Mapping Sites of O-Glycosylation and Fringe Elongation on Drosophila Notch
Mapping Sites of O-Glycosylation and Fringe Elongation on Drosophila Notch
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DOI：
10.1074/jbc.m116.732537
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发表时间：
2016-07-29
影响因子：
4.8
通讯作者：
Haltiwanger, Robert S.
中科院分区：
文献类型：
--
作者：
Harvey, Beth M.;Rana, Nadia A.;Haltiwanger, Robert S.
Notch受体的糖基化对其活性是必不可少的，并且作为信号传导的重要调节剂。预计在受体胞外结构域中表皮生长因子样重复序列内的共有位点发生三种主要形式的O-糖基化：O-岩藻糖基化、O-葡萄糖基化和O-GlcNAc酰化。我们对S2细胞中产生的果蝇Notch上的这三种类型的O-糖基化进行了全面的质谱分析，并鉴定了含有所有22个预测的O-岩藻糖位点、所有18个预测的O-葡萄糖位点和所有18个推定的O-GlcNAc位点的肽。使用半定量质谱方法，我们已经评估了在每个站点的聚糖的占用率和相对量。大多数的O-岩藻糖位点被修饰为高化学计量。在用Notch表达β 3-N-乙酰葡糖胺转移酶Fringe后，我们观察到超过O-岩藻糖单糖的不同程度的延伸，表明Fringe优先修饰某些位点。鲁米修改的O-葡萄糖的网站，以高化学计量，虽然延长的O-葡萄糖的位点特异性。尽管目前推测的O-GlcNAc化的共有序列预测Notch上有18个O-GlcNAc位点，但我们仅在5个位点观察到明显的O-GlcNAc修饰。此外，我们对从果蝇胚胎中纯化的内源性Notch进行了质谱分析，发现糖基化状态与S2细胞中Notch的糖基化状态相似。这些数据为进一步研究O-糖基化如何调节Notch活性的机制提供了基础信息。
Glycosylation of the Notch receptor is essential for its activity and serves as an important modulator of signaling. Three major forms of O-glycosylation are predicted to occur at consensus sites within the epidermal growth factor-like repeats in the extracellular domain of the receptor: O-fucosylation, O-glucosylation, and O-GlcNAcylation. We have performed comprehensive mass spectral analyses of these three types of O-glycosylation on Drosophila Notch produced in S2 cells and identified peptides containing all 22 predicted O-fucose sites, all 18 predicted O-glucose sites, and all 18 putative O-GlcNAc sites. Using semiquantitative mass spectral methods, we have evaluated the occupancy and relative amounts of glycans at each site. The majority of the O-fucose sites were modified to high stoichiometries. Upon expression of the beta 3-N-acetylglucosaminyltransferase Fringe with Notch, we observed varying degrees of elongation beyond O-fucose monosaccharide, indicating that Fringe preferentially modifies certain sites more than others. Rumi modified O-glucose sites to high stoichiometries, although elongation of the O-glucose was site-specific. Although the current putative consensus sequence for O-GlcNAcylation predicts 18 O-GlcNAc sites on Notch, we only observed apparent O-GlcNAc modification at five sites. In addition, we performed mass spectral analysis on endogenous Notch purified from Drosophila embryos and found that the glycosylation states were similar to those found on Notch from S2 cells. These data provide foundational information for future studies investigating the mechanisms of how O-glycosylation regulates Notch activity.