DOCSLIB.ORG

Recombinant Bovine Enteropeptidase/Enterokinase

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Recombinant Bovine Enteropeptidase/Enterokinase Recombinant Bovine Enteropeptidase/Enterokinase Catalog Number: 4139-SE DESCRIPTION Source E. coli­derived Cys788­Lys800 (heavy chain C­terminal fragment) with an N­terminal Ala, & Ile801­His1035 (light chain) Accession # P98072 N­terminal Sequence Ala & Ile801 Analysis Structure / Form Disulfide­linked heterodimer Predicted Molecular 1.5 kDa (heavy chain C­terminal fragment), 26 kDa (light chain) Mass SPECIFICATIONS SDS­PAGE 34 kDa, reducing conditions 30 kDa, non­reducing conditions Activity Measured by its ability to cleave a colorimetric peptide substrate, N­carbobenzyloxy­Lys­ThioBenzyl ester (Z­Lys­SBzl), in the presence of 5,5’Dithio­bis (2­nitrobenzoic acid) (DTNB). Lu, D. et al. (1997) J. Biol. Chem. 272:31293. The specific activity is >35 nmol/min/µg, as measured under the described conditions. Endotoxin Level <1.0 EU per 1 μg of the protein by the LAL method. Purity >90%, by SDS­PAGE under reducing conditions and visualized by silver stain. Formulation Supplied as a 0.2 μm filtered solution in Glycerol, NaCl and HEPES. See Certificate of Analysis for details. Activity Assay Protocol Materials l Assay Buffer: 50 mM Tris, pH 7.5 l Recombinant Bovine Enteropeptidase/Enterokinase (rbEnterokinase) (Catalog # 4139­SE) l Substrate: Z­Lys­SBZL (Bachem, Catalog # M­1300), 10 mM stock in DMSO l 5,5’­dithio­bis (2­nitrobenzoic acid) (DTNB) (Sigma, Catalog # D­8130), 10 mM stock in DMSO l 96 well Clear Plate (Costar, Catalog # 92592) l Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent Assay 1. Dilute rbEnterokinase to 0.04 µg/mL in Assay Buffer. 2. Dilute Substrate to 200 µM in Assay Buffer with 200 µM of DTNB. 3. Load into a 96 well clear plate 50 µL of the diluted rbEnterokinase. For a Substrate Blank, load 50 µL of the Assay Buffer. 4. Start the reaction by adding 50 µL of the Substrate/DTNB mixture to wells. 5. Read in kinetic mode for 5 minutes at an absorbance of 405 nm. 6. Calculate specific activity: Adjusted V * (OD/min) x well volume (L) x 109 nmol/M Specific Activity (nmol/min/µg) = max ext. coeff** (M­1cm­1) x path corr.*** (cm) x amount of enzyme (µg) *Adjusted for Substrate Blank **Using the extinction coefficient 13260 M­1cm­1 ***Using the path correction 0.32 cm Note: the output of many spectrophotometers is in mOD Final Assay Per Well: Conditions l rbEnterokinase: 0.002 µg l DTNB: 100 µM l Substrate: 100 µM PREPARATION AND STORAGE Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. Stability & Storage Use a manual defrost freezer and avoid repeated freeze­thaw cycles. l 6 months from date of receipt, ­20 to ­70 °C as supplied. l 3 months, ­20 to ­70 °C under sterile conditions after opening. BACKGROUND EK initiates activation of pancreatic proteases by converting trypsinogen to trypsin, which in turn activates chymotrypsin, carboxypeptidases and elastases. Located in intestinal brush border, it is a disulfide bond linked dimer of the heavy and light chains, which are derived from the same single­chain precursor. The multidomain­ containing heavy chain consists of a short cytoplasmic tail, a transmembrane, a SEA, a SRCR, a MAM, two CUB and two LDL­receptor class A domains. The light chain contains the catalytic domain of trypsin­like serine proteases. The purified recombinant bovine EK (residues 788­1035) corresponds to a disulfide bond­linked dimer that consists of the C­terminal fragment of the heavy chain (residues 788­800) and the light chain (residues 801­1035). rbEnterokinase can cleave fusion proteins having an accessible Enterokinase cleavage site (DDDDK). At an average ratio for fusion protein:rbEnterokinase of 1000:1 (w/w), cleavage up to 90% completion is achieved within one hour at room temperature. Non­specific cleavage at basic residues has also been observed for some proteins. It is recommended that cleavage reaction be optimized for each fusion protein. The reaction may be terminated by passing the sample through a soybean trypsin inhibitor (SBTI)­agarose affinity column (e.g. Sigma Catalog # T0637 ) to remove the rbEnterokinase from the reaction mixture. SBTI inhibits rbEnterokinase with a Ki of 1.6 nM. Rev. 2/6/2018 Page 1 of 1 .
