Immunochemical detection of a novel lysine adduct using an antibody to linoleic acid hydroperoxide-modified protein
2003; Elsevier BV; Volume: 44; Issue: 6 Linguagem: Inglês
10.1194/jlr.m200442-jlr200
ISSN1539-7262
AutoresYoshichika Kawai, Yoji Kato, Hiroyuki Fujii, Yuko Makino, Yoko Mori, Michitaka Naito, Toshihiko Osawa,
Tópico(s)Advanced Glycation End Products research
ResumoWe have previously prepared the polyclonal antibody to the 13-hydroperoxyoctadecadienoic acid-modified protein (13Ab) (Kato et al. 1997. J. Lipid Res. 38: 1334–1346), however, the epitopes have not yet been structurally identified. In this study, we identified a novel amide-type adduct as one of the major epitopes of 13Ab and characterized the endogenous formation. Upon incubation of the lysine derivative with peroxidized linoleic acid, the formation of N ε-(azelayl)lysine (AZL) was confirmed using liquid chromatography-mass spectrometry. The chemically synthesized azelayl protein was significantly recognized by 13Ab. The peroxidation products of different polyunsaturated fatty acids also generated several analogous carboxyalkylamide-type adducts to AZL by the reaction with the lysine derivative, whereas 13Ab specifically recognized AZL, suggesting that the AZL moiety may be one of the major epitopes of 13Ab. The immunoreactive materials of 13Ab were immunohistochemically detected in atherosclerotic lesions from hypercholesterolemic rabbits. More strikingly, the immunoreactivity was significantly enhanced when the sections were treated with alkali or phospholipase A2 for hydrolyzing the ester bonds prior to the staining.These results suggest that the lipid hydroperoxide-derived carboxylic adducts, such as AZL, and their esters linked with phospholipids may be generated in vivo and involved in the pathogenesis of atherosclerosis associated with oxidative stress. We have previously prepared the polyclonal antibody to the 13-hydroperoxyoctadecadienoic acid-modified protein (13Ab) (Kato et al. 1997. J. Lipid Res. 38: 1334–1346), however, the epitopes have not yet been structurally identified. In this study, we identified a novel amide-type adduct as one of the major epitopes of 13Ab and characterized the endogenous formation. Upon incubation of the lysine derivative with peroxidized linoleic acid, the formation of N ε-(azelayl)lysine (AZL) was confirmed using liquid chromatography-mass spectrometry. The chemically synthesized azelayl protein was significantly recognized by 13Ab. The peroxidation products of different polyunsaturated fatty acids also generated several analogous carboxyalkylamide-type adducts to AZL by the reaction with the lysine derivative, whereas 13Ab specifically recognized AZL, suggesting that the AZL moiety may be one of the major epitopes of 13Ab. The immunoreactive materials of 13Ab were immunohistochemically detected in atherosclerotic lesions from hypercholesterolemic rabbits. More strikingly, the immunoreactivity was significantly enhanced when the sections were treated with alkali or phospholipase A2 for hydrolyzing the ester bonds prior to the staining. These results suggest that the lipid hydroperoxide-derived carboxylic adducts, such as AZL, and their esters linked with phospholipids may be generated in vivo and involved in the pathogenesis of atherosclerosis associated with oxidative stress. Oxidative modification of an LDL is thought to be involved in the etiology of atherosclerosis (1Steinberg D. Role of oxidized LDL and antioxidants in atherosclerosis.Adv. Exp. Med. Biol. 1995; 369: 39-48Google Scholar, 2Steinberg D. Parthasarathy S. Carew T.E. Khoo J.C. Witztum J.L. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity.N. Engl. J. Med. 1989; 320: 915-924Google Scholar). The oxidation of LDL leads to the loss of ε-amino groups from the lysine residues in apolipoprotein B (apoB) due to the covalent adduction by the oxidative decomposed products of polyunsaturated fatty acid esters (3Steinbrecher U.P. Witztum J.L. Parthasarathy S. Steinberg D. Decrease in reactive amino groups during oxidation or endothelial cell modification of LDL. Correlation with changes in receptor-mediated catabolism.Arteriosclerosis. 1987; 7: 135-143Google Scholar, 4Steinbrecher U.P. Oxidation of human low density lipoprotein results in derivatization of lysine residues of apolipoprotein B by lipid peroxide decomposition products.J. Biol. Chem. 1987; 262: 3603-3608Google Scholar). Aldehydes such as malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) are highly reactive to ε-amino groups in proteins, and therefore, aldehyde-derived protein adducts have been extensively investigated. The adduction of aldehydes to apoB of LDL has been implicated in the uncontrolled uptake of these lipoproteins by macrophage scavenger receptors, and the following formation of foam cells leading to fatty streaks. Indeed, MDA-lysine (5Uchida K. Sakai K. Itakura K. Osawa T. Toyokuni S. Protein modification by lipid peroxidation products: formation of malondialdehyde-derived N ε-(2-propenal)lysine in proteins.Arch. Biochem. Biophys. 1997; 346: 45-52Google Scholar), HNE-lysine (6Uchida K. Toyokuni S. Nishikawa K. Kawakishi S. Oda H. Hiai H. Stadtman E.R. Michael addition-type 4-hydroxy-2-nonenal adducts in modified low-density lipoproteins: markers for atherosclerosis.Biochemistry. 1994; 33: 12487-12494Google Scholar, 7Uchida K. Itakura K. Kawakishi S. Hiai H. Toyokuni S. Stadtman E.R. Characterization of epitopes recognized by 4-hydroxy-2-nonenal specific antibodies.Arch. Biochem. Biophys. 