Structure and binding determinants of the recombinant kringle-2 domain of human plasminogen to an internal peptide from a group A Streptococcal surface protein 1 1Edited by R. Huber

2001 ◽  
Vol 308 (4) ◽  
pp. 705-719 ◽  
Author(s):  
Jorge L. Rios-Steiner ◽  
Mónica Schenone ◽  
Igor Mochalkin ◽  
Alexander Tulinsky ◽  
Francis J. Castellino
Keyword(s):  
Surface Protein ◽  
Group A ◽  
Human Plasminogen ◽  
Internal Peptide ◽  
Kringle 2 ◽  
Binding Determinants

scholarly journals Binding of the kringle-2 domain of human plasminogen to streptococcal PAM-type M-protein causes dissociation of PAM dimers

2021 ◽  
Author(s):  
Olawole Ayinuola ◽  
Yetunde Ayinuola ◽  
Cunjia Qiu ◽  
Shaun Lee ◽  
Victoria Ploplis ◽  
...  
Keyword(s):  
Group A Streptococcus ◽  
Tight Binding ◽  
Coiled Coil ◽  
M Protein ◽  
Temperature Dependent ◽  
Functional Monomers ◽  
Molecular Weights ◽  
Group A ◽  
Human Plasminogen ◽  
Kringle 2

M-protein (PAM) largely contributes to the pathogenesis of Pattern D Group A Streptococcus pyogenes (GAS). However, the mechanism of complex formation is unknown. In a system consisting of a Class II PAM from Pattern D GAS isolate NS88.2 (PAMNS88.2), with one K2hPg binding a-repeat in its A-domain, we employed biophysical techniques to analyze the mechanism of the K2hPg/PAMNS88.2 interaction. We show that apo-PAMNS88.2 is a coiled-coil homodimer (M.Wt. ~80 kDa) at 4°C - 25°C, and is monomeric (M.Wt. ~40 kDa) at 37°C, demonstrating a temperature-dependent dissociation of PAMNS88.2 over a narrow temperature range. PAMNS88.2 displayed a single tight binding site for K2hPg at 4°C, which progressively increased at 25°C through 37°C. We isolated the K2hPg/PAMNS88.2 complexes at 4°C, 25°C, and 37°C and found molecular weights of ~50 kDa at each temperature, corresponding to a 1:1 (m:m) K2hPg/PAMNS88.2 monomer complex. hPg activation experiments by streptokinase demonstrated that the hPg/PAMNS88.2 monomer complexes are fully functional. The data show that PAM dimers dissociate into functional monomers at physiological temperatures or when presented with the active hPg module (K2hPg) showing that PAM is a functional monomer at 37°C.

scholarly journals The Lack of Binding of VEK-30, an Internal Peptide from the Group A Streptococcal M-like Protein, PAM, to Murine Plasminogen Is due to Two Amino Acid Replacements in the Plasminogen Kringle-2 Domain

2007 ◽  
Vol 283 (3) ◽  
pp. 1580-1587 ◽  
Author(s):  
Qihua Fu ◽  
Mariana Figuera-Losada ◽  
Victoria A. Ploplis ◽  
Sara Cnudde ◽  
James H. Geiger ◽  
...  
Keyword(s):  
Amino Acid ◽  
Molecular Basis ◽  
Species Specificity ◽  
Therapeutic Interventions ◽  
Positive Group ◽  
High Affinity ◽  
Group A ◽  
High Conservation ◽  
Internal Peptide ◽  
Kringle 2

VEK-30, a 30-amino acid internal peptide present within a streptococcal M-like plasminogen (Pg)-binding protein (PAM) from Gram-positive group-A streptococci (GAS), represents an epitope within PAM that shows high affinity for the lysine binding site (LBS) of the kringle-2 (K2) domain of human (h)Pg. VEK-30 does not interact with this same region of mouse (m)Pg, despite the high conservation of the mK2- and hK2-LBS. To identify the molecular basis for the species specificity of this interaction, hPg and mPg variants were generated, including an hPg chimera with the mK2 sequence and an mPg chimera containing the hK2 sequence. The binding of synthetic VEK-30 to these variants was studied by surface plasmon resonance. The data revealed that, in otherwise intact Pg, the species specificity of VEK-30 binding in these two cases is entirely dictated by two K2 residues that are different between hPg and mPg, namely, Arg-220 of hPg, which is a Gly in mPg, and Leu-222 of hPg, which is a Pro in mPg, neither of which are members of the canonical K2-LBS. Neither the activation of hPg, nor the enzymatic activity of its activated product, plasmin (hPm), are compromised by replacing these two amino acids by their murine counterparts. It is also demonstrated that hPg is more susceptible to activation to hPm after complexation with VEK-30 and that this property is greatly reduced as a result of the R220G and L222P replacements in hPg. These mechanisms for accumulation of protease activity on GAS likely contribute to the virulence of PAM+-GAS strains and identify targets for new therapeutic interventions.

scholarly journals The M Protein Is Dispensable for Maturation of Streptococcal Cysteine Protease SpeB

