Platelet-Activating Factor-Acetylhydrolase Can Monodeacylate and Inactivate Lipoteichoic Acid

Abstract

Bacterial lipoteichoic acid (LTA) shares a structural motif with platelet-activating factor (PAF). Both molecules are strong inflammatory agents and have a glycerol backbone with two lipid chains at the sn-1 and sn-2 positions. PAF is normally inactivated by PAF-acetylhydrolase (PAF-AH), a phospholipase A2 (PLA2), which removes a short acyl group at the sn-2 position. To investigate whether PAF-AH can similarly degrade LTA, we studied the effects of porcine PLA2, bee venom PLA2, and recombinant human PAF-AH on pneumococcal LTA (PnLTA) and staphylococcal LTA (StLTA). After incubation with a porcine or bee venom PLA2, a large fraction of PnLTA lost 264 Da, which corresponds to the mass of the oleic acid group at the sn-2 position. After incubation with recombinant human PAF-AH, PnLTA lost 264 Da; the reduction did not occur when PAF-AH was exposed to Pefabloc SC, an irreversible inhibitor of the PAF-AH active site. Following PAF-AH treatment, PnLTA and StLTA were not able to stimulate mouse RAW 264.7 cells to produce tumor necrosis factor alpha but could stimulate CHO cells expressing human TLR2. This stimulation pattern has been observed with monoacyl PnLTA prepared by mild alkali hydrolysis (22). Taking these data together, we conclude that PAF-AH can remove one acyl chain at the sn-2 position of LTA and produce a monoacyl-LTA that is inactive against mouse cells.

Lipoprotein Lipase and Hydrofluoric Acid Deactivate Both Bacterial Lipoproteins and Lipoteichoic Acids, but Platelet-Activating Factor-Acetylhydrolase Degrades Only Lipoteichoic Acids

“…4A). Thus, PAF-AH specifically inactivates LTA, but not LPS or lipoproteins, as described previously (48,49). These findings also support the contention that our LTA preparations are clean and not contaminated with LPS or lipoproteins.…”

“…This approach uses methods to selectively inactivate either LTA or lipoproteins in bacterial culture supernatants or crude bacterial cell wall extracts (22)(23)(24)49). LTA inactivation is usually performed with hydrofluoric acid (HF) or platelet-activating factor-acetylhydrolase (PAF-AH) (23,48,49), which, respectively, hydrolyzes the phosphodiester bonds in the LTA or deacylates one of its acyl chains (17,28,36,55). Lipoprotein inactivation is commonly achieved by deacylation with a lipoprotein lipase (LPL) or by oxidation with hydrogen peroxide (H 2 O 2 ) (22,24,62).…”

“…The H 2 O 2 reaction was stopped by adding 1,000 U of catalase (at 37°C for 1 h), and the mixture was lyophilized. The PAF-AH in the reaction mixture was inactivated by adding 100 M Pefabloc SC, as reported previously (48).…”

Lipoteichoic Acid Is Important in Innate Immune Responses to Gram-Positive Bacteria

“…In the first approach, 9 volumes of bacterial supernatant was mixed with 1 volume of 2 N NaOH, incubated for 2 h at 37°C, and neutralized with 6 N HCl to obtain a pH of 6.5 to 7.5. In the second approach, the supernatants were incubated at 37°C for 2 h with various concentrations of PAF-AH and then mixed with Pefabloc SC (final concentration, 100 M), as we have described previously (44). In the third approach, LTA was removed from staphylococcal GPCS with an anti-LTA MAb (BD1701).…”

“…Selective inactivation was possible since we have shown that plateletactivating factor acetylhydrolase (PAF-AH), a phospholipase A 2 , selectively inactivates staphylococcal and pneumococcal LTAs by removing their acyl-2 chain (44) but does not affect other acylated bacterial molecules, such as LPS and phosphatidylcholine (44). We report here the effects of LTA inactivation on the inflammatory properties of gram-positive bacterial culture supernatants (GPCS).…”