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 of 18O 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 therapeutic targeting of VEGFR-2, ligand binding, trafficking and signaling.

Biological Functions and Analytical Strategies of Sialic Acids in Tumor

“…On the other hand, tumor cells can also regulate the sialic acid expression of surrounding cells by delivering ST6GAL1 through exosomes [80]. N-glycans with terminal α2,6-sialylation on the receptors of blood vessels such as VEGFR2 are required for the VEGF engagement and proangiogenic activation of endothelial cells [81,82]. α2,6-sialylation mediates the homophilic interaction of the platelet endothelial cell adhesion molecule (PECAM).…”

N-Glycosylation regulates ligand-dependent activation and signaling of vascular endothelial growth factor receptor 2 (VEGFR2)

“…Separately, VEGFR2 was immunoprecipitated from unlabeled lysates, and the high-and low-molecular-weight bands were resolved on a 7.5% SDS-polyacrylamide gel. All VEGFR2 samples were subsequently treated with trypsin and analyzed via nUPLC-MS/MS to assess VEGFR2 site-specific N-glycosylation, as described previously (47,67). Briefly, VEGFR2 glycopeptides were enriched, separated, and analyzed using a 6550 Q-TOF MS with a 1200 series nanoflow HPLC-Chip-ESI source fitted with a custom HPLC-Chip with 360 nl of TSK gel amide-80, 5-m trapping and enrichment column, and a 150-mm ϫ 75-m Polaris C18-A 3-m analytical column (all from Agilent Corp., Santa Clara, CA).…”

“…We previously surveyed the site-specific N-glycan occupancy and glycoform heterogeneity displayed by VEGFR2 and identified multiple sites bearing complex, sialylated N-linked glycans (47). Immune-mediated changes have been shown to alter the expression of ␣2,6-linked sialic acid-containing carbohydrate epitopes on the endothelial cell surface, as noted above (4).…”

The manifold roles of sialic acid for the biological functions of endothelial glycoproteins

“…2001 ; Koch and Claesson-Welsh 2012 ) ( Table II ). Its seven Ig-like domains are heavily N-glycosylated ( Chandler et al. 2017 ).…”