Roles of Glycoslyation in Notch Signaling

描述（由申请人提供）：Notch EGF重复序列上的聚糖是Notch信号传导的关键调节剂，从而控制许多细胞类型的发育命运。构成型和缺陷型Notch信号都会导致疾病和癌症。因此，确定所有促成稳健Notch信号的因素至关重要。Pofut1编码O-聚焦转移酶，将O-聚焦转移到Notch，其失活会导致Notch信号的严重破坏。Pofut1需要底物GDP-焦点，必须被运输到分泌途径。缺乏已知的GDP聚焦转运蛋白Slc35c1和假定的GDP聚焦转运蛋白Slc35c2的小鼠表现出相对轻微的Notch信号缺陷，这表明存在额外的、新型的GDP聚焦转运蛋白，对Notch信号传导至关重要。特异性目标1将确定哺乳动物Notch信号所需的GDP关键聚焦转运蛋白。我们将使用Slc35c1/Slc35c2双敲除突变成纤维细胞分离哺乳动物中最佳Notch信号所需的GDP聚焦转运蛋白，确定其在分泌途径中的位置，并建立GDP聚焦转运或转运蛋白伴侣活性。特异性目标2将确定lng、mng和Rfng糖基转移酶的单独和互补功能，将n -乙酰氨基葡萄糖（GlcNAc）转移到Notch上的O-聚焦，从而调节Notch信号传导。这三者对于T细胞的最佳发育都是必需的。小鼠表达单个Fng基因，所有三个Fng基因或不表达Fng基因将被用来揭示每个Fng在调节Notch1信号传导和决定T细胞命运中的结构/功能关系。这将确定Notch1的关键EGF重复序列，这些重复序列必须被O-聚焦聚糖修饰，以及这些O-聚焦聚糖在T细胞发育过程中的结构。第三个目标将集中于Notch EGF重复序列的新修饰- O-GlcNAc。我们最近发现了果蝇Notch信号基因与EGF特异性O-GlcNAc转移酶Eogt之间的遗传相互作用，并表明在HeLa细胞中，O-GlcNAc修饰Notch1 EGF重复序列是配体诱导Notch1激活的最佳条件。特异性Aim 3将确定O-GlcNAc在哺乳动物细胞和小鼠中对Notch1信号传导的重要作用。O-GlcNAc缺失对Notch受体运输到细胞表面、Notch配体结合以及共培养实验中Notch信号传导的影响将被定义。我们将在仅表达一个Notch1拷贝的Eogt缺失小鼠中研究胚胎发生和T细胞和B细胞发育过程中的Notch信号缺陷。果蝇和哺乳动物Notch之间保守的4个O-GlcNAc位点将发生突变，以确定O-GlcNAc的特定功能。综合结果将揭示聚糖如何调节Notch信号的重要新见解。

DESCRIPTION (provided by applicant): Glycans on Notch EGF repeats are critical regulators of Notch signaling and thus control the developmental fate of many cell types. Both constitutive and defective Notch signaling lead to disease and cancer. Therefore, it is critically important to determine all factors that contribute to robust Notch signaling. Inactivation of Pofut1, which encodes the protein O-fucosyltransferase that transfers O-fucose to Notch, causes severe disruption of Notch signaling. Pofut1 requires the substrate GDP-fucose that must be transported into the secretory pathway. Mice lacking the known GDP-fucose transporter Slc35c1, and the putative GDP-fucose transporter Slc35c2, exhibit comparatively mild Notch signaling defects, revealing the existence of an additional, novel GDP-fucose transporter essential for Notch signaling. Specific Aim 1 will identify the GDP key-fucose transporter(s) required for Notch signaling in mammals. We will use Slc35c1/Slc35c2 double knockout mutant fibroblasts to isolate the GDP-fucose transporter(s) required for optimal Notch signaling in mammals, define their location in the secretory pathway and establish GDP-fucose transport or transporter chaperone activity. Specific Aim 2 will identify individual and complementary functions of Lfng, Mfng and Rfng glycosyltransferases that transfer N-acetylglucosamine (GlcNAc) to O-fucose on Notch, and thereby regulate Notch signaling. All three are necessary for optimal T cell development. Mice expressing a single Fng gene, all three Fng genes or no Fng genes will be used to reveal structure/function relationships for each Fng in regulating Notch1 signaling and determining T cell fates. This will identify critical EGF repeats of Notch1 that must be modified by O-fucose glycans, and the structure of those O-fucose glycans during T cell development. The third aim will focus on a new modification of Notch EGF repeats - O-GlcNAc. We have recently identified genetic interactions between Drosophila Notch signaling genes and the EGF- specific O-GlcNAc-transferase Eogt, and shown that modification of Notch1 EGF repeats by O-GlcNAc is required for optimal ligand-induced activation of Notch1 in HeLa cells. Specific Aim 3 will determine how O-GlcNAc is important for Notch1 signaling in mammalian cells and in the mouse. The effects of the loss of O-GlcNAc on Notch receptor trafficking to, and stability at, the cell surface, for Notch ligand binding, and for Notch signalig in co-culture assays, will be defined. We will investigate Notch signaling defects during embryogenesis and T and B cell development in Eogt null mice expressing only one copy of Notch1. Four O-GlcNAc sites that are conserved between Drosophila and mammalian Notch will be mutated to identify specific functions for O-GlcNAc. The combined results will reveal important new insights into how Notch signaling is regulated by glycans.