Molecular cloning of the full-length cDNA encoding mouse neutral ceramidase - A novel but highly conserved gene family of neutral/alkaline ceramidases

We report here the molecular cloning, sequencing, and expression of the gen e encoding the mouse neutral ceramidase, which has been proposed to functio n in sphingolipid signaling. A full-length cDNA encoding the neutral cerami dase was cloned from a cDNA library of mouse liver using the partial amino acid sequences of the purified mouse liver ceramidase, The open reading fra me of 2,268 nucleotides encoded a polypeptide of 756 amino acids having nin e putative N-glycosylation sites. Northern blot analysis revealed that the mRNA of the ceramidase was expressed widely in mouse tissues, with especial ly strong signals found in the liver and kidney, The ceramidase activity of lysates of CHOP cells increased more than 900-fold when the cells were tra nsformed with a plasmid containing the cDNA encoding ceramidase, We also cl oned the ceramidase homologue from the cDNA library of mouse brain and foun d that the sequence of the open reading frame, but not the 5'-noncoding reg ion, was identical to that of the liver. Interestingly, phylogenetic analys is of various ceramidases clearly indicated that neutral/alkaline ceramidas es form a novel but highly conserved gene family that is evolutionarily dif ferent from lysosomal acid ceramidases.