes, P., Vetvicka, V., and Fusek, M. (2008) Cathepsin D--many functions of one . Crit Rev.Oncol.Hematol. 68, 12-28. 690. Benes, P., Vashishta, A., Saraswat-Ohri, S., Fusek, M., Pospisilova, S., Tichy, B., and Vetvicka, V. (2006) Effect of procathepsin D activation peptide on gene expression of breast cancer cells. Cancer Lett. 239, 46-54. 691. Cataldo, A. M. and Nixon, R. A. (1990) Enzymatically active lysosomal proteases are associated with amyloid deposits in Alzheimer brain. Proc.Natl.Acad.Sci.U.S.A 87, 3861-3865. 692. Nakanishi, H., Tominaga, K., Amano, T., Hirotsu, I., Inoue, T., and Yamamoto, K. (1994) Age-related changes in activities and localizations of cathepsins D, E, B, and L in the rat brain tissues. Exp.Neurol. 126, 119-128. 693. Nakanishi, H., Zhang, J., Koike, M., Nishioku, T., Okamoto, Y., Kominami, E., von Figura, K., Peters, C., Yamamoto, K., Saftig, P., and Uchiyama, Y. (2001) Involvement of nitric oxide released from microglia-macrophages in pathological changes of cathepsin D-deficient mice. J.Neurosci. 21, 7526-7533. 694. Nakanishi, H. (2003) Neuronal and microglial cathepsins in aging and age-related diseases. Ageing Res.Rev. 2, 367- 381. 695. Zaidi, N., Maurer, A., Nieke, S., and Kalbacher, H. (2008) Cathepsin D: a cellular roadmap. Biochem Biophys.Res.Commun. 376, 5-9. 696. Berglund, M. M., Schober, D. A., Statnick, M. A., McDonald, P. H., and Gehlert, D. R. (2003) The use of bioluminescence resonance energy transfer 2 to study neuropeptide Y receptor agonist-induced beta-arrestin 2 interaction. J.Pharmacol.Exp.Ther. 306, 147-156. 697. Lindner, D., Walther, C., Tennemann, A., and Beck-Sickinger, A. G. (2009) Functional role of the extracellular N- terminal domain of neuropeptide Y subfamily receptors in membrane integration and agonist-stimulated internalization. Cell Signal. 21, 61-68. 698. Bohme, I., Morl, K., Bamming, D., Meyer, C., and Beck-Sickinger, A. G. (2007) Tracking of human Y receptors in living cells--a fluorescence approach. Peptides 28, 226-234. 699. Bohme, I., Stichel, J., Walther, C., Morl, K., and Beck-Sickinger, A. G. (2008) Agonist induced receptor internalization of neuropeptide Y receptor subtypes depends on third intracellular loop and C-terminus. Cell Signal. 20, 1740-1749. 700. Walther, C., Nagel, S., Gimenez, L. E., Morl, K., Gurevich, V. V., and Beck-Sickinger, A. G. (2010) Ligand-induced internalization and recycling of the human neuropeptide Y2 receptor is regulated by its carboxyl-terminal tail. J Biol Chem 285, 41578-41590. 701. Walther, C., Morl, K., and Beck-Sickinger, A. G. (2011) Neuropeptide Y receptors: ligand binding and trafficking suggest novel approaches in drug development. J Pept.Sci. 17, 233-246. 702. Bowden, J. J., Garland, A. M., Baluk, P., Lefevre, P., Grady, E. F., Vigna, S. R., Bunnett, N. W., and McDonald, D. M. (1994) Direct observation of substance P-induced internalization of neurokinin 1 (NK1) receptors at sites of inflammation. Proc.Natl.Acad.Sci.U.S.A 91, 8964-8968. 703. Defea, K., Schmidlin, F., Dery, O., Grady, E. F., and Bunnett, N. W. (2000) Mechanisms of initiation and termination of signalling by neuropeptide receptors: a comparison with the proteinase-activated receptors. Biochem.Soc.Trans. 28, 419-426. 704. Garland, A. M., Grady, E. F., Lovett, M., Vigna, S. R., Frucht, M. M., Krause, J. E., and Bunnett, N. W. (1996) Mechanisms of desensitization and resensitization of G protein-coupled neurokinin1 and neurokinin2 receptors. Mol.Pharmacol. 49, 438-446. 705. Garland, A. M., Grady, E. F., Payan, D. G., Vigna, S. R., and Bunnett, N. W. (1994) Agonist-induced internalization of the substance P (NK1) receptor expressed in epithelial cells. Biochem.J. 303 ( Pt 1), 177-186. 706. Grady, E. F., Garland, A. M., Gamp, P. D., Lovett, M., Payan, D. G., and Bunnett, N. W. (1995) Delineation of the endocytic pathway of substance P and its seven-transmembrane domain NK1 receptor. Mol.Biol.Cell 6, 509-524. 707. Grady, E. F., Gamp, P. D., Jones, E., Baluk, P., McDonald, D. M., Payan, D. G., and Bunnett, N. W. (1996) Endocytosis and recycling of neurokinin 1 receptors in enteric neurons. Neuroscience 75, 1239-1254. 708. Minarowska, A., Gacko, M., Karwowska, A., and Minarowski, L. (2008) Human cathepsin D. Folia Histochem.Cytobiol. 46, 23-38. 709. Follo, C., Castino, R., Nicotra, G., Trincheri, N. F., and Isidoro, C. (2007) Folding, activity and targeting of mutated human cathepsin D that cannot be processed into the double-chain form. Int.J.Biochem.Cell Biol. 39, 638-649. 710. Wittlin, S., Rosel, J., Hofmann, F., and Stover, D. R. (1999) Mechanisms and kinetics of procathepsin D activation. Eur.J.Biochem. 265, 384-393.

132 Reference

711. Ekman, R., Juhasz, P., Heilig, M., Agren, H., and Costello, C. E. (1996) Novel neuropeptide Y processing in human cerebrospinal fluid from depressed patients. Peptides 17, 1107-1111. 712. Nilsson, C., Westman, A., Blennow, K., and Ekman, R. (1998) Processing of neuropeptide Y and somatostatin in human cerebrospinal fluid as monitored by radioimmunoassay and mass spectrometry. Peptides 19, 1137-1146. 713. Jackson, E. K., Dubinion, J. H., and Mi, Z. (2008) Effects of dipeptidyl peptidase iv inhibition on arterial blood pressure. Clin.Exp.Pharmacol.Physiol 35, 29-34. 714. Jackson, E. K. and Mi, Z. (2008) Sitagliptin augments sympathetic enhancement of the renovascular effects of angiotensin II in genetic hypertension. Hypertension 51, 1637-1642. 715. Jackson, E. K. (2010) Dipeptidyl Peptidase IV Inhibition Alters the Hemodynamic Response to Angiotensin- Converting Enzyme Inhibition in Humans With the Metabolic Syndrome. Hypertension 56, 581-583. 716. Marney, A., Kunchakarra, S., Byrne, L., and Brown, N. J. (2010) Interactive Hemodynamic Effects of Dipeptidyl Peptidase-IV Inhibition and Angiotensin-Converting Enzyme Inhibition in Humans. Hypertension 56, 728-733. 717. Khan, I. U., Reppich, R., and Beck-Sickinger, A. G. (2007) Identification of neuropeptide Y cleavage products in human blood to improve metabolic stability. Biopolymers 88, 182-189. 718. Abid, K., Rochat, B., Lassahn, P. G., Stocklin, R., Michalet, S., Brakch, N., Aubert, J. F., Vatansever, B., Tella, P., De, M., I, and Grouzmann, E. (2009) Kinetic study of neuropeptide Y (NPY) proteolysis in blood and identification of NPY3-35, a new peptide cleaved by plasma kallikrein. J.Biol.Chem. 284, 24715-24724. 719. Tans, G., Rosing, J., Berrettini, M., Lammle, B., and Griffin, J. H. (1987) Autoactivation of human plasma prekallikrein. J.Biol.Chem. 262, 11308-11314. 720. Schmaier, A. H. (2008) Assembly, activation, and physiologic influence of the plasma kallikrein/kinin system. Int.Immunopharmacol. 8, 161-165. 721. Holmes, A., Heilig, M., Rupniak, N. M., Steckler, T., and Griebel, G. (2003) Neuropeptide systems as novel therapeutic targets for depression and anxiety disorders. Trends Pharmacol.Sci. 24, 580-588. 722. Rotzinger, S., Lovejoy, D. A., and Tan, L. A. (2010) Behavioral effects of neuropeptides in rodent models of depression and anxiety. Peptides 31, 736-756. 723. Mentlein, R. (2004) Cell-surface peptidases. Int.Rev.Cytol. 235, 165-213. 724. Maes, M., Capuron, L., Ravaud, A., Gualde, N., Bosmans, E., Egyed, B., Dantzer, R., and Neveu, P. J. (2001) Lowered serum dipeptidyl peptidase IV activity is associated with depressive symptoms and cytokine production in cancer patients receiving interleukin-2-based immunotherapy. Neuropsychopharmacology. 24, 130-140. 725. Elgun, S., Keskinege, A., Akan, H., and Karaca, L. (1999) Serum dipeptidyl peptidase IV activity correlates with the T- cell CD26 antigen. Clin.Chem.Lab.Med. 37, 839-840. 726. Masuyama, J., Yoshio, T., Suzuki, K., Kitagawa, S., Iwamoto, M., Kamimura, T., Hirata, D., Takeda, A., Kano, S., and Minota, S. (1999) Characterization of the 4C8 antigen involved in transendothelial migration of CD26(hi) T cells after tight adhesion to human umbilical vein endothelial cell monolayers. J.Exp.Med. 189, 979-990. 727. Masuyama, J., Berman, J. S., Cruikshank, W. W., Morimoto, C., and Center, D. M. (1992) Evidence for recent as well as long term activation of T cells migrating through endothelial cell monolayers in vitro. J Immunol. 148, 1367-1374. 728. Mattern, T., Reich, C., Schonbeck, U., Ansorge, S., Demuth, H. U., Loppnow, H., Ulmer, A. J., and Flad, H. D. (1998) CD26 (dipeptidyl peptidase i.v.) on human T lymphocytes does not mediate adhesion of these cells to endothelial cells or fibroblasts. Immunobiol. 198, 465-475. 729. Lambeir, A. M., Diaz, P. J., Chacon, P., Vermeulen, G., Heremans, K., Devreese, B., Van Beeumen, J., De Meester, I., and Scharpe, S. (1997) A prediction of DPP IV/CD26 domain structure from a physico-chemical investigation of dipeptidyl peptidase IV (CD26) from human seminal plasma. Biochim.Biophys.Acta 1340, 215-226. 730. Aertgeerts, K., Ye, S., Shi, L., Prasad, S. G., Witmer, D., Chi, E., Sang, B. C., Wijnands, R. A., Webb, D. R., and Swanson, R. V. (2004) N-linked glycosylation of dipeptidyl peptidase IV (CD26): effects on enzyme activity, homodimer formation, and adenosine deaminase binding. Protein Sci. 13, 145-154. 731. Augustyns, K., Van der Veken, P., Senten, K., and Haemers, A. (2005) The therapeutic potential of inhibitors of dipeptidyl peptidase IV (DPP IV) and related proline-specific dipeptidyl aminopeptidases. Curr.Med.Chem. 12, 971- 998. 732. Van der Veken, P., Haemers, A., and Augustyns, K. (2007) Prolyl peptidases related to dipeptidyl peptidase IV: potential of specific inhibitors in drug discovery. Curr.Top.Med.Chem. 7, 621-635. 733. Ogata, S., Misumi, Y., Tsuji, E., Takami, N., Oda, K., and Ikehara, Y. (1992) Identification of the active site residues in dipeptidyl peptidase IV by affinity labeling and site-directed mutagenesis. Biochemistry 31, 2582-2587. 734. Fujiwara, T., Tsuji, E., Misumi, Y., Takami, N., and Ikehara, Y. (1992) Selective cell-surface expression of dipeptidyl peptidase IV with mutations at the active site sequence. Biochem.Biophys.Res.Commun. 185, 776-784. 735. David, F., Bernard, A. M., Pierres, M., and Marguet, D. (1993) Identification of serine 624, aspartic acid 702, and histidine 734 as the catalytic triad residues of mouse dipeptidyl-peptidase IV (CD26). A member of a novel family of nonclassical serine hydrolases. J.Biol.Chem. 268, 17247-17252. 736. Bjelke, J. R., Christensen, J., Branner, S., Wagtmann, N., Olsen, C., Kanstrup, A. B., and Rasmussen, H. B. (2004) Tyrosine 547 constitutes an essential part of the catalytic mechanism of dipeptidyl peptidase IV. J.Biol.Chem. 279, 34691-34697. 737. Abbott, C. A., McCaughan, G. W., and Gorrell, M. D. (1999) Two highly conserved glutamic acid residues in the predicted beta propeller domain of dipeptidyl peptidase IV are required for its enzyme activity. FEBS Lett. 458, 278- 284. 738. Gans, K. Untersuchung des EInflusses von Aminosäureresten im aktivem Zentrum der Dipeptidylpeptidase IV auf die Substrathydrolyse. Dipl., Martin-Luther-Universität Halle-Wittenberg, Germany, 2004. 739. Augustyns, K., Van der Veken, P., Senten, K., and Haemers, A. (2003) Dipeptidyl peptidase IV inhibitors as new therapeutic agents for the treatment of type 2 diabetes. Expert.Opin.Ther.Patents 13, 499-511. 740. McIntosh, C. H. (2008) Dipeptidyl peptidase IV inhibitors and diabetes therapy. Front Biosci. 13, 1753-1773. 741. Silva, A. P., Cavadas, C., Baisse-Agushi, B., Spertini, O., Brunner, H. R., and Grouzmann, E. (2003) NPY, NPY receptors, and DPP IV activity are modulated by LPS, TNF-alpha and IFN-gamma in HUVEC. Regul.Pept. 116, 71- 79. 742. Arndt, M., Lendeckel, U., Spiess, A., Faust, J., Neubert, K., Reinhold, D., and Ansorge, S. (2000) Dipeptidyl peptidase IV (DP IV/CD26) mRNA expression in PWM-stimulated T-cells is suppressed by specific DP IV inhibition, an effect mediated by TGF-beta(1). Biochem Biophys.Res.Commun. 274, 410-414. 133 Reference

743. Arndt, M., Reinhold, D., Lendeckel, U., Spiess, A., Faust, J., Neubert, K., and Ansorge, S. (2000) Specific inhibitors of dipeptidyl peptidase IV suppress mRNA expression of DP IV/CD26 and cytokines. Adv.Exp.Med.Biol. 477, 139-143. 744. Kahne, T., Wrenger, S., Manutscharow, A., and Reinhold, D. (2003) Cd26/DP IV in Hematopoietic Cells-Expression, Function Regulation, Clinical Aspects. In Langner J and Ansorge, S., editors. Ectopeptidases, Kluwer Acedemic/Plenum Publishers, New York, 197-222. 745. Kahne, T., Lendeckel, U., Wrenger, S., Neubert, K., Ansorge, S., and Reinhold, D. (1999) Dipeptidyl peptidase IV: a cell surface peptidase involved in regulating T cell growth (review). Int.J.Mol.Med. 4, 3-15. 746. Buhling, F., Kunz, D., Reinhold, D., Ulmer, A. J., Ernst, M., Flad, H. D., and Ansorge, S. (1994) Expression and functional role of dipeptidyl peptidase IV (CD26) on human natural killer cells. Nat.Immun. 13, 270-279. 747. Boonacker, E. P., Wierenga, E. A., Smits, H. H., and Van Noorden, C. J. (2002) CD26/DPPIV signal transduction function, but not proteolytic activity, is directly related to its expression level on human Th1 and Th2 cell lines as detected with living cell cytochemistry. J Histochem.Cytochem. 50, 1169-1177. 748. Schon, E., Jahn, S., Kiessig, S. T., Demuth, H. U., Neubert, K., Barth, A., Von Baehr, R., and Ansorge, S. (1987) The role of dipeptidyl peptidase IV in human T lymphocyte activation. Inhibitors and antibodies against dipeptidyl peptidase IV suppress lymphocyte proliferation and immunoglobulin synthesis in vitro. Eur.J.Immunol. 17, 1821-1826. 749. Danilov, A. V., Klein, A. K., Lee, H. J., Baez, D. V., and Huber, B. T. (2005) Differential control of G0 programme in chronic lymphocytic leukaemia: a novel prognostic factor. Br.J.Haematol. 128, 472-481. 750. Danilov, A. V., Danilova, O. V., Brown, J. R., Rabinowitz, A., Klein, A. K., and Huber, B. T. (2010) Dipeptidyl peptidase 2 apoptosis assay determines the B-cell activation stage and predicts prognosis in chronic lymphocytic leukemia. Exp.Hematol. 38, 1167-1177. 751. Mele, D. A., Sampson, J. F., and Huber, B. T. (2011) Th17 differentiation is the default program for DPP2-deficient T- cell differentiation. Eur.J.Immunol. 41, 1583-1593. 752. Kelly, T. (2005) Fibroblast activation protein-alpha and dipeptidyl peptidase IV (CD26): Cell-surface proteases that activate cell signaling and are potential targets for cancer therapy. Drug Resist.Updat. 8, 51-58. 753. Busek, P., Mali, k R, and Sedo, A. (2004) Dipeptidyl peptidase IV activity and/or structure homologues (DASH) and their substrates in cancer. Int.J Biochem.Cell Biol. 36, 408-421. 754. Kotackova, L., Balaziova, E., and Sedo, A. (2009) Expression pattern of dipeptidyl peptidase IV activity and/or structure homologues in cancer. Folia Biol.(Praha) 55, 77-84. 755. Balaziova, E., Busek, P., Stremenova, J., Sromova, L., Krepela, E., Lizcova, L., and Sedo, A. (2011) Coupled expression of dipeptidyl peptidase-IV and fibroblast activation protein-alpha in transformed astrocytic cells. Mol.Cell Biochem 354, 283-289. 756. Sulda, M. L., Abbott, C. A., and Hildebrandt, M. (2006) DPIV/CD26 and FAP in cancer: a tale of contradictions. Adv.Exp.Med.Biol. 575, 197-206. 757. Stremenova, J., Krepela, E., Mares, V., Trim, J., Dbaly, V., Marek, J., Vanickova, Z., Lisa, V., Yea, C., and Sedo, A. (2007) Expression and enzymatic activity of dipeptidyl peptidase-IV in human astrocytic tumours are associated with tumour grade. Int.J Oncol. 31, 785-792. 758. Stremenova, J., Mares, V., Lisa, V., Hilser, M., Krepela, E., Vanickova, Z., Syrucek, M., Soula, O., and Sedo, A. (2010) Expression of dipeptidyl peptidase-IV activity and/or structure homologs in human meningiomas. Int.J Oncol. 36, 351-358. 759. Maes, M. B., Lambeir, A. M., Van der Veken, P., De Winter, B., Augustyns, K., Scharpe, S., and De Meester, I. (2006) In vivo effects of a potent, selective DPPII inhibitor: UAMC00039 is a possible tool for the elucidation of the physiological function of DPPII. Adv.Exp.Med.Biol. 575, 73-85. 760. Chen, S. J. and Jiaang, W. T. (2011) Current advances and therapeutic potential of agents targeting dipeptidyl peptidases-IV, -II, 8/9 and fibroblast activation protein. Curr.Top.Med.Chem 11, 1447-1463. 761. Jiaang, W. T., Chen, Y. S., Hsu, T., Wu, S. H., Chien, C. H., Chang, C. N., Chang, S. P., Lee, S. J., and Chen, X. (2005) Novel isoindoline compounds for potent and selective inhibition of prolyl dipeptidase DPP8. Bioorg.Med.Chem.Lett. 15, 687-691. 762. Van Goethem, S., Matheeussen, V., Joossens, J., Lambeir, A. M., Chen, X., De, M., I, Haemers, A., Augustyns, K., and Van, D., V (2011) Structure-activity relationship studies on isoindoline inhibitors of dipeptidyl peptidases 8 and 9 (DPP8, DPP9): is DPP8-selectivity an attainable goal? J Med.Chem. 763. Van der Veken, P., De Meester, I., Dubois, V., Soroka, A., Van Goethem, S., Maes, M. B., Brandt, I., Lambeir, A. M., Chen, X., Haemers, A., Scharpe, S., and Augustyns, K. (2008) Inhibitors of dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Part 1: identification of dipeptide derived leads. Bioorg.Med.Chem.Lett. 18, 4154-4158. 764. Van Goethem, S., Van der Veken, P., Dubois, V., Soroka, A., Lambeir, A. M., Chen, X., Haemers, A., Scharpe, S., De Meester, I., and Augustyns, K. (2008) Inhibitors of dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Part 2: isoindoline containing inhibitors. Bioorg.Med.Chem.Lett. 18, 4159-4162. 765. Leiting, B., Nichols, E, Biftu, T, Edmond, S, Ok, H, Weber, A, Zaller, D, and Thornberry, N A. () Inhibition of Dipeptidyl Peptidase IV Does Not Attentuate T Cell Activation in vitro. Diabetes 766. Fleischer, B., Steeg, C., Huhn, J., and von Bonin, A. (1997) Molecular associations required for signalling via dipeptidyl peptidase IV (CD26). Adv.Exp.Med.Biol. 421:117-25, 117-125. 767. Kobayashi, S., Ohnuma, K., Uchiyama, M., Iino, K., Iwata, S., Dang, N. H., and Morimoto, C. (2004) Association of CD26 with CD45RA outside lipid rafts attenuates cord blood T-cell activation. Blood 103, 1002-1010. 768. Ohnuma, K., Hosono, O., Dang, N. H., and Morimoto, C. (2011) Dipeptidyl peptidase in autoimmune pathophysiology. Adv.Clin.Chem 53, 51-84. 769. Hegen, M., Kameoka, J., Dong, R. P., Morimoto, C., and Schlossman, S. F. (1997) Structure of CD26 (dipeptidyl peptidase IV) and function in human T cell activation. Adv.Exp.Med.Biol. 421, 109-116. 770. von Bonin, A., Huhn, J., and Fleischer, B. (1998) Dipeptidyl-peptidase IV/CD26 on T cells: analysis of an alternative T- cell activation pathway. Immunol.Rev. 161, 43-53. 771. Yu, D. M., Slaitini, L., Gysbers, V., Riekhoff, A. G., Kahne, T., Knott, H. M., De, M., I, Abbott, C. A., McCaughan, G. W., and Gorrell, M. D. (2011) Soluble CD26 / dipeptidyl peptidase IV enhances human lymphocyte proliferation in vitro independent of dipeptidyl peptidase enzyme activity and adenosine deaminase binding. Scand.J Immunol. 73, 102-111. 772. Hegen, M., Mittrücker, H.-W., Hug, R., Demuth, H. U., Neubert, K., Barth, A., and Fleischer, B. (1993) Enzymatic activity of CD26 (dipeptidylpeptidase IV) is not required for its signalling function in T cells. Immunobiol. 189, 483-493. 134 Reference

773. Augustyns, K., Bal, G., Thonus, G., Belyaev, A., Zhang, X. M., Bollaert, W., Lambeir, A. M., Durinx, C., Goossens, F., and Haemers, A. (1999) The unique properties of dipeptidyl-peptidase IV (DPP IV / CD26) and the therapeutic potential of DPP IV inhibitors. Curr.Med.Chem. 6, 311-327. 774. Thornberry, N. A. and Weber, A. E. (2007) Discovery of JANUVIA (Sitagliptin), a selective dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes. Curr.Top.Med.Chem. 7, 557-568. 775. Edmondson, S. D., Mastracchio, A., Beconi, M., Colwell, L. F., Jr., Habulihaz, B., He, H., Kumar, S., Leiting, B., Lyons, K. A., Mao, A., Marsilio, F., Patel, R. A., Wu, J. K., Zhu, L., Thornberry, N. A., Weber, A. E., and Parmee, E. R. (2004) Potent and selective proline derived dipeptidyl peptidase IV inhibitors. Bioorg.Med.Chem.Lett. 14, 5151-5155. 776. Kim, D., Wang, L., Beconi, M., Eiermann, G. J., Fisher, M. H., He, H., Hickey, G. J., Kowalchick, J. E., Leiting, B., Lyons, K., Marsilio, F., McCann, M. E., Patel, R. A., Petrov, A., Scapin, G., Patel, S. B., Roy, R. S., Wu, J. K., Wyvratt, M. J., Zhang, B. B., Zhu, L., Thornberry, N. A., and Weber, A. E. (2005) (2R)-4-oxo-4-[3-(trifluoromethyl)-5,6- dihydro[1,2,4]triazolo[4,3-a]pyrazin -7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine: a potent, orally active dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes. J Med.Chem. 48, 141-151. 777. Weber, A. E., Kim, D, Beconi, M, Eiermann, G, Fisher, M, He, H, Hickey, G, Jaspal, S, Leiting, B, Lyons, K, Marsilio, F, McCann, P, Moller, D E, Patel, A T, Petrov, A, Pryor, K, Sinha, R R, Wu, J K, Wyvratt, M, Zhang, B B, and Thornberry, N A. () MK-0431 is a Potent, Selective Dipeptidyl Peptidase IV Inhibitor for the Treatment of Type 2 Diabetes. Diabetes 778. Soisson, S. M., Patel, S. B., Abeywickrema, P. D., Byrne, N. J., Diehl, R. E., Hall, D. L., Ford, R. E., Reid, J. C., Rickert, K. W., Shipman, J. M., Sharma, S., and Lumb, K. J. (2010) Structural definition and substrate specificity of the S28 protease family: the crystal structure of human prolylcarboxypeptidase. BMC.Struct.Biol 10, 16. 779. Epstein, B. J. (2007) Drug evaluation: PSN-9301, a short-acting inhibitor of dipeptidyl peptidase IV. Curr.Opin.Investig.Drugs 8, 331-337. 780. Grouzmann, E., Livio, F., and Buclin, T. (2009) Angiotensin-Converting Enzyme and Dipeptidyl Peptidase IV Inhibitors. An Increased Risk of Angioedema. Hypertension. 781. Grouzmann, E., Monod, M., Landis, B., Wilk, S., Brakch, N., Nicoucar, K., Giger, R., Malis, D., Szalay-Quinodoz, I., Cavadas, C., Morel, D. R., and Lacroix, J. S. (2002) Loss of dipeptidylpeptidase IV activity in chronic rhinosinusitis contributes to the neurogenic inflammation induced by substance P in the nasal mucosa. FASEB J 16, 1132-1134. 782. Grouzmann, E., Monod, M., Landis, B. N., and Lacroix, J. S. (2007) Adverse effects of incretin therapy for type 2 diabetes. JAMA 298, 1759-1760. 783. Grouzmann, E., Buclin, T., and Biollaz, J. (2007) Gliptins. Lancet 369, 269. 784. Jackson, E. K., Zhang, M., Liu, W., and Mi, Z. (2007) Inhibition of Renal Dipeptidyl Peptidase IV Enhances Peptide YY1-36-Induced Potentiation of Angiotensin II-Mediated Renal Vasoconstriction in Spontaneously Hypertensive Rats. J Pharmacol.Exp.Ther. 323, 431-437. 785. Ahren, B., Simonsson, E., Larsson, H., Landin-Olsson, M., Torgeirsson, H., Jansson, P. A., Sandqvist, M., Bavenholm, P., Efendic, S., Eriksson, J. W., Dickinson, S., and Holmes, D. (2002) Inhibition of dipeptidyl peptidase IV improves metabolic control over a 4-week study period in type 2 diabetes. Diabetes Care 25, 869-875. 786. White, J. (2009) Efficacy and safety of incretin based therapies: clinical trial data. J Am.Pharm.Assoc.(2003.) 49 Suppl 1, S30-S40. 787. Barnett, A. (2006) DPP-4 inhibitors and their potential role in the management of type 2 diabetes. Int.J Clin.Pract. 60, 1454-1470. 788. Neumiller, J. J. (2009) Differential chemistry (structure), mechanism of action, and pharmacology of GLP-1 receptor agonists and DPP-4 inhibitors. J Am.Pharm.Assoc.(2003.) 49 Suppl 1, S16-S29. 789. Drucker, D. J. (2007) Dipeptidyl peptidase-4 inhibition and the treatment of type 2 diabetes: preclinical biology and mechanisms of action. Diabetes Care 30, 1335-1343. 790. FDA (2012) http://www.fda.gov/drugSafety/postmarketDrugSafetyInformationforPatientsandProviders/DrugSafety InformationforHealthCareProfessionals/ucm183764.htm. 791. Mas-Vidal, A., Santos-Juanes, J., Esteve-Martinez, A., Caminal-Montero, L., and Coto-Segura, P. (2012) Psoriasiform eruption triggered by a dipeptidyl peptidase IV inhibitor. Australas.J Dermatol. 53, 70-72. 792. Nishioka, T., Shinohara, M., Tanimoto, N., Kumagai, C., and Hashimoto, K. (2011) Sitagliptin, a Dipeptidyl Peptidase- IV Inhibitor, Improves Psoriasis. Dermatology. 793. Imai, Y., Patel, H. R., Hawkins, E. J., Doliba, N. M., Matschinsky, F. M., and Ahima, R. S. (2007) Insulin secretion is increased in pancreatic islets of neuropeptide Y-deficient mice. Endocrinology 148, 5716-5723. 794. Niijima, A. (1989) Neural mechanisms in the control of blood glucose concentration. J Nutr. 119, 833-840. 795. Gilon, P. and Henquin, J. C. (2001) Mechanisms and physiological significance of the cholinergic control of pancreatic beta-cell function. Endocr.Rev. 22, 565-604. 796. Weisz, O. A. (2003) Acidification and protein traffic. Int.Rev.Cytol. 226, 259-319. 797. Seidl, R., Mann, K., and Schafer, W. (1991) N-terminal amino-acid sequence of pig kidney dipeptidyl peptidase IV solubilized by autolysis. Biol.Chem.Hoppe Seyler 372, 213-214. 798. Riemann, D., Hansen, G. H., Niels-Christiansen, L., Thorsen, E., Immerdal, L., Santos, A. N., Kehlen, A., Langner, J., and Danielsen, E. M. (2001) Caveolae/lipid rafts in fibroblast-like synoviocytes: ectopeptidase-rich membrane microdomains. Biochem J 354, 47-55. 799. Dannies, P. S. (1999) Protein hormone storage in secretory granules: mechanisms for concentration and sorting. Endocr.Rev. 20, 3-21. 800. Burgoyne, R. D. and Morgan, A. (2003) Secretory granule exocytosis. Physiol Rev. 83, 581-632. 801. Nichols, B. (2003) Caveosomes and endocytosis of lipid rafts. J Cell Sci 116, 4707-4714. 802. Kiss, A. L. and Botos, E. (2009) Endocytosis via caveolae: alternative pathway with distinct cellular compartments to avoid lysosomal degradation? J Cell Mol.Med. 13, 1228-1237. 803. Mousavi, S. A., Malerod, L., Berg, T., and Kjeken, R. (2004) Clathrin-dependent endocytosis. Biochem J 377, 1-16. 804. Razani, B., Woodman, S. E., and Lisanti, M. P. (2002) Caveolae: from cell biology to animal physiology. Pharmacol.Rev. 54, 431-467. 805. Helms, J. B. and Zurzolo, C. (2004) Lipids as targeting signals: lipid rafts and intracellular trafficking. Traffic. 5, 247- 254.

