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

• Published in: The Journal of biological chemistry Vol. 275; no. 33; pp. 25585 - 25594
• Main Authors: Katsumi, Akira, Tuley, Elodee A., Bodó, Imre, Sadler, J. Evan
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18-08-2000; American Society for Biochemistry and Molecular Biology
• Subjects: Alanine - chemistry; Alkylation; Amino Acid Sequence; Animals; Chromatography, High Pressure Liquid; Computer Simulation; COS Cells; Cyanogen Bromide - pharmacology; Cystine - chemistry; Dimerization; Disulfides; Dithiothreitol - pharmacology; Ethylmaleimide - pharmacology; Glycosylation; Humans; Indicators and Reagents - pharmacology; Mass Spectrometry; Molecular Sequence Data; Mutagenesis, Site-Directed; Phosphines - pharmacology; Plasmids - metabolism; Protein Structure, Tertiary; Pyridines - pharmacology; Recombinant Proteins - chemistry; Sequence Analysis, Protein; Sequence Homology, Amino Acid; Sulfhydryl Reagents - pharmacology; Thermolysin - pharmacology; Time Factors; von Willebrand Factor - chemistry; von Willebrand Factor - genetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M002654200

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: Cys2008, Cys2010, and Cys2048. By partial reduction and alkylation, chemical and proteolytic digestion, mass spectrometry, and amino acid sequencing, the remaining intrachain disulfide bonds were characterized: Cys1961–Cys2011 (1-4), Cys1987–Cys2041 (2-5), Cys1991–Cys2043 (3-6), and Cys1976–Cys2025. The mutation C2008A or C2010A prevented dimerization, whereas the mutation C2048A did not. Symmetry considerations and molecular modeling based on the structure of transforming growth factor-β suggest that one or three of residues Cys2008, Cys2010, and Cys2048 in each subunit mediate the covalent dimerization of proVWF.