O-Glycosylation of Epidermal Growth Factor-like Modules

表皮生长因子样模块的 O-糖基化

• 批准号: 7744626
• 负责人: Robert S. Haltiwanger
• 金额: $ 62.15万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7744626
• 关键词: Ablation; Acute T Cell Leukemia; Affect; Alagille Syndrome; Arterial Disorder; Biological Assay; Carbohydrates; Cell Nucleus; Cell Surface Receptors; Cell membrane; Cells; Cerebrum; Cryoelectron Microscopy; Data; Defect; Development; Disease; Drosophila genus; Dysostoses; Enzymes; Epidermal Growth Factor; Event; Extracellular Domain; Family; Fucose; Genes; Genetic; Genetic Transcription; Glucose; Glucosyltransferase; Glucosyltransferases; Health; Homologous Gene; Human; Human Pathology; Infarction; Lead; Leukoencephalopathy; Ligand Binding; Ligands; Link; Malignant Neoplasms; Malignant neoplasm of cervix uteri; Mammalian Cell; Maps; Mediating; Methodology; Methods; Modification; Molecular; Molecular Conformation; Mus; Mutation; N-Acetylglucosaminyltransferases; Nature; Notch Signaling Pathway; Peptide Hydrolases; Phenotype; Play; Polysaccharides; Post-Translational Protein Processing; Proteins; Proteolysis; Role; Shapes; Signal Transduction; Site; Small Interfering RNA; Staging; Structure; System; Temperature; Testing; Trisaccharides; Vascular Diseases; Xylose; analytical ultracentrifugation; base; cancer type; design; developmental disease; flexibility; fly; glycosylation; human disease; innovation; melanoma; notch protein; protein structure; public health relevance; research study; secretase; sugar; xylosyltransferase

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.

描述（由申请人提供）：Notch受体在众多发育阶段起着至关重要的作用，并且对Notch信号的管制导致各种人类病理，包括癌症，血管疾病和发育障碍。 Notch细胞外域（ECD）包含多达36个串联表皮生长因子（EGF）重复序列，其中许多通过两种异常形式的糖基化来修饰：O-糖糖糖和O-葡萄糖。 O-凝血糖修饰对于Notch功能至关重要。酶的遗传消融添加了O-浓度为EGF重复的o糖含量会导致小鼠或蝇中的严重缺口样表型，并通过SS3-N-乙酰葡萄糖氨基氨基氨基氨基转移酶调节Notch信号传导的SS3-N-乙酰氨基葡萄糖氨基氨基氨基转移酶对O-糖糖的延伸。边缘通过更改Notch及其配体之间的相互作用来函数：Delta或Serrate/Jagged。有趣的是，边缘修改会增加Notch-delta的相互作用，同时抑制缺口serrate/锯齿状的相互作用。我们最近表明，O-葡萄糖修饰对于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（蛋白O-葡萄糖基转移酶或Rumi）的酶的基因中的突变会导致果蝇中温度敏感的凹槽样表型。尽管Notch功能都需要O-观糖和O-葡萄糖修饰，但尚不清楚它们如何介导其效应的分子细节。我们的假设是O-葡萄糖和O-葡萄糖聚糖通过影响Notch ECD的构象来影响Notch功能。该假设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。在第一个目的中，我们将分析条纹修改如何改变Notch-s-rigand结合。使用定量的质谱方法，我们将绘制条纹修饰位点，并将这些修饰与凹配合结合的变化相关联。这些研究将告诉我们哪些Notch ECD的区域参与调节凹痕结合。我们将研究Notch ECD的某些区域抑制配体结合的可能性，并且我们将测试边缘是否调节这种抑制活性。最后，我们将使用蛋白酶敏感性，分析性超速离心和创新的冷冻方法来检查边缘是否诱导Notch ECD形状的变化。在第二个目标中，我们将识别并表征Rumi的哺乳动物同源物，以及用木糖促进O-葡萄糖的木糖基转移酶。最终目的旨在测试消除哺乳动物细胞（使用siRNA策略）在AIM 2中鉴定的Rumi（或在AIM 2中鉴定出的二甲糖基转移酶）是否导致果蝇中所见的Notch活性的丧失。此外，我们将使用AIM 1中描述的相同方法对边缘对Notch的影响进行相同的方法，是否使用O-glucose的损失会导致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信号通路中的缺陷导致各种人类病理，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（糖修饰蛋白质）。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来设计与Notch相关疾病的潜在疗法的能力。