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Human Trypsinogens in the Pancreas and in Cancer

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HUMAN TRYPSINOGENS IN THE PANCREAS AND IN CANCER Outi Itkonen Department of Clinical Chemistry, University of Helsinki and Hospital District of Helsinki and Uusimaa - HUSLAB Helsinki, Finland ACADEMIC DISSERTATION To be presented, with the permission of the Medical Faculty of the University of Helsinki, for public criticism in lecture room I, Meilahti Hospital, Haartmaninkatu 4, Helsinki on September 12th at noon. Helsinki 2008 Supervised by Professor Ulf-Håkan Stenman Department of Clinical Chemistry, University of Helsinki, Finland Revised by Docent Jouko Lohi Department of Pathology, University of Helsinki, Finland and Docent Olli Saksela Department of Dermatology, University of Helsinki, Finland Examined by Professor Kim Pettersson Department of Biotechnology, University of Turku, Finland ISBN 978-952-92-3963-4 (paperback) ISBN 978-952-10-4732-9 (pdf) http://ethesis.helsnki.fi Yliopistopaino Helsinki 2008 2 Contents Original publications 5 Abbreviations 6 Abstract 7 Review of the literature 8 Properties and biochemical characterization of human pancreatic trypsinogens 8 Extrapancreatic trypsinogen expression 12 Trypsinogen genes 13 Regulation of pancreatic trypsinogen gene expression and secretion 15 Regulation of gene expression 15 Regulation of secretion 16 Activation of trypsinogen to trypsin 17 Activation by enteropeptidase 17 Autoactivation 17 Activation by cathepsin B 17 Factors affecting trypsinogen activation 18 Trypsinogen and trypsin structure and mechanism of catalysis 20 Structure 20 Substrate binding 21 Catalysis 22 Functions of trypsins 23 Digestion of food 23 Activation of protease-activated receptors 23 Trypsinogens in cancer 25 Other functions of trypsins 26 Trypsin inhibitors 27 α2-macroglobulin 28 α1-proteinase inhibitor 28 Inter-α-inhibitors 28 PSTI or TATI 29 Polyamines 31 Pancreatitis 31 Hereditary pancreatitis 32 Clinical value of trypsinogen determinations 34 Post-translational modification of proteins 36 Tyrosine O-sulfation 36 Effects of tyrosine-sulfation 37 Post-translational modification of pancreatic trypsinogens 38 Aims of the present study 39 Materials and methods 40 Samples, patients and cell lines (I- IV) 40 Monoclonal antibodies (I) 40 Time-resolved immunofluorometric assays (I) 40 Radioimmunoassays (I and II) 41 Characterization of trypsinogens by gel filtration chromatography (I and II) 42 Purification of trypsinogen (I, II and IV) 42 3 Separation of trypsinogen isoenzymes by anion exchange and RP HPLC (I, III and IV) 42 Activation of trypsinogen isoenzymes (I and II) 43 Alkylation and digestion of trypsinogens (IV) 43 Mass spectrometry (IV) 43 Data analysis (IV) 44 N-terminal sequence analysis (IV) 44 Electrophoresis and immunoblotting (II and IV) 44 Statistical analysis (II) 44 Results 45 Immunoassays for trypsinogens/trypsin and concentrations in serum samples (I, III) 45 TAT and TATI concentrations in ovarian tumor cyst fluids (II) 47 Characterization of trypsinogen immunoreactivity by gel filtration (I, II) 47 Trypsinogens in anion exchange chromatography (I, III) 48 Purification of trypsinogen by reverse-phase HPLC (II, IV) 49 MS-analysis of trypsin and trypsinogen isoenzymes (IV) 49 Identification of the tryptic peptide with 80 Da mass addition (IV) 50 Identification of Tyr154 sulfation in trypsinogen-1 and -2 (IV) 50 Discussion 54 TR-IFMAs for trypsinogen-1 and -2 54 Immunoreactive trypsinogen-1 and -2 in serum samples 54 Immunoreactive trypsinogen-1 and -2 in cancer 56 Immunoreactive TATI in cancer 56 Characterization of pancreatic and extra-pancreatic trypsinogens 57 Investigation of Tyr154 modification 58 Aromatic interactions in proteins 59 The effect of tyrosine sulfation on trypsin 59 Conclusions 61 Concluding remarks 62 Acknowledgements 64 References 66 4 Original publications This thesis is based on the following articles which are referred to in the text by their Roman numerals: I Itkonen O, Koivunen E, Hurme M, Alfthan H, Schröder T and Stenman UH. Time-resolved immunofluorometric assays for trypsinogen-1 and 2 in serum reveal preferential elevation of trypsinogen-2 in pancreatitis. J Lab Clin Med 115, 712-718 (1990). II Koivunen E, Itkonen O, Halila H and Stenman UH. Cyst fluid of ovarian cancer patients contains high concentrations of trypsinogen-2. Cancer Res 50, 2375-2378 (1990). III Itkonen O, Stenman UH, Osman S, Koivunen E, Halila H and Schröder T. Serum samples from pancreatectomized patients contain trypsinogen immunoreactivity. J Lab Clin Med 128, 98-102 (1996). IV Itkonen O, Helin J, Saarinen J, Kalkkinen N, Ivanov KI, Stenman UH and Valmu L. Mass spectrometric detection of tyrosine sulfation in human pancreatic trypsinogens, but not in tumor-associated trypsinogen. FEBS J 275, 289-301 (2008). Publication II was also included in the thesis entitled “Tumor-associated trypsinogen” by Ph.D, docent Erkki Koivunen, Department of Biological and Environmental Sciences, University of Helsinki, 1991. 