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

Abstract

The tumor microenvironment and proinflammatory signals significantly alter glycosylation of cell-surface proteins on endothelial cells. By altering the N -glycosylation machinery in the endoplasmic reticulum and Golgi, proinflammatory cytokines promote the modification of endothelial glycoproteins such as vascular endothelial growth factor receptor 2 (VEGFR2) with sialic acid–capped N -glycans. VEGFR2 is a highly N- glycosylated receptor tyrosine kinase involved in pro-angiogenic signaling in physiological and pathological contexts, including cancer. Here, using glycoside hydrolase and kinase assays and immunoprecipitation and MS-based analyses, we demonstrate that N -linked glycans at the Asn-247 site in VEGFR2 hinder VEGF ligand–mediated receptor activation and signaling in endothelial cells. We provide evidence that cell surface–associated VEGFR2 displays sialylated N -glycans at Asn-247 and, in contrast, that the nearby sites Asn-145 and Asn-160 contain lower levels of sialylated N -glycans and higher levels of high–mannose N -glycans, respectively. Furthermore, we report that VEGFR2 Asn-247–linked glycans capped with sialic acid oppose ligand-mediated VEGFR2 activation, whereas the uncapped asialo-glycans favor activation of this receptor. We propose that N -glycosylation, specifically the capping of N- glycans at Asn-247 by sialic acid, tunes ligand-dependent activation and signaling of VEGFR2 in endothelial cells.