Site-Specific N-Glycosylation of Endothelial Cell Receptor Tyrosine Kinase VEGFR-2.
Site-Specific N-Glycosylation of Endothelial Cell Receptor Tyrosine Kinase VEGFR-2.
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DOI：
10.1021/acs.jproteome.6b00738
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发表时间：
2017-02-03
影响因子：
4.4
通讯作者：
Costello CE
中科院分区：
文献类型：
--
作者：
Chandler KB;Leon DR;Meyer RD;Rahimi N;Costello CE
血管内皮生长因子受体-2(VEGFR-2)是一种重要的受体酪氨酸激酶(RTK)，在生理性和病理性血管生成中发挥重要作用。VEGFR-2的胞外结构域由7个免疫球蛋白样结构域组成，每个结构域都有多个潜在的N-糖基化位点(启动子)。N-糖基化在RTK配体的结合、转运和稳定性中起着核心作用。然而，尽管VEGFR-2的N-糖基化作用很重要，但其功能作用仍然知之甚少。本研究的目的是通过酶促释放糖链和将18O掺入先前的N-糖基化位点，然后通过串联质谱仪(MS/MS)分析来确定VEGFR-2糖基化位点的占有率和VEGFR-2糖肽的N-糖基化位点特异性的N-糖链异质性。这些数据表明，所有7个VEGFR-2免疫球蛋白样结构域都至少有一个占据的N-糖基化位点。MS/MS分析从猪主动脉内皮细胞(PAE)异位表达的VEGFR-2中提取的糖肽和去胺化、去糖化(PNGase F处理)的多肽，以位点特异性的方式鉴定了VEGFR-2上17个潜在的N-糖基化位点中的大多数N-糖链。这些数据为VEGFR-2上的位点特异性、异质性N-糖基化和N-糖基化位点的占位提供了直接证据。这项研究对VEGFR-2的治疗靶向、配体结合、转运和信号转导具有重要意义。
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.