Molecular and biochemical characterization of a Zeta-class glutathione S-transferase of the silkmoth

Pesticide Biochemistry and Physiology 94 (2009) 30–35

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Molecular and biochemical characterization of a Zeta-class glutathione S-transferase of the silkmoth Kohji Yamamoto *, Yuichi Shigeoka, Yoichi Aso, Yutaka Banno, Makoto Kimura, Takashi Nakashima Faculty of Agriculture, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

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Article history: Received 2 October 2008 Accepted 18 February 2009 Available online 6 March 2009 Keywords: Bombyx mori Glutathione Glutathione S-transferase Lepidoptera Lipid peroxidation

a b s t r a c t A cDNA encoding Zeta-class GST of the silkmoth, Bombyx mori (bmGSTZ), was cloned by a reverse transcriptase-polymerase chain reaction. The resulting clone was sequenced and deduced for amino acid sequence, which revealed 45–50% identities to Zeta-class GSTs from other organisms. A recombinant protein (rbmGSTZ) was functionally overexpressed in Bombyx mori cells in a soluble form and purified to homogeneity. rbmGSTZ was able to catalyze the biotranslation of glutathione with dichloroacetic acid. We found that the present GST-catalyzed dechlorination of permethrin and distributed abundantly in silkmoth strain exhibiting permethrin resistance. Our results suggest that bmGSTZ could contribute to permethrin resistance in lepidopteran. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction Glutathione conjugation is known to be a major pathway for the detoxification of xenobiotics as well as the homeostasis of endogenous compounds. Glutathione S-transferases [GSTs, EC 2.5.1.18] are enzymes widespread in both prokaryotic and eukaryotic cells and catalyze the glutathione conjugation reaction with reduced glutathione (GSH) [1,2]. There have been seven classes of mammalian GSTs, Alpha, Mu, Pi, Sigma, Theta, Omega, and Zeta [3]. Whereas, six different GST classes, designated Delta, Epsilon, Sigma, Theta, Omega, and Zeta [4,5], have been found in dipteran insects. Although GSTs have been identified from insect species mainly from the viewpoint of insecticide metabolism [6–8], the molecular identity of lepidoptera GSTs has not been investigated in comparison with those in the dipteran species. Detoxification of DDT with dehydrochlorination was shown to catalysis by GSTs [9], and GSTs detoxified lipid peroxidation products induced by pyrethroids [10]. However, GSTs have not been directly responsible for the metabolism or degradation of pyrethroid insecticides. We have characterized GSTs of the domesticated silkmoth, Bombyx mori, a lepidorteran model insect [11,12], and of the fall webworm, Hyphantria cunea, one of most serious lepidopteran pests for broad-leaved trees [13]. In this study, we focus on a novel GST (Zeta-class GST) of the silkmoth, B. mori (bmGSTZ). There has been no relationship between Zeta-class GST and insecticide metabolism in other species. A cDNA encoding this enzyme, abbreviated as bmGSTZ, was sequenced and overexpressed as a recombinant protein in Escherichia coli cells to investigate its * Corresponding author. Fax: +81 92 624 1011. E-mail address: [email protected] (K. Yamamoto). 0048-3575/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2009.02.008

properties. Furthermore, the expressions of bmGSTZ and its mRNA were examined. A lot of agricultural insect pests are lepidopteran. As the silkmoth is a model animal for lepidopteran insects, it is useful to obtain information concerning its detoxification capacity, in particular the novel GST identified in the current study, for application to other pests.

2. Materials and methods 2.1. Insects and tissue dissection Fifth-instar larvae of the silkmoth, B. mori were reared on mulberry leaves at the Institute of Genetic Resources, Kyushu University (Fukuoka, Japan). These were dissected on ice, and the fat body, midgut and silk gland were collected and kept at 80 °C until use. Total RNAs were rapidly extracted from the tissues dissected with Sepasol-RNA 1 (Nacalai Tesque) according to the manufacturer’s instructions. Total proteins were prepared by homogenization of the tissues dissected with PBS containing 0.1% Triton X-100. 2.2. Cloning and sequencing of the cDNA encoding bmGSTZ Total RNA isolated from the fat body of the larvae was subjected to the reverse transcriptase-polymerase chain reaction (RT-PCR). First-strand cDNA was produced using SuperScript II reverse transcriptase (Invitrogen) and an oligo-dT primer. The resulting cDNA was used as a template to amplify a DNA fragment by PCR with the following two oligonucleotide primers: 50 -GAA TTCATATGGTTGAAAATCGTGTGATTT-30 (sense) and 50 -CCGGATCCT

