scholarly journals Fibrinogen Cleavage by the Streptococcus pyogenes Extracellular Cysteine Protease and Generation of Antibodies That Inhibit Enzyme Proteolytic Activity

1999 ◽  
Vol 67 (9) ◽  
pp. 4326-4333 ◽  
Author(s):  
Yury V. Matsuka ◽  
Subramonia Pillai ◽  
Siddeswar Gubba ◽  
James M. Musser ◽  
Stephen B. Olmsted
Keyword(s):  
Proteolytic Activity ◽  
Streptococcus Pyogenes ◽  
Cysteine Protease ◽  
Active Site ◽  
Cysteine Residue ◽  
Human Infection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Fibrinogen ◽  
Enzyme Active Site ◽  
Enzyme Replacement

ABSTRACT The extracellular cysteine protease from Streptococcus pyogenes is a virulence factor that plays a significant role in host-pathogen interaction. Streptococcal protease is expressed as an inactive 40-kDa precursor that is autocatalytically converted into a 28-kDa mature (active) enzyme. Replacement of the single cysteine residue involved in formation of the enzyme active site with serine (C192S mutation) abolished detectable proteolytic activity and eliminated autocatalytic processing of zymogen to the mature form. In the present study, we investigated activity of the wild-type (wt) streptococcal protease toward human fibrinogen and bovine casein. The former is involved in blood coagulation, wound healing, and other aspects of hemostasis. Treatment with streptococcal protease resulted in degradation of the COOH-terminal region of fibrinogen α chain, indicating that fibrinogen may serve as an important substrate for this enzyme during the course of human infection. Polyclonal antibodies generated against recombinant 40- and 28-kDa (r40- and r28-kDa) forms of the C192S streptococcal protease mutant exhibited high enzyme-linked immunosorbent assay titers but demonstrated different inhibition activities toward proteolytic action of the wt enzyme. Activity of the wt protease was readily inhibited when the reaction was carried out in the presence of antibodies generated against r28-kDa C192S mutant. Antibodies produced against r40-kDa C192S mutant had no significant effect on proteolysis. These data suggest that the presence of the NH2-terminal prosegment prevents generation of functionally active antibodies and indicate that inhibition activity of antibodies most likely depends on their ability to bind the active-site region epitope(s) of the protein.

Streptococcus pyogenes Fibronectin-Binding Protein Is a Novel Prothrombin Activator.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1691-1691
Author(s):  
Jonathan I. Creamer ◽  
Peter R. Panizzi ◽  
Paul E. Bock
Keyword(s):  
Fusion Protein ◽  
Streptococcus Pyogenes ◽  
Active Site ◽  
Binding Protein ◽  
Catalytic Site ◽  
Fluorescence Probes ◽  
Binding Studies ◽  
Human Fibrinogen ◽  
Fibronectin Binding Protein ◽  
Fibronectin Binding

