DOCSLIB.ORG

Similarity with Serine Proteases (Primary Structure/Sequence Homology/Seminal Protease/Chymotrypsin-Like and Trypsin-Like Activity) KENNETH W

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Similarity with Serine Proteases (Primary Structure/Sequence Homology/Seminal Protease/Chymotrypsin-Like and Trypsin-Like Activity) KENNETH W Proc. Nati. Acad. Sci. USA Vol. 83, pp. 3166-3170, May 1986 Biochemistry Human prostate-specific antigen: Structural and functional similarity with serine proteases (primary structure/sequence homology/seminal protease/chymotrypsin-like and trypsin-like activity) KENNETH W. K. WATT*, PAUL-JANE LEE*, THABISO M'TIMKULUt, WAI-PAN CHAN*, AND RUEYMING LOORt Departments of *Protein Chemistry and tDiagnostics, Cetus Corp., 1400 Fifty-Third Street, Emeryville, CA 94608 Communicated by Russell F. Doolittle, December 31, 1985 ABSTRACT The complete amino acid sequence of the MATERIALS AND METHODS prostate-specific antigen (PA) from human seminal plasma has PA was purified from human seminal plasma as described (1). been determined from analyses of the peptides generated by PA was finally purified on a large-pore Vydac (Hesperia, CA) cyanogen bromide, hydroxylamine, endoproteinases Arg-C C4 column and eluted either with a linear gradient between and Lys-C. The single polypeptide chain of PA contains 70% (vol/vol) buffer A (buffer A = 0.1% trifluoroacetic acid 240-amino acid residues and has a calculated Mr of 26,496. An in H20) and 37% (vol/vol) buffer B (buffer B = 0.1% N-linked carbohydrate side chain is predicted at asparagine-45, trifluoroacetic acid in acetonitrile) in 280 min or 10% and O-linked carbohydrate side chains are possibly attached to (vol/vol) buffer B and 80% (vol/vol) buffer B in 60 min. serine-69, threonine-70, and serine-71. The primary structure The enzymatic activity ofPA was assessed on commercially of PA shows a high degree of sequence homology with other purified proteins including insulin A, insulin B, gelatin, serine proteases ofthe kallikrein family. The active site residues myoglobin, ovalbumin, fibrinogen (Sigma), and recombinant of histidine, aspartic acid, and serine comprising the charge- interleukin 2 (Ala-IL2, Cetus). The substrates (1 mg/ml) were relay system of typical serine proteases were found in similar dissolved in either 0.1 M ammonium bicarbonate or 50 mM positions in PA (histidine-41, aspartic acid-96, and serine-192). Tris HCl, pH 7.8, containing PA at 0.1 mg/ml. In some cases, At pH 7.8, PA hydrolyzed insulin A and B chains, recombinant a mass ratio (enzyme:substrate) of1:20 was used. After an 18-hr interleukin 2, and-to a lesser extent-gelatin, myoglobin, digestion at 37°C, the peptides were separated by HPLC using ovalbumin, and fibrinogen. The cleavage sites ofthese proteins a 60-min linear gradient of 0-80% (vol/vol) buffer B. To by PA were chemically analyzed as the a-carboxyl side of some determine the peptide bond specificity of PA, hydrazinolysis hydrophobic residues, tyrosine, leucine, valine, and phenylal- was performed on each ofthe PA/substrate digestion mixtures, anine, and of basic residues histidine, lysine, and arginine. The substrate alone, and intact PA as described (9). The free PA with the carboxyl-terminal