135 Reference

806. Blanco, A. M., Perez-Arago, A., Fernandez-Lizarbe, S., and Guerri, C. (2008) Ethanol mimics ligand-mediated activation and endocytosis of IL-1RI/TLR4 receptors via lipid rafts caveolae in astroglial cells. J Neurochem. 106, 625- 639. 807. Johannes, L. and Lamaze, C. (2002) Clathrin-dependent or not: is it still the question? Traffic. 3, 443-451. 808. Parton, R. G. and Richards, A. A. (2003) Lipid rafts and caveolae as portals for endocytosis: new insights and common mechanisms. Traffic. 4, 724-738. 809. Parton, R. G. (2003) Caveolae--from ultrastructure to molecular mechanisms. Nat.Rev.Mol.Cell Biol. 4, 162-167. 810. Nabi, I. R. and Le, P. U. (2003) Caveolae/raft-dependent endocytosis. J Cell Biol 161, 673-677. 811. Pelkmans, L. and Helenius, A. (2002) Endocytosis via caveolae. Traffic. 3, 311-320. 812. Schroeder, F., Gallegos, A. M., Atshaves, B. P., Storey, S. M., McIntosh, A. L., Petrescu, A. D., Huang, H., Starodub, O., Chao, H., Yang, H., Frolov, A., and Kier, A. B. (2001) Recent advances in membrane microdomains: rafts, caveolae, and intracellular cholesterol trafficking. Exp Biol Med (Maywood.) 226, 873-890. 813. Brown, D. A. and London, E. (2000) Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J.Biol.Chem. 275, 17221-17224. 814. Janes, P. W., Ley, S. C., Magee, A. I., and Kabouridis, P. S. (2000) The role of lipid rafts in T cell antigen receptor (TCR) signalling. Semin.Immunol. 12, 23-34. 815. Magee, T., Pirinen, N., Adler, J., Pagakis, S. N., and Parmryd, I. (2002) Lipid rafts: cell surface platforms for T cell signaling. Biol.Res. 35, 127-131. 816. Pike, L. J. (2003) Lipid rafts: bringing order to chaos. J.Lipid Res. 44, 655-667. 817. Ohnuma, K., Inoue, H., Uchiyama, M., Yamochi, T., Hosono, O., Dang, N. H., and Morimoto, C. (2006) T-cell activation via CD26 and caveolin-1 in rheumatoid synovium. Mod.Rheumatol. 16, 3-13. 818. Kirsch, S. (2010 ) http://www.nanodaten.de/uploads/downloads/58d4a17a05b84cee3051bda0de09678d.pdf. 819. Endocytosis (2010 ) http://herkules.oulu.fi/isbn9514252764/html/x492.html. 820. Endosome (2010 ) http://wapedia.mobi/en/Endosome. 821. Arancibia-Carcamo, L., Fairfax, B. P., Moss, S. M., and Kittler, J. T. (2006) Studying the Localization, Surface Stability and Endocytosis of Neurotransmitter Receptors by Antibody Labeling and Biotinylation Approaches. In Raton, B., editor. The dynamic Synapse: Molecular Methods in Inotropic Receptor Biology, Taylor & Francis Groups, London, 6.1-6.4. 822. Trugnan, G., Baricault, L., David, F., Garcia, M., and Sapin, S. (1995) Control of dipeptidyl peptidase IV/CD26 cell surface espression in intestinal cells. In Fleischer, B., editor. Dipeptidyl Peptidase IV (CD26) in Metabolism and the Immune Response, R.G. Landes Company, Austin, 79-98. 823. Smythe, E. (2003) Clathrin-coated vesicle formation: a paradigm for coated-vesicle formation. Biochem Soc.Trans. 31, 736-739. 824. Wu, M., Baumgart, T., Hammond, S., Holowka, D., and Baird, B. (2007) Differential targeting of secretory lysosomes and recycling endosomes in mast cells revealed by patterned antigen arrays. J Cell Sci. 120, 3147-3154. 825. Prekeris, R., Klumperman, J., Chen, Y. A., and Scheller, R. H. (1998) Syntaxin 13 mediates cycling of plasma membrane proteins via tubulovesicular recycling endosomes. J Cell Biol. 143, 957-971. 826. Prekeris, R., Yang, B., Oorschot, V., Klumperman, J., and Scheller, R. H. (1999) Differential roles of syntaxin 7 and syntaxin 8 in endosomal trafficking. Mol.Biol.Cell 10, 3891-3908. 827. Prekeris, R., Foletti, D. L., and Scheller, R. H. (1999) Dynamics of tubulovesicular recycling endosomes in hippocampal neurons. J Neurosci. 19, 10324-10337. 828. Woodman, P. G. and Futter, C. E. (2008) Multivesicular bodies: co-ordinated progression to maturity. Curr.Opin.Cell Biol. 20, 408-414. 829. Pryor, P. R. and Luzio, J. P. (2009) Delivery of endocytosed membrane proteins to the lysosome. Biochim.Biophys.Acta 1793, 615-624. 830. Luzio, J. P., Mullock, B. M., Pryor, P. R., Lindsay, M. R., James, D. E., and Piper, R. C. (2001) Relationship between endosomes and lysosomes. Biochem Soc.Trans. 29, 476-480. 831. Woodman, P. (2009) ESCRT proteins, endosome organization and mitogenic receptor down-regulation. Biochem Soc.Trans. 37, 146-150. 832. Casanova, J. E., Mishumi, Y., Ikehara, Y., Hubbard, A. L., and Mostov, K. E. (1991) Direct apical sorting of rat liver dipeptidylpeptidase IV expressed in Madin-Darby canine kidney cells. J.Biol.Chem. 266, 24428-24432. 833. Tuma, P. L., Finnegan, C. M., Yi, J. H., and Hubbard, A. L. (1999) Evidence for apical endocytosis in polarized hepatic cells: phosphoinositide 3-kinase inhibitors lead to the lysosomal accumulation of resident apical plasma membrane proteins. J.Cell Biol. 145, 1089-1102. 834. Tuma, P. L. and Hubbard, A. L. (2003) Transcytosis: crossing cellular barriers. Physiol Rev. 83, 871-932. 835. Tagawa, A., Mezzacasa, A., Hayer, A., Longatti, A., Pelkmans, L., and Helenius, A. (2005) Assembly and trafficking of caveolar domains in the cell: caveolae as stable, cargo-triggered, vesicular transporters. J Cell Biol. 170, 769-779. 836. Advani, R. J., Yang, B., Prekeris, R., Lee, K. C., Klumperman, J., and Scheller, R. H. (1999) VAMP-7 mediates vesicular transport from endosomes to lysosomes. J Cell Biol. 146, 765-776. 837. Claus, V., Jahraus, A., Tjelle, T., Berg, T., Kirschke, H., Faulstich, H., and Griffiths, G. (1998) Lysosomal enzyme trafficking between phagosomes, endosomes, and lysosomes in J774 macrophages. Enrichment of cathepsin H in early endosomes. J.Biol.Chem. 273, 9842-9851. 838. Taunton, J. (2001) Actin filament nucleation by endosomes, lysosomes and secretory vesicles. Curr.Opin.Cell Biol. 13, 85-91. 839. Griffiths, G. (2002) What's special about secretory lysosomes? Semin.Cell Dev.Biol. 13, 279-284. 840. Griffiths, G. M. (2003) Endocytosing the death sentence. J Cell Biol. 160, 155-156. 841. Kyouden, T., Himeno, M., Ishikawa, T., Ohsumi, Y., and Kato, K. (1992) Purification and characterization of dipeptidyl peptidase IV in rat liver lysosomal membranes. J.Biochem.(Tokyo.) 111, 770-777. 842. Blott, E. J. and Griffiths, G. M. (2002) Secretory lysosomes. Nat.Rev.Mol.Cell Biol. 3, 122-131. 843. Dahms, N. M., Lobel, P., and Kornfeld, S. (1989) Mannose 6-phosphate receptors and lysosomal enzyme targeting. J Biol.Chem. 264, 12115-12118. 844. Clark, R. and Griffiths, G. M. (2003) Lytic granules, secretory lysosomes and disease. Curr.Opin.Immunol. 15, 516- 521. 136 Reference

845. Page, L. J., Darmon, A. J., Uellner, R., and Griffiths, G. M. (1998) L is for lytic granules: lysosomes that kill. Biochim.Biophys.Acta 1401, 146-156. 846. Nuovo, G. J. (1996) The foundations of successful RT in situ PCR. Front Biosci. 1, c4-15. 847. Nuovo, G. J. (2001) Co-labeling using in situ PCR: a review. J Histochem.Cytochem. 49, 1329-1339. 848. Schmidt, H., Gelhaus, C., Nebendahl, M., Lettau, M., Lucius, R., Leippe, M., Kabelitz, D., and Janssen, O. (2011) Effector granules in human T lymphocytes: the luminal proteome of secretory lysosomes from human T cells. Cell Commun.Signal. 9, 4. 849. Lettau, M., Schmidt, H., Kabelitz, D., and Janssen, O. (2007) Secretory lysosomes and their cargo in T and NK cells. Immunol.Lett. 108, 10-19. 850. Casey, T. M., Meade, J. L., and Hewitt, E. W. (2007) Organelle proteomics: identification of the exocytic machinery associated with the natural killer cell secretory lysosome. Mol.Cell Proteomics. 6, 767-780. 851. McDonald, J. K. and Ohkubo, I. (2004) 603. Dipeptidyl-peptidase II. In Barrett, A. J., Rawlings, N. D., and Woessner, J. F., editors. Handbook of Proteolytic Enzymes, Elsevier Academic Press, London, 1938-1942. 852. McDonald, J. K. and Barrett, A. J. (1986) Mammalian Proteases: A Glossary and Bibliography, Academic Press, London 853. Sara, V. R., Carlsson-Skwirut, C., Bergman, T., Jornvall, H., Roberts, P. J., Crawford, M., Hakansson, L. N., Civalero, I., and Nordberg, A. (1989) Identification of Gly-Pro-Glu (GPE), the aminoterminal tripeptide of insulin-like growth factor 1 which is truncated in brain, as a novel neuroactive peptide. Biochem Biophys.Res.Commun. 165, 766-771. 854. Sara, V. R., Carlsson-Skwirut, C., Drakenberg, K., Giacobini, M. B., Hakansson, L., Mirmiran, M., Nordberg, A., Olson, L., Reinecke, M., Stahlbom, P. A., and . (1993) The biological role of truncated insulin-like growth factor-1 and the tripeptide GPE in the central nervous system. Ann.N.Y.Acad.Sci. 692, 183-191. 855. Nilsson-Hakansson, L., Civalero, I., Zhang, X., Carlsson-Skwirut, C., Sara, V. R., and Nordberg, A. (1993) Effects of IGF-1, truncated IGF-1 and the tripeptide Gly-Pro-Glu on acetylcholine release from parietal cortex of rat brain. Neuroreport 4, 1111-1114. 856. D'Alessio, D. A., Kieffer, T. J., Taborsky, G. J., Jr., and Havel, P. J. (2001) Activation of the parasympathetic nervous system is necessary for normal meal-induced insulin secretion in rhesus macaques. J Clin.Endocrinol.Metab 86, 1253-1259. 857. Guan, J. and Gluckman, P. D. (2009) IGF-1 derived small neuropeptides and analogues: a novel strategy for the development of pharmaceuticals for neurological conditions. Br.J Pharmacol. 157, 881-891. 858. Foster, M. C., Leapman, R. D., Li, M. X., and Atwater, I. (1993) Elemental composition of secretory granules in pancreatic islets of Langerhans. Biophys.J 64, 525-532. 859. Fukasawa, K., Fukasawa, K. M., Hiraoka, B. Y., and Harada, M. (1983) Purification and properties of dipeptidyl peptidase II from rat kidney. Biochim.Biophys.Acta 745, 6-11. 860. Lynn, K. R. (1991) The isolation and some properties of dipeptidyl peptidases II and III from porcine spleen. Int.J.Biochem. 23, 47-50. 861. Kwon, P. and Laurenceau, J. P. (2002) A longitudinal study of the hopelessness theory of depression: testing the diathesis-stress model within a differential reactivity and exposure framework. J Clin.Psychol. 58, 1305-1321. 862. Brozina, K. and Abela, J. R. (2006) Behavioural inhibition, anxious symptoms, and depressive symptoms: a short- term prospective examination of a diathesis-stress model. Behav.Res.Ther. 44, 1337-1346. 863. Jones, S. R. and Fernyhough, C. (2007) A new look at the neural diathesis--stress model of schizophrenia: the primacy of social-evaluative and uncontrollable situations. Schizophr.Bull. 33, 1171-1177. 864. Walker, E. F. and Diforio, D. (1997) Schizophrenia: a neural diathesis-stress model. Psychol.Rev. 104, 667-685. 865. Lee, K. N., Jackson, K. W., Christiansen, V. J., Dolence, E. K., and McKee, P. A. (2011) Enhancement of fibrinolysis by inhibiting enzymatic cleavage of precursor alpha2-antiplasmin. J Thromb.Haemost. 9, 987-996. 866. Wagner, L., Kaestner, F., Wolf, R., Stiller, H., Heiser, U., Gaertner, U.-T., Manhart, S., Cieslick, K., Hoffmann, T., Rahfeld, J., Demuth, H.-U., von Hoersten, S., and Rothermundt, M. (2012) Identifying neuropeptide Y (NPY) as the main stress-related substrate of dipeptidyl peptidase 4 in blood. Biol.Psychiatry (in preparation). 867. Wagner, L., Kaestner, F., Wolf, R., Stiller, H., Gans, K., Kohlstedt, B., Manhart, S., Niestroj, A.J., Hoffmann, T., Rahfeld, J.-U., Demuth, H.-U., Von Horsten, S., and Rothermundt, M. (2012) Galanin (GAL) is a substrate of prolyl endopeptidase (PEP) in the human blood. Peptides (in preparation). 868. Konno, J., Yoshida, S., Ina, A., Ohmomo, H., Shutoh, F., Nogami, H., and Hisano, S. (2008) Upregulated expression of neuropeptide Y in hypothalamic-pituitary system of rats by chronic dexamethasone administration. Neurosci.Res. 60, 259-265. 869. Rodriguez, E. M., Blazquez, J. L., Pastor, F. E., Pelaez, B., Pena, P., Peruzzo, B., and Amat, P. (2005) Hypothalamic tanycytes: a key component of brain-endocrine interaction. Int.Rev.Cytol. 247, 89-164. 870. Peruzzo, B., Pastor, F. E., Blazquez, J. L., Amat, P., and Rodriguez, E. M. (2004) Polarized endocytosis and transcytosis in the hypothalamic tanycytes of the rat. Cell Tissue Res. 317, 147-164. 871. Chauvet, N., Prieto, M., and Alonso, G. (1998) Tanycytes present in the adult rat mediobasal hypothalamus support the regeneration of monoaminergic axons. Exp.Neurol. 151, 1-13. 872. Ganong, W. F. (2000) Circumventricular organs: definition and role in the regulation of endocrine and autonomic function. Clin.Exp.Pharmacol.Physiol 27, 422-427. 873. Bartanusz, V. and Jezova, D. (1992) The role of some circumventricular organs in hormone action and secretion. Endocr.Regul. 26, 3-9. 874. Elenkov, I. J., Wilder, R. L., Chrousos, G. P., and Vizi, E. S. (2000) The sympathetic nerve--an integrative interface between two supersystems: the brain and the immune system. Pharmacol.Rev. 52, 595-638. 875. Engelhardt, B. and Ransohoff, R. M. (2005) The ins and outs of T-lymphocyte trafficking to the CNS: anatomical sites and molecular mechanisms. Trends Immunol. 26, 485-495. 876. Engelhardt, B. and Sorokin, L. (2009) The blood-brain and the blood-cerebrospinal fluid barriers: function and dysfunction. Semin.Immunopathol. 31, 497-511. 877. Ohnuma, K., Yamochi, T., Uchiyama, M., Nishibashi, K., Yoshikawa, N., Shimizu, N., Iwata, S., Tanaka, H., Dang, N. H., and Morimoto, C. (2004) CD26 up-regulates expression of CD86 on antigen-presenting cells by means of caveolin-1. Proc Natl.Acad Sci U.S A 101, 14186-14191. 878. Maolood, N. and Meister, B. (2009) Protein components of the blood-brain barrier (BBB) in the brainstem area postrema-nucleus tractus solitarius region. J Chem.Neuroanat. 37, 182-195. 137 Reference

879. Price, C. J., Hoyda, T. D., and Ferguson, A. V. (2008) The area postrema: a brain monitor and integrator of systemic autonomic state. Neuroscientist. 14, 182-194. 880. Bombardi, C., Grandis, A., Chiocchetti, R., Lucchi, M. L., Callegari, E., and Bortolami, R. (2004) Membrane-transport systems in the fenestrated capillaries of the area postrema in rat and calf. Anat.Rec.A Discov.Mol.Cell Evol.Biol. 279, 664-670. 881. Harris, K. F. and Matthews, K. A. (2004) Interactions between autonomic nervous system activity and endothelial function: a model for the development of cardiovascular disease. Psychosom.Med. 66, 153-164. 882. Kühn-Wache, K., Manhart, S., Rosche, F., Hoffmann, T., Pederson, R., and Demuth, H.-U. Extended substrate specificity of DP IV-role in processing bioactive peptides. 1999, Magdeburg, Germany. 883. Maes, M., Yirmyia, R., Noraberg, J., Brene, S., Hibbeln, J., Perini, G., Kubera, M., Bob, P., Lerer, B., and Maj, M. (2009) The inflammatory & neurodegenerative (I&ND) hypothesis of depression: leads for future research and new drug developments in depression. Metab Brain Dis. 24, 27-53. 884. Rothermundt, M., Arolt, V., Peters, M., Gutbrodt, H., Fenker, J., Kersting, A., and Kirchner, H. (2001) Inflammatory markers in major depression and melancholia. J Affect.Disord. 63, 93-102. 885. Rothermundt, M., Arolt, V., Weitzsch, C., Eckhoff, D., and Kirchner, H. (1998) Immunological dysfunction in schizophrenia: a systematic approach. Neuropsychobiology 37, 186-193. 886. Rothermundt, M., Arolt, V., and Bayer, T. A. (2001) Review of immunological and immunopathological findings in schizophrenia. Brain Behav.Immun. 15, 319-339. 887. Cunningham, M. J. (2004) Galanin-like peptide as a link between metabolism and reproduction. J Neuroendocrinol. 16, 717-723. 888. Lang, R., Gundlach, A. L., and Kofler, B. (2007) The galanin peptide family: receptor pharmacology, pleiotropic biological actions, and implications in health and disease. Pharmacol.Ther. 115, 177-207. 889. Mitsukawa, K., Lu, X., and Bartfai, T. (2008) Galanin, galanin receptors and drug targets. Cell Mol.Life Sci. 65, 1796- 1805. 890. Karl, T., Burne, T. H., and Herzog, H. (2006) Effect of Y1 receptor deficiency on motor activity, exploration, and anxiety. Behav.Brain Res 167, 87-93. 891. Karl, T., Lin, S., Schwarzer, C., Sainsbury, A., Couzens, M., Wittmann, W., Boey, D., Von Horsten, S., and Herzog, H. (2004) Y1 receptors regulate aggressive behavior by modulating serotonin pathways. Proc.Natl.Acad.Sci.U.S.A 101, 12742-12747. 892. Merten, N., Lindner, D., Rabe, N., Rompler, H., Morl, K., Schoneberg, T., and Beck-Sickinger, A. G. (2007) Receptor subtype-specific docking of Asp6.59 with C-terminal arginine residues in Y receptor ligands. J Biol.Chem. 282, 7543- 7551. 893. Furukawa, S., Kumagi, T., Miyake, T., Ueda, T., Niiya, T., Nishino, K., Murakami, S., Murakami, M., Matsuura, B., and Onji, M. (2012) Suicide attempt by an overdose of sitagliptin, an oral hypoglycemic agent: A case report and a review of the literature. Endocr.J. 894. Neumiller, J. J. (2012) Pharmacology, Efficacy, and Safety of Linagliptin for the Treatment of Type 2 Diabetes Mellitus (March). Ann.Pharmacother. 895. Augeri, D. J., Robl, J. A., Betebenner, D. A., Magnin, D. R., Khanna, A., Robertson, J. G., Wang, A., Simpkins, L. M., Taunk, P., Huang, Q., Han, S. P., Abboa-Offei, B., Cap, M., Xin, L., Tao, L., Tozzo, E., Welzel, G. E., Egan, D. M., Marcinkeviciene, J., Chang, S. Y., Biller, S. A., Kirby, M. S., Parker, R. A., and Hamann, L. G. (2005) Discovery and preclinical profile of Saxagliptin (BMS-477118): a highly potent, long-acting, orally active dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes. J Med.Chem. 48, 5025-5037. 896. Kim, Y. B., Kopcho, L. M., Kirby, M. S., Hamann, L. G., Weigelt, C. A., Metzler, W. J., and Marcinkeviciene, J. (2006) Mechanism of Gly-Pro-pNA cleavage catalyzed by dipeptidyl peptidase-IV and its inhibition by saxagliptin (BMS- 477118). Arch.Biochem.Biophys. 445, 9-18. 897. Magnin, D. R., Robl, J. A., Sulsky, R. B., Augeri, D. J., Huang, Y., Simpkins, L. M., Taunk, P. C., Betebenner, D. A., Robertson, J. G., Abboa-Offei, B. E., Wang, A., Cap, M., Xin, L., Tao, L., Sitkoff, D. F., Malley, M. F., Gougoutas, J. Z., Khanna, A., Huang, Q., Han, S. P., Parker, R. A., and Hamann, L. G. (2004) Synthesis of Novel Potent Dipeptidyl Peptidase IV Inhibitors with Enhanced Chemical Stability: Interplay between the N-Terminal Amino Acid Alkyl Side Chain and the Cyclopropyl Group of alpha-Aminoacyl-l-cis-4,5-methanoprolinenitrile-Based Inhibitors. J.Med.Chem. 47, 2587-2598. 898. Villhauer, E. B., Brinkman, J. A., Naderi, G. B., Burkey, B. F., Dunning, B. E., Prasad, K., Mangold, B. L., Russell, M. E., and Hughes, T. E. (2003) 1-[[(3-hydroxy-1-adamantyl)amino]acetyl]-2-cyano-(S)-pyrrolidine: a potent, selective, and orally bioavailable dipeptidyl peptidase IV inhibitor with antihyperglycemic properties. J.Med.Chem. 46, 2774- 2789. 899. Ahren, B., Schweizer, A., Dejager, S., Villhauer, E. B., Dunning, B. E., and Foley, J. E. (2011) Mechanisms of action of the dipeptidyl peptidase-4 inhibitor vildagliptin in humans. Diabetes Obes.Metab 13, 775-783. 900. Graefe-Mody, E. U., Padula, S., Ring, A., Withopf, B., and Dugi, K. A. (2009) Evaluation of the potential for steady- state pharmacokinetic and pharmacodynamic interactions between the DPP-4 inhibitor linagliptin and metformin in healthy subjects. Curr.Med.Res.Opin. 25, 1963-1972. 901. Fuchs, H., Binder, R., and Greischel, A. (2009) Tissue distribution of the novel DPP-4 inhibitor BI 1356 is dominated by saturable binding to its target in rats. Biopharm.Drug Dispos. 30, 229-240. 902. Fuchs, H., Runge, F., and Held, H. D. (2011) Excretion of the dipeptidyl peptidase-4 inhibitor linagliptin in rats is primarily by biliary excretion and P-gp-mediated efflux. Eur J Pharm.Sci. 903. Heise, T., Graefe-Mody, E. U., Huttner, S., Ring, A., Trommeshauser, D., and Dugi, K. A. (2009) Pharmacokinetics, pharmacodynamics and tolerability of multiple oral doses of linagliptin, a dipeptidyl peptidase-4 inhibitor in male type 2 diabetes patients. Diabetes Obes.Metab 11, 786-794. 904. Lim, K. S., Kim, J. R., Choi, Y. J., Shin, K. H., Kim, K. P., Hong, J. H., Cho, J. Y., Shin, H. S., Yu, K. S., Shin, S. G., Kwon, O. H., Hwang, D. M., Kim, J. A., and Jang, I. J. (2008) Pharmacokinetics, pharmacodynamics, and tolerability of the dipeptidyl peptidase IV inhibitor LC15-0444 in healthy Korean men: a dose-block-randomized, double-blind, placebo-controlled, ascending single-dose, Phase I study. Clin.Ther. 30, 1817-1830. 905. Kim, S. E., Yi, S., Shin, K. H., Kim, T. E., Kim, M. J., Kim, Y. H., Yoon, S. H., Cho, J. Y., Shin, S. G., Jang, I. J., and Yu, K. S. (2012) Evaluation of the pharmacokinetic interaction between the dipeptidyl peptidase IV inhibitor LC15- 0444 and pioglitazone in healthy volunteers. Int.J Clin.Pharmacol.Ther. 50, 17-23.