Load more

Recommended publications
  • Congenital Diarrheal Disorders: an Updated Diagnostic Approach
    4168 Int. J. Mol. Sci.2012, 13, 4168-4185; doi:10.3390/ijms13044168 OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Review Congenital Diarrheal Disorders: An Updated Diagnostic Approach Gianluca Terrin 1, Rossella Tomaiuolo 2,3,4, Annalisa Passariello 5, Ausilia Elce 2,3, Felice Amato 2,3, Margherita Di Costanzo 5, Giuseppe Castaldo 2,3 and Roberto Berni Canani 5,6,* 1 Department of Gynecology-Obstetrics and Perinatal Medicine, University of Rome “La Sapienza”, Viale del Policlinico 1, Rome 00161, Italy; E-Mail: [email protected] 2 CEINGE-Advanced Biotechnology, Via Comunale Margherita, Naples 80131, Italy; E-Mails: [email protected] (R.T.); [email protected] (A.E.); [email protected] (F.A.); [email protected] (G.C.) 3 Department of Biochemistry and Biotechnology, University of Naples “Federico II”, Via Pansini 5, Naples 80131, Italy 4 Biotechnology Science, University of Naples “Federico II”, Via De Amicis, Naples 80131, Italy 5 Department of Pediatrics, University of Naples “Federico II”, Via Pansini 5, Naples 80131, Italy; E-Mails: [email protected] (A.P.); [email protected] (M.D.C.) 6 European Laboratory for the Investigation of Food Induced Diseases, University of Naples “Federico II”, Via Pansini 5, Naples 80131, Italy * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel./Fax: +39-0817462680. Received: 18 February 2012; in revised form: 2 March 2012 / Accepted: 19 March 2012 / Published: 29 March 2012 Abstract: Congenital diarrheal disorders (CDDs) are a group of inherited enteropathies with a typical onset early in the life.
    [Show full text]
  • 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]
  • Trypsin Inhibitor from Glycine Max (Soybean) (T6522)
    Trypsin inhibitor from Glycine max (soybean) Cell Culture Tested Product Number T 6522 Storage Temperature 2-8 °C Product Description Precautions and Disclaimer CAS Number: 9035-81-8 For Laboratory Use Only. Not for drug, household or Extinction Coefficient: E1% = 9.94 (280 nm, other uses. pH 7.6 buffer) pI: 4.51 Preparation Instructions Synonyms: Kunitz Trypsin Inhibitor, Tia1, STI, and Trypsin inhibitor is soluble in water and phosphate SBT1 buffers at 10 mg/ml. It is soluble in balanced salt solutions (1 mg/ml) and in serum-free media. This product is cell culture tested and is appropriate Solutions at concentrations higher than 10 mg/ml may for use in cell culture applications. It is extensively be hazy and have a yellow to amber color. dialyzed against water. After dialysis, sodium phosphate buffer, pH 7.6, is added, and the inhibitor is Storage/Stability lyophilized. The final product consists of about 90% A 10 mg/ml sterile-filtered solution stored for greater protein and 10% sodium phosphate buffer salts (by than 3 years at 2-8 °C showed no loss in trypsin mass). inhibition activity. Solutions are stable in frozen aliquots at -20 °C, but freeze-thaw cycles should be 2 Soybean trypsin inhibitor was first isolated by Kunitz. avoided. This protein is reversibly denatured by short Several other related inhibitors are also found in heating to 80 °C and irreversibly inhibited by heating to 3 soybeans. Trypsin inhibitor from soybeans is a 90 °C.3 monomeric protein containing 181 amino acid residues in a single polypeptide chain crosslinked by two 4,5,6 Procedure disulfide bridges.
