O-GLYCOSYLATION OF EPIDERMAL GROWTH FACTOR-LIKE MODULES

描述：Notch蛋白是一种细胞表面受体， 在不同的发展和分化阶段。Notch参与了 细胞间信号传导，在与其配体结合后被激活， 是相邻细胞上的跨膜蛋白。Notch信号缺陷导致 从果蝇到哺乳动物， 包括人类疾病，如T细胞淋巴瘤，一种脑 动脉病（CADASIL）和Alagille综合征。我们最近的研究表明 Notch被两种不常见的0-连接糖基化形式修饰， 和0-葡萄糖，对表皮生长因子样（EGF）模块在其 胞外区Notch的36个串联EGF模块中有一半以上含有 添加这些糖的推定共有序列，以及 这些位点在进化上是保守的。更重要的是， 最近发现了0-岩藻糖修饰的生物学作用， 已知的Notch功能调节剂Fringe蛋白是一种0-岩藻糖， 特异性131，3 N-乙酰葡糖胺基转移酶。这些结果有力地表明 Fringe通过改变0-岩藻糖介导其对Notch功能的影响， Notch上的结构Notch信号转导的调节通过延长 0-岩藻糖为糖基化参与信号转导提供了新的范例 转导 我们的假设是0-连接的碳水化合物修饰的改变 EGF在Notch信号转导中起着关键作用。 我们的数据表明，通过改变O-岩藻糖， 结构强烈支持这一假设，这里描述的研究 我们将进一步探索它。在目标1中，使用标准的生物化学和分子生物学方法， 生物学方法，我们将绘制0-岩藻糖基化的实际位点 在Notch。特别是，我们将集中努力查明 0-受条纹影响的岩藻糖位点。在目标2中，我们将通过以下方式消除这些站点： 产生点突变，并确定其中哪些是必需的 边缘功能。然后，我们将利用这些突变来分析如何 Notch上0-岩藻糖的延长导致Notch信号传导的变化。在Aim中 3、我们将继续鉴定和表征负责 添加糖至EGF模块上的0-岩藻糖。根据我们的研究 Fringe，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被预测为用0-岩藻糖修饰，因此这些蛋白质被修饰。 酶可参与其它环境中信号传导事件的调节， 好.

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.