138 Reference

906. Deacon, C. F., Hughes, T. E., and Holst, J. J. (1998) Dipeptidyl peptidase IV inhibition potentiates the insulinotropic effect of glucagon-like peptide 1 in the anesthetized pig. Diabetes 47, 764-769. 907. Johnson, K. M. (2010) Dutogliptin, a dipeptidyl peptidase-4 inhibitor for the treatment of type 2 diabetes mellitus. Curr.Opin.Investig.Drugs 11, 455-463. 908. Li, J., Klemm, K., O'Farrell, A. M., Guler, H. P., Cherrington, J. M., Schwartz, S., and Boyea, T. (2010) Evaluation of the potential for pharmacokinetic and pharmacodynamic interactions between dutogliptin, a novel DPP4 inhibitor, and metformin, in type 2 diabetic patients. Curr.Med.Res.Opin. 26, 2003-2010. 909. Pattzi, H. M., Pitale, S., Alpizar, M., Bennett, C., O'Farrell, A. M., Li, J., Cherrington, J. M., and Guler, H. P. (2010) Dutogliptin, a selective DPP4 inhibitor, improves glycaemic control in patients with type 2 diabetes: a 12-week, double-blind, randomized, placebo-controlled, multicentre trial. Diabetes Obes.Metab 12, 348-355. 910. Melogliptin (2010 ) http://www.expresspharmaonline.com/20090715/market02.shtml. 911. Melogliptin (2010 ) http://www.chemblink.com/products/868771-57-7.htm. 912. Mattei, P., Boehringer, M., Di Giorgio, P., Fischer, H., Hennig, M., Huwyler, J., Kocer, B., Kuhn, B., Loeffler, B. M., Macdonald, A., Narquizian, R., Rauber, E., Sebokova, E., and Sprecher, U. (2010) Discovery of carmegliptin: a potent and long-acting dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes. Bioorg.Med.Chem Lett. 20, 1109- 1113. 913. Parmee, E. R., He, J., Mastracchio, A., Edmondson, S. D., Colwell, L., Eiermann, G., Feeney, W. P., Habulihaz, B., He, H., Kilburn, R., Leiting, B., Lyons, K., Marsilio, F., Patel, R. A., Petrov, A., Di Salvo, J., Wu, J. K., Thornberry, N. A., and Weber, A. E. (2004) 4-Amino cyclohexylglycine analogues as potent dipeptidyl peptidase IV inhibitors. Bioorg.Med.Chem.Lett. 14, 43-46. 914. Lin, J., Toscano, P. J., and Welch, J. T. (1998) Inhibition of dipeptidyl peptidase IV by fluoroolefin-containing N- peptidyl-O-hydroxylamine peptidomimetics. Proc.Natl.Acad.Sci.U.S.A. 95, 14020-14024. 915. Zhao, K., Lim, D. S., Funaki, T., and Welch, J. T. (2003) Inhibition of dipeptidyl peptidase IV (DPP IV) by 2-(2-amino- 1-fluoro-propylidene)-cyclopentanecarbonitrile, a fluoroolefin containing peptidomimetic. Bioorg.Med.Chem. 11, 207- 215. 916. Sorbera, L. A., Revel, L., and Castaner, J. (2001) P32/98.Antidiabetic, dipeptidyl-peptidase IV inhibitor. Drugs of the Future 26, 859-864. 917. Sudre, B., Broqua, P., White, R. B., Ashworth, D., Evans, D. M., Haigh, R., Junien, J. L., and Aubert, M. L. (2002) Chronic inhibition of circulating dipeptidyl peptidase IV by FE 999011 delays the occurrence of diabetes in male zucker diabetic fatty rats. Diabetes 51, 1461-1469. 918. Green, B. D., Flatt, P. R., and Bailey, C. J. (2006) Dipeptidyl peptidase IV (DPP IV) inhibitors: A newly emerging drug class for the treatment of type 2 diabetes. Diab.Vasc.Dis.Res 3, 159-165. 919. Peters, J. U., Weber, S., Kritter, S., Weiss, P., Wallier, A., Zimmerli, D., Boehringer, M., Steger, M., and Loeffler, B. M. (2004) Aminomethylpyridines as DPP-IV inhibitors. Bioorg.Med.Chem.Lett. 14, 3579-3580. 920. Peters, J. U., Hunziker, D., Fischer, H., Kansy, M., Weber, S., Kritter, S., Muller, A., Wallier, A., Ricklin, F., Boehringer, M., Poli, S. M., Csato, M., and Loeffler, B. M. (2004) An aminomethylpyrimidine DPP-IV inhibitor with improved properties. Bioorg.Med.Chem.Lett. 14, 3575-3578. 921. Peters, J. U., Weber, S., Kritter, S., Weiss, P., Wallier, A., Boehringer, M., Hennig, M., Kuhn, B., and Loeffler, B. M. (2004) Aminomethylpyrimidines as novel DPP-IV inhibitors: A 10(5)-fold activity increase by optimization of aromatic substituents. Bioorg.Med.Chem.Lett. 14, 1491-1493. 922. Lubbers, T., Bohringer, M., Gobbi, L., Hennig, M., Hunziker, D., Kuhn, B., Loffler, B., Mattei, P., Narquizian, R., Peters, J. U., Ruff, Y., Wessel, H. P., and Wyss, P. (2007) 1,3-disubstituted 4-aminopiperidines as useful tools in the optimization of the 2-aminobenzo[a]quinolizine dipeptidyl peptidase IV inhibitors. Bioorg.Med.Chem.Lett. 17, 2966- 2970. 923. Balkan, B., Kwasnik, L., Miserendino, R., Holst, J. J., and Li, X. (1999) Inhibition of dipeptidyl peptidase IV with NVP- DPP728 increases plasma GLP-1 (7-36 amide) concentrations and improves oral glucose tolerance in obese Zucker rats. Diabetologia 42, 1324-1331. 924. Hughes, T. E., Mone, M. D., Russell, M. E., Weldon, S. C., and Villhauer, E. B. (1999) NVP-DPP728 (1-[[[2-[(5- cyanopyridin-2-yl)amino]ethyl]amino]acetyl]-2- cyano-(S)- pyrrolidine), a slow-binding inhibitor of dipeptidyl peptidase IV. Biochemistry 38, 11597-11603. 925. Villhauer, E. B., Brinkman, J. A., Naderi, G. B., Dunning, B. E., Mangold, B. L., Mone, M. D., Russell, M. E., Weldon, S. C., and Hughes, T. E. (2002) 1-[2-[(5-Cyanopyridin-2-yl)amino]ethylamino]acetyl-2-(S)-pyrrolidinecarbon itrile: a potent, selective, and orally bioavailable dipeptidyl peptidase IV inhibitor with antihyperglycemic properties. J Med.Chem. 45, 2362-2365. 926. Coppola, G. M., Zhang, Y. L., Schuster, H. F., Russell, M. E., and Hughes, T. E. (2000) 1-Aminomethylisoquinoline-4- carboxylates as novel dipeptidylpeptidase IV inhibitors. Bioorg.Med.Chem.Lett. 10, 1555-1558. 927. Kanstrup, A.B., Christiansen, L.B., Lundbeck, J.M., Sams, C.K., and Kristiansen, M. (2002) Heterocyclic compounds, which are inhibitors of the enzyme DPP-IV. WO 02/02560 A2 928. Narra, K., Mullins, S. R., Lee, H. O., Strzemkowski-Brun, B., Magalong, K., Christiansen, V. J., McKee, P. A., Egleston, B., Cohen, S. J., Weiner, L. M., Meropol, N. J., and Cheng, J. D. (2007) Phase II trial of single agent Val- boroPro (Talabostat) inhibiting Fibroblast Activation Protein in patients with metastatic colorectal cancer. Cancer Biol.Ther. 6, 1691-1699. 929. Cunningham, C. C. (2007) Talabostat. Expert.Opin.Investig.Drugs 16, 1459-1465. 930. Lai, J.H., Liu, Y., Maw, H.H., Wu, W., Zhou, J., Poplaw, S., Sanford, D., and Bachovchin, W.W. Investigation of exo- and endopeptidase activity of fibroblast activation protein inhibitors. 2005, Magdeburg, Germany. 931. Flentke, G. R., Munoz, E., Huber, B. T., Plaut, A. G., Kettner, C. A., and Bachovchin, W. W. (1991) Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors to examine the role of DP-IV in T-cell function. Proc.Natl.Acad.Sci.U.S.A. 88, 1556-1559. 932. Coutts, S. J., Kelly, T. A., Snow, R. J., Kennedy, C. A., Barton, R. W., Adams, J., Krolikowski, D. A., Freeman, D. M., Campbell, S. J., Ksiazek, J. F., and Bachovchin, W. W. (1996) Structure-activity relationships of boronic acid inhibitors of dipeptidyl peptidase IV. 1. Variation of the P2 position of Xaa-boroPro dipeptides. J.Med.Chem. 39, 2087-2094. 933. Sudmeier, J. L., Gunther, U. L., Gutheil, W. G., Coutts, S. J., Snow, R. J., Barton, R. W., and Bachovchin, W. W. (1994) Solution structures of active and inactive forms of the DP IV (CD26) inhibitor Pro-boroPro determined by NMR spectroscopy. Biochemistry 33, 12427-12438. 139 Reference

934. Gutheil, W. G. and Bachovchin, W. W. (1993) Separation of L-Pro-DL-boroPro into its component diastereomers and kinetic analysis of their inhibition of dipeptidyl peptidase IV. A new method for the analysis of slow, tight-binding inhibition. Biochemistry 32, 8723-8731. 935. Poplawski, S. E., Lai, J. H., Sanford, D. G., Sudmeier, J. L., Wu, W., and Bachovchin, W. W. (2011) Pro-soft Val- boroPro: a strategy for enhancing in vivo performance of boronic acid inhibitors of serine proteases. J Med.Chem 54, 2022-2028. 936. Lambeir, A. M., Borloo, M., De Meester, I., Belyaev, A., Augustyns, K., Hendriks, D., Scharpe, S., and Haemers, A. (1996) Dipeptide-derived diphenyl phosphonate esters: mechanism-based inhibitors of dipeptidyl peptidase IV. Biochim.Biophys.Acta 1290, 76-82. 937. Senten, K., Daniels, L., Van der Veken, P., De Meester, I., Lambeir, A. M., Scharpe, S., Haemers, A., and Augustyns, K. (2003) Rapid parallel synthesis of dipeptide diphenyl phosphonate esters as inhibitors of dipeptidyl peptidases. J.Comb.Chem. 5, 336-344. 938. Ferraris, D., Ko, Y. S., Calvin, D., Chiou, T., Lautar, S., Thomas, B., Wozniak, K., Rojas, C., Kalish, V., and Belyakov, S. (2004) Ketopyrrolidines and ketoazetidines as potent dipeptidyl peptidase IV (DPP IV) inhibitors. Bioorg.Med.Chem.Lett. 14, 5579-5583. 939. Ferraris, D., Belyakov, S., Li, W., Oliver, E., Ko, Y. S., Calvin, D., Lautar, S., Thomas, B., and Rojas, C. (2007) Azetidine-based inhibitors of dipeptidyl peptidase IV (DPP IV). Curr.Top.Med.Chem. 7, 597-608. 940. Kuby, J. (1997) Cytokines. In Allen, D., editor. Immunology, W.H.Freeman and Company, New York, 313-334. 941. Janeway, C. A. J., Travers, P., Walport, M., and Shlomchik, M. J. (2004) The adaptive immune response. Immunobiology, Churchill Livingstone, New York, USA, 319-454. 942. Salgado, F. J., Lojo, J., Alonso-Lebrero, J. L., Lluis, C., Franco, R., Cordero, O. J., and Nogueira, M. (2003) A role for IL-12 in the regulation of T cell plasma membrane compartmentation. J Biol.Chem. 278, 24849-24857. 943. Deacon, C. F., Ahren, B., and Holst, J. J. (2004) Inhibitors of dipeptidyl peptidase IV: a novel approach for the prevention and treatment of Type 2 diabetes? Expert.Opin.Investig.Drugs 13, 1091-1102. 944. Senten, K., Van der Veken, P., De Meester, I., Lambeir, A. M., Scharpe, S., Haemers, A., and Augustyns, K. (2003) Design, synthesis, and SAR of potent and selective dipeptide-derived inhibitors for dipeptidyl peptidases. J Med.Chem. 46, 5005-5014. 945. Caldwell, C. G., Chen, P., He, J., Parmee, E. R., Leiting, B., Marsilio, F., Patel, R. A., Wu, J. K., Eiermann, G. J., Petrov, A., He, H., Lyons, K. A., Thornberry, N. A., and Weber, A. E. (2004) Fluoropyrrolidine amides as dipeptidyl peptidase IV inhibitors. Bioorg.Med.Chem.Lett. 14, 1265-1268. 946. Senten, K., Van der Veken, P., De Meester, I., Lambeir, A. M., Scharpe, S., Haemers, A., and Augustyns, K. (2004) Gamma-amino-substituted analogues of 1-[(S)-2,4-diaminobutanoyl]piperidine as highly potent and selective dipeptidyl peptidase II inhibitors. J.Med.Chem. 47, 2906-2916. 947. Akiyama, T., Sawa, R., Naganawa, H., Muraoka, Y., Aoyagi, T., and Takeuchi, T. (1998) Epostatin, new inhibitor of dipeptidyl peptidase II, produced by streptomyces sp. MJ995-OF5 II. Structure elucidation. J.Antibiot.(Tokyo) 51, 372- 373. 948. Thomas, L., Eckhardt, M., Langkopf, E., Tadayyon, M., Himmelsbach, F., and Mark, M. (2008) (R)-8-(3-amino- piperidin-1-yl)-7-but-2-ynyl-3-methyl-1-(4-methyl-quinazoli n-2-ylmethyl)-3,7-dihydro-purine-2,6-dione (BI 1356), a novel xanthine-based dipeptidyl peptidase 4 inhibitor, has a superior potency and longer duration of action compared with other dipeptidyl peptidase-4 inhibitors. J.Pharmacol.Exp.Ther. 325, 175-182. 949. Lee, B., Shi, L., Kassel, D. B., Asakawa, T., Takeuchi, K., and Christopher, R. J. (2008) Pharmacokinetic, pharmacodynamic, and efficacy profiles of alogliptin, a novel inhibitor of dipeptidyl peptidase-4, in rats, dogs, and monkeys. Eur J.Pharmacol. 589, 306-314. 950. Hwang, D.M., Kim, S.H., Yoon.M.K., Kwon, O.H., Koo, K.D., Min.C., Lee, S.H., Lee, J., Lee, C.S., and Yim, H.J. (2012) http://professional.diabetes.org/Content/Posters/2008/p502-P.pdf. 951. Van der Veken, P., Senten, K., Kertesz, I., De Meester, I., Lambeir, A. M., Maes, M. B., Scharpe, S., Haemers, A., and Augustyns, K. (2005) Fluoro-olefins as peptidomimetic inhibitors of dipeptidyl peptidases. J Med.Chem. 48, 1768- 1780. 952. Shreder, K. R., Wong, M. S., Corral, S., Yu, Z., Winn, D. T., Wu, M., Hu, Y., Nomanbhoy, T., Alemayehu, S., Fuller, S. R., Rosenblum, J. S., and Kozarich, J. W. (2005) Boro-norleucine as a P1 residue for the design of selective and potent DPP7 inhibitors. Bioorg.Med.Chem.Lett. 15, 4256-4260. 953. Tran, T., Quan, C., Edosada, C. Y., Mayeda, M., Wiesmann, C., Sutherlin, D., and Wolf, B. B. (2007) Synthesis and structure-activity relationship of N-acyl-Gly-, N-acyl-Sar- and N-blocked-boroPro inhibitors of FAP, DPP4, and POP. Bioorg.Med.Chem.Lett. 17, 1438-1442. 954. Hu, Y., Ma, L., Wu, M., Wong, M. S., Li, B., Corral, S., Yu, Z., Nomanbhoy, T., Alemayehu, S., Fuller, S. R., Rosenblum, J. S., Rozenkrants, N., Minimo, L. C., Ripka, W. C., Szardenings, A. K., Kozarich, J. W., and Shreder, K. R. (2005) Synthesis and structure-activity relationship of N-alkyl Gly-boro-Pro inhibitors of DPP4, FAP, and DPP7. Bioorg.Med.Chem.Lett. 15, 4239-4242. 955. Hnasko, R. and Lisanti, M. P. (2003) The biology of caveolae: lessons from caveolin knockout mice and implications for human disease. Mol Interv. 3, 445-464. 956. Brown, D. A. and London, E. (1998) Functions of lipid rafts in biological membranes. Annu.Rev.Cell Dev.Biol. 14, 111- 136. 957. Thomas, S., Kumar, R. S., and Brumeanu, T. D. (2004) Role of lipid rafts in T cells. Arch.Immunol.Ther.Exp.(Warsz.) 52, 215-224. 958. Edidin, M. (2003) The State of Lipid Rafts: From Model Membranes to Cells. Annu.Rev.Biophys.Biomol.Struct. 32, 257-283. 959. Simons, K. and Ikonen, E. (1997) Functional rafts in cell membranes. Nature 387, 569-572. 960. Simons, K. and Ehehalt, R. (2002) Cholesterol, lipid rafts, and disease. J.Clin.Invest 110, 597-603. 961. Luzio, J. P., Piper, S. C., Bowers, K., Parkinson, M. D., Lehner, P. J., and Bright, N. A. (2009) ESCRT proteins and the regulation of endocytic delivery to lysosomes. Biochem Soc.Trans. 37, 178-180. 962. Luzio, J. P., Parkinson, M. D., Gray, S. R., and Bright, N. A. (2009) The delivery of endocytosed cargo to lysosomes. Biochem Soc.Trans. 37, 1019-1021. 963. Weisz, O. A., Machamer, C. E., and Hubbard, A. L. (1992) Rat liver dipeptidylpeptidase IV contains competing apical and basolateral targeting information. J Biol Chem 267, 22282-22288. 140 Reference

964. Jaeger, P. A. and Wyss-Coray, T. (2009) All-you-can-eat: autophagy in neurodegeneration and neuroprotection. Mol.Neurodegener. 4, 16. 965. Mizushima, N., Ohsumi, Y., and Yoshimori, T. (2002) Autophagosome formation in mammalian cells. Cell Struct.Funct. 27, 421-429. 966. Rosmaninho-Salgado, J., Alvaro, A. R., Grouzmann, E., Duarte, E. P., and Cavadas, C. (2007) Neuropeptide Y regulates catecholamine release evoked by interleukin-1beta in mouse chromaffin cells. Peptides 28, 310-314. 967. Zukowska-Grojec, Z., Karwatowska-Prokopczuk, E., Rose, W., Rone, J., Movafagh, S., Ji, H., Yeh, Y., Chen, W. T., Kleinman, H. K., Grouzmann, E., and Grant, D. S. (1998) Neuropeptide Y: a novel angiogenic factor from the sympathetic nerves and endothelium. Circ.Res. 83, 187-195. 968. Saito, R., Takano, Y., and Kamiya, H. O. (2003) Roles of substance P and NK(1) receptor in the brainstem in the development of emesis. J Pharmacol.Sci. 91, 87-94. 969. Heilig, M. (2004) The NPY system in stress, anxiety and depression. Neuropeptides. 38, 213-224. 970. Eaton, K., Sallee, F. R., and Sah, R. (2007) Relevance of neuropeptide Y (NPY) in psychiatry. Curr.Top.Med.Chem 7, 1645-1659. 971. Sewards, T. V. and Sewards, M. A. (2003) Fear and power-dominance motivation: proposed contributions of peptide hormones present in cerebrospinal fluid and plasma. Neurosci.Biobehav.Rev. 27, 247-267. 972. Keire, D. A., Bowers, C. W., Solomon, T. E., and Reeve, J. R., Jr. (2002) Structure and receptor binding of PYY analogs. Peptides 23, 305-321. 973. Keire, D. A., Mannon, P., Kobayashi, M., Walsh, J. H., Solomon, T. E., and Reeve, J. R., Jr. (2000) Primary structures of PYY, [Pro(34)]PYY, and PYY-(3-36) confer different conformations and receptor selectivity. Am.J.Physiol Gastrointest.Liver Physiol 279, G126-G131. 974. Keire, D. A., Kobayashi, M., Solomon, T. E., and Reeve, J. R., Jr. (2000) Solution structure of monomeric peptide YY supports the functional significance of the PP-fold. Biochemistry 39, 9935-9942. 975. Lindner, D., Stichel, J., and Beck-Sickinger, A. G. (2008) Molecular recognition of the NPY hormone family by their receptors. Nutrition 24, 907-917. 976. Lindner, D., van Dieck, J., Merten, N., Morl, K., Gunther, R., Hofmann, H. J., and Beck-Sickinger, A. G. (2008) GPC receptors and not ligands decide the binding mode in neuropeptide Y multireceptor/multiligand system. Biochemistry 47, 5905-5914. 977. Akerberg, H., Fallmar, H., Sjodin, P., Boukharta, L., Gutierrez-de-Teran, H., Lundell, I., Mohell, N., and Larhammar, D. (2010) Mutagenesis of human neuropeptide Y/peptide YY receptor Y2 reveals additional differences to Y1 in interactions with highly conserved ligand positions. Regul.Pept. 163, 120-129. 978. Sjodin, P., Holmberg, S. K., Akerberg, H., Berglund, M. M., Mohell, N., and Larhammar, D. (2006) Re-evaluation of receptor-ligand interactions of the human neuropeptide Y receptor Y1: a site-directed mutagenesis study. Biochem.J. 393, 161-169.

141 Appendix

8 Appendix 8.1 Introduction 8.1.1 DP 4-like Enzymes Appendix 1: Substrate specificity of DP 4-like enzymes, DP 4 (A), FAP (B + C), DP 8 (D), DP 9 (E) and DP 2 (F). FAP displays both exopeptidase activity (B) and endopeptidase activity (C). As, badly tolerated; as, not cleaved. [Taken and adapted from (2;96;97;127;172;173;210)].

142 Appendix

Appendix 2: Half-lives of substrate hydrolysis of NPY, PYY, GLP-1 and GLP-2 by DP 4, DP 8 and DP 9. [Taken and adapted from (127)].

Appendix 3: Kintetic parameters of substrates Gly-Pro-pNA, Ala-Pro-pNA, Val-Ala-pNA and Arg- Pro-pNA for DP 4, DP 8, DP 9 and DP 2, respectively (127;173;732).

143 Appendix

8.2 Material and Methods 8.2.1 Materials Appendix 4: List of equipment used for the experiments in the present study. Apparatus Supplier 8 channel Impact2 Electronic Pipettor, 2-125 µl Matrix Technology Corporation, Wilmslow, UK 8 channel Impact2 Electronic Pipettor, 5-250 µl Matrix Technology Corporation, Wilmslow, UK 8 channel Impact2 Electronic Pipettor, 15-1250 µl Matrix Technology Corporation, Wilmslow, UK Autoclave Varioklav H+P Labortechnik, Oberschleißheim, Germany BioCad 700E Perspective Biosystems, Wiesbaden, Germany Blotting-Apparatus TRANS-BLOT SD BIO-RAD®, Herdforshire, UK Centrifuge Allegra 21R and Avanti J20 Beckman Coulter, Palo Alto, USA

CO2 incubator MCO-17 AI Sanyo, Gunma, Japan Cryostat HC/F30 Julabo Labortechnik GmbH, Seelbach, Germany Cryo-microtome 1206 Frigormobil Reichert-Jung, Heidelberg-Nussdorf, Germany DNA-gel electrophoresis GEB1A-UVT Hybaid AGS, Heidelberg, Germany Drying Cabinet WTC Binder, Tuttlingen, Germany Eppendorf PCR Mastercycle gradient Eppendorf, Hamburg, Germany Eppendorf Research Pipettes, Eppendorf, Hamburg, Germany 0.5-10 µl, 10-100 µl, 100-1000 µl Fine balance A120S-D1 Sartorius, Göttingen, Germany Fluorescence microscope Axiovert S100 Zeiss, Jena, Germany Fluostar BMG LABTECH, Offenburg, Germany Freezer Profiline ECC-2080-5 (-80° C) National Lab GmbH Mölln, Germany Freezer GT-300B (-20° C) BL-Alaska, Germany Freezer 234 L Energy Saver Privileg (-20° C) Quelle, Nuremberg, Germany Gel-electrophoresis system Mini-Protean II BIO-RAD®, Herdforshire, UK Gel-electrophoresis system NOVEX NOVEX, Invitrogen, Frankfurt (M), Germany Gilson Pipettes, 0.2-2 µl, 1-10 µl, 2-20 µl, Gilson, Viliers-Bel, France 20-100 µl, 50-200 µl, 200-1000 µl, 1000-5000 µl Horizontal Electrophoresis Apparatus Multiphore II Amersham, Pharmacia Biotech, Uppsala, Sweden Ice machine ZBE 30-10 ZIEGRA, Isernhagen, Germany Incubator Heraeus, Osterode, Germany LD-TOF system G2025 Helwett Packard, Waldbronn, Germany Magnetic stirrer MR3000 Heidolph, Schwabach, Germany MicroFire digital microsope camera Otronics, Goleta, USA Micro-plate reader HTS 7000 PLUS Perkin Elmer, Überlingen, Germany Micro-plate reader SUNRISE TECAN, Crailsheim, Germany Microscope camera MC80DX Zeiss, Jena, Germany Millipore Model 8003 (Ultrafiltration) Amicon, Witten, Germany Nicon Eclipse 80i microscope Nikon GmbH, Düsseldorf, Germany pH-meter Multilab, Berlin, Germany Power Supply, Power Pac 200, Power Pac 300 BIO-RAD, Hertforshire, UK Power Supply, Power PAC 3000 BIO-RAD, Hertforshire, UK Power Supply Consort E143 Biozyme Diagnostics GmbH, Oberndorf, Germany Rotofor® BIO-RAD Laboratories, Hercules, USA Sample preparation accessory HP G2024A Helwett Packard, Waldbronn, Germany Sonicator Sonoplus SH70G BANDELIN, Berlin, Germany Spectrophotometer Lamda 20 Applied Biosystems, Weiterstadt, Germany Spectrophotometer SmartSpec 3000 BIO-RAD, Hertforshire, UK Spectrophotometer U2000 Hitachi, Darmstadt, Germany Submarine Electroporesis Agagel Standard Whatman, Biometra, Göttingen, Germany Table centrifuge pico Heraeus, Osterode, Germany Taumel-Rollenmischer 35 rpm Labortechnik Fröbel GmbH, Lindau, Germany Teflon homogenizer Roth, Karlsruhe, Germany Thermomixer compact Eppendorf, Hamburg, Germany Ultra Torrax ® Janke and Kunkel, Staufen, Germany Ultracentrifuge Optima MAX Beckman Coulter, Palo Alto, USA UV-System INTAS, Göttingen, Germany Vortex 2 Genie Scientific Industries, Bohemia, USA Voyager-DE Pro Biospectrometry workstation Applied Biosystems, Weiterstadt, Germany Water purification apparatus PURELAB Plus UV USF SERAL, Ransbach-Baumbach, Germany Waterbath DC10 HAAKE, Karlsruhe, Germany Waterbath F25 Julabo Llabortechnik GmbH, Seelbach, Germany

144 Appendix

Appendix 5: List of consumable used for the experiments in the present study.