amino acids were lyophilized and analyzed chymotrypsin-like activity of exhibited chromo- on a Beckman 121MB amino acid analyzer. genic substrate N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phen- The kinetics ofPA hydrolytic activity on synthetic substrates ylalanine p-nitroanilide yielded a specific activity of 9.21 ,uM were studied by monitoring the absorbance change at room per min per mg of PA and Km and krt values of 15.3 mM and temperature using a Hewlett-Packard 8450A spectrophotome- 0.075 s-1, respectively. "Trypsin-like" activity of PA was also ter. The following substrates were used: Na-benzoyl-L-tyrosine detected with Na-benzoyl-DL-arginine p-nitroanilide and gave ethyl ester (Bz-Tyr-OEt), Na-succinyl-phenylalanine nitroani- a specific activity of 1.98 ,uM per min per mg of PA. Protease lide (N-Suc-Phe-NA), Na-benzoyl-arginine-13-naphthylamide inhibitors such as phenylmethylsulfonyl fluoride, diisopropyl (Bz-Arg-NHNpt), Na-benzoyl-DL-arginine-p-nitroanilide (Bz- fluorophosphate, L-1-tosylamido-2-phenylethyl chloromethyl Arg-p-NA), Na-tosyl-L-arginine methyl ester (Tos-Arg-OMe) ketone, aprotinin, leupeptin, soybean trypsin inhibitor as well (Sigma), and Na-succinyl-L-alanyl-L-alanyl-L-prolyl-Lphenyl- as Zn2+ and spermidine were effective inhibitors of PA alanine-p-nitroanilide (N-Suc-Ala-Ala-Pro-Phe-p-NA) (Vega enzymatic activity. Biochemical, Tucson, AZ). The Km and the kg of PA on N-Suc-Ala-Ala-Pro-Phe-p-NA were calculated from Line- Human prostate-specific antigen (PA) is a glycoprotein with weaver-Burke plots. Inhibitors at 2 mM in 0.1 M CaCl2/0.1 M a Mr of 33,000-34,000 containing approximately 7% (wt/wt) Tris HCl (pH 8.0) were used to affect the PA activity on these PA is in the cells substrates. The following inhibitors were tested: phenylmethyl- carbohydrate (1, 2). detected only epithelial sulfonyl fluoride (PhMeSO2F), diisopropylfluorophosphate of the prostatic ductal element by a specific antibody to PA (DFP), L-1-tosylamido-2-phenylethyl chloromethyl ketone (1, 3). Seminal plasma also contains PA identical to that ofthe (Tos-PheCH2Cl), aprotinin, soybean trypsin inhibitor, zinc prostate gland (3). PA is synthesized in the epithelial cells of acetate, spermidine (Sigma), and leupeptin (Boehringer Mann- the prostate gland and secreted into the seminal fluid (3). heim). Recently, PA has been demonstrated to be clinically impor- The primary structure ofPA was established by automated tant for the detection and monitoring ofprostate cancer (4, 5). sequence analyses of intact polypeptide and by character- The PA molecule was independently identified as p30 (6) and ization of CNBr, hydroxylamine, endoproteinase Arg-C, and was tested as a marker for postcoital detection in rape endoproteinase Lys-C peptides. CNBr reaction was conduct- investigations (7). Although the clinical utility ofPA has been shown, its biological function and chemical structure are not Abbreviations: N-Suc-Ala-Ala-Pro-Phe-p-NA, Na-succinyl-L- well characterized (8). We report here the complete amino alanyl-L-analyl-L-prolyl-L-phenylalanine-p-nitroanilide; Bz-Arg-p- acid sequence of PA and describe the characteristics of its NA, Na-benzoyl-DL-arginine-p-nitroanilide; PhMeSO2F, phenyl- enzymatic