Davies Sean S, Talati Megha, Wang Xiahong, Mernaugh Raymond L, Amarnath Venkataraman, Fessel Joshua, Meyrick Barbara O, Sheller James, Roberts L Jackson
Department of Pharmacology, Vanderbilt University, Nashville, TN 37232-6602, USA.
Free Radic Biol Med. 2004 May 1;36(9):1163-74. doi: 10.1016/j.freeradbiomed.2004.02.014.
Isoketals are highly reactive gamma-ketoaldehydes formed by the oxidation of arachidonic acid that rapidly adduct to proteins. To investigate the formation of isoketal adducts in vivo, we isolated and characterized a single-chain antibody from a phage displayed recombinant ScFv library that bound a model peptide adducted with synthetic 15-E2-isoketal. Recognition of isoketal adduct by this anti-isoketal adduct single-chain antibody was essentially independent of the amino acid sequence of adducted peptides or proteins. The antibody did not cross-react with 4-hydroxynonenal or 4-oxononanal adducts or with 15-F2t-isoprostane (8-iso-prostaglandin F2alpha). We investigated the formation of isoketal adducts in a well-established model of oxidative injury, hyperoxia. Exposure to >98% oxygen for 7 h dramatically increased both the number of immunoreactive airway epithelial cells and the intensity of immunoreactivity compared with animals exposed to normal room air (21% oxygen). We conclude that isoketal adducts form in epithelial cells as a result of high oxygen exposure and that this single-chain antibody provides a valuable tool to localize the formation of isoketal adducts in tissues in vivo.
异前列腺素是由花生四烯酸氧化形成的高反应性γ-酮醛，可迅速与蛋白质加合。为了研究体内异前列腺素加合物的形成，我们从噬菌体展示的重组单链抗体可变区（ScFv）文库中分离并鉴定了一种单链抗体，该抗体可与合成的15-E2-异前列腺素加合的模型肽结合。这种抗异前列腺素加合物单链抗体对异前列腺素加合物的识别基本上不依赖于加合肽或蛋白质的氨基酸序列。该抗体与4-羟基壬烯醛或4-氧代壬醛加合物或15-F2t-异前列腺素（8-异前列腺素F2α）无交叉反应。我们在一个成熟的氧化损伤模型——高氧模型中研究了异前列腺素加合物的形成。与暴露于正常室内空气（21%氧气）的动物相比，暴露于>98%氧气7小时显著增加了免疫反应性气道上皮细胞的数量和免疫反应强度。我们得出结论，高氧暴露导致上皮细胞中形成异前列腺素加合物，并且这种单链抗体为体内定位组织中异前列腺素加合物的形成提供了一种有价值的工具。
