The disulfide bond Cys2724-Cys2774 in the C-terminal cystine knot domain of von Willebrand factor is critical for its dimerization and secretion

Yuxin Zhang,Aizhen Yang,Jingyu Zhang,Xiaoying Wang,Yi Wu,Depei Wu,Yue Han,Fengwu Chen

doi:10.1186/s12959-021-00348-w

Abstract

BackgroundType 3 von Willebrand disease (VWD) exhibits severe hemorrhagic tendency with complicated pathogenesis. The C-terminal cystine knot (CTCK) domain plays an important role in the dimerization and secretion of von Willebrand factor (VWF). The CTCK domain has four intrachain disulfide bonds including Cys2724-Cys2774, Cys2739-Cys2788, Cys2750-Cys2804 and Cys2754-Cys2806, and the single cysteine mutation in Cys2739-Cys2788, Cys2750-Cys2804 and Cys2754-Cys2806 result in type 3 VWD, demonstrating the crucial role of these three disulfide bonds in VWF biosynthesis, however, the role of the remaining disulfide bond Cys2724-Cys2774 remains unclear.Method and resultsIn this study, by the next-generation sequencing we found a missense mutation a c.8171G>A (C2724Y) in the CTCK domain of VWF allele in a patient family with type 3 VWD. In vitro, VWF C2724Y protein was expressed normally in HEK-293T cells but did not form a dimer or secrete into cell culture medium, suggesting that C2724 is critical for the VWF dimerization, and thus for VWF multimerization and secretion.ConclusionsOur findings provide the first genetic evidence for the important role of Cys2724-Cys2774 in VWF biosynthesis and secretion. Therefore, all of the four intrachain disulfide bonds in CTCK monomer contribute to VWF dimerization and secretion.

Highlights

von Willebrand factor (VWF) is a large-molecular multimerized glycoprotein synthesized and secreted by endothelial cells and megakaryocytes
The results of laboratory tests showed that the proband had a prolonged activated partial thromboplastin time (APTT, 66.8 s), low plasma VWF Antigen level (VWF:Ag, 2%) and markedly reduced factor VIII (FVIII) coagulant activities (FVIII:C, 2%)
There was no expression of VWF C2724Y, normal expression and multimer pattern of VWF WT and coexpression of VWF WT and VWF C2724Y were detected (Fig. 4-B), consistent with the immunoblotting in the reduced condition. These results indicated that mutation of C2724Y causes defective VWF dimerization, which further leads to dysfunction of VWF multimerization and secretion

Summary

Introduction

VWF is a large-molecular multimerized glycoprotein synthesized and secreted by endothelial cells and megakaryocytes. Homozygous mutations of C2739Y[5], C2804Y[6], C2754W[7, 8], and C2806R[9] caused type 3 VWD These mutations impair C-terminal dimerization and preventing the formation of long multimers, demonstrating that these three disulfide bonds Cys2739-Cys2788, Cys2750-Cys2804, and Cys2754-Cys2806 are critical for VWF biosynthesis. The C-terminal cystine knot (CTCK) domain plays an important role in the dimerization and secretion of von Willebrand factor (VWF). The CTCK domain has four intrachain disulfide bonds including Cys2724-Cys2774, Cys2739-Cys2788, Cys2750-Cys2804 and Cys2754-Cys2806, and the single cysteine mutation in Cys2739-Cys2788, Cys2750-Cys2804 and Cys2754-Cys2806 result in type 3 VWD, demonstrating the crucial role of these three disulfide bonds in VWF biosynthesis, the role of the remaining disulfide bond Cys2724-Cys2774 remains unclear

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Results

Discussion

Conclusion