properties. methylsulfonyl fluoride; DFP, diisopropylfluorophosphate; Tos- PheCH2Cl, L-1-tosylamido-2-phenylethyl chloromethyl ketone; Bz- Tyr-OEt, Na-benzoyl-L-tyrosine ethyl ester; N-Suc-Phe-NA, Na- The publication costs of this article were defrayed in part by page charge succinyl-phenylalanine nitroanilide; Bz-Arg-NHNpt, Na-benzoyl- payment. This article must therefore be hereby marked "advertisement" arginine-,f-naphthylamide; Tos-Arg-OMe, Na-tosyl-L-arginine in accordance with 18 U.S.C. §1734 solely to indicate this fact. methyl ester; PA, prostate-specific antigen. 3166 Downloaded by guest on September 30, 2021 Biochemistry: Watt et al. Proc. Nail. Acad. Sci. USA 83 (1986) 3167 I C~~~e~ Is a eC ed by dissolving the protein (5 mg/mi) in 70% (vol/vol) formic ii. d SI, GI. I.. b.1 C., Si. L.4 M. So, Si.Pahey Si, eta Laow letI AlafSor Are SI, re Ala Vel acid with the addition of solid CNBr (2-fold excess by weight) for 24 hr at room temperature. Hydroxylamine cleavage was Inleet PA carried out to Borustein and Balian (10). Digestion CC VI according -7 --7 7 --7-7 --7-7-7- -7 -7 -7 -7 --7 --7 of PA (5 mg/mi) with endoproteinase Mrg-C and endopro- Arg-C II a teinase Lys-C (Boehringer Mannheim) was performed in 0.1 k - Lye-C IV - Lye-C V O-e e--LoLv-C OX M ammonium bicarbonate containing 0.2 mg of enzyme per ml at 370C for 18 hr. All peptides were purified by HPLC on a Vydac C4 column using the F3CCOOH/CH3CN buffer Cys Sly Sly Val Lew Val MsI Pro Sin lrp Vei Lee Oh, Ala Ala Ms Cy [lie Ae Meo Ly, e Vtale Ie See system with a linear gradient from 100% buffer A to 80% Inlec PA (vol/vol) buffer B in 60 min. Amino acid analyses were CN VI ________________ performed using a Beckman 121MB amino acid analyzer after 24,48, and 72 hr ofacid hydrolysis (5.7 M HCl, 0.1% phenol). Lye-C IX *I Lye-C XCIb-. Automated sequence analyses were conducted on a Beckman 890C sequencer and Applied Biosystems (Foster City, CA) 470A gas-phase sequencer as reported (11). Comparison of Inlec PA the primary structure of PA with the kallikrein family of CC VI serine protease was carried out according to Doolittle (12) and Dayhoff (13). * as w e in La. Tyr Meo Plt Sot Loe Lee Lye PA, leg Phl Leu lee Pto Sly Msp Msp ot Sot CI" My Lee pin Lee See RESULTS Inlacl PA -e Amino Acid Composition. The amino acid composition of PA is tabulated in Table 1. Our results agree well with a previous report and with the theoretical composition derived - ~Lye-C Mill, Lye-C X from the sequence reported here. 10511 It0 III 1CC Amino Acid Sequence. The complete amino acid sequence us of PA is given in Fig. 1. The sequence was primarily constructed through analysis of the reduced and alkylated chain, of fragments derived from partial endoproteolytic Ley-C X . Lye-C X11e cleavages, CNBr fragmentation, endoproteinase Arg-C, and 10 1Sn 100 1le SW Lys-C cleavages. Amino-terminal sequence analysis with Oht Ohr Cys Tyr Ale Mt Sly ITy Sly Mt lIe Slie Ptro S SimGluM Lee Low Tyt Myp 1. OtO Lye Lye Lee o-phthalaldehyde blocking at proline residues (11) yielded the sequence of R1-R87. The chemical cleavage of methionine residues and enzymatic digestion with endoproteinase Lys-C Lye-C Xllm VUL gave the sequences of R1-R170, R174-R198, and R225- Ole ~~~~100 100 lie 15S R240.
