Molecular cloning and functional analysis of zebrafish neutral ceramidase

Almost all observations on the functions of neutral ceramidase have been carried out at cellular levels but not 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' RACE-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 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 compared to that in control embryos. Simultaneous injection of synthetic znCD mRNA into 1 cell-stage embryos with the AMO rescued znCD activity and blood cell circulation. These results indicate that znCD is essential for the metabolism of ceramide and early development of zebrafish.