Two novel proteins homologous to human acid ceramidase from caenorhabditis elegans

Abstract

Acid ceramidase (EC 3.5.1.23; N-acylsphingosine amidohydrolase) is the lysosomal enzyme required for the hydrolysis of N-acyl linkage between the fatty acid and sphigosine moieties in ceramide. Inheritent deficiency of this enzymatic activity results in the lipid storage disorder, Farber disease. Computer-assisted database analysis showed that two genomic sequences (designated as K11D2.2 and F27E5.1, respectively) homologous to human acid ceramidase (hAC) from C. elegans may encode hypothetical proteins (393 and 401 amino acid, respectively) that have ~35% identity to hAC. We could measure acid and neutral ceramidase activity in worm lysates using 14C-labeled palimtoyl-sphingosine as a substrate. The acid and neutral ceramidase activities were about 200 pmol/mg/hr each. Based on the high sequence homology between the two hypothetical proteins and acid ceramidase, we started this study to characterize those two sequences further. From a C. elegans cDNA library, we could isolated cDNAs encoding the hypothetical proteins and then renamed the gene as cac-1 and cac-2. The full-length cAC-1 and cAC-2 cDNA were 1,274 bp and 1,295 bp, respectively, and contained an open-reading frame encoding a 393 and a 401 amino acid polypeptide. cAC-1 and cAC-2 showed 37 and 33% identity and 62 and 56% similararity to the hAC polypeptide over its entire length. Comparison of the cDNA and genomic sequences revealed that cac-1 and cac-2 consist of 4 exons and 8 exons, respectively. The 5```` and 3```` untranslated regions (UTR) of cac-1 were 11 and 81 bp, respectively and those of cac-2 were 7 and 56 bp, respectively. Northern blot analysis showed that 1.3 kb cac-1 transcripts were expressed only in the post-embyonic stages of worms. However, we could hardly detect cac-2 transcripts. Transient transfection of recombinant plasmids expressing cAC-1/Myc and cAC-2/Myc fusion proteins to human 293-T cells led to a 1.6- and 3-fold increase in acid ceramidase activity of 293-T cells. Western...