DOCSLIB.ORG

Stability Profiles of Nepenthesin in Urea and Guanidine Hydrochloride

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Stability Profiles of Nepenthesin in Urea and Guanidine Hydrochloride Biosci. Biotechnol. Biochem., 74 (11), 2323–2326, 2010 Note Stability Profiles of Nepenthesin in Urea and Guanidine Hydrochloride: Comparison with Porcine Pepsin A Keiko KUBOTA,1;* Yuya METOKI,1 Senarath B. P. ATHAUDA,1;2 y Chiaki SHIBATA,3 and Kenji TAKAHASHI1; 1Laboratory of Molecular Biochemistry, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan 2Department of Biochemistry, Faculty of Medicine, University of Peradeniya, Peradeniya, Sri Lanka 3Department of Biology, School of Life Dentistry, The Nippon Dental University, Chiyoda-ku, Tokyo 102-8159, Japan Received May 20, 2010; Accepted July 26, 2010; Online Publication, November 7, 2010 [doi:10.1271/bbb.100391] Nepenthesin, an aspartic endopeptidase from the When a crude digestive fluid of N. alata was pitcher fluid of Nepenthes, was found to be markedly incubated at different temperatures for 1 h, activity was less stable than porcine pepsin A when treated with urea stable up to about 53 C. Then it started to decrease, and or guanidine hydrochloride. This is in sharp contrast was lost completely at about 80 C.4) This result is with its remarkably high pH/temperature stability as similar to those obtained with nepenthesin I and crude compared with porcine pepsin A. No protein with such a fluid from N. distillatoria.2,3) On the other hand, when stability profile has been reported to date. crude fluid of N. alata was incubated at pH 2.9 at 37 C, activity decreased only very slowly on longer incuba- Key words: denaturation; guanidine hydrochloride; tion. About 60% and 30% of the original activity were nepenthesin; porcine pepsin A; urea retained after 30 d and 65 d of incubation respectively.4) Thus, the activity of nepenthesins in the crude digestive Nepenthesin is a novel aspartic endopeptidase pro- fluid of N. alata was also rather stable under the duced by the carnivorous plant Nepenthes (MEROPS conditions examined. Recently, nepenthesin I was also subfamily A1B).1,2) Previously, we purified two iso- cloned from N. alata and sequenced.5) The sequence forms of nepenthesin, nepenthesin I and II, to homoge- identity was 94.7% for nepenthesin I from N. gracilis. neity from the pitcher fluid of Nepenthes distillatoria, It also has 12 residues of cysteine per molecule,5) and determined their enzymatic and structural character- like those from N. gracilis. In order to obtain more istics, including cloning and sequencing of cDNAs of information on the stability of nepenthesins, in this study nepenthesins from N. gracilis.2,3) Although the enzymes we investigated the effects of the denaturing agents share active site motifs, including two catalytic aspartic urea and guanidine hydrochloride (HCl) on the stability acid residues and the flap tyrosine residue, with pepsin- of nepenthesins purified from the pitcher fluid of type aspartic peptidases (MEROPS subfamily A1A), N. alata by measuring changes in activity toward they are different in several other respects. Among hemoglobin. these, a higher content of cysteine residues and Nepenthesin was purified from the pitcher fluid of remarkable stability at relatively high temperatures over N. alata essentially as described previously2) for the a wide range of pH levels are notable. When nepenthe- enzymes from N. distillatoria, with various modifica- sin I from N. distillatoria was incubated at pH 3.0 at tions. Batchwise treatment with DEAE-cellulose and 60 C for 30 d, about 30% of the original activity chromatography on Sephacryl S-200 were omitted. In remained, whereas porcine pepsin A was completely DEAE-cellulose chromatography on a DE-32 column inactivated at pH 3.0 at 50 C within 7 d. Furthermore, (5:0 Â 15:0 cm), nepenthesin II was