Structural Diversity of Streptokinase and Activation of Human Plasminogen

Abstract

The ␤ domain of streptokinase is required for plasminogen activation and contains a region of sequence diversity associated with infection and disease in group A streptococci. We report that mutagenesis of this polymorphic region does not alter plasminogen activation, which suggests an alternative function for this molecular motif in streptococcal disease.Streptokinase (SK) is a plasminogen activator secreted by group A, C, and G streptococci. SK contributes to streptococcal virulence by generating plasmin, which leads to bacterial spread from a primary focus of infection by causing fibrinolysis and degradation of extracellular matrix and basement membrane components (6, 16). Plasmin also induces inflammation via complement activation, which may play a role in postinfectious diseases, such as glomerulonephritis (12).The extent to which SK is involved in streptococcal pathogenesis may depend on structural differences among SKs. SK is a 414-amino-acid (aa) protein composed of three structural domains: ␣ (aa 1 to 150), ␤ (aa 151 to 287), and ␥ (aa 288 to 414) (19). The highly conserved (Ն85% sequence identity) ␣ and ␥ domains provide most of the contact sites with the plasmin moiety (2, 19) and exhibit a synergistic effect on plasminogen activation (9). The ␤ domain provides no direct contact sites with the plasmin active-site moiety, yet it is required to dock plasminogen via a kringle-binding surface-exposed hairpin loop (termed the 250-loop) between residues 251 and 262 ( Fig. 1A) (1, 18). In contrast, a region between amino acids 144 and 218 which spans the second structural loop of the ␤ domain (170-loop) (Fig. 1A) is the major focus of sequence heterogeneity (4), yet its function is unknown. The side chains of nonconserved amino acids within this heterogenous loop are oriented towards the surface, away from the side that faces the plasmin moiety of the activator complex (18).This suggests that SK polymorphism may not be directly engaged in plasminogen binding and activation but instead may determine biological properties related to disease. Previous studies have linked sequence patterns within this polymorphic motif to specific strains of group A streptococci (GAS) that cause acute poststreptococcal glomerulonephritis (APSGN) (10). Also, phylogenetic analysis of ␤-domain sequences reveals a strong linkage disequilibrium (P Ͻ Ͻ 0.01) with plasminogen-binding group A streptococcal M protein (PAM) in strains that exhibit tissue tropism to the skin (5). Considering the fact that both SK and PAM cooperate to enhance streptococcal virulence through plasminogen activation (15), the specific association between ␤-domain polymorphism and tissue tropism further supports a role of this motif in disease.Despite these associations, the biological significance of sequence polymorphism in SK remains unknown. Studies on SK structure and function have focused entirely on therapeutic SK from a nonpathogenic group C streptococcus and have not considered the role of sequence polymorphism of the ␤ domain on plasminogen activation...

Allelic variants of streptokinase fromStreptococcus pyogenesdisplay functional differences in plasminogen activation

“…This suggests that variation within the β domain of these proteins may be responsible for the observed differences in plasminogen activation activities. Lizano and Johnston (42) have suggested that sequence polymorphism in the β-domain has little affect on plasminogen activation. The recombinant streptokinase produced from SF13013 (containing a cluster 2a ska allele) in that study displayed soluble plasminogen activation activity.…”

Identification of a New Exosite Involved in Catalytic Turnover by the Streptokinase-Plasmin Activator Complex during Human Plasminogen Activation

“…Interestingly, the two amino acid residues identified to be relatively vital for 170 loop functioning, namely Pro 177 and Lys 180 , are phylogenetically conserved across the various streptokinase allelic variant "classes" (Fig. 6) despite the generally diverse sequence heterogeneity in this region of streptokinase (34,35). This suggests the evolutionary significance of these amino acid residues in the 170 loop that impart high catalytic turnover to SK⅐plasmin.…”

Role of Streptococcal T Antigens in Superficial Skin Infection

“…Recombinant plasmids were used to transform Escherichia coli, and recombinant polypeptides were expressed as fusions with glutathione S-transferase (GST). Recombinant fusion polypeptides were purified by glutathione-Sepharose affinity chromatography (25). The primers used for PCR amplification are listed in Table S2 at http://www.nymc.edu/hotfiles/s3pdebra_bessen.pdf.…”