Ceramide glucosyltransferase of the nematode Caenorhabditis elegans is involved in oocyte formation and in early embryonic cell division
Ceramide glucosyltransferase of the nematode Caenorhabditis elegans is involved in oocyte formation and in early embryonic cell division
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DOI：
10.1093/glycob/cwr019
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发表时间：
2011-06-01
期刊：
影响因子：
4.3
通讯作者：
Nomura, Kazuya
中科院分区：
文献类型：
--
作者：
Nomura, Kazuko H.;Murata, Daisuke;Nomura, Kazuya
神经酰胺葡萄糖基转移酶(UGCG)[尿苷二磷酸(UDP)-葡萄糖：N-乙酰鞘氨醇-D-葡萄糖转移酶或UDP-葡萄糖神经酰胺葡萄糖转移酶(GlcT)：EC 2.4.1.80]催化神经酰胺和UDP-葡萄糖形成葡萄糖神经酰胺(GlcCer)。在小鼠基因组中只有一个UGcg基因，它在胚胎发育和脑发育中是必不可少的。秀丽线虫有3个UGcg基因(CGT-1、CGT-2和CGT-3)，CGT-1和CGT-3基因的双重RNAi导致L1幼虫死亡。在这项研究中，我们分离了这三个基因的敲除蠕虫，并对其基因功能进行了鉴定。每种基因产物在缺乏神经鞘糖脂(GSLS)的GM95细胞中表达时均显示出活性。当每个基因的功能被破坏时，动物的孵化仔数显著减少，并形成异常的卵母细胞和多核胚胎。CGT-3蛋白具有最高的UGcg活性，其基因敲除导致了最严重的表型。当对缺乏体细胞RNAi机制但具有完整种系RNAi机制的RRF-1蠕虫进行CGT-3 RNAi时，尽管CGT-1/CGT-3 RNAi的体细胞表型(即L1致死效应)完全被抑制，但仍观察到许多异常的卵母细胞和多核卵子。在RNAi处理的胚胎中，膜结构域的重要组成部分GSLS和鞘磷脂的细胞表面表达受到影响。在胚胎中，也观察到细胞质分裂的异常。根据这些结果，我们得出结论，UGcg基因在胚系中是不可或缺的，卵母细胞和受精卵需要充足的GlcCer来维持正常的膜和进行正常的细胞周期。
Ceramide glucosyltransferase (Ugcg) [uridine diphosphate (UDP)-glucose:N-acylsphingosine d-glucosyltransferase or UDP-glucose ceramide glucosyltransferase (GlcT): EC 2.4.1.80] catalyzes formation of glucosylceramide (GlcCer) from ceramide and UDP-glucose. There is only one Ugcg gene in the mouse genome, which is essential in embryogenesis and brain development. The nematode Caenorhabditis elegans has three Ugcg genes (cgt-1, cgt-2 and cgt-3), and double RNAi of the cgt-1 and cgt-3 genes results in lethality at the L1 larval stage. In this study, we isolated knockout worms for the three genes and characterized the gene functions. Each gene product showed active enzymatic activity when expressed in GM95 cells deficient in glycosphingolipids (GSLs). When each gene function was disrupted, the brood size of the animal markedly decreased, and abnormal oocytes and multinucleated embryos were formed. The CGT-3 protein had the highest Ugcg activity, and knockout of its gene resulted in the severest phenotype. When cgt-3 RNAi was performed on rrf-1 worms lacking somatic RNAi machinery but with intact germline RNAi machinery, a number of abnormal oocytes and multinucleated eggs were observed, although the somatic phenotype, i.e., L1 lethal effects of cgt-1/cgt-3 RNAi, was completely suppressed. Cell surface expression of GSLs and sphingomyelin, which are important components of membrane domains, was affected in the RNAi-treated embryos. In the embryos, an abnormality in cytokinesis was also observed. From these results, we concluded that the Ugcg gene is indispensable in the germline and that an ample supply of GlcCer is needed for oocytes and fertilized eggs to maintain normal membranes and to proceed through the normal cell cycle.