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• W2023784332 abstract "Paraoxonase-1 (PON1), an high density lipoprotein (HDL)-associated organophosphate triesterase, suppresses atherosclerosis in an unknown way. Purified PON1 protects lipoprotein particles from oxidative modification and hydrolyzes pro-atherogenic oxidized phospholipids and the inflammatory mediator platelet-activating factor (PAF). We find human PON1 acted as a phospholipase A2 but not as a phospholipase C or D through cleavage of phosphodiester bonds as expected. PON1 requires divalent cations, but EDTA did not block the phospholipase A2 activity of PON1. In contrast, a serine esterase inhibitor abolished phospholipase activity even though PON1 has no active-site serine residues. PAF acetylhydrolase, an oxidized phospholipid phospholipase A2, is a serine esterase associated with specific HDL particles. Western blotting did not reveal detectable amounts of PAF acetylhydrolase in PON1 preparations, although very low amounts of PAF acetylhydrolase might still account for PON1 phospholipase A2 activity. We revised the standard PON1 purification by first depleting HDL of PAF acetylhydrolase to find PON1 purified in this way no longer hydrolyzed oxidized phospholipids or PAF. Serum from a donor with an inactivating mutation in the PAF acetylhydrolase gene did not hydrolyze oxidized phospholipids or PAF, yet displayed full paraoxonase activity. We conclude that PAF acetylhydrolase is the sole phospholipase A2 of HDL and that PON1 has no phospholipase activity toward PAF or pro-atherogenic oxidized phospholipids. Paraoxonase-1 (PON1), an high density lipoprotein (HDL)-associated organophosphate triesterase, suppresses atherosclerosis in an unknown way. Purified PON1 protects lipoprotein particles from oxidative modification and hydrolyzes pro-atherogenic oxidized phospholipids and the inflammatory mediator platelet-activating factor (PAF). We find human PON1 acted as a phospholipase A2 but not as a phospholipase C or D through cleavage of phosphodiester bonds as expected. PON1 requires divalent cations, but EDTA did not block the phospholipase A2 activity of PON1. In contrast, a serine esterase inhibitor abolished phospholipase activity even though PON1 has no active-site serine residues. PAF acetylhydrolase, an oxidized phospholipid phospholipase A2, is a serine esterase associated with specific HDL particles. Western blotting did not reveal detectable amounts of PAF acetylhydrolase in PON1 preparations, although very low amounts of PAF acetylhydrolase might still account for PON1 phospholipase A2 activity. We revised the standard PON1 purification by first depleting HDL of PAF acetylhydrolase to find PON1 purified in this way no longer hydrolyzed oxidized phospholipids or PAF. Serum from a donor with an inactivating mutation in the PAF acetylhydrolase gene did not hydrolyze oxidized phospholipids or PAF, yet displayed full paraoxonase activity. We conclude that PAF acetylhydrolase is the sole phospholipase A2 of HDL and that PON1 has no phospholipase activity toward PAF or pro-atherogenic oxidized phospholipids. paraoxonase-1 high density lipoprotein low density lipoprotein platelet-activating factor polymorphonuclear leukocyte Paraoxonase-1 (PON1)1catalyzes the hydrolysis of organophosphorous triesters and aryl carboxyl esters; it is of interest because of its hydrolysis of paraoxon (a metabolite of the insecticide parathion) and neurotoxins such as sarin and diisopropylfluorophosphate. Insects and birds, in contrast to mammals, lack this activity, rendering them sensitive to various organophosphates (1Mackness M.I. Mackness B. Durrington P.N. Connelly P.W. Hegele R.A. Curr. Opin. Lipidol. 1996; 7: 69-76Crossref PubMed Scopus (420) Google Scholar). Hydrolysis of these substrates by PON1 is Ca2+-dependent, and the enzyme is extraordinarily sensitive to EDTA (2Kuo C.L. La Du B.N. Drug Metab. Dispos. 