title: Site-directed mutagenesis of a soluble recombinant fragment of platelet glycoprotein Ib alpha demonstrating negatively charged residues involved in von Willebrand factor binding.
abstract: We have expressed in mammalian cells a fragment (residues 1-302) of the alpha 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 Ib alpha 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 Ib alpha 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 Ib alpha 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 Ib alpha synthesize a fully functional vWF-binding site; acidic residues in the sequence 252-287 are essential for normal function.
authors.affiliation: Department of Molecular and Experimental Medicine, Scripps Clinic and Research Foundation, La Jolla, California 92037.
authors.forename: M
authors.initials: M
authors.lastname: Murata
authors.forename: J
authors.initials: J
authors.lastname: Ware
authors.forename: Z M
authors.initials: ZM
authors.lastname: Ruggeri
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015153:Blotting, Western; D006224:Cricetinae; D003412:Cricetulus; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014841:von Willebrand Factor / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D014841:von Willebrand Factor; C028254:von Willebrand factor receptor