Abstract Streptococcus pyogenes fibronectin-binding Protein (SfbX), expressed by emm12 and emm49 strains of S. pyogenes, binds fibronectin through COOH-terminal interactions of the protein.1 SfbX NH2-terminal residues 54–229 share 21% identity to staphylocoagulase (SC) residues 107–321 from Tager 104 strain, which increases to 35% homology when conservative substitutions are considered. SC is a non-proteolytic activator of the central coagulation zymogen prothrombin (ProT). Our structure of the prethrombin 2 complex with a fully active SC fragment (SC(1-325)) showed that SC(1-325) consisted of two α-helical bundle domains and that the NH2-terminal dipeptide is critical for ProT activation via the molecular sexuality mechanism.2 In this mechanism, SC inserts its NH2-terminal Ile1 into the Asp194 pocket of the ProT catalytic domain, inducing conformational activation of the catalytic site. On the basis of its homology, SfbX has been postulated to be a member of the SC family of zymogen activator and adhesion proteins. Plasmids encoding a His6-tagged SfbX(1-312) tobacco etch virus proteinase-cleavable fusion protein and the viral proteinase were co-transformed into E. coli to enable generation of the native SfbX NH2-terminus (Ile-Ser-Asn) during purification. SfbX(1-312) was purified by affinity chromatography on ProT-Affigel and Ni2+-iminodiacetic acid-Sepharose. Active site-specific fluorescent probe labeling of a mixture of ProT and SfbX(1-312) showed covalent labeling of the ProT zymogen, demonstrating that SfbX(1-312) is a non-proteolytic activator of ProT. Incubation of ProT with the NH2-terminally blocked SfbX(1-312) fusion protein did not result in labeling of the active site, indicating that the native NH2-terminus is required for activation, and suggesting that SfbX(1-312) activates ProT through the molecular sexuality mechanism. In ProT activation assays measured by the appearance of chromogenic substrate activity, SfbX(1-312) activated ProT weakly and was more effective in activating prethrombin 1, lacking the fragment 1 domain of ProT. Preliminary binding studies using ProT and thrombin labeled at the catalytic site with fluorescence probes demonstrated binding of SfbX(1-312). SfbX(1-312) decreased thrombin activity toward D-Phe-Pip-Arg-pNA by 75%, with an apparent KD of ~20 nM, indicating that the thrombin catalytic site is perturbed by SfbX binding. Clotting assays showed that neither SfbX(1-312) nor a mixture of SfbX(1-312) and prethrombin 1 clotted human fibrinogen or plasma. Addition of 5 μM SfbX(1-312) to a factor V-dependent plasma clotting assay increased clotting times, indicating that SfbX(1-312) has an inhibitory effect. We conclude that SfbX(1-312) activates ProT conformationally, possibly through the molecular sexuality mechanism. SfbX(1-312) binds tightly to thrombin, perturbing its catalytic site, but does not clot human fibrinogen or plasma. Further studies are needed to determine whether ProT is the pathophysiological target zymogen of SfbX(1-312), to delineate the natural substrate of the SfbX(1-312)·ProT complex, and to elucidate the role of SfbX in the pathology of S. pyogenes infection.

scholarly journals Inhibition of glutathione S-transferase 3-3 by glutathione derivatives that bind covalently to the active site

1991 ◽  
Vol 278 (1) ◽  
pp. 63-68 ◽  
Author(s):  
A E P Adang ◽  
W J Moree ◽  
J Brussee ◽  
G J Mulder ◽  
A van der Gen
Keyword(s):  
Active Site ◽  
Current Knowledge ◽  
Control Level ◽  
Gel Filtration ◽  
Cysteine Residue ◽  
Covalent Modification ◽  
British Library ◽  
Glutathione S Transferase ◽  
Enzyme Active Site ◽  
Active Site Cysteine

In all, 13 GSH derivatives have been synthesized and tested for their potency to inhibit glutathione S-transferase (GST) 3-3. All of these derivatives contained a reactive group that could potentially react with the enzyme active site. Best results were obtained with the phenylthiosulphonate derivative of GSH, GSSO2Ph. Preincubation of GST 3-3 with a 100 microM concentration of this inhibitor resulted in a time-dependent loss of activity: after 30 min at pH 6.5 and 25 degrees C, 51% of the activity was lost. At more alkaline pH, the activity is more rapidly inhibited: at pH 8.0 the 90%-inhibition level is already reached after 10 min preincubation. Separation of enzyme and excess unbound GSSO2Ph after preincubation by gel-filtration chromatography did not result in a reappearance of enzyme activity. If 100 microM-GSH was added to the preincubation mixture at pH 7.4, inhibition was almost completely prevented. Addition of S-(hexyl)glutathione (20 microM) could delay the inhibition but, ultimately, not prevent it. The inhibited enzyme could be re-activated by addition of 10 mM-2-mercaptoethanol: 60 min after this thiol was added, the inhibited GST-3- activity was bacxk to the control level. GSH at the same concentration could not re-activate the enzyme. On the basis of these results, on the known reactivity of thiosulphonate compounds, and on current knowledge about the amino acid residues involved in GST catalysis, a covalent modification of an active-site cysteine residue by mixed-disulphide formation between enzyme and the cosubstrate GSH is postulated. Information on the synthesis and characterization of the GSH derivatives is given in Supplementary Publication SUP 50166 (5 pages) which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273, 5.