1995; 324: 241-248Google Scholar), and acrolein-lysine adducts (8Uchida K. Kanematsu M. Sakai K. Matsuda T. Hattori N. Mizuno Y. Suzuki D. Miyata T. Noguchi N. Niki E. Osawa T. Protein-bound acrolein: potential markers for oxidative stress.Proc. Natl. Acad. Sci. USA. 1998; 95: 4882-4887Google Scholar, 9Uchida K. Kanematsu M. Morimitsu Y. Osawa T. Noguchi N. Niki E. Acrolein is a product of lipid peroxidation reaction. Formation of free acrolein and its conjugate with lysine residues in oxidized low density lipoproteins.J. Biol. Chem. 1998; 273: 16058-16066Google Scholar) have been detected in oxidized LDL (oxLDL) and atherosclerotic lesions. On the other hand, the esterified aldehydes bound to phospholipids or cholesteryl esters (called “core aldehydes”) are generated during the lipid peroxidation as well as “free” aldehydes. For example, 9-oxononanoyl esters and 5-oxovaleroyl esters have been detected during the oxidation of linoleic acid and arachidonic acid (AA) esters, respectively (10Tanaka T. Minamino H. Unezaki S. Tsukatani H. Tokumura A. Formation of platelet-activating factor-like phospholipids by Fe2+/ascorbate/EDTA-induced lipid peroxidation.Biochim. Biophys. Acta. 1993; 1166: 264-274Google Scholar, 11Kamido H. Kuksis A. Marai L. Myher J.J. Identification of core aldehydes among in vitro peroxidation products of cholesteryl esters.Lipids. 1993; 28: 331-336Google Scholar). These core aldehydes, i.e., phospholipids/cholesteryl esters in which there are aldehydic fatty acid fragments still esterified to the glycerol/cholesterol backbone, can also react with the amino groups of proteins and form the esterified lipid-protein adducts (12Hoppe G. Ravandi A. Herrera D. Kuksis A. Hoff H.F. Oxidation products of cholesteryl linoleate are resistant to hydrolysis in macrophages, form complexes with proteins, and are present in human atherosclerotic lesions.J. Lipid Res. 1997; 38: 1347-1360Google Scholar). Protein adducts from levuglandin E2 esters (13Salomon R.G. Subbanagounder G. Singh U. O'Neil J. Hoff H.F. Oxidation of low-density lipoproteins produces levuglandin-protein adducts.Chem. Res. Toxicol. 1997; 10: 750-759Google Scholar) and hydroxyalkenal esters (14Kaur K. Salomon R.G. O'Neil J. Hoff H.F. (Carboxyalkyl)pyrroles in human plasma and oxidized low-density lipoproteins.Chem. Res. Toxicol. 1997; 10: 1387-1396Google Scholar) of the phospholipids have also been discovered in oxLDL. In contrast to the reactivity of aldehydes, the lipid hydroperoxides, the primary products of lipid peroxidation, can also modify the amino groups of proteins. Kim et al. have shown the first demonstration that the immuno-positive materials of a polyclonal antibody to lipid hydroperoxide-modified proteins were generated in atherosclerotic lesions (15Kim J.G. Sabbagh F. Santanam N. Wilcox J.N. Medford R.M. Parthasarathy S. Generation of a polyclonal antibody against lipid peroxide-modified proteins.Free Radic. Biol. Med. 1997; 23: 251-259Google Scholar). We have also prepared the polyclonal antibodies to the 13-hydroperoxyoctadecadienoic acid (13-HPODE)-modified protein (16Kato Y. Makino Y. Osawa T. Characterization of a specific polyclonal antibody against 13-hydroperoxyoctadecadienoic acid-modified protein: formation of lipid hydroperoxide-modified apoB-100 in oxidized LDL.J. Lipid Res. 1997; 38: 1334-1346Google Scholar) and 15-hydroperoxyeicosatetraenoic acid (15-HPETE)-modified protein (17Kato Y. Osawa T. Detection of oxidized phospholipid-protein adducts using anti-15-hydroperoxyeicosatetraenoic acid-modified protein antibody: contribution of esterified fatty acid-protein adduct to oxidative modification of LDL.Arch. Biochem. Biophys. 1998; 351: 106-114Google Scholar). Although the precise epitopes of these antibodies have not been identified, several aldehyde-modified proteins could not be recognized by these antibodies, suggesting the existence of specific epitopes derived from the lipid hydroperoxides. These polyclonal antibodies (named 13Ab and 15Ab) weakly recognized the intact oxLDL, whereas the immunoreactivity was enhanced by the alkali-treatment of oxLDL for hydrolyzing the carboxyl esters (16Kato Y. Makino Y. Osawa T. Characterization of a specific polyclonal antibody against 13-hydroperoxyoctadecadienoic acid-modified protein: formation of lipid hydroperoxide-modified apoB-100 in oxidized LDL.J. Lipid Res. 1997; 38: 1334-1346Google Scholar, 17Kato Y. Osawa T. Detection of oxidized phospholipid-protein adducts using anti-15-hydroperoxyeicosatetraenoic acid-modified protein antibody: contribution of esterified fatty acid-protein adduct to oxidative modification of LDL.Arch. Biochem. Biophys. 1998; 351: 106-114Google Scholar). These characterizations suggest that the lipid hydroperoxide-derived esterified adducts are generated in oxLDL. During the reaction of 13-HPODE with lysine derivatives, we have recently identified the formation of N ε-(hexanoyl) lysine (HEL), a unique amide-type lysine adduct (18Kato Y. Mori Y. Makino Y. Morimitsu Y. Hiroi S. Ishikawa T. Osawa T. Formation of N ε-(hexanonyl)lysine in protein exposed to lipid hydroperoxide. A plausible marker for lipid hydroperoxide-derived protein modification.J. Biol. Chem. 1999; 274: 20406-20414Google Scholar). The amide-type adduct is a new class of protein adducts derived from lipid peroxidation. The presence of the HEL moiety in oxLDL and human atherosclerotic lesions has been revealed by using a specific polyclonal antibody to the HEL moiety (18Kato Y. Mori Y. Makino Y. Morimitsu Y. Hiroi S. Ishikawa T. Osawa T. Formation of N ε-(hexanonyl)lysine in protein exposed to lipid hydroperoxide. A plausible marker for lipid hydroperoxide-derived protein modification.J. Biol. Chem. 1999; 274: 20406-20414Google Scholar). In view of the analogy between the action of free aldehydes and core aldehydes, it is possible that the esterified amide adducts are generated during the reaction of lipid hydroperoxide-esters with proteins as well as HEL formation. In the present study, we characterized the epitopes of 13Ab for assessing the implication of the lipid hydroperoxide-derived esterified adduct formation in atherosclerosis, and discovered the formation of carboxyalkylamide-type novel lysine adducts. The results in this study suggest that the lipid hydroperoxide-derived esterified adducts are endogenously generated and may be implicated in the pathogenesis of atherosclerosis. N α-benzoyl-glycyl-l-lysine (Bz-Gly-Lys) was obtained from Peptide, Inc. (Osaka, Japan). Monomethylazelaic acid, monomethylglutaric acid, and monomethylsuccinic acid were obtained from Fluka (Buchs, Switzerland). Bovine serum albumin (BSA) and phospholipase A2 (EC 3.1.1.4) from bee venom were obtained from Sigma Chemical Co. (St. Louis, MO). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) were obtained from Pierce Chemical Co. (Rockford, IL). 13-HPODE was prepared by the lipoxygenase-catalyzed enzymatic oxidation of linoleic acid as previously described (16Kato Y. Makino Y. Osawa T. Characterization of a specific polyclonal antibody against 13-hydroperoxyoctadecadienoic acid-modified protein: formation of lipid hydroperoxide-modified apoB-100 in oxidized LDL.J. Lipid Res. 1997; 38: 1334-1346Google Scholar). The polyclonal antibodies to 13-HPODE-modified protein (13Ab) and 15-HPETE-modified protein (15Ab) were prepared as previously described (16Kato Y. Makino Y. Osawa T. Characterization of a specific polyclonal antibody against 13-hydroperoxyoctadecadienoic acid-modified protein: formation of lipid hydroperoxide-modified apoB-100 in oxidized LDL.J. Lipid Res. 1997; 38: 1334-1346Google Scholar, 17Kato Y. Osawa T. Detection of oxidized phospholipid-protein adducts using anti-15-hydroperoxyeicosatetraenoic acid-modified protein antibody: contribution of esterified fatty acid-protein adduct to oxidative modification of LDL.Arch. Biochem. Biophys. 1998; 351: 106-114Google Scholar). The competitive indirect enzyme-linked immunosorbent assay (ELISA) was done as described (16Kato Y. Makino Y. Osawa T. Characterization of a specific polyclonal antibody against 13-hydroperoxyoctadecadienoic acid-modified protein: formation of lipid hydroperoxide-modified apoB-100 in oxidized LDL.J. Lipid Res. 1997; 38: 1334-1346Google Scholar) with some modification. Briefly, 100 μl of 13-HPODE-modified BSA [0.5 μg/well in phosphate-buffered saline (PBS)] was coated into the wells and kept at 4°C overnight. The 13-HPODE-modified BSA was prepared by incubating 13-HPODE (5 mM) with BSA (1 mg/ml) in phosphate buffer (pH 7.4) at 37°C for 3 days. At the same time, 50 μl of 13Ab [1:500 in PBS containing 0.05% Tween 20 (TPBS)] and 50 μl of competitor (in PBS) were mixed in a tube and reacted overnight at 4°C. The plate was washed and blocked with 4% Block Ace (Dainihon Seiyaku Co., Osaka), and 90 μl of the competitor solution was then added to a well and further incubated. The binding of the residual antibody on the coated modified BSA was indirectly estimated by the binding of the peroxidase-labeled anti-rabbit IgG secondary antibody. The color development reaction was performed by adding ο-phenylenediamine solution (0.5 mg/ml containing 0.03% hydrogen peroxide), and the optical density was measured at 490 nm. The result of the competitive ELISA was expressed as B/B0, where B is the amount of antibody bound in the presence and B0 is the amount in the absence of the competitor. Each point represents the mean of duplicate determinations. The noncompetitive indirect ELISA was performed as described (16Kato Y. Makino Y. Osawa T. Characterization of a specific polyclonal antibody against 13-hydroperoxyoctadecadienoic acid-modified protein: formation of lipid hydroperoxide-modified apoB-100 in oxidized LDL.J. Lipid Res. 1997; 38: 1334-1346Google Scholar) with some modifications. Briefly, 50 μl of protein (0.06–5.55 μg/ml) was dispensed into wells and kept at 4°C overnight. The plate was then incubated with 13Ab (1:1,000, v/v in TPBS), and the binding of the antibody to the protein was evaluated by incubation with the peroxidase-labeled anti-rabbit IgG antibody. The data represent the mean of duplicate determinations. The N ε-(azelayl)lysine (AZL) derivative [N α-benzoyl-glycyl-N ε-(azelayl)lysine] was prepared by the carbodiimide method. Briefly, monomethylazelaic acid (50 mg), EDC (52.2 mg), and sulfo-NHS (31.3 mg) in dimethylformamide (800 μl) were magnetically stirred for 24 h at room temperature. After the incubation, Bz-Gly-Lys (83.3 mg) in 2.2 ml phosphate buffer (pH 7.4) was added and further stirred for 4 h at room temperature. The formed monomethylazelayl derivative was extracted by ethyl acetate and separated by reverse-phase high performance liquid chromatography (HPLC) equipped with a Develosil ODS-HG-5 column (8 × 250 mm, Nomura Chemical Co., Aichi, Japan) and isocratic conditions, with 37.5% aqueous CH3CN containing 0.1% trifluoroacetic acid as the eluent at a flow rate of 2.0 ml/min (detected at UV 234 nm). The obtained monomethylazelayl derivative was incubated in 1 N NaOH at 37°C for 1 h, and the formed AZL derivative was separated by reverse-phase HPLC as described above. The purified AZL was identified by 1H-nuclear magnetic resonance (NMR) (Bruker ARX-400) and liquid chromatography-mass spectrometry (LC-MS, VG Biotech PLATFORM II) in the electrospray ionization positive (ESP+) mode. The spectral data were as follows: 1H-NMR (CD3OD) (ppm) 1.33 (m, 6H), 1.43 (m, 2H), 1.51 (m, 2H), 1.59 (m, 4H), 1.75 (m, 1H), 1.92 (m, 1H), 2.16 (t, J = 7.6, 2H), 2.27 (t, J = 7.4, 2H), 3.16 (t, J = 6.8, 2H), 4.11 (m, 2H), 4.44 (m, 1H), 7.47 (t, J = 7.4, 2H), 7.55 (t, J = 7.4, 1H), 7.87 (d, J = 7.2, 2H); LC-MS (ESP+) m/z 478 (M+H)+. Other alkyl lengths of the carboxyalkylamide-type lysine adducts were also synthesized using the corresponding alkyl length of the monomethylcarboxylic acid. These synthetic derivatives were identified by 1H-NMR and