Von Willebrand factor (VWF) dimerizes through C-terminal CK domains, and VWF dimers assemble into multimers in the Golgi by forming intersubunit disulfide bonds between D3 domains. This unusual oxidoreductase reaction requires the VWF propeptide (domains D1D2), which acts as an endogenous pH-dependent chaperone. The cysteines involved in multimer assembly were characterized by using a VWF construct that encodes the N-terminal D1D2D′D3 domains. Modification with thiol-specific reagents demonstrated that secreted D′D3 monomer contained reduced Cys, whereas D′D3 dimer and propeptide did not. Reduced Cys in the D′D3 monomer were alkylated with N-ethylmaleimide and analyzed by mass spectrometry. All 52 Cys within the D′D3 region were observed, and only Cys1099 and Cys 1142 were modified by N-ethylmaleimide. When introduced into the D1D2D′D3 construct, the mutation C1099A or C1142A markedly impaired the formation of D′D3 dimers, and the double mutation prevented dimerization. In full-length VWF, the mutations C1099A and C1099A/C1142A prevented multimer assembly; the mutation C1142A allowed the formation of almost exclusively dimers, with few tetramers and no multimers larger than hexamers. Therefore, Cys1099 and Cys1142 are essential for the oxidoreductase mechanism of VWF multimerization. Cys1142 is reported to form a Cys1142-Cys1142 intersubunit bond, suggesting that Cys1099 also participates in a Cys1099-Cys1099 disulfide bond between D3 domains. This arrangement of intersubunit disulfide bonds implies that the dimeric N-terminal D′D3 domains of VWF subunits align in a parallel orientation within VWF multimers.
|Number of pages||6|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - Oct 2 2007|
- Disulfide bond
- Mass spectrometry