轻链二硫键缺失突变促进链间重组二硫键BDD-FVⅢ变构体分泌  

Elimination of a disulfide bond in the light chain of coagulation factor VⅢ improves secretion of a BDD-FVⅢ variant with an engineered inter-chain disulfide

作　　者：刘泽隆[1] 缪静[1] 屈慧鸽[1] 迟晓艳[1] 朱甫祥[1] LIU Ze-long;MIAO Jing;QU Hui-ge;CHI Xiao-yan;ZHU Fu-xiang(Life Science College,Ludong University,Yantai 264025,China)

机构地区：[1]鲁东大学生命科学学院

出　　处：《药学学报》2020年第1期54-59,共6页Acta Pharmaceutica Sinica

基　　金：山东省自然科学基金资助项目(ZR2010CM061);教育部留学回国人员科研启动基金(20071108)

摘　　要：凝血VⅢ因子(coagulation factor VⅢ, FVⅢ)分子内含有8对二硫键,参与维持其结构和功能,轻链A3结构域中Cys1899/Cys1903间的二硫键阻碍分泌,本室曾通过在FVⅢ重链和轻链间引入重组二硫键,证明其可促进消除B结构域的FVⅢ (B domain-deleted FVⅢ, BDD-FVⅢ)重、轻链异源二聚体的组装和分泌。本文在此基础上将BDD-FVⅢ的重链A2区Met662和轻链A3区Asp1828突变为Cys,从而构建可形成链间二硫键的变构体F8C。将F8C轻链A3区Cys1899和Cys1903突变为Gly,从而消除内源性二硫键,得到变构体F8CG,并探索这两种BDD-FVⅢ变构体的分泌促进作用。通过HEK293和COS-7细胞的基因瞬时转染实验,检测细胞表达变构体多肽的二硫键形成情况和培养上清中变构体多肽的分泌量和凝血活性,并检测变构体的血管性血友病因子(von Willebrand factor, vWF)结合力。结果显示,细胞转基因表达产物F8C和F8CG均以二硫键连接的异源二聚体多肽为主,而野生型BDD-FVⅢ以易于解离的异源二聚体多肽为主。F8C分泌量和活性明显高于野生型BDD-FVⅢ, F8CG的分泌量和活性相对于F8C得到进一步提高,显示轻链二硫键的破坏对分泌具有促进作用,而对vWF的结合力无显著性影响。本文为进一步的变构体体内转基因研究奠定了基础。The coagulation VⅢ factor(FVⅢ) contains eight pairs of disulfide bonds, which are involved in maintaining its structure and function. It has been demonstrated that the disulfide bond between Cys1899/Cys1903 of the A3 domain in the light chain impedes secretion. In our previous work, an engineered inter-chain disulfide in the B domain-deleted FVⅢ(BDD-FVⅢ) promoted heterodimer assembly and secretion of separately expressed heavy and light chains. In this study, we constructed two BDD-FVⅢ variants, one of which contains an engineered interchain disulfide bond(F8 C) between Met662> Cys and Asp1828> Cys mutations and another contains an endogenous A3 domain with a disrupted disulfide bond from F8 C(F8 CG) by replacement of Cys1899 and Cys1903 with Gly in F8 C. We explored their function and secretion. By transducing F8 C and F8 CG into HEK293 and COS-7 cells, the formation of disulfide bonds and the secretion and coagulation activity of the two variants in the culture media and their binding affinity for von Willebrand factor(vWF) could be observed. The results show that variants F8 C and F8 CG are mainly the disulfide bonded heavy and light chain dimer, while the wild type BDD-FVⅢ(F8) is dominated by the easily dissociated heavy and light chain dimer. The secretion and activity of F8 C was significantly higher than that of F8, while the secretion and activity of F8 CG was significantly higher than that of F8 C. The vWF binding of the two variants is similar to F8. This indicates that the BDD-FVⅢ variant F8 CG may be attractive molecule for protein replacement and as a transgene in gene-therapy strategies. These findings are encouraging for future studies targeting disulfide bond elimination for further enhancement of FVⅢ secretion.

关 键 词：凝血VⅢ因子 二硫键突变 转基因 分泌 

分 类 号：Q812[生物学—生物工程]