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

Abstract

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.

Figure 1. Identification of hydrolytic products of PAF by TLC

[³H]PAF was incubated without (upper panel) or with rat intestinal alk-SMase (bottom panel) in 50 mM Tris/HCl buffer (pH 7.5) containing 0.15 M NaCl, 0.25 mM ZnCl₂ and 6 mM TC (P buffer) at 37 °C for 2 h. Lipids were extracted, separated by TLC and stained with iodine vapour. Authentic PAF and DG were used as standards. After TLC, each lane was divided into 18 fractions and each fraction was scraped, and the radioactivity was assayed by liquid scintillation. Results shown are means±S.E.M. for three separate experiments.

Figure 2. Identification of hydrolytic product of PAF by MS

PAF (5 nmol) was treated with 1.5 ng of rat intestinal alk-SMase (lower panel) or without (upper panel). The hydrolytic products were extracted and subjected to LC-MS. The m/z values 524.5 and 546.5 in the Figure are consistent with the protonated ion mass and sodium ion mass of PAF, and 381.4 and 359.3 are consistent with the sodium ion mass and protonated ion mass of AAG.

Figure 3. PAF hydrolytic activity in COS-7 cells transfected with human alk-SMase cDNA

COS-7 cells were transfected with human alk-SMase cDNA in the presence of Lipofectamine™ 2000, followed by incubation of the cells for 48 h. The medium was collected and the cell-free extracts were prepared. The activity against PAF and SM was determined. (A, B) The activity against PAF and SM in the cell-free extracts. (C, D) The activity against PAF and SM in culture medium. Results shown are means±S.E.M. for three separate experiments. ***P<0.001 compared with control.

Figure 4. Comparison of the effects of alk-SMase and neutral SMase

Both rat alk-SMase and bacterial neutral SMase were diluted to equivalent hydrolytic capacity and incubated with PAF in different buffers. P buffer is optimal for the activity against PAF; K and N buffers are optimal for alk-SMase and neutral SMase against SM respectively as described in the Materials and methods section. The formation of AAG was analysed by TLC. Results shown are means±S.E.M. for three separate experiments.

Figure 5. Competition between PAF and SM for hydrolysis by alk-SMase

In the upper panel, PAF hydrolysis by alk-SMase (1.5 ng) was determined in P buffer containing 100 pmol of PAF and different amounts of SM. After a 2 h incubation, the lipids were extracted and the hydrolytic products were separated by TLC. The radioactivities of AAG were assayed by liquid scintillation. In the lower panel, the activity against SM was determined in the K buffer containing 100 pmol of [¹⁴C]SM and different amounts of PAF. After incubation at 37 °C for 30 min, the reaction was terminated by adding 0.4 ml of chloroform/methanol (2:1, v/v) followed by centrifugation at 9000 g for 10 s. An aliquot (100 μl) of the upper phase containing the cleaved phosphocholine was analysed for radioactivity by liquid scintillation. Results shown are means±S.E.M. for three separate experiments. *P<0.05 and **P<0.01.

Figure 6. Characterization of the PAF hydrolytic effects of alk-SMase

In the top left panel, 1.5 ng of rat intestinal alk-SMase was incubated with 100 pmol of [³H]PAF in 50 mM Tris/maleate buffer at pH 5–7 or Tris/HCl buffer at pH 7.5–9.0, containing 0.15 M NaCl, 0.25 mM ZnCl₂ and 6 mM TC, at 37 °C for 1 h. In the top right panel, rat intestinal alk-SMase was incubated with 100 pmol of [³H]PAF in P buffer at 37 °C and the reaction was terminated at different times. In the bottom left panel, 0–3 ng of alk-SMase was incubated with 100 pmol of [³H]PAF at 37 °C for 60 min. The production of AAG was separated with TLC and radioactivity was analysed by liquid scintillation. Results shown are means±S.E.M. for three separate experiments. In the bottom right panel, 1 ng of purified rat alk-SMase was incubated with different amounts of [³H]PAF ranging from 80 to 40000 pmol at 37 °C for 60 min. The production of AAG was analysed and the hydrolytic capacity was determined by Lineweaver–Burk plot. A similar Figure was obtained from another experiment.

Figure 7. Effect of bile salts on PAF hydrolytic activity of alk-SMase

In the upper panel, the PAF hydrolytic activity was assayed in P buffer containing different concentrations of TC and TDC. In the bottom panel, the activity was assayed in the same buffer with different bile salts and synthetic detergents at CMCs: 6 mM for cholate (CA), glycocholate (GC), TC and CHAPS, 1.5 mM for TDC and TCDC and 0.12% for Triton X-100 in the presence of 100 pmol of [³H]PAF. Results shown are mean±S.E.M. for three separate experiments.

Figure 8. Effect of metal ion on PAF hydrolytic activity of alk-SMase

The PAF hydrolytic activities of purified rat alk-SMase were assayed in 50 mM Tris/HCl (pH 7.5), with 100 pmol of [³H]PAF, 0.15 M NaCl, 6 mM TC and 2 mM EDTA in the presence of 0–2 mM Zn²⁺ (top left panel) 0–4 mM Ca²⁺ (top right panel), or 0–4 mM Mg²⁺ (bottom left panel). Bottom right panel: COS-7 cells were transfected with wild-type human alk-SMase cDNA (WT), or with D199A or D246A mutant cDNA. After expression for 48 h, the cells were lysed and the PAF hydrolytic activity in cell-free extraction was assayed in P buffer and the SM hydrolytic activity was assayed in K buffer. Western blotting for alk-SMase is shown under the panel. All the results shown are means±S.E.M. for three separate experiments. *P<0.05, **P<0.01 and ***P<0.001 compared with the one indicated.

Figure 9. Effect of intestinal alk-SMase on PAF-induced chemotaxis, MAPK activation and IL-8 release

PAF was preincubated with or without rat intestinal alk-SMase for 2 h, then extracted and dissolved in PBS with 0.25% BSA. In the top panel, HL-60 cells were stimulated with 1 nM PAF treated with or without alk-SMase. The chemotaxis of the cells was determined. In the middle panel, 100 nM PAF treated with or without alk-SMase was incubated with HT-29 cells for 5 min, and the phosphorylated MAPK was determined by Western-blot analysis. The membrane was stripped and reblotted with anti-actin antibody as a loading control. In the lower panel, PAF treated with or without alk-SMase was incubated with HT-29 cells in a 96-well plate at the concentration of 100 nM for 16 h. The release of IL-8 was assayed by an IL-8 ELISA kit. Results shown are means±S.E.M. for three separate experiments. *P<0.05 compared with control; #P<0.05 compared with PAF.