Preferential acquisition and activation of plasminogen glycoform II by PAM positive group A streptococcal isolates

Plasminogen (Plg) circulates in the host as two predominant glycoforms. Glycoform I Plg (GI-Plg) contains glycosylation sites at Asn(289) and Thr(346), whereas glycoform II Plg (GII-Plg) is exclusively glycosylated at Thr(346). Surface plasmon resonance experiments demonstrated that Plg binding group A streptococcal M protein (PAM) exhibits comparative equal affinity for GI- and GII-Plg in the closed conformation (for GII-Plg, K-D = 27.4 nM; for GI-Plg, K-D = 37.0 nM). When Plg was in the open conformation, PAM exhibited an 11-fold increase in affinity for GII-Plg (K-D = 2.8 nM) compared with that for GI-Plg (K-D = 33.2 nM). The interaction of PAM with Plg is believed to be mediated by lysine binding sites within kringle (KR) 2 of Plg. PAMGI-Plg interactions were fully inhibited with 100 mM lysine analogue epsilon-aminocaproic acid (epsilon ACA), whereas PAMGII-Plg interactions were shown to be weakened but not inhibited in the presence of 400 mM epsilon ACA. In contrast, binding to the KR13 domains of GII-Plg (angiostatin) by PAM was completely inhibited in the presence 5 mM epsilon ACA. Along with PAM, emm pattern D GAS isolates express a phenotypically distinct SK variant (type 2b SK) that requires Plg ligands such as PAM to activate Plg. Type 2b SK was able to generate an active site and activate GII-Plg at a rate significantly higher than that of GI-Plg when bound to PAM. Taken together, these data suggest that GAS selectively recruits and activates GII-Plg. Furthermore, we propose that the interaction between PAM and Plg may be partially mediated by a secondary binding site outside of KR2, affected by glycosylation at Asn(289)