Comparative genomics identifies new alpha class genes within the avian glutathione S-transferase gene cluster
Introduction
Glutathione S-transferases (GSTs; E.C.2.5.1.18), a superfamily of multifunctional dimeric proteins, are important phase II biotransformation enzymes involved in cellular detoxification and excretion of a variety of xenobiotic substances (Eaton and Bammler, 1999, Frova, 2006). Carcinogens, environmental toxins and products of oxidative stress are detoxified by GSTs which principally catalyze the conjugation of reactive, electrophilic atoms with reduced glutathione (GSH) (Konishi et al., 2005, Salinas and Wong, 1999). Because of their importance in disease resistance, cancer susceptibility, and responsiveness to drug therapy, mammalian GSTs have been intensively studied. GSTs are primarily cytosolic enzymes, but microsomal forms also exist (Kelner et al., 1996). Cytosolic GSTs exist as dimeric subunits of 23–30 k Da with an average length of 199–244 amino acids (Hayes and Pulford, 1995, Mannervik and Danielson, 1988). Each subunit is composed of two spatially distinct domains. The N-terminal domain I has an α/β structure consisting of four β-strands and three α-helices. Domain II contains a larger α domain with five to six α-helices. There are two ligand-binding sites per subunit: a specific GSH-binding site (G-site) and the hydrophobic substrate binding site (H-site) (Frova, 2006, Sun et al., 1998).
Cytosolic GSTs from human, rat, and mouse have been well studied and are assigned to one of seven classes [alpha (α), mu (μ), pi (π), theta (τ), sigma (σ), zeta (ζ), omega (ο)] based on amino acid similarities (Frova, 2006, Hayes et al., 2005). Human GSTs are diverse and most abundantly expressed in the liver. Members of each class tend to have high sequence identity (> 60%)(Board, 1998) and individual genes for each human GST class are clustered together on the same chromosome (Board and Webb, 1987). Human α-class GSTs (hGSTA) are well documented with five functional genes (hGSTA1-hGSTA5) and seven pseudogenes on chromosome 6p12.1-6p12.2 (Coles and Kadlubar, 2005, Morel et al., 2002).
Avian GSTs comprise a complex isoenzyme system that has received much less attention (Yeung and Gidari, 1980). According to electrophoretic mobility on SDS/PAGE, five groups of GST subunits (designated CL1–CL5) have been identified in the cytosolic fraction of Leghorn chick livers (Chang et al., 1990). Searches of Expressed Sequence Tag (EST) databases have isolated α (Chang et al., 1990, Chang et al., 1992, Liu et al., 1993), μ (Liu and Tam, 1991, Sun et al., 1998), τ (Hsiao et al., 1995) and σ (Thomson et al., 1998) classes from cDNA sequences of the domestic chicken. Full-length cDNA of α-class GSTs was isolated and heterologously expressed in baculovirus and Escherichia coli system using GST-specific substrates (Liu et al., 1997, Liu et al., 1993). Nine class-alpha isozymes with distinctive molecular masses were affinity purified from chicken livers and partially cloned and characterized (Hsieh et al., 1999); clustering of chicken ESTs accessioned in Genbank suggests expression of six separate α-class transcripts. The nomenclature of chicken α-class GSTs was recently re-named (GenBank accession nos.) as cGSTA1 (NM_001001777), cGSTA2 (NM_001001776), cGSTA3 (NM_204818), and cGSTA-CL3 (M38219) based on subunit nomenclature proposed by (Mannervik et al., 1992).
In nearly all animals studied, GSTs are the principal detoxification enzymes for aflatoxin B1 (AFB1), a ubiquitous food and feed-borne mycotoxin that is a potent animal and human hepatotoxin and carcinogen (Coulombe, 1993, Newberne and Butler, 1969). To exert its toxic and carcinogenic effects, AFB1 requires metabolic activation to the highly reactive electrophilic and carcinogenic intermediate, the exo-AFB1-8,9-epoxide (AFBO), catalyzed by hepatic cytochromes P450 (CYPs) (Hayes et al., 1991b, Swenson et al., 1975). When functional, GSTs catalyze the conjugation of GSH to AFBO, thereby rendering it non-toxic and easily excretable. We recently amplified and cloned from turkey CYP1A5 and CYP3A37, high-affinity enzymes mostly responsible for AFB1 bioactivation in turkey liver (Rawal et al., 2009, Yip and Coulombe, 2006).