Material Supplier Agarose gel bonds Amersham, Pharmacia Biotech, Uppsala, Sweden Amicon Ultra-4, 30 K Millipore, Bedford, USA C18 ZipTip Millipore, Bedford, USA Cell culture flasks 25 cm2 for suspension culture Greiner Bio-One, Frickenhausen, Germany Cell culture petri dish, d = 3 cm Greiner Bio-One, Frickenhausen, Germany Centrifuge Quick-Seal 4 ml, 8 ml Beckmann, Palo Alto, USA Costar 96-well microplates, half area, clear, flat Corning Life Sciences Dialysis Tubing ø = 3 cm Roth, Karlsruhe, Germany DismTipTM syringes 125 µl, 12.5 ml Gilson, Villers-Bel, France Disposable syringes Omnix, 10, 20 and 60 ml Roth, Karlsruhe, Germany Disposable cuvettes (Polystyrene) Roth, Karlsruhe, Germany Eppendorf tubes 2.0 ml, -sterilised Eppendorf, Hamburg, Germany Eppendorf tubes 0.65 ml, -sterilised Eppendorf, Hamburg, Germany Filter pipette tips 1000 µl, 200 µl, 100 µl, 10 µl PeqLab Biotechnology, Erlangen, Germany Kodak KB-film Ektachrome EliteSelect 400 Nordphot, Norderstedt, Germany Mµlti Safety Reaction Vials, colourful sorted, 0.65 ml Roth, Karlsruhe, Germany Mµlti Safety Reaction Vials, colourful sorted, 1.7 ml Roth, Karlsruhe, Germany Mµlti Safety Reaction Vials, colourful sorted, 2.0 ml Roth, Karlsruhe, Germany Mµlti Safety Reaction Vials, 0.65 ml Roth, Karlsruhe, Germany Mµlti Safety Reaction Vials, 1.7 ml Roth, Karlsruhe, Germany Mµlti Safety Reaction Vials, 2.0 ml Roth, Karlsruhe, Germany Matrix Pipette tips, 250 µl, 1250 µl Matrix Technology Corporation, Wilmslow, UK Micro-Flo butterfly canule 0.6 mm x 30 cm Heiland Medical Vertriebs-GmbH, Viena, Austria Micro tubes 0.5 ml, 1.5 ml Roth, Karlsruhe, Germany Microman pipette tips, 100 µl, 1000 µl Gilson, Viliers-Bel, France Microplates, 96-well, clear, flat Nunc, Roskilde, Denmark Microscopic cover slides 20 x 20 mm Roth, Karlsruhe, Germany Microscopic slides 76 x 76 mm Roth, Karlsruhe, Germany Multifly-canule 0.8 x 9 mm Heiland Medical Vertriebs-GmbH, Viena, Austria Nalgene FastCap Bottle top filter units, -sterilised (0.2 µm) Sigma-Aldrich, Steinheim, Germany Naturlatex, latex disposable gloves, size small Roth, Karlsruhe, Germany Nitrocellulose membrane PROTRAN BA83 Schleicher & Schuel, Dassel, Germany Pasteur pipettes Schubert, Laborfachhandel, Espenhain, Germany PCR-tubes, 0.2 ml, 0.5 ml, PeqLab Biotechnology, Erlangen, Germany Pipette tips 5000 µl, 1000 µl, 200 µl, 10 µl Roth, Karlsruhe, Germany Plastic Pasteur pipettes Roth, Karsruhe, Germany PP-centrifuge tubes 15 ml, 50 ml, -sterilised Schuber Laborhandel, Espenhain, Germany Serological pipettes 5 ml, 10 ml and 25 ml Greiner Bio-one, Frickenhausen, Germany S-Monovette® EDTA K for haematologie 2.7 ml Heiland Medical Vertriebs-GmbH, Viena, Austria S-Monovette® Plasma /Li Heparin 2.7 ml Heiland Medical Vertriebs-GmbH, Viena, Austria S-Monovette® Serum 4.5 ml Heiland Medical Vertriebs-GmbH, Viena, Austria Sterican canules 0.9 x 40 mm, 0.9 x 70 mm, 0.6 x 80 mm Heiland Medical Vertriebs-GmbH, Viena, Austria Supremeo nitrile examination gloves, size small Roth, Karlsruhe, Germany Tris-Glycine Native-gradient gel, 4-12 % Novex, Invitrogen, Germany Ultrafiltration membrane YM 100 for 100 000 Da Millipore, Bedford, USA Vivaspin 15 10,000 MWCO PES VivaScience, Satorius Group, Göttingen, Germany Vivaspin 20 5,000 MWCO PES VivaScience, Satorius Group, Göttingen, Germany Vivaspin, Tube Concnetrator 500-3, 500-10 Novagen, Merck Biosciences, Darmstadt, Germany Vasculon Plus 0.9 x 25 mm Heiland Medical Vertriebs-GmbH, Viena, Austria

145 Appendix

Appendix 6: Summary of chemicals used for the purification and isolation of DP 4 isoforms. Substance Supplier -mercaptoethanol Sigma-Aldrich, Steinheim, Germany 2´, 6´-dihydroxyacetonephenone (DHAP) Sigma-Aldrich, Taufenkirchen, Germany Acetonitrile (ACN) Baker, Atlantic Labo, France Acidic IEF standards Amersham Pharmacia Biotech, Uppsala, Sweden Acrylamide solution 30 % (w/v) Serva, Invitrogen, Heidelberg, Germany Ammonium sulphate ICN Biomedicals, Aurora, USA Ammoniumpersulphate BIO-RAD, Hertfortshire, UK BlueSlick Serva, Invitrogen, Heidelberg Germany Bradford reagent Sigma-Aldrich, Steinheim, Germany DEAE-Sephacel Amersham Pharmacia Biotech, Uppsala, Sweden Diammonium hydrogen citrate (DAHC) Sigma-Aldrich, Taufenkirchen, Germany DMF Rathburn, Walkerburn, Scotland Ethanolamine Fluka, Seelze, Germany Fast Blue B Fluka, Seelze, Germany Glycine Roth, Karlsruhe, Germany Glycerol ICN Biomedicals, Eschwege, Germany HEPES Merck, Darmstadt, Germany IEF agarose Amersham Pharmacia Biotech, Uppsala, Sweden Kerosene Serva, Invitrogen, Heidelberg, Germany

Na2HPO4 Roth, Karlsruhe, Germany NaOH, K < 0.0002% Fluka, Sigma-Aldrich, Seelze, Germany Ponçeau S Fluka, Sigma-Aldrich, Seelze, Germany SDS ICN Biomedicals, Eschwege, Germany SDS high molecular standards Sigma, Deisenhofen, Germany Sepharose 6B Amersham Pharmacia Biotech, Uppsala, Sweden Serine Merck, Darmstadt, Germany Serum Bovine Albumin Sigma-Aldrich, Steinheim, Germany Serva Blue R250 Serva, Invitrogen, Heidelberg, Germany Servalyt® carrier ampholytes, pH 3-6, 4-6, 5-6, 3-7 Serva, Invitrogen, Heidelberg, Germany Sodium Azide Serva, Invitrogen, Heidelberg, Germany Sorbitol Sigma-Aldrich, Steinheim, Germany Sucrose Merck, Darmstadt, Germany Trichloroacetic acid Roth, Karlsruhe, Germany TEMED Pharmacia, Freiburg, Germany Trifluor acetic acid Baker, Atlantic Labo, France Tris ICN Biomedicals, Aurora, USA Tris-Glycine native running buffer Novex, Invitrogen, Frankfurt (M), Germany Tris-Glycine native sample buffer Novex, Invitrogen, Frankfurt (M), Germany Triton X-100 Serva, Invitrogen, Heidelberg, Germany Uno Q BIO-RAD Laboratories Munich, Germany

Appendix 7: Reagents used for the analysis of the distribution of DP 4-like activity enzymes in various porcine and canine tissues.

Reagent Supplier Agarose Qualex Gold Hybaid-AGS, Heidelberg, Germany Roth, Karlsruhe, Germany, distilled by the Department of Medicinal Chloroform (distilled) Chemistry, probiodrug AG, Halle, Germany DEPC Sigma-Aldrich, Steinheim, Germany DNA oligonucleotides mix Promega, Mannheim, Germany DNA-bioladder standards 100 bp PeqLab Biotechnologie GmbH, Erlangen Germany DNA-bioladder standards 100 bp plus PeqLab Biotechnologie GmbH, Erlangen Germany DNA-bioladder standards 1Kb PeqLab Biotechnologie GmbH, Erlangen Germany Ethanol Roth, Karlsruhe, Germany Ethidium bromide Roth, Karlsruhe, Germany Herculase polymerase Stratagene, Karlsruhe, Germany Isopropanol Roth, Karlsruhe, Germany Sample buffer for agarose gel electrophoresis PeqLab Biotechnologie GmbH, Erlangen Germany Superscript II transcriptase Invitrogen, Life Technologies, Amsterdam, Netherlands Taq-DNA polymerase Promega, Mannheim, Germany TRIzol Invitrogen, life technologies, Amsterdam, Netherlands

146 Appendix

Appendix 8: Reagents used for the analysis of DP 4-like Enzymes in porcine islets of Langerhans. Reagent Supplier BSA Sigma-Aldrich, Steinheim, Germany Citifluor Plano, Wetzlar, Germany CMRL-1066 Sigma-Aldrich, Steinheim, Germany Collagenase Pan Plus from Serva,Invitrogen, Heidelberg, Germany D-Mannitol Sigma-Aldrich, Steinheim, Germany FBS Gibco BRL, Neu Isenburg, Germany Ficoll Sigma-Aldrich, Steinheim, Germany Fluorescein diacetate Sigma-Aldrich, Steinheim, Germany Glycerol ICN Biomedicals, Eschwege, Germany Gentamycin Gibco BRL, Neu Isenburg, Germany HBSS Sigma-Aldrich, Steinheim, Germany HEPES Sigma-Aldrich, Steinheim, Germany HiTrap protein A HP (5 ml) Amersham Bioscience, Uppsala, Sweden Lactobionic acid Aldrich, Taufenkirchen, Germany Magnesium sulphate Sigma-Aldrich, Steinheim, Germany

NaHCO3 Sigma-Aldrich, Steinheim, Germany NCS Sigma-Aldrich, Steinheim, Germany Niacinamide Sigma-Aldrich, Steinheim, Germany NTB Sigma-Aldrich, Steinheim, Germany Paraformaldehyde Sigma-Aldrich, Steinheim, Germany PBS Invitrogen Life Technologies, Amsterdam, Netherlands 1000 x stock solution of L-glutamine, penicillin Sigma-Aldrich, Steinheim, Germany and streptomycin Phenanzine methosulfate Sigma-Aldrich, Steinheim, Germany pNBA Sigma-Aldrich, Taufenkirchen, Germany Propidium iodide Sigma-Aldrich, Steinheim, Germany Potassium phosphate monobasic Sigma-Aldrich, Steinheim, Germany Superoxide Dismutase Sigma-Aldrich, Steinheim, Germany Trypsin Inhibitor from soya bean Fluka, Sigma-Aldrich, Seelze, Germany

Appendix 9A: Summary of all substrates, and inhibitors employed in the present study. Substrate /Inhibitor Function Supplier Gly-Pro-pNA.HCl Substrate Dr. Manhart, Department of Peptide Synthesis, probiodrug AG, Halle Gly-Pro-4-methoxy--naphthylamide HBr Substrate Bachem, Bubendorf, Switzerland Ala-pNA HCl Substrate Dr. Manhart, Department of Peptide Synthesis, probiodrug AG, Halle Gly-Pro-AMC HCl Substrate Bachem, Bubendorf, Switzerland Ala-Pro-AMC.HCl Substrate Dr. Manhart, Department of Peptide Synthesis, probiodrug AG, Halle Gly-Pro-1-hydroxy-4-naphthylamide Substrate Dr. A. Niestroj, Department of Medical Chemistry, probiodrug AG, Halle Lys-Ala-pNA HCl Substrate Bachem, Bubendorf, Switzerland Lys-Ala-AMC HCl Substrate Bachem, Bubendorf, Switzerland Lys-Ala-CAH HBr Substrate Dr. A. Niestroj, Department of Medical Chemistry, probiodrug AG, Halle Neuropeptide Y (NPY1-36) Substrate Dr. Manhart, Department of Peptide Synthesis, probiodrug AG, Halle NPY3-36 Substrate Dr. Manhart, Department of Peptide Synthesis, probiodrug AG, Halle NPY2-36 Substrate Dr. Manhart, Department of Peptide Synthesis, probiodrug AG, Halle NPY1-30 Substrate Dr. Manhart, Department of Peptide Synthesis, probiodrug AG, Halle NPY1-25 Substrate Dr. Manhart, Department of Peptide Synthesis, probiodrug AG, Halle NPY1-20 Substrate Dr. Manhart, Department of Peptide Synthesis, probiodrug AG, Halle NPY1-36-Biotin Substrate Dr. Manhart, Department of Peptide Synthesis, probiodrug AG, Halle NPY3-36-Biotin Substrate Dr. Manhart, Department of Peptide Synthesis, probiodrug AG, Halle Galanin-like peptide Substrate Dr. Manhart, Department of Peptide Synthesis, probiodrug AG, Halle Galanin Substrate Dr. Manhart, Department of Peptide Synthesis, probiodrug AG, Halle CRF Substrate Bachem, Bubendorf, Switzerland ACTH Substrate Bachem, Bubendorf, Switzerland Oxytocin Substrate Bachem, Bubendorf, Switzerland Arg-Vasopressin Substrate Bachem, Bubendorf, Switzerland Neurotensin Substrate Bachem, Bubendorf, Switzerland Angiotensin Substrate Bachem, Bubendorf, Switzerland Orexin A Substrate Bachem, Bubendorf, Switzerland Substance P Substrate Dr. Manhart, Department of Peptide Synthesis, probiodrug AG, Halle Peptide YY Substrate Dr. Manhart, Department of Peptide Synthesis, probiodrug AG, Halle PACAP Substrate Dr. Manhart, Department of Peptide Synthesis, probiodrug AG, Halle Rat Orexin B Substrate Dr. Manhart, Department of Peptide Synthesis, probiodrug AG, Halle Human Orexin B Substrate Dr. Manhart, Department of Peptide Synthesis, probiodrug AG, Halle P32/98 Inhibitor Dr. Manhart, Department of Peptide Synthesis, probiodrug AG, Halle Pefabloc Inhibitor Merck, Darmstadt, Germany Ile-CN-Pyr Inhibitor Dr. U. Heiser, Department of Medical Chemistry, probiodrug AG, Halle

147 Appendix

Appendix 9B: Summary of all recombinant and purified enzymes employed in the present study. Enzyme Supplier Human recombinant DP 2 N. Jänckel, Department of Molecular biology, probiodrug AG, Halle Human recombinant DP 4 J. Bär and A. Weber, Department of Molecular biology, probiodrug AG, Halle Human recombinant DP 9 A. Weber, Department of Molecular biology, probiodrug AG, Halle Human recombinant FAP K. Gans, Department of Molecular biology, probiodrug AG, Halle Human recombinant PEP Dr D. Ruiz-Carrillo Department of Molecular Biology, probiodrug AG, Halle Human recombinant Neprilysin R&D Systems GmbH, Wiesbaden-Nordstadt, Germany Human recombinant Meprin A R&D Systems GmbH, Wiesbaden-Nordstadt, Germany Human recombinant Meprin B R&D Systems GmbH, Wiesbaden-Nordstadt, Germany Human purified Cathepsin D Calbiochem, Merck Bioscience, Darmstadt, Germany Human recombinant Cathepsin B R&D Systems GmbH, Wiesbaden-Nordstadt, Germany Human Cathepsin G Sigma-Aldrich, Steinheim, Germany Human recombinant Cathepsin L R&D Systems GmbH, Wiesbaden-Nordstadt, Germany Human recombinant Cathepsin S R&D Systems GmbH, Wiesbaden-Nordstadt, Germany Human recombinant ACE R&D Systems GmbH, Wiesbaden-Nordstadt, Germany Human recombinant ACE2 R&D Systems GmbH, Wiesbaden-Nordstadt, Germany Human Plasma Kallikrein Sigma-Aldrich, Steinheim, Germany Human Trypsin Sigma-Aldrich, Steinheim, Germany Human Plasmin Calbiochem, Merck Bioscience, Darmstadt, Germany Human Thrombin Calbiochem, Merck Bioscience, Darmstadt, Germany Human Urokinase Calbiochem, Merck Bioscience, Darmstadt, Germany Human Tissue Kallikrein Biopur AG, Bubendorf, Switzerland Hek-129 cells tranfected with Cathepsin D Dr. D. Schlenzig, Department of Enzymology, probiodrug AG, Halle

Appendix 10: Commercial inhibitors implemented in the pesent study. Inhibitor Targetenzym Firma 6-Aminohexanoic acid Chymorypsin, Faktor VIIa, Plasmin, Plasminogen Sigma-Aldrich, Steinheim, Germany Actinonin Meprin, MMP’s, Aminopeptidases Fluka, Sigma-Aldrich, Seelze, Germany AEBSF Serinproteasen Sigma-Aldrich, Steinheim, Germany Amiloride uPA, ATPase Inhibitor Calbiochem, Merck Bioscience, Darmstadt, Germany

2- Antiplasmin Plasmin Calbiochem, Merck Bioscience, Darmstadt, Germany Aprotinin Serinproteasen Sigma-Aldrich, Steinheim, Germany Apstatin Aminopeptidase P MobiTec GmbH, Göttingen, Germany Bestatin Aminopeptidases Sigma-Aldrich, Steinheim, Germany Captopril ACE, Meprins Fluka, Sigma-Aldrich, Seelze, Germany Chlorpromazine Phosphordiesterase Calbiochem, Merck Bioscience, Darmstadt, Germany Collagenase Inhibitor I Collagenases Calbiochem, Merck Bioscience, Darmstadt, Germany Cpp-AAF-pAb Thimet Oligopeptidase Bachem, Bubendorf, Switzerland Dichloroisocoumarin Cathepsin G Sigma-Aldrich, Steinheim, Germany H-DAB-pyrrolidideHCl DP 2 Calbiochem, Merck Bioscience, Darmstadt, Germany E-64 Cysteinproteasen Sigma-Aldrich, Steinheim, Germany EGTA Metalloproteases Sigma-Aldrich, Steinheim, Germany Gabexate mesylate Plasma Kallikrein Sigma-Aldrich, Steinheim, Germany Galardin MMP’s, Meprins Calbiochem, Merck Bioscience, Darmstadt, Germany GEGFLG(D)-FL Cathepsin D Bachem, Bubendorf, Switzerland GEMSA Carboypeptidase N Calbiochem, Merck Bioscience, Darmstadt, Germany Heparin Anticoagulant Sigma-Aldrich, Steinheim, Germany Hirudin Thrombin Sigma-Aldrich, Steinheim, Germany Leupeptin Serine- + Thiolproteases Sigma-Aldrich, Steinheim, Germany MGTA , Carboxypeptidase H Calbiochem, Merck Bioscience, Darmstadt, Germany Pepstatin A Cathepsin D, Pepsin, Renin Sigma-Aldrich, Steinheim, Germany 1,10-Phenanthroline Metalloproteases Sigma-Aldrich, Steinheim, Germany Phosphoramidon Neprilysin Sigma-Aldrich, Steinheim, Germany Pro-Ile Neurolysin Bachem, Bubendorf, Switzerland PKI-127 Plasma Kallikrein, Kallikreins EnzoLife Sciences GmbH, Lörrach, Germany Pro-Leu-Gly-NHOH Collagenases, MMP, Meprin Bachem, Bubendorf, Switzerland Sitagliptin N-sulfate DP 4-selective inhibitor Santa-Cruz, Biotechnology Inc. Heidelberg, Germany Soyabean Trypsin Inhibitor Trypsin Sigma-Aldrich, Steinheim, Germany Complete mini Protease Inhibitor Mix Roche Diagnostics GmbH, Prenzberg, Germany Thiorphan Neprilysin, ACE Fluka, Sigma-Aldrich, Seelze, Germany Z-Pro-Pro-aldehyde-dimethyl PEP Bachem, Bubendorf, Switzerland acetal

148 Appendix

Appendix 11: Primary and secondary antibodies used for immunohistochemical analysis of pancreas. Antibody Supplier Class

Anti DPIVpig Ab (rabbit, polyclonal) probiodrug AG, Halle, Germany Primary Anti-Glucagon Ab (guinea pig, serum) Linco Research, St. Charles, USA Primary

Anti-Insulinhuman Ab (mouse, monoclonal) ICN Biomedicals, Aurora, USA Primary Anti-IgMouse Ab-TRITC (goat, purified antiserum) Dianova, Hamburg, Germany Secondary Anti-Igrabbit Ab-FITC (goat, purified antiserum) Dianova, Hamburg, Germany Secondary Anti-Igguinea pig Ab-FITC (rabbit, purified antiserum) Dianova, Hamburg, Germany Secondary Anti-Ig Ab-Rhodamine Red X guinea pig Dianova, Hamburg, Germany Secondary (donkey, purified antiserum)

Appendix 12: List of chemicals used for identification of DP 4 as a target for psychological disorders. Reagent Supplier List of neuropeptides  Appendix 9 List of commercial enzymes  Appendix 9 List of inhibitors  Appendices 9 and 10 -Cyano-4-hydroxycinnamic acid Sigma-Aldrich,Taufenkirchen, Germany Acetonitrile Baker, Atlantic Labo, France Anti-Cathepsin D pAb Cell-Signalling Inc., New England Biolabs GmbH, Frankfurt, Germany Thermo Fisher Scientific Germany Ltd. & Co. KG Anti-Rabbit-IgG-HRP-linked Ab Perbio Science, Bonn Germany Ferula Fluka, Sigma-Aldrich, Sleeze, Germany Sinapic acid Fluka, Sigma-Aldrich, Sleeze, Germany Trifluor acetic acid Baker, Atlantic Labo, France

Appendix 13: List of all the kits employed for the present study. Kit Supplier Thermo Fisher Scientific Germany Ltd. & Co. KG BCA Protein Determination Kit Perbio Science, Bonn Germany Cytotox 96® Non-radioactive cytotoxicity Assay Promega, Mannheim Germany 3’ RACE Stratagene, Cedar Creek, USA Histopaque®-1077 Sigma-Aldrich, Steinheim, Germany JETquick GelExtraction Spin kit Genomed, Oeynhausen, Germany JETquick PCR Purification Kit Genomed, Oeynhausen, Germany sCD26 ELISA Bender MedSystems® GmbH, Vienna, Austria Thermo Fisher Scientific Germany Ltd. & Co. KG Supersignal WestFemto Maximum Sensitivity Substrate Perbio Science, Bonn Germany Thermo Fisher Scientific Germany Ltd. & Co. KG Supersignal WestPico Chemilumniscent substrate Perbio Science, Bonn Germany

8.2.2 Methods 8.2.2.1 Isolation of DP 4 Isoforms from Porcine Kidney Appendix 14: BSA-Standard Curve according to the Bradford method.

149 Appendix

Appendix 15: Staining protocol of proteins separated by SDS-PAGE or native PAGE Proteins were detected with a Serva Blue G dye following a standard method. i. Fixation: 10 minutes in 50% (v/v) methanol, 10% (v/v) acetic acid. ii. Staining: 30 minutes in 50% (v/v) methanol, 10% acetic acid and 0.025% (w/v) Serve Blue G. iii. Destaining: 10 % acetic acid, with changing the destain solution 2-4 times until no background visible.

Appendix 16: Staining protocol of proteins separated by agarose analytical IEF The protein staining of analytical IEF-gels consisted of the following 6 steps: i. Fixing: 30 min in 20% (w/v) TCA. ii. Washing: The gel was washed for 2 x 15 min in 200 ml of fresh washing solution consisting of 10% glacial acetic acid and 25% methanol. iii. Drying: 3 layers of filter paper were placed on the gel and a 1 to 2 kg weight was put on top. After 10 min everything was removed and drying was completed in the heating chamber. iv. Staining: Staining was achieved by soaking the gel for 10 min in 0.5% (w/v) Serva Blue R- 350 in 10% glacial acetic acid and 25% methanol. v. Destaining: Destaining was performed with a 10% glacial acetic acid and 25 % methanol solution until background was clear. vi. Drying: Finally the gel was dried in the heating cabinet.

Appendix 17: Protocol of DP 4 activity staining separated by agarose analytical IEF A Blotting was performed according to the following capillary method: i. Gel was placed on a clean glass plate and a wet nitro-cellulose membrane was arranged over it. ii. Two fibre-less filter papers were added, followed by 4 layers of normal filter paper. iii. A 5 kg weight was put on top to enhance capillary forces. iv. After 10 min, filter papers were changed and a 5 kg weight was placed again on top for further 10 min. v. Weight and filter papers were removed and the nitro-cellulose was immediately soaked in PBS to maintain DP 4 activity. B Activity staining was performed as followed: i. 2 mg of Gly-Pro 4-methoxy--naphtylamide and 1 mg of Fast Blue, dissolved in 200 µl and 100 µl DMF, respectively. ii. Substrate and Fast Blue were added to 7 ml PBS. iii. PBS was removed from nitro-cellulose membrane, substrate solution was added and incubation proceeded until red-violet staining became visible. iv. Reaction was stopped with water.

8.2.2.2 Distribution of DP 4 – like Enzymes in Porcine and Canine Tissues Appendix 18 : Scheme of primer annealing sites on their respective templates for the various DP 4- like enzymes. CT/C=cytoplasmic tail; TM = transmembrane region; N-α/βH = N-terminal portion of /-hydrolase domain; Propeller = propeller domain; C-α/β-Hydrolase = /- hydrolase domain; N-T = N-Terminus; SP = signal peptide; P = propeptide; SKS = helical bundle domain; , forward primer; , reverse primer (131).    C TM N-H Propeller C--Hydrolase DP 4  N-T N-H Propeller C--Hydrolase DP 8   N-T N-H Propeller C- -Hydrolase DP 9  C TM N-H Propeller C--Hydrolase FAP    SPPN-H) SKS C-- DP 2

Hydrolase

150 Appendix

Appendix 19: Summay of primers for the DP 4- like enzymes applied during RT-PCR (131;578). Enzyme Primer Type Tm GC% Sequence TaOpt PCR product DP4-24 Forward 50 47.4 GCAGTACCCAAAGACTGTGC DP 4 54.1 798 p-DP 4-2 Reverse 49.6 55.0 TTTGAGGGCATCTGGACAT DP8-1 Forward 44.5 45.0 CCTTTAGAGCATCACCTGTA DP 8 51.7 398 DP8-2 Reverse 54.1 57.9 GCCCCAGCAATAGCAACCT DP9-5 Forward 52.5 55.0 CAGGGCTACGATTGGAGTGA DP 9 59.3 651 DP9-11 Reverse 56.5 55.0 CCTGCCGTCAATCACAACCA hFAP-1 Forward 52.8 50.0 GCATTGTCTTACGCCCTTCA FAP 51.6 410 hFAP-2 Reverse 58.1 60.0 CCCAACAGGCGACCAGCATA hQPP-11 Forward 54.9 57.9 CCAACAACTCGGGCTTCGT DP 2 62.0 1094 hQPP-2 Reverse 56.0 55.0 ATGGTGACGGCGATGACTGA

Appendix 20: Summary of primers for the DP 4 like enzymes applied during nested PCR (131;578). Enzyme Primer Type Tm GC (%) Sequence TaOpt PCR product p-DP 4-1 Forward 50.0 47.4 TGGCTGGGTTGGAAGATTTA DP4 53.1 483 p-DP 4-2 Reverse 49.6 55.0 TTTGAGGGCATCTGGACAT DP8-1 Forward 44.5 45.0 CCTTTAGAGCATCACCTGTA DP8 50.1 364 DP8-3 Reverse 40.5 42.1 TGAACAGCACAGTAGGATA DP9-1 Forward 42.6 55.6 GTTCGTCAGCCACTACAG DP9 58.6 367 DP9-11 Reverse 56.5 55.0 CCTGCCGTCAATCACAACCA hFAP-1 Forward 52.8 50.0 GCATTGTCTTACGCCCTTCA FAP 49.4 264 hFAP-3 Reverse 49.2 45.0 ATTGCCGATCAGGTGATAAG hQPP-1 Forward 60.0 53.1 GGGATGCTCAGTGCCTACCT DP2 60.9 821 hQPP-2 Reverse 56.0 55.0 ATGGTGACGGCGATGACTGA

Appendix 21: Accession numbers, exons included in the RT-PCR fragment and number of alternative spliced variants of DP 4-like enzymes and control templates. Exons included in the Number of alternative Enzyme Accession number RT-PCR fragment spliced variants Porcine DP 4 AY198323 9-17 - Human DP 8 AF221634 4-6 5 Human DP 9 AF452102 4-7 3 Human FAP NM_004460 2-7 2 Human DP 2 AF154502 3-12 - Porcine -actin AF054837 - Canine-actin Z70044 -

Appendix 22: Properties of designed primers for the controls. Enzyme Source Primer Type Tm GC% Sequence TaOpt PCR product p-actin-1 Sense 51.4 50.0 ATGGTGGGTATGGGTCAGAA β-Actin Porcine 54.9 151 p-actin-2 Antisense 50.9 52.4 GCTCGTTGTAGAAGGTGTGGT c-actin-1 Sense 51.4 55.0 GGCATCCTGACCCTGAAGTA β -Actin Canine 57.6 209 c-actin-2 Antisense 52.9 50.0 ATGGTTGGGGTGTTGAAGGT

Appendix 23: Program of Eppendorf PCR mastercycler used for the first PCR. Steps Temperature Time (sec) Cycles 1 Denaturation 95° C 2 min 1 x Denaturation 95° C 30 sec 2 Annealing 57° ± 5.5° C 30 sec 10 x Elongation 72° C 80 sec Denaturation 94° C 30 sec 3 Annealing 57° ± 5.5° C 30 sec 20 x Elongation 72° C 80 sec + 10 sec/cycle 4 Extension 72° C 7 min 1 x 5 Kept on hold 4° C  1 x

Appendix 24: Distribution of 0.2 ml PCR tubes within temperature gradient. 1 2 3 4 5 6 7 8 9 10 11 12 51.5 51.7 52.3 53.3 54.5 56.0 57.5 59.0 60.4 61.6 62.5 63.0 FAP DP 8 DP 4 p- c- DP 9 DP 2 Actin Actin 151 Appendix

Appendix 25: Program of Eppendorf PCR mastercycler used for the nested PCR. Steps Temperature Time (sec) Cycles 1 Denaturation 95° C 5 min 1 x Denaturation 95° C 45 sec 2 Annealing 55° ± 5° C 45 sec 35 x Elongation 68° C 50 sec 4 Extension 72° C 10 min 1 x 5 Kept on hold 4° C  1 x

Appendix 26: Temperature gradient and distribution of 0.5 ml PCR tubes for nested PCR.

