Functional analysis of sphingolipid-signaling enzymes and its application for lipid biology

(1)用5‘和3’RACE-PCR方法克隆了一个编码znCD的cDNA克隆。开放阅读率为2229个碱基对，编码743个氨基酸。用该基因转化中国仓鼠CHOP和斑马鱼BRF41细胞后，中性pH条件下的酶活力明显提高。过表达的酶分布在内质网/高尔基体和质膜上。根据ZnCD mRNA序列设计的反义吗啉寡核苷酸(AMO)成功地阻断了ZnCD在小麦胚芽体外翻译系统中的翻译。用AMO基因敲除ZnCD基因后，受精后36h斑马鱼胚胎数量增加，头部和尾部形态发生异常，心包水肿，血细胞循环障碍，凋亡细胞增多，尤其是头部和神经管区。注射AMO的胚胎中神经酰胺水平显著增加COMP…在对照胚胎中更倾向于这一点。在1细胞期胚胎中同时注射AMO和合成的ZnCD mRNA，可挽救ZnCD活性和血细胞循环。这些结果表明，ZnCD对于神经酰胺的代谢和斑马鱼的早期发育是必不可少的。(2)神经酰胺内切酶(EGCase；EC 3.2.1.123)是一种能够切断各种神经鞘糖脂的寡糖和神经酰胺之间的糖苷键的酶。该酶由517个氨基酸残基组成，与红球菌和水母EGCase的同源性分别为19.2%和50.2%。在CHOP细胞中表达的重组水合酶能降解[^&lt；14&gt；C]GM1a生成[^&lt；14&gt；C]神经酰胺，最适pH为3.0~3.5。整装原位杂交和免疫细胞化学分析表明，EGCase广泛表达于内胚层，尤其是消化细胞。注射入胃腔的GM1a被EGCase直接降解生成GM1a寡糖和神经酰胺，分别被外切糖苷酶和神经酰胺酶进一步降解。然而，水合胞外糖苷酶并不直接水解GM1a。这些结果表明，EGCase在水产动物饲料中神经鞘糖脂的分解过程中是必不可少的，这表明了GSLS在动物中独特的分解代谢途径。较少

(1) We isolated a cDNA clone encoding znCD by 5' and 3' RACE-PCR. It possessed an open reading fame of 2,229 base pairs encoding 743 amino acids. The enzyme activity at neutral pH markedly increased after transformation of Chinese hamster CHOP and zebrafish BRF41 cells with the cDNA. The overexpressed enzyme was found to be distributed in ER/Golgi compartments as well as the plasma membranes. Antisense morpholino oligonucleotide (AMO), which was designed based on the sequence of znCD mRNA, successfully blocked the translation of znCD in a wheat germ in vitro translation system. The knockdown of znCD with AMO led to an increase in the number of zebrafish embryos with severe morphological and cellular abnormalities ; abnormal morphogenesis in the head and tail, pericardiac edema, defect of blood cell circulation, and increase of apoptotic cells especially in the head and neural tube regions at 36 h postfertilization. The ceramide level in AMO-injected embryos significantly increased comp … More ared to that in control embryos. Simultaneous injection of both AMO and synthetic znCD mRNA into 1 cell-stage embryos rescued znCD activity and blood cell circulation. These results indicate that znCD is essential for the metabolism of ceramide and early development of zebrafish.(2) Endoglycoceramidase (EGCase ; EC 3.2.1.123) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides of various glycosphingolipids. The hydra EGCase, consisting of 517 amino acid residues, showed 19.2% and 50.2% identity to the Rhodococcus and jellyfish EGCases, respectively. The recombinant hydra enzyme, expressed in CHOP cells, hydrolyzed [^<14>C]GM1a to produce [^<14>C]ceramide with a pH optimum at 3.0-3.5. Whole mount in situ hybridization and immunocytochemical analysis revealed that EGCase was widely expressed in the endodermal layer, especially in digestive cells. GM1a injected into the gastric cavity was incorporated and then directly catabolized by EGCase to produce GM1a-oligosaccharide and ceramide, which were further degraded by exoglycosidases and ceramidase, respectively. However, hydra exoglycosidases did not hydrolyze GM1a directly. These results indicate that the EGCase is indispensable for the catabolic processing of dietary glycosphingolipids in hydra, demonstrating the unique catabolic pathway for GSLs in the animal. Less