K. Yamamoto et al. / Pesticide Biochemistry and Physiology 94 (2009) 30–35

TACAAATTGATTTTCAACTTT-30 (antisense). These were designed based on the partial sequence obtained from the SilkBase EST database [14]. The underlined and double-underlined regions are NdeI and BamHI restriction enzyme sites respectively, incorporated for the purpose of subcloning the PCR product into an expression plasmid vector. PCR was conducted for one cycle at 94 °C for 2 min, then 35 cycles at 94 °C for 1 min, 54 °C for 1 min, and 72 °C for 2 min, followed by one cycle at 72 °C for 10 min. The bmGSTZ cDNA (bmgstz) obtained was ligated into pGEM-T Easy vector (Promega). DNASIS software (ver. 3.4) was used for sequence analysis, using the Sanger method. Homology alignment was performed by CLUSTALW (ver. 1.83), with 10 and 0.2 to the values of the gap creation penalty and the gap extension, respectively. Preparation of the phylogenetic tree was done by the UPGMA method in GENETYX-MAC software (GENETYX). 2.3. Overexpression and purification of recombinant protein The bmgstz cDNA was cloned into pGEM-T Easy vector, as described above. After digestion of the PCR product with NdeI and BamHI, the obtained fragment was subcloned into the NdeI–BamHI site of the expression vector pET-11b (Novagen). Competent E. coli Rosetta (DE3) pLysS cells (Novagen) was transformed with the prepared expression plasmid harboring bmgstz, which were grown at 37 °C on Luria-Bertani (LB) media containing 100 lg/ml ampicillin. After the cell density reached 0.7 OD600, isopropyl 1-thio-b-Dgalactoside (IPTG) was added to a final concentration of 1 mM to induce the production of recombinant protein. After further incubation for 3 h, cells were harvested by centrifugation, homogenized in 20 mM Tris–HCl buffer (pH 8.0) containing 0.5 M NaCl, 4 mg/ml of lysozyme and 1 mM phenylmethanesulfonyl fluoride, and disrupted by sonication. Unless otherwise noted, all operations described below were conducted at 4 °C. The supernatant was clarified by centrifugation at 10,000g for 15 min. Salting out was carried out as the first step of the protein purification. Ammonium sulfate was added to the supernatant up to 30% saturation and the precipitate was removed by centrifugation at 10,000g for 20 min. Additional ammonium sulfate was added to the supernatant up to 70% saturation. The pellet was collected by centrifugation and was suspended in 20 mM Tris–HCl (pH 8). After dialysis against the same buffer, the supernatant was loaded onto anionexchange chromatography on DEAE-Sepharose (GE Healthcare) column that had been equilibrated with the Tris buffer. After being washed with the Tris buffer, bound proteins were eluted with a linear gradient of NaCl from 0 to 0.3 M. Fractions containing the enzyme were determined as described below and pooled. After dialysis against the Tris buffer containing 0.2 M NaCl, the resultant was subjected to gel filtration chromatography on a Superdex 200 (GE Healthcare) column, which was eluted with the Tris buffer containing 0.2 M NaCl. SDS–PAGE was conducted with a 15% polyacrylamide slab gel containing 0.1% SDS according to the method described by Laemmli (1970) [15]. Protein sample (10 ll) was mixed with the same volume of 0.2 M Tris–HCl buffer (pH 6.8) containing 2% SDS, 2% 2-mercaptoethanol, 20% glycerol, and 2  103% bromophenol blue and boiled for 3 min. Protein bands were visualized by staining with Coomassie Brilliant Blue R250 (CBB). Protein concentration was measured using a Protein Assay kit (Bio-Rad) with bovine serum albumin as a standard. 2.4. Measurements of enzyme activity Dehalogenase activity was measured with purified bmGSTZ as described previously [16]. The reaction was carried out at 37 °C in 1 ml of phosphate/acetate/borate buffer (pH 7) containing 1 mM EDTA, 1 mM GSH, 1 mM dichloroacetic acid (DCA), 0.2 mM NADH, 2 U of lactate dehydrogenase (Sigma), and en-

