Dataset Information

VEGFR N-glycosylation

ABSTRACT: Vascular endothelial growth factor receptor-2 (VEGFR2) is a highly N-glycosylated receptor tyrosine kinase involved in pro-angiogenic signaling in physiological and pathological contexts, including cancer. VEGFR2 post-translational modifications (PTMs) and their roles in receptor function and signaling have been extensively studied. However, until recently, co- and post-translational N-glycosylation of RTKs has been regarded as decorative rather than functional. N-glycosylation is a non-template based process that generates heterogeneously modified proteins. Emerging evidence suggests that the tumor microenvironment modulates expression of glycosyltransferases involved in the trimming and remodeling of nascent N-linked glycans into their heterogeneous mature forms, and that these changes alter the functions of modified proteins and thereby impact cellular signaling and adhesion. We sought to understand the consequences of altered glycosylation on VEGFR2 signaling. We used multiple strategies, including: enzymatic removal of N-linked glycans from the receptor, site-directed mutagenesis of specific N-glycosylation sites near the VEGF ligand binding site of VEGFR2, mass spectrometry of VEGFR2 glycopeptides, and concurrent measurement of receptor activation, signaling, and dimerization to interrogate how N-glycosylation modulates VEGFR2 ligand-dependent pro-angiogenic signaling. We demonstrate that VEGFR2 glycosylation at site N247 regulates ligand-dependent receptor activation and signaling. Complex N-glycans with α2-6 linked sialic acid residues at site N247 hinder receptor activation, while asialo-glycans favor VEGFR2 ligand-mediated activation and signaling.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

SUBMITTER: Kevin Chandler

LAB HEAD: Catherine E Costello

PROVIDER: PXD012629 | Pride | 2021-09-08

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	01_NonSurface.xlsx	Xlsx
	01_Surface.xlsx	Xlsx
	02142018_PAEFlk1_PRM_InclList_mzRangeStarts100_180214211132.mzML	Mzml
	02142018_PAEFlk1_PRM_InclList_mzRangeStarts100_180214211132.raw	Raw
	02_NonSurface.xlsx	Xlsx

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Publications

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

Chandler Kevin Brown KB Leon Deborah R DR Kuang Jenevieve J Meyer Rosana D RD Rahimi Nader N Costello Catherine E CE

The Journal of biological chemistry 20190715 35

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-angiogen ...[more]

PMID: 31308178

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Project description:MET, the receptor for hepatocyte growth factor (HGF), is involved in wide variety of biological events and its aberrant activation is implicated in the pathogenesis of cancer. Especially, MET signaling is associated with resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), key drugs in therapy of non-small cell lung cancer. MET has 11 potential N-glycosylation sites, however, the site-specific roles of N-glycans have not been elucidated. Here, we report that N-glycans regulate proteolytic processing of MET and HGF induced MET signaling, site specifically. N-glycosylation inhibitors suppressed processing and trafficking of endogenous MET in H1975 and EBC-1 lung cancer cells and exogenous MET in CHO cells. We purified the recombinant extracellular domain of human MET and determined the site-specific N-glycan structures and occupancy using nanoflow liquid chromatography-electrospray ionization mass spectrometry. The results indicated that most sites were fully glycosylated and the dominant population were complex-type which were rich in sialic acids and core fucoses. To examine the effects of N-glycan deletion of MET, we prepared endogenous-MET-knockout Flp-In CHO cells and transfected with series of N-glycan deletion mutants of MET. It was found that several N-glycans are implicated in processing of MET. It was also suggested that the N-glycans of -subunit of MET positively regulate HGF signaling, and the N-glycans of β-subunit negatively regulate HGF signaling. In all-N-glycan deletion mutant, processing and signaling were significantly suppressed. It was observed that the cell proliferation rate was suppressed in all-N-glycan deletion mutant, although the cell surface expression levels of MET were not completely diminished. We examined the HGF biding affinity for the recombinant extracellular domain of MET and found that peptide N-glycosidase F treatment did not affect the ligand binding affinity. It was suggested that N-glycans of MET affect the status and the function of the receptor site-specifically.

Dataset Information

VEGFR N-glycosylation

Dataset's files

Publications

<i>N</i>-Glycosylation regulates ligand-dependent activation and signaling of vascular endothelial growth factor receptor 2 (VEGFR2).

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