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PCYT2 synthesizes CDP-glycerol in mammals and reduced PCYT2 enhances the expression of functionally glycosylated α-dystroglycan
The Journal of Biochemistry ( IF 2.1 ) Pub Date : 2021-07-13 , DOI: 10.1093/jb/mvab069
Rieko Imae 1 , Hiroshi Manya 1 , Hiroki Tsumoto 2 , Yuri Miura 2 , Tamao Endo 1
Affiliation  

α-Dystroglycan (α-DG) is a highly glycosylated cell-surface protein. Defective O-mannosyl glycan on α-DG is associated with muscular dystrophies and cancer. In the biosynthetic pathway of the O-mannosyl glycan, fukutin (FKTN) and fukutin-related protein (FKRP) transfer ribitol phosphate (RboP). Previously, we reported that FKTN and FKRP can also transfer glycerol phosphate (GroP) from CDP-glycerol (CDP-Gro) and showed the inhibitory effects of CDP-Gro on functional glycan synthesis by preventing glycan elongation in vitro. However, whether mammalian cells have CDP-Gro or associated synthetic machinery has not been elucidated. Therefore, the function of CDP-Gro in mammals is largely unknown. Here, we reveal that cultured human cells and mouse tissues contain CDP-Gro using liquid chromatography tandem-mass spectrometry (LC-MS/MS). By performing the enzyme activity assay of candidate recombinant proteins, we found that ethanolamine-phosphate cytidylyltransferase (PCYT2), the key enzyme in de novo phosphatidylethanolamine biosynthesis, has CDP-Gro synthetic activity from glycerol-3-phosphate (Gro3P) and CTP. In addition, knockdown of PCYT2 dramatically reduced cellular CDP-Gro. These results indicate that PCYT2 is a CDP-Gro synthase in mammals. Furthermore, we found that the expression of functionally glycosylated α-DG is increased by reducing PCYT2 expression. Our results suggest an important role for CDP-Gro in the regulation of α-DG function in mammals.

中文翻译:

PCYT2 在哺乳动物中合成 CDP-甘油,减少的 PCYT2 增强功能性糖基化 α-dystroglycan 的表达

α-Dystroglycan (α-DG) 是一种高度糖基化的细胞表面蛋白。α-DG 上有缺陷的O-甘露糖基聚糖与肌营养不良和癌症有关。在O-甘露糖基聚糖的生物合成途径中,福丁 (FKTN) 和福丁相关蛋白 (FKRP) 转移磷酸核糖醇 (RboP)。之前,我们报道了 FKTN 和 FKRP 还可以从 CDP-甘油 (CDP-Gro) 中转移磷酸甘油 (GroP),并通过在体外阻止聚糖延伸显示 CDP-Gro 对功能性聚糖合成的抑制作用. 然而,哺乳动物细胞是否具有 CDP-Gro 或相关的合成机制尚未阐明。因此,CDP-Gro 在哺乳动物中的功能在很大程度上是未知的。在这里,我们使用液相色谱串联质谱 (LC-MS/MS) 揭示培养的人类细胞和小鼠组织含有 CDP-Gro。通过对候选重组蛋白进行酶活性测定,我们发现乙醇胺-磷酸胞苷酰转移酶(PCYT2)是从头合成的关键酶。磷脂酰乙醇胺生物合成,具有来自 3-磷酸甘油 (Gro3P) 和 CTP 的 CDP-Gro 合成活性。此外,PCYT2 的敲低显着降低了细胞 CDP-Gro。这些结果表明 PCYT2 是哺乳动物中的 CDP-Gro 合酶。此外,我们发现功能性糖基化 α-DG 的表达通过降低 PCYT2 表达而增加。我们的结果表明 CDP-Gro 在调节哺乳动物的 α-DG 功能中具有重要作用。
更新日期:2021-07-14
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