title: Necessity of conserved asparagine residues in the leucine-rich repeats of platelet glycoprotein Ib alpha for the proper conformation and function of the ligand-binding region.
abstract: The polypeptides of the platelet von Willebrand factor (vWf) receptor, the GP Ib-IX-V complex, each contain tandem repeats of a sequence that assigns them to the leucine-rich repeat protein family. Here, we studied the role of conserved Asn residues in the leucine-rich repeats of GP Ib alpha, the ligand-binding subunit of the complex. We replaced the Asn residue in the sixth position of the first or sixth leucine-rich repeat (of seven) either with a bulky, charged Lys residue or with a Ser residue (sometimes found in the same position of other leucine-rich repeats) and studied the effect of the mutations on complex expression, modulator-dependent vWf binding, and interactions with immobilized vWf under fluid shear stress. As predicted, the Lys substitutions yielded more severe phenotypes, producing proteins that either were rapidly degraded within the cell (mutant N158K) or failed to bind vWf in the presence of ristocetin or roll on immobilized vWf under fluid shear stress (mutant N41K). The binding of function-blocking GP Ib alpha antibodies to the N41K mutant was either significantly reduced (AK2 and SZ2) or abolished (AN51 and CLB-MB45). Ser mutations were tolerated much better, although both mutants demonstrated subtle defects in vWf binding. These results suggest a vital role for the conserved asparagine residues in the leucine-rich repeats of GP Ib alpha for the structure and functions of this polypeptide. The finding that mutations in the first leucine-rich repeat had a much more profound effect on vWf binding indicates that the more N-terminal repeats may be directly involved in this interaction.
authors.affiliation: Division of Hematology/Oncology, Department of Internal Medicine, Baylor College of Medicine and Veterans Affairs Medical Center, Houston, Texas 77030, USA.
authors.forename: V
authors.initials: V
authors.lastname: Afshar-Kharghan
authors.forename: G
authors.initials: G
authors.lastname: Gineys
authors.forename: A J
authors.initials: AJ
authors.lastname: Schade
authors.forename: L
authors.initials: L
authors.lastname: Sun
authors.forename: C Q
authors.initials: CQ
authors.lastname: Li
authors.forename: L V
authors.initials: LV
authors.lastname: McIntire
authors.forename: J F
authors.initials: JF
authors.lastname: Dong
authors.forename: J A
authors.initials: JA
authors.lastname: López
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001216:Asparagine / Q000235:genetics / Q000378:metabolism*; D001666:Binding Sites, Antibody / Q000235:genetics; D016466:CHO Cells; D002448:Cell Adhesion / Q000235:genetics; D002465:Cell Movement / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D006224:Cricetinae; D006801:Humans; D007930:Leucine / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics; D014162:Transfection; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D008024:Ligands; D019038:Platelet Glycoprotein GPIb-IX Complex; D014841:von Willebrand Factor; D001216:Asparagine; D007930:Leucine