CD133 Protein N-Glycosylation Processing Contributes to Cell Surface Recognition of the Primitive Cell Marker AC133 Epitope
CD133 Protein N-Glycosylation Processing Contributes to Cell Surface Recognition of the Primitive Cell Marker AC133 Epitope
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DOI：
10.1074/jbc.m111.261545
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发表时间：
2011-11-25
影响因子：
4.8
通讯作者：
Moffat, Jason
中科院分区：
文献类型：
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作者：
Mak, Anthony B.;Blakely, Kim M.;Moffat, Jason
在CD 133糖蛋白上表达的AC 133表位已被广泛用作许多干细胞和癌症干细胞类型的细胞表面标志物。最近有人提出，CD 133的翻译后修饰和调节可能支配细胞表面AC 133的识别。因此，我们进行了大规模的合并RNA干扰（RNAi）筛选，以确定参与细胞表面AC 133表达的基因。在使用正交RNAi系统的二次测定中，基因命中可以以70.5%的比率被验证，这表明我们的初级RNAi筛选充当了强大的遗传筛选方法。在来自初步筛选的命中列表中，参与N-聚糖生物合成的基因显著富集，如通过Incidity Canonical Pathway分析所确定的。实际上，使用小分子衣霉素抑制N-聚糖前体的生物合成或通过产生N-聚糖缺陷型CD 133突变体抑制其转移至CD 133导致不可检测的细胞表面AC 133。在参与N-糖基化的筛选命中中，有参与复杂N-聚糖加工的基因，包括表征不佳的MGAT 4C，我们证明其是细胞表面AC 133表达的正调控因子。我们的研究确定了一组参与CD 133 N-糖基化的基因作为细胞表面AC 133识别的直接贡献因子，并为CD 133 N-聚糖的功能和结构提供了生化证据。
The AC133 epitope expressed on the CD 133 glycoprotein has been widely used as a cell surface marker of numerous stem cell and cancer stem cell types. It has been recently proposed that posttranslational modification and regulation of CD133 may govern cell surface AC133 recognition. Therefore, we performed a large scale pooled RNA interference (RNAi) screen to identify genes involved in cell surface AC133 expression. Gene hits could be validated at a rate of 70.5% in a secondary assay using an orthogonal RNAi system, demonstrating that our primary RNAi screen served as a powerful genetic screening approach. Within the list of hits from the primary screen, genes involved in N-glycan biosynthesis were significantly enriched as determined by Ingenuity Canonical Pathway analyses. Indeed, inhibiting biosynthesis of the N-glycan precursor using the small molecule tunicamycin or inhibiting its transfer to CD 133 by generating N-glycan-deficient CD133 mutants resulted in undetectable cell surface AC133. Among the screen hits involved in N-glycosylation were genes involved in complex N-glycan processing, including the poorly characterized MGAT4C, which we demonstrate to be a positive regulator of cell surface AC133 expression. Our study identifies a set of genes involved in CD133 N-glycosylation as a direct contributing factor to cell surface AC133 recognition and provides biochemical evidence for the function and structure of CD133 N-glycans.