Load more

Recommended publications
  • Chymotrypsin: a Serine Protease Reaction Mechanism Step
    CHEM464/Medh,J.D. Catalytic Strategies Chymotrypsin: A serine protease • Covalent catalysis: temporary covalent modification of reactive • Hydrolyzes peptide bonds on the carboxyl side of group on enzyme active site Tyr, Phe, Trp, Met, Leu • Acid-Base catalysis: A molecule other than water is proton • Since peptide bond is highly unreactive, a strong donor or acceptor (nucleophilic or electrophilic attack) nucleophile is required for its hydrolysis • Metal ion catalysis: Involvement of metal ion in catalysis. A metal ion is an electrophile and (i) may stabilize a negative • Catalytic strategy is covalent modification and charge on an intermediate; (ii) by attracting electrons from acid-base catalysis water, renders water more acidic (prone to loose a proton); (iii) • Contains catalytic triad of Ser, His and Asp. Ser is may bind to substrate and reduce activation energy a nucleophile and participates in covalent • Catalysis by approximation: In reactions requiring more than modification, His is a proton acceptor (base), Asp one substrate, the enzyme facilitates their interaction by serving stabilizes His (and active site) by electrostatic as an adapter that increases proximity of the substrates to each interactions other Reaction Mechanism Step-wise reaction • Hydrolysis by chymotrypsin is a 2-step process • Enzyme active site is stabilized by ionic interactions • Step 1: serine reacts with substrate to form covalent between Asp and His and H-bond between His and Ser. ES complex • In the presence of a substrate, His accepts a proton from • Step 2: release of products from ES complex and Ser, Ser makes a nucleophilic attack on the peptide’s regeneration of enzyme carbonyl C converting its geometry to tetrahedral.
    [Show full text]
  • NS3 Protease from Flavivirus As a Target for Designing Antiviral Inhibitors Against Dengue Virus
    Genetics and Molecular Biology, 33, 2, 214-219 (2010) Copyright © 2010, Sociedade Brasileira de Genética. Printed in Brazil www.sbg.org.br Review Article NS3 protease from flavivirus as a target for designing antiviral inhibitors against dengue virus Satheesh Natarajan Department of Biochemistry, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malayasia. Abstract The development of novel therapeutic agents is essential for combating the increasing number of cases of dengue fever in endemic countries and among a large number of travelers from non-endemic countries. The dengue virus has three structural proteins and seven non-structural (NS) proteins. NS3 is a multifunctional protein with an N-terminal protease domain (NS3pro) that is responsible for proteolytic processing of the viral polyprotein, and a C-terminal region that contains an RNA triphosphatase, RNA helicase and RNA-stimulated NTPase domain that are essential for RNA replication. The serine protease domain of NS3 plays a central role in the replicative cycle of den- gue virus. This review discusses the recent structural and biological studies on the NS2B-NS3 protease-helicase and considers the prospects for the development of small molecules as antiviral drugs to target this fascinating, multifunctional protein. Key words: antiviral inhibitor, drug discovery, multifunctional protein, NS3, protease. Received: March 4, 2009; Accepted: November 1, 2009. Introduction seven non-structural proteins involved in viral replication The genus Flavivirus in the family Flaviviridae con- and maturation (Henchal and Putnak, 1990; Kautner et al., tains a large number of viral pathogens that cause severe 1996). The virus-encoded protease complex NS2B-NS3 is morbidity and mortality in humans and animals (Bancroft, responsible for cleaving the NS2A/NS2B, NS2B/NS3, 1996).
    [Show full text]
  • Human Elastase 1: Evidence for Expression in the Skin and the Identi®Cation of a Frequent Frameshift Polymorphism
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Human Elastase 1: Evidence for Expression in the Skin and the Identi®cation of a Frequent Frameshift Polymorphism Ulvi Talas, John Dunlop,1 Sahera Khalaf , Irene M. Leigh, and David P. Kelsell Center for Cutaneous Research, St. Bartholomew's and the Royal London School of Medicine and Dentistry, Whitechapel, London, U.K. Human pancreatic elastase 1 is a serine protease individuals with/without the keratoderma revealed a which maps to the chromosomal region 12q13 close sequence variant, which would result in a premature to a locus for an autosomal dominant skin disease, truncation of the protein. This sequence variant, diffuse nonepidermolytic palmoplantar keratoderma, however, did not segregate with the skin disease and, and was investigated as a possible candidate gene for indeed, was found to occur at a relatively high fre- this disorder. Expression of two elastase inhibitors, quency in the population. Individuals homozygous ela®n and SLPI, has been related to several hyper- for the variant do not have any obvious skin proliferative skin conditions. elastase 1 is functionally abnormalities. Based on the analysis of the secondary silent in the human pancreas but elastase 1 expres- structure of the translated putative protein, the sion at the mRNA level was detected in human truncation is unlikely to result in knock-out of the cultured primary keratinocytes. Antibody staining elastase, but may cause destabilization of the localized the protein to the basal cell layer of the enzyme±inhibitor complex. Key words: ela®n/keratino- human epidermis at a number of sites includingthe cyte/protein truncation/serine protease.