1 2 3 4 5 6 7 8 9 10 11 50.1 50.4 51.1 52.2 53.4 54.7 56.1 57.4 58.6 59.6 60.2 FAP DP 8 DP 4 p-Actin c-Actin DP 9 DP 2

8.2.2.3 Specific Activities of DP 4-like Enzymes in Porcine and Canine Tissues Appendix 27: Summary of assay composition applied for the determination of specific activities of DP 4-like enzymes DP 4, DP 8 /DP 9 and DP 2 in various tissues at pH 7.6. Volume (µl) Final Concentration Sample (tissue extract) 20 µl - 104 mM Hepes, pH 7.6, 70 mM KCl (DP 4, DP 8 + DP 9) 100 µl 40 mM

H2O/Inhibitor 100 µl - Ala-Pro-AMC (0.675 mM) 50 µl 125 µM

Appendix 28: Summary of assay composition applied for the determination of specific activities of DP 2 in various tissues at pH 5.5. Volume (µl) Final Concentration Sample (tissue extract) 20 µl - 0.2 mM NaAcetate, pH 5.5 (DP 2) 100 µl 80 mM H2O/Inhibitor 100 µl - Ala-Pro-AMC (1.22 mM) 50 µl 225 µM

Appendix 29: Selectivity, IC50 and concentrations of selective DP 4-like inhibitors, applied in the inhibition studies of tissue extracts (131;135;170;622;622;732). Inhibitor Sitagliptin UG93 Dab-Pip Selectivity IC50 IC50 IC50 DP 4 8.8e-9 M 1.2e-5 M >1e-3 M DP 8 2.6e-5 M 2.0e-10 M 9.5e-5 M DP 9 1.1e-4 M 3.6e-9 M 2.2e-5 M DP 2 2.7e-4 M 5.3e-6 M 1.3e-7 M PEP 1.2e-4 M none ? [Inhibitor] applied in assay 1e-6 M 1e-7 M 1e-5 M

Appendix 30: AMC-Standard curves required for the conversion of RFU/min into enzymatic units at gain 100. Conversion factor for AMC standard + Hepes + phosphate buffer: 7.77E-05; AMC standard + NaAc + Phosphate: 7.79E-05; AMC standard + Hepes + phosphate buffer + sucrose: 7.35E-05; AMC standard + NaAc + phosphate buffer + sucrose: 7.29E-05.

152 Appendix

Appendix 31: BSA-Standard curve according to the BCA method adapted for microplates.

8.2.2.4 DP 4-like enzymes in pancreas and islets of Langerhans Appendix 32: Solutions required for the isolation of porcine and canine islets of Langerhans. Weigh off (g) Concentration M Solution Reagents r In volume (ml) pH Reference (mol/l) (g/mol) Volume added to (ml) HBSS Bottle - 1000 HBSS Na2HCO3 0.35 7.1 – 7.3 (581) FBS 10 % 100 HBSS - - 200

HBSS Na2HCO3 0.35 Digestion BSA 0.1 % 1 7.1 - 7.3 (581) Solution Collagenase 1 mg/ml - 0.2 Trypsin Inhibitor 0.1 mg/ml - 0.02

H2O - - 1000 D-Mannitol 30 mM 182.2 5,47 K-lactobionate 100 mM 358.3 35,83 LAP-1 KH2PO4 15 mM 136.1 2,042 7.4 (583) MgSO4 5 mM 246.5 1,23 Nicotonamide 10 mM 122.1 1,22 3 Superoxide Dismutase 30 x 10 Units/l - dissolve in 1 ml H2O CMRL 1066 Package 1000 Hepes 10 mM 238,3 2.383 Na HCO 0.35 CMRL- 2 3 Glutamine 2 mM 7.1 – 7.3 (581) 1066 Streptomycin 100 Units Penicilline 10 µg/ml 200 µg Gentamycin HBSS Bottle - 400 25 % Na HCO 0.14 7.1 – 7.3 Ficoll 2 3 Ficoll 25 g/ 100 ml - 100 23 % 25 % Ficoll 23 7.1 – 7.3 Ficoll HBSS/ Na2HCO3 2 (581) 20.5 % 25 % Ficoll 20.5 7.1 – 7.3 Ficoll HBSS/ Na2HCO3 4.5 11 % 25 % Ficoll 11 7.1 – 7.3 Ficoll HBSS/ Na2HCO3 14

153 Appendix

Appendix 33: Primers for the controls of pancreatic tissues and cells of islets of Langerhans (661). # bp of Tissue Animal Enzyme Source Primer Type Sequence TaOpt PCR controls Applied product Amy-1 Forward TGGGTTGACATTGCTCTTGAA Amylase Pig Pig 53.6 183 Amy-13 Reverse CATCTTCTCTCCACTCCACTTG Exocrine Amy-2 Forward CTTCACAACCTAAACACCAACTG Amylase Dog Pig 54.5 762 Amy-13 Reverse CATCTTCTCTCCACTCCACTTG CA-4 Forward ACTTTCACTGGGGCTCATCT CAII Pig Mouse 52.8 143 CA-1 Reverse GGCCAGTCCATCAGGTTGCT Ductal CA-3 Forward GCCCCTGTCTGTTTCCTATGAT CAII Dog Human 55.0 289 CA-1 Reverse GGCCAGTCCATCAGGTTGCT Islet Pig/ Pig/ Insulin-21 Forward GGGCTTCTTCTACACGCCTAA Insulin 56.6 195 β-cells dog dog Insulin-16 Reverse TAGTTGCAGTAGTTCTCCAG c-ppg-2 Forward CAGGACACAGCACAGCCAAA Glucagon Pig Dog 54.3 243 Islet c-ppg-3 Reverse TTCTTGTTCCTCTTGGTGTTCA α-cells c-ppg-1 Forward CTTTGTGGCTGGATTGTTTGTA Glucagon Dog Dog 53.0 465 c-ppg-3 Reverse TTCTTGTTCCTCTTGGTGTTCA pCD31-4 Forward TCTAATCCCAAGTTCCACATCA CD31 Pig Pig 55.9 340 pCD31-5 Reverse ACCACAGTCTCAGGCTTTCAC Capillaries pCD31-2 Forward GGTGGTCGTGGAAGGAGTGT CD31 Dog Pig 56.2 668 pCD31-5 Reverse ACCACAGTCTCAGGCTTTCAC

Appendix 34: Summary of the dilution factor of primary and secondary antibodies. Primary Antibody Secondary antibody Dilution in Dilution in Antibody Antibody PBS/1% BSA PBS/1% BSA Rabbit anti-porcine DP 4 pAb 1000 Goat anti-rabbit IgG Ab-FITC 200 Donkey anti-Guinea pig IgG Ab-Rhodamine Red X 100 Guinea pig anti-glucagon pAb 40 Rabbit anti-Guinea pig IgG Ab-FITC 200 Mouse anti-human insulin mAb 40 Goat anti-mouse IgG Ab-TRITC 100

Appendix 35: Chemical structures of Gly-Pro-1-hydroxy-4 naphtylamide hydrochloride (A) and competitive inhibitor P32/98 (B), used in the histochemical activity staining of DP 4 activity (28).

A B S S N N O Cl +H N H 3 O N OH NH + +H3N N 3 COO- O -OOC O

Gly-Pro-1-hydroxy-4-naphtylamide 3-Ile-thiazolidine fumarate hydrochloride

Appendix 36: Chemical structure of substrate, used in the histochemical activity staining of DP 2 activity. A, Substrate Lys-Ala-5-chloro-1-anthraquinonylhydrazide trifluoracetate.

CF3COO- O NH + H 3 H N N N H +H3N O O O CF3COO- Cl

Lys-Ala-5-chloro-1-anthraquinonylhydrazide trifluoracetate

154 Appendix

Appendix 37: Chemical structure of sodium nitroprusside.

2- O N N N + (II) 2 Na + 2 H O Fe 2 N N

N

Appendix 38: Summary of media components or stimmulants interfering with DP 4 activity. Component Function Interference Assessment Concentrations BSA Mainatainance of viscosity contaminated DP 4 DP 4 activity 0.31 – 2.0 mM (RIA grade) in perfusion and perifusion activity dependence on [BSA]

Arginine Stimulation of glucaon Inhibition of DP 4 Ki-determination 0.16 – 10 mM

Nitroprusside Stimulation of glucagon Inhibition of DP 4 Ki-determination 0.625 – 40 µM

Appendix 39: Stimulants to initiate glucagon and DP 4-like activity enzyme sectretion. Stimulant Concentration(s) or electrical intensity Sodium nitroprusside 10 µM Norepinephrine 10 ng/ml or 100 ng/ml Splanchic nerve 10 Hz 8 msec 7 V / 20 Hz 5 msec 10 V

Appendix 40: Conditions of perfusion.  Modified Krebs Ringer bicarbonate buffer  3 % Dextran  0.2 % BSA (fraction V, RIA grade, Sigma)  gassed with with 95 % O2/5 % CO2 until pH 7.4 is achieved and maintained  Perfusion rate: 4 ml/min  Collection of portal venous outflow: 1 min/tube

8.2.2.5 Identification of DP 4 as a Pharmaceutical Target for Stress Disorders

Appendix 41: Experimental conditions applied for the histochemical DP 4-like staining of brain and kidney sections from F344/Ztm and F344/Crl(Wiga)SvH-Dpp4 rats. Reagent /Experimental Step Brain Kidney Rats F344/Ztm + F344/Crl(Wiga)SvH-Dpp4 F344/Ztm + F344/Crl(Wiga)SvH-Dpp4 Thickness of sections 30 µm, 50 µm 10 µm [H-Gly-L-Pro-1-OH-4-naphthylamide . HCl] 0.25 mM 0.25 mM NBT 0.25 mM 0.25 mM Incubation times 1, 6, 12, 15, 20 h 10, 20, 30, 60, 90 min Glycergel Mounting medium Glycergel Mounting medium Embedding medium (DAKO, Hamburg) (DAKO, Hamburg) Nikon Eclipse 80i microscope Nikon Eclipse 80i microscope Light Microscopy (Nikon GmbH, Duesseldorf) (Nikon GmbH, Duesseldorf) MicroFire digital microscope camera MicroFire digital microscope camera Camera (Optronics, Goleta) (Optronics, Goleta)

155 Appendix

Appendix 42: Protocol of neuropeptide hydrolysis by MALDI-TOF-MS. Reagent Volume (µl) Final Concnetration Sample 25 µl - 100 mM Tris/HCl, pH 7.6 25 µl 20 mM H2O/Inhibitor 25 µl - 62.5 µM Neuropeptide 50 µl 25 µM Assay Incubation @ 37° C (2 min, 10 min, 20 min, 30 min, 60 min, 90 min, 2 h, 3 h, 6 h, 12 h and 24 h) Stop reaction by taking 10 µl aliquots and mix with 10 µl 0.1 % TFA by vortexing Add 20 µl matrix (sinapinic acid/-cyano-4-hydroxy-cinnamic acid) Load 1 µl on sample plate and read on MALDI-TOF-MS 0.1 % TFA 10 µl Sample 2 µl 10 min incubation @ room temperature to allow sample denaturation 100 mM Tris/HCl, pH 7.6 2 µl 20 mM t0 = 0 H2O/Inhibitor 2 µl - 62.5 µM Neuropeptide 4 µl 25 µM Add 20 µl matrix (sinapinic acid/-cyano-4-hydroxy-cinnamic acid) Load 1 µl on sample plate and read on MALDI-TOF-MS 0.1 % TFA 10 µl 20 mM Tris/HCl, pH 7.6 2 µl 100 mM Tris/HCl, pH 7.6 2 µl 20 mM

Blank H2O/Inhibitor 2 µl - 62.5 µM Neuropeptide 4 µl 25 µM Add 20 µl matrix (sinapinic acid/-cyano-4-hydroxy-cinnamic acid) and centrifuge Load 1 µl on sample plate and read on MALDI-TOF-MS

Appendix 43: Summary of buffers employed for the capillary depletion. Conc. Weigh-off Weigh-off Weigh-off Weigh-off Buffer Reagent M (g/mol) (mM) (100 ml) (mg) (250 ml) (500 ml) (l) (g) NaCl 58.44 118.0 690 1.725 g 3.45 g 6.9

CaCl2 110.99 2.5 27.75 69.375 mg 138.75 mg 0.278 KCl 74.55 4.7 35.04 87.6 mg 175.2 mg 0.35

MgSO4 x 7 H2O 246.5 1.2 29.58 73.95 mg 147.9 mg 0.296 Henseleit Henseleit

- KH2PO4 136.09 1.2 16.33 40.825 mg 81.65 mg 0.163 NaHCO3 x 2 H2O/ 84.01 25 210.03 525.08 mg 1.05 g 2.10

buffer, pH 7.4 pH buffer, D-Glucose 180.2 10 180.2 450.5 mg 901 mg 1.8 Krebs BSA 3 g/dl 3 g 7.5 g 15 g 30 Hepes 238.3 10 238.3 595.75 mg 1.19 g 2.38

NaCl 58.44 141.0 824.3 2.061 g 4.12 g 8.24

CaCl2 110.99 2.8 31.08 77.69 mg 155.39 mg 310.78 KCl 74.55 4 29.8 74.5 mg 149 mg 298

pH.7.4 MgSO4 x 7 H2O 246.5 1 24.65 61.63 mg 123.25 mg 246.50

NaH2PO4 x 2 H2O/ NaH2PO4 156.01/119.98 1 15.60/ 2.00 39/30 mg 78/60 mg 0.156/0.120

Hepes buffer, buffer, Hepes Physiological Physiological D-Glucose 180.2 10 180.2 450.5 mg 901 mg 1.80

Appendix 44: Composition of 10 x conservation solution implemented in the capillary depletion.

Reagent Weigh off Dissolve in + add Final Concentration

Aprotinin 0.8 ± 0.1 mg 1 ml H2O + 20 Units/ml

Sodium azide 4 mg 1 ml H2O + 200 µg/ml

Glutathion 1 g 11 ml H2O + 50 mg/ml Streptomycinsulphate 4 mg 1 ml H2O + 200 µg/ml EDTA Na4 607 mg 2 ml H2O + 500 µM Val-Pyrr 10 mM add 2 ml + 500 µM Dithiothreitol 4 mg 500 µl EtOH + 200 µg/ ml PMSF 10 mg 500 µl EtOH + 500 µg/ml Chloramphenicol 40 mg 1 ml EtOH + 2 mg/ml Saccharose 1.2 g Add to final solution 60 mg/ml Final Volume 20 ml vortex solution vigorously

156 Appendix

Appendix 45: Summary of experiments, methods and analysis of capillary depletion. Experiment Analysis Methods Aim Wildetype F344/Ztm + Perfusion Brain Metabolism of NPY Rats DP 4-deficient F344/Crl(Wiga)SvH-Dpp4 Perfusion Brain  DP 4 truncation of NPY

Sample Sample NPY1-36-Biotin IP + MALDI-TOF-MS (624;625) Metabolism of NPY

Perfusion Perfusate NPY1-36-Biotin IP + MALDI-TOF-MS (624;625) Endothlial cleavage of NPY Positive Control Chlorpromazine HPLC-MS  BBB Controls Negative Control Fluorescein Fluorescence  BBB Inhibitor PD31 IP + MALDI-TOF-MS (624;625)  DP 4 truncation of NPY DP 4-like enzymes Activity/inhibition Assays Which DP 4-like Enzyme? Capillaries Alkaline Phosphatase Activity Assay EC-purity Analysis NPY-Metabolite IP + MALDI-TOF-MS (624;625) Adsorption capillaries Capillary DP 4-like enzymes Activity/inhibition Assays Which DP 4-like Enzyme? Depletion Brain Alkaline Phosphatase Activity Assay EC contamination Parenchyma NPY-Metabolite IP + MALDI-TOF-MS (624;625) NPY-Metabolite ? Distribution Brain Brain Sections NPY-Biotin Immunohistochemistry  BBB of NPY

Appendix 46: Experimental conditions to investigate neuropeptidases for NPY hydrolysis. [Inibitor] [Enzyme] Assay buffer pH Enzyme activation DP 4 PD31 1 µM 1 µg/ml 20 mM Tris/HCl 7.6 - Neprilysin Phosphoramidon 10 – 200 µM 1 µg/ml 20 mM Tris/HCl 7.6 - Meprin A Actinonin 200 µM 0.4 µg/ml – 1µg/ml 20 mM Tris/HCl 7.6 Trypsin, Pl, uPA, hK1 Meprin B Actinonin 200 µM 1 µg/ml 20 mM Tris/HCl 7.6 Trypsin, hK1 20 mM Tris/HCl 7.6 Cathepsin D Pepstatin A 50 – 200 µM 1 µg/ml - 10 mM Mes 5.5 Cathepsin B E64 50 µM 1 µg/ml 10 mM Mes 5.5 - Cathepsin G DIC 1 mM 1 µg/ml 20 mM Tris/HCl 7.6 - Cathepsin L E64 50 µM 1 µg/ml 10 mM Mes 5.5 - Cathepsin S E64 50 µM 1 µg/ml 20 mM NaAcetate 6.0 - Neurolysin Pro-Ile 1 mM - 20 mM Tris/HCl 7.6 - AEBSF 1 mM Trypsin 1 µg/ml 20 mM Tris/HCl 7.6 - soyabean trypsin inhibitor 4 mg/ml

Plasmin (Pl) 2-antiplasmin 0.2 µg/ml 5 µg/ml 20 mM Tris/HCl 7.6 - Urokinase (uPA) Amiloride 1 mM 7.5 µg/ml 20 mM Tris/HCl 7.6 - Thrombin Hirudin 40 µg/ml 10 µg/ml 20 mM Tris/HCl 7.6 - Plasma Kallikrein (Kal) PKI-527 200 µM 1 µg/ml 20 mM Tris/HCl 7.6 - Tissue Kallikrein (hK1) Gabexate mesylate 200 µM 1 µg/ml 20 mM Tris/HCl 7.6 -

Appendix 47: Activation of Mep-A and Mep-B by trypsin, plasmin, uPA and hKlk1.

Activation buffer 50 mM Tris-HCl, 10 mM CaCl2, 0.15 M NaCl, 0.05% Brij, pH 7,5. Enzyme Mep A Mep-B Activating Enzyme Trypsin Plasmin uPA hK1 Trypsin hK1 [Activating Enzyme] 0.1 µg/ml 0.1 µg/ml 0.1 µg/ml 0.1 µg/ml 0.1 µg/ml 0.1 µg/ml Incubtation time (h) 1 h 3 h 5 h 3 h 30 min 3 h

Stopping inhibitor AEBSF/sbTI 2-antiplasmin Amiloride Gabexate mesylate AEBSF/sbTI Gabexate mesylate [Stopping inhibitor] 1 mM/2 mg/ml 0.2 µg/ml 200 µM 1 mM 1 mM/2 mg/ml 1 mM

Appendix 48: Composition of improved 10 x conservation solution for capillary depletion. Reagent Weigh off Dissolve in + add Final Concentration

Aprotinin 0.8 ± 0.1 mg 1 ml H2O + 200 Units/ml

Sodium azide 4 mg 1 ml H2O + 200 µg/ml

Glutathion 1 g 7 ml H2O + 50 mg/ml Streptomycinsulphate 4 mg 1 ml H2O + 200 µg/ml EDTA Na4 1.214 2 ml H2O + 1 mM Actinonin 3.5 mg 1 ml H2O + 175 µg/ml Pepstatin A 3 mg 1 ml H2O + 150 µg/ml E64 1.5 mg 1 ml H2O + 75 µg/ml

Phosphoramidon 1 mg 1 ml H2O + 50 µg/ml Val-Pyrr 10 mM add 2 ml + 500 µM Dithiothreitol 4 mg 500 µl EtOH + 200 µg/ ml PMSF 10 mg 500 µl EtOH + 500 µg/ml Chloramphenicol 40 mg 1 ml EtOH + 2 mg/ml Saccharose 1.2 g Add to final solution 60 mg/ml Final Volume 20 ml vortex solution vigorously

157 Appendix

Appendix 49: Cruor diluted 200-1,000,000-fold with PBS to assess haemolysis on AMC Standard curves.

y = 3514,3x y = 2987,9x y = 3216,8x y = 3221x y = 3237,9x R² = 0,9992 R² = 0,9992 R² = 0,9981 R² = 0,9982 R2 = 0,9968 y = 3334,1x y = 3389,6x R² = 0,9965 y = 3295,4x y = 3348,3x y = 3344,1x y = 3334,5x R2 = 0,9995 R² = 0,999 R2 = 0,9998 R2 = 0,9972 R² = 0,9991

40000 Serum 1x

35000 Haemolysis 200x

30000 Haemolysis 500x 25000 Haemolysis 1000x 20000 Haemolysis 1500x Haemolysis 2000x 15000 PBS 10000 RFU RFU (380nm/460nm) Haemolysis 20 000x

5000 Haemolysis 200 000 0 Haemolysis 500 000 0 2 4 6 8 10 Haemolysis 1 000 000 [AMC] (µM)

Appendix 50: Influence of haemolysis on the slopes of the various AMC standard curves. Dilution Cruor Abs 405 nm Abs 405nm – Abs 690nm Slope (RFU/µM) Conversion factor PBS 0.053-0.112 0-0.001 3514 0.00384 Serum 0.1755 – 0.199 0.0125 - 0.035 3334 0.00405 200 0.411 -0.0.447 0.2285 -0.255 2988 0.00452 500 0.2875-0.3135 0.0975-0.1255 3217 0.00420 Limit 1,000 0.2385-0.2755 0.065 -0.0885 3221 0.0419 1,500 0.213 -0.26 0.0445 -0.081 3238 0.00416 2,000 0.24 -0.268 0.0525-0.0715 3295 0.00410 20,000 0.194 -0.2385 0.0275-0.0435 3348 0.00403 200,000 0.194- 0.223 0.0165-0.0405 3344 0.00404 500,000 0.182 -0.219 0 - 0.0065 3335 0.00405 1,000,000 0.1805-0.2185 0 - 0.002 3390 0.00398

Appendix 51: Summary of stress-related neuropeptides and the corresponding matrices.

Neuropeptide N-terminus Xaa-Pron-Xaa # aa Matrix

Neuropeptide Y (NPY) Tyr-Pro… P2, P5, P8, P13 36 Sinapinic acid Galanin-like peptide (GALP) Ala-Pro… P2, P21, P26, P52, P56, P57, P59 60 Sinapinic acid Corticotropic-Releasing Factor (CRF) Ser-Glu… P4, P5 41 Sinapinic acid Adrenocorticotropic hormone (ACTH) Ser-Tyr… P12, P19, P24, P36 39 Sinapinic acid Orexin A (OrxA) pGlu-Pro… P2, P4 32 Sinapinic acid Human Orexin B (hOrxB) Arg-Pro… P4, P5 28 -cyano-4-hydroxy-cinnamic acid Rat Orexin B (rOrxB) Arg-Ser… P2, P4, P5 28 -cyano-4-hydroxy-cinnamic acid Galanin (Gal) Gly-Trp… P13 30 -cyano-4-hydroxy-cinnamic acid -Endorphin (-Endo) Tyr-Gly... P13 31 -cyano-4-hydroxy-cinnamic acid Substance P (SP) Arg-Pro… P2, P4 11 -cyano-4-hydroxy-cinnamic acid Neurotensin (NT) pGlu-Leu… P7, P10 16 -cyano-4-hydroxy-cinnamic acid Oxytocin (Oxy) Cys-Tyr… P7 9 -cyano-4-hydroxy-cinnamic acid Arg-Vasopressin (AVP) Cys-Tyr… P7 9 -cyano-4-hydroxy-cinnamic acid

Appendix 52: Protocol for desalting by C18 ZipTips (Millipore). 1. Wetting ZipTip: Aspirate 3 x 10 µl with 50 % ACN, dispensing ACN into waste. 2. Equilibration: Aspirate 3 x 10 µl with 0.1 % TFA, dispensing TFA into waste. 3. Sampling: Aspirate and dispense 10 x 10 µl of sample. 4. Washing: Aspirate 3 x 10 µl with 0.1 % TFA, dispensing TFA into waste. 5. Elution: Aspirate and dispense 10 x 5 µl with 50 % ACN. 6. Matrix: Add 5 µl matrix to desalted sample

158 Appendix

Appendix 53: Summary of ex vivo hydrolysis of neuropeptides by human blood

Reagents Assay t0 = 0 Blank (Substrate) Blank (Blood)

20 mM Tris/HCl, pH 7.6, 0.353 M Sucrose 204 µl 8.5 µl 8.5 µl 8.5 µl

Blood 24µl 1 µl - 1 µl fold fold

- 1 mM Substrate (NPY, SP, GALP) 12 µl 0.5 µl 0.5 µl -

10 H2O - - - 0.5 µl Dilution Dilution of Blood of 20 mM Tris/HCl, pH 7.6 - - 1 µl - 20 mM Tris/HCl, pH 7.6, 0.4 M Sucrose 180 µl 7.5 µl 7.5 µl 7.5 µl

Blood 48 µl 2 µl - 2 µl 1 mM Substrate

fold fold 12 µl 0.5 µl 0.5 µl -

- (NPY, SP, GALP)

5 Blood

H2O - - - 0.5 µl Dilution of Dilution 20 mM Tris/HCl, pH 7.6 - - 2 µl -

Appendix 54: Summary of major episode of clinical depression§. Definition Symptoms Classification Treatment Depression Despondence Non-melancholic Depression: Current: = lat. deprimere No interests Reactive Depression Antidepressants: “feeling down“ Sadness, anxiety, Cause: Reuptake-Inhibitors Diagnosis: Passiveness Traumatic Event and/or Chronic Stress (SSRIs,SNRIs, NRIs, NDRIs)* ICD10 No appetite Melancholic Depression: Tricyclics DSM IV Insomnia Endogeneous Depression MAOIs# Psychomotoric Disorders Cause: Genetic Novel pharmacological targets: Physical/mental exhaustion Inflammation Feelings of guilt (IL-6, IL-1β, TNF-α)  Concentrations/Decisions Neuropeptides  Self-confidence (NPY, SP, CRF, Gal, Oxy, AVP, Suicide/suicidal thoughts Nt, OrxA, OrxB, ACTH) § Taken from (2010): http://en.wikipedia.org/wiki/Major_depressive_disorder; ICD10, F32, F33; http://omim.org/entry/608516; Holmes et al., 2003; Rotzinger et al., 2010; Maes et al., 2009 (721,722,883); * SSRI, selective serotonine reuptake inhibitors; SNRIs, serotonine-norepinephrine reuptake inhibitors; NRIs, norepinephrine-reupake inhibitors; NDRIs, norepinephrine- dopamine-reuptake inhibitors ; # MAOI, Monamine Oxidase Inhibitors.

Appendix 55: sCD26 standard curve of ELISA to compute the protein concentrations of serum DP 4.

8.3 Results 8.3.1 Isolation of DP 4 Isoforms from Porcine Kidneys for Crystallisation Appendix 56: Summary of potential glycosylation sites of different mammalian DP 4’s.

Potential glycosylation sites Species # N-Glyc.* aa 1-200 aa 201-400 aa 401-766 Mouse 9 83 90 113 213 223 315 328 514 679 Rat 8 83 90 148 237 247 319 521 686 Cat 11 84 91 149 178 228 280 320 330 331 519 685 Cattle 11 84 91 149 219 228 271 280 320 392 495 684 Pig 10 85 92 150 218 229 263 279 321 393 684 Human 9 85 92 150 219 229 281 321 520 684 *, number of potential N-glycosylation 159 Appendix

Appendix 57: Chronological presentation of DP 4 crystal structures from 2002-2012

25

20

15

10

5 # # 4DP Crystal Structures

0 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012

8.3.2 Results of RT-PCR from Porcine and Canine Tissues Appendix 58: Results of nested RT-PCR of DP 4-like enzymes obtained from porcine and canine kidney tissue. Kidney tissue was divided into kidney cortex, outer and inner stripe of outer medulla (131;578).

160 Appendix

Appendix 59: Results of nested RT-PCR of DP 4-like enzymes obtained from porcine and canine lung tissue. Lung tissue was divided into bronchial epithelium and alveolar tissue (131;578).

Appendix 60: Results of nested RT-PCR of DP 4-like enzymes obtained from porcine and canine small intestine, divided into duodenum, jejunum and ileum (131).

