Structural basis of protein-bound endogenous aldehydes - Chemical and immunochemical characterizations of configurational isomers of a 4-hydroxy-2-nonenal-histidine adduct
Structural basis of protein-bound endogenous aldehydes - Chemical and immunochemical characterizations of configurational isomers of a 4-hydroxy-2-nonenal-histidine adduct
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DOI：
10.1074/jbc.m210129200
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发表时间：
2003-02-14
影响因子：
4.8
通讯作者：
Uchida, K
中科院分区：
文献类型：
--
作者：
Hashimoto, M;Sibata, T;Uchida, K
4-羟基-2-壬烯醛（HNE）是脂质过氧化反应的主要外消旋产物，它与组氨酸反应生成稳定的HNE-组氨酸Michael加成型加合物，该加合物在环状半缩醛结构中具有三个手性中心。在本研究中，我们通过NMR光谱和分子轨道计算表征了HNE-Nalpha-乙酰组氨酸加合物的构型异构体。此外，我们提出了针对（R）-HNE-组氨酸和（S）-HNE-组氨酸加合物的单克隆抗体，其特征在于它们的特异性，并检查在氧化应激下的每个加合物的体内定位。为了促进HNE-组氨酸加合物的构型异构体的结构表征，我们通过将N-α-乙酰组氨酸与每种HNE对映异构体孵育来制备（R）-HNE-组氨酸和（S）-HNE-组氨酸加合物，这两者在反相高效液相色谱中均提供两个峰（来自（R）-HNE-组氨酸的Ra和Rb以及来自（S）-HNE-组氨酸加合物的Sa和Sb）。核磁共振分析表明每个峰都是两种非对映体的混合物。此外，核奥弗豪泽效应的分析，使八个异构体的配置的确定。这些异构体在NMR分析中的相对量与通过分子轨道方法计算的相对能量相关。另一方面，使用（R）-HNE修饰的和（S）-HNE修饰的钥孔血蓝蛋白作为抗原，我们制备了单克隆抗体mAbR 310和mAbS 412，其分别对映选择性地识别（R）-HNE-组氨酸和（S）-HNE-组氨酸加合物。在非对映异构体的混合物（Ra、Rb、Sa和Sb）中，mAbR 310对Rb（2 R，4S，5 R和2S，4S，5 R异构体的混合物）显示出最高的免疫反应性，而mAbS 412优先识别Sa（2 R，4S，5S和2S，4S，5S异构体的混合物）。在暴露于肾致癌物次氮基三乙酸铁的大鼠肾脏中，用化学方法检测体内（R）-HNE和（S）-HNE表位的存在，通过分别用mAbR 310和mAbS 412检测核和胞质染色。
4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, reacts with histidine to form a stable HNE-histidine Michael addition-type adduct possessing three chiral centers in the cyclic hemiacetal structure. In the present study, we characterized configurational isomers of a HNE-Nalpha-acetylhistidine adduct by NMR spectroscopy and by molecular orbital calculations. In addition, we raised monoclonal antibodies against (R)-HNE-histidine and (S)-HNE-histidine adducts, characterized their specificities, and examined in vivo localizations of each adduct under oxidative stress. To facilitate structural characterization of the configurational isomers of an HNE-histidine adduct, we prepared the (R)-HNE-histidine and (S)-HNE-histidine adducts by incubating N-alpha-acetylhistidine with each HNE enantiomer, both of which provided two peaks (Ra and Rb from (R)-HNE-histidine and Sa and Sb from (S)-HNE-histidine adducts) in reversed-phase high-performance liquid chromatography. The NMR analysis showed that each peak was a mixture of two diastereomers. In addition, the analysis of the nuclear Overhauser effect enabled the determination of configurations of the eight isomers. The relative amounts of these isomers in the NMR analysis correlated with the relative energies calculated by molecular orbital methods. On the other hand, using (R)-HNE-modified and (S)-HNE-modified keyhole limpet hemocyanins as the antigens, we raised the monoclonal antibodies, mAbR310 and mAbS412, which enantioselectively recognized the (R)-HNE-histidine and (S)-HNE-histidine adducts, respectively. Among the mixtures (Ra, Rb, Sa, and Sb) of diastereomers, mAbR310 showed the highest immunoreactivity to Rb (the mixture of 2R,4S,5R and 2S,4S,5R isomers), whereas mAbS412 preferentially recognized Sa (the mixture of 2R,4S,5S and 2S,4S,5S isomers). The presence of (R)-HNE and (S)-HNE epitopes in vivo was immunohistochemically examined in the kidney of rats exposed to the renal carcinogen, ferric nitrilotriatcetate, by which nuclear and cytosolic stainings with mAbR310 and mAbS412, respectively, were detected.