LC-MS measurements (unpublished observations). The HEL derivative was synthesized as previously described (18Kato Y. Mori Y. Makino Y. Morimitsu Y. Hiroi S. Ishikawa T. Osawa T. Formation of N ε-(hexanonyl)lysine in protein exposed to lipid hydroperoxide. A plausible marker for lipid hydroperoxide-derived protein modification.J. Biol. Chem. 1999; 274: 20406-20414Google Scholar). The fatty acid (final concentration, 5 mM) was oxidized in the presence of 0.5 mM ascorbate and 0.05 mM FeSO4 for 24 h at 37°C in phosphate buffer (pH 7.4) containing 20% methanol. To the solution, Bz-Gly-Lys (final concentration, 5 mM) was added and further incubated at 37°C for 3 days. After the incubation, the reaction mixtures were analyzed by LC-MS. The gradient elution of HPLC was done using two solvents: solvent A, 10% CH3CN containing 0.1% acetate; and solvent B, 100% CH3CN containing 0.1% acetic acid. The sample was injected into a Develosil ODS HG-5 column (4.6 × 250 mm) and eluted with a linear gradient from 100% A to 65% B for 40 min at 0.8 ml/min. For the quantification of AZL and HEL, tert-butoxycarbonyl isoleucine was added to the samples at a final concentration of 0.1 mM as the internal standard. Analyses were performed by monitoring ions of m/z 478 (AZL), 406 (HEL), and 232 (internal standard). A standard curve was produced by plotting the synthetic adduct level against the quotient of the peak area of the adduct divided by the peak areas of the internal standard. The azelayl BSA (AZL-BSA), N ε-(glutaroyl)lysine BSA (GLL-BSA), and N ε-(succinyl)lysine BSA (SUL-BSA) were prepared as follows. Monomethylcarboxylic acid (monomethylazelaic acid, monomethylglutaroic acid, or monomethylsuccinic acid) was conjugated with BSA using the EDC/sulfo-NHS system as previously described (18Kato Y. Mori Y. Makino Y. Morimitsu Y. Hiroi S. Ishikawa T. Osawa T. Formation of N ε-(hexanonyl)lysine in protein exposed to lipid hydroperoxide. A plausible marker for lipid hydroperoxide-derived protein modification.J. Biol. Chem. 1999; 274: 20406-20414Google Scholar). The methyl esters of the adducts in the proteins were hydrolyzed by incubation in 0.5 N NaOH for 1 h at room temperature. After the incubation, the mixture was neutralized with an equal volume of 0.5 N HCl. These modified proteins were then dialyzed against PBS for 3 days at 4°C. The preparation of the carboxymethyl BSA was performed as previously reported (19Kato Y. Tokunaga K. Osawa T. Immunochemical detection of carboxymethylated Apo B-100 in copper-oxidized LDL.Biochem. Biophys. Res. Commun. 1996; 226: 923-927Google Scholar). Male New Zealand White rabbits (2.5 kg; Kitayama, Japan) were allowed free access to water and a commercial rabbit diet containing 1% cholesterol (20Kang M.H. Kawai Y. Naito M. Osawa T. Dietary defatted sesame flour decreases susceptibility to oxidative stress in hypercholesterolemic rabbits.J. Nutr. 1999; 129: 1885-1890Google Scholar). After 12 weeks, the rabbits were sacrificed and their aortas were removed. Formalin-fixed, paraffin-embedded aorta sections (5 μm thick) were prepared and used for the immunohistochemical analyses. Briefly, deparaffinized sections were incubated with normal serum for 30 min to block the nonspecific binding of the secondary antibody and then with a primary antibody (1:50, v/v) at 4°C overnight. For the ester bond hydrolysis with alkali, prior to blocking, the sections were incubated with 0.25 N NaOH for 1 h at room temperature. For the enzymatic hydrolysis, the sections were incubated with phospholipase A2 (1.0 U/μl) for 6 h at room temperature. Immunostaining was performed using the avidin-biotin complex method with the Vectastain ABC-AP (alkaline phosphatase) kit and Vector Alkaline Phosphatase Substrate Kit II (Vector Laboratories, Inc., Burlingame, CA). A competitive experiment was performed by staining with 13Ab preincubated with an excess of AZL derivative (25 mM) at 37°C for 1 h. To examine the lipid hydroperoxide-derived protein modification, we previously prepared the polyclonal antibody 13Ab against the 13-HPODE-modified protein (16Kato Y. Makino Y. Osawa T. Characterization of a specific polyclonal antibody against 13-hydroperoxyoctadecadienoic acid-modified protein: formation of lipid hydroperoxide-modified apoB-100 in oxidized LDL.J. Lipid Res. 1997; 38: 1334-1346Google Scholar); however, the structural identification of the epitope(s) has not yet been performed. Amino acid analyses of the lipid hydroperoxide-modifed proteins have shown that lysine residues are the potential targets by the reaction of lipid hydroperoxides (16Kato Y. Makino Y. Osawa T. Characterization of a specific polyclonal antibody against 13-hydroperoxyoctadecadienoic acid-modified protein: formation of lipid hydroperoxide-modified apoB-100 in oxidized LDL.J. Lipid Res. 1997; 38: 1334-1346Google Scholar, 17Kato Y. Osawa T. Detection of oxidized phospholipid-protein adducts using anti-15-hydroperoxyeicosatetraenoic acid-modified protein antibody: contribution of esterified fatty acid-protein adduct to oxidative modification of LDL.Arch. Biochem. Biophys. 