Lipid Peroxidation Modification of Protein Generates Nε-(4-Oxononanoyl)lysine as a Pro-inflammatory Ligand
Lipid Peroxidation Modification of Protein Generates Nε-(4-Oxononanoyl)lysine as a Pro-inflammatory Ligand
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DOI：
10.1074/jbc.m110.187047
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发表时间：
2011-06-03
影响因子：
4.8
通讯作者：
Uchida, Koji
中科院分区：
文献类型：
--
作者：
Shibata, Takahiro;Shimozu, Yuuki;Uchida, Koji
欧米伽-6多不饱和脂肪酸的过氧化产物4-氧代-2(E)-壬烯醛(ONE)与赖氨酸残基共价反应，生成4-酮酰胺型1-赖氨酸加合物N-epsilon-(4-oxonanoyl)赖氨酸(ONL)。以ONL偶联蛋白为免疫原，制备了针对ONL的单抗9K3，证实ONL是在体外氧化修饰低密度脂蛋白(LDL)过程中产生的。此外，我们观察到ONL存在于动脉粥样硬化病变中，其中强烈的免疫反应主要定位于血管内皮细胞、巨噬细胞和血管平滑肌细胞来源的泡沫细胞。利用高效液相色谱在线电喷雾电离串联质谱仪，建立了一种高灵敏的ONL定量方法，证实ONL确实是在体内和体外脂质过氧化修饰蛋白质的过程中形成的。为了评估ONL形成的生物学意义，我们检测了清道夫受体凝集素样氧化型低密度脂蛋白受体-1(LOX-1)对ONL的识别。利用稳定表达LOX-1的CHO细胞，我们评估了ONL与乙酰化低密度脂蛋白竞争的能力，发现一个修饰的和ONL偶联的蛋白都抑制了修饰的低密度脂蛋白的结合和摄取。此外，我们还证明了ONL偶联蛋白通过LOX-1整合到分化的THP-1细胞中。最后，我们检测了ONL对THP-1炎症相关基因表达的影响，观察到ONL偶联蛋白以LOX-1依赖的方式显著诱导动脉粥样硬化相关基因的表达，如单核细胞趋化蛋白-1和肿瘤坏死因子-α。因此，ONL被确定为LOX-1的潜在内源性配体。
4-Oxo-2(E)-nonenal (ONE), a peroxidation product of omega-6 polyunsaturated fatty acids, covalently reacts with lysine residues to generate a 4-ketoamide-type ONE-lysine adduct, N-epsilon-(4oxononanoyl) 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-alpha, in a LOX-1-dependent manner. Thus, ONL was identified to be a potential endogenous ligand for LOX-1.