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Comprehensive analysis of platelet glycoprotein Ibα ectodomain glycosylation

2023; Elsevier BV; Volume: 21; Issue: 4 Linguagem: Inglês

10.1016/j.jtha.2023.01.009

1538-7933

Marie A. Hollenhorst, Katherine H. Tiemeyer, Keira E. Mahoney, Kazuhiro Aoki, Mayumi Ishihara, Sarah Lowery, Valentina Rangel-Angarita, Carolyn R. Bertozzi, Stacy A. Malaker,

Glycosylation and Glycoproteins Research

Background Platelet glycoprotein (GP) Ibα is the major ligand-binding subunit of the GPIb-IX-V complex that binds von Willebrand factor. GPIbα is heavily glycosylated, and its glycans have been proposed to play key roles in platelet clearance, von Willebrand factor binding, and as target antigens in immune thrombocytopenia syndromes. Despite its importance in platelet biology, the glycosylation profile of GPIbα is not well characterized. Objectives The aim of this study was to comprehensively analyze GPIbα amino acid sites of glycosylation (glycosites) and glycan structures. Methods GPIbα ectodomain that was recombinantly expressed or that was purified from human platelets was analyzed by Western blot, mass spectrometry glycomics, and mass spectrometry glycopeptide analysis to define glycosites and the structures of the attached glycans. Results We identified a diverse repertoire of N- and O-glycans, including sialoglycans, Tn antigen, T antigen, and ABO(H) blood group antigens. In the analysis of the recombinant protein, we identified 62 unique O-glycosites. In the analysis of the endogenous protein purified from platelets, we identified 48 unique O-glycosites and 1 N-glycosite. The GPIbα mucin domain is densely O-glycosylated. Glycosites are also located within the macroglycopeptide domain and mechanosensory domain. Conclusions This comprehensive analysis of GPIbα glycosylation lays the foundation for further studies to determine the functional and structural roles of GPIbα glycans.