药学学报, 2020, 55(1): 54-59
引用本文:
刘泽隆, 缪静, 屈慧鸽, 迟晓艳, 朱甫祥. 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[J]. 药学学报, 2020, 55(1): 54-59.
LIU Ze-long, MIAO Jing, QU Hui-ge, CHI Xiao-yan, ZHU Fu-xiang. 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[J]. Acta Pharmaceutica Sinica, 2020, 55(1): 54-59.

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
刘泽隆, 缪静, 屈慧鸽, 迟晓艳, 朱甫祥
鲁东大学生命科学学院, 山东 烟台 264025
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。
关键词:    凝血VIII因子      二硫键突变      转基因      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
LIU Ze-long, MIAO Jing, QU Hui-ge, CHI Xiao-yan, ZHU Fu-xiang
Life Science College, Ludong University, Yantai 264025, China
Abstract:
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 inter-chain disulfide bond (F8C) between Met662 > Cys and Asp1828 > Cys mutations and another contains an endogenous A3 domain with a disrupted disulfide bond from F8C (F8CG) by replacement of Cys1899 and Cys1903 with Gly in F8C. We explored their function and secretion. By transducing F8C and F8CG 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 F8C and F8CG 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 F8C was significantly higher than that of F8, while the secretion and activity of F8CG was significantly higher than that of F8C. The vWF binding of the two variants is similar to F8. This indicates that the BDD-FVIII variant F8CG 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.
Key words:    coagulation factor VIII    disulfide bond mutation    gene transfer    secretion   
收稿日期: 2019-10-21
DOI: 10.16438/j.0513-4870.2019-0822
基金项目: 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).
通讯作者: 朱甫祥,Tel:86-535-6693825,E-mail:fuxiangmail@163.com
Email: fuxiangmail@163.com
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