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zyme. The activity was monitored as the change in NADH absorption at 340 nm. The specific activity of GST was expressed as mol DCA biotransformed to glyoxylic acid per min per mg protein at 37 °C using the milli-molar extinction coefficient of NADH (6.2 mM1 cm1). Alternatively, the colorimetric assay for dehalogenase activity of GST was carried out [17]. The concentration of released Cl ions was estimated with phenol red as a pH marker. The reaction mixture included phosphate/acetate/borate buffer (pH 7), 1 mM GSH and various concentrations of substrates. After incubation at 25 °C for 1 h, 5 ll of the reaction mixture was transferred to a plastic tube. Subsequent pH measurements were performed by adding 200 ll of assay buffer containing phosphate/acetate/borate buffer (pH 7), 20 mM sodium sulfate, 1 mM EDTA, and 25 lg/ml phenol red. Control tubes had the same reagent mixture without the recombinant enzyme. The color that has absorbency at 550 nm was read and the amount of released Cl ion was estimated by comparing it with the standard prepared using HCl. 2.5. Immunoblotting analysis For preparation of peptide antibodies, two peptides found in bmGSTZ were synthesized by Operon (Tokyo, Japan). One peptide sequence, HAKSIPFEERPVDI, corresponded to amino acids 25–38 and the other peptide, THPKATKEKLKINL, corresponded to amino acids 203–216, which were used to produce rabbit polyclonal antibodies. Fat body was homogenized in phosphate buffered saline (PBS) containing 0.1% Triton X-100 and the resulting sample was resolved by SDS–PAGE under the same conditions as described above. The proteins were then transferred to a PVDF membrane (Bio-Rad Laboratories). After blocking for 1 h in 5% fat free milk in PBS, the membrane was incubated with anti-bmGSTZ polyclonal antibodies (primary antibodies) for 1 h, followed by goat anti-rabbit IgG secondary antibodies (Cappel) for 1 h. Detection was performed using ECL Plus Western Blotting Detection Reagents (GE Healthcare).

3. Results and discussion 3.1. Cloning and sequencing of cDNA encoding bmGSTZ The cDNA encoding bmGSTZ was obtained by RT-PCR using total RNA from B. mori (p50 strain). The nucleotide sequence was determined and deposited in GenBank under accession No. AB456582. It contains an open reading frame of 651 bp, encoding 216 amino acid residues (Fig. 1), whose theoretical molecular mass and pI were evaluated to be 24,727 and 8.52, respectively. The deduced amino acid sequence of this putative GST showed 49%, 46%, and 45% homogeneous identities to Zeta-class GSTs from Denio rerio, Drosophila melanogaster and Anopheles gambiae, respectively. In contrast, the sequence of bmGSTZ showed lower homology with other classes such as Sigma-class GST (bmGSTS) and Delta- (bmGSTD): 22.3% and 21.8% homologies to the enzymes of B. mori. The GST class shared more than 40% amino acid identity with other organisms [5]. Highly conserved in the protein was an SSC-motif of the N-terminal domain [18]. It has been reported that the serine residue at position 14 in the SSC-motif of the N-terminal region is catalytically essential [19] and that it is well conserved in Zeta-class GST of mammalian as well as for Sigma-class GST. On the other hand, tyrosine is essential residue in Theta-class GST [20,21]. The sites involved in the GSH-binding of GST were conserved in the sequence of bmGSTZ (boxed in Fig. 1) [19]. On the basis of the phylogenetic tree generated from the aligned amino acid sequences of GSTs, the present GST was closest to those of Zeta-class GSTs of mos-

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K. Yamamoto et al. / Pesticide Biochemistry and Physiology 94 (2009) 30–35

Fig. 1. Alignment of amino acid sequences of Zeta-class GSTs. Sequences of GSTs from different organisms were obtained from Swiss-Prot databases: silkmoth (determined in the present study); A. gambiae (No. Q8MUQ5); D. melanogaster (No. Q9VHD3); D. rerio (No. Q6DGL3). An asterisk represents identical amino acids and a dash denotes a deletion. Conserved GSH-binding site residues are boxed. The bold letters indicate conserved SSC-motif.

quitoes, flies and zebra fish among the three GST classes (Fig. 2). As seen in the phylogenetic tree and amino acid identities homogeneous to the enzymes from various species, we concluded that the clone represented a Zeta-class enzyme (bmGSTZ).