    [Show full text]
  • IL-33 Is Processed Into Mature Bioactive Forms by Neutrophil Elastase and Cathepsin G
    IL-33 is processed into mature bioactive forms by neutrophil elastase and cathepsin G Emma Lefrançais, Stephane Roga, Violette Gautier, Anne Gonzalez-de-Peredo, Bernard Monsarrat, Jean-Philippe Girard1,2, and Corinne Cayrol1,2 Centre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale, F-31077 Toulouse, France; Université de Toulouse, Université Paul Sabatier, Institut de Pharmacologie et de Biologie Structurale, F-31077 Toulouse, France Edited* by Charles A. Dinarello, University of Colorado Denver, Aurora, CO, and approved December 19, 2011 (received for review October 3, 2011) Interleukin-33 (IL-33) (NF-HEV) is a chromatin-associated nuclear activity (4). However, we (23) and others (24–26) demonstrated cytokine from the IL-1 family, which has been linked to important that full-length IL-33 is biologically active and that processing of diseases, including asthma, rheumatoid arthritis, ulcerative colitis, IL-33 by caspases results in its inactivation, rather than its activa- and cardiovascular diseases. IL-33 signals through the ST2 receptor tion. Further analyses revealed that IL-33 is constitutively and drives cytokine production in type 2 innate lymphoid cells (ILCs) expressed to high levels in the nuclei of endothelial and epithelial (natural helper cells, nuocytes), T-helper (Th)2 lymphocytes, mast cells in vivo (27) and that it can be released in the extracellular cells, basophils, eosinophils, invariant natural killer T (iNKT), and space after cellular damage (23, 24). IL-33 was, thus, proposed (23, natural killer (NK) cells. We and others recently reported that, unlike 24, 27) to function as an endogenous danger signal or alarmin, IL-1β and IL-18, full-length IL-33 is biologically active independently similar to IL-1α and high-mobility group box 1 protein (HMGB1) of caspase-1 cleavage and that processing by caspases results in IL-33 (28–32), to alert cells of the innate immune system of tissue inactivation.
    [Show full text]
  • Serine Protease (Chymotrypsin) from Nocardiopsis Prasina Expressed in Bacillus Licheniformis
    SERINE PROTEASE (CHYMOTRYPSIN) FROM NOCARDIOPSIS PRASINA EXPRESSED IN BACILLUS LICHENIFORMIS Chemical and Technical Assessment Prepared by Jannavi R. Srinivasan, Ph.D., Reviewed by Dr. Inge Meyland, Ph. D. 1. Summary This Chemical and Technical Assessment (CTA) summarizes data and information on the serine protease with chymotrypsin specificity from Nocardiopsis prasina expressed in Bacillus Licheniformis enzyme preparation submitted to the Joint FAO/WHO Expert Committee on Food Additives (JECFA) by Novozymes A/S in a dossier dated November 25, 2011 (Novozymes, 2011)a. In this CTA, the expression ‘serine protease (chymotrypsin)’ is used when referring to the serine protease with chymotrypsin specificity enzyme and its amino acid sequence, whereas the expression ‘serine protease (chymotrypsin) enzyme preparation’ is used when referring to the enzyme preparation. This document also discusses published information relevant to serine protease (chymotrypsin), the B. licheniformis production organism, and the N. prasina organism that is the source for the serine protease (chymotrypsin) gene. Serine protease (chymotrypsin) catalyses the hydrolysis of peptide bonds in a protein, preferably at the carboxyl end of Tyr (Tyr-X), Phe (Phe-X), Trp (Trp-X), when X is not proline. It also catalyses the hydrolysis of peptide bonds at the carboxyl end of other amino acids, primarily Met and Leu, albeit at a slower rate. It is intended for use in the production of hydrolysed animal and vegetable proteins including casein, whey, soy isolate, soy concentrate, wheat gluten and corn gluten. These hydrolysed proteins will be used for various applications as ingredients in food, protein-fortified food, and beverages. The serine protease (chymotrypsin) enzyme preparation is expected to be inactivated during food processing.