1995; 23: 935-944PubMed Google Scholar); for instance EDTA used as an anti-coagulant destabilizes and irreversibly inactivates the enzyme (3Mackness M.I. Atherosclerosis. 1998; 136: 195-196Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). PON1 is of interest for a second reason documented in a broad range of literature (4Mackness M.I. Arrol S. Durrington P.N. FEBS Lett. 1991; 286: 152-154Crossref PubMed Scopus (829) Google Scholar, 5Watson A.D. Berliner J.A. Hama S.Y., La Du, B.N. Faull K.F. Fogelman A.M. Navab M. J. Clin. Invest. 1995; 96: 2882-2891Crossref PubMed Scopus (1033) Google Scholar, 6Aviram M. Rosenblat M. Bisgaier C.L. Newton R.S. Primo-Parmo S.L. La Du B.N. J. Clin. Invest. 1998; 101: 1581-1590Crossref PubMed Scopus (1025) Google Scholar, 7Shih D.M., Gu, L. Xia Y.R. Navab M., Li, W.F. Hama S. Castellani L.W. Furlong C.E. Costa L.G. Fogelman A.M. Lusis A.J. Nature. 1998; 394: 284-287Crossref PubMed Scopus (956) Google Scholar, 8Subbanagounder G. Leitinger N. Shih P.T. Faull K.F. Berliner J.A. Circ. Res. 1999; 85: 311-318Crossref PubMed Scopus (127) Google Scholar, 9Navab M. Hama S.Y. Anantharamaiah G.M. Hassan K. Hough G.P. Watson A.D. Reddy S.T. Sevanian A. Fonarow G.C. Fogelman A.M. J. Lipid Res. 2000; 41: 1495-1508Abstract Full Text Full Text PDF PubMed Google Scholar, 10Aviram M. Hardak E. Vaya J. Mahmood S. Milo S. Hoffman A. Billicke S. Draganov D. Rosenblat M. Circulation. 2000; 101: 2510-2517Crossref PubMed Scopus (415) Google Scholar, 11Durrington P.N. Mackness B. Mackness M.I. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 473-480Crossref PubMed Scopus (718) Google Scholar, 12Ahmed Z. Ravandi A. Maguire G.F. Emili A. Draganov D., La Du, B.N. Kuksis A. Connelly P.W. Biochem. Biophys. Res. Commun. 2002; 290: 391-396Crossref PubMed Scopus (72) Google Scholar, 13Ahmed Z. Ravandi A. Maguire G.F. Emili A. Draganov D., La Du, B.N. Kuksis A. Connelly P.W. J. Biol. Chem. 2001; 276: 24473-24481Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) that associates PON1 with a suppression of atherogenesis. For example, mice lacking PON1 are more susceptible to organophosphate toxicity and are also more susceptible to atherosclerosis when fed a high fat diet (7Shih D.M., Gu, L. Xia Y.R. Navab M., Li, W.F. Hama S. Castellani L.W. Furlong C.E. Costa L.G. Fogelman A.M. Lusis A.J. Nature. 1998; 394: 284-287Crossref PubMed Scopus (956) Google Scholar). Additionally PON1 has two common phenotypes that affect catalysis with certain substrates, and one phenotype correlates with a propensity toward vascular disease (14Jarvik G.P. Rozek L.S. Brophy V.H. Hatsukami T.S. Richter R.J. Schellenberg G.D. Furlong C.E. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2441-2447Crossref PubMed Scopus (287) Google Scholar). Additionally, there are polymorphisms that affect circulating PON1 levels (15Furlong C.E. Cole T.B. Jarvik G.P. Costa L.G. Pharmacogenomics. 2002; 3: 341-348Crossref PubMed Scopus (41) Google Scholar) that may alter the propensity to develop vascular disease.Despite the association of PON1 with less vascular disease, two essential components in defining a biologic role for PON1 remain mysterious. The biologic substrate of PON1 is unknown, because it did not evolve to detoxify organophosphorous pesticides and neurotoxins and, at least in part because of the uncertainty over its substrates, the way by which PON1 lessens atherogenesis is also unknown. The search for a mechanistic role for PON1 reveals that it acts as an antioxidant (11Durrington P.N. Mackness B. Mackness M.I. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 473-480Crossref PubMed Scopus (718) Google Scholar) and has peroxidase activity (6Aviram M. Rosenblat M. Bisgaier C.L. Newton R.S. Primo-Parmo S.L. La Du B.N. J. Clin. Invest. 1998; 101: 1581-1590Crossref PubMed Scopus (1025) Google Scholar). However, the antioxidant activity of PON1 is independent of its esterase activity (16Cao H. Girard-Globa A. Berthezene F. Moulin P. J. Lipid Res. 1999; 40: 133-139Abstract Full Text Full Text PDF PubMed Google Scholar), is at variance with its aryl esterase activity in isoforms arising from genetic polymorphisms (11Durrington P.N. Mackness B. Mackness M.I. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 473-480Crossref PubMed Scopus (718) Google Scholar, 16Cao H. Girard-Globa A. Berthezene F. Moulin P. J. Lipid Res. 1999; 40: 133-139Abstract Full Text Full Text PDF PubMed Google Scholar), and is unaffected by either heat or EDTA treatment that destroy aryl esterase activity (16Cao H. Girard-Globa A. Berthezene F. Moulin P. J. Lipid Res. 1999; 40: 133-139Abstract Full Text Full Text PDF PubMed Google Scholar). PON1 has a single unpaired cysteine residue that mutagenesis shows has no role in catalysis or stability (17Sorenson R.C. Primo-Parmo S.L. Kuo C.L. Adkins S. Lockridge O. La Du B.N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7187-7191Crossref PubMed Scopus (123) Google Scholar), but this residue is absolutely essential for the antioxidant effect of PON1 (10Aviram M. Hardak E. Vaya J. Mahmood S. Milo S. Hoffman A. Billicke S. Draganov D. Rosenblat M. Circulation. 2000; 101: 2510-2517Crossref PubMed Scopus (415) Google Scholar, 18Aviram M. Billecke S. Sorenson R. Bisgaier C. Newton R. Rosenblat M. Erogul J. Hsu C. Dunlop C. La Du B. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1617-1624Crossref PubMed Scopus (407) Google Scholar). The possibility that PON1 has two active sites that separately catalyze its esterase and antioxidant activities has been raised (11Durrington P.N. Mackness B. Mackness M.I. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 473-480Crossref PubMed Scopus (718) Google Scholar).Oxidized lipoprotein particles contain pro-inflammatory lipid mediators that include ones that functionally mimic the potent phospholipid mediator platelet-activating factor (PAF) (19McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Biol. Chem. 1999; 274: 25189-25192Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). PAF initiates physiologic inflammation and is active at picomolar levels. PAF is recognized by a single molecularly characterized receptor found on a number of cells that comprise the innate immune system (20Izumi T. Shimizu T. Biochim. Biophys. Acta. 1995; 1259: 317-333Crossref PubMed Scopus (208) Google Scholar). High affinity recognition of PAF by the PAF receptor depends on ansn-1 ether bond, the short acetyl sn-2 residue, and the choline head group of this phospholipid mediator. Oxidation ofsn-2 polyunsaturated fatty acyl residues of ether phosphatidylcholines, as occurs during oxidation of lipoprotein particles, generates phospholipid products with shortenedsn-2 residues that are inflammatory agents (21Silva A.R. de Assis E.F. Caiado L.F. Marathe G.K. Bozza M.T. McIntyre T.M. Zimmerman G.A. Prescott S.M. Bozza P.T. Castro-Faria-Neto H.C. J. Immunol. 2002; 168: 4112-4120Crossref PubMed Scopus (71) Google Scholar) because they are PAF receptor agonists (22Heery J.M. Kozak M. Stafforini D.M. Jones D.A. Zimmerman G.A. McIntyre T.M. Prescott S.M. J. Clin. Invest. 1995; 96: 2322-2330Crossref PubMed Scopus (275) Google Scholar, 23Marathe G.K. Davies S.S. Harrison K.A. Silva A.R. Murphy R.C. Castro-Faria Neto H. Prescott S.M. Zimmerman G.A. McIntyre T.M. J. Biol. Chem. 1999; 274: 28395-28405Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). The oxidative formation of PAF-like lipids results from an unregulated chemical reaction, in contrast to the tight regulation of PAF synthesis (24Prescott S.M. Zimmerman G.A. Stafforini D.M. McIntyre T.M. Ann. Rev. Biochem. 2000; 69: 419-445Crossref PubMed Scopus (579) Google Scholar), and this process occurs in atherosclerotic lesions (25Watson A.D. Leitinger N. Navab M. Faull K.F. Horkko S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar) and in the circulation after exposure to cigarette smoke (26Imaizumi T. Satoh K. Yoshida H. Kawamura H. Hiramoto M. Takamatsu S. Atherosclerosis. 