A Covalent Oxidoreductase Intermediate in Propeptide-dependent von Willebrand Factor Multimerization

Abstract

The assembly of von Willebrand factor multimers in the Golgi apparatus requires D1D2 domains of the von Willebrand factor propeptide, which may act as an oxidoreductase to promote disulfide bond formation or rearrangement between two D3 domains in the mature subunit. This mechanism predicts that the propeptide should form a transient intrachain disulfide bond with the D3 domain before multimerization. Such an intermediate was detected using truncated subunits that simplify the analysis of the multimerization process. When only the D1D2DD3 region of von Willebrand factor was expressed in baby hamster kidney cells, the propeptide and DD3 formed an intrachain disulfidelinked species in the endoplasmic reticulum that could be identified by two-dimensional gel electrophoresis after cleavage with thrombin or furin. This intermediate rearranged in the Golgi to form free propeptide and DD3 dimers that were secreted. A similar intracellular disulfide-linked species was identified in cells expressing the propeptide and DD3 as separate proteins and in cells expressing full-length von Willebrand factor. These results support a model in which the propeptide acts as an oxidoreductase to promote von Willebrand factor multimerization in the Golgi apparatus.

Phylogenetic and Functional Analysis of Histidine Residues Essential for pH-dependent Multimerization of von Willebrand Factor

“…This pH-dependent assembly process can be studied conveniently with constructs of VWF that are truncated after the D3 domain (4,36). For example, the expression of VWF D1D2DЈD3 domains in BHK cells results in the secretion of DЈD3 55-kDa monomers and 110-kDa dimers, as well as cleaved 85-kDa VWF propeptide (domains D1D2) (4), and treatment of cells expressing this construct with chloroquine prevented DЈD3 dimerization ( Fig.…”

“…Mutations were prepared in pSVHVWF1.1, which encodes full-length human VWF (21), in plasmid pSVH-propeptide-FLAGNT, which encodes the VWF propeptide (amino acid residues 1-763) with an N-terminal FLAG tag between the signal peptide and domain D1 (4), or in plasmid pSVH-D3-FLAGNT/ cMycCT, which encodes the VWF D1D2DЈD3 domains (residues 1-1241) with an N-terminal FLAG tag and a C-terminal c-Myc tag (4) , which prevents cleavage by furin (22). Plasmid pSVH-DЈD3⌬pro-c-Myc encodes the VWF signal peptide (residues 1-22) followed by domains DЈD3 (residues 764 -1220) and a c-Myc tag (4).…”

“…insertion of an extra Gly into the CGLC motif of domain D1 blocks multimer assembly without affecting storage in rodshaped Weibel-Palade bodies (5). Whether specific pH-sensing His residues participate in one or both of these processes will be established by additional analyses that discriminate between the noncovalent dimerization of the VWF propeptide (D1D2), binding of propeptide to DЈD3 domains, disulfide-mediated multimerization, and packing of VWF into tubules (4,10).…”

“…In the ER, the VWF propeptide forms a transient intrachain disulfide-linked intermediate with the D3 domain that rearranges in the Golgi to yield interchain disulfide bonds linking VWF subunits into multimers (4). In addition, the D1 and D2 domains both contain vicinal cysteines in a CGLC motif similar to the active site of disulfide isomerases, and insertion of an additional Gly into either motif inhibits VWF multimerization (5).…”

Structural organization of Weibel-Palade bodies revealed by cryo-EM of vitrified endothelial cells

“…Proteolytic cleavage of proVWF in the Golgi gives rise to the N-terminal propolypeptide (a 100-kDa protein called proregion) and to mature VWF dimers that form large homo-oligomers through disulfide-links near each of its mature N-termini, a process catalyzed by proregion (2,3). VWF and proregion remain non-covalently associated and are stored together in specialized secretory organelles called WeibelPalade bodies (WPBs), first identified by EM of fixed tissue sections as rod-shaped organelles containing fine tubules (4).…”