Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan and.
Department of Pathology, Tokyo Women's Medical University, Tokyo 162-8666, Japan.
J Biol Chem. 2011 Aug 19;286(33):29313-29324. doi: 10.1074/jbc.M111.255737. Epub 2011 Jun 20.
α,β-Unsaturated aldehydes generated during lipid peroxidation, such as 4-oxoalkenals and 4-hydroxyalkenals, can give rise to protein degeneration in a variety of pathological states. Although the covalent modification of proteins by these end products has been well studied, the reactivity of unstable intermediates possessing a hydroperoxy group, such as 4-hydroperoxy-2-nonenal (HPNE), with protein has received little attention. We have now established a unique protein modification in which the 4-hydroperoxy group of HPNE is involved in the formation of structurally unusual lysine adducts. In addition, we showed that one of the HPNE-specific lysine adducts constitutes the epitope of a monoclonal antibody raised against the HPNE-modified protein. Upon incubation with bovine serum albumin, HPNE preferentially reacted with the lysine residues. By employing N(α)-benzoylglycyl-lysine, we detected two major products containing one HPNE molecule per peptide. Based on the chemical and spectroscopic evidence, the products were identified to be the N(α)-benzoylglycyl derivatives of N(ε)-4-hydroxynonanoic acid-lysine and N(ε)-4-hydroxy-(2Z)-nonenoyllysine, both of which are suggested to be formed through mechanisms in which the initial HPNE-lysine adducts undergo Baeyer-Villiger-like reactions proceeding through an intramolecular oxidation catalyzed by the hydroperoxy group. On the other hand, using an HPNE-modified protein as the immunogen, we raised a monoclonal antibody against the HPNE-modified protein and identified one of the HPNE-specific lysine adducts, N(ε)-4-hydroxynonanoic acid-lysine, as an intrinsic epitope of the monoclonal antibody. Furthermore, we demonstrated that the HPNE-specific epitopes were produced not only in the oxidized low density lipoprotein in vitro but also in the atherosclerotic lesions. These results indicated that HPNE is not just an intermediate but also a reactive molecule that could covalently modify proteins in biological systems.
脂质过氧化过程中产生的α,β-不饱和醛，如 4-氧代烯醛和 4-羟基烯醛，可以在多种病理状态下导致蛋白质变性。尽管这些终产物对蛋白质的共价修饰已经得到了很好的研究，但具有过氧基的不稳定中间体的反应性，如 4-过氧基-2-壬烯醛（HPNE）与蛋白质的反应性却很少受到关注。我们现在已经建立了一种独特的蛋白质修饰方式，其中 HPNE 的 4-过氧基参与形成结构异常的赖氨酸加合物。此外，我们还表明，HPNE 特异性赖氨酸加合物之一构成了针对 HPNE 修饰蛋白的单克隆抗体的抗原决定簇。在与牛血清白蛋白孵育时，HPNE 优先与赖氨酸残基反应。通过使用 N(α)-苯甲酰基甘氨酸-赖氨酸，我们检测到两种主要产物，每个肽中含有一个 HPNE 分子。基于化学和光谱证据，这些产物被鉴定为 N(ε)-4-羟基壬酸-赖氨酸和 N(ε)-4-羟基-(2Z)-壬烯酰基赖氨酸的 N(α)-苯甲酰基衍生物，这两种产物都被认为是通过初始 HPNE-赖氨酸加合物通过过氧基催化的分子内氧化反应形成的。另一方面，我们使用 HPNE 修饰的蛋白质作为免疫原，针对 HPNE 修饰的蛋白质产生了一种单克隆抗体，并鉴定出 HPNE 特异性赖氨酸加合物之一，N(ε)-4-羟基壬酸-赖氨酸，是单克隆抗体的内在抗原决定簇。此外，我们还证明，HPNE 特异性表位不仅在体外氧化的低密度脂蛋白中产生，而且在动脉粥样硬化病变中也产生。这些结果表明，HPNE 不仅是一种中间体，而且是一种能够在生物系统中共价修饰蛋白质的反应性分子。
