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

Abstract

Background: Type 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 results: In 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.

Conclusions: Our 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.

Keywords: cystine knot domain; disulfide bond; multimerization; von Willebrand disease (VWD); von Willebrand factor (VWF).

Fig. 1

VWF multimer analysis of plasma from the family with type 3 VWD. Plasma VWF multimers were assessed by 1.5% SDS-agarose gel non-reducing electrophoresis and Western blotting. All plasma samples were diluted 1:5 with sample dilution buffer. NHP, normal human plasma; P, proband; PF, proband’s father; PM, proband’s mother

Fig. 2

Identification of candidate VWF gene mutations. A Results of first-generation sequencing showed that one VWF allele of the proband had a c.8171G>A:p.C2724Y mutation in exon 51. B The location of the C2724Y mutation site in the CK domain of VWF molecular

Fig. 3

Expression of recombinant VWF protein in HEK-293T cells. A The results of genetic sequencing showed a G >A mutation at position 8171 of VWF. B Western blotting analysis of concentrated lysate and supernatant from cultured cells transfected with the plasmids of VWF WT, VWF C2724Y, and mixture of VWF WT and VWF C2724Y. Protein in the lysate and supernatant was separated by 8% reducing SDS-PAGE and immunoblotted with anti-VWF antibody

Fig. 4

Multimer analysis of recombinant VWF protein in HEK-293T cells. Multimer analysis of concentrated lysate (A) and supernatant (B) of cultured cells transfected with the plasmid of vector, VWF WT, VWF C2724Y, and mixture of VWF WT and VWF C2724Y (1:2). Protein in the lysate and culture supernatant was separated by non-reducing electrophoresis on a 1.5% agarose gel and immunoblotted with anti-VWF antibody. NHP, normal human plasma