烙铁头蛇毒中一个具有抗补体活性金属蛋白酶的纯化和性质研究

Purification and characterization of a metalloproteinase with anticomplementary activity from Trimeresurus mucrosquamatus venom

目的从烙铁头蛇毒中筛选分离抗补体活性蛋白,并对其部分生物学活性开展研究,以了解其在蛇伤中的病理生理作用和潜在的应用价值。方法采用蛋白层析技术分离纯化抗补体活性蛋白,测定其分子量、等电点、抗补体作用、多种蛋白水解酶活性、水肿活性及出血活性。结果从烙铁头蛇毒中分离纯化出一个抗补体活性蛋白TMAC-1,表观分子量约为25ku,等电点为9.0。TMAC-1能够抑制补体经典途径和替代途径的溶血,预孵条件下,其IC50分别为62、29mg·L-1;不预孵条件下,其IC50为263、246mg·L-1。TMAC-1能够裂解C3、C5,但裂解产物不能诱导内皮细胞P-selectin的表达。TMAC-1能依次降解纤维蛋白原的Aα、Bβ、γ链,该活性能被EDTA、1,10-phenanthroline、EGTA完全抑制,不受PMSF、SBTI的抑制。TMAC-1具有水肿活性和微弱的偶氮酪蛋白水解活性,没有精氨酸酯酶水解活性和皮下出血活性。结论 TMAC-1是一个非出血性的金属蛋白酶,它可通过酶切补体特定成分抑制补体激活。

Aim To screen and purify anticomplementary protein from Trimeresurus mucrosquamatus venom,then further to investigate how it works physiologically and pathologically in snakebite wound,and explore potential application.Methods The anticomplementary protein was isolated by sequentical chromatography.Assays were made to determine the molecular weight,isoelectric point,anti-complementary effect,proteolytic activity,edema-inducing activity and hemorrhagic activity.Results An anti-complementary protein,named TMAC-1,was purified from Trimeresurus mucrosquamatus venom.It was a single chain basic protein with molecular weight of about 25 ku,and an isoelectric point of pH 9.0.TMAC-1 inhibited classical pathway and alternative pathway of complement activation.After preincubation,IC50 values of classsical pathway and al-ternative pathway were 62,29 mg·L-1;and 263,246 mg·L-1 without preincubation,respectively.TMAC-1 cleaved C3 and C5,but the cleavage products could not induce HMEC to release P-selectin.TMAC-1 degraded Aα,Bβ and γ chains of fibrinogen.This fibrinogenolytic activity was inhibited by EDTA,1,10-phenanthroline and EGTA,but not by PMSF and soybean trysin inhibitior.TMAC-1 showed edema-inducing activity and weak azocaseinolytic activity,but it did not show arginine esterase activity and hemorrhagic activity.Conclusion TMAC-1 is a non-hemorrhagic metalloprotease,which inhibits complement by degrading certain complement components.