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
10.16438/j.0513-4870.2019-0822
- VernacularTitle: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
- Author:
Ze-long LIU
1
;
Jing MIAO
1
;
Hui-ge QU
1
;
Xiao-yan CHI
1
;
Fu-xiang ZHU
1
Author Information
1. Life Science College, Ludong University, Yantai 264025, China
- Publication Type:Research Article
- Keywords:
coagulation factor VIII;
isulfide bond mutation;
gene transfer;
secretion
- From:
Acta Pharmaceutica Sinica
2020;55(1):54-59
- CountryChina
- Language:Chinese
-
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.