1998; 351: 106-114Google Scholar). We have previously identified the formation of HEL (Fig. 1A)as one of the major products formed by the reaction of 13-HPODE with a lysine derivative (18Kato Y. Mori Y. Makino Y. Morimitsu Y. Hiroi S. Ishikawa T. Osawa T. Formation of N ε-(hexanonyl)lysine in protein exposed to lipid hydroperoxide. A plausible marker for lipid hydroperoxide-derived protein modification.J. Biol. Chem. 1999; 274: 20406-20414Google Scholar). HEL is a unique amide-type adduct, which has the C6 saturated aliphatic chain containing the CH3-terminal of 13-HPODE; however, the HEL moiety was scarcely recognized by the antibody (Fig. 1B), showing that HEL could not be the epitope of 13Ab. This result supports our previous characterization of 13Ab that the carboxyl groups derived from 13-HPODE are important for the recognition of 13Ab (16Kato Y. Makino Y. Osawa T. Characterization of a specific polyclonal antibody against 13-hydroperoxyoctadecadienoic acid-modified protein: formation of lipid hydroperoxide-modified apoB-100 in oxidized LDL.J. Lipid Res. 1997; 38: 1334-1346Google Scholar). Based on the information of HEL generation during the reaction of 13-HPODE with lysine derivatives (18Kato Y. Mori Y. Makino Y. Morimitsu Y. Hiroi S. Ishikawa T. Osawa T. Formation of N ε-(hexanonyl)lysine in protein exposed to lipid hydroperoxide. A plausible marker for lipid hydroperoxide-derived protein modification.J. Biol. Chem. 1999; 274: 20406-20414Google Scholar) and the characterization of 13Ab (16Kato Y. Makino Y. Osawa T. Characterization of a specific polyclonal antibody against 13-hydroperoxyoctadecadienoic acid-modified protein: formation of lipid hydroperoxide-modified apoB-100 in oxidized LDL.J. Lipid Res. 1997; 38: 1334-1346Google Scholar) (Fig. 1), we presumed the formation of AZL, a novel amide-type adduct containing a carboxyl group derived from linoleic acid, as a plausible epitope of the antibody. The scheme for the proposed formation of AZL and the synthetic scheme for this adduct are illustrated in Fig. 2. To examine the formation of AZL, the reaction of the peroxidized linoleic acid with a lysine derivative (Bz-Gly-Lys) was carried out as the model system, and the reaction mixture was then analyzed by LC-MS. As expected, the molecular ion peak at m/z 478 was detected from the reaction mixture (Fig. 3A). Compared with the retention time and the molecular ion peak of the synthetic AZL derivative, the obtained peak was identified as AZL. The quantitative analyses using LC-MS showed that the amount of AZL generated in the reaction mixture was comparable to that of HEL (Fig. 3B). We have previously shown that the conversion yield of HEL from the loss of Bz-Gly-Lys after a 3 day incubation with 13-HPODE was 5.6% (18Kato Y. Mori Y. Makino Y. Morimitsu Y. Hiroi S. Ishikawa T. Osawa T. Formation of N ε-(hexanonyl)lysine in protein exposed to lipid hydroperoxide. A plausible marker for lipid hydroperoxide-derived protein modification.J. Biol. Chem. 1999; 274: 20406-20414Google Scholar). These observations suggest that AZL may be one of the major lysine adducts generated from the reaction with peroxidized linoleic acid as well as HEL.Fig. 3Formation of AZL as one of the major products from the reaction of oxidized linoleic acid with Bz-Gly-Lys. A: Analysis of AZL adduct by liquid chromatography-mass spectrometry (LC-MS). Linoleic acid (5 mM) was oxidized by Fe2+-ascorbate for 24 h at 37°C, and then incubated with Bz-Gly-Lys (5 mM) for 3 days at 37°C. LC-MS measurements were performed by monitoring ions at m/z 478. The authentic standard was synthesized as described in Experimental Procedures. B: Time-dependent formation of AZL and HEL during the reaction of oxidized linoleic acid with Bz-Gly-Lys, estimated by LC-MS. The amount of adducts was calculated from the standard curves developed by using synthetic standards with tert-butoxycarbonyl isoleucine as the internal standard.View Large Image Figure ViewerDownload (PPT) The alkyl length of the azelayl moiety of AZL (C9) corresponds to the alkyl length from the COOH terminal to the first double bond on the original fatty acid. To examine the structural correlation between the formation of the carboxyalkylamide-type adducts such as AZL and the fatty acid sources, various polyunsaturated fatty acids were used for the reaction with the lysine derivative instead of linoleic acid. As shown in Fig. 4, as expected, AZL (m/z 478) was generated from the reaction of Bz-Gly-Lys with oxidized α-linolenic acid (α-LNA), which has a C9 carboxyalkyl moiety. The result exhibited a positive correlation with our previous immunochemical observations that 13Ab significantly reacted with the oxidized α-LNA-modified protein (16Kato Y. Makino Y. Osawa T. Characterization of a specific polyclonal antibody against 13-hydroperoxyoctadecadienoic acid-modified protein: formation of lipid hydroperoxide-modified apoB-100 in oxidized LDL.J. Lipid Res. 1997; 38: 1334-1346Google Scholar). The GLL (m/z 422) was generated from the reaction of Bz-Gly-Lys with oxidized AA or eicosapentaenoic acid (EPA). In addition, the SUL (m/z 408) was also generated from the reaction of Bz-Gly-Lys with oxidized docosahexaenoic acid (DHA). By comparison with authentic standards, the formation of these amide-type adducts was confirmed. The formation of AZL could not be observed from the reaction of the oxidized oleic acid containing the C9 carboxyalkyl moiety (unpublished observations), showing that monounsaturated fatty acids could not be the sources of the carboxyalkylamide-type adducts. These results show that these characteristic carboxyalkylamide-type lysine adducts are universal for the modification of lysine residues by various oxidized polyunsaturated fatty acids. To confirm whether 13Ab can recognize the AZL adducts in protein, the azelayl BSA containing a large amount of AZL in the ε-amino groups of lysine was chemically synthesized and the cross-reactivity of 13Ab was examined. We also synthesized carboxymethyl BSA to examine the cross-reactivity of 13Ab to N ε-(carboxymethyl)lysine (CML), because CML is a representative carboxylic lysine adduct and is reported to be formed not only during the Maillard reaction but also during lipid peroxidation in the presence of protein (21Fu M-X. Requena J.R. Jenkins A.J. Lyons T.J. Baynes J.W. Thorpe S.R. The advanced glycation end product, N ε-(carboxymethyl)lysine, is a product of both lipid peroxidation and glycoxidation reactions.J. Biol. Chem. 1996; 271: 9982-9986Google Scholar). Figure 