scholarly journals Bifunctional Role of theTreponema pallidumExtracellular Matrix Binding Adhesin Tp0751

2010 ◽  
Vol 79 (3) ◽  
pp. 1386-1398 ◽  
Author(s):  
Simon Houston ◽  
Rebecca Hof ◽  
Teresa Francescutti ◽  
Aaron Hawkes ◽  
Martin J. Boulanger ◽  
...  
Keyword(s):  
Proteolytic Activity ◽  
Specific Binding ◽  
Circulatory System ◽  
Attachment Site ◽  
Site Directed Mutagenesis ◽  
Host Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Active Site Residues ◽  
Human Fibrinogen

ABSTRACTTreponema pallidum, the causative agent of syphilis, is a highly invasive pathogenic spirochete capable of attaching to host cells, invading the tissue barrier, and undergoing rapid widespread dissemination via the circulatory system. TheT. pallidumadhesin Tp0751 was previously shown to bind laminin, the most abundant component of the basement membrane, suggesting a role for this adhesin in host tissue colonization and bacterial dissemination. We hypothesized that similar to that of other invasive pathogens, the interaction ofT. pallidumwith host coagulation proteins, such as fibrinogen, may also be crucial for dissemination via the circulatory system. To test this prediction, we used enzyme-linked immunosorbent assay (ELISA) methodology to demonstrate specific binding of soluble recombinant Tp0751 to human fibrinogen. Click-chemistry-based palmitoylation profiling of heterologously expressed Tp0751 confirmed the presence of a lipid attachment site within this adhesin. Analysis of the Tp0751 primary sequence revealed the presence of a C-terminal putative HEXXH metalloprotease motif, andin vitrodegradation assays confirmed that recombinant Tp0751 purified from both insect andEscherichia coliexpression systems degrades human fibrinogen and laminin. The proteolytic activity of Tp0751 was abolished by the presence of the metalloprotease inhibitor 1,10-phenanthroline. Further, inductively coupled plasma-mass spectrometry showed that Tp0751 binds zinc and calcium. Collectively, these results indicate that Tp0751 is a zinc-dependent, membrane-associated protease that exhibits metalloprotease-like characteristics. However, site-directed mutagenesis of the HEXXH motif to HQXXH did not abolish the proteolytic activity of Tp0751, indicating that further mutagenesis studies are required to elucidate the critical active site residues associated with this protein. This study represents the first published description of aT. pallidumprotease capable of degrading host components and thus provides novel insight into the mechanism ofT. pallidumdissemination.

Cysteine protease activity of streptococcal pyrogenic exotoxin B

1994 ◽  
Vol 40 (11) ◽  
pp. 930-936 ◽  
Author(s):  
Yuko Ohara-Nemoto ◽  
Minoru Sasaki ◽  
Masaru Kaneko ◽  
Takayuki Nemoto ◽  
Minoru Ota
Keyword(s):  
Protease Inhibitors ◽  
Streptococcus Pyogenes ◽  
Cysteine Protease ◽  
Protease Activity ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Cysteine Residue ◽  
Peripheral Blood Mononuclear ◽  
Streptococcal Pyrogenic Exotoxin B ◽  
Spe B

