title: Lipid peroxidation modification of protein generates Nepsilon-(4-oxononanoyl)lysine as a pro-inflammatory ligand.
abstract: 4-Oxo-2(E)-nonenal (ONE), a peroxidation product of ω-6 polyunsaturated fatty acids, covalently reacts with lysine residues to generate a 4-ketoamide-type ONE-lysine adduct, N(ε)-(4-oxononanoyl)lysine (ONL). Using an ONL-coupled protein as the immunogen, we raised the monoclonal antibody (mAb) 9K3 directed to the ONL and conclusively demonstrated that the ONL was produced during the oxidative modification of a low density lipoprotein (LDL) in vitro. In addition, we observed that the ONL was present in atherosclerotic lesions, in which an intense immunoreactivity was mainly localized in the vascular endothelial cells and macrophage- and vascular smooth muscle cell-derived foam cells. Using liquid chromatography with on-line electrospray ionization tandem mass spectrometry, we also established a highly sensitive method for quantification of the ONL and confirmed that the ONL was indeed formed during the lipid peroxidation-mediated modification of protein in vitro and in vivo. To evaluate the biological implications for ONL formation, we examined the recognition of ONL by the scavenger receptor lectin-like oxidized LDL receptor-1 (LOX-1). Using CHO cells stably expressing LOX-1, we evaluated the ability of ONL to compete with the acetylated LDL and found that both the ONE-modified and ONL-coupled proteins inhibited the binding and uptake of the modified LDL. In addition, we demonstrated that the ONL-coupled protein was incorporated into differentiated THP-1 cells via LOX-1. Finally, we examined the effect of ONL on the expression of the inflammation-associated gene in THP-1 and observed that the ONL-coupled proteins significantly induced the expression of atherogenesis-related genes, such as the monocyte chemoattractant protein-1 and tumor necrosis factor-α, in a LOX-1-dependent manner. Thus, ONL was identified to be a potential endogenous ligand for LOX-1.
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Shibata
authors.forename: Yuuki
authors.initials: Y
authors.lastname: Shimozu
authors.forename: Chika
authors.initials: C
authors.lastname: Wakita
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Shibata
authors.forename: Makio
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Sachiko
authors.initials: S
authors.lastname: Machida
authors.forename: Rina
authors.initials: R
authors.lastname: Kato
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Itabe
authors.forename: Xiaochun
authors.initials: X
authors.lastname: Zhu
authors.forename: Lawrence M
authors.initials: LM
authors.lastname: Sayre
authors.forename: Koji
authors.initials: K
authors.lastname: Uchida
mesh_terms: D000818:Animals; D050197:Atherosclerosis / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D018932:Chemokine CCL2 / Q000096:biosynthesis / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D018836:Inflammation Mediators / Q000276:immunology; D015227:Lipid Peroxidation; D008077:Lipoproteins, LDL / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D051127:Scavenger Receptors, Class E / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics
chemical_list: C111835:CCL2 protein, human; D018932:Chemokine CCL2; D018836:Inflammation Mediators; D008077:Lipoproteins, LDL; C495137:OLR1 protein, human; D051127:Scavenger Receptors, Class E; D014409:Tumor Necrosis Factor-alpha; D008239:Lysine