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，因此控制许多细胞类型的发育命运。组成型和缺陷型Notch信号传导均导致疾病和癌症。因此，至关重要的是确定有助于稳健Notch信号传导的所有因素。Pofut 1编码将O-岩藻糖转移到Notch的蛋白质O-岩藻糖基转移酶，其失活导致Notch信号传导严重中断。Pofut 1需要必须被转运到分泌途径中的底物GDP-岩藻糖。缺乏已知的GDP-岩藻糖转运蛋白Slc 35 c1和推定的GDP-岩藻糖转运蛋白Slc 35 c2的小鼠表现出相对轻微的Notch信号传导缺陷，揭示了Notch信号传导所必需的额外的新型GDP-岩藻糖转运蛋白的存在。具体目标1将鉴定哺乳动物中Notch信号传导所需的GDP关键岩藻糖转运蛋白。我们将使用Slc 35 c1/Slc 35 c2双敲除突变体成纤维细胞来分离哺乳动物中最佳Notch信号传导所需的GDP-岩藻糖转运蛋白，确定它们在分泌途径中的位置并建立GDP-岩藻糖转运或转运蛋白伴侣活性。具体目标2将鉴定Lfng、Mfng和Rfng糖基转移酶的单独和互补功能，这些糖基转移酶将N-乙酰葡糖胺（GlcNAc）转移到Notch上的O-岩藻糖，从而调节Notch信号传导。所有这三个都是最佳T细胞发育所必需的。表达单个Fng基因、所有三个Fng基因或无Fng基因的小鼠将用于揭示每个Fng在调节Notch 1信号传导和确定T细胞命运中的结构/功能关系。这将确定Notch 1的关键EGF重复序列，这些重复序列必须被O-岩藻糖聚糖修饰，以及T细胞发育过程中这些O-岩藻糖聚糖的结构。第三个目标将集中在Notch EGF重复序列的新修饰- O-GlcNAc。我们最近已经确定了果蝇Notch信号传导基因和EGF特异性O-GlcNAc-转移酶Eogt之间的遗传相互作用，并表明Notch 1 EGF重复序列被O-GlcNAc修饰是HeLa细胞中Notch 1最佳配体诱导激活所必需的。具体目标3将确定O-GlcNAc对哺乳动物细胞和小鼠中Notch 1信号传导的重要性。将定义O-GlcNAc损失对Notch受体运输至细胞表面和在细胞表面的稳定性、Notch配体结合和共培养测定中的Notch信号转导的影响。我们将研究Notch信号缺陷在胚胎发育和T和B细胞的发展Eogt裸小鼠只表达一个拷贝的Notch 1。果蝇和哺乳动物Notch之间保守的四个O-GlcNAc位点将被突变以鉴定O-GlcNAc的特异性功能。综合结果将揭示重要的新见解Notch信号是如何调节聚糖。

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.