Streptococcal pyrogenic exotoxin B (SPE B) was purified and its protease and mitogenic activities were investigated. The apparent molecular mass of SPE B purified in the presence of iodoacetic acid was 42 kDa, whereas 29-kDa SPE B was predominant without the reagent. A polyclonal antibody raised against the 29-kDa species reacted with both species, indicating that the 42-kDa species was a precursor of the 29-kDa entity. Both the 42- and 29-kDa species enhanced [3H]thymidine incorporation into human peripheral blood mononuclear cells, whereas neither had any effect on T cell depleted mononuclear cells. The 29-kDa SPE B possessed caseinolytic activity, with an optimal pH of 8, and the activity was specifically suppressed by the antibody. A group of cysteine protease inhibitors, but no serine-, metallo-, or acidic-protease inhibitors, limited the protease activity, whereas dithiothreitol increased the activity. The DNA sequence around a putative active cysteine residue was identical among the speB genes from Streptococcus pyogenes R70, NY-5, and T 19. Taken together, these results indicate that SPE B is identical to a cysteine protease, streptopain (EC 3.4.22.10).Key words: streptococcal pyrogenic exotoxin B, streptopain, Streptococcus pyogenes, cysteine protease.

An Enzyme-Linked Immunosorbent Assay for Urokinase-Type Plasminogen Activator (u-PA) and Mutants and Chimeras Containing the Serine Protease Domain of u-PA

1992 ◽  
Vol 67 (01) ◽  
pp. 095-100 ◽  
Author(s):  
Paul J Declerck ◽  
Leen Van Keer ◽  
Maria Verstreken ◽  
Désiré Collen
Keyword(s):  
Serine Protease ◽  
Plasminogen Activator ◽  
Active Site ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Protease Domain ◽  
Serine Protease Domain ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Normal Human

SummaryAn enzyme-linked immunosorbent assay (ELISA) for quantitation of natural and recombinant plasminogen activators containing the serine protease domain (B-chain) of urokinase-type plasminogen activator (u-PA) was developed, based on two murine monoclonal antibodies, MA-4D1E8 and MA-2L3, raised against u-PA and reacting with non-overlapping epitopes in the B-chain. MA-4D1E8 was coated on microtiter plates and bound antigen was quantitated with MA-2L3 conjugated with horseradish peroxidase. The intra-assay, inter-assay and inter-dilution coefficients of variation of the assay were 6%, 15% and 9%, respectively. Using recombinant single-chain u-PA (rscu-PA) as a standard, the u-PA-related antigen level in normal human plasma was 1.4 ± 0.6 ng/ml (mean ± SD, n = 27).The ELISA recognized the following compounds with comparable sensitivity: intact scu-PA (amino acids, AA, 1 to 411), scu-PA-32k (AA 144 to 411), a truncated (thrombin-derived) scu-PA comprising A A 157 to 411, and chimeric t-PA/u-PA molecules including t-PA(AA1-263)/scu-PA(AA144-411), t-PA(AA1-274)/scu-PA(AA138-411) and t-PA(AA87-274)/scu-PA(AA138-411). Conversion of single-chain to two-chain forms of u-PA or inhibition of active two-chain forms with plasminogen activator inhibitor-1 or with the active site serine inhibitor phenyl-methyl-sulfonyl fluoride, did not alter the reactivity in the assay. In contrast, inactivation with α2-antiplasmin or with the active site histidine inhibitor Glu-Gly-Arg-CH2Cl resulted in a 3- to 5-fold reduction of the reactivity. When purified scu-PA-32k was added to pooled normal human plasma at final concentrations ranging from 20 to 1,000 ng/ml, recoveries in the ELISA were between 84 and 110%.The assay was successfully applied for the quantitation of pharmacological levels of scu-PA and t-PA(AA87_274)/scu-PA(AA138-411) in plasma during experimental thrombolysis in baboons.Thus the present ELISA, which is specifically dependent on the presence of the serine protease part of u-PA, is useful for measurement of a wide variety of variants and chimeras of u-PA which are presently being developed for improved thrombolytic therapy.

scholarly journals Conformation-Specific Inhibitory Anti-MMP-7 Monoclonal Antibody Sensitizes Pancreatic Ductal Adenocarcinoma Cells to Chemotherapeutic Cell Kill

