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 alkSMase. 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 Zn 2؉ . 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 ectoenzyme related to the NPP family with specific features essential for its SMase activity. Sphingomyelin (SM)1 is a component of all mammalian cell membranes particularly the plasma membrane and the lysosomal membrane. SM is also a dietary component and is mainly present in milk, eggs, meat, and marine products (1, 2). Hydrolysis of SM generates ceramide, sphingosine, and sphingosine 1-phosphate that have regulatory effects on numerous cellular functions such as proliferation, differentiation, and apoptosis (3, 4). At least five types of sphingomyelinase (SMase) have been identified, of which acid and neutral SMases have been cloned (5-9). An enzyme that catalyzes hydrolysis of SM with optimal alkaline pH was first identified in the intestinal content of human and intestinal mucosa of rat and pig by Nilsson (10) and was named alkaline SMase (alkSMase) thereafter (11). Previous studies indicated that alkSMase may be responsible for digestion of dietary SM and for hydrolysis of endogenous SM derived from bile and from the brush borders of sloughed mucosal cells.SM metabolism in the intestine may have implications in colon cancer development. Dietary supplement with SM and ceramide analogues...

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Sphingolipids are abundant in the microvillar membrane of intestinal epithelial cells where they are essential for structural integrity and may act as receptors for toxins, virus and bacteria. Metabolism of dietary and membrane sphingolipids in the intestine generates ceramide, sphingosine, sphingosine-1-phosphate, and ceramide-1-phosphate, via the action of alkaline sphingomyelinase, neutral ceramidase, sphingosine-1-kinase, and ceramide-1-kinase. These intermediary metabolites act as bioactive lipid messengers, influencing numerous cellular functions including growth, differentiation and apoptosis of both epithelial and immunocompetent cells in the gastrointestinal tract, and also the progress of inflammation and responsiveness of the mucosal cells to pathogens. This review summarizes background and recent progress in the metabolism of dietary and endogenous sphingolipids in the gut and its pathophysiological implications.

SummaryEscherichia coli express fimbriae-associated adhesins through which they attach to mucosal cells and activate a cytokine response. The receptors for E. coli P fimbriae are the globoseries of glycosphingolipids; Galc~l--)4Gall3-containing oligosaccharides bound to ceramide in the outer leaflet of the lipid bilayer. The receptors for type 1 fimbriae are mannosylated glycoproteins rather than glycolipids. This study tested the hypothesis that P-fimbriated E. coli elicit a cytokine response through the release of ceramide in the receptor-bearing cell. We used the A498 human kidney cell line, which expressed functional receptors for P and type 1 fimbriae and secreted higher levels of interleukin (IL)-6 when exposed to the fimbriated strains than to isogenic nonfimbriated controls. P-fimbriated E. coli caused the release of ceramide and increased the phosphorylation of ceramide to ceramide 1-phosphate. The IL-6 response to P-fimbriated E. coli was reduced by inhibitors ofserine/threonine kinases but not by other protein kinase inhibitors. In contrast, ceramide levels were not influenced by type 1-fimbriated E. coli, and the IL-6 response was insensitive to the serine/threonine kinase inhibitors. These results demonstrate that the ceramide-signaling pathway is activated by P-fimbriated E. coli, and that the receptor specificity of the P fimbriae influences this process. We propose that this activation pathway contributes to the cytokine induction by P-fimbriated E. coli in epithelial cells.

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