Snake venom metalloproteinases (SVMPs) are enzymes with multiple domains classified in classes PI to PIV, and they are widely distributed in venoms of Viperidae snakes. Their main toxic effects are due to disruption of the hemostatic system and result in symptoms of human envenomation. Some of them are glycoproteins with N-linked glycosylation. However, the role of N-linked glycan and its relationship to the enzymatic activity is still unclear. We purified two novel SVMPs from Taiwan cobra (Naja atra) venom and they were identified by MS and full length sequence. Results indicated they were both PIII class SVMPs with N-linked glycan and designated as Vansigin and Atragin. After De-N-glycosylation, the enzymatic activity of Vansigin increased but Atragin decreased. According to their X-ray crystal structures and N-linked glycan locations, we suggest N-linked glycosylation play an important role in the enzymatic activity and it can also explain why the enzyme specificity of Vansigin is higher than Atragin. Based on previous research, we found there is geographic variation of CTX family expression between eastern and western origin of Taiwan cobra venom. We used chromatography and tandem MS technique to show the expression of Vansigin and Atragin also involved in this phenomenon. Surprisingly, their terminal sugars of N-linked glycan were also found variational modification in the geographic variation issue. This indicates that eastern origin of Taiwan cobra venom gland may lack gene of sialyltransferase or its N-linked glycosylation biosynthetic pathway of Vansigin has been regulated. Overall, we suggest that N-linked glycosylation in different domains will have different influences in enzymatic activity, and the same protein can have variational modification on N-linked glycan due to regional evolution.

蛇毒金屬蛋白水解酵素為具有多個功能區域的酵素並依此區分為四大類型PI到PIV，一般廣泛存在於腹蛇科及響尾蛇科，其主要的毒性及症狀來自於破壞生理平衡系統。已有許多文獻指出此酵素具有N鍵結醣基(N-linked glycan)，然而其與酵素活性之間的關聯仍未明朗。台灣眼鏡蛇毒中的兩個蛇毒金屬蛋白水解酵素Vansigin及Atragin，核苷酸序列及質譜儀分析顯示兩者皆為第三類型且皆具有N鍵結醣基；移除N鍵結醣基後Vansigin活性增強而Atragin活性減弱。伴隨兩者晶體結構解出及N鍵結醣基位置的確認，我們可以合理推測N鍵結醣基在酵素活性中扮演的重要角色，同時也解釋Vansigin為何具有比Atragin更高的受質專一性。
實驗室之前研究指出，台灣東西部眼鏡蛇毒在低分子量心臟毒蛋白家族的表達有所差異，顯示了地理區域分布及環境對同種眼鏡蛇在蛇毒蛋白的分泌及表達上有所影響。此論文另一部分討論高分子量蛋白如Vansigin及Atragin是否有上述的現象。藉由層析純化來自台灣東西部個體眼鏡蛇毒，並運用串聯式質譜儀分析個體眼鏡蛇毒蛋白Vansigin及Atragin的N鍵結醣基，發現台灣東西部眼鏡蛇除了在高分子量蛋白的表達量有差異外，N鍵結醣基末端醣修飾也有變異性存在。此結果隱含分子生物學層面上，台灣東部眼鏡蛇毒腺體可能缺乏sialyltransferase的基因，或在生合成的路徑受到抑制性的調控。
綜觀以上結果，我們認為N鍵結醣基位於不同的功能區域會造成酵素活性不同的影響，且在相同蛋白上的N鍵結醣基可能因演化而造成修飾差異。

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