Site-directed mutagenesis of a soluble recombinant fragment of platelet glycoprotein Ibα demonstrating negatively charged residues involved in von Willebrand factor binding

M. Murata, J. Ware, Z. M. Ruggeri

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102 Citations (Scopus)

Abstract

We have expressed in mammalian cells a fragment (residues 1-302) of the α chain of platelet glycoprotein (GP) Ib containing the von Willebrand factor- (vWF) binding site. The secreted soluble protein had an apparent molecular mass of 45 kDa and reacted with conformation-dependent monoclonal antibodies that bind only to native GP Ib, thus demonstrating its proper folding. After insolubilization on nitrocellulose membrane, the recombinant GP Ibα fragment bound soluble vWF in the presence of ristocetin or botrocetin with a dissociation constant similar to that exhibited by GP Ib · IX complex on platelets. Moreover, the interaction was blocked by anti-GP Ib monoclonal antibodies known to inhibit vWF binding to platelets. The sequence of GP Ibα between residues 269-287 has a strong net negative charge due to the presence of 10 glutamic or aspartic acid residues; 5 of these are contained in the sequence of a synthetic peptide (residues 251-279) previously shown to inhibit vWF-platelet interaction. In order to evaluate the possible functional role of these acidic residues, we employed site-directed mutagenesis to express two mutant GP Ibα fragments containing asparagine or glutamine instead of aspartic or glutamic acid, respectively. Mutant 1, with substitutions between residues 251-279, failed to bind vWF whether in the presence of ristocetin or botrocetin; in contrast, vWF binding to Mutant 2, with substitutions between residues 280-302, was nearly normal in the presence of ristocetin, but markedly decreased in the presence of botrocetin. Thus, mammalian cells transfected with a truncated cDNA sequence encoding the amino-terminal domain of GP Ibα synthesize a fully functional vWF-binding site; acidic residues in the sequence 252-287 are essential for normal function.

Original languageEnglish
Pages (from-to)15474-15480
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number23
Publication statusPublished - 1991

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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