Comprehensive analysis of platelet glycoprotein Ibα ectodomain glycosylation

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

doi:10.1016/j.jtha.2023.01.009

Comprehensive analysis of platelet glycoprotein Ibα ectodomain glycosylation

J Thromb Haemost. 2023 Apr;21(4):995-1009. doi: 10.1016/j.jtha.2023.01.009. Epub 2023 Jan 14.

Authors

Marie A Hollenhorst¹, Katherine H Tiemeyer², Keira E Mahoney³, Kazuhiro Aoki⁴, Mayumi Ishihara⁵, Sarah C Lowery³, Valentina Rangel-Angarita³, Carolyn R Bertozzi⁶, Stacy A Malaker⁷

Affiliations

¹ Sarafan ChEM-H, Stanford University, Stanford, California, USA; Department of Pathology, Stanford University, Stanford, California, USA; Department of Medicine, Division of Hematology, Stanford University, Stanford, California, USA. Electronic address: https://twitter.com/HollenhorstM.
² Sarafan ChEM-H, Stanford University, Stanford, California, USA.
³ Department of Chemistry, Yale University, New Haven, Connecticut, USA.
⁴ Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
⁵ Cancer Center, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
⁶ Sarafan ChEM-H, Stanford University, Stanford, California, USA; Department of Chemistry, Stanford University, Stanford, California, USA; Howard Hughes Medical Institute, Stanford University, Stanford, California, USA.
⁷ Department of Chemistry, Yale University, New Haven, Connecticut, USA. Electronic address: [email protected].

Abstract

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.

Keywords: blood group antigens; glycomics; glycosylation; platelet glycoprotein GPIb-IX complex; platelet membrane glycoproteins.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Blood Platelets / metabolism
Glycosylation
Humans
Platelet Glycoprotein GPIb-IX Complex* / chemistry
Protein Binding
Protein Structure, Tertiary
Recombinant Proteins / metabolism
von Willebrand Factor* / metabolism

Substances

von Willebrand Factor
Platelet Glycoprotein GPIb-IX Complex
Recombinant Proteins

Abstract

Publication types

MeSH terms

Substances

Grants and funding