5Ashows that azelayl BSA was strongly recognized by 13Ab, whereas carboxymethyl BSA was not recognized. The cross-reactivity of 13Ab to the purified AZL derivative was also confirmed by competitive ELISA (unpublished observations). In addition, Fig. 4 showed that the peroxidation of other polyunsaturated fatty acids also resulted in the formation of carboxyalkylamide-type adducts such as GLL and SUL upon the reaction with lysine residues. To investigate the specificity of 13Ab to these analogous carboxyalkylamide adducts, the cross-reactivity with the AZL-, GLL-, or SUL-containing proteins was examined. As shown in Fig. 5B, 13Ab recognized the AZL-conjugated protein, but not the GLL- and SUL-conjugated proteins. These results suggest that the AZL moiety should be one of the major epitopes of the antibody. To evaluate the endogenous generation of the epitopes of 13Ab, the antibody was applied to the immunohistochemical staining in aorta specimens from the hypercholesterolemic rabbits, an experimental animal model for atherosclerosis. The lipid peroxidation levels in the tissues and serum of the hypercholesterolemic rabbits were significantly increased compared with the control rabbits (unpublished observations). When the immunostaining using 13Ab was performed with the aorta sections of the hypercholesterolemic rabbits, immunopositive materials were observed, showing that the epitopes of the antibody, containing AZL, may be generated in vivo (Fig. 6A). More strikingly, alkali treatment of the sections prior to use of the primary antibody significantly enhanced the immunoreactivity of the antibody (Fig. 6B). Furthermore, when sections were enzymatically hydrolyzed with phospholipase A2, which specifically cleaves the ester bonds of the sn-2 polyunsaturated fatty acids of glycerophospholipids, the enhanced immunoreactivity was also observed (Fig. 6C). The treatments of sections with hydrolyzing agents may saponify the ester bonds and expose the carboxylic epitopes of the antibody (Scheme 1). These results suggest that the esterified lipid-protein adducts, such as the AZL esters, were also generated in the rabbit atherosclerotic lesions, as well as the free carboxylic adducts. In addition, no positive staining was observed in the experiments using 13Ab preabsorbed by an excess of the AZL derivative (Fig. 6D) or using normal rabbit serum (Fig. 6E), suggesting the specificity of the immunostaining. Furthermore, no positive staining was observed in the nonatherosclerotic aortas obtained from normal rabbits (Fig. 6F), suggesting the possibility that the formation of the epitopes may be closely associated with the formation of atherosclerotic lesions.Scheme 1Formation of free and/or phospholipid-esterifed N ε-(azelayl)lysine adduct and the immunochemical detection by 13Ab.View Large Image Figure ViewerDownload (PPT) We have previously prepared the polyclonal antibody against the 13-HPODE-modified protein (13Ab) for assessing lipid hydroperoxide-derived protein modifications. Characterization of this antibody indicated that i) the fatty acid-derived carboxyl group is essential for the recognition, and ii) the epitopes are generated in oxLDL as the esterified form (16Kato Y. Makino Y. Osawa T. Characterization of a specific polyclonal antibody against 13-hydroperoxyoctadecadienoic acid-modified protein: formation of lipid hydroperoxide-modified apoB-100 in oxidized LDL.J. Lipid Res. 1997; 38: 1334-1346Google Scholar). In this study, we identified the formation of AZL, a carboxyalkylamide-type novel lysine adduct, as one of the major epitopes of 13Ab, and characterized the endogenous generation using the antibody. Covalent modifications of apoB in LDL by the oxidation products of the polyunsaturated fatty acids are believed to be involved in the pathogenesis of atherosclerosis (1Steinberg D. Role of oxidized LDL and antioxidants in atherosclerosis.Adv. Exp. Med. Biol. 1995; 369: 39-48Google Scholar, 2Steinberg D. Parthasarathy S. Carew T.E. Khoo J.C. Witztum J.L. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity.N. Engl. J. Med. 1989; 320: 915-924Google Scholar, 3Steinbrecher U.P. Witztum J.L. Parthasarathy S. Steinberg D. Decrease in reactive amino groups during oxidation or endothelial cell modification of LDL. Correlation with changes in receptor-mediated catabolism.Arteriosclerosis. 1987; 7: 135-143Google Scholar, 4Steinbrecher U.P. Oxidation of human low density lipoprotein results in derivatization of lysine residues of apolipoprotein B by lipid peroxide decomposition products.J. Biol. Chem. 1987; 262: 3603-3608Google Scholar). It has been reported that about 250 mol of amino groups per mole of apoB decreased by overnight copper-catalyzed oxidation (22Gillotte K.L. Ho¨rkko¨ S. Witztum J.L. Steinberg D. Oxidized phospholipids, linked to apolipoprotein B of oxidized LDL, are ligands for macrophage scavenger receptors.J. Lipid Res. 2000; 41: 824-833Google Scholar). Among them, ∼30% of the decreased lysine appeared to be due to the conjugation of the phospholipids. It has generally been assumed that these phospholipid-protein conjugations were due to the attachment of the phospholipid core aldehydes. As well as the aldehydes, several lines of evidence have suggested that the lipid hydroperoxides, the primary products of lipid peroxidation, can modify proteins (15Kim J.G. Sabbagh F. Santanam N. Wilcox J.N. Medford R.M. Parthasarathy S. Generation of a polyclonal antibody against lipid peroxide-modified proteins.Free Radic. Biol. Med. 1997; 23: 251-259Google Scholar, 23Fruebis J. Parthasarathy S. Steinberg D. Evidence for a concerted reaction between lipid hydroperoxides and polypeptides.Proc. Natl. Acad. Sci. USA. 1992; 89: 10588-10592Google Scholar, 24Kim J.G. Taylor W.R. Parthasarathy S. Demonstration of the presence of lipid peroxide-modified proteins in human atherosclerotic lesions using a novel lipid peroxide-modified anti-peptide antibody.Atherosclerosis. 