Contribution of Streptokinase-Domains from Groups G and A (SK2a) Streptococci in Amidolytic/Proteolytic Activities and Fibrin-Dependent Plasminogen Activation: A Domain-Exchange Study

BACKGROUND: SK, a heterogeneous PA protein from groups A, C, and G streptococci (GAS, GCS, GGS, respectively) contains three structural domains (SKα, SKβ, and SK). Based on the variable region of SKβ, GAS-SK (ska) are clustered as SK1 and SK2 (including SK2a/SK2b), which show low and high FG-dependent Plg activation properties, respectively. Despite being co-clustered as SK2a, GCS/GGS-SK (skcg) variants display properties similar to SK1. Herein, by SKβ exchange between GGS (G88) and GAS-SK2a (STAB902) variants, the potential roles of SK domains in amidolytic/proteolytic activity and FG-bound-Plg activation are represented. METHODS: Two parental SK(G88) and SK(STAB902 )genes were cloned into the NdeI/XhoI site of pET26b expression vector. The two chimeric SKβ-exchanged constructs (SK(C1): α(G88)-β(STAB)-γ(G88) and SK(C2); α(STAB)-β(G88)-γ(STAB)) were constructed by BstEII/BsiWI digestion/cross-ligation in parental plasmids. SK were expressed in E. coli and purified by Ni-NTA chromatography. PA potencies of SK were measured by colorimetric assay. RESULTS: SDS-PAGE and Western-blot analyses confirmed the proper expression of 47-kDa SK. Analyses indicated that the catalytic efficiency (K(cat)/K(m)) for amidolytic and proteolytic activity were less and moderately dependent on SKβ, respectively. The increase of FG-bound-Plg activation for SK(STAB902)/SK(C1) containing SK2aβ was around six times, whereas for SK(G88)/SK(C2) containing skcgβ, it was four times. CONCLUSION: Although SKβ has noticeable contribution in FG-bound-Plg activation activity, it had minor contribution in fibrin-independent, amidolytic activity. These data might be of interest for engineering fibrin-specific versions of SK.