Cloning of alkaline sphingomyelinase from rat intestinal mucosa and adjusting of the hypothetical protein XP_221184 in GenBank.
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.
Wu
Jun
J
Gastroenterology Lab, Biomedical Center, B11, Lund University, S-221 84 Lund, Sweden.
Cheng
Yajun
Y
Palmberg
Carina
C
Bergman
Tomas
T
Nilsson
Ake
A
Duan
Rui-Dong
RD
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D030561:Databases, Nucleic Acid; D005260:Female; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology*; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013108:Sphingomyelin Phosphodiesterase / Q000235:genetics* / Q000378:metabolism*; D014018:Tissue Distribution
D013108:Sphingomyelin Phosphodiesterase