    [Show full text]
  • (12) Patent Application Publication (10) Pub. No.: US 2016/0346364 A1 BRUNS Et Al
    US 2016.0346364A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0346364 A1 BRUNS et al. (43) Pub. Date: Dec. 1, 2016 (54) MEDICAMENT AND METHOD FOR (52) U.S. Cl. TREATING INNATE IMMUNE RESPONSE CPC ........... A61K 38/488 (2013.01); A61K 38/482 DISEASES (2013.01); C12Y 304/23019 (2013.01); C12Y 304/21026 (2013.01); C12Y 304/23018 (71) Applicant: DSM IPASSETS B.V., Heerlen (NL) (2013.01); A61K 9/0053 (2013.01); C12N 9/62 (2013.01); A23L 29/06 (2016.08); A2ID 8/042 (72) Inventors: Maaike Johanna BRUINS, Kaiseraugst (2013.01); A23L 5/25 (2016.08); A23V (CH); Luppo EDENS, Kaiseraugst 2002/00 (2013.01) (CH); Lenneke NAN, Kaiseraugst (CH) (57) ABSTRACT (21) Appl. No.: 15/101,630 This invention relates to a medicament or a dietary Supple (22) PCT Filed: Dec. 11, 2014 ment comprising the Aspergillus niger aspergilloglutamic peptidase that is capable of hydrolyzing plant food allergens, (86). PCT No.: PCT/EP2014/077355 and more particularly, alpha-amylase/trypsin inhibitors, thereby treating diseases due to an innate immune response S 371 (c)(1), in humans, and/or allowing to delay the onset of said (2) Date: Jun. 3, 2016 diseases. The present invention relates to the discovery that (30) Foreign Application Priority Data the Aspergillus niger aspergilloglutamic peptidase is capable of hydrolyzing alpha-amylase/trypsin inhibitors that are Dec. 11, 2013 (EP) .................................. 13196580.8 present in wheat and related cereals said inhibitors being strong inducers of innate immune response. Furthermore, Publication Classification the present invention relates to a method for hydrolyzing alpha-amylase/trypsin inhibitors comprising incubating a (51) Int.
    [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]
  • Highly Potent and Selective Plasmin Inhibitors Based on the Sunflower Trypsin Inhibitor-1 Scaffold Attenuate Fibrinolysis in Plasma
    Highly Potent and Selective Plasmin Inhibitors Based on the Sunflower Trypsin Inhibitor-1 Scaffold Attenuate Fibrinolysis in Plasma Joakim E. Swedberg,‡† Guojie Wu,§† Tunjung Mahatmanto,‡# Thomas Durek,‡ Tom T. Caradoc-Davies,∥ James C. Whisstock,§* Ruby H.P. Law§* and David J. Craik‡* ‡Institute for Molecular Bioscience, The University of Queensland, Brisbane QLD 4072, Australia §ARC Centre of Excellence in Advanced Molecular Imaging, Department of Biochemistry and Molecular Biology, Biomedical Discovery Institute, Monash University, VIC 3800, Australia. ∥Australian Synchrotron, 800 Blackburn Road, Clayton, Melbourne, VIC 3168, Australia. †J.E.S. and G.W. contributed equally to this work. Keywords: Antifibrinolytics; Fibrinolysis; Inhibitors; Peptides; Plasmin ABSTRACT Antifibrinolytic drugs provide important pharmacological interventions to reduce morbidity and mortality from excessive bleeding during surgery and after trauma. Current drugs used for inhibiting the dissolution of fibrin, the main structural component of blood clots, are associated with adverse events due to lack of potency, high doses and non-selective inhibition mechanisms. These deficiencies warrant the development of a new generation highly potent and selective fibrinolysis inhibitors. Here we use the 14-amino acid backbone-cyclic sunflower trypsin inhibitor-1 scaffold to design a highly potent (Ki = 0.05 nM) inhibitor of the primary serine protease in fibrinolysis, plasmin. This compound displays a million-fold selectivity over other serine proteases in blood, inhibits fibrinolysis in plasma more effectively than the gold-standard therapeutic inhibitor aprotinin and is a promising candidate for development of highly specific fibrinolysis inhibitors with reduced side effects. 1 INTRODUCTION The physiological process of fibrinolysis regulates the dissolution of blood clots and thrombosis.
    [Show full text]
  • Characterization of a Bowman–Birk Type Trypsin Inhibitor Purified From
    www.nature.com/scientificreports OPEN Characterization of a Bowman–Birk type trypsin inhibitor purifed from seeds of Solanum surattense Abhijeet P. Herwade1, Sainath S. Kasar1,2, Niraj R. Rane3, Shadab Ahmed4, Jaswinder Singh Maras5 & Pankaj K. Pawar6* A Bowman–Birk type trypsin inhibitor protein (SSTI) from seeds of the medicinal plant Solanum surattense was isolated, purifed and characterized. SSTI showed a single band on SDS-PAGE corresponding to 11.4 kDa molecular weight. It is a glycoprotein (2.8% glycosylation) that diferentially interacted with trypsin and chymotrypsin in a concentration-dependent manner. Its peptide sequence is similar to other Bowman–Birk type protease inhibitors found in Glycine max and Phaseolus acutifolius. The inhibitory activity was stable over a wide range of pH (1–10) and temperatures (10–100° C). Far-UV Circular Dichroism (CD) studies showed that SSTI contains β sheets (~ 23%) and α helix (~ 6%) and demonstrated structural stability at wide pH and high temperature. The kinetic analysis revealed a noncompetitive (mixed) type nature of SSTI and low inhibitor constant (Ki) −8 values (16.6 × ­10 M) suggested strong inhibitory activity. Isothermal titration calorimetric analysis revealed its high afnity towards trypsin with dissociation constant (Kd) 2.28 µM. Biotic stress induces the generation and accumulation of phenolic compounds and pathogenesis-related (PR) proteins which subsequently prevent an invasion of pests like insects and microbial pathogens1. Most of the plant PR proteins are acid-soluble, low molecular weight and protease enzyme inhibitors 2,3. Protease inhibitors are mainly harbored by four plant families’ viz. Fabaceae, Gramineae, Leguminosae, and Solanaceae4,5.