5 Abbreviations AA amino acid API α1-proteinase inhibitor CCK cholecystokinin CF cystic fibrosis CTSB cathepsin B ECM extracellular matrix ESI electrospray ionization HP hereditary pancreatitis IRT immunoreactive trypsinogen LC liquid chromatography MAb monoclonal antibody MMP matrix metalloproteinase MS mass spectrometry MSMS tandem mass spectrometry PTM post-translational modification RIA radioimmunoassay RP reverse-phase TAP trypsinogen activation peptide TAT tumor-associated trypsinogen TATI tumor-associated trypsin inhibitor TIMP tissue inhibitor of metalloproteinase TPST tyrosylprotein sulfotransferase TR-IFMA time-resolved immunofluorometric assay 6 Abstract Human pancreatic juice contains two major Serum samples from patients who had trypsinogen isoenzymes called trypsinogen-1 undergone pancreatoduodenectomy contained and -2, or cationic and anionic trypsinogen, trypsinogen-2. Trypsinogen-1 was detected respectively. Trypsinogen isoenzymes are also in only one of nine samples. These results expressed in various normal and malignant suggested that the expression of trypsinogen tissues. We aimed at developing monoclonal is not restricted to the pancreas. antibodies (MAbs) and time-resolved immunofluorometric assays recognizing Determination of the isoenzyme pattern by ion human trypsinogen-1 and -2, respectively. exchange chromatography revealed isoelectric Using these MAbs and assays we purified, variants of trypsinogen isoenzymes in serum characterized and quantitated trypsinogen samples. Intact trypsinogen isoenzymes isoenzymes in serum samples, ovarian cyst and tryptic and chymotryptic trypsinogen fluids and conditioned cell culture media. peptides were purified and characterized by mass spectrometry, Western blot analysis and In sera from healthy subjects and patients N-terminal sequencing. The results showed with extrapancreatic disease the concentration that pancreatic trypsinogen-1 and -2 are of trypsinogen-1 is higher than that of sulfated at tyrosine 154 (Tyr154), whereas trypsinogen-2. However, in acute pancreatitis TAT-2 from a colon carcinoma cell line is not. we found that the concentration of serum Tyr154 is located within the primary substrate trypsinogen-2 is 50-fold higher than in controls, binding pocket of trypsin, thus Tyr154 whereas the difference in trypsinogen-1 sulfation is likely to influence substrate concentration is only 15-fold. This suggested binding. The previously known differences that trypsinogen-2 could be used as a diagnostic in charge, substrate specificity and inhibitor marker for acute pancreatitis. binding between pancreatic and tumor- associated trypsinogens are suggested to be In human ovarian cyst fluids tumor-associated caused by sulfation of Tyr154 in pancreatic trypsinogen-2 (TAT-2) is the predominant trypsinogens. isoenzyme. Most notably, in mucinous cyst fluids the levels of TAT-2 were higher in borderline and malignant than in benign cases. The increased levels in association with malignancy suggested that TAT could be involved in ovarian tumor dissemination and breakage of tissue barriers. 7 Review of the literature The terms “enzyme” (from the Greek en - in corresponded to those of pancreatic and zume - yeast) and “trypsin” were first trypsinogen-1 and -2, respectively. However, suggested by a german scientist, Wilhelm the isoenzymes had different specificities for (Willy) Friedrich Kühne (1837-1900), when p-nitroanilide substrates, responded differently he found a substance in bovine pancreatic to various protease inhibitors and had different juice that degraded other biological isoelectric points from those of trypsinogen-1 substances. He proposed the term “enzyme” and -2. Therefore, they were named tumor- for non-organized ferments and “trypsin” for associated trypsinogen-1 and -2 (TAT-1 and the enzyme that breaks down proteins. Kühne TAT-2) (Koivunen et al., 1989). presented this paper in the 4th February 1876 to the Heidelberger Naturhistorischen und Properties and biochemical Medizinische Verein, and was reprinted in characterization of human 1976 (Kühne, 1976). pancreatic trypsinogens Trypsinogen was first characterized from cattle Trypsinogen (Enzyme Commission (EC) pancreatic extracts (Kunitz and Northrop, number 3.4.21.4) was first reported to occur 1934, Northrop and Kunitz, 1932). In a study in human pancreatic juice by Haverback et of Kunitz and Northrop (Kunitz and Northrop, al. (Haverback et al., 1960) and was among 1935) bovine trypsinogen was shown to be the first human enzymes to be purified activated either by enteropeptidase or active and characterized (Buck et al., 1962). trypsin, indicating
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