- 學位論文
整合素和醣胺素對台灣眼鏡蛇毒金屬酶蛋白的酵素活性和專一性之影響
The effects of integrin and heparin binding on the enzymatic activity and specificity of Taiwan cobra SVMPs
摘要
PIII類型蛇毒金屬酶蛋白擁有金屬水解酵素區域、去整合素相似區域、多半胱胺酸區域，且為主要在蛇毒毒液中的主要酵素，且被發現和傷患病人的凝血機制相關。最近我們實驗室找到兩個像似序列 (~60%)、3D結構的蛇毒金屬酶蛋白，分別為atragin和k-like，並且由之前實驗知道atragin會和整合素αvβ3結合進而去抑制NIH 3T3纖維母細胞移動，且擁有醣胺素結合能力，然而我們並不清楚atragin和整合素αvβ3、醣胺素之前的結合模式及atragin 和它們結合之後在蛇毒金屬酶蛋白酵素功能上所扮演的角色。 （一）因此我們利用表面電漿共振技術去分別測量atragin和整合素αvβ3還有atragin和醣胺素之間的親和力，實驗發現atragin和整合素αvβ3、醣胺素的解離常數分別大約為53nM、17nM，相較於k-like有較強的結合力，這個和利用醣胺素管柱分析的現象一致。由於，重組表現atragin多半胱胺酸區域蛋白和整合素αvβ3、醣胺素的親和力比整個蛋白比較分別弱約8倍和3倍，此實驗顯示除了atragin的多半胱胺酸區域，其他蛋白區域也可能參與在整合素、醣胺素結合模式。（二）利用RGD胜肽去和atragin競爭實驗，顯示atragin結合在整合素的RGD結合區位上。根據我們利用電腦模擬的結合模式發現不只多半胱胺酸區域結合在RGD結合區位上，還有金屬水解酵素區域也有可能參與atragin和整合素的結合模式，此和我們SPR所得到的結果一致。（三）在水解纖維蛋白原的實驗中，整合素αvβ3和atragin結合後會抑制atragin水解纖維蛋白原的活性，這可能顯示atragin干擾整合素和其配體結合且atragin 的c形結構可以藉由和整合素結合去調節atragin的活性。此外，atragin利用和醣胺素結合，使得atragin可以額外水解纖維蛋白原的β鍊，此現象表示與醣胺素結合可以改變atragin的酵素專一性並且可能影響和纖維蛋白原β鍊相關的生物活性，例如：新血管新生。 上述結果顯示蛇毒金屬酶蛋白的醣胺素結合能力具有生物意義，而且非RGD的蛋白亦有可能與整合素αvβ3結合。
並列摘要
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 toαvβ3 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.