Heterologous expression and functional characterization of avian mu-class glutathione S-transferases Brett R. Bunderson a , Ji Eun Kim a,1 , Amanda Croasdell a , Kristelle M. Mendoza b , Kent M. Reed b , Roger A. Coulombe Jr. a, ⁎ a Graduate Toxicology Program, Department of Veterinary Sciences, Utah State University, Logan, UT 84322, United States b Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, MN 55108, United States abstract article info Article history: Received 12 April 2013 Received in revised form 21 May 2013 Accepted 21 May 2013 Available online 25 May 2013 Keywords: Glutathione S-transferases mu-Class GST Aflatoxin B 1 Turkey Detoxification 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 determi- nant of species susceptibility to the toxic and carcinogenic mycotoxin aflatoxin B 1 (AFB 1 ), which in resistant animals efficiently detoxifies the toxic intermediate produced by hepatic cytochrome P450 bioactivation, the exo-AFB 1 -8,9-epoxide (AFBO). Domestic turkeys (Meleagris gallopavo) are one of the most sensitive animals known to AFB 1 , 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 AFB 1 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 25.8 kDa, 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. © 2013 Published by Elsevier Inc. 1. Introduction Glutathione S-transferases (GSTs) (EC 2.5.2.18) are a family of multifunctional enzymes important in detoxification of electrophilic intermediates through conjugation with the endogenous peptide glutathione (GSH) (Hayes et al., 2005). There are three major types of GSTs: cytosolic, mitochondrial, and membrane-associated proteins in eicosanoid and glutathione metabolism, however the cy- tosolic forms are primarily associated with detoxification (MAPEG) (Hayes et al., 2005). Further classification of cytosolic GSTs based on functional and sequence similarities places the enzymes in separate classes, the members of which generally exist as dimers composed of subunits within the same class (Wilce and Parker, 1994). Mammalian cytosolic GSTs have been well studied and include alpha, mu, pi, omega, sigma, theta, and zeta classes (Frova, 2006). Modern domestic turkeys are one of the most susceptible animals known to the ubiquitous worldwide food and feed contaminant AFB 1 , an important cause of liver cancer worldwide in humans. This suscep- tibility is historically important because it was instrumental to the discovery of AFB 1 in the etiology of “Turkey X Disease,” responsible for the deaths of hundreds of thousands of domestic turkeys in Europe traced to contaminated feed (Rawal et al., 2010a). In most animals studied, GSTs are central to detoxifying the hepato- toxic and hepatocarcinogenic mycotoxin aflatoxin B 1 (AFB 1 ), a common contaminant of corn and peanut-based foods and feeds (Eaton and Comparative Biochemistry and Physiology, Part C 158 (2013) 109–116 ⁎ Corresponding author. Tel.: +1 435 797 1598. E-mail address: roger@usu.edu (R.A. Coulombe). 1 Present address: Avian Research Center, University of British Columbia, 2357 Main Mall, Vancouver, BC V6T 1Z4, Canada. 1532-0456/$ – see front matter © 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.cbpc.2013.05.007 Contents lists available at SciVerse ScienceDirect Comparative Biochemistry and Physiology, Part C journal homepage: www.elsevier.com/locate/cbpc