Divergence in the Plasminogen-binding Group A Streptococcal M Protein Family

Abstract

Group A streptococci (GAS) display receptors for the human zymogen plasminogen on the cell surface, one of which is the plasminogen-binding group A streptococcal M protein (PAM). Characterization of PAM genes from 12 GAS isolates showed significant variation within the plasminogen-binding repeat motifs (a1/a2) of this protein. To determine the impact of sequence variation on protein function, recombinant proteins representing five naturally occurring variants of PAM, together with a recombinant M1 protein, were expressed and purified. Equilibrium dissociation constants for the interaction of PAM variants with biotinylated Gluplasminogen ranged from 1.58 to 4.99 nM. Effective concentrations of prototype PAM required for 50% inhibition of plasminogen binding to immobilized PAM variants ranged from 0.68 to 22.06 nM. These results suggest that although variation in the a1/a2 region of the PAM protein does affect the comparative affinity of PAM variants, the functional capacity to bind plasminogen is conserved. Additionally, a potential role for the a1 region of PAM in eliciting a protective immune response was investigated by using a mouse model for GAS infection. The a1 region of PAM was found to protect immunized mice challenged with a PAM-positive GAS strain. These data suggest a link between selective immune pressure against the plasminogen-binding repeats and the functional conservation of the binding domain in PAM variants.

The Maintenance of High Affinity Plasminogen Binding by Group A Streptococcal Plasminogen-binding M-like Protein Is Mediated by Arginine and Histidine Residues within the a1 and a2 Repeat Domains

“…However, in the Northern Territory of Australia, where streptococcal skin infection is endemic and high rates of invasive infection such as bacteremia have been reported (10), PAM-positive GAS are associated with a variety of disease states (11). The plasminogen binding domain of PAM has been shown to be highly variable; however, this diversity does not abrogate plasminogen binding by naturally occurring PAM variants (12). This finding may, in part, be due to the conservation of key amino acid residues within the plasminogen binding domain (11,12).…”

“…Site-directed Mutagenesis-The PAM gene from streptococcal strain NS13 (PAM NS13 GenBank TM AY351851), which is identical to the prototype PAM sequence in the a1 and a2 repeat domain, had previously been cloned into the expression vector pGEX2T (12). To assess the role of specific-binding site residues in the interaction of PAM with plasminogen, site-specific mutations were introduced into the PAM NS13 -binding site.…”

“…Recombinant proteins were expressed and purified essentially as described previously (12,19), from 1 liter of E. coli cultures, using glutathione-agarose (Sigma-Aldrich) and native nickel-nitrilotriacetic acid-agarose (Qiagen, Germany) affinity chromatography. Prior to elution from the glutathione-agarose column, the glutathione S-transferase tag was removed from the N terminus of recombinant fusion proteins by the addition of one column volume of thrombin solution (1 unit of thrombin/l, phosphate-buffered saline, pH 8.0).…”

“…The plasminogen binding domain of PAM has been shown to be highly variable; however, this diversity does not abrogate plasminogen binding by naturally occurring PAM variants (12). This finding may, in part, be due to the conservation of key amino acid residues within the plasminogen binding domain (11,12). Plasmin-(ogen) is known to interact with its ligands via pre-formed lysine-binding sites within the N-terminally located tripledisulfide-bonded kringle domains (4).…”

A Processed Multidomain Mycoplasma hyopneumoniae Adhesin Binds Fibronectin, Plasminogen, and Swine Respiratory Cilia

“…Porcine Plasminogen Purification-Plasminogen was isolated from porcine plasma as described previously (27,28). MALDI Q-TOF tandem MS, performed according to Henrich et al (26), confirmed the purified protein as plasminogen.…”