2005 ◽  
Vol 73 (2) ◽  
pp. 859-864 ◽  
Author(s):  
Björn Zimmerlein ◽  
Hae-Sun Park ◽  
Shaoying Li ◽  
Andreas Podbielski ◽  
P. Patrick Cleary
Keyword(s):  
Cysteine Protease ◽  
Protease Activity ◽  
Active Form ◽  
Surface Protein ◽  
M Protein ◽  
Cysteine Protease Activity ◽  
Group A ◽  
Streptococcal Pyrogenic Exotoxin B ◽  
Major Surface ◽  
Culture Supernatants

ABSTRACT The streptococcal pyrogenic exotoxin B (SpeB) is an important virulence factor of group A streptococci (GAS) with cysteine protease activity. Maturation of SpeB to a proteolytically active form was suggested to be dependent on cell-wall-anchored M1 protein, the major surface protein of GAS (M. Collin and A. Olsén, Mol. Microbiol. 36:1306-1318, 2000). Collin and Olsén showed that mutant GAS strains expressing truncated M protein secrete a conformationally different form of unprocessed SpeB with no proteolytic activity. Alternatively, we hypothesized that a truncated M protein may interfere with processing of this secreted protease, and therefore we tested cysteine protease activity in genetically defined mutant strains that express either no M protein or membrane-anchored M protein with an in-frame deletion of the AB repeat region. Measurements of SpeB activity by cleavage of a substrate n-benzoyl-Pro-Phe-Arg-p-nitroanilide hydrochloride showed that the proteolytic activities in culture supernatants of both mutants were similar to those from the wild-type strain. In addition, Western blot analysis of culture supernatants showed that SpeB expression and processing to a mature form was unaffected by either deletion mutation. Therefore, we conclude that M protein is not required for maturation of the streptococcal cysteine protease SpeB.

scholarly journals A Novel Sortase, SrtC2, from Streptococcus pyogenes Anchors a Surface Protein Containing a QVPTGV Motif to the Cell Wall

2004 ◽  
Vol 186 (17) ◽  
pp. 5865-5875 ◽  
Author(s):  
Timothy C. Barnett ◽  
Aman R. Patel ◽  
June R. Scott
Keyword(s):  
Cell Wall ◽  
Streptococcus Pyogenes ◽  
Group A Streptococcus ◽  
Surface Protein ◽  
Surface Proteins ◽  
Human Pathogen ◽  
Hydrophobic Region ◽  
C Terminus ◽  
Group A ◽  
Covalently Linked

ABSTRACT The important human pathogen Streptococcus pyogenes (group A streptococcus GAS), requires several surface proteins to interact with its human host. Many of these are covalently linked by a sortase enzyme to the cell wall via a C-terminal LPXTG motif. This motif is followed by a hydrophobic region and charged C terminus, which are thought to retard the protein in the cell membrane to facilitate recognition by the membrane-localized sortase. Previously, we identified two sortase enzymes in GAS. SrtA is found in all GAS strains and anchors most proteins containing LPXTG, while SrtB is present only in some strains and anchors a subset of LPXTG-containing proteins. We now report the presence of a third sortase in most strains of GAS, SrtC. We show that SrtC mediates attachment of a protein with a QVPTGV motif preceding a hydrophobic region and charged tail. We also demonstrate that the QVPTGV sequence is a substrate for anchoring of this protein by SrtC. Furthermore, replacing this motif with LPSTGE, found in the SrtA-anchored M protein of GAS, leads to SrtA-dependent secretion of the protein but does not lead to its anchoring by SrtA. We conclude that srtC encodes a novel sortase that anchors a protein containing a QVPTGV motif to the surface of GAS.

scholarly journals Reduced thrombin generation increases host susceptibility to group A streptococcal infection

Blood ◽  
2009 ◽  
Vol 113 (6) ◽  
pp. 1358-1364 ◽  
Author(s):  
Hongmin Sun ◽  
Xixi Wang ◽  
Jay L. Degen ◽  
David Ginsburg
Keyword(s):  
Host Defense ◽  
Thrombin Generation ◽  
Host Susceptibility ◽  
Microbial Pathogens ◽  
Factor V ◽  
Significant Survival ◽  
Hemostatic Factors ◽  
Group A ◽  
Human Plasminogen ◽  
Fv Leiden

AbstractBacterial plasminogen activators are commonplace among microbial pathogens, implying a central role of host plasmin in supporting bacterial virulence. Group A streptococci (GAS) secrete streptokinase, a specific activator of human plasminogen (PLG). The critical contribution of the streptokinase-PLG interaction to GAS pathogenicity was recently demonstrated using mice expressing human PLG. To examine the importance of thrombin generation in antimicrobial host defense, we challenged mice with deficiency of factor V (FV) in either the plasma or platelet compartment. Reduction of FV in either pool resulted in markedly increased mortality after GAS infection, with comparison to heterozygous F5-deficient mice suggesting a previously unappreciated role for the platelet FV pool in host defense. Mice with complete deficiency of fibrinogen also demonstrated markedly increased mortality to GAS infection relative to controls. Although FV Leiden may be protective in the setting of severe sepsis in humans, no significant survival advantage was observed in GAS-infected mice carrying the FV Leiden mutation. Taken together, our data support the hypothesis that local thrombosis/fibrin deposition limits the survival and dissemination of at least a subset of microbial pathogens and suggest that common variation in hemostatic factors among humans could affect host susceptibility to a variety of infectious diseases.

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