title: Identification of human intestinal alkaline sphingomyelinase as a novel ecto-enzyme related to the nucleotide phosphodiesterase family.
abstract: Alkaline sphingomyelinase (alk-SMase) hydrolyzes dietary sphingomyelin and generates sphingolipid messengers in the gut. In the present study, we purified the enzyme, identified a part of the amino acid sequence, and found a cDNA in the GenBank coding for the protein. The cDNA contains 1841 bp, and the open reading frame encodes 458 amino acids. Transient expression of the cDNA linked to a Myc tag in COS-7 cells increased alk-SMase activity in the cell extract by 689-fold and in the medium by 27-fold. High activity was also identified in the anti-Myc immunoprecipitated proteins and the proteins cross-reacted with anti-human alk-SMase. Northern blotting of human intestinal tissues found high levels of alk-SMase mRNA in the intestine and liver. The amino acid sequence shared no similarity with acid and neutral SMases but was related to the ecto-nucleotide phosphodiesterase (NPP) family with 30-36% identity to human NPPs. Alk-SMase has a predicted signal peptide domain at the N terminus and a signal anchor domain at the C terminus. The ion-binding sites and the catalytic residue of NPPs were conserved, but the substrate specificity domain was modified. Alk-SMase had no detectable nucleotidase activity, but its activity against sphingomyelin could be inhibited by orthovanadate, imidazole, and ATP. In contrast to NPPs, alk-SMase activity was not stimulated by divalent metal ions but inhibited by Zn2+. Differing from NPP2, the alk-SMase cleaved phosphocholine but not choline from lysophosphatidylcholine. Phylogenetic tree indicated that the enzyme is a new branch derived from the NPP family. Two cDNA sequences of mouse and rat that shared 83% identity to human alk-SMase were identified in the GenBank. In conclusion, we identified the amino acid and cDNA sequences of human intestinal alk-SMase, and found that it is a novel ecto-enzyme related to the NPP family with specific features essential for its SMase activity.
authors.affiliation: Gastroenterology Laboratory, Biomedical Center, B11, Lund University, S-221 84 Lund, Sweden. Rui-dong.duan@med.lu.se
authors.forename: Rui-Dong
authors.initials: RD
authors.lastname: Duan
authors.forename: Tomas
authors.initials: T
authors.lastname: Bergman
authors.forename: Ning
authors.initials: N
authors.lastname: Xu
authors.forename: Jun
authors.initials: J
authors.lastname: Wu
authors.forename: Yajun
authors.initials: Y
authors.lastname: Cheng
authors.forename: Jianxin
authors.initials: J
authors.lastname: Duan
authors.forename: Sven
authors.initials: S
authors.lastname: Nelander
authors.forename: Carina
authors.initials: C
authors.lastname: Palmberg
authors.forename: Ake
authors.initials: A
authors.lastname: Nilsson
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D019556:COS Cells; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D007422:Intestines / Q000201:enzymology*; D008244:Lysophosphatidylcholines / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry*; D010767:Phosphorylcholine / Q000737:chemistry; D010802:Phylogeny; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D014018:Tissue Distribution; D014162:Transfection; D014638:Vanadates / Q000494:pharmacology; D015032:Zinc / Q000737:chemistry
chemical_list: D018076:DNA, Complementary; D007093:Imidazoles; D008244:Lysophosphatidylcholines; D012333:RNA, Messenger; D010767:Phosphorylcholine; D014638:Vanadates; C029899:imidazole; D000255:Adenosine Triphosphate; D010727:Phosphoric Diester Hydrolases; D013108:Sphingomyelin Phosphodiesterase; D015032:Zinc