3.2. Overexpression and purification of rbmGSTZ The bmGSTZ was overexpressed as a recombinant protein (rbmGSTZ) using the E. coli expression vector. An SDS–PAGE anal-

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Fig. 2. Phylogenetic analysis of GST amino acid sequences. The phylogenetic tree was made with Neighbor-joining plot software using GST sequences cited from Swiss-Prot: these include Delta-class GSTs from B. mori (No. Q60GK5), M. domestica (No. P28338), and D. melanogaster (No. P20432), and Sigma-class GSTs from B. mori (No. Q5CCJ4), H. cunea (No. Q4ACU7), and M. sexta (No. P46429) and the other organisms cited in the legend in Fig. 1. Numbers attached indicate branch length.

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K. Yamamoto et al. / Pesticide Biochemistry and Physiology 94 (2009) 30–35

ysis of E. coli cell lysate revealed that rbmGSTZ was in a soluble form (Fig. 3A). The specific activity of GST (towards CDNB) of the lysate containing rbmGSTZ was about eight-fold higher than that from the E. coli cells without bmgstz. The rbmGSTZ was purified to homogeneity by ammonium sulfate fractionation, anion-exchange chromatography, and gel filtration. The purified protein migrated with an apparent molecular weight of 24,000 (Fig. 3B). There were insignificant differences in the molecular sizes of rbmGSTZ between the value calculated from the deduced amino acid sequence and that measured by the SDS–PAGE: 24,727 and 24,000 from the sequence and SDS–PAGE, respectively. These sizes were similar to those of lepidopteran GSTs isolated. Finally, we obtained 4.8 mg of highly purified rbmGSTZ from 250 ml of an LB medium. The specific activity of the final preparation toward DCA was 0.11 lmol/min/mg. Thus, active rbmGSTZ was successfully overexpressed in a soluble form in E. coli cells.

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3.3. Localization of bmGSTZ transcript and bmGSTZ Knowledge of the tissue distributions of bmGSTZ mRNA and bmGSTZ could be used to better understand the tissue’s physiology. As shown in Fig. 4A, RT-PCR revealed only a single band of approximately 0.7 kilo bases were obtained in RNA samples from ovaries, midgut, fat body and testis, whereas relatively little amounts of the mRNA were detected in silk glands. Regarding the protein levels of bmGSTZ, immunoblotting showed that bmGSTZ was distributed in B. mori fat body, although little amount of bmGSTZ was present in homogenates from ovaries, silk glands, midgut, and testis. The question why there was a difference in levels between bmGSTZ mRNA and bmGSTZ remains unanswered. Further understanding of the physiology of bmGSTZ requires comprehensive studies on developmental changes of activity, protein, and mRNA in various tissues. 3.4. Characterization of rbmGSTZ Enzymatic properties of bmGSTZ were determined by measuring mol DCA biotransformed to glyoxylic acid. The pH optimum of rbmGSTZ was found to be around 6 (Fig. 4A), lower than those

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Fig. 4. Localization of bmGSTZ transcript and bmGSTZ. (A) RT-PCR was performed to detect bmGSTZ transcripts in various tissues. The PCR products were resolved in 1% agarose gel. Lane 1, ovaries; lane 2, silk glands; lane 3, midgut; lane 4, fat body; lane 5, testis. (B) Total protein (5.6 lg) was subjected to SDS–PAGE followed by immunodetection. Lane 1, ovaries; lane 2, silk glands; lane 3, midgut; lane 4, fat body; lane 5, testis. Zymograms were given, after the three independent experiments were repeated.

