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

ABSTRACT: Background:Streptokinase (SK), a heterogeneous plasminogen activator (PA) protein from groups A, C, and G streptococci (GAS, GCS, GGS, respectively) contains three structural domains (SK?, SK?, and SK?). Based on the variable region of SK?, GAS-SK (ska) are clustered as SK1 and SK2 (including cluster2-streptokinase (SK2a)/SK2b), which show low and high fibrinogen (FG)-dependent plasminogen (Plg) activation properties, respectively. Despite being co-clustered as SK2a, GCS/GGS-SK (skcg) variants display properties similar to SK1. Herein, by SK? 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 SKG88 and SKSTAB902 genes were cloned into the NdeI/XhoI site of pET26b expression vector. The two chimeric SK?-exchanged constructs (SKC1: ?G88-?STAB-?G88 and SKC2; ?STAB-?G88-?STAB) were constructed by BstEII/BsiWI digestion/cross-ligation in parental plasmids. SK were expressed in E. coli and purified by nickel-nitriloacetic acid chromatography. PA potencies of SKs 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 (Kcat/Km) for amidolytic and proteolytic activity were less and moderately dependent on SK?, respectively. The increase of FG-bound-Plg activation for SKSTAB902/SKC1 containing SK2a? was around six times, whereas for SKG88/SKC2 containing skcg?, it was four times. Conclusion:Although SK? 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.

SUBMITTER: Rafipour M 

PROVIDER: S-EPMC6900475 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature