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: Streptokinase (SK), a heterogeneous plasminogen activator (PA) protein from groups A, C, and G streptococci (GAS, GCS, GGS, respectively) contains three structural domains (SKα, SKβ, and SKg). Based on the variable region of SKβ, GAS-SKs (ska) are clustered as SK1 and SK2 (including clus...
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| Format: | Article |
| Language: | English |
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Pasteur Institute of Iran
2020-01-01
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| Series: | Iranian Biomedical Journal |
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| Online Access: | http://ibj.pasteur.ac.ir/article-1-2859-en.html |
| _version_ | 1818217694447534080 |
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| author | Maryam Rafipour Malihe Keramati Mohammad Mehdi Aslani Arash Arashkia Farzin Roohvand |
| author_facet | Maryam Rafipour Malihe Keramati Mohammad Mehdi Aslani Arash Arashkia Farzin Roohvand |
| author_sort | Maryam Rafipour |
| collection | DOAJ |
| description | Background: Streptokinase (SK), a heterogeneous plasminogen activator (PA) protein from groups A, C, and G streptococci (GAS, GCS, GGS, respectively) contains three structural domains (SKα, SKβ, and SKg). Based on the variable region of SKβ, GAS-SKs (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 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 SKG88 and SKSTAB902 genes were cloned into the NdeI/XhoI site of pET26b expression vector. The two chimeric SKβ-exchanged constructs (SKC1: αG88-βSTAB-γG88 and SKC2; αSTAB-βG88-γSTAB) 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 SKs. Analyses indicated that the catalytic efficiency (Kcat/Km) for amidolytic and proteolytic activity were less and moderately dependent on SKβ, respectively. The increase of FG-bound-Plg activation for SKSTAB902/SKC1 containing SK2aβ was around six times, whereas for SKG88/SKC2 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. |
| first_indexed | 2024-12-12T07:11:56Z |
| format | Article |
| id | doaj.art-fac0cc35a6e948f08422e80e6368fa73 |
| institution | Directory Open Access Journal |
| issn | 1028-852X 2008-823X |
| language | English |
| last_indexed | 2024-12-12T07:11:56Z |
| publishDate | 2020-01-01 |
| publisher | Pasteur Institute of Iran |
| record_format | Article |
| series | Iranian Biomedical Journal |
| spelling | doaj.art-fac0cc35a6e948f08422e80e6368fa732022-12-22T00:33:36ZengPasteur Institute of IranIranian Biomedical Journal1028-852X2008-823X2020-01-012411523Contribution of Streptokinase-Domains from Groups G and A (SK2a) Streptococci in Amidolytic/Proteolytic Activities and Fibrin-Dependent Plasminogen Activation: A Domain-Exchange StudyMaryam Rafipour0Malihe Keramati1Mohammad Mehdi Aslani2Arash Arashkia3Farzin Roohvand4 Department of Virology, Pasteur Institute of Iran, Tehran, Iran Department of Nanobiotechnology, Pasteur Institute of Iran, Tehran, Iran Department of Microbiology, Pasteur Institute of Iran, Tehran, Iran Department of Virology, Pasteur Institute of Iran, Tehran, Iran Department of Virology, Pasteur Institute of Iran, Tehran, Iran Background: Streptokinase (SK), a heterogeneous plasminogen activator (PA) protein from groups A, C, and G streptococci (GAS, GCS, GGS, respectively) contains three structural domains (SKα, SKβ, and SKg). Based on the variable region of SKβ, GAS-SKs (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 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 SKG88 and SKSTAB902 genes were cloned into the NdeI/XhoI site of pET26b expression vector. The two chimeric SKβ-exchanged constructs (SKC1: αG88-βSTAB-γG88 and SKC2; αSTAB-βG88-γSTAB) 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 SKs. Analyses indicated that the catalytic efficiency (Kcat/Km) for amidolytic and proteolytic activity were less and moderately dependent on SKβ, respectively. The increase of FG-bound-Plg activation for SKSTAB902/SKC1 containing SK2aβ was around six times, whereas for SKG88/SKC2 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.http://ibj.pasteur.ac.ir/article-1-2859-en.htmlplasminogenstreptokinasethrombolytic therapy |
| spellingShingle | Maryam Rafipour Malihe Keramati Mohammad Mehdi Aslani Arash Arashkia Farzin Roohvand Contribution of Streptokinase-Domains from Groups G and A (SK2a) Streptococci in Amidolytic/Proteolytic Activities and Fibrin-Dependent Plasminogen Activation: A Domain-Exchange Study Iranian Biomedical Journal plasminogen streptokinase thrombolytic therapy |
| title | Contribution of Streptokinase-Domains from Groups G and A (SK2a) Streptococci in Amidolytic/Proteolytic Activities and Fibrin-Dependent Plasminogen Activation: A Domain-Exchange Study |
| title_full | Contribution of Streptokinase-Domains from Groups G and A (SK2a) Streptococci in Amidolytic/Proteolytic Activities and Fibrin-Dependent Plasminogen Activation: A Domain-Exchange Study |
| title_fullStr | Contribution of Streptokinase-Domains from Groups G and A (SK2a) Streptococci in Amidolytic/Proteolytic Activities and Fibrin-Dependent Plasminogen Activation: A Domain-Exchange Study |
| title_full_unstemmed | Contribution of Streptokinase-Domains from Groups G and A (SK2a) Streptococci in Amidolytic/Proteolytic Activities and Fibrin-Dependent Plasminogen Activation: A Domain-Exchange Study |
| title_short | Contribution of Streptokinase-Domains from Groups G and A (SK2a) Streptococci in Amidolytic/Proteolytic Activities and Fibrin-Dependent Plasminogen Activation: A Domain-Exchange Study |
| title_sort | contribution of streptokinase domains from groups g and a sk2a streptococci in amidolytic proteolytic activities and fibrin dependent plasminogen activation a domain exchange study |
| topic | plasminogen streptokinase thrombolytic therapy |
| url | http://ibj.pasteur.ac.ir/article-1-2859-en.html |
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