161 Appendix

Appendix 61: Alignment of porcine and canine nested RT-PCR product of FAP (131). 301 350 FAP human (301) TCATAACTCTGAAGAAAATACAATGAGAGCACTCACACTGAAGGATATTT FAP dog (1) ------GAGGANGTACA-TGAGAGCACTCACACTGNAGGATATTT FAP pig (1) ------CTCTGAAG-AAATACAATGAGAGCACTCACACTGAAGGATATTT Consensus (301) CTCTGAAG AAATACAATGAGAGCACTCACACTGAAGGATATTT 351 400 FAP human (351) TAAATGGAACATTTTCTTATAAAACATTTTTTCCAAACTGGATTTCAGGA FAP dog (39) TAAATGGNACATTTNCNTATAAAACATTTTTTCCAAACTGGATTTCAGGA FAP pig (44) TAAATGGAACATTTTCTTATAAAACATTTTTTCCAAACTGGATTTCAGGA Consensus (351) TAAATGGAACATTTTCTTATAAAACATTTTTTCCAAACTGGATTTCAGGA 401 450 FAP human (401) CAAGAATATCTTCATCAATCTGCAGATAACAATATAGTACTTTATAATAT FAP dog (89) CAAGAATATCTTCATCAATCTGCAGATAACNATATAGTACTTTANAATAT FAP pig (94) CAAGAATATCTTCATCAATCTGCAGATAACAATATAGTACTTTATAATAT Consensus (401) CAAGAATATCTTCATCAATCTGCAGATAACAATATAGTACTTTATAATAT 451 500 FAP human (451) TGAAACAGGACAATCATATACCATTTTGAGTAATAGAACCATGAAAAGTG FAP dog (139) TGAAACAGGACAATCATATACCATTTTGAGTAATGCNACCATGAAAAGTG FAP pig (144) TGAAACAGGACAATCATATACCATTTTGAGTAATAGAACCATGAAAAGTG Consensus (451) TGAAACAGGACAATCATATACCATTTTGAGTAATAGAACCATGAAAAGTG 501 550 FAP human (501) TGAATGCTTCAAATTACGGCTTATCACCTGATCGGCAATTTGTATATCTA FAP dog (189) TGAATGCTTCAAATTACGGCTTATCACCTGATCGGGAATANNNNNNNNNN FAP pig (194) TGAATGCTTCAAATTACGGCTTATCACCTGATCGGGAATANNNNNNNNNN Consensus (501) TGAATGCTTCAAATTACGGCTTATCACCTGATCGGGAATANNNNNNNNNN

Appendix 62: Percentage of homology of the nested RT-PCR products with their respective templates. Enzyme Porcine Canine DP 4 100* 88.3 DP 8 98.7 98.7 DP 9 99.1 98.2 FAP 98.3 91.1 DP 2 97.9 85.5 *Based on porcine template

8.3.3 DP 4-like Activities in various Porcine and Caninine Tissues Appendix 63: % Remaining Activity at pH 5.5 after inhibition with Dab-Pip, a DP 2 selective inhibitor. A, % remaining activity at pH 5.5 after inhibition with Dab-Pip, using porcine tissues extracts. B, % remaining activity at pH 5.5 after inhibition with DAP, using canine tissues extracts (131).

A

B

162 Appendix

Appendix 64: Results of nested RT-PCR of DP 4-like enzymes obtained from porcine and canine pancreas. Pancreatic tissue was divided into body, head and tail (131;661).

Appendix 65: Residual DP 4 activity found in BSA (RIA grade) . A, Michaleis-Menten curves, , 0.2% BSA; , 0.1% BSA; , without BSA; n = 3. B, Correlation between concentration of BSA and DP 4 activity, r² = 0.999, n = 3, error bars represent SEM.

A B

Appendix 66: Inhibition of DP 4 by argine and sodium nitroprusside. A, IC50 of DP 4 inhibition by arginine. Graph represents inhibition with 0.033 mM Gly-Pro-AMC (), resulting in an IC50 of 7.13 mM, n = 3. B, Dixon-plot of DP 4 inhibition by sodium nitroprusside. , 0.025 mM Gly-Pro-AMC; ▼, 0.033 mM; ▲, 0.05 mM Gly-Pro-AMC; , 0.1 mM Gly-Pro-AMC substrate. KI = 8.48 µM, n = 3, error bars represent SEM

A B

10

8

6 Activity

4 (Units/l/h) 2

0 2 1 101 10 [Arginine] (mM) 163 Appendix

8.3.4 DP 4 and NPY as a Pharmaceutical Target for Psychiatric Diseases Appendix 67: Specific Actvities of DP 4-like enzymes in brain areas corresponding to stress and neuronal circuits of NPY (443).

164 Appendix

Appendix 68: Preliminary results of Capillary Depletion. A, fluorescein (perfusion control) in endothelial capillaries (Cap.), brain parenchyma (BP) and perfusate (P) fractions; B, Chlorpromazine, BBB control in the various fractions of the capillary depletion; C, activity of alkaline phosphatase, an endothelial marker, in the various fractions of the capillary depletion; D, specific activity of DP 4 in the various fractions of the capillary depletion. Specifi activity of DP 4 = Total specific activity – specific activity of selectively DP 4- inhibition.

165 Appendix

Appendix 69: Time dependent degradation of NPY by the trypsin-like enzymes trypsin, plasmin (Pl), urokinase (uPA), thrombin, plasma kallikrein (Kal) and tissue kallikrein (hK1) in presence or absence of selective inhibitors, using MALDI-TOF-MS analysis. A, 1 µg/ml trypsin, inhibited by 4 mg/ml trypsin inhibitor; B, 5 µg/ml plasmin, inhibited by 20 µg/ml 2-antiplasmin; C, 7.5 µg/ml uPA, inhibited by 200 µM amiloride; D, 10 µg/ml thrombin, inhibited by 40 µg/ml hirudin. E, 1 µg/ml activated plasma kallikrein, inhibited by 200 µg/ml PKSI-527. F, 1 µg/ml activated tissue kallikrein, inhibited by 1 mM µg/ml Gabexate mesylate. A-D, was analysed using -cyano-4-hydroxycinnamic acid as matrix, in E + F, metabolites were measured with sinapinic acid.

166 Appendix

Appendix 70: NPY hydrolysis by Mep-A, previously activated by uPA and Pl, applying MALDI- TOF-MS. A, NPY hydrolysis by 7.5 µg/ml Mep-A, previously activated by 0.1 µg/ml uPA for 5 h and stopped with 180 µM amiloride. B, NPY hydrolysis by 5 µg/ml Mep-A,

previously activated by 0.1 µg/ml Pl for 3 h and stopped with 0.2 µg/ml 2-antiplasmin. Mep-A specific cleavage was inhibited by 200 µM actinonin. C, NPY hydrolysis of 0.5 µg/ml MepA, previously activated with 0.1 µg/ml hklk1 for 3 h and stopped with 1 mM Gabexate mesylate. D, NPY hydrolysis of 0.5 µg/ml MepB, previously activated with 0.1 µg/ml hklk1 for 3 h and stopped with 1 mM Gabexate mesylate.

Appendix 71: Summary of stability of neuropeptides in rat and human serum, involved in stress-related disorders.

Type of proteolysis Serum ex vivo blood DP 4 truncation NPY, GALP, rOrxB, SP NPY, GALP, SP PEP-hydrolysis Gal Gal N-terminal truncation hOrxB Endoproteolytical degradation -Endo, SP, PACAP, NT, GALP, SP, GALP, Gal rOrxB, hOrxB C-terminal truncation AVP, ACTH, AngI, NPY NPY Stable OrxA, Oxy, CRF

167 Appendix

Appendix 72: Hydrolysis of Galanin (A), human Orexin B (C) and neurotensin (D) by human recombinant PEP as well as Galanin by human recombinant FAP (B).

A C

B D

8.3.5 Characterisation of NPY Hydrolysis in Sera from Depressed Patients Appendix 73: Time dependent hydrolysis of neuropeptide Y (NPY) in sera of depressed patients and control subjects, using MALDI-TOF-MS. A-C, time-dependent hydrolysis of NPY in sera obtained from patients diagnosed with non-melancholic depression. A, before treatment; B, after treatment; C, healthy control subject matching with respect to age and sex. D-F, time- dependent hydrolysis of NPY in sera obtained from patients diagnosed with melancholic depression. D, before treatment; E, after treatment; F, healthy control subject matching with respect to age and sex. Matrix, sinapic acid; NPY1-36, untruncated NPY; NPY3-36, DP 4- truncated NPY; NPY2-36, Amp P-cleaved NPY; NPY1-34, ACE-like cleavage of NPY; NPY1-30, cathepsin D-truncated NPY. Range of mass (X-axis): 3300-4400 Da.

168 Appendix

8.4 Conclusion 8.4.1 DP 4- and DP 4-like Inhibitors Appendix 74: Summary of DP 4 inhibitors with their chemical structure, mechanism of action and Ki currently implemented in vivo as drug targets.

Inhibitor Type of inhibition Ki Molecular Structure Disease Ref

F F NH O MK-0431 2 N Sitagliptin 18 nM F N (774;776)  Competitive N Diabetes Januvia (Merck) (IC50) N (777) FDA admission F F F

BMS-477118 N Mixed type, N Saxagliptin 0.6 nM substrate analogue, O Diabetes (895-897) Onglyza (BMS) (IC ) slow tight binding 50 FDA admission (2006) NH2 HO NVP-LAF-237 HO Vildagliptin H 3.5 nM N O CN Galvus(Novartis) Competitive Diabetes (898;899) (IC ) EMEA admission (2007) 50 N FDA pending SYR-322 O Alogliptin N ® Nesina H2N Competitive 6.9 nM N N O Diabetes (617) (Takeda Pharamceuticals) FDA application

Admission in Japan (2009) N BI 1356

Linagliptin O ® Ondero 4 nM N N Slow tight binding N Diabetes (900-903) (Boehringer Ingelheim) (IC50) N N FDA admission (2011) O N N

NH2

O NH2 O F F F LC-15-0444 N Gemigliptin 16 nM F N F (409) Competitive N Diabetes LG-Life Sciences (IC50) (904;905) KFDA admission (2011) F F F

F Denagliptin N (GSK) Competitive 22 nM O Diabetes (906) Phase III N F H2N

HO PHX1149T OH Competitive, O B Dutogliptin 25 nM H transition state analogue, N Diabetes (907-909) (Phenomix Corporation) (IC ) HN N slow tight binding 50 Phase III N

GRC-8200 O H Melogliptin Competitive 1.6 nM N N Diabetes (910;911) Phase III N N N F

H2N O R1579 Carmegliptin H N Competitive 9.3 nM O Diabetes (912) Roche N F

Phase II O

NH F 2 2,4 difuorobenzene- O Competitive, 88 nM O sulfonamide-4-amino- S Diabetes (913) product analogue (IC50) N N cyclohexylglycine pyrrolidine F O H

NO 2 (Z)-fluoroolefin N-peptidyl-O- Competitive, H N (4-nitro-benzoyl)hydroxyl- 0.16 µM 2 Diabetes (914;915) mechanism unknown O amine F N O H O

169 Appendix

P32/98 NH S Competitive, 2 Diabetes Isoleucinylthiazolidine 0.1 µM N (916) product analogue Anxiety O Diabetes Competitive, Valylpyrrolidine 48 µM N Bone marrow (906) product analogue H2N O transplantation Mixed type,

substrate analogue, 3.8 nM N Diabetes (917) CN FE 999011 H N slow tight binding 2 O O

OEt N H O Irreversible, leading to a N O AB207 phosphorylated serine at 6 nM P O Diabetes (732) N O O the active site H OEt

NH O O NH2

NH2 N R 1438 O (740;918- reversible 0.1 nM N Diabetes (Roche) 922) Cl Cl O NVP-DPP728 Mixed type, NC CN substrate analogue, 22 nM N N (Novartis) H Diabetes (923-925) (IC ) N slow tight binding 50 N O discontinued H

H3C CH3 SSR-16239 O N (Sanofi Aventis Slow tight binding 2 nM N Diabetes (732) H Deutschland GMBH) O O N N H NH

CH3 O O 0.32 µM O SDZ 029-576 Mixed type Diabetes (926) (IC50) O NH2

BDMX O Competitive 0.55 µM N N Diabetes (739;927) (Novo Nordisc) N NH O N N L-Val-LboroPro Competitive, (126;144) PT-100 Cancer transition state analogue, 2 nM N (311;400) Talabostat (Point Therapeutics) H2N AIDS slow tight binding O B (928;929) Phase II-III, discontinued HO OH Competitive, Autoimmune L-Pro-L-boroPro transition state analogue, 16 pM N AIDS (930-935) B OH slow tight binding N O HO Cancer H

O OH OH HO OH

TMC-2A Uncompetitive 5.3 µM OH Arthritis (323;418) N N N O H H H N O O 2

TSL-225 Uncompetitive 3.6 µM N Arthritis (323) N

H2N OH O O

O NH Pro-Pro--diphenyl Irreversible, leading to a t½ = 5 h phosphonate phosphorylated serine at kinact/Kd = Graft rejection (936;937) O O O O P H (Prodipine) the active site 3000 N N O N H

NO 2 Autoimmune (266) Competitive, Multiple 4.01 µM N O (413;416) Lys[Z(NO )]-pyrrolidine product analogue sklerosis 2 O N NH Psoriaris H 2

C N 30 nM Psychosis (419) AMAC Competitive (IC50) O Schizophrenia (938;939)

NH2

170 Appendix

Appendix 75: Involvement of DP 4 /CD26 in TH1- and TH2- response (197;199;200;207;258;262)

TH1 TH2 High expression on CD26bright memory T-cell subset Binding of Caveolin-1 on dentritic APC cells to CD26 on TH1 cells, results in binding of Carma-1 cytoplasmic tail of CD26  CD26 Expression Slight upregulation of CD26 expression recruitment of CARMA-1, CD26, Bcl10 and IkappaB kinase complex to lipid rafts  signal transduction

 upregulation of CD26 expression upon induction of TH1 response Association of CD26 to lipid rafts Binding of CD26 to M6P/IGFIIR  Sub-cellular + Binding of CD26 to CD45R0 on lipid rafts, resulting in signal internalisation + T-cell activation compartmentation transduction; followed by disassembly of CD45R0 from lipid rafts Association of CD26 + CXCR4  internalisation

CXCR4  SDF-11-68 > SDF-13-68 CXCR3  IP-10 CCR3  eotaxin > eotaxin Chemokine 1-74 3-74 CCR1  RANTES > RANTES DP 4 substrates 1-68 3-68 CCR4  MDC1-67  MDC3-67 CCR5  RANTES3-68 > RANTES1-68 CCR1  RANTES1-68 > RANTES3-68 CCR3  RANTES3-68 > RANTES1-68 Truncation of SDF-1α by DP 4 Diseases Rheumatoid Arthritis associated with Multiple Sclerosis  protection against HIV entry CD26+ T cells involved in the pathogenesis of DP 4/CD26 Truncation of RANTES by DP 4   protection against HIV entry asthma correlating to IgE titre of antigen

Appendix 76:Involvement of DP 4-like enzymes in T-cell effector response. A, naïve CD4 T-cells are protected by DP 2. Antigen Presenting Cell (APC) stimulate and activate Naïve CD4 T-cell. B, secretion of IL-2 results in clonal expansion, yielding TH0-cell. Enzymatic activities of DP 8 and DP 9 are required for this process. C, secretion of IL-12 and IL-4 results in the differentiation of TH1-and TH2-effector cells, respectively. Differentiation into TH1-cells initiates the upregulation of bright DP 4 expression. A small subset of CD26 memory T-cells already expresses high amounts bright of CD26. Upon antigen stimulation, CD26 memory T-cells augment the TH1-and TH2- response by secreting IFN-γ, IL-12 and IL-4, IL-5, IL-10, respectively. Differentiation into TH2- cells results only into a slight upregulation of CD26. The DP 4 activity is equal in both T-effector cells, probably due to specific DP 4 isoforms or DP 8 and/or DP 9. D, differentiated T-effector cells secrete specific cytokines that induce differentiation of leucocytes, resulting in cellular response by TH1 and humoral by TH2-effector cells. Leukocytes expressing DP 4, DP 2 or DP 8/9 are indicated. Green arrow, stimulation, red arrow suppression. [Taken and adapted by Boonacker et al., 2003; Kuby, 1997; Ohnuma et al., 2004, Geiss-Friedlander et al., 2009, Vora et el., 2009; Lankas et al., 2005; Maes et al., 2007; Reinhold et al., 2009; Yazbeck et al., 2009 and Janeway et al., 2004 (132;140;142;143;190;200;255;309;940-942)].

171 Appendix

Appendix 77: Summary of the selectivity of DP 4 and DP 2 inhibitors. Inhibitor DP 4 DP 2 Ref.

142,000-fold higher selectivity over DP 2, (898) LAF-237 IC50 > 500,000 nM (IC50 = 3.5 nM) (943) 5,000-fold higher selectivity over DP 2 (924) NVP-DPP728 IC50 = 110,000 nM

(IC50 = 22 nM) (924)

Aminoacylpyrrolidine-2-nitriles High selectivity (IC50 = 0.2 µM) Ki = 110 µM (944) DP4 4-amino cyclohexylglycine 100 - 5,800-fold selectivity over IC50 : 8,600 – 15,000 nM (913) analogues DP 2 (IC50 : 2.6 - 88 nM) 100 - 250-fold selectivity over DP 2 Highly selective for selective Highly Fluoropyrrolidine amides IC50 : 660 - 25,000 nM (945) (IC50 : 48 - 340 mM)

Aminoacyl pyrrolidides 116-fold selectivity over DP 2 (Ki = 0.218 µM) Ki = 24.7 µM (591)

65-fold selectivity over DP 2 Aminoacyl thiazolidides Ki = 8.17 µM (591)

(Ki = 0.126 µM) Slightly Slightly

for DP 4 for 63-fold selectivity over DP 2 selective selective L-Pro-L-boroPro Ki = 125 µM (591)

(Ki = 2 nM)

33 fold higher potency (936) Pro-Pro-diphenyl IC > 125 µM over DP 4 (937) phosphonate esters 50

(IC = 3.8 µM) for DP 2 DP for

Slightly Slightly 50

selective

7,700 fold higher potency over (591) 2,4- Dab-Piperidinamide IC50 > 1 mM

DP 4 (IC50 = 130 nM) (946)

Epostatin O

H NH2 N O No inhibition of DP 4 K 1.44 µM (947) for DP 2 DP for i OH O O N

Highly selective Highly H

Appendix 78: Selective and non – selective inhibitors of DP 4 -like enzymes.

IC50 (KI) Inhibitor Ref DP 4 FAP DP 8 DP 9 DP 2 2-(799)}-amino)-methoxyl]- phenoxy}-3-methyl-butyric acid F CF COO- 3 O O COOH 0.48 nM 21 µM >100 µM 86 µM >100 µM (775) O N

NH + 3 N F MK-0431 (Sitagliptin) 18 nM >100 µM 48 µM >100 µM >100 µM (776) NVP-LAF-237 (Vildagliptin) 3 nM > 100 µM 810 nM 95 nM - (145) BI1356 (Linagliptin) 4 nM 89 nM 40 µM 10 µM > 100 µM (948) DP 4 selective DP4 AB207 14 nM 530 nM > 100 µM (732) Alogliptin 6.9 nM > 100 µM > 100 µM > 100 µM > 100 µM (949) Saxagliptin 1.3 nM > 5.2 µM 520 nM 97.5 nM > 5.2 µM (147) LC15-0444 16 nM 169 µM 47 µM > 400 µM (950) (S)-cyclohexylglycine-isoquinoline

15 µM - 24 nM - 16.5 µM (761)

N H2N O Cyclohexylglycine-(2S)- cyanopyrolidine 12 nM - 27 nM - 40 µM (761)

N H2N

O CN Ile-isoindoline O N (777) 30 µM >100 µM 38 nM 55 nM 14 µM (142) NH2

DP 8 /DP 9 selective /DP DP9 8 1-(4,4’-difluor-benzhydryl)- piperazin-2- F isoindoline >100 µM - 14 nM - 38.4 µM (761)

N N N F O

O NH2

Epostatin

none 1.4 µM (947)

DP 2 DP2 selective

172 Appendix

2,4-diaminobutanoyl-piperidine (DAB)-Pip NH2 (591;946) > 1 mM 130 nM

N H2N

O UAMC00039

Cl N 165 µM 142 µM 0.48 nM (759;951) H N O

NH2 1-Benzyl-N-[2-oxo-2-(1- piperidinyl)ethyl]-4-piperidinamine

N > 1 mM 3.1 µM (951)

N N H O

1-Benzyl-N-(2cyclohexylidine-2- fluoroethyl)-4-piperidinamine

N > 250 µM 3.8 µM (951)

N H F 2,4-diaminobutanoyl-boro-

DP 2 selective norleucine NH2 > 33 µM none 4.3 µM 800 nM 0.48 nM (952)

H2N

O B HO OH N-cyclo-pentylglycyl- diphenylphosphonate

H N > 125 µM 3.8 µM (937) O N H O P O O

2-amino-2-[1-(4-iodo-benzene sulfonyl)-pyrrolidin-3-yl]-1- thiazolindin-3-yl-ethane O 1.9 µM >100 µM 11 µM 22 µM 19 nM (142) O S N O N NH I 2 S N-acetyl-Gly-boroPro O N 377 nM 23 nM 19.1 µM 8.8 µM 125 µM (97;953) N H B O OH HO N-acetyl-Gly-Pro-chloromethyl- O -1 ketone k2 = 0.017 min N > 500 µM (96) N H Cl KI = 156 µM O O N-(1-naphtalencarboxyl)-Gly- boroPro O 27 µM 2.17 nM (930) N N H

B O OH HO N-cycloheptyl-Gly-boroPro

N 7.8 nM 150 nM 1.8 nM (954) N H B O OH HO

FAP selectiveve FAP N-(1-adamantyl)-Gly-boroPro

N 8.0 nM 46 nM 420 nM (954) N H B O OH HO N-(2-adamantyl)-Gly-boroPro

N 12 nM 99 nM 120 nM (954) B OH N H O HO N-cyclohexyl-Val-boroPro

N 16 µM - - (954) N H B O OH HO

173 Appendix

4-amino cyclohexylglycine 7.2 nM >100 µM 0.45 µM 4.1 µM >100 µM (775;913) analogue

threo-Ile-Thia (P32/98) 0.42 µM > 100 µM 2.18 µM 1.6 µM 14 µM (141;142) allo-Ile-Thia 0.46 µM > 100 µM 0.22 µM 0.32 µM 18 µM (141;142)

Val-L-boroPro (Talabostat) > 4 nM 560 nM 4 nM 11 nM 310 nM (141;142)

selective -

Lys[Z(NO2)]-pyrrolidine 1.3 µM 51 µM 0.154 µM 1.65 µM 1.21 µM (141;142) Non Lys[Z(NO2)]-thiazolidide 0.401 µM 33 µM 0.21 µM 75 nM 0.21 µM (141;142)

Lys[Z(NO2)]-piperidide 17 µM >100 µM 1.1 µM 0.67 µM 0.71 µM (141;142)

8.4.2 Characterisation of DP 4-like Enzymes in Porcine Pancreas

Appendix 79: Influence of DP 4 on neuronal, endocrinal and paracrinal regulation of insulin secretion (2). , inactivation of peptide / neuropeptide by DP 4;  enhanced insulin secretion via via stimulation of peptide/ neuropeptide receptor; , binding of neuropeptide/peptide to its receptor results in decreased insulin secretion. [Taken and adapted from Lambeir et al., 2003 (2)]

Appendix 80: Localisation of DP 4 in granules of pancreatic -cells co-localising with glucagon (A, arrow) or DP 4 found in more lucent granules (B, bent arrow) compared to glucagon in more dense granules. [Taken and adapted from Poulsen et al., 1993 (A) and and Grondin et al., 1999 (B) (447;448)].

174 Appendix

Appendix 81: Summary of endocrine pancreatic cells, their hormone peptides and secretory granules. Cell % Immunochemistry Granule Infrastructure Type Frequency Hormone Peptide Size Range (nm) Morphology  15-20 Glucagon, Glicentin, GIP, Endorphin?, Corticotropin, Cholecytokinin 190-310  60-80 Insulin/Proinsulin 225-375  5-15 Somatostatin 190-370 pp ?-2 PP 110-170

1 2-5 VIP 140-190 S ? Sectretin 170-260 ? G ? Gastrin - ? P ? Bombesin? 100-140 EC Rare Biogenic aminopeptides variable pleomorphic

Appendix 82: Summary of endocytic and exocytic vescicles/granules. Organelle Size (nm) pH Marker Morphology/Function DP 4-like Enzyme Ref. Hormone/ DP 2 found to be distinct to Secretory vesicle 50-80 5.5 neuropeptide/ Electron-dense spherical vesicles (175;176) lysosomes in secretory vesicles (796;799;800) SNARE/rab3 Hormone/ DP 4 co-localise with glucagon in - (447;448) Secretory granules 80-350 5.5 neuropeptide/ Electron-dense spherical vesicles cells of pancreatic islets/granules (796;799;800) SNARE/ rab3 distinct of glucagon in -cells flask-shape; grape-like clusters; DP 4 in caveolae of synovocytes Caveolae 50-100 7.0-7.4 Caveolin-1 (255;262;798;817) rosettes Association of DP 4 to Caveolin-1 (804;809;955) Cholersterol; DP 4/CD26 in T-cells  recruitment (199;798;805) sphingolipids; of CD45 to lipid rafts; CD26 isoform Lipid rafts 10-200 - Flat, membrane associated (767;808;812- detergent dependent + involves IL-12; Signal 816;942;956-960) resistant transduction grape-like heteromorphic multi- DP 4 in caveolae/caveosomes of Caveosome 500 -2000 7.0-7.4 Caveolin-1 (798;801;802;806) caveolar complex synovocytes (808;810;811;818) Clathrin-dependent Spherical with internal Binding and subsequent internalisation 100-150 < 6.0 Clathrin, AP-1 (196;796;803;807) endocytosis membranes of DP 4 to CXCR4/SDF- complex (819-821) Early endosome Internalisation of DP 4 to be recycled 500-800 5.9 – 6.0 EEA1; rab4 Tubulo-vesicular, cisternal (75;796;803;819- /sorting endosome or transcytosed 823) Recycling Tubulo-vesicular (large, fast); Recycling of DP 4 for terminal 500-2000 6.4-6.5 rab11 (70-72;75;819-822) endosome oval/spherical (small, slow) glycosylation; (824-827) ESCRTII; Round/oval structures, containing Multivesicular body 200-1000 5 – 6.0 ? (819-821) ESCRTIII multiple vesicles (828-831;961;962) Transcytotic 100-150 < 6.0 (1) 1. Clathrin coated (100-150 nm) Cellular trafficking of DP 4 dependent rab17 (76;77;822;832) vesicles 50-100 7.0-7.4 (2) 2. flask-shape, grape-like on glycolysation (834;835;963) M6P-R; rab7; Complex structure with internal Association of CD26 with M6P-R/ (198;796;807) Late endosome 500-1000 5.6-6.0 (819-822;828- VAMP-7 membranes IGFIIR and subsequent internalisation 830;836;837) Lucent/electron-dense spherical DP 4 in lucent granules of -cells (447;448;839) Secretory lysosome 100-1200 5.0-5.5 Rab27a, CD63 distinct from glucagon; DP 4 in secretory (842-845;848;850) vesicles lysosomes of NK- and Tc-cells Variable: tubular or similar to late DP 2 suggested to be lysosomal (176;185;821;822) Lysosome 100-1250 4.5-5.5 LAMP-1/ rab9 (828-830;837- endosomes DP 4 purified from lysosomal membrane 845;961;962) Double membrane surrounding Autophagosome 500-1500 7.0-7.4 LC-3-II ? May involve DP 8/DP 9/PEP? (964;965) cytosolic organelle LAMP-2A / Variable morphology, double AAutophagolysosome 500-1500 5.0-5.5 ? (964;965) LC-3-II membrane

Appendix 83: Comparison of porcine and canine islets of Langerhans (672). Markers Pig Dog Three types of islets: (i) In the pancreatic head, the -cells are present Insulin (i) small islets with low numbers of -cells either as single cells or as clusters (-cells) (ii) large islets with -cells in the core of islets (ii) In the pancreatic tail, intra-insular -cells occupy (iii) large islets with -cells at the periphery the central part or the periphery. (i) In the pancreatic head occasionally single -cell Glucagon (i) In the pancreatic head, small number of -cell (ii) In the pancreatic tail -cell in the central part or (-cells) (ii) In the pancreatic tail, high number of -cell at the periphery Somatostatin (i) only few -cells, mostly at the periphery of islets or (i) -cells between acinar cells or intrainsular; more (-cells) between acinar cells numerous in the pancreatic tail Pancreatic (i) Approximately 90 % of pp-cells in the pancreatic (i) all pp-cells are found in the pancreatic head, as single polypeptide head as single cells or in clusters cells, clusters of cells or at the periphery of islets (pp-cells) (ii) Only 10 % of pp-cells in the pancreatic tail Microcirculation No vascular plexus, but corrosive cast Vascular plexus, flow:     -cells

175 Appendix

8.4.3 Identification of DP 4 as a Target for Psychiatric Disorders Appendix 84: Sub-cellular localisation of DP 4-like enzymes and NPY, indicating physiological accessibility of NPY (A) as well as catalytic efficiency (B) with respect to of NPY hydrolysis (94;95;123;127;135;179;210).

Appendix 85: The CVO’s median eminence (EM) and area postrema (AP) form the interphase between the neuronal, endocrinal and immunological system, involving the two stress axis hypothalamic-pituitary-adrenal (HPA) and the neuro-sympathico-axis (NSA). Activation of HPA and NSA results in the release of stress hormones, cytokines and NPY (A). Cortisol at the median eminence increases the expression of NPY and peripheral NPY enters the brain via the AP or EM (B) (562;571;868;874;966;967).

Appendix 86: Anatomy of median eminence (A) and distribution of its various tanycytes (B) (869;871). ArcM, medial nucleus arcuate; ArcL, lateral nucleus arcuate; MEE, outer zone of median eminence; MEI, inner zone of median eminence; 3V, third ventricle.

176 Appendix

Appendix 87: Differential properties of 1, 2, 1, 2-tanycytes at the median eminence (869).

1-Tanycyte 2-Tanycyte 1-Tanycyte 2-Tanycyte Facing the dorsomedial Facing the arcuate Lateral evaginations of Localisation of cell body Floor of infundibular recess and ventromedial nuclei nucleus infundibular recess Zone of projection of basal Dorsomedial and Lateral region of external Medial region of external Arcuate nucleus processes ventromedial nuclei zone of median eminence zone of median eminence Bridging arrangements CSF  hypothalamus CSF  hypothalamus CSF  portal blood CSF  portal blood Reaction to adrenalectomy No reaction No reaction  metabolic reactivity  metabolic reactivity Caveolin-1 Missing Missing Present Present Rab4 Missing Missing Missing Present Transcytosis of WGA from CSF Yes No Yes Yes -catenin At ventricular cell pole At ventricular cell pole Throughout tanycyte Throughout tanycyte -cadherin At ventricular cell pole At ventricular cell pole At ventricular cell pole Throughout tanycyte Zonula adherens At ventricular cell pole At ventricular cell pole Throughout the cell body Throughout the cell body Tight junctions Missing Between basal processes Between basal processes At ventricular cell pole Permeability HRP injected to CSF Yes Yes Yes No At the preterminal region of Innervation Missing Missing Throughout basal process basal process Neurogenic Properties Not evident Evident Not evident Not evident

Appendix 88: Summary of BBB, fenestrated capillaries, tight junctions and cells at the nucleus tractus solitary (NTS) and area postrema (AP) (878). -, none; +/-, few; +, present; ++, strongly expressed; n.d., not determined. Area Postrema Marker Function NTS Ref. mantle zone central zone funiculus separans Claudin Tight junction + - - + (878) ZO-1 Tight junction + + - + (878) Laminin Basal lamina + ++ ++ ++ (878) RECA1 EC + + + + (878) EBA EC BBB + - - - (878) Glut1 EC BBB + + - ++ (878) Transferrin-R EC CNS +/- +/- - +/- (878) SMA Smooth muscle +/- +/- +/- +/- (878) NG2 Pericytes + + + + (878) GFAP Astrocytes + + - + (878) DARPP-32 Tanycytes - + + + (878) CD11b Microglia + + + + (878) NPY Peptideric neurons + - - - (878) -MSH POMC +/- +/- +/- +/- (878) Caveolin-1 Transcytosis via caveolae n.d. + (880) Caveolin-2 Transcytosis via caveolae n.d. + (880) P-glycoprotein Transport protein n.d. + (880)

Y1-R, Y2-R, Y4-R +/- ++ (571) Receptors AT1A, V1, ET A, CL-RAMP3, CCK-1, GLP-1R, (565;879) ? + GHSR, Adipo R1/R2, IL-1R1, Ox-R, NK1-R (968)

Appendix 89: Compartmentation of area postrema (AP) and its projections. A, Schematic diagram of a coronal section through the rat brainstem, showing the AP and surrounding neuronal tissue. The AP can be divided into several zones, which differ in the predominant cell type (neurons vs. tanycytes) and their respective projections. The funiculus separans represents a layer of tanycytes that function much like the blood-brain barrier separating the AP from the underlying nucleus tractus solitary (NTS). GR, gracilis nucleus; DMNX, dorsal motor nucleus of the vagus; HN, hypoglossal nucleus; CC, central canal. B, sagittal section, showing the projections of AP from either efferent neurons (blue), afferent neurons (orange) or both (green). 4V, 4th ventricle; DMH, dorsomedial hypothalamus; DMNV, dorsal motor nucleus vagus; DTN, dorsal tegmental nucleus; NA, nucleus ambiguous; PBN, parabrachial nucleus; PVN, paraventricular nucleus; SON, supraoptic nucleus. [Taken and adapted from Price et al., 2008 (879)].

A B

177 Appendix

Appendix 90: Hypothetical scheme of NPY neuronal circuits in anxiey (A) and schizophrenia (B) involving DP 4 at the circumventricular organs (CVO’s) median eminence (ME), area postrema (AP), organum vasculosum of lamina terminalis (OVLT) and subfornical organ (SFO). 3V, 3rd ventricle; 4V, 4th ventricle; AMY, amygdala; Arc, nucleus arcuate; CNS, central nervous system; CRF, corticotropin-releasing factor; CSF, cerebrospinal fluid; GABA, -butyric acid; IGF-1, insulin-growth factor 1; HPA, hypothalamic-pituitary adrenal; LC, locus coerulus; LS, lateral septum; NE, norepinephrine; NPY, neuropeptide Y; NSA, neuro- sympathetic axis; NTS, nucleus tractus solitary; OVLT, organum vasculosum of lamina terminalis; PAG, periaqueductal gray; Pb, parabrachial nucleus; PVN, paraventricular nucleus; PVS, perivascular space and SFO, subfornical organ (470;480;969-971).

178 Appendix

Appendix 91: Influence of N-terminal truncation of neuropeptides by dipeptidyl peptidase 4 (DP 4) on receptor binding. A, truncation of NPY to NPY3-36 alters receptor selectivity; B, successive cleavage of SP to SP5-11 causes loss of NK1-R selectivity; C, DP 4 inactivates PACAP1-27/PACAP1-38; D, DP 4-derived metabolite GALP3-32 has increased affinities with respect to Gal1-3-R’s (2;208;476;477;479;481-483;510;888;889;892).

179 Appendix

Appendix 92:Proposed interaction of NPY1-36 and NPY3-36 with Y1-R and Y2-R, respectively. DP 4 hydroysis results in changes of tertiary conformation and subsequent loss of selectivity 35 towards Y1-receptor, but equal binding towards Y2-R. R of NPY and E6.59 of Y1-R as well 33 25 as R and R of NPY and E6.59 and E5.77 of Y2-R are involved in ligand binding, respectively. [Taken and adapted from (2;892;972-978)].

Appendix 93: Summary of anxiety disorders§. Definition Symptoms Classification Treatment Anxiety Generalised anxiety disorder (GAD) Benzodiazepines “unpleasant Headache Panic disorder GABAA-receptors Sweating emotional state out Phobias Muscle spasm of control/no Agarophobia Antidepressants Palpitation causative origin“ Social anxiety disorder Reuptake-inhibitors Hypertension Obsessive compulsive disorder (OCD) (SSRI, SNRIs, NRIs, NDRIs)* Classification: Fatigue ICD10, DSM IV § Taken from (2010): http://en.wikipedia.org/wiki/Anxiety_disorder; ICD10, F40, F42; * SSRI, selective serotonine reuptake inhibitors; SNRIs, serotonine-norepinephrine reuptake inhibitors; NRIs, norepinephrine-reuptake inhibitors; NDRIs, norepinephrine-dopamine-reuptake inhibitors.

Appendix 94: Summary of schizophrenia§. Definition Symptoms Classification Treatment 1. Characteristic symptoms Positive Symptoms: Paranoid Schizophrenia Antipsychotic Schizophrenia Hallucinations Disorganised Schizophrenia (Hebephrenic) Typical: Greek “split mind“ Paranoia Catatonic Schizophrenia Delusion D2-receptor antagonist Undifferentiated Schizophrenia Classification: Disorganised speech/thinking ICD10 DSM IV- Negative Symptoms: Post-schizophrenic Depression Atypical: TR Catonia Residual Schizophrenia D2-receptor antagonist Apathy Simple Schizophrenia + 5HT2A, 5HTT1A 2. Social/occupational dysfunctions 3. Duration § Taken from (2010): http://en.wikipedia.org/wiki/Schizophrenia; ICD10, F20; http://omim.org/entry/181500

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Figure 1: Substrate specificity of DP 4. Xaa and Yaa indicate any amino acid. Decreasing font of amino acid at P1 position represents declining rate of hydrolysis. Amino acids crossed out must not occupy P1’. Arrow indicates site of cleavage (2;10). Figure 2: Primary and quaternary structure of human DP 4, based on pdb: 1N1M. A, primary structure of DP 4 subunit, consisting of an intracellular tail (aa 1 - 6), transmembrane region (aa 7 - 28), flexible stalk (aa 29 - 39), glycosylated-rich region (aa 101 - 350), cysteine rich region (aa 55 - 100, 351 - 497) and catalytic region (aa 506 - 766). , N- glycosylation; , potential unoccupied N-glycosylation; , cysteine residues involved in S-bridges; red numbers and letters indicate the catalytic triad. B, quaternary structure of homodimeric human recombinant DP 4 as determined by Rassmussen et al., 2003, (11) showing the /-hydrolase domain (aa 39 - 51 and 506 - 766) in green and propeller domain (aa 55 - 497) with the glycosylation-rich subdomain (red) and the cystein-rich subdomain (blue). Inhibitor Val-Pyr is presented in spheres, indicating that catalysis occurs at the interface of the two domains. C, Propeller domain viewed from the top, illustrating the 8 propeller blades designated with roman numbers and two subdomains. D, active site zoomed in, depicting the residues involved in catalysis, catalytic triad Ser630, Asp708, His740 are shown in red, Tyr547 responsible for oxyanion hole in pale green, Tyr666 forming the hydrophobic pocket in beige, Arg125 and Asn710, contributing to an electrostatic sink in yellow and orange, respectively, and Glu205 and Glu206 ensuring N-terminal anchoring in pink; inhibitor Val-Pyr is presented in cyano. S-S bridges are illustrated in yellow and carbohydrates in orange. The broken line separates propeller from /-hydrolase domain. Structures were drawn with pymol, using pdb: 1N1M. Figure 3: Structure, properties and functions of DP 4 sialylation. Figure 4: Phylogenetic relationship between the members of the S 9b subfamily (DP 4-gene family). A, percent of amino acid identity (bold) and similarity (90); B, dentogram showing the phylogenetic relationship of human members of the S9b subfamily. The analysis was based on the cDNA sequences of DP 4, FAP, DP 8, DP 9, DPL 1-long, DPL 1-short and DPL 2, using standard parameters in Vector NTI 6. Figure 5: Schematic presentation of the proteins belonging to the DP 4-gene family of S9b. The arrangement of structural domains is depicted. Approximate positions of N- glycosylation sites, cysteine residues and some residues required for enzyme activity and ADA or antibody sites are shown. Not to scale. [Taken and adapted from Gorrell, 2005 (92)]. Figure 6: Cellular compartmentation of DP 4-like enzymes. DP 4 and FAP are located either as homodimers or heteromeric complex on the plasma membrane or shedded into the serum. DP 2, a homodimer, is distributed as a zymogen in secretory vesicles or lysosome. DP 8 and DP 9 are also homodimers and located cytosolically. , glycosylation. Figure 7: Physiological processes influenced by DP 4 or/and DP 4-like enzymes (2;190). Figure 8: Pathophysiological role of DP 4 with either altered expression and/or activity (2;190). Figure 9: Schematic presentation of the enteroinsular axis, illustrating the release and insulinotropic action of GLP-1 and GIP as well as the influence of DP 4 and the effect of DP 4 inhibition (401). Figure 10: Anatomical presentation of the pancreas, illustrating the exocrine and endocine tissue. Islets of Langerhans consist predominantly of 3 different cells: blue, -cells secreting glucagon; yellow, -cells secreting insulin; green, -cells secreting somatostatin and VIP. Figure 11: Influence of N-terminal truncation of neuropeptides by dipeptidyl peptidase 4 (DP 4) on receptor binding. A, truncation of NPY to NPY3-36 alters receptor selectivity; B, subquential cleavage of SP to SP5-11 causes loss of NK1-R selectivity; C, DP 4 inactivates PACAP1-27/ PACAP1-38 (436;439-447) Figure 12: Scheme of methods employed to analyse DP 4-like enzymes in porcine and canine tissues.

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Figure 13: Scheme of Rotofor cell device, involving loading, focusing and elution under vacuum. Figure 14: Schematic overview of the isolation of DP 4 isoforms comprised of parallel Rotofor separation runs, 1st refractionation and 2nd refractionation steps. Figure 15: Schematic overview of the islet isolation from porcine pancreas. Figure 16: Schematic outline of the differential fractionation to isolate secretory granules from porcine islets of Langerhans. Buffer: 5 mM Na2HPO4/NaH2PO4, pH 6.0, 300 mM sucrose. Figure 17: Principles of sample preparation and detection by MALDI-TOF-MS using Voyager-DE Pro Biospectrometry workstation from Applied Biosystem (insert photo). [Taken and adapted from http://www.ncbi.nlm.nih.gov/books/NBK22410/figure/A481/, Berg JM, Tymoczko JL, Stryer L. (2002) Biochemistry. 5th edition. New York: W H Freeman] Figure 18: Scheme illustrating the method of capillary depletion, comprised of 1st surgery, 2nd perfusion and 3rd capillary Depletion (537). Figure 19: Analysis of purified porcine DP 4 from kidney cortex. A, Native PAGE with gradient 4- 12 %; a, purified porcine DP 4. B, 7.5 % SDS-PAGE of purified DP 4, b, marker; c, purified porcine DP 4; C, analytical 0.8 % agarose IEF, pH 4 - 6; d, activity stained with Gly-Pro-4- methoxy-naphtylamide; e, Serva Blue stained, f, IEF standards. D, MALDI-TOF mass spectra of purified porcine DP 4 from kidney cortex. Peak 1 - 5 correspond to the mass of 6-, 4-, 3-, 2-and 1- fold charged molecule, respectively. The mass spectrum represents the cumulative m/z signal for 184 out of 185 single laser shots with a laser power of 1.0 - 2.5 µJ. Figure 20: Scheme of initial parallel separation runs of Rotofor 1 - 3. Sample composition of purified DP 4, 2 % Servalytes pH 5 - 6 and triple distilled water to a volume of 56 ml. Running conditions: 15 W for 5 h at 0° C. Figure 21: Analysis of fractions obtained from initial parallel separation runs of Rotofor 1 - 3, using purified DP 4. A + B, Analysis of the Rotofor 1, using purified porcine DP 4 from DEAE-Sephacel-Pool 1. A, analytical agarose IEF, pH 4 - 6, Serva Blue stained of Rotofor 1; B, elution profile representing Rotofor 1; solid line, protein concentration; ▲,specific activity;■, pH profile of Rotofor fractions. C - E, represent analytical IEF profiles, pH 4 - 6 from Rotofor’s 1 - 3 respectively, activity stained with Gly-Pro-4-methoxy--naphtylamide; dotted lines indicate most acidic-, acidic- and slightly acidic clusters pooled together for subsequent refractionation. F, mean pH profile with standard error of mean from Rotofor’s 1 - 3. Figure 22: pH-gradient of parallel Rotofor separation runs using 2 independent DP 4 preparations. ▲, first DP 4 preparation for preparative IEF, n = 3; , second DP 4 preparation for preparative IEF, n = 4. Figure 23: Characterisation of first refractionation step. A, analyical agarose IEF, pH 4 - 6 of Rotofor 4 Pool 1. DP 4 isoforms are stained with Gly-Pro-4 methoxy--naphtylamide. B, elution profile of Rotofor 4 Pool 1; solid line, protein concentration; ▲, specific activity; , pH profile of Rotofor fractions. C, analyical agarose IEF, pH 4 - 6 of Rotofor 5 Pool 2. DP 4 isoforms are stained with Gly-Pro-4-methoxy--naphtylamide. D, elution profile of Rotofor 5 Pool 2; solid line, protein concentration; ▲, specific activity; , pH profile of Rotofor fractions. Figure 24: Analysis of fractions obtained from Rotofor separation runs of the second refractionation step. A, analytical IEF, pH 4-6 of Rotofor 6 Pool 3 representing fraction 1 - 20, activity stained with Gly-Pro-4-methoxy--naphtylamide. B, elution profile of Rotofor 6 Pool 3, solid line, protein; ▲, specific activity (Units/mg); , pH; frame indicate pooled isolated isoforms used for crystallisation. C, analytical IEF, pH 4 - 6 of Rotofor 7 Pool 4 representing fraction 1 - 20, activity stained with Gly-Pro-4-methoxy--naphtylamide. D, elution profile of Rotofor 7 Pool 4, solid line, protein; ▲, specific activity (Units/mg); , pH; frame indicate pooled isolated isoforms used for crystallisation. E, analytical IEF, pH 4 - 6 of Rotofor 8 Pool 5 representing fraction 1 - 20, activity stained with Gly-Pro-4-methoxy-- naphtylamide. F, elution profile of Rotofor 8 Pool 5, solid line, protein; ▲, specific activity

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(Units/mg); , pH. Frame indicates pooled isoform fractions sent to Martinsried, Germany for crystallisation. Figure 25: DP 4 crystals obtained from the isoforms by the collaboration partners of the working group led by Prof. Bode, MPI, Martinsried, Germany. Crystallography data acquired from DP 4 isoforms by the collaboration partners of the working group led by Prof. Bode, MPI, Martinsried, Germany, resulting in the first native DP 4 crystal pdb: 1ORV (13). Figure 26:Example of pH refinement of Rotofor runs, comprised of initial parallel Rotofors, first refractionation and second refractionation. , mean pH of Rotofor 1 - 3 with SEM; , Rotofor 4 Pool 1; , Rotofor 5 Pool 2; , Rotofor 6 Pool 3; , Rotofor 7 Pool 4; , Rotofor 8 Pool 5. Figure 27: Schematic presentation of crystal structure of native porcine DP 4 from kidney cortex (pdb 1orv). A, soluble DP 4 forms a 222 symmetric assembly as a dimer of dimers. Arrows indicate respective propeller and side opening. Each subunit consists of a catalytic /-hydrolase (green shades) and -propeller domain as indicated. The -propeller is again subdivided into a carbohydrate rich sub-domain (red shades) and cystein-rich sub-domain (blue shades). Carbohydrates and disulfite bonds at the -propeller tunnel are indicated in orange and yellow, respectively. B, tetramerisation interphase of -propepller domains of sub-unit A and sub-unit C as viewed from the top, illustrating opposite facing of the sub- units. C, tetramerisation cavity suggesting that glycosylation at Asn-279 and Asn-229 appears to play a crucial role in the tetramerisation of DP 4. The transmembrane helices and their orientation to the membrane were drawn in to illustrate how tetramerisation of DP 4 may mediate cell–cell contacts (11). Structures were drawn with Pymol, using pdb 1orv. Figure 28: Analytical IEF, pH 3 - 7 of purified porcine and human recombinant DP 4 activity stained with Gly-Pro-4 methoxy--naphtylamide. A, porcine DP 4 purified from kidney cortex; (pDP 4) 1, pDP 4 + pNGNASE; 2, pDP 4 + Endo H; 3, pDP 4 + Neuramidase; 4, pDP 4 dephosphorylated; 5, native pDP 4. B, human recombinant DP 4 expressed in Pichia pastoris; C, Serum DP 4-like activity, 1, without inhibitor; 2, + DP 4-selective inhibitor; 3, + DP 8/9-selective inhibitor; 4, + Dab-Pip; 5, + P32/98; 6, + Inhibitor mix. D, diagram of DP 4-like activity in serum. DP 4-I, DP 4 selective inhibitor; DP 8/9 selective inhibitor; DP 2-I, DP 2 selective inhibitor; Mix, DP 4-I + DP 8/9-I + DP 2-I; P32/98, non- selective DP 4-like inhibitor. Figure 29: Influence of selective DP 4-like inhibitors on the activity of porcine and canine tissue extracts, at pH 7.6. A, remainng DP 4-like activity of porcine tissue extracts after incubation with selective inhibitors against DP 4, DP 8/DP 9 and DP 2 as well as inhibitor mix and P32/98. B, remaining DP 4-like activity of canine tissue extracts after incubation with selective inhibitors against DP 4, DP 8/DP 9 and DP 2 as well as inhibitor mix and P32/98. , DP 4 inhibitor Sitagliptin; , DP 8/DP 9 inhibitor UG93; , DP 2 inhibitor Dab-Pip; , inhibitor mix (Sitagliptin, UG93, Dab-Pip; , P32/98. Concentrations and IC50 of the selective DP 4-like inhibitors are summarised in Appendix 29. Overall DP 4 activity, Sitagliptin, UG93, Dab-Pip, inhibitor mix and P32/98 were analysed with 0.1 mM Ala-Pro-AMC, in 40 mM Hepes, pH 7.6; n = 6; error bars = SEM (131). Figure 30: Purity, morphology and viability of isolated islets of Langerhans from porcine pancreas. A, single islet under phase contrast microscopy; B, islets obtained from the top– phase of Ficoll density gradient and observed under phase contrast microscopy. C, viable single islet stained for fluorescein diacetate; D, viable islets obtained from the top–phase of Ficoll density gradient and stained for fluorescein diacetate. Figure 31: DP 4-like activity in fractions obtained from the Ficoll density gradient after islet isolation. A, Specific activity of DP 4-like enzymes in the various fractions. B, Percentage of total DP 4-like activity in the various fractions (661). n = 6; error bars = SEM. Figure 32: Analysis of DP 4-like enzymes in fractions obtained after centrifugation with Ficoll density gradient as a final step of islet isolation, using selective inhibitors against DP 4, DP 8/DP 9 and DP 2, respectively. , selective DP 4 inhibitor; , selective DP 8/DP 9 inhibitor; , selective DP 2 inhibitor; , inhibitor mix. % remaining activity in presence of DP 4 inhibitor, DP 8/ DP 9 inhibitor and inhibitor mix was determined with respect to overall DP 4-like activity at pH 7.6, whereas remaining activity in presence of DP 2 inhibitor was determined with respect to overall DP 4-like activity at pH 5.5. n = 3; error bars = SEM.

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Figure 33: Analysis of DP 4-like enzymes by selective DP 4-like inhibitors in cytosolic and membrane fractions of porcine isolated islets, obtained after ultracentriugation at 105,000 g for 1 h at 4° C. A, specific activities in presence and absence of selective inhibitors specific for DP 4, DP 8/DP 9 and DP 2, respectively as well as non-selective DP 4-like inhibitor P32/98. , overall DP 4-like activity; , selective DP 4 inhibitor Sitagliptin; , selective DP 8/9 inhibitor UG93; , DP 4- like activity at pH 5.5; , selective DP 2 inhibitor Dab-Pip; , inhibitor mix; , P32/98. DP 4, Sitagliptin, UG93, inhibitor mix and P32/98 were determined with 0.1 mM Ala-Pro-AMC, pH 7.6, whereas DP 2 and Dab-Pip were analysed with 0.2 mM Ala-Pro-AMC, pH 5.5. n = 6; error bars = SEM. B, Specific activity of lactate dehydrogenase (A/h/mg), a cytosolic marker. C, specific activity of alkaline phosphodiesterase I (mUnits/h/mg), a plasma membrane marker. Cyt, cytosol; mem, membrane, PH, pancreatic head (131;661). Figure 34: Results of nested RT-PCR of DP 4-like enzymes from porcine and canine pancreas as well as isolated islets. Figure 35: Results obtained from the histochemical staining for DP 4-like activity in porcine kidney and pancreas, using Gly-Pro-1-OH-4-naphtylamide. A, staining of porcine kidney after 10 min; B, staining of porcine kidney after 30 min, showing strong DP 4 activity at the brush border of proximal tubules; C, staining of kidney in presence of 1 mM P32/98. D, staining of kidney in presence of 4 mM Pefabloc; E, interlobular duct of porcine pancreas; F, intercalated ducts linking to intralobular duct of pancreas; G, endothelial DP 4 surrounding a small islet in pancreas. H, islets in pancreas, showing differential staining of DP 4-like activity; I, staining of pancreas in presence of 1 mM P32/98. Figure 36:Histochemical staining for DP 4-like activity in isolated porcine islets. A, staining of porcine islet after 30 min, showing DP 4-like activity at surface of the central islet cells as indicated by arrows and intra-cellular staining of peripheral islet cells marked with arrow head; B, positive staining of DP 4-like activity in peripheral islet cells after 30 min; C, staining of DP 4-like activity in presence of 1 mM P32/98 after 30 min; D, staining of DP 4-like activity in presence of 4 mM Pefabloc after 30 min. Figure 37: Histochemical staining for DP 2 activity in porcine kidney, pancreas and isolated islets, using KA-CAH, pH 5.5. A, staining of porcine pancreas after 30 min, implying specific cell-type staining of possible -cells in islets; B, staining of porcine pancreas after 60 min, indicating additional peripheral staining of islets; C+D, islet in porcine pancreas after 90 min, showing staining of islet cells and most likely its surrounding neuronal network; E, staining of isolatetd islets after 90 min; F, staining of porcine kidney after 30 min, showing strong DP 2 activity at the brush border of distal tubules. Figure 38: Immunohistochemical analysis of porcine pancreas, staining various tissue sections either with anti-DP 4 pAb, anti-insulin mAb or anti-glucagon pAb or in combination of two. A, staining of a large islet for insulin (red). B, staining of the islet for glucagon (green). C, super-imposing the islet, stained for insulin (red) and glucagon (green). D, staining of an islet for insulin (red). E, staining of the islet for DP 4 (green). F, super-imposing the islet, stained for insulin (red) and surrounded by capillaries expressing DP 4 (green). G, staining of exocrine tissue, locating DP 4 mainly to intercalated- (arrow) and intralobular-ducts and more faintly to acinar cells. H, DP 4 staining of an interlobular- (arrow head) and intralobular- (arrow) duct. I, double-staining of a small islet for insulin (red) surrounded by exocrine tissue expressing DP 4 (green). J, double-staining of an islet for glucagon (red) surrounded by capillaries expressing DP 4 (green). In collaboration with Dr. Hause, Biocentre, Halle. Figure 39: Perfusion studies of rat pancreas, showing secretion of DP 4-like enzymes and glucagon after stimulation of splanchic nerve in Wistar rats. ▲, DP 4 activity, measured with 0,1 M Gly-Pro-AMC after ultracentrifugating samples at 105 000 g for 1 h and deducting residual DP 4 activity in BSA (RIA grade); -, glucagon concentration; black arrow, splanchic nerve stimulation; blue arrow, 10 µM sodium nitroprusside (SNP) stimulation. n = 3; error bars = SEM. Figure 40: Analysis of specific activities, contributed by the various DP 4-like enzymes in brain, neuronal and glial tissues, using selective inhibitors against the DP 4-like enzymes. A, Analysis of the specific activities of the DP 4-like enzymes in brain extracts obtained from wildtype Fischer rats (F344/Ztm) and DP 4-deficient Fischer rats (F344/Crl(Wiga)SvH-Dpp4) as well as cell extracts from primary rat neurons. B, Analysis of the specific activities of the DP 4-like enzymes in brain extracts as well as cell extracts from primary rat neurons, astrocytes and microglia. Specific activities were determined using 0.1 mM Ala-Pro-AMC, in 184 List of Tables

