Intestinal alkaline sphingomyelinase hydrolyses and inactivates platelet-activating factor by a phospholipase C activity.
Alkaline sphingomyelinase (alk-SMase) is a new member of the NPP (nucleotide pyrophosphatase/phosphodiesterase) family that hydrolyses SM (sphingomyelin) to generate ceramide in the intestinal tract. The enzyme may protect the intestinal mucosa from inflammation and tumorigenesis. PAF (platelet-activating factor) is a pro-inflammatory phospholipid involved in pathogenesis of inflammatory bowel diseases. We examined whether alk-SMase can hydrolyse and inactivate PAF. [3H]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 alkSMase 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 mumol x h(-1) x (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.
Wu
Jun
J
Gastroenterology Laboratory, Biomedical Centre, B11, Lund University, S-221 84 Lund, Sweden.
Nilsson
Ake
A
Jönsson
Bo A G
BA
Stenstad
Hanna
H
Agace
William
W
Cheng
Yajun
Y
Duan
Rui-Dong
RD
D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism; D019556:COS Cells; D002118:Calcium; D002522:Chlorocebus aethiops; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007422:Intestines / Q000201:enzymology*; D008274:Magnesium; D010972:Platelet Activating Factor / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D013109:Sphingomyelins / Q000378:metabolism; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism*; D015032:Zinc
D001647:Bile Acids and Salts; D010972:Platelet Activating Factor; D013109:Sphingomyelins; D010738:Type C Phospholipases; C508908:ENPP7 protein, human; D013108:Sphingomyelin Phosphodiesterase; C508907:intestinal alkaline sphingomyelinase, rat; D008274:Magnesium; D015032:Zinc; D002118:Calcium