1991; 87: 47-55Abstract Full Text PDF PubMed Scopus (65) Google Scholar, 27Lehr H.A. Weyrich A.S. Saetzler R.K. Jurek A. Arfors K.E. Zimmerman G.A. Prescott S.M. McIntyre T.M. J. Clin. Invest. 1997; 99: 2358-2364Crossref PubMed Scopus (150) Google Scholar).Oxidatively fragmented phospholipids, like PAF, are substrates for PAF acetylhydrolase (28Stremler K.E. Stafforini D.M. Prescott S.M. Zimmerman G.A. McIntyre T.M. J. Biol. Chem. 1989; 264: 5331-5334Abstract Full Text PDF PubMed Google Scholar, 29Stremler K.E. Stafforini D.M. Prescott S.M. McIntyre T.M. J. Biol. Chem. 1991; 266: 11095-11103Abstract Full Text PDF PubMed Google Scholar), a phospholipase A2 that only hydrolyzes phospholipids with sn-2 residues that are short or contain an oxy function introduced during oxidative fragmentation. This enzyme is a serine esterase with the signature catalytic triad (30Tjoelker L.W. Eberhardt C. Unger J. Trong H.L. Zimmerman G.A. McIntyre T.M. Stafforini D.M. Prescott S.M. Gray P.W. J. Biol. Chem. 1995; 270: 25481-25487Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar), and so is sensitive to serine esterase inhibitors but not divalent ion chelators. PAF acetylhydrolase circulates in association with LDL and HDL particles and can migrate between them in a pH-dependent fashion (31Stafforini D.M. McIntyre T.M. Carter M.E. Prescott S.M. J. Biol. Chem. 1987; 262: 4215-4222Abstract Full Text PDF PubMed Google Scholar).PON1 is reported to hydrolyze PAF (32Rodrigo L. Mackness B. Durrington P.N. Hernandez A. Mackness M.I. Biochem. J. 2001; 354: 1-7Crossref PubMed Scopus (133) Google Scholar) and oxidatively fragmented phospholipids (5Watson A.D. Berliner J.A. Hama S.Y., La Du, B.N. Faull K.F. Fogelman A.M. Navab M. J. Clin. Invest. 1995; 96: 2882-2891Crossref PubMed Scopus (1033) Google Scholar, 9Navab M. Hama S.Y. Anantharamaiah G.M. Hassan K. Hough G.P. Watson A.D. Reddy S.T. Sevanian A. Fonarow G.C. Fogelman A.M. J. Lipid Res. 2000; 41: 1495-1508Abstract Full Text Full Text PDF PubMed Google Scholar, 12Ahmed Z. Ravandi A. Maguire G.F. Emili A. Draganov D., La Du, B.N. Kuksis A. Connelly P.W. Biochem. Biophys. Res. Commun. 2002; 290: 391-396Crossref PubMed Scopus (72) Google Scholar, 13Ahmed Z. Ravandi A. Maguire G.F. Emili A. Draganov D., La Du, B.N. Kuksis A. Connelly P.W. J. Biol. Chem. 2001; 276: 24473-24481Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), suggesting a potential mechanism that could account for the vascular protective effect of PON1. The existence of two distinct enzymes in the same lipoprotein particle apparently catalyzing the same reaction has yet to be tested directly. Here, we show that trace amounts of PAF acetylhydrolase contaminate PON1 preparations and that PON1 lacking PAF acetylhydrolase displays no phospholipase activity.MATERIALS AND METHODSCibacron blue 3GA-agarose (type 3000-CL), DEAE-Sepharose 6B, concanavalin A-Sepharose 4B, deoxycholate, methyl-α-d-mannopyranoside, IGEPAL CA-630, potassium bromide, phenyl acetate, phospholipase A2 (bee venom), phospholipase C (Bacillus cereus), and phospholipase D (cabbage) were from Sigma, and Extracti-gel was from Pierce. Octadecylsilica cartridges, aminopropyl columns, and high pressure liquid chromatography grade solvents were from JT Baker Inc. (Phillipsburg, NJ), and Pefabloc was from Pentapharm AG (Basel, Switzerland). Human serum albumin was the product of Baxter Healthcare Corp. (Glendale, CA), whereas Hank's balanced salt solution was from BioWhittaker (Walkersville, MD). PAF, carbamyl-PAF, 2-O-methyl PAF, and the PAF receptor antagonist BN52021 were from BioMol Research Laboratories, Inc. (Plymouth Meeting, PA). Fura-2/AM was from Molecular Probes (Eugene, OR). ECL kits were fromAmersham Biosciences, and polyclonal anti-PAF acetylhydrolase antiserum and cognate blocking peptide were from Cayman Chemical Co. (Ann Arbor, MI). Recombinant human PAF acetylhydrolase was from ICOS Corp. (Bothell, WA). [3H]PAF (10 Ci/mmol) was the product of American Radiolabeled Chemicals, Inc. (St. Louis, MO). PAF acetylhydrolase-deficient plasma (0.078 units of PAF acetylhydrolase/mlversus 1.5 units/ml from a healthy control) was a kind gift from Drs. Kei Satoh and Tada-atsu Imaizumi (Hirosaki University, Hirosaki, Japan). We prepared oxidized phospholipids from low density lipoprotein isolated from normolipidic subjects as described (22Heery J.M. Kozak M. Stafforini D.M. Jones D.A. Zimmerman G.A. McIntyre T.M. Prescott S.M. J. Clin. Invest. 