In rodents, α-class GSTs are the most efficient isozymes in detoxifying the AFBO (Hayes et al., 1991a, Hayes et al., 1991b). Mouse liver cytosol almost exclusively conjugates the exo-AFBO through the activity of its α-class GST (Raney et al., 1992), designated Gsta3, which has a high affinity toward AFBO, has been shown to be critical in the relative resistance of mice toward AFB1 (Ramsdell and Eaton, 1990). Rat constitutively express only small amount of α-class GST with high AFBO activity (rGSTA5-5) and thus are sensitive to AFB1-induced hepatocarcinogenesis (Hayes and Pulford, 1995, Wang et al., 2002). In contrast to rodents, constitutively expressed human hepatic α-class GSTs has little or no AFBO detoxifying activity(Raney et al., 1992, Slone et al., 1995).
Turkeys are one of the most susceptible animals known to AFB1 (Giambrone et al., 1985, Hamilton et al., 1972). Even small amounts in the diet cause severe hepatotoxicosis and reduction in growth rate, feed efficiency and hatchability, acute hepatic necrosis, and increased susceptibility to bacterial and viral diseases(Kubena et al., 1995, Pier et al., 1980).
There is currently little information available on the nature of GSTs in turkeys. Using prototype substrates we have demonstrated that turkey liver possesses active GST activity, but none with measurable GST-mediated detoxification of AFB1 (Klein et al., 2000, Klein et al., 2002, Klein et al., 2003). The purpose of this study was to fully characterize the α-class GSTs of the turkey. We amplified six α-class GSTs (tGSTs) from turkey liver mRNA by RACE and genetically mapped them to turkey chromosome MGA2. The CHORI-260 turkey BAC library was screened to identify clones containing the α-class gene cluster and one clone was fully sequenced and assembled. The six α-class GST genes were annotated according to gene structure, sequence similarity and synteny with chicken and human α-class GSTs. This study provides the complete sequence of the α-class genes and genetic markers that will be important in future studies of AFB1 susceptibility and resistance in turkeys.
Section snippets
RNA extraction and Rapid Amplification of cDNA Ends (RACE)
Male day-old turkey poults (Nicholas commercial strain) were obtained from Moroni Feed Co. (Moroni, UT). Freshly isolated turkey livers were stored in RNAlater (Ambion). Samples were homogenized using a Polytron (Brinkman) and mRNA was extracted using Oligotex Direct mRNA kit (Qiagen). The first strand cDNA was synthesized using MMLV reverse transcriptase (Clontech) and each 5′-CDS primer and 3′-CDS primer provided in SMART™ RACE cDNA Amplification Kit (Clontech), respectively, to carry out
Amplification of turkey α-class GST genes
The full-length cDNAs of six α-class GST genes were isolated and amplified from turkey liver using 5′- and 3′-RACE. Availability of the extensive chicken EST database which is genetically close to turkey (http://www.ncbi.nlm.nih.gov/genome/guide/chicken/ enabled the design of gene-specific primers to amplify turkey α-class GST genes (Table 1, Table 2). Primers for cGSTA1 amplified the three related A1 genes, tGSTA1.1, tGSTA1.2 and tGSTA1.3. Predicted open reading frames (tGSTA1.1, 743 bp with
Conclusions
The extreme sensitivity of turkeys is strongly associated with unresponsiveness of hepatic GSTs toward AFBO. We have shown that while turkey livers contain catalytically-active GSTs, none of which possess affinity toward AFBO (Klein et al., 2000, Klein et al., 2002, Klein et al., 2003), a situation similar in humans where constitutively expressed hepatic α-class GSTs have little or no AFBO detoxifying activity (Raney et al., 1992, Slone et al., 1995). Despite the large impact of this mycotoxin
Acknowledgements
The authors thank L.D. Chaves for assistance in BAC screening and sequence annotation. We also thank Dr. Lynn Bagley of the Moroni Feed Cooperative for generously providing turkeys and feed for this study. This research was supported in part by NRI competitive grant 2004-35205-14217 from the USDA-CSREES, by competitive grant 2007-35205-17880 from the USDA CSREES, Animal Genome program, and support from the Minnesota, and Utah and Agricultural Experiment Stations, where this is published as
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