partially separated when incubated at pH 3.0–10.0 at 37 C for 30 d, into two peaks, designated nepenthesins IIa and IIb, and nepenthesin I retained 80% or more of activity, whereas these were purified separately in the following steps of porcine pepsin A was completely inactivated under the chromatography on a pepstatin-agarose column (1:5 Â same conditions. Porcine pepsin A was extremely 3:0 cm) and a MonoQ column (0:5 Â 5:0 cm). Thus unstable at 7.0 and above, at which it was irreversibly 4.25 mg, 0.47 mg, and 0.35 mg of purified nepenthesins inactivated almost instantly. Nepenthesin II was also I, IIa, and IIb respectively were obtained from 500 ml of very stable, although it was slightly less stable than crude digestive fluid. Nepenthesins IIa and IIb showed nepenthesin I. Nepenthesins contain 12 residues of the same behavior on pepstatin-agarose chromatography cysteine per molecule, twice as many as porcine pepsin and native polyacrylamide gel electrophoresis followed A, that presumably form six disulfide bonds, and these by activity staining. Part of each pooled fraction was disulfide bonds are thought to contribute greatly to their dialyzed against 0.1 M glycine/HCl buffer, pH 2.0, in unusual pH/temperature stability. the subsequent studies. y To whom correspondence should be addressed. Fax: +81-49-297-8168; E-mail: [email protected] * Present address: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Abbreviations: DTT, dithiothreitol; 3D, three-dimensional 2324 K. KUBOTA et al. Figure 1b shows the effects of guanidine HCl on the a activity of nepenthesins and porcine pepsin A. Porcine pepsin A was fairly stable. It was stable up to 3.6 M guanidine HCl, and then started to lose activity, and was completely inactivated at 5.0 M guanidine HCl. These results are also consistent with those reported previously.14) On the other hand, nepenthesins were found to be much less stable, and were completely inactivated at 3.0 M guanidine HCl. The midpoints of denaturation were estimated to be 4.3 M, 0.4 M, 1.2 M, and 1.8 M guanidine HCl for porcine pepsin A and nepenthesins I, IIa, and IIb respectively. The midpoints b of denaturation of nepenthesins I, IIa, and IIb are different as between urea and guanidine HCl. The reason for this difference is not clear at present. Further molecular characterization of these enzymes is neces- sary in this regard. DTT, a typical reducing agent for proteins, was found more or less to enhance denaturation of the various enzymes. In the absence of urea or guanidine HCl, however, DTT alone did not much affect their stability. On the other hand, in the presence of urea or guanidine HCl, denaturation of the enzymes was markedly accerelated by DTT. Porcine pepsin A and nepenthesins Fig. 1. Effects of Urea (a) and Guanidine HCl (b) on the Activity of I, IIa, and IIb retained over 90% of the original activity Nepenthesins and Porcine Pepsin A. in the presence of 3.59 M guanidine HCl, 0.67 M urea, , Nepenthesin I; , nepenthesin IIa; , nepenthesin IIb; , 1.46 M urea, and 0.67 M urea respectively, as shown in crude digestive fluid; , porcine pepsin A. Fig. 1. These activities were completely or almost completely lost when 40–70 mM DTT was present (data The enzyme assay was performed at pH 2.0 with not shown). This is clearly due to breakage of the hemoglobin as substrate, essentially as described pre- disulfide bonds by DTT in the presence of urea or viously.2) Urea, guanidine HCl, and dithiothreitol (DTT) guanidine HCl, and it shows their importance for were purchased from Wako Pure Chemical Industries stability. It also confirms that the cysteine residues in (Tokyo), and porcine pepsin A from Sigma (St. Louis, nepenthesins form disulfide bonds in the native state. Mo). Other reagents used were of analytical grade. From these results, however, it is difficult to evaluate the The effects of urea and guanidine HCl on activity relative importance of the disulfide bonds for protein were measured by incubating the enzyme (approximately stability as between porcine pepsin A and nepenthesins. 