Loeber Rachel, Rajesh Mathur, Fang Qingming, Pegg Anthony E, Tretyakova Natalia
University of Minnesota Cancer Center and Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Chem Res Toxicol. 2006 May;19(5):645-54. doi: 10.1021/tx0600088.
1,2,3,4-Diepoxybutane (DEB) is a key carcinogenic metabolite of the important industrial chemical 1,3-butadiene. DEB is a bifunctional alkylating agent capable of reacting with DNA and proteins. Initial DNA alkylation by DEB produces N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-guanine monoadducts, which can react with another nucleophilic site to form cross-linked adducts. A recent report revealed a strong correlation between cellular expression of the DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) and the cytotoxic and mutagenic activity of DEB, suggesting that DEB induces AGT-DNA cross-links (Valadez, J. G., et al. (2004) Activation of bis-electrophiles to mutagenic conjugates by human O6-alkylguanine-DNA alkyltransferase. Chem. Res. Toxicol. 17, 972-982). The purpose of our study was to analyze the formation and structures of DEB-induced AGT-DNA conjugates and to identify specific amino acid residues within the protein involved in cross-linking. DNA-protein cross-link formation was detected by SDS-PAGE when 32P-labeled double-stranded oligodeoxynucleotides were exposed to DEB in the presence of either wild-type hAGT or a C145A hAGT mutant. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of hAGT that had been treated with N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-deoxyguanosine (dG monoepoxide) revealed the ability of the protein to form either one or two butanediol-dG cross-links, corresponding to mass shifts of +353 and +706 Da, respectively. HPLC-ESI+ -MS/MS sequencing of the tryptic peptides obtained from dG monoepoxide-treated protein indicated that the two cross-linking sites were the alkyl acceptor site, Cys145, and a neighboring active site residue, Cys150. The same two amino acid residues of hAGT became covalently cross-linked to DNA following DEB treatment. Modification of Cys145 was further confirmed by HPLC-ESI+ -MS/MS analysis of dG monoepoxide-treated synthetic peptide GNPVPILIPCHR which represents the active site tryptic fragment of hAGT (C = Cys145). The replacement of the catalytic cysteine residue with alanine in the C145A hAGT mutant abolished DEB-induced cross-linking at this site, while the formation of conjugates via neighboring Cys150 was retained. The exact chemical structure of the cross-linked lesion was established as 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol by HPLC-ESI+ -MS/MS analysis of the amino acids resulting from the total digestion of modified proteins analyzed in parallel with an authentic standard. AGT-DNA cross-linking is a likely mechanism of DEB-mediated cytotoxicity in cells expressing this important repair protein.
1,2,3,4-二环氧丁烷（DEB）是重要工业化学品1,3-丁二烯的关键致癌代谢产物。DEB是一种双功能烷基化剂，能够与DNA和蛋白质发生反应。DEB对DNA的初始烷基化作用会产生N7-（2'-羟基-3',4'-环氧丁-1'-基）-鸟嘌呤单加合物，该单加合物可与另一个亲核位点反应形成交联加合物。最近的一份报告显示，DNA修复蛋白O6-烷基鸟嘌呤-DNA烷基转移酶（AGT）的细胞表达与DEB的细胞毒性和诱变活性之间存在很强的相关性，这表明DEB可诱导AGT-DNA交联（瓦拉德兹，J.G.等人，（2004年）人O6-烷基鸟嘌呤-DNA烷基转移酶将双亲电子试剂激活为诱变共轭物。《化学研究毒理学》17卷，972 - 982页）。我们研究的目的是分析DEB诱导的AGT-DNA共轭物的形成和结构，并确定蛋白质中参与交联的特定氨基酸残基。当32P标记的双链寡脱氧核苷酸在野生型hAGT或C145A hAGT突变体存在的情况下暴露于DEB时，通过SDS-PAGE检测到DNA-蛋白质交联的形成。对用N7-（2'-羟基-3',4'-环氧丁-1'-基）-脱氧鸟苷（dG单环氧化物）处理过的hAGT进行毛细管HPLC-电喷雾电离质谱（ESI-MS）分析，结果显示该蛋白质能够形成一个或两个丁二醇-dG交联，分别对应于质量偏移+353和+706 Da。对从dG单环氧化物处理过的蛋白质中获得的胰蛋白酶肽段进行HPLC-ESI + -MS/MS测序表明，两个交联位点分别是烷基接受位点Cys145和相邻的活性位点残基Cys150。在DEB处理后，hAGT的相同两个氨基酸残基与DNA发生了共价交联。通过对dG单环氧化物处理的合成肽GNPVPILIPCHR（代表hAGT的活性位点胰蛋白酶片段，C = Cys145）进行HPLC-ESI + -MS/MS分析，进一步证实了Cys145的修饰。在C145A hAGT突变体中，用丙氨酸取代催化性半胱氨酸残基消除了DEB在此位点诱导的交联，而通过相邻的Cys150形成共轭物的过程得以保留。通过对与真实标准品平行分析的修饰蛋白质完全消化后产生的氨基酸进行HPLC-ESI + -MS/MS分析，确定交联损伤的确切化学结构为1-（S-半胱氨酰基）-4-（鸟嘌呤-7-基）-2,3-丁二醇。在表达这种重要修复蛋白的细胞中，AGT-DNA交联可能是DEB介导细胞毒性的一种机制。