1999; 143: 335-340Google Scholar). Alkali saponification of the oxidized phosphatidylcholine-modified BSA and oxLDL resulted in an increase in the immunoreactivity of 13Ab (16Kato Y. Makino Y. Osawa T. Characterization of a specific polyclonal antibody against 13-hydroperoxyoctadecadienoic acid-modified protein: formation of lipid hydroperoxide-modified apoB-100 in oxidized LDL.J. Lipid Res. 1997; 38: 1334-1346Google Scholar). The immunoreactivity of 13Ab was also observed when oxLDL was treated with phospholipase A2 (unpublished observations). These results strongly suggest the occurrence of esterified lipid hydroperoxide-derived protein modifications in LDL. The importance of the esterified lipid-protein adducts was strongly suggested by the demonstration that a monoclonal autoantibody against oxLDL obtained from apoE-deficient mice could react with both the protein and lipid moieties of oxLDL and inhibit the macrophage binding of oxLDL (25Bird D.A. Gillotte K.L. Ho¨rkko¨ S. Friedman P. Dennis E.A. Witztum J.L. Steinberg D. Receptors for oxidized low-density lipoprotein on elicited mouse peritoneal macrophages can recognize both the modified lipid moieties and the modified protein moieties: implications with respect to macrophage recognition of apoptotic cells.Proc. Natl. Acad. Sci. USA. 1999; 96: 6347-6352Google Scholar, 26Ho¨rkko¨ S. Bird D.A. Miller E. Itabe H. Leitinger N. Subbanagounder G. Berliner J.A. Friedman P. Dennis E.A. Curtiss L.K. Palinski W. Witztum J.L. Monoclonal autoantibodies specific for oxidized phospholipids or oxidized phospholipid-protein adducts inhibit macrophage uptake of oxidized low-density lipoproteins.J. Clin. Invest. 1999; 103: 117-128Google Scholar). In addition, the covalent conjugation of oxidized phospholipids with apoB is suggested to be one of the major ligands for macrophage scavenger receptors (22Gillotte K.L. Ho¨rkko¨ S. Witztum J.L. Steinberg D. Oxidized phospholipids, linked to apolipoprotein B of oxidized LDL, are ligands for macrophage scavenger receptors.J. Lipid Res. 2000; 41: 824-833Google Scholar). To characterize the formation of the esterified lipid hydroperoxide-derived protein adducts in vivo, 13Ab was applied to the immunohistochemical staining in the atherosclerotic aortas from hypercholesterolemic rabbits. Enhanced positive staining was obtained in the alkali-saponified sections from the atherosclerotic aortas, suggesting the contribution of the esterified lipid hydroperoxides as well as the free lipid hydroperoxides to the generation of the antigenic epitopes in vivo (Fig. 6). By using not only the alkali treatment but also enzymatic treatments for the hydrolysis of the sections, it was confirmed that the epitopes were linked with phospholipids in the lesions (Fig. 6C). As mentioned above, the esterified lipid-protein adducts may contribute to the pathogenesis of atherosclerosis. Therefore, the formation of the lipid hydroperoxide-derived esterified adducts may possess some consequence to atherosclerosis, and 13Ab may be a good tool for the characterization of these esterified adducts in vivo. During the reaction of 13-HPODE with the lysine residues, HEL, a nonesterified amide-type lysine adduct, was identified as one of the major reaction products (18Kato Y. Mori Y. Makino Y. Morimitsu Y. Hiroi S. Ishikawa T. Osawa T. Formation of N ε-(hexanonyl)lysine in protein exposed to lipid hydroperoxide. A plausible marker for lipid hydroperoxide-derived protein modification.J. Biol. Chem. 1999; 274: 20406-20414Google Scholar). HEL is also generated by the reaction of various oxidized ω-6 polyunsaturated fatty acids (18Kato Y. Mori Y. Makino Y. Morimitsu Y. Hiroi S. Ishikawa T. Osawa T. Formation of N ε-(hexanonyl)lysine in protein exposed to lipid hydroperoxide. A plausible marker for lipid hydroperoxide-derived protein modification.J. Biol. Chem. 1999; 274: 20406-20414Google Scholar). Similarly, the oxidation of the ω-3 polyunsaturated fatty acids such as α-LNA, EPA, and DHA can also generate an amide-type adduct, N ε-(propanoyl)lysine, by reacting with lysine residues (Kawai et al., unpublished observations). As well as the generation of amide-type adducts containing the CH3-terminal moiety of fatty acids, the amide-type adducts containing the fatty acid-derived COOH moiety are likely generated from the reaction of oxidized polyunsaturated fatty acids. Indeed, AZL was chemically identified from the reaction of the oxidized linoleic acid with a lysine derivative (Fig. 3). In addition, the oxidation of α-LNA, AA, EPA, and DHA in the presence of lysine residues could also generate carboxyalkylamide-type adducts (Fig. 4); however, 13Ab specifically recognized AZL but not other carboxyalkylamide-type adducts (Fig. 5B). We also confirmed that the GLL adduct formed from the reaction of the oxidized AA may be one of the major epitopes of the previously prepared 15Ab (ref. 17 and unpublished observations). Although linoleic acid and AA are the major polyunsaturated fatty acids in normal human serum phospholipids (27Salomon R.G. Sha W. Brame C. Kaur K. Subbanagounder G. O'Neil J. Hoff H.F. Roberts II, L.J. Protein adducts of iso[4]levuglandin E2, a product of the isoprostane pathway, in oxidized low density lipoprotein.J. Biol. Chem. 1999; 274: 20271-20280Google Scholar), the ω-3 polyunsaturated fatty acids such as EPA and DHA are highly susceptible to oxidation, and therefore may contribute to the oxidative modifications of protein in vivo. Studies on the DHA-derived SUL formation in DHA-administrated rodents are now in progress in our laboratories. Thus, these amide-type lysine adducts may be very useful markers for estimating the protein damage by lipid peroxidation. Undoubtedly, other lipid-protein adducts and other oxidized lipids may play an important role in the LDL oxidation. For example, a high concentration of HNE (150 mM) has been detected in the lipid phase of LDL (28Esterbauer H. Ramos P. Chemistry and pathophysiology of oxidation of LDL.Rev. Physiol. Biochem. Pharmacol. 