1995; 96: 2322-2330Crossref PubMed Scopus (275) Google Scholar,23Marathe G.K. Davies S.S. Harrison K.A. Silva A.R. Murphy R.C. Castro-Faria Neto H. Prescott S.M. Zimmerman G.A. McIntyre T.M. J. Biol. Chem. 1999; 274: 28395-28405Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar).PON1 was purified from healthy human volunteers essentially as described by Gan et al. (33Gan K.N. Smolen A. Eckerson H.W. La Du B.N. Drug Metab. Dispos. 1991; 19: 100-106PubMed Google Scholar) with minor modifications. Human blood was collected in heparin, and plasma was generated from this and then calcified with CaCl2 to a final concentration of 1 mm before the resulting fibrin clot was separated by centrifugation. In few experiments the plasma pH was adjusted to either to pH 6.0 or to pH 9.0 with 1 n HCl or NaOH prior to an overnight incubation. These plasma samples (9 ml) were adjusted to a density of 1.3 with potassium bromide and then layered with 27 ml of saline in a centrifuge tube prior to ultracentrifugation for 3 h at 150,000 × g to separate HDL and LDL. HDL particles enriched with PON1 were mixed with Cibacron blue 3GA-agarose in 50 mm Tris-HCl buffer (pH 8.0), and loosely bound proteins were removed with 4 m NaCl buffer. PON1 bound to the Cibacron blue was then eluted with 0.1% deoxycholate and mixed with either DEAE-biogel or DEAE-Sepharose 6B in 0.1% IGEPAL CA-630 (rather than Nonidet P-40) before PON1 with was eluted with a linear gradient of NaCl (0.0–0.5 m) in 0.1% IGEPAL CA-630. DEAE fractions containing aryl esterase activity were diluted and rechromatographed on a DEAE-Sepharose 6B column. The final purification step used a concanavalin A-Sepharose column, and material from this step was concentrated with a Centricon apparatus. We resolved the proteins in the various fractions by 12% SDS-PAGE. PON1 was made detergent-free by passing it over Extracti-gel when the products of its reaction(s) were to be analyzed in a bioassay.Aryl esterase activity of PON1 was determined spectrophotometrically using phenyl acetate as the substrate (2Kuo C.L. La Du B.N. Drug Metab. Dispos. 1995; 23: 935-944PubMed Google Scholar). The assay contained 1 mm phenyl acetate, 20 mm Tris-Cl (pH 8.0), and 1 mm CaCl2. Blanks without enzyme were used to correct for spontaneous hydrolysis. Activity was calculated from the molar extinction coefficient at 270 nm (using differences in the absorbance of phenol versus phenyl acetate) of 1310m−1 cm−1. One unit of aryl esterase activity was defined as a micromole of phenyl acetate hydrolyzed per min.We measured PAF acetylhydrolase activity using [3H]acetate-PAF as described by Stafforini et al. (31Stafforini D.M. McIntyre T.M. Carter M.E. Prescott S.M. J. Biol. Chem. 1987; 262: 4215-4222Abstract Full Text PDF PubMed Google Scholar, 34Stafforini D.M. Prescott S.M. McIntyre T.M. J. Biol. Chem. 1987; 262: 4223-4230Abstract Full Text PDF PubMed Google Scholar) and also by bioassay. For this, human neutrophils were isolated by dextran sedimentation and centrifugation over Ficoll as before (35Zimmerman G.A. McIntyre T.M. Prescott S.M. J. Clin. Investig. 