    [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]
  • Uterine-Associated Serine Protease Inhibitors Stimulate Deoxyribonucleic Acid Synthesis in Porcine Endometrial Glandular Epithelial Cells of Pregnancy 1
    BIOLOGY OF REPRODUCTION 61, 380±387 (1999) Uterine-Associated Serine Protease Inhibitors Stimulate Deoxyribonucleic Acid Synthesis in Porcine Endometrial Glandular Epithelial Cells of Pregnancy 1 Lokenga Badinga, Frank J. Michel, and Rosalia C.M. Simmen2 Animal Molecular and Cell Biology Interdisciplinary Concentration, Department of Animal Science, University of Florida, Gainesville, Florida 32611-0910 ABSTRACT Consistent with this, uteri from mammalian species with distinct placentation types express common classes of pro- Protease inhibitors are major secretory components of the tease inhibitors (e.g., tissue inhibitors of metalloproteases, Downloaded from https://academic.oup.com/biolreprod/article/61/2/380/2734487 by guest on 24 September 2021 mammalian uterus that are thought to mediate pregnancy-as- TIMPs) as well as distinct ones (e.g., secretory leukocyte sociated events primarily by regulating the activity of proteolytic protease inhibitor, SLPI, and uterine plasmin/trypsin inhib- enzymes. In the present study, we examined the mitogenic po- tentials of two serine protease inhibitors, namely secretory leu- itor, UPTI) [4, 8, 9]. Since embryos from all species, re- kocyte protease inhibitor (SLPI) and uterine plasmin/trypsin in- gardless of placentation type, exhibit invasive properties hibitor (UPTI) in primary cultures of glandular epithelial (GE) when placed into ectopic sites [10], the limiting of blasto- cells isolated from early pregnant (Day 12) pig endometrium, cyst invasiveness, albeit to varying extents, is most likely
    [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]
  • N-Glycosylation in the Protease Domain of Trypsin-Like Serine Proteases Mediates Calnexin-Assisted Protein Folding
    RESEARCH ARTICLE N-glycosylation in the protease domain of trypsin-like serine proteases mediates calnexin-assisted protein folding Hao Wang1,2, Shuo Li1, Juejin Wang1†, Shenghan Chen1‡, Xue-Long Sun1,2,3,4, Qingyu Wu1,2,5* 1Molecular Cardiology, Cleveland Clinic, Cleveland, United States; 2Department of Chemistry, Cleveland State University, Cleveland, United States; 3Chemical and Biomedical Engineering, Cleveland State University, Cleveland, United States; 4Center for Gene Regulation of Health and Disease, Cleveland State University, Cleveland, United States; 5Cyrus Tang Hematology Center, State Key Laboratory of Radiation Medicine and Prevention, Soochow University, Suzhou, China Abstract Trypsin-like serine proteases are essential in physiological processes. Studies have shown that N-glycans are important for serine protease expression and secretion, but the underlying mechanisms are poorly understood. Here, we report a common mechanism of N-glycosylation in the protease domains of corin, enteropeptidase and prothrombin in calnexin- mediated glycoprotein folding and extracellular expression. This mechanism, which is independent *For correspondence: of calreticulin and operates in a domain-autonomous manner, involves two steps: direct calnexin [email protected] binding to target proteins and subsequent calnexin binding to monoglucosylated N-glycans. Elimination of N-glycosylation sites in the protease domains of corin, enteropeptidase and Present address: †Department prothrombin inhibits corin and enteropeptidase cell surface expression and prothrombin secretion of Physiology, Nanjing Medical in transfected HEK293 cells. Similarly, knocking down calnexin expression in cultured University, Nanjing, China; ‡Human Aging Research cardiomyocytes and hepatocytes reduced corin cell surface expression and prothrombin secretion, Institute, School of Life Sciences, respectively. Our results suggest that this may be a general mechanism in the trypsin-like serine Nanchang University, Nanchang, proteases with N-glycosylation sites in their protease domains.
    [Show full text]