OC 77.5 - Comprehensive analysis of human glycoprotein Ibα O-glycosylation.

Wednesday, July 13, 2022

11:15 AM – 11:30 AM

Location: ExCel Center, ICC Capital Suite Room 17

Presentation Format: In-Person

Presenting Author(s)

Marie A. Hollenhorst, MD, PhD

Clinical Instructor and Research Scientist
Stanford University
Palo Alto, California, United States

Co-Author(s)

Katherine H. Tiemeyer

Research Assistant
Stanford University
Palo Alto, California, United States
KM

Keira E. Mahoney

Postdoctoral Fellow
Yale University
New Haven, Connecticut, United States
KA

Kazuhiro Aoki

Senior Research Scientist
University of Georgia
MI

Mayumi Ishihara

Research Professional
University of Georgia
VR

Valentina Rangel-Angarita

Graduate Student
Yale University
New Haven, Connecticut, United States
CB

Carolyn R. Bertozzi

Professor of Chemistry
Stanford University
Stanford, California, United States
SM

Stacy A. Malaker

Assistant Professor of Chemistry
Yale University
New Haven, Connecticut, United States

Background: Platelet glycoprotein (GP) Ibα is the major ligand-binding subunit of the platelet GPIb-IX-V complex that binds von Willebrand Factor (VWF). Human GPIbα is heavily glycosylated, with 58 predicted O-glycosites (amino acid sites of O-glycosylation). GPIbα O-glycans have been hypothesized to play key roles in platelet biology, possibly modulating VWF binding and regulating platelet clearance. The O-glycosylation profile of GPIbα has previously been largely uncharacterized.

Aims: The aim of this study was to comprehensively analyze GpIbα O-glycosylation in a site-specific manner.

Methods: Two different preparations of the human GpIbα ectodomain were analyzed: 1) commercially available recombinant protein, and 2) endogenous protein purified from platelets. GpIbα ectodomain was digested using a combination of commercially available proteases and bacterial mucinase enzymes. Glycopeptides were analyzed by higher-energy collisional dissociation (HCD) triggered electron-transfer/higher-energy collision dissociation (EThcD) mass spectrometry.

Results: We identified a total of 65 unique O-glycosites in recombinant GpIbα (Figure 1). 12 O-glycosites are located within the mucin domain and 11 within the VWF ligand binding domain (LBD). We identified a total of 30 O-glycosites in endogenous GpIbα, including 11 within the mucin domain and one within the VWF LBD. In the analysis of the endogenous protein, intact O-glycan masses were observed that were consistent with both sialylated and non-sialylated core 1, core 2, Tn antigen, and ABO blood group antigens.

Conclusion(s): This is the first study to report a comprehensive analysis of GpIbα O-glycosylation. This information lays the foundation for further studies to determine the functional and structural implications of GpIbα O-glycosylation.