Molecular cloning and functional analysis of zebrafish neutral ceramidase
Molecular cloning and functional analysis of zebrafish neutral ceramidase
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DOI：
10.1074/jbc.m405598200
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发表时间：
2004-10-15
影响因子：
4.8
通讯作者：
Ito, M
中科院分区：
文献类型：
--
作者：
Yoshimura, Y;Tani, M;Ito, M
几乎所有关于中性神经酰胺酶功能的观察都是在细胞水平上进行的，而不是在个体水平上进行的。本文报道了斑马鱼中性神经酰胺酶（znCD）的分子克隆及其胚胎发育过程中的功能分析。利用cDNA末端快速扩增技术（5′和3′）分离到一个编码znCD的cDNA克隆。其开放阅读框包含2229个碱基对，编码743个氨基酸。在推测的序列中，在N端附近发现了一个可能的信号/锚定序列，并发现了4个潜在的o糖基化位点和8个潜在的N糖基化位点。用cDNA转化中国仓鼠CHOP和斑马鱼BRF41细胞后，在中性pH下酶活性显著升高。发现过表达酶分布在内质网/高尔基区室以及质膜中。基于znCD mRNA序列设计的反义morpholino oligonucleotide （AMO）在小麦胚芽体外翻译系统中成功阻断了znCD的翻译。用AMO敲低znCD导致斑马鱼胚胎数量增加，在受精后36 h出现头尾形态发生异常、心包水肿、血细胞循环缺陷、凋亡细胞增多，尤其是头部和神经管区域凋亡细胞增多等严重的形态和细胞异常。注射amo的胚胎神经酰胺水平显著高于对照胚胎。在单细胞期胚胎中同时注射AMO和合成znCD mRNA可恢复znCD活性和血细胞循环。这些结果表明，znCD对神经酰胺代谢和斑马鱼的早期发育至关重要。
Almost all observations on the functions of neutral ceramidase have been carried out at cellular levels but not at an individual level. Here, we report the molecular cloning of zebrafish neutral ceramidase (znCD) and its functional analysis during embryogenesis. We isolated a cDNA clone encoding znCD by 5' and 3' rapid amplification of cDNA ends-PCR. It possessed an open reading frame of 2,229 base pairs encoding 743 amino acids. A possible signal/anchor sequence near the N terminus and four potential O-glycosylation and eight potential N-glycosylation sites were found in the putative sequence. The enzyme activity at neutral pH increased markedly after transformation of Chinese hamster CHOP and zebrafish BRF41 cells with the cDNA. The overexpressed enzyme was found to be distributed in endoplasmic reticulum/Golgi compartments as well as the plasma membranes. The 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 such as abnormal morphogenesis in the head and tail, pericardiac edema, defect of blood cell circulation, and an increase of apoptotic cells, especially in the head and neural tube regions, at 36 h post-fertilization. The ceramide level in AMO-injected embryos increased significantly compared with that in control embryos. Simultaneous injection of both AMO and synthetic znCD mRNA into one-cell-stage embryos rescued znCD activity and blood cell circulation. These results indicate that znCD is essential for the metabolism of ceramide and the early development of zebrafish.