O-Glycosylation of Epidermal Growth Factor-like Motifs

描述（由申请人提供）：Notch信号对于许多组织的发育至关重要，Notch活性的失调导致多种疾病，包括几种癌症（如t细胞急性淋巴细胞白血病）和发育障碍，如Alagille综合征和先天性心脏缺陷。Notch细胞外结构域（ECD）的糖基化是调控Notch活性的关键机制。O-聚焦或O-葡萄糖聚糖的缺失阻断了Notch信号传导，而O-聚焦的延伸通过Fringe家族的¿3-N-乙酰氨基葡萄糖转移酶调节了Notch对配体的特异性。Notch ECD包含多达36串表皮生长因子样（EGF）重复序列，其中大多数在预测的一致序列上被这些聚糖修饰。利用基于细胞的Notch信号分析来评估小鼠Notch1中单个O-聚焦和O-葡萄糖修饰位点的重要性，我们确定了两个功能区域，即配体结合域（EGF11-12）和Abruptex区域（EGF24-29），它们介导这些聚糖的作用。有趣的是，已知Abruptex区域与果蝇的边缘基因有关。我们还开发了高灵敏度的半定量纳米lc -MS/MS糖蛋白组学方法，以检测细胞系统中过度表达的Notch蛋白上单个位点的聚糖结构。值得注意的是，Notch1配体结合区和Abruptex区的O-聚焦位点在高化学计量学上被修饰，并被条纹有效地拉长。最后，已发表的结构研究和我们初步的电镜结构研究表明，聚糖的分布和结构影响Notch构象。基于这些观察结果，我们提出Notch1的功能是由Notch1细胞外区域的配体结合区和Abruptex区的聚糖结构调节的。在这里，我们将通过检查相同区域的糖基化是否也影响Notch2来改进和测试我们的模型，Notch2对发育至关重要，但与Notch1起着不同的非冗余作用。在目的1中，我们使用基于细胞的方法评估了小鼠Notch2中预测的O-聚焦位点和O-葡萄糖位点突变的影响。如果聚糖通过一种共同的机制调节所有Notch蛋白，我们预测配体结合区和Abruptex区糖基化位点的消除将影响Notch2的活性，就像Notch1一样。另外，Notch1和Notch2负责调节的一个或多个糖基化位点可能不同。在Aim 2中，我们使用我们高度敏感的糖蛋白组学方法来检查这些功能重要的位点在B细胞和T细胞发育过程中是否在体内被修饰，其中已知边缘可以调节Notch活性。最后，在Aim 3中，我们与几位世界级的结构生物学家合作，确定聚糖的位点特异性变化如何影响Notch的结构和功能。这些研究将明确体内聚糖分布和结构对Notch功能的贡献，并将为未来开发利用糖基化调节Notch的新治疗策略提供基础。

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 ¿3-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.