3–7 ng/500 ml) at pH 2.0 at room temperature (about Nepenthesins have been found to exhibit marked pH/ 25 C) for 15 min in the presence of various concen- temperature stability as compared with porcine pepsin trations of urea or guanidine HCl, followed by enzyme A.2,3) The higher content of disulfide bonds is thought to assay. When necessary, various concentrations of DTT contribute greatly to stabilization of the native con- were added to the mixtures. In the assays, the hemo- formation by rendering the enzymes more resistant to globin assay mixture contained the same concentration denaturation. Figure 2 shows the location of the disul- of various denaturants and/or DTT as in the preceding fide bonds in the 3D structures of nepenthesin I (Fig. 2a, incubation, and the time of digestion was prolonged to model structure) and porcine pepsin A15) (Fig. 2b, PDB 3 h. Porcine pepsin A was treated in the same manner ID PEP5). It is notable that five disulfide bonds were for comparison. present in the N-terminal lobe (the right half of the The three-dimensional (3D) structure of nepenthesin I molecule) of nepenthesin I, whereas only one disulfide was predicted by the homology modeling method of bond was present in that of porcine pepsin A. As for CPHmodels,6) using the crystal structure of human porcine pepsin A, it was reported that in the course of gastricsin (pepsin C, PDB ID lavf).7) The secondary thermal denaturation disruption of the N-terminal lobe structures were predicted by the GOR4 program8,9) and (residues 1–179, 307–327) is the first event, which leads according to Chou and Fasman.10) A hydropathy plot to subsequent desruption of the C-terminal lobe (the left was obtained by the method of Kyte and Doolittle.11,12) half of the molecule, residues 180–306).16) Since the Figure 1a shows the effects of urea on the activity of N-terminal lobe of nepenthesin I is thought to be much nepenthesins and porcine pepsin A.
Load more

Recommended publications
  • Membrane Proteins • Cofactors – Plimstex • Membranes • Dna • Small Molecules/Gas • Large Complexes
    Structural mass spectrometry hydrogen/deuterium exchange Petr Man Structural Biology and Cell Signalling Institute of Microbiology, Czech Academy of Sciences Structural biology methods Low-resolution methods High-resolution methods Rigid SAXS IR Raman CD ITC MST Cryo-EM AUC SPR MS X-ray crystallography Chemical cross-linking H/D exchange Native ESI + ion mobility Oxidative labelling Small Large NMR Dynamic Structural biology approaches Simple MS, quantitative MS Cross-linking, top-down, native MS+dissociation native MS+ion mobility Cross-linking Structural MS What can we get using mass spectrometry IM – ion mobility CXL – chemical cross-linking AP – afinity purification OFP – oxidative footprinting HDX – hydrogen/deuterium exchange ISOTOPE EXCHANGE IN PROTEINS 1H 2H 3H occurence [%] 99.988 0.0115 trace 5 …Kaj Ulrik Linderstrøm-Lang „Cartesian diver“ Proteins are migrating in tubes with density gradient until they stop at the point where the densities are equal 1H 2H 3H % 99.9885 0.0115 trace density [g/cm3] 1.000 1.106 1.215 Methods of detection IR: β-: NMR: 1 n = 1.6749 × 10-27 kg MS: 1H 2H 3H výskyt% [%] 99.9885 0.0115 trace hustotadensity vody [g/cm [g/cm3] 3] 1.000 1.106 1.215 jadernýspinspin ½+ 1+ ½+ mass [u] 1.00783 2.01410 3.01605 Factors affecting H/D exchange hydrogen bonding solvent accessibility Factors affecting H/D exchange Side chains (acidity, steric shielding) Bai et al.: Proteins (1993) Glasoe, Long: J. Phys. Chem. (1960) Factors affecting H/D exchange – side chain effects Inductive effect – electron density is Downward shift due to withdrawn from peptide steric hindrance effect of bond (S, O).
