Regulation of N-linked glycosylation in Mammalian Cells

项目总结： 天冬酰胺(N)连接的糖基化(NLG)是最常见的膜和蛋白质修饰 真核生物中的分泌蛋白。然而，糖生物学中的一个主要挑战是识别和 了解调控NLG的细胞机制和非必需基因产物。此外，尽管 这一途径靶向丰富，至少涉及34种基因产物，以及调节这种作用的药物抑制剂。 进程不可用。我们设计并实施了一种生物发光成像策略来 在蜂窝级别检测NLG站点占有率的变化。该平台可以集成到实验中， 范围从微孔板检测到活体成像，高通量筛查努力已经提供了两个 结构上独立的NLG抑制剂类。第一种抑制物，NGI-1，阻断寡糖转移酶 这种多亚单位酶复合体的活性和保真度的丧失。我们计划确定特定的氨基 酸和NGI-1结合口袋以便更准确地了解小分子OST的机制 抑制力。第二种抑制物的细胞靶点尚未确定，因此我们建议 研究和确定这一抑制NLG的新作用机制的方法。我们还有 设计并验证了一种荧光成像方法，它可以检测异常糖基化，并且可以 用于在单个细胞水平上检测多糖位置占有率的异质性。我们建议将这一点提前 技术应用于体外和体内模型，以研究调节NLG和NLG的遗传因素 以检测小鼠器官和组织中糖基化的差异。这项工作将共同提供新的 深入了解遗传因素或小分子抑制剂对NLG的调节，并提供一种技术 定量NLG在体外和体内特定细胞中的动态变化和异质性。

PROJECT SUMMARY: Asparagine (N) linked glycosylation (NLG) is the most common protein modification of membrane and secretory proteins in eukaryotes. However, A major challenge in glycobiology has been to identify and understand the cellular mechanisms and non-essential gene products that regulate NLG. Furthermore, though this pathway is target rich and involves at least 34 gene products, pharmacologic inhibitors that regulate this process have not been available. We have designed and implemented a bioluminescent imaging strategy to detect changes in NLG site occupancy at the cellular level. This platform can be integrated in experiments that range from microplate detection to in vivo imaging, and high throughput screening efforts have delivered two structurally independent classes of NLG inhibitors. The first inhibitor, NGI-1, blocks oligosaccharyltransferase activity and causes loss of fidelity for this multi-subunit enzyme complex. We plan to identify the specific amino acids and NGI-1 binding pocket in order to more precisely understand the mechanism of small molecule OST inhibition. The cellular target for the second inhibitor has yet to be defined and we therefore propose methodologies to investigate and identify this novel mechanism of action for NLG inhibition. We have also designed and validated a fluorescent imaging approach which detects abnormal glycosylation and that can be used to detect heterogeneity of glycan site occupancy at the single cell level. We propose to advance this technique to both in vitro and in vivo models in order to both investigate genetic factors that regulate NLG and to examine glycosylation differences in mouse organs and tissues. Together this work will provide new insights into the regulation of NLG by genetic factors or small molecule inhibitors, and provide a technique for quantifying dynamic changes and the heterogeneity of NLG in specific cells both in vitro and in vivo.