1985; 76: 2235-2246Crossref PubMed Scopus (274) Google Scholar). Neutrophils (2.25 × 106/ml) were labeled with Fura-2/AM as before (23Marathe G.K. Davies S.S. Harrison K.A. Silva A.R. Murphy R.C. Castro-Faria Neto H. Prescott S.M. Zimmerman G.A. McIntyre T.M. J. Biol. Chem. 1999; 274: 28395-28405Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), and changes in intracellular calcium concentration were measured by dual excitation at 340 and 380 nm with emission collected at 510 nm. The amount of PAF stated in the figures (typically at 10−10m) was pretreated with detergent-free PON1 or recombinant PAF acetylhydrolase for the specified time at 37 °C before the entire reaction was tested in the Ca2+ mobilization assay. We find as little as 5 to 10 ng of PAF acetylhydrolase in 15 min abolishes the calcium signal generated by 0.1 nm PAF. In some experiments, purified PON1 was pretreated for 1 h at 37 °C with either EDTA (100 μm) or the serine esterase inhibitor Pefabloc (100 μm) prior to assay. In some experiments PAF or 2-O-methyl PAF were treated with 10 μg of bee venom phospholipase A2 (in 0.5% human serum albumin in Hank's balanced salt solution with 10 mm Ca2+), 10 units of phospholipase C (B. cereus), or 10 units of phospholipase D (cabbage) for 2 h at 37 °C and then tested in the Ca2+-flux bioassay. The analysis of Ca2+flux in PMN was as described (23Marathe G.K. Davies S.S. Harrison K.A. Silva A.R. Murphy R.C. Castro-Faria Neto H. Prescott S.M. Zimmerman G.A. McIntyre T.M. J. Biol. Chem. 1999; 274: 28395-28405Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 36Marathe G.K. Silva A.R. de Castro Faria Neto H.C. Tjoelker L.W. Prescott S.M. Zimmerman G.A. McIntyre T.M. J. Lipid Res. 2001; 42: 1430-1437Abstract Full Text Full Text PDF PubMed Google Scholar).We probed for PAF acetylhydrolase by immunoblotting after electrophoretic separation by SDS-PAGE. The resolved proteins were transferred to an Immobilon-P membrane (Millipore Corp., Bedford, MA) and probed with PAF acetylhydrolase polyclonal antibody (diluted 1:1000). A standard of truncated, non-glycosylated (the form approved for clinical trials) recombinant PAF acetylhydrolase was examined in a separate lane of the gel. Peroxidase-conjugated goat anti-rabbit IgG antibody was the secondary antibody (diluted 1:10000) with enhanced chemiluminescence reagent used for visualization. In some experiments, the primary anti-PAF acetylhydrolase antibody was pre-incubated with 200 μg per blot of the cognate blocking peptide.DISCUSSIONWe discovered that highly purified preparations of human PON1 inactivate PAF and PAF-like oxidatively fragmented phospholipids by hydrolyzing the sn-2 residue to produce two inactive products, lysoPAF and a short chain free fatty acid. This reaction suppresses atherogenesis, and it protects lipoprotein particles from chemically reactive phospholipids. Genetic deletion of PON1 activity in mice creates animals that are susceptible to organophosphate intoxication and, importantly, demonstrate an increased propensity to form atherosclerotic lesions when placed on a high fat diet or an apoE knock-out background (7Shih D.M., Gu, L. Xia Y.R. Navab M., Li, W.F. Hama S. Castellani L.W. Furlong C.E. Costa L.G. Fogelman A.M. Lusis A.J. Nature. 1998; 394: 284-287Crossref PubMed Scopus (956) Google Scholar, 40Shih D.M., Gu, L. Hama S. Xia Y.R. Navab M. Fogelman A.M. Lusis A.J. J. Clin. Invest. 1996; 97: 1630-1639Crossref PubMed Scopus (234) Google Scholar, 41Shih D.M. Xia Y.R. Wang X.P. Miller E. Castellani L.W. Subbanagounder G. Cheroutre H. Faull K.F. Berliner J.A. Witztum J.L. Lusis A.J. J. Biol. Chem. 2000; 275: 17527-17535Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). Conversely, transgenic mice with increased levels of circulating PON1 are less susceptible to developing atherosclerosis (42Oda M.N. Bielicki J.K., Ho, T.T. Berger T. Rubin E.M. Forte T.M. Biochem. Biophys. Res. Commun. 2002; 290: 921-927Crossref PubMed Scopus (95) Google Scholar, 43Tward A. Xia Y.R. Wang X.P. Shi Y.S. Park C. Castellani L.W. Lusis A.J. Shih D.M. Circulation. 2002; 106: 484-490Crossref PubMed Scopus (381) Google Scholar), as are mice expressing PAF acetylhydrolase as a transgene (44Quarck R., De Geest B. Stengel D. Mertens A. Lox M. Theilmeier G. Michiels C. Raes M. Bult H. Collen D. Van Veldhoven P. Ninio E. Holvoet P. Circulation. 