    [Show full text]
  • Discovery of Digestive Enzymes in Carnivorous Plants with Focus on Proteases
    A peer-reviewed version of this preprint was published in PeerJ on 5 June 2018. View the peer-reviewed version (peerj.com/articles/4914), which is the preferred citable publication unless you specifically need to cite this preprint. Ravee R, Mohd Salleh F‘, Goh H. 2018. Discovery of digestive enzymes in carnivorous plants with focus on proteases. PeerJ 6:e4914 https://doi.org/10.7717/peerj.4914 Discovery of digestive enzymes in carnivorous plants with focus on proteases Rishiesvari Ravee 1 , Faris ‘Imadi Mohd Salleh 1 , Hoe-Han Goh Corresp. 1 1 Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia Corresponding Author: Hoe-Han Goh Email address: [email protected] Background. Carnivorous plants have been fascinating researchers with their unique characters and bioinspired applications. These include medicinal trait of some carnivorous plants with potentials for pharmaceutical industry. Methods. This review will cover recent progress based on current studies on digestive enzymes secreted by different genera of carnivorous plants: Drosera (sundews), Dionaea (Venus flytrap), Nepenthes (tropical pitcher plants), Sarracenia (North American pitcher plants), Cephalotus (Australian pitcher plants), Genlisea (corkscrew plants), and Utricularia (bladderworts). Results. Since the discovery of secreted protease nepenthesin in Nepenthes pitcher, digestive enzymes from carnivorous plants have been the focus of many studies. Recent genomics approaches have accelerated digestive enzyme discovery. Furthermore, the advancement in recombinant technology and protein purification helped in the identification and characterisation of enzymes in carnivorous plants. Discussion. These different aspects will be described and discussed in this review with focus on the role of secreted plant proteases and their potential industrial applications.
    [Show full text]
  • Serine Proteases with Altered Sensitivity to Activity-Modulating
    (19) & (11) EP 2 045 321 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 08.04.2009 Bulletin 2009/15 C12N 9/00 (2006.01) C12N 15/00 (2006.01) C12Q 1/37 (2006.01) (21) Application number: 09150549.5 (22) Date of filing: 26.05.2006 (84) Designated Contracting States: • Haupts, Ulrich AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 51519 Odenthal (DE) HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI • Coco, Wayne SK TR 50737 Köln (DE) •Tebbe, Jan (30) Priority: 27.05.2005 EP 05104543 50733 Köln (DE) • Votsmeier, Christian (62) Document number(s) of the earlier application(s) in 50259 Pulheim (DE) accordance with Art. 76 EPC: • Scheidig, Andreas 06763303.2 / 1 883 696 50823 Köln (DE) (71) Applicant: Direvo Biotech AG (74) Representative: von Kreisler Selting Werner 50829 Köln (DE) Patentanwälte P.O. Box 10 22 41 (72) Inventors: 50462 Köln (DE) • Koltermann, André 82057 Icking (DE) Remarks: • Kettling, Ulrich This application was filed on 14-01-2009 as a 81477 München (DE) divisional application to the application mentioned under INID code 62. (54) Serine proteases with altered sensitivity to activity-modulating substances (57) The present invention provides variants of ser- screening of the library in the presence of one or several ine proteases of the S1 class with altered sensitivity to activity-modulating substances, selection of variants with one or more activity-modulating substances. A method altered sensitivity to one or several activity-modulating for the generation of such proteases is disclosed, com- substances and isolation of those polynucleotide se- prising the provision of a protease library encoding poly- quences that encode for the selected variants.
    [Show full text]
  • The Coordinate Regulation of Digestive Enzymes in the Pitchers of Nepenthes Ventricosa
    Rollins College Rollins Scholarship Online Honors Program Theses Spring 2020 The Coordinate Regulation of Digestive Enzymes in the Pitchers of Nepenthes ventricosa Zephyr Anne Lenninger [email protected] Follow this and additional works at: https://scholarship.rollins.edu/honors Part of the Plant Biology Commons Recommended Citation Lenninger, Zephyr Anne, "The Coordinate Regulation of Digestive Enzymes in the Pitchers of Nepenthes ventricosa" (2020). Honors Program Theses. 120. https://scholarship.rollins.edu/honors/120 This Open Access is brought to you for free and open access by Rollins Scholarship Online. It has been accepted for inclusion in Honors Program Theses by an authorized administrator of Rollins Scholarship Online. For more information, please contact [email protected]. The Coordinate Regulation of Digestive Enzymes in the Pitchers of Nepenthes ventricosa Zephyr Lenninger Rollins College 2020 Abstract Many species of plants have adopted carnivory as a way to obtain supplementary nutrients from otherwise nutrient deficient environments. One such species, Nepenthes ventricosa, is characterized by a pitcher shaped passive trap. This trap is filled with a digestive fluid that consists of many different digestive enzymes, the majority of which seem to have been recruited from pathogen resistance systems. The present study attempted to determine whether the introduction of a prey stimulus will coordinately upregulate the enzymatic expression of a chitinase and a protease while also identifying specific chitinases that are expressed by Nepenthes ventricosa. We were able to successfully clone NrCHIT1 from a mature Nepenthes ventricosa pitcher via a TOPO-vector system. In order to asses enzymatic expression, we utilized RT-qPCR on pitchers treated with chitin, BSA, or water.
