Localization of Disulfide Bonds in the Cystine Knot Domain of Human von Willebrand Factor

Abstract

von Willebrand factor (VWF) is a multimeric glycoprotein that is required for normal hemostasis. After translocation into the endoplasmic reticulum, proVWF subunits dimerize through disulfide bonds between their C-terminal cystine knot-like (CK) domains. CK domains are characterized by six conserved cysteines. Disulfide bonds between cysteines 2 and 5 and between cysteines 3 and 6 define a ring that is penetrated by a disulfide bond between cysteines 1 and 4. Dimerization often is mediated by additional cysteines that differ among CK domain subfamilies. When expressed in a baculovirus system, recombinant VWF CK domains (residues 1957-2050) were secreted as dimers that were converted to monomers by selective reduction and alkylation of three unconserved cysteine residues: Cys 2008 , Cys

The recombinant C-terminus of the human MUC2 mucin forms dimers in Chinese-hamster ovary cells and heterodimers with full-length MUC2 in LS 174T cells

“…This proposes that this cysteine might be involved also in the dimerization of mucins. Studies on rat Muc2 [11,12] point out that this cysteine residue is essential for dimer formation, whereas studies on vWF [15], PSM [16] and Norrin [17] indicate that additional cysteine residues may be of importance. The mucin genes MUC2, MUC5AC, MUC5B and MUC6 are clustered on chromosome 11p15.5 [18], all of which contain the cystine-knot motif and show large similarities in the positions of other cysteine residues also outside of the cystine-knot motif, indicating that these mucin genes are part of a family having a common ancestral gene.…”

A von Willebrand factor fragment containing the D′D3 domains is sufficient to stabilize coagulation factor VIII in mice

“…The set of VWF fragments cloned into pLIVE-EF are shown in Figure 1. SS contains only the 22-amino acid VWF signal sequence (M1-C22); D9D3 directs expression of the VWF signal peptide fused to a monomeric fragment encompassing the VWF D9 and D3 domains (S764-P1247); truncD9D3 encodes the signal peptide fused to VWF amino acids S764-R1035; D1-D3 includes the signal peptide and VWF propeptide through the D3 domain (M1-P1247), directing expression of a dimeric fragment; and D1D3-GS-CK adds the C-terminal CK domain to D1-D3 (M1-P1247, G2713-K2813), separated by a glycine-serine rich linker [GGRG (G 3 S) 3 (EG 3 S) 6 (G 3 S) 1 GSGGRG]. Expression of D1D3-GS-CK should result in multimers of VWF lacking the A1-C2 domains.…”

“…In the endoplasmic reticulum, proVWF subunits dimerize via intermolecular disulfide bonds at the C-terminal CK domain. 6 In the trans-Golgi and post-Golgi compartments, the VWF propeptide catalyzes the formation of VWF multimers via disulfide bonds near the VWF N terminus (C1099-C1099 and C1142-C1142) and is cleaved from proVWF by furin. [7][8][9] The resulting mature VWF multimers condense into long, helical structures that characterize the shape of WeibelPalade bodies and are released into the circulation as long, linear polymers with multiple concatemerized subunits.…”

Specific N-linked glycosylation sites modulate synthesis and secretion of von Willebrand factor

“…The pro-VWF monomer is composed of 4 types of domains (A-D) arranged as follows: NH 2 -D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK-COOH. 1 VWF multimers are formed by C-and N-terminal intermolecular disulphide bonds, 4,5 with the largest multimers exceeding 2 ϫ 10 4 kDa and having the greatest adhesive activity. 6 During synthesis, VWF undergoes extensive posttranslational modification resulting in the addition of 12 N-linked and 10 O-linked glycosylation sites per mature monomer.…”