O-GLYCOSYLATION OF EPIDERMAL GROWTH FACTOR-LIKE MODULES

描述：Notch蛋白是一种细胞表面受体，在细胞表面起关键作用 在发展和分化的多个阶段。Noch参与了 细胞间的信号，当与其配体结合时被激活 是相邻细胞上的跨膜蛋白。Notch信令中的缺陷导致 从果蝇到哺乳动物，生物体中的许多发育畸形， 包括人类疾病，如T细胞淋巴瘤，一种脑部疾病 动脉病(CADASIL)和阿拉格尔综合征。我们最近已经证明， Noch被两种不寻常的0-连接糖基化形式--0-岩藻糖修饰 和0-葡萄糖，在其体内的表皮生长因子样模块上 胞外域。Notch的36个串联EGF模块中有一半以上包含 这些糖添加的推定共识序列，以及大多数 这些位置在进化上是保守的。更重要的是，我们有非常多的 最近发现了0-岩藻糖修饰的生物学作用 已知的Notch功能调节器--边缘蛋白是一种0-岩藻糖 特异性的131，3N-乙酰氨基葡萄糖转移酶。这些结果强烈地表明 该条纹通过改变0-岩藻糖来调节其对Notch功能的影响 凹槽上的结构。细胞伸长对Notch信号的调制 0-岩藻糖为糖基化参与信号转导提供了新的范式 转导。 我们的假设是，0连接的碳水化合物修饰的变化 Notch的EGF模块在Notch信号的调节中起着关键作用。 我们的数据显示了通过O-岩藻糖的变化来实现条纹功能 Structures强烈支持这一假设，这里描述的研究 将进一步探索这一问题。在目标1中，使用标准的生化和分子 生物学方法，我们将绘制0-岩藻糖糖基化的实际位置图 在诺奇身上。特别是，我们将集中精力识别 0-受边缘影响的岩藻糖站点。在目标2中，我们将通过以下方式消除这些网站 产生点突变，并确定哪些点突变是必需的 刘海起作用了。然后，我们将利用这些突变来分析 Notch上0-岩藻糖的延长导致了Notch信号的改变。在AIM 3，我们将继续鉴定和表征负责的酶 在EGF模块上向0-岩藻糖中添加糖。基于我们对 边缘，任何修饰0-岩藻糖的酶都可能调节Notch 功能。由于许多蛋白质预计会被0-岩藻糖修饰，这些 在其他环境中，酶可能参与信号事件的调节，如 井。

DESCRIPTION: The Notch protein is a cell surface receptor that plays a key role in numerous phases of development and differentiation. Notch participates in cell-to-cell signaling, becoming activated upon binding to its ligands which are transmembrane proteins on adjacent cells. Defects in Notch signaling cause numerous developmental deformities in organisms from Drosophila to mammals, including human diseases such as T cell lymphomas, a type of cerebral arteriopathy (CADASIL), and Alagille syndrome. We have recently shown that Notch is modified with two unusual forms of 0-linked glycosylation, 0-fucose and 0-glucose, on the epidermal growth factor-like (EGF) modules in its extracellular domain. Over half of Notch's 36 tandem EGF modules contain putative consensus sequences for the addition of these sugars, and most of these sites are evolutionary conserved. Even more significantly, we have very recently discovered a biological role for the 0-fucose modifications by showing that the Fringe protein, a known modulator of Notch function, is an 0-fucose specific 131,3 N-acetylglucosaminyltransferase. These results strongly suggest that Fringe mediates its affects on Notch function by altering the 0-fucose structures on Notch. The modulation of Notch signaling by elongation of 0-fucose provides a new paradigm for the involvement of glycosylation in signal transduction. Our hypothesis is that alterations in the 0-linked carbohydrate modifications on the EGF modules of Notch play a key role in regulation of Notch Signaling. Our data demonstrating Fringe functions through alterations in O-fucose structures strongly supports this hypothesis, and the studies described here will explore it further. In Aim 1, using standard biochemical and molecular biological approaches, we will map the actual sites of 0-fucose glycosylation on Notch. In particular, we will focus our efforts on identification of the 0-fucose sites affected by Fringe. In Aim 2, we will eliminate these sites by generation of point mutations and determine which of them are required for Fringe to function. Then, we will utilize these mutations to analyze how elongation of 0-fucose on Notch results in a change in Notch signaling. In Aim 3, we will continue to identify and characterize enzymes responsible for addition of sugars to 0-fucose on EGF modules. Based on our studies with Fringe, any enzyme which modifies 0-fucose could potentially regulate Notch function. Since many proteins are predicted to be modified with 0-fucose, these enzymes may be involved in regulation of signaling events in other contexts as well.