    [Show full text]
  • ( 12 ) United States Patent
    US009745565B2 (12 ) United States Patent (10 ) Patent No. : US 9 , 745 , 565 B2 Schriemer (45 ) Date of Patent: * Aug. 29 , 2017 ( 54 ) TREATMENT OF GLUTEN INTOLERANCE FOREIGN PATENT DOCUMENTS AND RELATED CONDITIONS EP 2 090 662 A2 8 /2009 WO WO - 2010 /021752 2 / 2010 ( 71 ) Applicant: Nepety , LLC , Destin , FL (US ) WO WO - 2011 /097266 8 / 2011 ( 72 ) Inventor: David Schriemer , Chestermere (CA ) WO WO - 2011 / 126873 10 / 2011 ( 73 ) Assignee : NEPETX , LLC , Destin , FL (US ) OTHER PUBLICATIONS Adlassnig W et al . Traps of carnivorous pitcher plants as a habitat: ( * ) Notice : Subject to any disclaimer, the term of this composition of the fluid , biodiversity and mutualistic activities . patent is extended or adjusted under 35 2011. Annals of Botany . 107 : 181 - 194 . * U . S . C . 154 (b ) by 98 days . Amagase , et al . , “ Acid Protease in Nepenthes, " The Journal of Biochemistry , ( 1969 ) , 66 ( 4 ) :431 -439 . This patent is subject to a terminal dis Athauda , et al . “ Enzymic and structural characterization of claimer . nepenthesin , a unique member of a novel subfamily of aspartic proteinases , ” Biochemical Journal ( 2004 ) 381 ( 1 ) :295 - 306 . (21 ) Appl. No. : 14/ 506 ,456 Bennett et al. , “ Discovery and Characterization of the Laulimalide Microtubule Binding Mode by Mass Shift Perturbation Mapping ," Chemistry & Biology , (2010 ) , 17 :725 - 734 . ( 22 ) Filed : Oct. 3 , 2014 Bethune, et al. , " Oral enzyme therapy for celiac sprue ,” Methods Enzymol. , (2012 ) , 502 :241 - 271 . (65 65) Prior Publication Data Blonder et al. , “ Proteomic investigation of natural killer cell US 2015 /0265686 A1 Sep . 24 , 2015 microsomes using gas -phase fractionation by mass spectrometry, " Biochimica et Biophysica Acta , (2004 ) , 1698 :87 -95 .
    [Show full text]
  • 20Th International Mass Spectrometry Conference
    IMSC 2014 20th International Mass Spectrometry Conference August 24-29, 2014 Geneva, Switzerland PROGRAM v. 17.09.2014 More targets. More accurately. Faster than ever. Analytical challenges grow in quantity and complexity. Quantify a larger number of compounds and more complex analytes faster and more accurately with our new portfolio of LC-MS instruments, sample prep solutions and software. High-resolution, accurate mass solutions using Thermo Scientific™ Orbitrap™ MS quantifies all detectable compounds with high specificity, and triple quadrupole MS delivers SRM sensitivity and speed to detect targeted compounds more quickly. Join us in meeting today’s challenges. Together we’ll transform quantitative science. Quantitation transformed. • Discover more at thermoscientific.com/quan-transformed • Visit thermoscientific.com/imsc or booth 23 for more information © 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are © 2014 Thermo Fisher Scientific Inc. All rights the property of Thermo Fisher Scientific and its subsidiaries. the property Thermo Scientific™ Q Exactive™ HF MS Thermo Scientific™ TSQ Quantiva™ MS Thermo Scientific™ TSQ Endura™ MS Screen and quantify known and unknown targets Leading SRM sensitivity and speed Ultimate SRM quantitative value and with HRAM Orbitrap technology in a triple quadrupole MS/MS unprecedented usability TABLE OF CONTENTS th 1. Welcome from the Chairs of the 20 IMSC ............................................................................................................................
