Biosynthesis of platelet-activating factor in cultured mast cells. Involvement of the CoA-independent transacylase demonstrated by analysis of the molecular species of platelet-activating factor.
We have recently demonstrated that arachidonate [20:4(5,8,11,14)] was primarily linked to the hexadecyl (16:0) and octadecenyl (18:1) species of alkylacyl derivatives of glycerolphosphocholine (GroPCho). Consistent with the involvement of arachidonate-specific CoA-independent transacylase in the synthesis of platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-GroPCho), 16:0 and 18:1 PAF species were formed upon antigen stimulation [Joly, F., Breton, M., Wolf, C., Ninio, E. & Colard, O. (1992) Biochim. Biophys. Acta 1125, 305-312]. In the present work, addition of lyso-PAF to mast cells resulted in PAF production. We analyzed the PAF species formed in the presence of a defined lyso-PAF molecular species in order to differentiate between either direct acetylation or involvement of the membrane precursor. The 18:1 lyso-PAF was more effective than the 16:0 in producing PAF which was composed of 95% 18:1 PAF, the balance being 16:0, indicating that part of the acetylated lyso-PAF originated from the cellular pool of alkyl-arachidonyl-GroPCho in resting cells. Consistent with alkyl-arachidonyl-GroPCho species content and acetyltransferase specificity, similar amounts of 16:0 and 18:1 PAF species were formed when mast cells were stimulated with antigen. Supplemented with 16:0 or 18:1 lyso-PAF, antigen-stimulated mast cells responded by 230% and 125% increase in PAF synthesis, respectively. As expected, the amount of the PAF species corresponding to the added lyso-PAF was increased. More interestingly, addition of 16:0 lyso-PAF almost doubled the amount of 18:1 PAF content as compared to antigen alone, thus indicating that the lyso-PAF formed via the CoA-independent transacylase was significantly used for PAF synthesis, despite a large excess of exogenous lyso-PAF. The CoA-independent transacylase, measured using [3H]lyso-PAF as a substrate in sonicates from antigen-stimulated cells, was decreased concurrently with PAF formation. In conclusion, we show that when lyso-PAF is added to mast cells, a direct acetylation may occur. However, PAF is preferentially synthesized through a mechanism involving the CoA-independent transacylase reaction.
Colard
O
O
CNRS URA 1283, CHU Saint-Antoine, Paris, France.
Bidault
J
J
Breton
M
M
Ninio
E
E
D000217:Acyltransferases / Q000378:metabolism*; D000818:Animals; D000941:Antigens / Q000494:pharmacology; D002478:Cells, Cultured; D003065:Coenzyme A / Q000378:metabolism*; D008407:Mast Cells / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D010972:Platelet Activating Factor / Q000031:analogs & derivatives / Q000096:biosynthesis* / Q000494:pharmacology; D013379:Substrate Specificity
D000941:Antigens; C029271:O-deacetyl platelet activating factor; D010972:Platelet Activating Factor; D000217:Acyltransferases; C039766:arachidonyl transacylase; D003065:Coenzyme A