In this study, we demonstrate the presence of a transacetylase activity in human plasma low-density lipoprotein (LDL) that transfers short-chain fatty acids from platelet-activating factor (PAF) and its close ether- and ester-linked analogues to ether/ester-linked lysophospholipids (lyso-PL). We show evidence that both PAF acetylhydrolase (PAF-AH) and transacetylase activities are inhibited to the same extent by serine esterase inhibitors, are resistant to heat treatment, and exhibit identical distributions in lipoprotein classes and in LDL subfractions. Additionally, the competitive inhibition of PAF-AH by lyso-PL, and the evidence that the recombinant PAF-AH also showed a similar transacetylase activity, suggest that PAF-AH is responsible for both activities. Using PAF as a donor molecule and lyso-PAF (1-O-alkyl-sn-glycero-3-phosphocholine) as an acceptor, the transacetylase activity showed typical allosteric kinetics, due to the positive co-operativity of the substrates, with apparent Vmax = 19.6±3.4nmol/min per mg of protein, apparent h = 2.0±0.3 and apparent [S]0.5 = 9.4±2.3μM at saturation for the concentration of lyso-PAF. The substrate specificity of the donor molecules was decreased by increasing the chain length of the acyl moiety in the sn-2 position of the glycerol. The ether linkage in the sn-1 position of the substrate was 30% more effective than the ester bond; cholesteryl acetate was inactive as an acetyl donor. The two acceptors tested, lyso-PAF and the ester-linked lyso-PC (1-acyl-sn-glycero-3-phosphocholine), showed similar specificity. Addition of exogenous lyso-PAF induced the transient formation of PAF-like aggregating activity predominately in small dense LDL subfractions upon oxidation. We conclude that PAF-AH possesses both transacetylase and acetylhydrolase activities which remove PAF and its ether-linked analogues from LDL particles upon LDL oxidation. However, in atherogenic small dense LDL-5 particles, the transacetylase activity may acetylate extracellular lyso-PAF into biologically active PAF.
Abbreviations used: PAF, platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine); lyso-PAF, 1-O-alkyl-sn-glycero-3-phosphocholine; lyso-PC, 1-acyl-sn-glycero-3-phosphocholine; lyso-PL, lysophospholipids; C16:0 PAF, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine; C16:0 lyso-PAF, 1-O-hexadecyl-sn-glycero-3-phosphocholine; C16:0 lyso-PC, 1-hexadecanoyl-sn-glycero-3-phosphocholine; HDL, high-density lipoprotein; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; VHDL, very-high-density lipoprotein-1; VLDL, very-low-density lipoprotein; LCAT, lecithin–cholesterol acyltransferase; PAF-AH, PAF acetylhydrolase; DFP di-isopropylfluorophosphate; Pefabloc, 4-(2-aminoethyl)benzenesulphonyl fluoride.
Present address: INSERM U525 Génétique Epidémiologique et Moléculaire des Pathologies Cardiovasculaires, IFR 14 ‘Coeur Muscle et Vaisseaux’ and Faculté de Médecine Pitié-Salpêtrière/Universitè P.M. Curie Paris 6, 91 Bvd. de l'Hôpital, 75634 Paris Cedex 13, France.