Graduate Toxicology Program, Department of Veterinary Sciences, Utah State University, Logan, UT 84322, USA.
Comp Biochem Physiol C Toxicol Pharmacol. 2013 Aug;158(2):109-16. doi: 10.1016/j.cbpc.2013.05.007. Epub 2013 May 25.
Hepatic glutathione S-transferases (GSTs: EC2.5.1.1.8) catalyze the detoxification of reactive electrophilic compounds, many of which are toxic and carcinogenic intermediates, via conjugation with the endogenous tripeptide glutathione (GSH). Glutathione S-transferase (GST)-mediated detoxification is a critical determinant of species susceptibility to the toxic and carcinogenic mycotoxin aflatoxin B1 (AFB1), which in resistant animals efficiently detoxifies the toxic intermediate produced by hepatic cytochrome P450 bioactivation, the exo-AFB1-8,9-epoxide (AFBO). Domestic turkeys (Meleagris gallopavo) are one of the most sensitive animals known to AFB1, a condition associated with a deficiency of hepatic GST-mediated detoxification of AFBO. We have recently shown that unlike their domestic counterparts, wild turkeys (Meleagris gallopavo silvestris), which are relatively resistant, express hepatic GST-mediated detoxification activity toward AFBO. Because of the importance of GSTs in species susceptibility, and to explore possible GST classes involved in AFB1 detoxification, we amplified, cloned, expressed and functionally characterized the hepatic mu-class GSTs tGSTM3 (GenBank accession no. JF340152), tGSTM4 (JF340153) from domestic turkeys, and a GSTM4 variant (ewGSTM4, JF340154) from Eastern wild turkeys. Predicted molecular masses of tGSTM3 and two tGSTM4 variants were 25.6 and 25.8kDa, respectively. Multiple sequence comparisons revealed four GSTM motifs and the mu-loop in both proteins. tGSTM4 has 89% amino acid sequence identity to chicken GSTM2, while tGSTM3 has 73% sequence identity to human GSTM3 (hGSTM3). Specific activities of Escherichia coli-expressed tGSTM3 toward 1-chloro-2,4-dinitrobenzene (CDNB) and peroxidase activity toward cumene hydroperoxide were five-fold greater than tGSTM4 while tGSTM4 possessed more than three-fold greater activity toward 1,2-dichloro-4-nitrobenzene (DCNB). The two enzymes displayed equal activity toward ethacrynic acid (ECA). However, none of the GSTM proteins had AFBO detoxification capability, in contrast to recombinant alpha-class GSTs shown in our recent study to possess this important activity. In total, our data indicate that although turkey hepatic GSTMs may contribute to xenobiotic detoxification, they probably play no role in detoxification of AFBO in the liver.
肝谷胱甘肽 S-转移酶（GSTs：EC2.5.1.1.8）催化反应性亲电化合物的解毒，其中许多是有毒和致癌的中间产物，通过与内源性三肽谷胱甘肽（GSH）缀合。GST 介导的解毒是物种对黄曲霉毒素 B1（AFB1）的毒性和致癌性的易感性的关键决定因素，在抗性动物中，AFB1 有效地解毒由肝细胞色素 P450 生物活化产生的有毒中间产物，即外-AFB1-8,9-环氧化物（AFBO）。火鸡（Meleagris gallopavo）是对 AFB1 最敏感的动物之一，这种情况与肝 GST 介导的 AFBO 解毒缺陷有关。我们最近发现，与国内同类动物不同，野生火鸡（Meleagris gallopavo silvestris）相对具有抵抗力，它们对 AFBO 具有肝 GST 介导的解毒活性。由于 GST 在物种易感性中的重要性，以及探索可能参与 AFB1 解毒的 GST 类别，我们从家养火鸡中扩增、克隆、表达和功能表征了肝 mu 类 GSTs tGSTM3（GenBank 登录号 JF340152）、tGSTM4（JF340153），并从东部野生火鸡中分离出 GSTM4 变体（ewGSTM4，JF340154）。tGSTM3 和两种 tGSTM4 变体的预测分子量分别为 25.6 和 25.8kDa。多重序列比较显示两种蛋白均具有四个 GST 基序和 mu 环。tGSTM4 与鸡 GSTM2 的氨基酸序列同一性为 89%，而 tGSTM3 与人类 GSTM3（hGSTM3）的序列同一性为 73%。大肠杆菌表达的 tGSTM3 对 1-氯-2,4-二硝基苯（CDNB）的比活性和对过氧化枯烯的过氧化物酶活性分别比 tGSTM4 高五倍，而 tGSTM4 对 1,2-二氯-4-硝基苯（DCNB）的活性则高出三倍以上。两种酶对 ethacrynic acid（ECA）的活性相同。然而，与我们最近研究中显示具有这种重要活性的重组α类 GST 相反，没有一种 GST 蛋白具有 AFBO 解毒能力。总的来说，我们的数据表明，尽管火鸡肝 GSTMs 可能有助于外来物解毒，但它们可能在肝脏中对 AFBO 的解毒没有作用。
