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bioRxiv · 10.1101/2022.07.19.500646

Comprehensive analysis of platelet glycoprotein Ibα glycosylation.

Hollenhorst, M. A.·Tiemeyer, K. H.·Mahoney, K. E.·Aoki, K.·Ishihara, M.·Lowery, S. C.·Rangel-Angarita, V.·Bertozzi, C. R.·Malaker, S. A.
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Abstract

BackgroundPlatelet glycoprotein (GP) Ib is the major ligand-binding subunit of the GPIb-IX-V complex that binds von Willebrand Factor (VWF). GPIb is heavily glycosylated, and its glycans have been proposed to play key roles in platelet clearance, VWF binding, and as target antigens in immune thrombocytopenia syndromes. Despite its importance in platelet biology, the glycosylation profile of GPIb is not well characterized. ObjectivesThe aim of this study was to comprehensively analyze GPIb amino acid sites of glycosylation (glycosites) and glycan structures. MethodsGPIb ectodomain that was recombinantly expressed or that was purified from human platelets was analyzed by Western blot, mass spectrometry (MS) glycomics, and MS glycoproteomics to define glycosites and the structures of the attached glycans. ResultsWe identified a diverse repertoire of N- and O-glycans, including sialoglycans, Tn antigen, T antigen, and ABH 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 at least 48 unique O-glycosites and 1 N-glycosite. The GPIb mucin domain is densely O-glycosylated. Glycosites are also located within the macroglycopeptide domain and mechanosensory domain (MSD). ConclusionsThis comprehensive analysis of GPIb glycosylation lays the foundation for further studies to determine the functional and structural roles of GPIb glycans. Essentials- Glycosylation of glycoprotein Ib (GPIb) is important for platelet function. - We report a comprehensive and site-specific analysis of human GPIb glycosylation. - GPIb carries sialoglycans, Tn antigen, T antigen, and ABO blood group (ABH) antigens. - We experimentally determined 48 O-glycosites and 1 N-glycosite by mass spectrometry.

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Hollenhorst, M. A., Tiemeyer, K. H., Mahoney, K. E., Aoki, K., Ishihara, M., Lowery, S. C., Rangel-Angarita, V., Bertozzi, C. R., Malaker, S. A.. 2022-07-20. Comprehensive analysis of platelet glycoprotein Ibα glycosylation.. https://doi.org/10.1101/2022.07.19.500646

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