Global inhibition of N-linked glycosylation broadly reduces glycan occupancy on glycoproteins, but identifying how this inhibition functionally impacts specific glycoproteins is challenging. This limits our understanding of pathogenesis in the congenital disorders of glycosylation (CDG). We used selective exo-enzymatic labeling of cells deficient in the two catalytic subunits of oligosaccharyltransferase - STT3A and STT3B - to monitor the presence and glycosylation status of cell surface glycoproteins. We show reduced abundance of two canonical tyrosine receptor kinases - the insulin receptor and insulin-like growth factor 1 receptor (IGF-1R) - at the cell surface in STT3A-null cells, due to decreased N-linked glycan site occupancy and proteolytic processing in combination with increased endoplasmic reticulum localization. Providing cDNA for Golgi-resident proprotein convertase subtilisin/kexin type 5a (PCSK5a) and furin cDNA to wild-type and mutant cells produced under-glycosylated forms of PCSK5a, but not furin, in cells lacking STT3A. Reduced glycosylation of PCSK5a in STT3A-null cells or cells treated with the oligosaccharyltransferase inhibitor NGI-1 corresponded with failure to rescue receptor processing, implying that alterations in the glycosylation of this convertase have functional consequences. Collectively, our findings show that STT3A-dependent inhibition of N-linked glycosylation on receptor tyrosine kinases and their convertases combines to impair receptor processing and surface localization. These results provide new insight into CDG pathogenesis and highlight how the surface abundance of some glycoproteins can be dually impacted by abnormal glycosylation.

中文翻译：

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N-联糖基化的选择性抑制损害受体酪氨酸激酶的加工。

对N-联糖基化的整体抑制作用广泛地降低了糖蛋白上的聚糖占有率，但是鉴定这种抑制作用如何影响特定糖蛋白具有挑战性。这限制了我们对先天性糖基化疾病（CDG）发病机理的了解。我们使用选择性的外切酶标记的缺乏寡糖基转移酶的两个催化亚基的细胞-STT3A和STT3B-来监测细胞表面糖蛋白的存在和糖基化状态。我们在STT3A的细胞表面显示出两种典型的酪氨酸受体激酶（胰岛素受体和胰岛素样生长因子1受体（IGF-1R））的丰度降低-空细胞，这是由于N-连接的聚糖位点占有率降低和蛋白水解加工，以及内质网定位增加所致。在缺乏STT3A的细胞中，为高尔基体原蛋白转化酶枯草杆菌蛋白酶/ kexin型5a（PCSK5a）和弗林蛋白酶cDNA提供给野生型和突变细胞，以产生糖基化形式的PCSK5a而不是弗林蛋白酶。降低STT3A中PCSK5a的糖基化-空细胞或经寡糖基转移酶抑制剂NGI-1处理的细胞与抢救受体加工失败相对应，这暗示该转化酶糖基化的改变具有功能性后果。总的来说，我们的研究结果表明，对受体酪氨酸激酶及其转化酶的N-连接糖基化的STT3A依赖性抑制作用联合起来会损害受体的加工和表面定位。这些结果为CDG的发病机理提供了新的见识，并突出了异常糖基化如何能双重影响某些糖蛋白的表面丰度。