    [Show full text]
  • Arabidopsis Thaliana Atypical Aspartic Proteases Involved in Primary Root Development and Lateral Root Formation
    André Filipe Marques Soares RLR1 and RLR2, two novel Arabidopsis thaliana atypical aspartic proteases involved in primary root development and lateral root formation 2016 Thesis submitted to the Institute for Interdisciplinary Research of the University of Coimbra to apply for the degree of Doctor in Philosophy in the area of Experimental Biology and Biomedicine, specialization in Molecular, Cell and Developmental Biology This work was conducted at the Center for Neuroscience and Cell Biology (CNC) of University of Coimbra and at Biocant - Technology Transfer Association, under the scientific supervision of Doctor Isaura Simões and at the Department of Biochemistry of University of Massachusetts, Amherst, under the scientific supervision of Doctor Alice Y. Cheung. Part of this work was also performed at the Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, under the scientific supervision of Doctor Herta Steinkellner and also at the Central Institute for Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich, Jülich, under the schientific supervision of Doctor Pitter F. Huesgen. André Filipe Marques Soares was a student of the Doctoral Programme in Experimental Biology and Biomedicine coordinated by the Center for Neuroscience and Cell Biology (CNC) of the University of Coimbra and a recipient of the fellowship SFRH/BD/51676/2011 from the Portuguese Foundation for Science and Technology (FCT). The execution of this work was supported by a PPP grant of the German Academic Exchange Service with funding from the Federal Ministry of Education and Research (Project-ID 57128819 to PFH) and the Fundação para a Ciência e a Tecnologia (FCT) (grant: Scientific and Technological Bilateral Agreement 2015/2016 to IS) Agradecimentos/Acknowledgments Esta tese e todo o percurso que culminou na sua escrita não teriam sido possíveis sem o apoio, o carinho e a amizade de várias pessoas que ainda estão ou estiveram presentes na minha vida.
    [Show full text]
  • Molecular Properties and New Potentials of Plant Nepenthesins
    plants Review Molecular Properties and New Potentials of Plant Nepenthesins Zelalem Eshetu Bekalu *, Giuseppe Dionisio and Henrik Brinch-Pedersen Department of Agroecology, Research Center Flakkebjerg, Aarhus University, DK-4200 Slagelse, Denmark; [email protected] (G.D.); [email protected] (H.B.-P.) * Correspondence: [email protected] Received: 10 April 2020; Accepted: 28 April 2020; Published: 30 April 2020 Abstract: Nepenthesins are aspartic proteases (APs) categorized under the A1B subfamily. Due to nepenthesin-specific sequence features, the A1B subfamily is also named nepenthesin-type aspartic proteases (NEPs). Nepenthesins are mostly known from the pitcher fluid of the carnivorous plant Nepenthes, where they are availed for the hydrolyzation of insect protein required for the assimilation of insect nitrogen resources. However, nepenthesins are widely distributed within the plant kingdom and play significant roles in plant species other than Nepenthes. Although they have received limited attention when compared to other members of the subfamily, current data indicates that they have exceptional molecular and biochemical properties and new potentials as fungal-resistance genes. In the current review, we provide insights into the current knowledge on the molecular and biochemical properties of plant nepenthesins and highlights that future focus on them may have strong potentials for industrial applications and crop trait improvement. Keywords: Plants; aspartic proteases; nepenthesins; molecular properties; industrial applications; trait improvement 1. Introduction Aspartic proteases (APs, EC 3.4.23) comprise the second largest family of plant proteases. Plant APs have been identified from a wide range of plant species and tissues as extensively reviewed in Simões and Faro [1].