    [Show full text]
  • Trypsin-Like Proteases and Their Role in Muco-Obstructive Lung Diseases
    International Journal of Molecular Sciences Review Trypsin-Like Proteases and Their Role in Muco-Obstructive Lung Diseases Emma L. Carroll 1,†, Mariarca Bailo 2,†, James A. Reihill 1 , Anne Crilly 2 , John C. Lockhart 2, Gary J. Litherland 2, Fionnuala T. Lundy 3 , Lorcan P. McGarvey 3, Mark A. Hollywood 4 and S. Lorraine Martin 1,* 1 School of Pharmacy, Queen’s University, Belfast BT9 7BL, UK; [email protected] (E.L.C.); [email protected] (J.A.R.) 2 Institute for Biomedical and Environmental Health Research, School of Health and Life Sciences, University of the West of Scotland, Paisley PA1 2BE, UK; [email protected] (M.B.); [email protected] (A.C.); [email protected] (J.C.L.); [email protected] (G.J.L.) 3 Wellcome-Wolfson Institute for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University, Belfast BT9 7BL, UK; [email protected] (F.T.L.); [email protected] (L.P.M.) 4 Smooth Muscle Research Centre, Dundalk Institute of Technology, A91 HRK2 Dundalk, Ireland; [email protected] * Correspondence: [email protected] † These authors contributed equally to this work. Abstract: Trypsin-like proteases (TLPs) belong to a family of serine enzymes with primary substrate specificities for the basic residues, lysine and arginine, in the P1 position. Whilst initially perceived as soluble enzymes that are extracellularly secreted, a number of novel TLPs that are anchored in the cell membrane have since been discovered. Muco-obstructive lung diseases (MucOLDs) are Citation: Carroll, E.L.; Bailo, M.; characterised by the accumulation of hyper-concentrated mucus in the small airways, leading to Reihill, J.A.; Crilly, A.; Lockhart, J.C.; Litherland, G.J.; Lundy, F.T.; persistent inflammation, infection and dysregulated protease activity.
    [Show full text]
  • Characterization and Expression Profiling of Serine Protease
    Lin et al. BMC Genomics (2017) 18:162 DOI 10.1186/s12864-017-3583-z RESEARCH ARTICLE Open Access Characterization and expression profiling of serine protease inhibitors in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) Hailan Lin1,2,3,4, Xijian Lin1,2,3,4, Jiwei Zhu1,2,3,4, Xiao-Qiang Yu1,2,5, Xiaofeng Xia1,2,3,4, Fengluan Yao6, Guang Yang1,2,3,4 and Minsheng You1,2,3,4* Abstract Background: Serine protease inhibitors (SPIs) have been found in all living organisms and play significant roles in digestion, development and innate immunity. In this study, we present a genome-wide identification and expression profiling of SPI genes in the diamondback moth, Plutella xylostella (L.), a major pest of cruciferous crops with global distribution and broad resistance to different types of insecticides. Results: A total of 61 potential SPI genes were identified in the P. xylostella genome, and these SPIs were classified into serpins, canonical inhibitors, and alpha-2-macroglobulins based on their modes of action. Sequence alignments showed that amino acid residues in the hinge region of known inhibitory serpins from other insect species were conserved in most P. xylostella serpins, suggesting that these P. xylostella serpins may be functionally active. Phylogenetic analysis confirmed that P. xylostella inhibitory serpins were clustered with known inhibitory serpins from six other insect species. More interestingly, nine serpins were highly similar to the orthologues in Manduca sexta which have been demonstrated to participate in regulating the prophenoloxidase activation cascade, an important innate immune response in insects. Of the 61 P.xylostella SPI genes, 33 were canonical SPIs containing seven types of inhibitor domains, including Kunitz, Kazal, TIL, amfpi, Antistasin, WAP and Pacifastin.
