Studies on a novel human gene encoding a protein homologous to acid ceramidase

Abstract

Acid ceramidase (EC 3.5.1.23) is a lysosomal enzyme that hydrolyses ceramide to sphingosine and fatty acid. Ceramidase is supposed to be a key regulator in sphingosine generation and ceramide degradation, both of which are known as intracellular second messengers. Using the reported human acid ceramidase sequence (hAC), we tried to find any related sequence from database. Computer-assisted database search of sequences homologous to hAC revealed a 1,233 bp sequence (previously designated as cPj-LTR) which encoded a 266 amino acid open reading frame that was ~36% identical to this lysosomal enzyme. Based on this high degree of homology, we started this study to characterize the sequence further. From placenta cDNA library screening, cDNA clones were isolated and sequenced, and then renamed it as hACH for human acid ceramidase homologous sequence. The full-length hACH cDNA was 1,825 bp and contained an open-reading frame encoding a 359 amino acid polypeptide that was 33% identical and 69% similar to the hAC polypeptide over its entire length. Numerous short regions of complete identity were observed between these two sequences and two sequences available from the C. elegans genomic database. Northern blotting experiments revealed a major 2.0 kb hACH transcript that was expressed at high levels in the liver and kidney, but at relatively low levels in other tissues such as the lung, heart, and brain. Glycosylated flag/hACH, hACH/myc and hACH/GFP fusion proteins (48, 52 and 74 kDa, respectively) was expressed in COS-1 cells and found to be localized in perinuclear and cytoplasm showing punctuated pattern. Treatment of the fusion protein with peptido-N-glycosidase F reduced the molecular weights to 39, 42.5 and 64 kDa. Using an assay system employing fluorescently conjugated bodipy-ceramide (C12) as a substrate, hACH did not express ceramidase activity. Fast-atom bombardment (FAB) mass spectrometry revealed that hACH expression in COS-1 cells changed the lipid content...