Abstract：
N-glycosylation and proper processing of N-glycans are required for the function of membrane proteins including cell surface receptors. Fibroblast growth factor receptor (FGFR) is involved in a wide variety of biological processes including embryonic development, osteogenesis, angiogenesis, and cell proliferation. Human FGFR3 contains six potential N-glycosylation sites, however, the roles of glycosylation have not been elucidated. The site-specific profiles of N-glycans of the FGFR3 extracellular domain expressed and secreted by CHO-K1 cells were examined, and glycan occupancies and structures of four sites were determined. The results indicated that most sites were fully occupied by glycans, and the dominant populations were the complex type. By examining single N-glycan deletion mutants of FGFR3, it was found that N262Q mutation significantly increased the population with oligomannose-type N-glycans, which was localized in the endoplasmic reticulum. Protein stability assay suggested that fraction with oligomannose-type N-glycans in the N262Q mutant is more stable than those in the wild type and other mutants. Furthermore, it was found that ligand-independent phosphorylation was significantly upregulated in N262Q mutants with complex type N-glycans. The findings suggest that N-glycans on N262 of FGFR3 affect the intracellular localization and phosphorylation status of the receptor.
摘要：
包括细胞表面受体在内的膜蛋白的功能需要N-糖基化和N-聚糖的适当加工。成纤维细胞生长因子受体（FGFR）参与多种生物过程，包括胚胎发育，成骨,血管生成,和细胞增殖。人FGFR3含有六个潜在的N-糖基化位点，然而,糖基化的作用尚未阐明。检查了CHO-K1细胞表达和分泌的FGFR3胞外域的N-聚糖的位点特异性谱，并确定了四个位点的聚糖占用率和结构。结果表明，大多数位点完全被聚糖占据，优势种群是复杂类型。通过检查FGFR3的单个N-聚糖缺失突变体，发现N262Q突变显着增加了寡甘露糖型N-聚糖的群体，位于内质网。蛋白质稳定性测定表明，N262Q突变体中具有寡甘露糖型N-聚糖的部分比野生型和其他突变体中的部分更稳定。此外，发现在具有复合型N-聚糖的N262Q突变体中,不依赖配体的磷酸化显著上调。研究结果表明，FGFR3的N262上的N-聚糖会影响受体的细胞内定位和磷酸化状态。
