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J. Biol. Chem., Vol. 279, Issue 42, 44012-44022, October 15, 2004

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Molecular Cloning and Functional Analysis of Zebrafish Neutral Ceramidase^*

Yukihiro Yoshimura, Motohiro Tani, Nozomu Okino, Hiroshi Iida, and Makoto Ito¶

From the Departments of Bioscience and Biotechnology and the Applied Genetics and Pest Management, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

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.

Received for publication, May 19, 2004 , and in revised form, July 19, 2004.

^* This work was supported in part by Grants-in-aid for scientific research on priority area 12140204 and research B 15380073 promoting science and technology from the Ministry of Education, Culture, Sport, Science and Technology, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a movie dealing with a defective blood circulation phenotype in zebrafish.

^¶ To whom correspondence should be addressed. Tel.: 81-92-642-2898; Fax: 81-92-642-2907; E-mail: makotoi{at}agr.kyushu-u.ac.jp.

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