Ceramidase is an enzyme that is responsible for ceramide hydrolysis to sphingosine and fatty acid. Different forms of ceramidase activity have been identified, including an acid, alkaline and neutral one, which are sequenced recently at mouse and human and so on. Acid ceramidase is the lysosomal enzyme and Inheritent deficiency of this enzymatic activity results in the lipid storage disorder, Farber disease. Using Computer-assisted database search of sequence homologous to hAC(human acid ceramidase), hACH(human acid ceramidase homologue) gene was found and studied. Like that, Novel mouse gene encoding a protein homologous to mouse acid acramidase was found by using computer-assited database search and library screening of mouse spleen cDNA library, mouse genomic library in our lab, and then renamed it as mACH for mouse acid ceramidase homologue sequence. The 2.4kb cDNA contained an open reading frame encoding a 362 amino acid polypeptide that was 76 and 36% identical to the hACH and hAC respectively. The gene is localized to approximately 51cM distal to the centromere of chromosome5 using interspecific backcross panels. The mapped region is syntenic to human chromosome 4q13-q21, to which hACH (4q21.1) has been mapped by FISH. Sequence analysis of the 5`-flanking region of mACH gene revealed a putative promoter region that lacked a TATA box and CCAAT box, but was GC rich and contained SP1 transcription factor binding sites, typical feature of a housekeeping gene promoter. The functional promoter activity of this region using luciferase reporter system revealed promoter activity. Northern blot analysis showed that mACH mRNAs were widely expressed in various mouse tissue, but the amount of transcripts are not so much. They were expressed at high levels in the spleen, brain, lung, followed by kidney. The expression was rarely detected in muscle. In situ hybridization of mACH in mouse kidney revealed that mACH were highly expressed in glomerulus. Glycosylated mACH/GFP fusion proteins (72kDa) was expressed in 293-T cells and found to be localized in cytoplasm showing punctuated pattern. Western blot of mACH-V5, which was expressed in 293-T cell, showed two major band, 45kDa and 32kDa. Treatment of the mACH-V5 with PNGase F reduced the molecular weights to 40kDa, 29kDa. We think 45kDa and 32kDa proteins are N-glycosylated protein, 32kDa of the two is the glycosylated proteolytic cleavage form (β-subunit) at +391 site. The proteolytic cleavage site is conserved at hAC, mAC, hACH, mACH, C.elegans acid ceramidase. Using an in vivo assay system employing $^{14}C$-N-palmitoyl-D-sphingosine$ as a substrate, mACH did not express ceramidase activity. In this study, we characterized a mouse gene encoding a protein homologous to acid ceramidase. Analysis of this novel protein, mACH could facilitate understanding of lipid metabolism and possibly lipid-mediated intracellular signaling.