40 mM Hepes, pH 7.6 for DP 4 and DP 8/9 as well as 0.2 mM Ala-Pro-AMC in 80 mM NaAcetate, pH 5.5 for DP 2, respectively. Except for the inhibition by non-selective DP 4-like inhibitor P32/98, the specific activities of the DP 4-like enzymes are presented as the difference of total specific activity - selectively inhibited specific activity. , brain extract F344/Ztm; , brain extract F344/Crl(Wiga)SvH-Dpp4; , rat neuronal cell extract; , rat microglial cell extract; , rat astrocytic cell extract. n = 6; error bars = SEM. Figure 41: Histochemical analysis of DP 4-like enzymes in 5 µm kidney sections from F344/Ztm and F344/Crl(Wiga)SvH-Dpp4 Fischer rats after 30 minutes, using Gly-Pro-4-OH-1-NA and NBT as substrate, and selective inhibitors against DP 4 and DP 8/9 as well as non-selective DP 4-like inhibitor P32/98. A, staining of kidney section from wildtype Fischer rats F344/Ztm in absence or presence of DP 4- or DP 8/9-selective or non- selective P32/98 inhibitors, showing predominantly staining of brushborders of proximal tubules and glomerulus by DP 4 and only weak intra-cellular staining of tubular cells by DP 8/9. B, staining of kidney sections from DP 4-deficient F344/Crl(Wiga)SvH-Dpp4 Fischer rats in absence or presence of DP 4- or DP 8/9-selective or non-selective P32/98 inhibitors, resulting in no or hardly any staining due to lack of DP 4. Figure 42: Histochemical analysis of DP 4-like enzymes in 50 µm brain sections from F344/Ztm and F344/Crl(Wiga)SvH-Dpp4 Fischer rats after 12 and 1 h, using Gly-Pro-4-OH-1-NA and NBT as substrate. A, staining of brain sections from F344/Ztm and F344/Crl(Wiga)SvH-Dpp4 Fischer rats, respectively, indicating ubiquitous activity of DP 8/9 in brain. B, staining of meninges in F344/Ztm and F344/Crl(Wiga)SvH-Dpp4 Fischer rats after 1 hour, results in exclusively staining of the meninges in wildtype only. WT, wildtype F344/Ztm; Mut, DP 4-deficient F344/Crl(Wiga)SvH-Dpp4. Figure 43: Histochemical analysis of DP 4-like enzymes in brain sections from F344/Ztm and F344/Crl(Wiga)SvH-Dpp4 Fischer rats after 1 h, using Gly-Pro-4-Met-2-NA and Fast Mut Blue B as substrate. A, staining of meninges from wildtype F344/Ztm rat at 10 x magnification, showing strong DP 4-like activity in the arachoidal membrane of meninges. (NBT) B, staining of meninges of wildtype F344/Ztm rat in presence of DP 4-selective inhibitor, indicating DP 2 activity in subarachnoidal space and arachnoidal membrane. C, staining of meninges from DP 4-deficient F344/Crl(Wiga)SvH-Dpp4 rat, pointing to DP 2 activity in subarachnoidal space. D, staining of meninges in wildtype F344/Ztm rats at 60 x magnification. E, staining of meninges from wildtype F344/Ztm rat in presence of DP 2- selective inhibitor Dab-Pip, showing strong activity of DP 4 in arachnoidal membrane and pia mater. F, no staining of meninges from DP 4-deficient F344/Crl(Wiga)SvH-Dpp4 rat in presence of Dab-Pip. DM, dura mater; AM, arachnoidal membrane; SAS, subarachnoidal space; PM, pia mater. Figure 44: Specific activities of DP 4 in brain and circumventricular organs (CVO’s). A, specific activities of DP 4 in extracts from brain and CVO’s as determined with 0.1 mM Ala-Pro-AMC in 40 mM Hepes pH 7.6 in presence or absence of DP 4 selective inhibitor Sitagliptin. n = 4, error bars = SEM. B, anatomical presentation of CVO’s in a sagittal section of rat brain. Ant.Pit., anterior pituitary; EM, median eminence; OVLT, organ vasculosum of lamina terminalis; SCO, subcommissural organ; SFO, subfornical organ; Ch.P, choroids plexus; PiG, pineal gland; AP, area postrema (679). Figure 45: Proteolytic studies of NPY and SP by human recombinant DP 2 and DP 4 using MALDI-TOF-MS. A, 25 µM NPY in presence of human recombinant DP 2 results in no truncation after 6 h. B, 25 µM SP in presence of human recombinant DP 2 results in no truncation after 6 h. C, 25 µM NPY is readily truncated to NPY3-36 by human recombinant DP 4 after 30 min. D, 25 µM SP is truncated to SP3-11 by human recombinant DP 4 after 60 min. Matrices: NPY, sinapinic acid; SP, -cyano-4-hydroxycinnamic acid. Figure 46: Hydrolysis of 25 µM NPY by 40 µg/ml tissue extracts from either rat brain, neurons, astroytes or microglia after 6 h and 24 h using MALDI-TOF-MS. Matrix, sinapinic acid. Figure 47: Hydrolysis of 25 µM NPY by 400 µg/ml tissue extracts from either rat brain or rat primary neurons, using MALDI-TOF-MS. A, hydrolysis of 25 µM NPY by 400 µg/ml brain extracts from rat after 30 min. B, hydrolysis of 25 µM NPY by 400 µg/ml brain extracts from rat after 2 h. C, hydrolysis of 25 µM NPY by 400 µg/ml cell extracts from rat primary neurons after 6 h. Sitagliptin, DP 4-selective inhibitor; P32/98, non-selective DP 4-like inhibitor. Matrix, sinapinic acid.

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Figure 48: Analysis of C-terminally labelled NPY-biotin and its metabolites after perfusion and capillary depletion of rat brain. A, N-terminal truncation of 50 µM NPY-biotin by endothelial DP 4 (NPY3-36-biotin) and mAmpP (NPY2-36-biotin) after brain perfusion corresponding to ± 3 min blood circulation. The MALDI-TOF-MS spectra was performed after immune precipitation of NPY-biotin with streptavidin-Ab and subsequent C18 zip-tip. B, complete degradation of NPY-biotin in brain parenchyma in presence of conservation solution with inhibitors aprotinin, PMSF, Val-Pyr, EDTA, phosphoramidon and DTT, as no NPY-metabolite could be detected after immune precipitation of NPY-biotin with streptavidin-Ab and subsequent C18 zip-tip, using MADLI-TOF-MS. C, N-terminal truncation of 50 µM NPY-biotin by soluble DP 4 (NPY3-36-biotin) and mAmpP (NPY2-36-biotin) in undiluted serum after ± 3 min. D, endoproteolytical degradation of unlabelled NPY spiked in brain parenchyma in presence of commercial protease inhibitor mix complete mini (Roche). Figure 49: NPY-hydrolysis by secreted Mep-A and lysosomal CTSD from rat brain, investigated by MALDI-TOF-MS. A, metabolism of 25 µM NPY by 500 µg/ml rat tissue extract in presence or absence of pepstatin A and actinonin. B, metabolism of 25 µM NPY by cytosolic and membrane fractions from extracts of rat brain. Figure 50:Improved conservation solution comprised of aprotinin, PMSF, Val-Pyr, EDTA, E64, pepstatin A, actinonin, phosphoramidon and DTT enabled stability of NPY for more than 24 h as determined by MALDI-TOF-MS. Matrix, sinapinic acid. Figure 51: Hydrolysis of 25 µM NPY by human recombinant/purified enzymes, using MALDI- TOF-MS analysis reveals a meprin-A-mediated degradation and an endoproteolytic inactivation of NPY by cathepsin D and Neprilysin. In addition, the cathepsin D mediated effect is highly pH-dependent. A, DP 4 (1 µg/ml, human recombinant). B, NEP (1 µg/ml, human recombinant; inhibitor: phosphoramidon). C, CTSD, pH 7.6 (1 µg/ml, purified from human spleen; inhibitor: pepstatin A). D, CTSD, pH 5.5 (1 µg/ml, purified from human spleen; inhibitor: pepstatin A). E, MepA (400 ng/ml, human recombinant, previously activated with 100 ng/ml trypsin for 1 h, activation stopped by AEBSF achieving a 1 mM final concentration; 1 µg/ml activated MepA resulted in rapid degradation of undetectable peaks after 2 min; inhibitor: actinonin). F, MepB (400 ng/ml, human recombinant, previously activated with 100 ng/ml trypsin for 30 min, activation stopped by AEBSF achieving a 1 mM final concentration; inhibitor: actinonin). Figure 52: Hydrolysis of 25 µM NPY by 1 µg/ml Cathepsins B, G, L and S as analysed by MALDI- TOF-MS. As control NPY cleavage was inhibited by cysteine protease inhibitor E64 (A-C) or DIC (D). A, CTSB, pH 5.5; B, CTSL, pH 5.5; C, CTSS, pH 6.0; D, GTSG, pH 7.6. Matrix, sinapinic acid. Figure 53: Hydrolysis of NPY in CSF as analysed by MALDI-TOF-MS. A, Western blot of CTSD. M, marker, lane 1, medium of HEK-cells transfected with CTSD, lane 2, human CTSD purified from spleen, lane 3, CTSD from 20 µl CSF. B, Rapid hydrolysis of NPY by human CSF results in CTSD-specific metabolite NPY1-30 after 30 min at physiological pH 7.4, as detected by MALDI-TOF-MS. Samples were kindly supplied by Dr. B. Leavitt and Prof. M. Haydn from University of British Columbia, Vancouver, Canada. Figure 54: Hydrolysis of NPY by human serum analysed by MALDI-TOF-MS. A, time-dependent hydrolysis of NPY in human serum revealing additional C-terminal truncation by unknown enzyme. B, identification of C-terminal truncation by ACE-like activity. Note the compensatory increase of ACE-like activity after inhibition of DP 4 and the increased half- life of vasoconstrictive full-length NPY1-36 after combined inhibition of ACE + DP 4. Data represent MALDI-TOF-MS analysis after 90 min. Matrix, sinapinic acid. Figure 55: Time-dependent hydrolysis of 50 µM galanin-like peptide (GALP) by human recombinant DP 4 and human serum as well as hydrolysis of 50 µM rat Orexin B (OrxB) by DP 4 and rat serum, using MALDI-TOF-MS. A, time-dependent hydrolysis of GALP by human recombinant DP 4. B, time-dependent hydrolysis of GALP by human serum. C, time-dependent hydrolysis of rat Orx B by human recombinant DP 4. D, time- dependent hydrolysis of rat OrxB by rat serum. Sitagliptin, DP 4 selective inhibitor; matrix, sinapinic acid for GALP; -cyano-4-hydroxycinnamic acid for rat OrxB. Figure 56: MALDI-TOF-MS spectra, showing ex vivo hydrolysis of neuropeptide Y (NPY), galanin-like peptide (GALP) and substance P (SP) in blood. A, MALDI-TOF-MS

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spectra, showing ex vivo hydrolysis of 25 µM NPY in blood diluted 1:4 with 20 mM Tris- HCl, pH 7.6 and 0.3 M sucrose after 30 min.. B, MALDI-TOF-MS spectra showing ex vivo hydrolysis of 25 µM GALP in blood diluted 1:4 with 20 mM Tris-HCl, pH 7.6 and 0.3 M sucrose after 6 h. C, MALDI-TOF-MS spectra showing ex vivo hydrolysis of 25 µM SP in blood diluted 1:4 with 20 mM Tris-HCl, pH 7.6 and 0.3 M sucrose after 3 h. D, MALDI-TOF- MS spectra showing in vitro hydrolysis of 25 µM SP in blood diluted 1:4 with hypotonic 20 mM Tris-HCl, pH 7.6 after 5 min. Matrices, sinapinic acid for NPY and GALP; -cyano-4- hydroxycinnamic acid for SP. Figure 57: MALDI-TOF-MS spectra comparing the hydrolysis of 25 µM neuropeptide Y (NPY) ex vivo blood versus in vitro haemolytic blood. A, MALDI-TOF-MS spectra showing the time- dependent ex vivo hydrolysis of 25 µM NPY in blood diluted 1:9 with isotonic 20 mM Tris-HCl, pH 7.6 and 0.3 M sucrose. B, MALDI-TOF-MS spectra showing the time-dependent in vitro hydrolysis of 25 µM NPY in haemolytic blood diluted 1:9 with hypotonic 20 mM Tris-HCl, pH 7.6, indicating additional NPY3-36 truncation by cytosolic DP 8 and/or DP 9. Matrix, sinapinic acid. Figure 58: DP 4-like activity in sera obtained from depressed patients, using 100 µM Gly-Pro-AMC. A, DP 4-like activity of sera obtained from patients diagnosed with clinical depression (n = 32 for depressed patient and n = 30 for control subjects). B, DP 4-like activity of sera obtained from patients diagnosed with non-melancholic depression (n = 16 for depressed patients and n = 15 for control subjects). C, DP 4-like activity of sera obtained from patients diagnosed with melancholic depression (n = 16 for depressed patients and n = 15 for control subjects). Sitagliptin, selective DP 4 inhibitor; P32/98, non-selective DP 4-like inhibitor; error bars = SEM. Samples were kindly supplied by PD Dr. M. Rothermundt and Dr. F. Kästner (597;598). Figure 59: Characterisation of DP 4 in sera from depressed patients and control subjects. A, correlation of DP 4 activity with CD26 (DP 4) protein concentration as determined by ELISA, p < 0.0001, n = 94. B, analytical agarose IEF pH 3 - 7 stained with Gly-Pro-4-Met- 2-NA, displaying DP 4 isoforms due to heterogeneity in sialynation. Lane 1, non- melancholic depressed patient before treatment; lane 2, non-melancholic depressed patient after treatment; lane 3, healthy control subject matching with respect to age and sex; lane 4, melancholic depressed patient before treatment; lane 5, melancholic depressed patient after treatment; lane 6, healthy control subject matching with respect to age and sex. Figure 60: Schematic presentation of crystal structure of native porcine DP 4 from kidney cortex, depicting the residues important for tetramerisation (pdb: 1ORV). A, Soluble DP 4 forms a 222 symmetric assembly as a dimer of dimers. Arrows indicate respective propeller and side opening. Each subunit consists of a catalytic /-hydrolase (green shades) and -propeller domain as indicated. The -propeller is again subdivided into a carbohydrate rich sub-domain (red shades) and cystein-rich sub-domain (blue shades). Carbohydrates and disulfite bonds at the -propeller tunnel are indicated in orange and yellow, respectively. B, Tetramerisation interphase of -propepller domains of subunit A and subunit C as viewed from the bottom, illustrating the involvement of the strands Asn279-Gln286 to form an antiparallel sheet. C, residues of strands Asn279-Gln286 of subunits A and C forming an antiparallel sheet, responsible for tetramerisation and stabilised by H-bonds (gray dots). The transmembrane helices and their orientation to the membrane were drawn in to illustrate how tetramerization of DP 4 may mediate cell–cell contacts (11). Figures were prepared with Pymol, using pdb: 1ORV (13). Figure 61: Superimposition of the space around the catalytic active serine 630 of human DP 4 (orange) and porcine DP 4 (green). Amino acids of the active centre were used for the comparison of both structures. For the calculation of the RMS value (root mean square), the amino acids of the human DP 4 (pdb: 1N1M) were used as the target molecule. Afterwards, each atom (without hydrogen atoms) of the porcine DP 4 (pdb: 1ORV) was put on the atom of the target molecule and a good match was found, (RMS-value: 0.34 Å). This value is in the range of the X-ray determination accuracy. The catalytic triad is shown in red. [Taken from Bär et al., 2003, (566)].

Figure 62: Binding of reversible tight binding inhibitor p-Jodo-Phe-Pyr-CN trifluoroacetate (Ki = 25.1 ± 0.9 nM) to the active site (Ser-630) of porcine DP 4 (13). A, presentation of active site showing covalently binding of Ser-630 to the inhibitor, anchoring of free N- terminus of the inhibitor by Glu-205 and Glu-206 and the P2 oxyanion trap by Arg-125 and

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Asn-710. Drawn with Pymol, using pdb: 1ORW. B, chemical structure of inhibitor p-Jodo- Phe-Pyr-CN trifluoroacetate. Figure 63: Schematic diagram of the Diathesis-Stress Model. Under an existing genetic disposition and in combination with chronic/acute stress, a psychiatric disorder of the hypothalamic- pituitary-adrenal axis (HPA) or neuro-sympathico-axis (NSA) will be developed, resulting in either depression, anxiety or psychosis/schizophrenia. Figure 64: Psycho-neuro-endocrino-immunological role of DP 4 at the CVO’s median eminence (EM) and area postrema (AP). EM and AP form the interphase between the neuronal, endocrinal and immunological systems, involving the two stress axis hypothalamic- pituitary-adrenal (HPA) and the neuro-sympathico-axis (NSA). Activation of HPA and NSA results in the release of stress hormones cortisol and epinephrine, cytokines and NPY, which in turn interact with the brain via EM and AP as well as with the lymph organs such as the spleen (449;510-513;516;623;789-794). Figure 65: Expression of DP 4 and NPY at the median eminence. A, immunohistochemical staining of DP 4 (blue) and NPY (purple) at the median eminence, showing mainly staining of

terminal ends of 1,2-tanycytes at the portal blood and/or endothelial cells of capillaries (blue arrow) and some staining of basal processes of 1,2-tanycytes of the inner (MEI) and outer zone (MEE) of median eminence (black arrow). Co-localisation of DP 4 and NPY is found on

lining of third ventricle corresponding to cell-bodies of 1,2-tanycytes (purple arrow head). B, schematic presentation of the median eminence, showing a -tanycyte bridging the CSF with portal blood. C, schematic presentation of blood brain barrier (BBB), illustrating the tight junction of overlapping endothelial cells, the tight adherens of astrocytes and pericytes to the capillary. D, schematic presentation of interaction between CVO and a capillary, demonstrating the fenestrated endothelial cells, loose astrocytes, large perivascular space and terminal ends of tanycytes picking up/passing on neuroendocrine signals to and from the periphery such as blood and perivascular space. BBB, blood brain barrier; CVO, circumventricular organ; EM, median eminence; MEE, exterior zone of median eminence; MEI, interior zone of median eminence; ArcL, lateral nucleus arcuate; ArcM, medial nucleus arcuate (790;791). Figure 66: Metabolism of NPY before (A) and after the present study (B), showing the sub- cellular and tissue distribution of NPY-cleaving enzymes. A, metabolism of NPY before the present study, illustrating the N-terminal modulating enzymes mAmpP, sAmpP, DP 4, DP 8 and DP 9, that alter receptor selectivity (1) as well as the proteolytical degrading enzymes NEP, Mep-B, Mep-A, uPA, Pl and Kal (2). B, metabolism of NPY including novel NPY-cleaving enzymes identified in the present study. In addition to the modulating enzymes, six additional degrading enzymes and one C-terminal inactivating enzyme were identified, including lysosomal enzymes CTSD, CTSB, CTSL, CTSS and CTSG, trypsin-like peptidase hK1 and ACE. Furthermore cleavage sites of Mep-A and Kal were discovered and novel Mep-A activating enzymes found. Novel NPY-cleaving enzymes are high-lighted in colour, arrows show cleavage sites (124;132;448;626;653;656).

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10 List of Tables Table 1: Tissue distribution of DP 4 in various species, detected by different methods. Table 2: Order of catalytic triad and arrangement around serine recognition sites of DP 4-like enzymes (90;91). Table 3: Summary of the DP 4-like enzymes comprised of the human DP 4-gene family (S9b) and DP 2 (90). Table 4: Relative abundace of the expression of DP 4-like enzymes by Northern blot analysis. Table 5: Summary of natural substrates known to be hydrolysed by DP 4. Table 6: Summary of molecules known to associate with DP 4. Table 7: Summary of phenotypes obtained from DP 4-knock-out (k.o.), DP 4-deficient and DP 4-transgenic animal models as well as FAP-/- k.o.-, DPL1-/- k.o.- and DP 2-/- k.o.- mice. Table 8: Summary of altered CD26 (DP 4) activity and expression in human pathogenic sera (2;92;190;323;324). Table 9: Summary of disease conditions, being potential targets for DP 4 and/or FAP inhibition or modulated by anti-DP 4-mAb (370). Table 10: Distribution of DP 4 (-like enzymes) in the pancreas. Table 11: Summary of insulinotropic receptors for natural DP 4 substrates found in the islets of Langerhans. Table 12: Natural DP 4 substrates in pancreas with neuronal, paracrine or endocrine functions. Table 13: Various animal models used for investigations of DP 4 as a psyhcopharmaceutical target. Table 14: Summary of neuropeptides/peptides hydrolysed by DP 4 and found in the brain. Table 15: Composition of Pools 1 - 5, consisting of fractions from Rotofor 1 - 5. Table 16: Tissue sections, used to investigate the distribution of DP 4-like enzymes. Table 17: Properties of human CSF, used to analyse DP 4-like activity and hydrolysis of 25 µM NPY. Table 18: Summary of depressed patients and the respective controls subjects for serum analysis. Table 19: Purification table of porcine DP 4, isolated from 1 200 g kidney cortex. Table 20: Statistical analysis of parallel Rotofor runs from 2 independent DP 4 preparations with respect to pH-gradient. Table 21: Composition of pooled fractions derived from the three parallel Rotofor runs and used for the first refraction. Table 22: Properties of pooled fractions derived from parallel Rotofor runs 1 - 3, diluted to 56 ml and employed for the first refractionation. Table 23: Composition of pooled fractions used for the second refrationation. Fractions were either derived from the first refractionation or parallel Rotofor runs as indicated. Table 24: Properties of pooled fractions derived either from first refractionation step or parallel Rotofor 1 - 3, diluted to 56 ml and applied for the second refractionation. Table 25: Properties of purified and pooled DP 4-isoforms containing only 2-3 isoforms per pool, which were sent to the working group led by Prof. Bode, Martinsried, Germany for further processing of crystallisation. All pools of purified DP 4-isoforms were obtained from the second refractionation. Table 26: Comparison of glycosylation sites occupied in various DP 4 crystal structures. Table 27: Specific activities of various porcine and canine tissues, using 0.1 mM Ala-Pro- AMC, pH 7.6. 189 List of Tables

Table 28: Summary of natural DP 4 substrates in the small intestine. Table 29: Specific activities of DP 4 (GP-AMC, pH 7.6) and DP 2 (KA-AMC, pH 5.5) after granule isolation. Table 30: Summary of tissue controls used for nested RT-PCR in pancreas and isolated islets. Table 31: Comparison of cell units obtained from crystal structures of native porcine and various human recombinant DP 4’s. Table 32: Important residues of the active site present/absent in the members of the DP 4- gene family as determined by sequence alignment with respect to DP 4. Table 33: Current medication for the treatment of depression, anxiety and schizophrenia§.

190 Acknowledgement

11 Acknowledgement First of all, the author would like to thank Prof. Hans-Ulrich Demuth, Dr. Konrad Glund and Hendrik Liebers for giving the opportunity to pursue a Ph.D. study at probiodrug AG, Halle. In addition, the patience and encouragement provided by Prof. H.-U. Demuth to finally complete the dissertation is greatly appreciated. Furthermore, the author would like to express her gratitude to Dr. Jens-Ulrich Rahfeld and Dr. Bernd Gerhartz from the Department of Molecular Biology at probiodrug AG as well as Dr. Torsten Hoffmann from the Department of Enzymology for proposing the topics of the present study and guidance during the course of the work. Special thanks is owed to Prof. Dr. Stephan von Hörsten from the Franz-Penzoldt Centre, FAU and Department of Experimental Therapy at the UKE for his supervision, the many great scientific discussions at day and night, for organising the supply of rat tissue sections and various rat organs, for performing the surgery in the experiment of capillary depletion and finally for co-ordinating many of the collaboration partners to supply biological materials. It was the bundling of the expertise of medical, biochemical and analytical background that led to the success of the project concerning DP 4 in stress-related diseases. In this context, the author would also like to express her gratitude towards Dr. Nadine Frerker and Dr. Jutta Schade from the Department of Functional and Applied Anatomy of the Medical School Hanover for the help with histochemical studies of rat brain sections and the supply of rat tissue as well as Dr. Kerstin A. Raber and Jenny Stiller from the Department of Experimental Therapy, UKE at the Friedrich-Alexander University of Erlangen-Nuremberg for providing fresh rat sera, dissecting brain areas/organs and the excellent assistance during the experiment of capillary depletion. The author was privileged to be provided with several biological materials for analysis and would therefore express her thanks to the following people:  Prof. Dr. Steffen Rossner and Dr. Ulrike Zeitschel from the Paul-Flechsig Institute of Brain Research of University of Leipzig for the supply of isolated primary neurons, microglia and astrocytes obtained from brains of neonatal Spargue Dawley rats.  PD Dr. Matthias Rothermundt and Dr. Florian Kästner from the Department of Psychiatry at the University of Münster for providing various sera from patients diagnosed with clinical depression.  Dr. Blaire Leavitt and Prof. Michael Haydn from the Centre for Molecular Medicine and Therapeutics at the Child and Family Research Institute of the University of British Columbia, Vancouver, Canada as well as Prof. Åsa Petersén from the Translational Neuroendocrine Research Unit of the Department of Experimental Medical Science at the University of Lund, Sweden for making available human cerebrospinal fluids (CSF) of very high quality.  Dr. Jörg R. Aschaffenbach from the Institute for Veternary Physiology of the Veternary Faculty at University of Leipzig, Germany for the porcine tissues.  Prof. Ray A. Petersen and Prof. Christopher H.S. McIntosh from the Department of Cellular and Physiological Science at the University of British Columbia (UBC), Canada for the opportunity of learning about islet isolation and perfusion studies. Prof. Ray Petersen and Dr. Nadine Pamir for performing the surgery on rats and Prof. Christopher H.S. McIntosh for the many scientific discussions held on the corridor as well as the supply of publications.  Prof. Wolfram Bode, Dr. Stefan Strobl, Dr. Michael Engel, Prof. Hans Brandstetter and Marianne Braun from the Department of Structural Research of the Max Planck Institute of Biochemistry in Martinsried for the collaboration and solving the crystal structure of porcine DP 4.  Dr. Hause from the Department of Applied Imaging of the Biocentre at the Martin-Luther University Halle for the help and collaboration in immunohistochemical and enzymatic histochemical analysis on porcine pancreas and kidney.

191 Acknowledgement

The assistance of master student Daniel Gündel and bachelor student Harald Stiller during the course of the study has been greatly appreciated. Teaching them and trying to solve the scientific problems together step by step has been very enjoyable. A great number of colleagues from probiodrug AG helped with the present study by either synthesising substrates, neuropeptides or inhibitors, supplying with human recombinant enzymes or providing technical advice and assistance. Therefore the author would like to express her gratitude to the following colleagues:  Michael Wermann for the advice and assistance in purification of porcine DP 4.  Madeleine Speck and Mercedes Scharfe for the assistance at the abattoir of the Veterinary Faculty at the University of Leipzig as well as isolation of islets of Langerhans from porcine pancreas. It was indeed very hard work.  Dr. André J. Niestroj and Antje Hamann for the synthesis of the enzymatic histochemical substrates for DP 4 and DP 2, respectively.  Dr. Ulrich Heiser and Ulf-Torsten Gärtner for the synthesis of many inhibitors.  Dr. David Ruiz-Carrillo for supplying PEP, being supportive at difficult times and teaching how to use pymol. Nadine Jänckel and Anja Weber for supplying DP 2 and DP 4, respectively.  Dr. Susanne Manhart, Hans-Henning Ludwig and Christina Schnittka for the synthesis of many neuropeptides and substrates. Hans-Henning Ludwig for critically reading the dissertation, particularly for the thorough help with the German summary and being in general of moral support.  Dr. Dagmar Schlenzig for supplying cell extracts/medium of CTSD transfected HEK-293 cells as well as for the exchange of information on cathepsins/meprins and provision of commercial cathepsins.  Dr. Astrid Kehlen for the nice collaboration regarding galanin and general cell biological issues.  Dr. Fred Rosche for the MALDI-TOF MS analysis of purified DP 4 and isolated isoforms.  There has been an excellent collaboration with Dr. Raik Wolf, Beate Kohlstedt as well as Katrin Klemm. Without them, the great number of MALDI-TOF-MS spectra could not have been achieved in the present study. Thanks are owed to Dr. Raik Wolf for teaching how to operate the MALDI-TOF MS Voyager-DE Pro Biospectrometry workstation. Special gratitude is given to Beate Kohlstedt and Katrin Klemm for their patience and preparing many of the samples, sometimes being pushed to the limits. It has been a great pleasure of working together. Beate Kohlstedt has also been of tremendous support at difficult times. Generally, the author would like to thank all the colleagues being sincere and respectful. Thus, the late scientific and philosophical discussions with Dr. Ingo Schulz were most enjoyable and very inspiring. Birgit Koch and Diane Meitzner have proven to be honest colleagues and have been great company during the many night-shifts at work. Steffen Böhme has been very helpful with computer and technical issues. Dr. Fabio Canneva has been of great support with submission of the thesis. In addition, the great friendships of Prof. Dr. Carminita Frost from the Nelson Mandela Metropolitan University, Port Elizabeth, South Africa as well as Margit Schumann, Halle have been very much appreciated. The moral support of my swimming group from the Deutsche Rheuma-Liga, AG Halle under the supervision of hydrotherapist Maika Waclawczyk has been indespensible. Finally, the author is very grateful for the encouragement brought forward by her parents Jürgen and Ingeborg Wagner as well as her sister Melanie Wagner, who had a never-ending patience to listen and gave an outstanding moral back up and were very much uplifting in harsh times.

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