Vicinal cysteines in the prosequence play a role in von Willebrand factor multimer assembly.
von Willebrand factor (vWf) is synthesized as a large precursor that dimerizes in the endoplasmic reticulum and forms multimers in the trans- and post-Golgi compartments of megakaryocytes and endothelial cells. The disulfide-bonded multimers are stored in alpha granules of platelets and Weibel-Palade bodies of endothelial cells. The prosequence, composed of two homologous D domains, is required for vWf multimerization and storage. Each D domain contains vicinal cysteines (159Cys-Gly-Leu-162Cys and 521Cys-Gly-Leu-524Cys) that are similar to those at the active site of disulfide isomerases that catalyze thiol protein disulfide interchange. As in disulfide isomerases, a positively charged amino acid (lysine) is also found in close proximity to the vicinal cysteines. Although conserved, the lysine present in thioredoxin was shown not to be essential for its redox activity. We investigated the role of the vicinal cysteines and the lysine residue in the vWf propolypeptide by site-directed mutagenesis and expression of the resulting constructs in mammalian cells. Insertion of an extra glycine between the vicinal cysteines in either D domain inhibited multimerization of dimers, whereas alteration of lysine to glycine in both domains (residues 157 and 519) had no effect. This suggests the importance of the vicinal cysteines but not the lysines in vWf multimerization. Expression of the mutant with an additional glycine in the D1 domain in AtT-20 cells, a mouse pituitary cell line that can store vWf, led to the storage of the resulting dimers. This demonstrates that the mutation did not effect the capacity of the propolypeptide to direct vWf storage while its ability to promote interchain disulfide bonding was eliminated.
Mayadas
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TN
Center for Hemostasis and Thrombosis Research, New England Medical Center, Boston, MA.
Wagner
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DD
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D003545:Cysteine; D004247:DNA / Q000235:genetics; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015202:Protein Engineering; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection; D014841:von Willebrand Factor / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D046911:Macromolecular Substances; D011498:Protein Precursors; D011994:Recombinant Proteins; D014841:von Willebrand Factor; C067003:von Willebrand factor propolypeptide; D004247:DNA; D003545:Cysteine