Abstract
Alkaline sphingomyelinase (alk-SMase) is a new member of the NPP (nucleotide pyrophophatase/phosphodiesterase) family that hydrolyses SM (sphingomyelin) to generate ceramide ill the intestinal tract. The enzyme may protect the intestinal mucosa front inflammation and tumorigenesis. PAF (platelet-activating factor) is a pro-inflammatory phospholipid involved in pathogenesis of inflammatory bowel diseases. We examined whether alk-SMase call hydrolyse and inactivate PAF. [H-3]Octadecyl-labelled PAF was incubated With purified rat intestinal alk-SMase or recombinant human alk-SMase expressed in COS-7 cells. The hydrolytic products were assayed with TLC and MS. We found that alk-SMase cleaved the phosphocholine head group from PAF and generated 1-O-alkyl-2-acetyl-sn-glycerol. Differing from the activity against SM, the activity against PAF was optimal at pH 7.5, inhibited by EDTA and stimulated by 0.1-0.25 mM Zn2+. The activity was abolished by site Mutation of the predicted metal-binding sites that are conserved in all NPP members. Similar to the activity against SM, the activity against PAF was dependent on bile salt, particularly taurocholate and taurochenodeoxycholate. The V-max for PAF hydrolysis was 374 mu mol . h(-1) . (mg of protein)(-1). The hydrolysis of PAF and SM could be inhibited by the presence of SM and PAF respectively, the inhibition of PAF hydrolysis by SM being stronger. The PAF-induced MAPK (mitogen-activated protein kinase) activation and IL-8 (interleukin 8) release in HT-29 cells, and chemotaxis in leucocytes were abolished by alk-SMase treatment. In Conclusion, alk-SMase hydrolyses and inactivates PAF by a phospholipase C activity. The finding reveals a novel function, by which alk-SMase may counteract the development of intestinal inflammation and colon cancer.
Original language | English |
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Pages (from-to) | 299-308 |
Journal | Biochemical Journal |
Volume | 394 |
DOIs | |
Publication status | Published - 2006 |
Subject classification (UKÄ)
- Biochemistry and Molecular Biology
Free keywords
- platelet-activating factor
- mitogen-activated protein kinase (MAPK)
- inflammatory bowel disease
- alkaline sphingomyelinase
- colon cancer
- phospholipase C