Cloning of alkaline sphingomyelinase from rat intestinal mucosa and adjusting of the hypothetical protein XP_221184 in GenBank

• Published in: Biochimica et biophysica acta Vol. 1687; no. 1; pp. 94 - 102
• Main Authors: Wu, Jun, Cheng, Yajun, Palmberg, Carina, Bergman, Tomas, Nilsson, Åke, Duan, Rui-Dong
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 21.02.2005
• Subjects: Alkaline sphingomyelinase; Amino Acid Sequence; Animals; Annan klinisk medicin; Cercopithecus aethiops; Clinical Medicine; Cloning; Cloning, Molecular; COS Cells; Databases, Nucleic Acid; Female; Humans; Intestinal Mucosa - enzymology; Klinisk medicin; Medical and Health Sciences; Medicin och hälsovetenskap; Molecular Sequence Data; Other Clinical Medicine; Rats; Rats, Sprague-Dawley; Sequence Alignment; Sequence Homology, Nucleic Acid; Sphingomyelin Phosphodiesterase - genetics; Sphingomyelin Phosphodiesterase - metabolism; Tissue Distribution; XP_221184; Alkaline sphingomyelinase; XP_221184; Cloning
• ISSN: 1388-1981; 0006-3002; 1879-2618
• DOI: 10.1016/j.bbalip.2004.11.006

Intestinal alkaline sphingomyelinase (alk-SMase) digests sphingomyelin and the process may influence colonic tumorigenesis and cholesterol absorption. We recently identified the gene of human alk-SMase and cloned the cDNA. Cross-species screening of homology in GenBank found a hypothetical rat protein, XP_221184, with 491 amino acid residues, which shares 73% identity with human alk-SMase. Based on the cDNA sequence of this protein, we cloned a cDNA from rat intestinal mucosa by RT-PCR. The cloned cDNA encodes a protein with 439 amino acid residues and higher (85%) identity with human alk-SMase. The cloned cDNA differed from the XP_221184 cDNA in splice sites linking exons 2 and 3, and exons 3 and 4, respectively. In the sequence of the cloned protein, the predicted activity motif, sphingomyelin binding sites, and potential glycosylation sites in human alk-SMase are all conserved. To confirm the cloned protein is the real form of alk-SMase, native alk-SMase was purified from rat intestine and subjected to proteolytic digestion followed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and electrospray ionization (ESI) tandem mass spectrometry. Seven tryptic peptides were found to match the cloned protein sequence. Transient expression of the cloned cDNA linked with a myc tag in COS-7 cells demonstrated high SMase activity, with an optimal pH at 9.0 and a specific dependence on taurocholate and taurochenodeoxycholate. The expressed protein reacted with both anti-myc and anti-human alk-SMase antibodies. Northern blotting of rat tissues revealed high levels of mRNA in jejunum but not in other tissues. In conclusion, we cloned rat alk-SMase cDNA from rat intestine, adjusted the putative rat alk-SMase protein in GenBank, and confirmed the specific expression of the gene in the small intestine.