Two Cys residues essential for Von Willebrand factor multimer assembly in the Golgi

被引:66
作者
Purvis, Angie R.
Gross, Julia
Dang, Luke T.
Huang, Ren-Huai
Kapadia, Milan
Townsend, R. Reid
Sadler, J. Evan
机构
[1] Washington Univ, Howard Hughes Med Inst, Sch Med, St Louis, MO 63110 USA
[2] Washington Univ, Sch Med, Dept Med, St Louis, MO 63110 USA
[3] Washington Univ, Sch Med, Dept Biochem, St Louis, MO 63110 USA
[4] Washington Univ, Sch Med, Dept Mol Biophys, St Louis, MO 63110 USA
关键词
disulfide bond; mass spectrometry; oxidoreductase;
D O I
10.1073/pnas.0705175104
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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 Cys(1099) and CyS1142 were modified by N-ethylmaleimide. When introduced into the D1D2D'D3 construct, the mutation C1099A or C1142A markedly impaired the formation of D'D3 dinners, 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, Cys(1099) and Cys(1142) are essential for the oxidoreductase mechanism of VWF multimerization. Cys(1142) is reported to form a Cys(1142)-Cys(1142) intersubunit bond, suggesting that Cys(1099) also participates in a Cys(1099)-Cys(1099) 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.
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页码:15647 / 15652
页数:6
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