    [Show full text]
  • Inhibition of Human Matriptase by Eglin C Variants
    FEBS Letters 580 (2006) 2227–2232 Inhibition of human matriptase by eglin c variants Antoine De´silets, Jean-Michel Longpre´, Marie-E` ve Beaulieu, Richard Leduc* Department of Pharmacology, Faculty of Medicine and Health Sciences, Universite´ de Sherbrooke, Sherbrooke, Que., Canada J1H 5N4 Received 2 February 2006; revised 2 March 2006; accepted 9 March 2006 Available online 20 March 2006 Edited by Michael R. Bubb inhibitor eglin c [14,15]. Further studies based on the three- Abstract Based on the enzyme specificity of matriptase, a type II transmembrane serine protease (TTSP) overexpressed in epi- dimensional structure of eglin c have found that optimization thelial tumors, we screened a cDNA library expressing variants of interaction between enzyme and inhibitor could be ad- of the protease inhibitor eglin c in order to identify potent dressed by screening eglin c cDNA libraries containing ran- 0 matriptase inhibitors. The most potent of these, R1K4-eglin, domly substituted residues within projected adventitious which had the wild-type Pro45 (P1 position) and Tyr49 (P40 posi- contact sites outside the reactive site [16]. The similarity in tion) residues replaced with Arg and Lys, respectively, led to the specificity between matriptase and furin, which preferentially production of a selective, high affinity (Ki of 4 nM) and proteo- recognizes the R–X–X–R (P4 to P1 position) sequence [17], lytically stable inhibitor of matriptase. Screening for eglin c vari- led us to the hypothesis that matriptase and other members ants could yield specific, potent and stable inhibitors to of the TTSP family could be targets of genetically engineered matriptase and to other members of the TTSP family.
    [Show full text]
  • Schneider-Dissertation-2019
    Biosynthetic Investigation, Synthesis, and Bioactivity Evaluation of Putative Peptide Aldehyde Natural Products From the Human Gut Microbiota The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Schneider, Benjamin Aaron. 2019. Biosynthetic Investigation, Synthesis, and Bioactivity Evaluation of Putative Peptide Aldehyde Natural Products From the Human Gut Microbiota. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:42029686 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use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
    [Show full text]
  • Chymotrypsin C (Caldecrin) Promotes Degradation of Human Cationic Trypsin: Identity with Rinderknecht’S Enzyme Y
    Chymotrypsin C (caldecrin) promotes degradation of human cationic trypsin: Identity with Rinderknecht’s enzyme Y Richa´ rd Szmola and Miklo´ s Sahin-To´ th* Department of Molecular and Cell Biology, Goldman School of Dental Medicine, Boston University, Boston, MA 02118 Communicated by Phillips W. Robbins, Boston Medical Center, Boston, MA, May 2, 2007 (received for review March 19, 2007) Digestive trypsins undergo proteolytic breakdown during their further tryptic sites (10). Autolysis was also proposed to play an transit in the human alimentary tract, which has been assumed to essential role in physiological trypsin degradation in the lower occur through trypsin-mediated cleavages, termed autolysis. Au- intestines. A number of studies in humans have demonstrated tolysis was also postulated to play a protective role against that trypsin becomes inactivated during its intestinal transit, and pancreatitis by eliminating prematurely activated intrapancreatic in the terminal ileum only Ϸ20% of the duodenal trypsin activity trypsin. However, autolysis of human cationic trypsin is very slow is detectable (12–14). On the basis of in vitro experiments, a in vitro, which is inconsistent with the documented intestinal theory was put forth that digestive enzymes are generally resis- trypsin degradation or a putative protective role. Here we report tant to each other and undergo degradation through autolysis that degradation of human cationic trypsin is triggered by chymo- only (10, 15). However, human cationic trypsin was shown to be trypsin C, which selectively cleaves the Leu81-Glu82 peptide bond highly resistant to autolysis, and appreciable autodegradation ؉ within the Ca2 binding loop. Further degradation and inactivation was observed only with extended incubation times in the com- of cationic trypsin is then achieved through tryptic cleavage of the plete absence of Ca2ϩ and salts (16–18).
