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 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...

Intestinal alkaline sphingomyelinase hydrolyses and inactivates platelet-activating factor by a phospholipase C activity

“…A general property of NPPs is the dependency of the activity on metal ions, particularly Zn 2+ . There are six amino acid residues forming two predicted metal-binding sites in this enzyme family, which were conserved also in alk-SMase [17,26]. An interesting finding that reveals a unique property of alk-SMase is that the activity against SM is not dependent on metal ions, but is inhibited by Zn 2+ and also by mutations within the metal-binding site.…”

“…Transient expression of alk-SMase in COS-7 cells was performed as described previously [17]. The medium of the cell culture was collected 48 h after transfection, and the cells were scraped and lysed in 50 mM Tris/HCl buffer (pH 7.4) containing 1 mM PMSF, 2 mM EDTA, 0.5 mM DTT (dithiothreitol), 10 µg/ml leupeptin, 10 µg/ml aprotinin and 10 mM TC, followed by sonication for 10 s. After centrifugation at 12 000 g for 10 min at 4 • C, the cellfree extract was collected.…”

“…The enzyme was recently purified and cloned from both human and rat by our group and found to be a novel member of the NPP (nucleotide pyrophosphatase/ phosphodiesterase) family, sharing no structural similarities with other SMases [15][16][17]. Although it belongs to the NPP family, the enzyme has no activity against nucleotide phosphodiesters, but was characterized as an SMase with some activity against phosphatidylcholine and lysophosphatidylcholine [17]. Alk-SMase is an ectoenzyme bound on the surface of intestinal microvilli with its C-terminal anchor, and is released to the intestinal lumen in an active form by pancreatic trypsin [18].…”

“…Alk-SMase is an ectoenzyme bound on the surface of intestinal microvilli with its C-terminal anchor, and is released to the intestinal lumen in an active form by pancreatic trypsin [18]. Previous studies showed that the physiological function of alk-SMase is to hydrolyse dietary SM (sphingomyelin), which is abundant in milk, egg, meat and fish, and also SM in the plasma membrane of epithelial cells [17]. Recent studies have shown decreased activity of the enzyme in the tissues of human long-standing ulcerative colitis and colonic adenoma and carcinomas, suggesting a potential role of the enzyme in both colonic inflammation and carcinogenesis [19][20][21].…”

“…The specific radioactivity of the labelled SM was 56 µCi/mg. The plasmids for wild-type human alk-SMase expression were constructed as described previously [17,24]. Rat intestinal alk-SMase was purified from rat intestine in our laboratory [15].…”