    [Show full text]
  • Handbook of Proteolytic Enzymes Second Edition Volume 1 Aspartic and Metallo Peptidases
    Handbook of Proteolytic Enzymes Second Edition Volume 1 Aspartic and Metallo Peptidases Alan J. Barrett Neil D. Rawlings J. Fred Woessner Editor biographies xxi Contributors xxiii Preface xxxi Introduction ' Abbreviations xxxvii ASPARTIC PEPTIDASES Introduction 1 Aspartic peptidases and their clans 3 2 Catalytic pathway of aspartic peptidases 12 Clan AA Family Al 3 Pepsin A 19 4 Pepsin B 28 5 Chymosin 29 6 Cathepsin E 33 7 Gastricsin 38 8 Cathepsin D 43 9 Napsin A 52 10 Renin 54 11 Mouse submandibular renin 62 12 Memapsin 1 64 13 Memapsin 2 66 14 Plasmepsins 70 15 Plasmepsin II 73 16 Tick heme-binding aspartic proteinase 76 17 Phytepsin 77 18 Nepenthesin 85 19 Saccharopepsin 87 20 Neurosporapepsin 90 21 Acrocylindropepsin 9 1 22 Aspergillopepsin I 92 23 Penicillopepsin 99 24 Endothiapepsin 104 25 Rhizopuspepsin 108 26 Mucorpepsin 11 1 27 Polyporopepsin 113 28 Candidapepsin 115 29 Candiparapsin 120 30 Canditropsin 123 31 Syncephapepsin 125 32 Barrierpepsin 126 33 Yapsin 1 128 34 Yapsin 2 132 35 Yapsin A 133 36 Pregnancy-associated glycoproteins 135 37 Pepsin F 137 38 Rhodotorulapepsin 139 39 Cladosporopepsin 140 40 Pycnoporopepsin 141 Family A2 and others 41 Human immunodeficiency virus 1 retropepsin 144 42 Human immunodeficiency virus 2 retropepsin 154 43 Simian immunodeficiency virus retropepsin 158 44 Equine infectious anemia virus retropepsin 160 45 Rous sarcoma virus retropepsin and avian myeloblastosis virus retropepsin 163 46 Human T-cell leukemia virus type I (HTLV-I) retropepsin 166 47 Bovine leukemia virus retropepsin 169 48
    [Show full text]
  • Orphan Enzymes Could Be an Unexplored Reservoir of New Drug Targets
    Drug Discovery Today Volume 11, Numbers 7/8 April 2006 REVIEWS Reviews GENE TO SCREEN Orphan enzymes could be an unexplored reservoir of new drug targets Olivier Lespinet and Bernard Labedan Institut de Ge´ne´tique et Microbiologie, CNRS UMR 8621, Universite´ Paris Sud, Baˆtiment 400, 91405 Orsay Cedex, France Despite the immense progress of genomics, and the current availability of several hundreds of thousands of amino acid sequences, >39% of well-defined enzyme activities (as represented by enzyme commission, EC, numbers) are not associated with any sequence. There is an urgent need to explore the 1525 orphan enzymes (enzymes having EC numbers without an associated sequence) to bridge the wide gap that separates knowledge of biochemical function and sequence information. Strikingly, orphan enzymes can even be found among enzymatic activities successfully used as drug targets. Here, knowledge of sequence would help to develop molecular-targeted therapies, suppressing many drug-related side-effects. Biology is exploring numerous and diverse fields, each of which discovered before, despite intensive research by thousands of is very complex and difficult to study in its entirety. For many people studying the genetics and biochemistry of Saccharomyces years, immense advances in disclosing molecular functions have cerevisiae over the past 50 years. This observation has been con- been made using reductionist approaches, such as molecular firmed repeatedly, with the cohort of genomes that have been biology. Combining genetic, biophysical and biochemical con- sequenced, at a steady pace, over the past ten years. We now know cepts and methodologies has helped to disclose details of com- that many genes have been missed by reductionist approaches plex molecular mechanisms such as DNA replication.
