Slc35c2 Promotes Notch1 Fucosylation and Is Required for Optimal Notch Signaling in Mammalian Cells
Slc35c2 Promotes Notch1 Fucosylation and Is Required for Optimal Notch Signaling in Mammalian Cells
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DOI：
10.1074/jbc.m110.126003
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发表时间：
2010-11-12
影响因子：
4.8
通讯作者：
Stanley, Pamela
中科院分区：
文献类型：
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作者：
Lu, Linchao;Hou, Xinghua;Stanley, Pamela
哺乳动物Notch受体需要在其细胞外结构域的表皮生长因子样（EGF）重复序列上通过岩藻糖进行修饰，以最佳地响应典型Notch配体的信号诱导。在小鼠或人中高尔基体GDP-岩藻糖转运蛋白Slc 35 c1的失活在发育期间不会引起Notch信号传导的显著缺陷，并且显示出比在不能合成GDP-岩藻糖的小鼠中观察到的那些更温和的岩藻糖基化缺陷，表明存在另一种将GDP-岩藻糖转运到分泌途径中的机制。我们在这里显示，缺乏Slc 35 c1的小鼠或人的成纤维细胞在共培养信号传导测定中表现出强大的Notch信号传导。第二个GDP-岩藻糖转运蛋白的潜在候选者是相关基因Slc 35 c2。Slc 35 c2的过表达降低CHO细胞中岩藻糖基化表位刘易斯X和唾液酸化刘易斯X的表达，表明与Slc 35 c1的竞争。在过表达Slc 35 c2的CHO转染子中，内质网中发生的Notch 1 EGF重复片段的岩藻糖基化增加。在具有低水平Slc 35 c2的CHO细胞中，Delta 1和Jagged 1诱导的Notch信号传导均降低，并且Notch 1片段的岩藻糖基化也降低。大鼠肠上皮细胞和HeLa细胞的免疫荧光显微镜，和大鼠肝膜组分的分析表明，Slc 35 c2主要是共定位的标记物的顺式高尔基体网络和内质网高尔基体中间室（ERGIC）。组合的结果表明，Slc 35 c2是与Slc 35 c1竞争GDP-岩藻糖的GDP-岩藻糖转运蛋白，或者是以其他方式增强Notch的岩藻糖基化的因子，并且是哺乳动物细胞中最佳Notch信号传导所需的。
Mammalian Notch receptors require modification by fucose on epidermal growth factor-like (EGF) repeats of their extracellular domain to respond optimally to signal induction by canonical Notch ligands. Inactivation of the Golgi GDP-fucose transporter Slc35c1 in mouse or human does not cause marked defects in Notch signaling during development, and shows milder fucosylation defects than those observed in mice unable to synthesize GDP-fucose, indicating the existence of another mechanism for GDP-fucose transport into the secretory pathway. We show here that fibroblasts from mice or humans lacking Slc35c1 exhibit robust Notch signaling in co-culture signaling assays. A potential candidate for a second GDP-fucose transporter is the related gene Slc35c2. Overexpression of Slc35c2 reduces expression of the fucosylated epitopes Lewis X and sialylated Lewis X in CHO cells, indicating competition with Slc35c1. The fucosylation of a Notch1 EGF repeat fragment that occurs in the endoplasmic reticulum was increased in CHO transfectants overexpressing Slc35c2. In CHO cells with low levels of Slc35c2, both Delta1- and Jagged1-induced Notch signaling were reduced, and the fucosylation of a Notch1 fragment was also decreased. Immunofluorescence microscopy of rat intestinal epithelial cells and HeLa cells, and analysis of rat liver membrane fractions showed that Slc35c2 is primarily colocalized with markers of the cis-Golgi network and endoplasmic reticulum-Golgi intermediate compartment (ERGIC). The combined results suggest that Slc35c2 is either a GDP-fucose transporter that competes with Slc35c1 for GDP-fucose, or a factor that otherwise enhances the fucosylation of Notch and is required for optimal Notch signaling in mammalian cells.