O-Glycosylation of Epidermal Growth Factor-like Motifs

表皮生长因子样基序的 O-糖基化

• 批准号: 9102203
• 负责人: Robert S. Haltiwanger
• 金额: $ 62.24万
• 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 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9102203
• 关键词: Acute T Cell Leukemia; Affect; Alagille Syndrome; B-Cell Development; B-Lymphocytes; Biological Assay; Blood; Carbohydrates; Cardiovascular system; Cell Maturation; Cells; Congenital Heart Defects; Consensus Sequence; Data; Defect; Development; Disease; Dysostoses; Electron Microscopy; Electrons; Enzymes; Epidermal Growth Factor; Extracellular Domain; Family; Foundations; Fucose; Future; Glucose; Health; Heart Abnormalities; Individual; Laboratories; Lead; Ligand Binding; Ligand Binding Domain; Ligands; Link; Lung; MFNG gene; Malignant Neoplasms; Mediating; Methods; Microscopic; Modeling; Modification; Molecular; Molecular Conformation; Mus; Mutate; Mutation; N-Acetylglucosaminyltransferases; Notch Signaling Pathway; Pattern; Play; Polysaccharides; Publishing; Regulation; Roentgen Rays; Role; Signal Transduction; Site; Specificity; Staging; Structural Biologist; Structure; System; T-Cell Development; T-Lymphocyte; Testing; Tetralogy of Fallot; Tissues; Work; base; cardiogenesis; defined contribution; developmental disease; fly; glycoproteomics; glycosylation; in vivo; malformation; nano; notch protein; novel therapeutics; overexpression; progenitor; skeletal; stoichiometry

DESCRIPTION (provided by applicant): Notch signaling is essential for development of numerous tissues, and dysregulation of Notch activity results in a wide variety of diseases including several cancers (e.g. T-cell acute lymphoblastic leukemia), and developmental disorders such as Alagille Syndrome and congenital heart defects. Glycosylation of the Notch extracellular domain (ECD) provides a critical mechanism for regulating Notch activity. Loss of O-fucose or O- glucose glycans blocks Notch signaling, and extension of O-fucose by the Fringe family of ß3-N- acetylglucosaminyltransferases modulates Notch specificity for ligand. The Notch ECD contains up to 36 tandems Epidermal Growth Factor-like (EGF) repeats, most of which are decorated with these glycans at predicted consensus sequences. Using cell-based Notch signaling assays to evaluate the importance of individual O-fucose and O-glucose modification sites in mouse Notch1, we identified two functional regions, the ligand-binding domain (EGF11-12) and the Abruptex region (EGF24-29), that mediate the effects of these glycans. Interestingly, the Abruptex region is known to be genetically linked to Fringe in flies. We also developed highly sensitive semi-quantitative nano-LC-MS/MS glycoproteomic methods to examine the structure of glycans at individual sites on Notch proteins overexpressed in cell-based systems. Significantly, O- fucose sites in the ligand-binding and Abruptex regions of Notch1 are modified at high stoichiometries and are efficiently elongated by Fringes. Finally, published structural studies and our preliminary electron microscopic structural studies suggest that glycan distribution and structure influence Notch conformation. Based on these observations, we have proposed that Notch1 function is regulated by glycan structures in the ligand- binding and Abruptex regions of the Notch1 extracellular domain. Here we will refine and test our model by examining whether glycosylation of the same regions also affect Notch2, which is essential for development but plays distinct non-redundant roles with Notch1. In Aim 1 we evaluate the effects of mutations in predicted O-fucose and O-glucose sites in mouse Notch2 using cell-based assays. If glycans regulate all Notch proteins through a common mechanism, we predict that elimination of glycosylation sites in the ligand-binding and Abruptex regions will affect Notch2 activity as they do in Notch1. Alternately, one or more of the glycosylation sites responsible for regulation may differ for Notch1 and Notch2. In Aim 2 we use our highly sensitive glycoproteomic methods to examine whether these functionally important sites are modified in vivo during development of B and T cells, where Fringes are known to modulate Notch activity. Finally, in Aim 3 we collaborate with several world-class structural biologists to determine how site-specific changes in glycans affect Notch structure and function. These studies will define the contribution of glycan distribution and structure to Notch function i vivo and will provide the foundation for future development of novel therapeutic strategies taking advantage of Notch regulation by glycosylation.

描述(申请人提供)：Notch信号对许多组织的发育是必不可少的，Notch活性的失调会导致多种疾病，包括几种癌症(例如T细胞急性淋巴细胞性白血病)，以及发育障碍，如Alagille综合征和先天性心脏缺陷。Notch胞外区(ECD)的糖基化为调节Notch活性提供了一个关键机制。O-岩藻糖或O-葡萄糖多糖的丢失阻断了Notch信号转导，而O-岩藻糖被3-N-乙酰氨基葡萄糖基转移酶的边缘家族延伸而调节了Notch对配体的特异性。Notch ECD包含多达36个类似表皮生长因子(EGF)的重复序列，其中大多数重复序列都是用这些预测的共识序列来装饰的。使用基于细胞的Notch信号分析来评估小鼠Notch1中单个O-岩藻糖和O-葡萄糖修饰位点的重要性，我们确定了两个功能区域，即配体结合区域(EGF11-12)和Abruptex区域(EGF24-29)，它们介导了这些多糖的影响。有趣的是，Abruptex区域已知与果蝇的条纹有遗传联系。我们还开发了高灵敏的半定量纳米LC-MS/MS糖蛋白组学方法，以检测细胞系统中过度表达的Notch蛋白上个别位置的糖链结构。值得注意的是，Notch1的配体结合区和Abruptex区的O-岩藻糖位置在高化学计量比下被修饰，并被条纹有效地拉长。最后，已发表的结构研究和我们的初步电子显微镜结构研究表明，多糖的分布和结构影响Notch构象。基于这些观察，我们提出了Notch1的功能受Notch1胞外区配体结合区和Abruptex区的糖链结构的调控。在这里，我们将通过检测相同区域的糖基化是否也影响Notch2来完善和测试我们的模型，Notch2对于发育是必不可少的，但与Notch1起着独特的非冗余作用。在目标1中，我们使用基于细胞的分析来评估小鼠Notch2中预测的O-岩藻糖和O-葡萄糖位点突变的影响。如果多糖通过一个共同的机制调节所有的Notch蛋白，我们预测消除配体结合区和Abruptex区的糖基化位点将影响Notch2的活性，就像它们在Notch1中所做的那样。或者，Notch1和Notch2的一个或多个负责调节的糖基化位点可能不同。在目标2中，我们使用我们的高度敏感的糖蛋白组学方法来检查这些功能重要的部位在体内是否在B细胞和T细胞的发育过程中被修饰，其中已知的条纹调节Notch的活性。最后，在目标3中，我们与几位世界级的结构生物学家合作，确定多糖的特定部位变化如何影响Notch结构和功能。这些研究将确定糖的分布和结构对Notch体内功能的贡献，并将为未来利用Notch糖基化调节的新治疗策略的开发提供基础。