    [Show full text]
  • Proteolytic Cleavage—Mechanisms, Function
    Review Cite This: Chem. Rev. 2018, 118, 1137−1168 pubs.acs.org/CR Proteolytic CleavageMechanisms, Function, and “Omic” Approaches for a Near-Ubiquitous Posttranslational Modification Theo Klein,†,⊥ Ulrich Eckhard,†,§ Antoine Dufour,†,¶ Nestor Solis,† and Christopher M. Overall*,†,‡ † ‡ Life Sciences Institute, Department of Oral Biological and Medical Sciences, and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada ABSTRACT: Proteases enzymatically hydrolyze peptide bonds in substrate proteins, resulting in a widespread, irreversible posttranslational modification of the protein’s structure and biological function. Often regarded as a mere degradative mechanism in destruction of proteins or turnover in maintaining physiological homeostasis, recent research in the field of degradomics has led to the recognition of two main yet unexpected concepts. First, that targeted, limited proteolytic cleavage events by a wide repertoire of proteases are pivotal regulators of most, if not all, physiological and pathological processes. Second, an unexpected in vivo abundance of stable cleaved proteins revealed pervasive, functionally relevant protein processing in normal and diseased tissuefrom 40 to 70% of proteins also occur in vivo as distinct stable proteoforms with undocumented N- or C- termini, meaning these proteoforms are stable functional cleavage products, most with unknown functional implications. In this Review, we discuss the structural biology aspects and mechanisms
    [Show full text]
  • Fecal Elastase-1 Is Superior to Fecal Chymotrypsin in the Assessment of Pancreatic Involvement in Cystic Fibrosis
    Fecal Elastase-1 Is Superior to Fecal Chymotrypsin in the Assessment of Pancreatic Involvement in Cystic Fibrosis Jaroslaw Walkowiak, MD, PhD*; Karl-Heinz Herzig, MD, PhD§; Krystyna Strzykala, M ChemA, Dr Nat Sci*; Juliusz Przyslawski, M Phar, Dr Phar‡; and Marian Krawczynski, MD, PhD* ABSTRACT. Objective. Exocrine pancreatic function ystic fibrosis (CF) is the most common cause in patients with cystic fibrosis (CF) can be evaluated by of exocrine pancreatic insufficiency in child- direct and indirect tests. In pediatric patients, indirect hood. Approximately 85% of CF patients are tests are preferred because of their less invasive charac- C 1,2 pancreatic insufficient (PI). Thus, the assessment of ter, especially in CF patients with respiratory disease. exocrine pancreatic function in CF patients is of great Fecal tests are noninvasive and have been shown to have clinical importance. For the evaluation, both direct a high sensitivity and specificity. However, there is no 3,4 comparative study in CF patients. Therefore, the aim of and indirect tests are used. The gold standard is the present study was to compare the sensitivity and the the secretin-pancreozymin test (SPT) or one of its specificity of the fecal elastase-1 (E1) test with the fecal modifications. However, this test is invasive, time chymotrypsin (ChT) test in a large cohort of CF patients consuming, expensive, and not well standardized in and healthy subjects (HS). children. Therefore, its use is limited to qualified Design. One hundred twenty-three CF patients and gastroenterologic centers. 105 HS were evaluated. In all subjects, E1 concentration Several indirect tests, such as serum tests—amy- and ChT activity were measured.
    [Show full text]