Molecular cloning and characterization of a human cDNA and gene encoding anovel acid ceramidase-like protein

Computer-assisted database analysis of sequences homologous to human acid c eramidase (ASAH) revealed a 1233-bp cDNA (previously designated cPj-LTR) wh ose 266-amino-acid open reading frame had similar to 36% identity with the ASAH polypeptide. Based on this high degree of homology, we undertook furth er molecular characterization of cPj-LTR and now report the full-length cDN A sequence, complete gene structure (renamed human ASAHL since it is a huma n acid ceramidase-like sequence), chromosomal location, primer extension an d promoter analysis, and transient expression results. The full-length huma n ASAHL cDNA was 1825 bp and contained an open-reading frame encoding a 359 -amino-acid polypeptide that was 33% identical and 69% similar to the ASAH polypeptide over its entire length. Numerous short regions of complete iden tity mere observed between these two sequences and two sequences obtained f rom the Caenorhabditis elegans genome database. The 30-kb human ASAHL genom ic sequence contained 11 exons, which ranged in size from 26 to 671 bp, and 10 introns, which ranged from 150 bp to 6.4 kb. The gene was localized to the chromosomal region 4q21.1 by fluorescence in situ hybridization analysi s. Northern blotting experiments revealed a major 2.0-kb ASAHL transcript t hat 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. Sequence a nalysis of the 5'-flanking region of the human ASAHL gene revealed a putati ve promoter region that lacked a TATA box and was GC rich, typical features of a housekeeping gene promoter, as well as several tissue-specific and/or hormone-induced transcription regulatory sites. 5'-Deletion analysis local ized the promoter activity to a 1.1-kb fragment within this region. A major transcription start site also was located 72 bp upstream from the ATG tran slation initiation site by primer extension analysis. Expression analysis o f a green fluorescence protein/ASAHL fusion protein in COS-1 cells revealed a punctate, perinuclear distribution, although no acid ceramidase activity was detected in the transfected cells using a fluorescence-based in vitro assay System. (C) 1999 Academic Press.