Glycoprotein Ibalpha forms disulfide bonds with 2 glycoprotein Ibbeta subunits in the resting platelet

Abstract

It is widely accepted that glycoprotein (GP) Ib contains one Ibalpha and one Ibbeta subunit that are connected by a disulfide bond. It is unclear which Cys residue in Ibalpha, C484 or C485, forms the disulfide bond with Ibbeta. Using mutagenesis studies in transfected Chinese hamster ovary (CHO) cells, we found that both C484 and C485 formed a disulfide bond with C122 in Ibbeta. In the context of isolated peptides containing the Ibalpha or Ibbeta transmembrane domain and nearby Cys residue, C484 and C485 in the Ibalpha peptide were both capable of forming a disulfide bond with the Ibbeta peptide. Furthermore, coimmunoprecipitation of epitope-tagged subunits showed that at least 2 Ibbeta subunits but only 1 Ibalpha 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 Ibalpha, not the number of Ibbeta subunits therein. Overall, these results demonstrate that Ibalpha is covalently connected to 2 Ibbeta subunits in the resting platelet, necessitating revision of the subunit stoichiometry of the GP Ib-IX-V complex. The alphabeta2 composition in GP Ib may provide the basis for possible disulfide rearrangement in the receptor complex.

Figure 1

Cysteine residues in GP Ibα and GP Ibβ. The disulfide bonds in each subunit are marked by connected lines. The transmembrane domains are marked by filled boxes. C65 in GP Ibα is buried in the N-terminal domain and is unpaired.^, GP Ibα has 2 membrane-proximal Cys residues (C484 and C485), whereas GP Ibβ has one (C122). C122 forms a disulfide bond with a Cys residue in GP Ibα, the identity of which remains unclear.

Figure 2

Effects of Cys mutations on formation of disulfide bonds between GP Ibα and GP Ibβ. (A) Illustration of wild-type and Cys mutant constructs of GP Ibα and GP Ibβ. (B-C) GP Ibα from the lysates of various transfected CHO cells that had been resolved in a 4-12% Bis-Tris SDS gel (Invitrogen) under nonreducing (N.R.) and reducing (R.) conditions. Molecular markers in kDa are labeled on the left. GP Ibα was blotted by WM23 antibody. (D) GP Ibα from various cell lysates that had been resolved in a 5% Tris-glycine SDS gel under nonreducing (N.R.) conditions. Dashed lines were added to show the difference between the protein bands. The figure is representative of 3 independent experiments.

Figure 3

Thiol-disulfide exchange assay of the TM peptides derived from GP Ibα and GP Ibβ. (A) Sequences of the Ibα and Ibβ TM peptides. The TM domain in each construct is boxed. Residue C148 in the Ibβ cytoplasmic domain was changed to Ser (the residue in the rat sequence) to avoid complication in data analysis. A GGG sequence was added between the thrombin cleavage site and the Ibβ sequence to facilitate thrombin digestion. (B) A reaction scheme for the Ibβ TM peptide. The TM-TM interaction mediates the dimerization step, which is coupled to formation of a disulfide bond between 2 Ibβ peptides. GSH indicates reduced glutathione; GSSG, oxidized glutathione; β-SG, Ibβ peptide linked to glutathione; β-SH, reduced monomeric Ibβ peptide; β-SS-β, disulfide-linked Ibβ homodimer. (C) The thiol-disulfide exchange reaction of the Ibβ TM peptide with the Ibα TM peptides containing one Cys residue. The TM peptides were mixed with DPC micelles and incubated at room temperature for the reaction to reach equilibrium. Acid was then added to quench the reaction, and the reaction products were analyzed by reverse-phase analytic HPLC using 2 different columns. DTT in the reaction prevented formation of any disulfides, and their chromatograms (dashed line) helped to assign the eluted peaks. Peak 1 indicates β-SG; peak 2, β-SH; peak 3, β-SS-β; peak 4, α_SC-SG; peak 5, α_SC-SH; peak 6, α_SC-SS-α_SC; peak 7, α_SC-SS-β; peak 8, α_CS-SG; peak 9, α_CS-SH; peak 10, α_CS-SS-α_CS; peak 11, α_CS-SS-β. (D) The thiol-disulfide exchange reaction of the Ibβ TM peptide with the α_CC TM peptide, at the 2:1 molar ratio. Peak 12 indicates oxidized monomeric α_CC with an intramolecular disulfide; peak 13, reduced monomeric α_CC; peak 14, oxidized dimeric α_CC with 2 intermolecular disulfides; peak 15, β-SS-α_CC-SS-β; peak 16, α_CC-SS-β; peak 17, β-SS-α_CC-SS-α_CC-SS-β.

Figure 4

Co-IP of tagged GP Ibα, GP Ibβ, and GP IX in the GP Ib-IX complex from CHO cells. The HA and myc tags were, respectively, appended to the C-terminus of (A) GP Ibβ, (B) GP Ibα, or (C) GP IX. The CHO cells were transiently transfected with indicated subunits, and cell lysates were immunoprecipitated with antibodies, resolved in a 4% to 12% Bis-Tris SDS gel under nonreducing (N.R.) or reducing (R.) conditions, and blotted with HRP-conjugated anti-HA antibody. In the lanes marked by —, the lysates were loaded directly onto the SDS gel.

Figure 5

Comparison of GP Ib, GP Ibα, and GP Ibβ from transfected CHO cells and human platelets. (A) GP Ib and GP Ibα from CHO cells and platelets of 2 donors (plt1, plt2) that had been resolved in a 5% Tris-glycine SDS gel under either nonreducing (N.R.) or reducing (R.) conditions and blotted by WM23. (B) GP Ibβ from CHO cells and platelets that had been resolved in a 4% to 12% Bis-Tris SDS gel under reducing (R.) conditions and blotted by Gi27. (C) Analysis of genomic Ibα gene sequences from the 2 donors. Genomic DNA was prepared from the buffy coat, and the VNTR-containing gene fragment was amplified by PCR along with various Ibα expression vectors. The amplified fragments were resolved in a 2.3% agarose gel. The 100 bp (base pair) DNA ladder was shown on the left. Four pcDNA-based vectors expressing GP Ibα with type A-D VNTR were included as standards on the right. (D) GP Ibα (type C VNTR) from platelets (plt2) and CHO cells before and after deglycosylation. Both Ibα were coimmunoprecipitated with SZ2 antibody, eluted from protein G-agarose beads, treated with a mixture of glycanases, resolved in a 5% Tris-glycine SDS gel under reducing conditions, and blotted by WM23.

Figure 6

Schematic drawing of the GP Ib-IX-V complex with the revised stoichiometry. Four GP Ibβ subunits, as well as 4 intermolecular disulfides, are included in the complex. Illustrations of individual subunits are adapted from earlier publications.^,