Chen Hauh-Jyun Candy, Chiu Wei-Loong, Lin Wen-Peng, Yang Siou-Siou
Department of Chemistry and Biochemistry, National Chung Cheng University, 168 University Road, Chia-Yi 62142, Taiwan.
Chembiochem. 2008 May 5;9(7):1074-81. doi: 10.1002/cbic.200700686.
Reactive nitrogen species are implicated in inflammatory diseases and cancers. Oxanine (Oxa) is a DNA lesion product originating from the guanine base through exposure to nitric oxide, nitrous acid, or N-nitrosoindoles. Oxanine was found to mediate formation of DNA-protein cross-links (DPCs) in the cell extract. We have previously characterized two DNA-protein cross-links from the reaction between Oxa and glutathione: namely, the thioester and the amide. In this study, lysozyme was used to study site-specific modification on protein by Oxa moieties in DNA. With the aid of nanoLC coupled with nanospray ionization tandem mass spectrometry, addition of Oxa was found at Lys13, Lys97, Lys116, Ser85, and Ser86 of lysozyme when it was treated with 2'-deoxyoxanosine (dOxo). Furthermore, incubation of lysozyme with Oxa-containing calf thymus DNA, produced by treating DNA with nitrous acid, led to lysozyme modification at Lys116, Ser85, and Ser86. Interestingly, none of the cysteine residues was modified by dOxo, in contrast with our previous findings that dOxo reacted with oxidized glutathione disulfide, forming the thioester. This might be due to the half-life of the dOxo-derived thioester being 2.2 days at the pH of incubation. Furthermore, the sites of modifications on lysozyme are in good agreement with the solvent accessibility of the residues. Since repair of Oxa-derived DPCs has not been extensively investigated, these results suggest that these stable DPCs might represent important forms of cellular damage caused by reactive nitrogen species involved in inflammationrelated diseases.
活性氮物质与炎症性疾病和癌症有关。氧杂尿嘧啶（Oxa）是一种DNA损伤产物，它由鸟嘌呤碱基通过暴露于一氧化氮、亚硝酸或N-亚硝基吲哚而产生。研究发现氧杂尿嘧啶能在细胞提取物中介导DNA-蛋白质交联（DPCs）的形成。我们之前已经鉴定了氧杂尿嘧啶与谷胱甘肽反应产生的两种DNA-蛋白质交联：即硫酯和酰胺。在本研究中，使用溶菌酶来研究DNA中氧杂尿嘧啶部分对蛋白质的位点特异性修饰。借助纳升液相色谱与纳喷雾电离串联质谱联用技术，发现用2'-脱氧氧杂尿苷（dOxo）处理溶菌酶时，在溶菌酶的Lys13、Lys97、Lys116、Ser85和Ser86位点有氧杂尿嘧啶的添加。此外，用亚硝酸处理小牛胸腺DNA产生含氧杂尿嘧啶的小牛胸腺DNA与溶菌酶孵育，导致溶菌酶在Lys116、Ser85和Ser86位点发生修饰。有趣的是，与我们之前发现dOxo与氧化型谷胱甘肽二硫化物反应形成硫酯的结果相反，没有半胱氨酸残基被dOxo修饰。这可能是由于在孵育pH值下，dOxo衍生的硫酯半衰期为2.2天。此外，溶菌酶上的修饰位点与这些残基的溶剂可及性高度一致。由于氧杂尿嘧啶衍生的DPCs的修复尚未得到广泛研究，这些结果表明这些稳定的DPCs可能代表了炎症相关疾病中活性氮物质引起的细胞损伤的重要形式。
