Uchida K, Itakura K, Kawakishi S, Hiai H, Toyokuni S, Stadtman E R
Laboratory of Food and Biodynamics, Nagoya University School of Agriculture, Japan.
Arch Biochem Biophys. 1995 Dec 20;324(2):241-8. doi: 10.1006/abbi.1995.0036.
In the present study, we have raised anti-peptide antibodies directed to the major membrane lipid peroxidation product 4-hydroxy-2-nonenal (HNE) attached covalently to histidine, and their specificities were compared with those of the polyclonal antibodies (anti-HNE-protein antibodies) raised against HNE-treated keyhole limpet hemocyanin (K. Uchida et al. (1993) Proc. Natl. Acad. Sci. USA 90, 8742-8746). The anti-HNE-histidyl peptide antibodies (anti-HNE-histidine antibodies) were prepared by immunizing rabbits with a HNE-conjugated heptapeptide (Gly3-His-Gly3 amide) coupled to the carrier protein. The antisera were purified on an affinity gel prepared by covalent attachment of a HNE-conjugated heptapeptide (Ala3-His-Ala3 amide). Among the structurally defined 4-hydroxy-2-alkenal-amino acid adducts tested, binding of anti-HNE-histidine antibodies to the HNE-treated protein was not only inhibited by HNE-histidine, HNE-cysteine, and HNE-lysine, but also by 4-hydroxy-2-octenal-histidine and 4-hydroxy-2-decenal-histidine adducts. Cross-reactivity studies revealed that both anti-HNE-protein antibodies had the highest affinity for the HNE-treated protein and that neither of the antibodies cross-reacted with the protein treated with aldehydes including malondialdehyde, 1-hexanal, 2-hexenal, or 2-nonenal. These results suggest that the dominant epitope recognized by antibodies is the 2-CH3(CH2)n-5-hydroxytetrahydrofuran (n > or = 3) moiety of the Michael adducts. The immunohistochemical analysis of atherosclerotic lesions of human aorta demonstrated that these antibodies reacted strongly with granular cytoplasmic elements of foam cells and weakly with elements in the surrounding sclerotic stroma.
在本研究中，我们制备了针对与组氨酸共价连接的主要膜脂质过氧化产物4-羟基-2-壬烯醛（HNE）的抗肽抗体，并将其特异性与针对经HNE处理的钥孔戚血蓝蛋白产生的多克隆抗体（抗HNE-蛋白质抗体）的特异性进行了比较（K. 内田等人（1993年）《美国国家科学院院刊》90，8742 - 8746）。抗HNE-组氨酰肽抗体（抗HNE-组氨酸抗体）是通过用与载体蛋白偶联的HNE-缀合七肽（Gly3-His-Gly3酰胺）免疫兔子制备的。抗血清在通过共价连接HNE-缀合七肽（Ala3-His-Ala3酰胺）制备的亲和凝胶上进行纯化。在所测试的结构明确的4-羟基-2-烯醛-氨基酸加合物中，抗HNE-组氨酸抗体与经HNE处理的蛋白质的结合不仅受到HNE-组氨酸、HNE-半胱氨酸和HNE-赖氨酸的抑制，还受到4-羟基-2-辛烯醛-组氨酸和4-羟基-2-癸烯醛-组氨酸加合物的抑制。交叉反应性研究表明，两种抗HNE-蛋白质抗体对经HNE处理的蛋白质具有最高亲和力，并且这两种抗体均未与用包括丙二醛、1-己醛、2-己烯醛或2-壬烯醛在内的醛处理的蛋白质发生交叉反应。这些结果表明，抗体识别的主要表位是迈克尔加成物的2-CH3(CH2)n-5-羟基四氢呋喃（n≥3）部分。对人主动脉动脉粥样硬化病变的免疫组织化学分析表明，这些抗体与泡沫细胞的颗粒状细胞质成分强烈反应，而与周围硬化基质中的成分反应较弱。
