Oxidation of LDL may contribute to atherogenesis, though the nature of the in vivo oxidant(s) remains obscure. Myeloperoxidase, the enzyme responsible for hypochlorous acid/hypochlorite (HOCl) production in vivo, is present in active form in human atherosclerotic lesions, and HOCl aggregates and transforms LDL into a high-uptake form for macrophages in vitro. Here we demonstrate HOCl-modified proteins in human lesions using an mAb raised against HOCl-modified LDL that recognizes HOCl-oxidized proteins but does not cross-react with Cu
Oxidation of low-density lipoprotein (LDL) lipid is thought to represent the initial step in a series of oxidative modification reactions that ultimately transform this lipoprotein into an atherogenic high-uptake form that can cause lipid accumulation in cells. We have studied the effects of hypochlorite, a powerful oxidant released by activated monocytes and neutrophils, on isolated LDL. Exposure of LDL to reagent hypochlorite (NaOCl) at 4 degrees C resulted in immediate and preferential oxidation of amino acid residues of apoprotein B-100, the single protein associated with LDL. Neither lipoprotein lipid nor LDL-associated antioxidants, except ubiquinol-10, represented major targets for this oxidant. Even when high concentrations of NaOCl were used, only low levels of lipid hydroperoxides could be detected with the highly sensitive h.p.l.c. post-column chemiluminescence detection method. Lysine residues of apoprotein B-100 quantitatively represented the major target, scavenging some 68% of the NaOCl added, with tryptophan and cysteine together accounting for an additional 10% of the oxidant. Concomitant with the loss of LDL's amino groups, chloramines were formed and the anionic surface charge of the lipoprotein particle increased, indicated by a 3-4-fold increase in electrophoretic mobility above that of native LDL on agarose gels. While both these changes could be initially reversed by physiological reductants such as ascorbic acid and methionine, incubation of the NaOCl-modified LDL at 37 degrees C resulted in increasing resistance of the modified lysine residues against reductive reversal. Exposure of mouse peritoneal macrophages to NaOCl-oxidized LDL resulted in increased intracellular concentrations of cholesterol and cholesteryl esters. These findings suggest that lipid-soluble antioxidants associated with LDL do not efficiently protect the lipoprotein against oxidative damage mediated by hypochlorite, and that extensive lipid oxidation is not a necessary requirement for oxidative LDL modification that leads to a high-uptake form of the lipoprotein.
Peroxidation of low-density lipoprotein (LDL) lipid is generally thought to represent the initial step in a series of modification reactions that ultimately transform the protein moiety of the lipoprotein into a form recognized by receptors different from those that bind native LDL. Uptake of LDL via these alternative receptors can lead to the formation of lipid-laden cells, which are typical for the early stages of atherogenesis. We have studied the oxidative modification of LDL by hypochlorite (-OCl), a powerful oxidant produced from H2O2 and chloride via the action of myeloperoxidase which is released from activated neutrophils and monocytes. Exposure of LDL to reagent or enzymically generated -OCl at 4 or 37 degrees C resulted in immediate and preferential oxidation of amino acid residues of apolipoprotein B-100, the single protein associated with LDL. Lysine residues quantitatively represented the major target and, like tryptophan, were oxidized to approximately the same extent with reagent or enzymically generated -OCl. In contrast, LDL lipid oxidation was less favoured than protein oxidation, as judged by the amounts of lipid hydroperoxides, chlorohydrins, cholesterol or fatty acid oxidation products formed. Treatment with -OCl caused aggregation of LDL, as shown by an increased turbidity of the oxidized LDL solution and elution from a size-exclusion h.p.l.c. column of high-molecular-mass LDL complexes. Chemical modification of lysine residues before oxidation with -OCl prevented aggregation, while it enhanced the extent of lipid peroxidation. Treatment of LDL with -OCl also caused the formation of carbonyl groups and release of ammonia; both these modifications were inhibited by lysine-residue modification before oxidation. These results demonstrate that aggregation reactions are dependent on initial lysine oxidation by -OCl, followed by deamination and carbonyl formation, but do not involve lipid (per)oxidation. We propose that the observed -OCl-mediated aggregation of LDL is caused, at least in part, by cross-linking of apoproteins by Schiff-base formation independently of lipid peroxidation.
Oxidation of LDL is thought to contribute to the early stages of atherogenesis. Because myeloperoxidase is present in atherosclerotic lesions and can produce the strong oxidant hypochlorous acid (HOCl), which converts LDL into its high-uptake atherogenic form in vitro, we raised polyclonal and monoclonal antibodies (MoAbs) against HOCl-modified LDL (HOCl-LDL). Characterization of the polyclonal anti-human HOCl-LDL Abs showed that they cross-reacted strongly with 4-hydroxynonenal-, malondialdehyde-, and Cu(2+)-oxidized LDL. Similarly, polyclonal and some monoclonal Abs against aldehyde- and Cu(2+)-modified LDL cross-reacted with HOCl-LDL. In contrast to the polyclonal Abs, two selected hybridoma cell line supernatants containing MoAbs raised against HOCl-LDL (MoAb-A and MoAb-B) did not cross-react with either native LDL or aldehyde- or Cu(2+)-modified LDL. MoAb-A (clone 1B10A11, subtype IgG1 kappa) recognized an epitope that appeared to be specific for HOCl-LDL and depended on the tertiary structure of the (lipo)protein, as judged by a lack of cross-reactivity with HOCl-modified human and bovine serum albumin and a loss of reactivity associated with lipoprotein denaturation. MoAb-B (clone 2D10G9, subtype IgG2b kappa), on the other hand, gave identical titration curves with HOCl-LDL and HOCl-modified albumins, suggesting that this antibody recognized epitopes that are commonly generated on proteins that have been oxidized with HOCl. Thus, MoAb-A and MoAb-B may be useful tools for the investigation of a possible role for HOCl-mediated damage to (lipo)proteins in atherosclerosis and other inflammatory diseases.
Oxidation of low-density lipoproteins (LDL) is thought to contribute to atherogenesis. Although there is increasing evidence for a role of myeloperoxidase-derived oxidants such as hypochlorite (HOCl), the mechanism by which HOCl modifies LDL remains controversial. Some studies report the protein component to be the major site of attack, whereas others describe extensive lipid peroxidation. The present study addresses this controversy. The results obtained are consistent with the hypothesis that radical-induced oxidation of LDL's lipids by HOCl is a secondary reaction, with most HOCl consumed via rapid, non-radical reaction with apolipoprotein B-100. Subsequent incubation of HOCl-treated LDL gives rise to lipid peroxidation and antioxidant consumption in a time-dependent manner. Similarly, with myeloperoxidase/H2O2/Cl- (the source of HOCl in vivo), protein oxidation is rapid and followed by an extended period of lipid peroxidation during which further protein oxidation does not occur. The secondary lipid peroxidation process involves EPR-detectable radicals, is attenuated by a radical trap or treatment of HOCl-oxidized LDL with methionine, and occurs less rapidly when the lipoprotein was depleted of alpha-tocopherol. The initial reaction of low concentrations of HOCl (400-fold or 800-fold molar excess) with LDL therefore seems to occur primarily by two-electron reactions with side-chain sites on apolipoprotein B-100. Some of the initial reaction products, identified as lysine-residue-derived chloramines, subsequently undergo homolytic (one-electron) reactions to give radicals that initiate antioxidant consumption and lipid oxidation via tocopherol-mediated peroxidation. The identification of these chloramines, and the radicals derived from them, as initiating agents in LDL lipid peroxidation offers potential new targets for antioxidative therapy in atherogenesis.
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