2001; 103: 2495-2500Crossref PubMed Scopus (200) Google Scholar). These observations, coupled with extensive literature (4Mackness M.I. Arrol S. Durrington P.N. FEBS Lett. 1991; 286: 152-154Crossref PubMed Scopus (829) Google Scholar, 5Watson A.D. Berliner J.A. Hama S.Y., La Du, B.N. Faull K.F. Fogelman A.M. Navab M. J. Clin. Invest. 1995; 96: 2882-2891Crossref PubMed Scopus (1033) Google Scholar, 6Aviram M. Rosenblat M. Bisgaier C.L. Newton R.S. Primo-Parmo S.L. La Du B.N. J. Clin. Invest. 1998; 101: 1581-1590Crossref PubMed Scopus (1025) Google Scholar, 7Shih D.M., Gu, L. Xia Y.R. Navab M., Li, W.F. Hama S. Castellani L.W. Furlong C.E. Costa L.G. Fogelman A.M. Lusis A.J. Nature. 1998; 394: 284-287Crossref PubMed Scopus (956) Google Scholar, 8Subbanagounder G. Leitinger N. Shih P.T. Faull K.F. Berliner J.A. Circ. Res. 1999; 85: 311-318Crossref PubMed Scopus (127) Google Scholar, 9Navab M. Hama S.Y. Anantharamaiah G.M. Hassan K. Hough G.P. Watson A.D. Reddy S.T. Sevanian A. Fonarow G.C. Fogelman A.M. J. Lipid Res. 2000; 41: 1495-1508Abstract Full Text Full Text PDF PubMed Google Scholar, 10Aviram M. Hardak E. Vaya J. Mahmood S. Milo S. Hoffman A. Billicke S. Draganov D. Rosenblat M. Circulation. 2000; 101: 2510-2517Crossref PubMed Scopus (415) Google Scholar, 11Durrington P.N. Mackness B. Mackness M.I. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 473-480Crossref PubMed Scopus (718) Google Scholar, 12Ahmed Z. Ravandi A. Maguire G.F. Emili A. Draganov D., La Du, B.N. Kuksis A. Connelly P.W. Biochem. Biophys. Res. Commun. 2002; 290: 391-396Crossref PubMed Scopus (72) Google Scholar, 13Ahmed Z. Ravandi A. Maguire G.F. Emili A. Draganov D., La Du, B.N. Kuksis A. Connelly P.W. J. Biol. Chem. 2001; 276: 24473-24481Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) indicating that paraoxonase is beneficial and hydrolyzes PAF-like phospholipid oxidation products that are formed during LDL oxidation (19McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Biol. Chem. 1999; 274: 25189-25192Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 23Marathe G.K. Davies S.S. Harrison K.A. Silva A.R. Murphy R.C. Castro-Faria Neto H. Prescott S.M. Zimmerman G.A. McIntyre T.M. J. Biol. Chem. 1999; 274: 28395-28405Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) to inactive products, suggest that this is the way PON1 exerts its salubrious effects. Hydrolysis of oxidatively modified and fragmented phospholipids that are PAF receptor agonists (19McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Biol. Chem. 1999; 274: 25189-25192Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 23Marathe G.K. Davies S.S. Harrison K.A. Silva A.R. Murphy R.C. Castro-Faria Neto H. Prescott S.M. Zimmerman G.A. McIntyre T.M. J. Biol. Chem. 1999; 274: 28395-28405Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar,45Smiley P.L. Stremler K.E. Prescott S.M. Zimmerman G.A. McIntyre T.M. J. Biol. Chem. 1991; 266: 11104-11110Abstract Full Text PDF PubMed Google Scholar), peroxisome proliferator-activated receptor ligands and agonists (46Davies S.S. Pontsler A.V. Marathe G.K. Harrison K.A. Murphy R.C. Hinshaw J.C. Prestwich G.D., St Hilaire A. Prescott S.M. Zimmerman G.A. McIntyre T.M. J. Biol. Chem. 2001; 276: 16015-16023Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar), and structurally inappropriate and disruptive phospholipids (47Dousset N. Dousset J.C. Douste-Blazy L. Biochim. Biophys. Acta. 1981; 641: 1-10Crossref PubMed Scopus (10) Google Scholar, 48Itabe H. Kobayashi T. Inoue K. Biochim. Biophys. Acta. 1988; 961: 13-21Crossref PubMed Scopus (37) Google Scholar, 49Itabe H. Kushi Y. Handa S. Inoue K. Biochim. Biophys. Acta. 1988; 962: 8-15Crossref PubMed Scopus (45) Google Scholar, 50Tokumura A. Sumida T. Toujima M. Kogure K. Fukuzawa K. Takahashi Y. Yamamoto S. J. Lipid Res. 2000; 41: 953-962Abstract Full Text Full Text PDF PubMed Google Scholar) are of unquestioned value in protecting against atherosclerosis, but this reaction is not accomplished by PON1.PON1 is an organophosphatase (51Costa L.G., Li, W.F. Richter R.J. Shih D.M. Lusis A. Furlong C.E. Chem. Biol. Interact. 