Glycoprotein Ibα forms disulfide bonds with 2 glycoprotein Ibβ subunits in the resting platelet

Abstract

AbstractIt is widely accepted that glycoprotein (GP) Ib contains one Ibα and one Ibβ subunit that are connected by a disulfide bond. It is unclear which Cys residue in Ibα, C484 or C485, forms the disulfide bond with Ibβ. Using mutagenesis studies in transfected Chinese hamster ovary (CHO) cells, we found that both C484 and C485 formed a disulfide bond with C122 in Ibβ. In the context of isolated peptides containing the Ibα or Ibβ transmembrane domain and nearby Cys residue, C484 and C485 in the Ibα peptide were both capable of forming a disulfide bond with the Ibβ peptide. Furthermore, coimmunoprecipitation of epitope-tagged subunits showed that at least 2 Ibβ subunits but only 1 Ibα and 1 IX subunit were present in the GP Ib-IX complex. Finally, the size difference between GP Ib from transfected CHO cells and human platelets was attributed to a combination of sequence polymorphism and glycosylation difference in Ibα, not the number of Ibβ subunits therein. Overall, these results demonstrate that Ibα is covalently connected to 2 Ibβ subunits in the resting platelet, necessitating revision of the subunit stoichiometry of the GP Ib-IX-V complex. The αβ2 composition in GP Ib may provide the basis for possible disulfide rearrangement in the receptor complex.