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

(1)我们通过5'和3' RACE-PCR分离了编码znCD的cDNA克隆。该基因的开放阅读码为2,229个碱基对，编码743个氨基酸。用该cDNA转化中国仓鼠CHOP细胞和斑马鱼BRF 41细胞后，中性pH下的酶活性显著增加。过表达的酶被发现分布在ER/高尔基室以及质膜。根据znCD mRNA序列设计的反义morpholino寡核苷酸（AMO）成功地阻断了znCD在小麦胚体外翻译系统中的翻译。用AMO敲低znCD导致斑马鱼胚胎数量增加，出现严重的形态和细胞异常;在受精后36 h，头部和尾部的形态发生异常，心包水肿，血细胞循环缺陷，以及凋亡细胞增加，特别是在头部和神经管区域。在注射AMO-injected胚胎中，神经酰胺水平显著增加， ...更多信息 与对照组胚胎相似。同时注射AMO和合成的znCD mRNA到1细胞期胚胎救出znCD活性和血细胞循环。这些结果表明，znCD是必不可少的神经酰胺的代谢和早期发育的斑马鱼。(2)神经酰胺糖苷内切酶（EGCase ; EC 3.2.1.123）是能够切割各种鞘糖脂的寡糖和神经酰胺之间的糖苷键的酶。水螅EGCase由517个氨基酸组成，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螅酶水解[^<14>C] GM 1a以产生[^<14>C]神经酰胺，最佳pH为3.0-3.5。整体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。注射到胃腔中的GM 1a被EGCase掺入，然后被EGCase直接分解代谢产生GM 1a-寡糖和神经酰胺，其分别被外切糖苷酶和神经酰胺酶进一步降解。然而，水螅外切糖苷酶不直接水解GM 1a。这些结果表明EGCase对于水螅中膳食鞘糖脂的分解代谢加工是不可或缺的，证明了动物中GSL的独特分解代谢途径。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