title: Site-Specific N-Glycosylation of Endothelial Cell Receptor Tyrosine Kinase VEGFR-2.
abstract: Vascular endothelial growth factor receptor-2 (VEGFR-2) is an important receptor tyrosine kinase (RTK) that plays critical roles in both physiologic and pathologic angiogenesis. The extracellular domain of VEGFR-2 is composed of seven immunoglobulin-like domains, each with multiple potential N-glycosylation sites (sequons). N-glycosylation plays a central role in RTK ligand binding, trafficking, and stability. However, despite its importance, the functional role of N-glycosylation of VEGFR-2 remains poorly understood. The objectives of the present study were to characterize N-glycosylation sites in VEGFR-2 via enzymatic release of the glycans and concomitant incorporation of18O into formerly N-glycosylated sites followed by tandem mass spectrometry (MS/MS) analysis to determine N-glycosylation site occupancy and the site-specific N-glycan heterogeneity of VEGFR-2 glycopeptides. The data demonstrated that all seven VEGFR-2 immunoglobulin-like domains have at least one occupied N-glycosylation site. MS/MS analyses of glycopeptides and deamidated, deglycosylated (PNGase F-treated) peptides from ectopically expressed VEGFR-2 in porcine aortic endothelial (PAE) cells identified N-glycans at the majority of the 17 potential N-glycosylation sites on VEGFR-2 in a site-specific manner. The data presented here provide direct evidence for site-specific, heterogeneous N-glycosylation and N-glycosylation site occupancy on VEGFR-2. The study has important implications for the therapeutic targeting of VEGFR-2, ligand binding, trafficking, and signaling.
authors.affiliation: Center for Biomedical Mass Spectrometry, Department of Biochemistry and ‡Department of Pathology and Laboratory Medicine, Boston University School of Medicine , Boston, Massachusetts 02118, United States.
authors.forename: Kevin Brown
authors.identifier: 0000-0001-5514-9652
authors.initials: KB
authors.lastname: Chandler
authors.affiliation: Center for Biomedical Mass Spectrometry, Department of Biochemistry and ‡Department of Pathology and Laboratory Medicine, Boston University School of Medicine , Boston, Massachusetts 02118, United States.
authors.forename: Deborah R
authors.initials: DR
authors.lastname: Leon
authors.affiliation: Center for Biomedical Mass Spectrometry, Department of Biochemistry and ‡Department of Pathology and Laboratory Medicine, Boston University School of Medicine , Boston, Massachusetts 02118, United States.
authors.forename: Rosana D
authors.initials: RD
authors.lastname: Meyer
authors.affiliation: Center for Biomedical Mass Spectrometry, Department of Biochemistry and ‡Department of Pathology and Laboratory Medicine, Boston University School of Medicine , Boston, Massachusetts 02118, United States.
authors.forename: Nader
authors.initials: N
authors.lastname: Rahimi
authors.affiliation: Center for Biomedical Mass Spectrometry, Department of Biochemistry and ‡Department of Pathology and Laboratory Medicine, Boston University School of Medicine , Boston, Massachusetts 02118, United States.
authors.forename: Catherine E
authors.identifier: 0000-0003-1594-5122
authors.initials: CE
authors.lastname: Costello
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001011:Aorta / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism*; D006020:Glycopeptides / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D010455:Peptides; D011134:Polysaccharides / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D013552:Swine; D053719:Tandem Mass Spectrometry; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics* / Q000378:metabolism
chemical_list: D006020:Glycopeptides; D010455:Peptides; D011134:Polysaccharides; D040301:Vascular Endothelial Growth Factor Receptor-2
keywords: N-glycosylation; RTK; VEGFR-2; receptor tyrosine kinase; vascular endothelial growth factor receptor-2