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      Paraoxonase Active Site Required for Protection Against LDL Oxidation Involves Its Free Sulfhydryl Group and Is Different From That Required for Its Arylesterase/Paraoxonase Activities : Selective Action of Human Paraoxonase Allozymes Q and R

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          Abstract

          Human serum paraoxonase (PON 1) exists in 2 major polymorphic forms (Q and R), which differ in the amino acid at position 191 (glutamine and arginine, respectively). These PON allozymes hydrolyze organophosphates and aromatic esters, and both also protect LDL from copper ion-induced oxidation. We have compared purified serum PONs of both forms and evaluated their effects on LDL oxidation, in respect to their arylesterase/paraoxonase activities. Copper ion-induced LDL oxidation, measured by the production of peroxides and aldehydes after 4 hours of incubation, were reduced up to 61% and 58%, respectively, by PON Q, but only up to 46% and 38%, respectively, by an equivalent concentration of PON R. These phenomena were PON-concentration dependent. Recombinant PON Q and PON R demonstrated similar patterns to that shown for the purified serum allozymes. PON Q and PON R differences in protection of LDL against oxidation were further evaluated in the presence of glutathione peroxidase (GPx). GPx (0.1 U/mL) alone reduced copper ion-induced LDL oxidation by 20% after 4 hours of incubation. The addition of PON R to the above system resulted in an additive inhibitory effect on LDL oxidation, whereas PON Q had no such additive effect. The 2 PON allozymes also differed by their ability to inhibit initiation, as well as propagation, of LDL oxidation. PON Q was more efficient in blocking LDL oxidation if added when oxidation was initiated, whereas PON R was more potent when added 1 hour after the initiation of LDL oxidation. These data suggest that the 2 allozymes act on different substrates. Both PON allozymes were also able to reduce the oxidation of phospholipids and cholesteryl ester. PON Q arylesterase activity was reduced after 4 hours of LDL oxidation by only 28%, whereas the arylesterase activity of PON R was reduced by up to 55%. Inactivation of the calcium-dependent PON arylesterase activity by using the metal chelator EDTA, or by calcium ion removal on a Chelex column, did not alter PON's ability to inhibit LDL oxidation. However, blockage of the PON free sulfhydryl group at position 283 with p-hydroxymercuribenzoate inhibited both its arylesterase activity and its protection of LDL from oxidation. Recombinant PON mutants in which the PON free sulfhydryl group was replaced by either alanine or serine were no longer able to protect against LDL oxidation, even though they retained paraoxonase and arylesterase activities. Overall, these studies demonstrate that PON's arylesterase/paraoxonase activities and the protection against LDL oxidation do not involve the active site on the enzyme in exactly the same way, and PON's ability to protect LDL from oxidation requires the cysteine residue at position 283.

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          Paraoxonase inhibits high-density lipoprotein oxidation and preserves its functions. A possible peroxidative role for paraoxonase.

          HDL levels are inversely related to the risk of developing atherosclerosis. In serum, paraoxonase (PON) is associated with HDL, and was shown to inhibit LDL oxidation. Whether PON also protects HDL from oxidation is unknown, and was determined in the present study. In humans, we found serum HDL PON activity and HDL susceptibility to oxidation to be inversely correlated (r2 = 0.77, n = 15). Supplementing human HDL with purified PON inhibited copper-induced HDL oxidation in a concentration-dependent manner. Adding PON to HDL prolonged the oxidation lag phase and reduced HDL peroxide and aldehyde formation by up to 95%. This inhibitory effect was most pronounced when PON was added before oxidation initiation. When purified PON was added to whole serum, essentially all of it became HDL-associated. The PON-enriched HDL was more resistant to copper ion-induced oxidation than was control HDL. Compared with control HDL, HDL from PON-treated serum showed a 66% prolongation in the lag phase of its oxidation, and up to a 40% reduction in peroxide and aldehyde content. In contrast, in the presence of various PON inhibitors, HDL oxidation induced by either copper ions or by a free radical generating system was markedly enhanced. As PON inhibited HDL oxidation, two major functions of HDL were assessed: macrophage cholesterol efflux, and LDL protection from oxidation. Compared with oxidized untreated HDL, oxidized PON-treated HDL caused a 45% increase in cellular cholesterol efflux from J-774 A.1 macrophages. Both HDL-associated PON and purified PON were potent inhibitors of LDL oxidation. Searching for a possible mechanism for PON-induced inhibition of HDL oxidation revealed PON (2 paraoxonase U/ml)-mediated hydrolysis of lipid peroxides (by 19%) and of cholesteryl linoleate hydroperoxides (by 90%) in oxidized HDL. HDL-associated PON, as well as purified PON, were also able to substantially hydrolyze (up to 25%) hydrogen peroxide (H2O2), a major reactive oxygen species produced under oxidative stress during atherogenesis. Finally, we analyzed serum PON activity in the atherosclerotic apolipoprotein E-deficient mice during aging and development of atherosclerotic lesions. With age, serum lipid peroxidation and lesion size increased, whereas serum PON activity decreased. We thus conclude that HDL-associated PON possesses peroxidase-like activity that can contribute to the protective effect of PON against lipoprotein oxidation. The presence of PON in HDL may thus be a major contributor to the antiatherogenicity of this lipoprotein.
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            Protection of low-density lipoprotein against oxidative modification by high-density lipoprotein associated paraoxonase.

            We have investigated the Cu2+ induced generation of lipid peroxides in low density lipoprotein (LDL) incubated with high density lipoprotein (HDL) and with purified paraoxonase, an enzyme normally resident on HDL. HDL (1.5 mg) and paraoxonase (20 micrograms) inhibited lipid peroxide generation in LDL by 32% and 25%, respectively after 24 h of incubation (both P < 0.01). The decrease in LDL lipid peroxides both with HDL and with paraoxonase were concentration dependent. The degree of protection offered by HDL tended to relate to its paraoxonase activity (R = 0.47; P < 0.06). Neither purified paraoxonase nor HDL chelated Cu2+ sufficiently to account for the decrease in LDL oxidation. Purified paraoxonase did not affect LDL oxidation when it had been heat inactivated. Mass transfer of lipid peroxides from LDL to HDL did not explain the protection of LDL against oxidation: the total lipid peroxides accumulating during incubation was decreased both by HDL and by paraoxonase. These results suggest a direct role for HDL in preventing atherosclerosis probably by an enzymic process which prevents the accumulation of lipid peroxides on LDL. Paraoxonase is an example of an enzyme which might possibly be involved.
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              Continuous Monitoring ofin VztroOxidation of Human Low Density Lipoprotein

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                Author and article information

                Journal
                Arteriosclerosis, Thrombosis, and Vascular Biology
                Arterioscler Thromb Vasc Biol
                Ovid Technologies (Wolters Kluwer Health)
                1079-5642
                1524-4636
                October 1998
                October 1998
                : 18
                : 10
                : 1617-1624
                Affiliations
                [1 ]From the Lipid Research Laboratory, Technion Faculty of Medicine, the Rappaport Family Institute for Research in the Medical Sciences and Rambam Medical Center, Haifa, Israel (M.A., M.R.); and the Department of Vascular and Cardiac Diseases, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, (C.B., R.N.), and the Departments of Pharmacology and Anesthesiology, The University of Michigan Medical School (S.B., R.S., J.E., C.H., C.D., B.L.D.), Ann Arbor, Mich.
                Article
                10.1161/01.ATV.18.10.1617
                9763535
                dcee4858-c26d-480c-af45-8bc7119764e5
                © 1998
                History

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