1996; 127: 31-64Google Scholar), and the presence of the HNE-modified protein adduct, the HNE-histidine Michael-type adduct (7–9 mol/mol LDL), has been estimated in oxLDL (6Uchida K. Toyokuni S. Nishikawa K. Kawakishi S. Oda H. Hiai H. Stadtman E.R. Michael addition-type 4-hydroxy-2-nonenal adducts in modified low-density lipoproteins: markers for atherosclerosis.Biochemistry. 1994; 33: 12487-12494Google Scholar). Although amide-type adducts, such as HEL and AZL, have not yet been quantified in oxLDL, the predominance of the amide-type lysine adducts during lipid peroxidation was supported by the demonstration of Onorato et al. that the major products from the reaction of oxidized linoleic acid with pyridoxamine, an inhibitor of glycation and lipid peroxidation, are hexanoyl-pyridoxamine and azelayl-pyridoxamine (29Onorato J.M. Jenkins A.J. Thorpe S.R. Baynes J.W. Pyridoxamine, an inhibitor of advanced glycation reactions, also inhibits advanced lipoxidation reactions. Mechanism of action of pyridoxamine.J. Biol. Chem. 2000; 275: 21177-21184Google Scholar). This raises the possibility that HEL and AZL may be one of the major lysine adducts in oxLDL, as well as the aldehyde-derived lysine adducts. The precise mechanism for the formation of amide-type lysine adducts has not yet been elucidated. It is interesting that the generation of amide-type adducts such as HEL and AZL was attenuated by preincubating 13-HPODE prior to the reaction with a lysine derivative (ref. 18 and unpublished observations). Similarly, the immunoreactivity of 13Ab to 13-HPODE-modified protein was also attenuated by preincubating 13-HPODE (unpublished observations). These observations suggest that the initial decomposed intermediate(s), but not stable end-products such as aliphatic aldehydes, may contribute to the formation of these amide-type adducts/antigenic epitopes. Fruebis et al. (23Fruebis J. Parthasarathy S. Steinberg D. Evidence for a concerted reaction between lipid hydroperoxides and polypeptides.Proc. Natl. Acad. Sci. USA. 1992; 89: 10588-10592Google Scholar) have reported the formation of fluorescent products upon the reaction of linoleic acid hydroperoxide with polylysine, although the structures of the presumed products remain to be proven. The report suggested a reaction mechanism initiated by the interaction of the amino group with the hydroperoxide group or the peroxy radical derived from a lipid hydroperoxide. This may in part support our prediction that initially formed unstable intermediate(s) may contribute to the formation of amide-type adducts, although amide-type adducts identified in this study were not fluorescent products. In addition to the modification of lysine residues, histidine and cysteine residues were also modified during the incubation with lipid hydroperoxides (16Kato Y. Makino Y. Osawa T. Characterization of a specific polyclonal antibody against 13-hydroperoxyoctadecadienoic acid-modified protein: formation of lipid hydroperoxide-modified apoB-100 in oxidized LDL.J. Lipid Res. 1997; 38: 1334-1346Google Scholar, 17Kato Y. Osawa T. Detection of oxidized phospholipid-protein adducts using anti-15-hydroperoxyeicosatetraenoic acid-modified protein antibody: contribution of esterified fatty acid-protein adduct to oxidative modification of LDL.Arch. Biochem. Biophys. 1998; 351: 106-114Google Scholar), and the reaction mixtures of 13-HPODE with the histidine or cysteine derivatives were also recognized by 13Ab (unpublished observations), suggesting that 13Ab recognizes multiple epitopes other than the AZL adduct, although the epitope structures in these reaction mixtures have not yet been identified. The mechanism of the decomposition processes of lipid hydroperoxides under physiological conditions is complicated, and therefore not completely resolved. In order to assess the biological consequence of lipid hydroperoxides, the detailed mechanism for the formation of amide-type adducts (and other unidentified histidine/cysteine epitopes) must be examined in the future. In summary, we found that one of the major epitopes of the antibody to the 13-HPODE-modified protein, 13Ab, was the novel AZL adduct. Using the antibody, we showed that the epitopes of 13Ab containing AZL and the esters were generated in rabbit atherosclerotic lesions. Carboxyalkylamide-type adducts, such as AZL, were universally generated from the reaction of oxidized polyunsaturated fatty acids with lysine residues. These results suggest that carboxyalkylamide-type adducts may be potential markers for the lipid peroxidation-derived modification of proteins associated with atherosclerosis and other oxidative stress-related diseases, and 13Ab may be a good tool for the detection of these lesions. The authors thank Dr. Koji Uchida (Nagoya University) for his helpful discussions. The authors also thank Yoshiaki Hongo for the preliminary study and Xiaohong Wu for the technical support of the animal experiments. arachidonic acid α-linolenic acid N ε-(azelayl)lysine Nα-benzoyl-glycyl-l-lysine N ε-(carboxymethyl)lysine docosahexaenoic acid 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide eicosapentaenoic acid N ε-(glutaroyl)lysine N ε-(hexanoyl)lysine 4-hydroxy-2-nonenal 15-hydroperoxyeicosatetraenoic acid 13-hydroperoxyoctadecadienoic acid liquid chromatography-mass spectrometry malondialdehyde N-hydroxysulfosuccinimide nuclear magnetic resonance N ε-(succinyl)lysine
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