Analysis of Vascular Endothelial Growth Factor Receptor 2 (VEGFR‐2) Glycosylation and its Role in Angiogenesis

Abstract

Angiogenesis, the formation of new blood vessels from pre‐existing vessels, is required for tumor growth and metastasis. Vascular endothelial growth factor receptor‐2 (VEGFR‐2) is one of the most important receptor tyrosine kinases (RTKs) among the VEGF receptor subfamily, and activation of VEGFR‐2 is essential for tumor angiogenesis. The extracellular domain of VEGFR‐2 contains seven immunoglobulin‐like (Ig) domains, each with multiple potential N‐glycosylation sites, and N‐glycosylation is thought to play a central role in receptor stability, ligand binding and trafficking. However, to date the N‐glycosylation sites and their putative role(s) in VEGFR‐2 function remain largely unknown. The objective of this study is to characterize VEGFR‐2 N‐glycosylation sites using tandem mass spectrometry (MS/MS) and investigate the functional importance of these modifications on VEGFR‐2 angiogenic signaling.VEGFR‐2 isolated from porcine aortic endothelial cell (PAEC) lysate was immunoprecipitated with a polyclonal anti‐VEGFR2 antibody. Evaluation of the purity and site occupancy of VEGFR‐2 were carried out by gel electrophoresis, followed by protease digestion, PNGaseF/H218O treatment, and MS/MS analysis. MS/MS data were processed using Proteome Discoverer 1.4. To obtain site‐specific glycosylation information we performed proteolysis of VEGFR‐2, glycopeptide enrichment via hydrophilic interaction liquid chromatography (HILIC) and subsequent analysis of glycopeptides via an Agilent 6550 Quadrupole Time‐of‐Flight (Q‐TOF) MS using collision‐induced dissociation.MS/MS analysis of the VEGFR‐2 tryptic digest achieved 56% sequence coverage. We detected candidate N‐glycosylated peptides in the extracellular domain of VEGFR‐2 after treatment with PNGaseF/H218O, including N98, N160, N247, N297, N521, N578, N702 and N719. MS/MS spectra were used to define the locations of the glycosylation sites. These results are the first evidence for which potential VEGFR‐2 N‐glycosylation sites are occupied by N‐linked glycans. In addition, VEGFR‐2 glycopeptide MS/MS data provide information on the glycoform distributions at each occupied site. All seven VEGFR‐2 immunoglobulin domains contain at least one occupied N‐glycosylation site. Observed glycosylation on Ig domain 2, involved in VEGFR‐2 ligand binding, and on Ig domain 7, involved in receptor dimerization and activation, could have important implications for protein folding, ligand binding and receptor dimerization. These analyses are essential for determination of the role of glycosylation in VEGFR‐2 function and trafficking.Support or Funding InformationThis research is supported by NIH grants P41 GM104603, R01 CA191970, and F32 CA196157.