| Summary: | 碩士 === 國立清華大學 === 生物資訊與結構生物研究所 === 97 === P-III type snake venom metalloproteases(SVMPs) are major enzymes with metalloprotease/disintegrin/cysteine-rich(MDC) domains in viper venom and have been implicated to disrupt the haemostatic system for the envenomed victims of viper snakebite. Recently, our laboratory identified two novel SVMPs, atragin and k-like, with similar primary sequence (~60%) and 3D structure. Atragin binds to αvβ3 integrin, inhibits NIH3T3 cell migration and possesses heparin binding ability. However, the binding model of atragin/αvβ3 integrin and the role of heparin binding on SVMP enzymatic activity were still unclear.
(i) We determined the binding affinity between SVMPs/integrin and SVMPs/heparin by using SPR, respectively. We found that atragin bind to αvβ3 integrin and heparin stronger than k-like with apparent dissociation constants ~53nM and ~17nM, respectively. It is consistent with the observation that atragin binding to heparin affinity column stronger than k-like. In addition, the affinity of the recombinant Cys-rich domain of atragin binding to αvβ3 integrin and heparin were ~8 and ~3 times weaker than whole protein, indicating that other domains might also be involved in the binding process. (ii) The RGD peptide competed with atragin binding αvβ3 integrin, indicating that atragin bind to RGD binding site of integrin. According to the molecular docking model, not only Cys-rich domain binds to the RGD site of αvβ3 integrin but also M domain of atragin may be involved in atragin/integrin interaction in consistent with our SPR studies. (iii) In fibrinogen digestion assay, αvβ3 integrin inhibited the enzymatic activity of atragin by binding to αvβ3 integrin. It suggested that atragin binding to αvβ3 integrin could interrupt natural ligands binding to αvβ3 integrin and the C-shape structure of atragin in binding integrin modulates the enzymatic activity. Beside, atragin can digest additional β chain of fibrinogen in the presence of heparin. This phenomenon indicated heparin binding could change the enzymatic specificity of atragin, therefore, may affect biological activity such as angiogenesis.
These results suggest that SVMPs binds to heparin with biological significance and non-RGD protein could be able to bind to integrin αvβ3.
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