    [Show full text]
  • Molecular Cloning and Characterization of Procirsin, an Active Aspartic Protease Precursor from Cirsium Vulgare (Asteraceae)
    Phytochemistry 81 (2012) 7–18 Contents lists available at SciVerse ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Molecular cloning and characterization of procirsin, an active aspartic protease precursor from Cirsium vulgare (Asteraceae) Daniela Lufrano a, Rosário Faro b, Pedro Castanheira c, Gustavo Parisi d, Paula Veríssimo b,e, ⇑ Sandra Vairo-Cavalli a, Isaura Simões b,c, , Carlos Faro b,c,e a Laboratorio de Investigación de Proteínas Vegetales (LIPROVE), Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, C.C. 711, 1900 La Plata, Argentina b CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal c Biocant, Biotechnology Innovation Center, 3060-197 Cantanhede, Portugal d Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Roque Sáenz Peña 352, Bernal, Buenos Aires, B1876BXD, Argentina e Department of Life Sciences, University of Coimbra, 3004-517 Coimbra, Portugal article info abstract Article history: Typical aspartic proteinases from plants of the Astereaceae family like cardosins and cyprosins are well- Received 16 February 2012 known milk-clotting enzymes. Their effectiveness in cheesemaking has encouraged several studies on Received in revised form 23 May 2012 other Astereaceae plant species for identification of new vegetable rennets. Here we report on the clon- Available online 21 June 2012 ing, expression and characterization of a novel aspartic proteinase precursor from the flowers of Cirsium vulgare (Savi) Ten. The isolated cDNA encoded a protein product with 509 amino acids, termed cirsin, Keywords: with the characteristic primary structure organization of plant typical aspartic proteinases. The pro form Cirsium vulgare of cirsin was expressed in Escherichia coli and shown to be active without autocatalytically cleaving its Asteraceae pro domain.
    [Show full text]
  • Nepenthesin Protease Activity Indicates Digestive Fluid Dynamics in Carnivorous Nepenthes Plants
    RESEARCH ARTICLE Nepenthesin Protease Activity Indicates Digestive Fluid Dynamics in Carnivorous Nepenthes Plants Franziska Buch1, Wendy E. Kaman2,3, Floris J. Bikker3, Ayufu Yilamujiang1, Axel Mithöfer1* 1 Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Hans Knöll Straße 8, D- 07745, Jena, Germany, 2 Department of Medical Microbiology and Infectious Diseases, Erasmus MC, `s- Gravendijkwal 230, 3015 CE, Rotterdam, The Netherlands, 3 Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam, Gustav Mahlerlaan 3004, 1081 LA, Amsterdam, The Netherlands * [email protected] Abstract Carnivorous plants use different morphological features to attract, trap and digest prey, mainly insects. Plants from the genus Nepenthes possess specialized leaves called pitch- ers that function as pitfall-traps. These pitchers are filled with a digestive fluid that is gener- ated by the plants themselves. In order to digest caught prey in their pitchers, Nepenthes OPEN ACCESS plants produce various hydrolytic enzymes including aspartic proteases, nepenthesins (Nep). Knowledge about the generation and induction of these proteases is limited. Here, Citation: Buch F, Kaman WE, Bikker FJ, Yilamujiang A, Mithöfer A (2015) Nepenthesin Protease Activity by employing a FRET (fluorescent resonance energy transfer)-based technique that uses a Indicates Digestive Fluid Dynamics in Carnivorous synthetic fluorescent substrate an easy and rapid detection of protease activities in the di- Nepenthes Plants. PLoS ONE 10(3): e0118853. gestive fluids of various Nepenthes species was feasible. Biochemical studies and the het- doi:10.1371/journal.pone.0118853 erologously expressed Nep II from Nepenthes mirabilis proved that the proteolytic activity Academic Editor: Dawn Sywassink Luthe, relied on aspartic proteases, however an acid-mediated auto-activation mechanism was Pennsylvania State University, UNITED STATES necessary.
    [Show full text]