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J. Biol. Chem., Vol. 278, Issue 40, 38528-38536, October 3, 2003

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Identification of Human Intestinal Alkaline Sphingomyelinase as a Novel Ecto-enzyme Related to the Nucleotide Phosphodiesterase Family^*

Rui-Dong Duan  , Tomas Bergman ¶, Ning Xu ||, Jun Wu , Yajun Cheng , Jianxin Duan **, Sven Nelander , Carina Palmberg ¶ and Åke Nilsson 

From the Gastroenterology Laboratory, Biomedical Center, B11, Lund University, S-221 84 Lund, the ^¶Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, the ^||Department of Clinical Chemistry, Lund University Hospital, S-221 85 Lund, the ^**Department of Biosciences at Novum, Structural Biochemistry Center, Karolinska Institutet, S-141 57 Huddinge, and the Department of Medical Biochemistry, Göteborg University, P. O. Box 440, S-405 30, Göteborg, Sweden

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^TM 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^TM. 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.

Received for publication, May 23, 2003 , and in revised form, July 8, 2003.

^* This work was supported by grants from the Swedish Cancer Society, the Swedish Medical Research Council, Albert Påhlsson Foundation, Gunnar Nilsson Cancer Foundation, Thelma Zoegas Foundation, and the Research Foundation of Lund University Hospital. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Gastroenterology Lab, Biomedical Center, B11, Lund University S-221 84 Lund, Sweden. Tel.: 46-46-222-0709; Fax: 46-46-137277; E-mail: Rui-dong.duan{at}med.lu.se.

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