O-Glycosylation of Epidermal Growth Factor-like Modules

描述（由申请人提供）：Notch受体在许多发育阶段中发挥重要作用，Notch信号传导的失调导致多种人类病理学，包括癌症、血管疾病和发育障碍。Notch细胞外结构域（ECD）包含多达36个串联的表皮生长因子样（EGF）重复序列，其中许多被两种不寻常的糖基化形式修饰：O-岩藻糖和O-葡萄糖。O-岩藻糖修饰对于Notch功能是必不可少的。将O-岩藻糖添加到EGF重复序列的酶的遗传消融在小鼠或苍蝇中引起严重的Notch样表型，并且β 3-N-乙酰葡糖胺转移酶的Fringe家族对O-岩藻糖的延长调节Notch信号传导。Fringe通过改变Notch及其配体之间的相互作用发挥作用：Delta或Serrate/Jagged。有趣的是，条纹修饰增加Notch-Delta相互作用，同时抑制Notch-Serrate/Jagged相互作用。我们最近表明，O-葡萄糖修饰对Notch功能也是必不可少的。编码负责将O-葡萄糖添加到EGF重复序列（蛋白质O-葡萄糖基转移酶或Rumi）的酶的基因突变会导致果蝇中的温度敏感性Notch样表型。虽然O-岩藻糖和O-葡萄糖修饰都是Notch功能所必需的，但它们如何介导其作用的分子细节尚不清楚。我们的假设是O-岩藻糖和O-葡萄糖聚糖通过影响Notch ECD的构象来影响Notch功能。这一假设是基于令人兴奋的最近的结构研究表明，Notch ECD具有进化保守的灵活性区域。在第一个目标中，我们将分析条纹修饰如何改变Notch-配体结合。使用定量质谱方法，我们将绘制条纹修饰位点，并将这些修饰与Notch-配体结合的变化相关联。这些研究将告诉我们Notch ECD的哪些区域参与调节Notch-配体结合。我们将研究Notch ECD的某些区域抑制配体结合的可能性，并且我们将测试Fringe是否调节这种抑制活性。最后，我们将研究是否条纹诱导的Notch ECD的形状变化，使用蛋白酶的敏感性，分析ultracenthesis，和一个创新的cryoEM方法。在第二个目标中，我们将确定和表征哺乳动物同源的鲁米，以及木糖基转移酶，延长O-葡萄糖与木糖。最终目的是测试哺乳动物细胞中Rumi（或Aim 2中鉴定的木糖基转移酶）的消除（使用siRNA策略）是否会导致如在果蝇中所见的Notch活性丧失。此外，我们将使用目标1中描述的Fringe对Notch影响的相同方法来检查O-葡萄糖的损失是否会导致Notch ECD的构象变化。这些实验将为这些不寻常的碳水化合物修饰如何改变Notch功能提供分子细节。公共卫生相关性：Notch信号通路的缺陷导致多种人类病理学，包括几种类型的癌症、血管疾病和发育障碍。Notch在许多水平上受到调节，包括通过糖基化（糖对蛋白质的修饰）。我们的研究旨在了解糖基化如何影响Notch活性，以便我们可以利用其调节Notch的能力来设计Notch相关疾病的潜在疗法。

DESCRIPTION (provided by applicant): Notch receptors play an essential role in numerous stages of development, and deregulation of Notch signaling leads to a variety of human pathologies, including cancers, vascular disorders, and developmental disorders. The Notch extracellular domain (ECD) contains up to 36 tandem epidermal growth factor-like (EGF) repeats, many of which are modified by two unusual forms of glycosylation: O-fucose and O-glucose. O-Fucose modifications are essential for Notch function. Genetic ablation of the enzyme that adds O-fucose to EGF repeats causes severe Notch-like phenotypes in either mice or flies, and elongation of O-fucose by the Fringe family of ss3-N-acetylglucosaminyltransferases modulates Notch signaling. Fringe functions by altering the interaction between Notch and its ligands: Delta or Serrate/Jagged. Interestingly, Fringe modification increases Notch-Delta interactions while inhibiting Notch-Serrate/Jagged interactions. We have recently shown that O-glucose modifications are also essential for Notch function. Mutations in the gene encoding the enzyme responsible for addition of O-glucose to EGF repeats (protein O-glucosyltransferase, or Rumi), cause a temperature sensitive Notch-like phenotype in flies. Although both O-fucose and O-glucose modifications are required for Notch function, the molecular details for how they mediate their effects are not known. Our hypothesis is that the O-fucose and O-glucose glycans affect Notch function by affecting the conformation of the Notch ECD. This hypothesis is based on exciting recent structural studies suggesting that the Notch ECD has regions of flexibility that are evolutionarily conserved. In the first aim we will analyze how Fringe modification alters Notch-ligand binding. Using quantitative mass spectral methods, we will map Fringe modification sites, and correlate these modifications with changes in Notch-ligand binding. These studies will tell us which regions of the Notch ECD are involved in regulating Notch-ligand binding. We will examine the possibility that some regions of the Notch ECD inhibit ligand binding, and we will test whether Fringe modulates this inhibitory activity. Finally, we will examine whether Fringe induces changes in the shape of the Notch ECD using protease sensitivity, analytical ultracentrifugation, and an innovative cryoEM method. In the second aim, we will identify and characterize mammalian homologue of Rumi, as well as the xylosyltransferases that elongate O-glucose with xyloses. The final aim is designed to test whether elimination of Rumi (or the xylosyltransferases identified in Aim 2) in mammalian cells (using siRNA strategies) causes a loss of Notch activity as was seen in flies. In addition, we will examine whether loss of O-glucose causes conformational changes in the Notch ECD using the same methodologies described in Aim 1 for the effects of Fringe on Notch. These experiments will provide molecular details for how these unusual carbohydrate modifications alter Notch function. Public Health Relevance: Defects in the Notch signaling pathway lead to a variety of human pathologies, including several types of cancer, vascular disorders, and developmental disorders. Notch is regulated at numerous levels, including by glycosylation (modification of proteins by sugars). Our studies are aimed at understanding how glycosylation affects Notch activity so that we can take advantage of its ability to regulate Notch to design potential therapies for Notch-related diseases.