1999; 119–120: 429-438Crossref PubMed Scopus (156) Google Scholar) that protects animals that express it against intoxication by organophosphate insecticides (7Shih D.M., Gu, L. Xia Y.R. Navab M., Li, W.F. Hama S. Castellani L.W. Furlong C.E. Costa L.G. Fogelman A.M. Lusis A.J. Nature. 1998; 394: 284-287Crossref PubMed Scopus (956) Google Scholar). If PON1 attacks the phosphodiester of biologically active phosphatidylcholines then it would inactivate 1-O-hexadecyl-2-O-methyl-sn-glycero-3-phosphocholine, a PAF analog that is not a substrate for the esterolytic activity of phospholipases A1 or A2. It did not. Instead we find that purified human PON1 inactivated PAF, as reported (32Rodrigo L. Mackness B. Durrington P.N. Hernandez A. Mackness M.I. Biochem. J. 2001; 354: 1-7Crossref PubMed Scopus (133) Google Scholar), where the single bond susceptible to esterolytic activity resides at thesn-2 position. This is the same type of activity displayed by the PAF acetylhydrolase that is also a component of certain HDL particles (31Stafforini D.M. McIntyre T.M. Carter M.E. Prescott S.M. J. Biol. Chem. 1987; 262: 4215-4222Abstract Full Text PDF PubMed Google Scholar).Physical resolution of PON1 and PAF acetylhydrolase has been found previously to be challenging (2Kuo C.L. La Du B.N. Drug Metab. Dispos. 1995; 23: 935-944PubMed Google Scholar, 6Aviram M. Rosenblat M. Bisgaier C.L. Newton R.S. Primo-Parmo S.L. La Du B.N. J. Clin. Invest. 1998; 101: 1581-1590Crossref PubMed Scopus (1025) Google Scholar, 32Rodrigo L. Mackness B. Durrington P.N. Hernandez A. Mackness M.I. Biochem. J. 2001; 354: 1-7Crossref PubMed Scopus (133) Google Scholar). We, like other investigators (32Rodrigo L. Mackness B. Durrington P.N. Hernandez A" @default.
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• W2023784332 cites W1513212892 @default.
• W2023784332 cites W1547302919 @default.
• W2023784332 cites W1585449429 @default.
• W2023784332 cites W1589395925 @default.
• W2023784332 cites W1589823408 @default.
• W2023784332 cites W1648812418 @default.
• W2023784332 cites W1671050155 @default.
• W2023784332 cites W1977363772 @default.
• W2023784332 cites W1983151078 @default.
• W2023784332 cites W1984396607 @default.
• W2023784332 cites W1990051093 @default.
• W2023784332 cites W1990949779 @default.
• W2023784332 cites W1992761305 @default.
• W2023784332 cites W2006679177 @default.
• W2023784332 cites W2008302027 @default.
• W2023784332 cites W2008378566 @default.
• W2023784332 cites W2008895292 @default.
• W2023784332 cites W2012639879 @default.
• W2023784332 cites W2025679035 @default.
• W2023784332 cites W2028461731 @default.
• W2023784332 cites W2030069587 @default.
• W2023784332 cites W2035182697 @default.
• W2023784332 cites W2049007654 @default.
• W2023784332 cites W2050082784 @default.
• W2023784332 cites W2050601245 @default.
• W2023784332 cites W2061663562 @default.
• W2023784332 cites W2061978337 @default.
• W2023784332 cites W2063559998 @default.
• W2023784332 cites W2071816313 @default.
• W2023784332 cites W2074800421 @default.
• W2023784332 cites W2077160008 @default.
• W2023784332 cites W2078503590 @default.
• W2023784332 cites W2078910490 @default.
• W2023784332 cites W2079270586 @default.
• W2023784332 cites W2084075677 @default.
• W2023784332 cites W2106809354 @default.
• W2023784332 cites W2111891584 @default.
• W2023784332 cites W2117394230 @default.
• W2023784332 cites W2119889870 @default.
• W2023784332 cites W2122626660 @default.
• W2023784332 cites W2122751858 @default.
• W2023784332 cites W2136995454 @default.
• W2023784332 cites W2137511741 @default.
• W2023784332 cites W2146892797 @default.
• W2023784332 cites W2150545098 @default.
• W2023784332 cites W2154168373 @default.
• W2023784332 cites W2155556672 @default.
• W2023784332 cites W2168115093 @default.
• W2023784332 cites W2189174822 @default.
• W2023784332 cites W4240818984 @default.
• W2023784332 cites W4251722549 @default.
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