Nε-(3-methylpyridinium) lysine, a major antigenic adduct generated in acrolein-modified protein
Nε-(3-methylpyridinium) lysine, a major antigenic adduct generated in acrolein-modified protein
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DOI：
10.1074/jbc.m309401200
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发表时间：
2003-12-05
影响因子：
4.8
通讯作者：
Uchida, K
中科院分区：
文献类型：
--
作者：
Furuhata, A;Ishii, T;Uchida, K
丙烯醛是一种具有代表性的致癌醛，在氧化应激下在生物系统中普遍产生，与亲核试剂如蛋白质具有易反应性。为了更好地理解丙烯醛修饰蛋白质的分子基础，我们采用电喷雾离子化-液相色谱/质谱联用技术对模型肽（胰岛素氧化B链）进行了丙烯醛修饰，建立了一种新的丙烯醛-赖氨酸缩合反应。此外，我们发现，这种缩合加合物代表了丙烯醛修饰的蛋白质中产生的主要抗原加合物。为了鉴定丙烯醛修饰的胰岛素B链中产生的加合物的修饰位点和结构，将丙烯醛预处理和未处理的肽用V8蛋白酶消化，并将所得肽进行电喷雾电离-液相色谱/质谱分析。该技术鉴定了9种肽，其在Lys-29和N末端含有丙烯醛加合物，并揭示了胰岛素B链与丙烯醛的反应产生多种加合物，包括一种未知的加合物，其每个赖氨酸含有两个丙烯醛分子。为了鉴定这种加合物，我们将N-α-乙酰赖氨酸与丙烯醛孵育，并分离出与未知的丙烯醛-赖氨酸加合物具有相同分子量的产物。根据化学和光谱证据，确定该加合物是一种新的吡啶鎓型赖氨酸加合物，N-α-（3-甲基吡啶鎓）赖氨酸（MP-赖氨酸）。MP-赖氨酸的形成通过用丙烯醛处理的蛋白质的氨基酸分析来证实。更值得注意的是，该缩合加合物似乎是针对丙烯醛修饰的蛋白质产生的单克隆抗体5 F6的固有表位。
Acrolein, a representative carcinogenic aldehyde, that could be ubiquitously generated in biological systems under oxidative stress shows facile reactivity with a nucleophile such as a protein. In this study, to gain a better understanding of the molecular basis of acrolein modification of protein, we characterized the acrolein modification of a model peptide ( the oxidized B chain of insulin) by electrospray ionization-liquid chromatography/ mass spectrometry method and established a novel acrolein-lysine condensation reaction. In addition, we found that this condensation adduct represented the major antigenic adduct generated in acrolein-modified protein. To identify the modification site and structures of adducts generated in the acrolein-modified insulin B chain, both the acrolein-pretreated and untreated peptides were digested with V8 protease and the resulting peptides were subjected to electrospray ionization-liquid chromatography/ mass spectrometry. This technique identified nine peptides, which contained the acrolein adducts at Lys-29 and the N terminus, and revealed that the reaction of the insulin B chain with acrolein gave multiple adducts, including an unknown adduct containing two molecules of acrolein per lysine. To identify this adduct, we incubated N-alpha-acetyllysine with acrolein and isolated a product having the same molecular mass as the unknown acrolein-lysine adduct. On the basis of the chemical and spectroscopic evidence, the adduct was determined to be a novel pyridinium-type lysine adduct, N-epsilon-(3-methylpyridinium) lysine (MP-lysine). The formation of MP-lysine was confirmed by amino acid analysis of proteins treated with acrolein. More notably, this condensation adduct appeared to be an intrinsic epitope of a monoclonal antibody 5F6 that had been raised against acrolein-modified protein.