Cancers ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1679
Author(s):  
Vishnu Mohan ◽  
Jean P. Gaffney ◽  
Inna Solomonov ◽  
Maxim Levin ◽  
Mordehay Klepfish ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Active Site ◽  
Drug Targets ◽  
Fas Ligand ◽  
Specific Activity ◽  
Matrix Metalloproteases ◽  
Ductal Adenocarcinoma ◽  
Enzyme Active Site ◽  
Induced Apoptosis

Matrix metalloproteases (MMPs) undergo post-translational modifications including pro-domain shedding. The activated forms of these enzymes are effective drug targets, but generating potent biological inhibitors against them remains challenging. We report the generation of anti-MMP-7 inhibitory monoclonal antibody (GSM-192), using an alternating immunization strategy with an active site mimicry antigen and the activated enzyme. Our protocol yielded highly selective anti-MMP-7 monoclonal antibody, which specifically inhibits MMP-7′s enzyme activity with high affinity (IC50 = 132 ± 10 nM). The atomic model of the MMP-7-GSM-192 Fab complex exhibited antibody binding to unique epitopes at the rim of the enzyme active site, sterically preventing entry of substrates into the catalytic cleft. In human PDAC biopsies, tissue staining with GSM-192 showed characteristic spatial distribution of activated MMP-7. Treatment with GSM-192 in vitro induced apoptosis via stabilization of cell surface Fas ligand and retarded cell migration. Co-treatment with GSM-192 and chemotherapeutics, gemcitabine and oxaliplatin elicited a synergistic effect. Our data illustrate the advantage of precisely targeting catalytic MMP-7 mediated disease specific activity.

scholarly journals Toward the Identification of Potential α-Ketoamide Covalent Inhibitors for SARS-CoV-2 Main Protease: Fragment-Based Drug Design and MM-PBSA Calculations

Processes ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 1004
Author(s):  
Mahmoud A. El Hassab ◽  
Mohamed Fares ◽  
Mohammed K. Abdel-Hamid Amin ◽  
Sara T. Al-Rashood ◽  
Amal Alharbi ◽  
...  
Keyword(s):  
Drug Design ◽  
Active Site ◽  
Binding Free Energy ◽  
Stable Complex ◽  
Active Site Residues ◽  
Structure Based Drug Design ◽  
Enzyme Active Site ◽  
Potential Inhibitor ◽  
Main Protease ◽  
Covalent Docking

Since December 2019, the world has been facing the outbreak of the SARS-CoV-2 pandemic that has infected more than 149 million and killed 3.1 million people by 27 April 2021, according to WHO statistics. Safety measures and precautions taken by many countries seem insufficient, especially with no specific approved drugs against the virus. This has created an urgent need to fast track the development of new medication against the virus in order to alleviate the problem and meet public expectations. The SARS-CoV-2 3CL main protease (Mpro) is one of the most attractive targets in the virus life cycle, which is responsible for the processing of the viral polyprotein and is a key for the ribosomal translation of the SARS-CoV-2 genome. In this work, we targeted this enzyme through a structure-based drug design (SBDD) protocol, which aimed at the design of a new potential inhibitor for Mpro. The protocol involves three major steps: fragment-based drug design (FBDD), covalent docking and molecular dynamics (MD) simulation with the calculation of the designed molecule binding free energy at a high level of theory. The FBDD step identified five molecular fragments, which were linked via a suitable carbon linker, to construct our designed compound RMH148. The mode of binding and initial interactions between RMH148 and the enzyme active site was established in the second step of our protocol via covalent docking. The final step involved the use of MD simulations to test for the stability of the docked RMH148 into the Mpro active site and included precise calculations for potential interactions with active site residues and binding free energies. The results introduced RMH148 as a potential inhibitor for the SARS-CoV-2 Mpro enzyme, which was able to achieve various interactions with the enzyme and forms a highly stable complex at the active site even better than the co-crystalized reference.

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