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15 10 10 Fig. 3. Electropherogams of rbmGSTZ overexpressed in E. coli cells and after purification. E. coli cell extracts or purified protein were subjected to 15% SDS–PAGE followed by staining with CBB. (A) Lane 1, protein molecular size markers; lane 2, extract of E. coli cells carrying bmGSTZ expression vector without IPTG induction; lane 3, the same with IPTG induction. (B) Lane 1, protein molecular size markers; lane 2, rbmGSTZ purified by the methods described in the text.

of bmGSTD and bmGSTS, as well as H. cunea Sigma-class GST. With respect to thermostability, rbmGSTZ was stable at temperatures below 50 °C (Fig. 4B), similar to bmGSTD and hcGSTS, whereas bmGSTS was stable at temperatures below 40 °C. Analysis of pH stability (Fig. 4C) indicated that rbmGSTZ retained more than 75% of it’s original activities at pHs 4–10, similar to those of bmGSTS, bmGSTD, and hcGSTS (Fig. 5). We found that bmGSTZ exhibited GSH-dependent activities in the biotransformation of DCA. In the case of humans and rats, Zeta-class GSTs were able to catalyze glutathione-dependent oxygenation of DCA [22]. It has been described that DCA is a carcinogenic contaminant in common drinking-water [18]. Cys16 residue of human Zeta-class GST, conserved in an SSC-motif of bmGSTZ, influenced the binding of GST to DCA [23]. 4-HNE is a cytotoxic product of lipid peroxidation under conditions of oxidative stress [24], but it is associated with signaling functions, cell proliferation, and apoptosis [25,26]. For other classes of GST of B. mori and H. cunea, CDNB and 4-HNE were good substrates [11–13]. When ECA and 4-NPA were used as a substrate, the activities of bmGSTZ were undetectable. ECA is a substrate for Pi, Mu, and Alpha-class GSTs, but not for bmGSTS. On the other hand, the bmGSTD conjugated GSH to ECA [11].

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Fig. 5. Enzymatic properties of rbmGSTZ were assayed with CDNB and GSH as substrates. GST activity was assayed under the standard conditions, as described in Section 2, unless otherwise indicated. The maximum value obtained was set to 100%. (A) Optimum pH for the activities was assayed using citrate–phosphate–borate buffer at various pHs with a fixed ionic strength of 0.25. (B) Thermostability was determined by preincubation of the enzyme solution at various temperatures for 30 min before residual activity was assayed. (C) pH stability was assessed by preincubation of the enzyme solution at various pHs at 4 °C for 24 h before residual activity was assayed.

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Fig. 6. Spectrophotometric assay of released Cl ions. (A) The concentration of Cl ions was determined by pH changes. Lane 1; without Cl ions, lane 2, with Cl ions. Two independent experiments were repeated. (B) The calibration curve was constructed by measurement of absorbance at 550 nm.

3.5. Determination of dehalogenase activity of bmGSTZ using alternative assay We determined dehalogenase activity of bmGSTZ by colorimetric assay using HCl as the control. In this assay, detection was based on the pH change by the amount of released Cl ions from DCA or substrates containing Cl during the GST-catalyzed dehydrochlorination reaction, and was monitored calorimetrically in the presence of phenol red as a pH marker (Fig. 6A). Elimination of red color was associated with the change of absorbency at 550 nm. Based on a calibration curve (Fig. 6B), we calculated the amounts of released Cl ions from DCA and permethrin, one of model pyrethroids. The amount of released Cl ions were 0.06/10 nmol DCA and 0.03/10 nmol permethrin, respectively. Although not enough information on the degradation of permethrin on GST is yet available, previous reports indicate that GSTs participate in resistance to 1,1,1-dichloro-2,2-bis (p-chlorophenyl) ethylene (DDT) in insect species such as Aedes aegypti [27]. By using the related assay, dechlorination of xenobiotics including Cl such as hexachlocyclohexane, haloalkane, and DDT were detected [17,28,29]. Permethrin is a pyrethroid insecticide popular worldwide, and this situation has promoted the occurrence of insect species that have acquired high resistance [30–32]. In this context, the prospects for chemical control are concerned.

Fig. 7. Immunoblotting of bmGSTZ. Western blotting was carried out under the conditions described in Section 2. The concentration of protein loaded on SDS–PAGE gel was 10 ll containing 5.6 lg protein. The bmGSTZ was detected in silkworm fat body of R1 (lane 1) and S1 strain (lane 2).

3.6. Expression analysis of bmGSTZ in fat bodies between insecticide susceptible- and resistant-silkmoth strain For the toxicity tests, LD50 values with permethrin in various strains of silkmoth were measured (data not shown). The value in the R1 strain was 0.43 lg/g larvae, which was about 20-fold higher compared with that in the S1 strain. Comparison of bmGSTZ expression in fat bodies of both strains was investigated by the immunoblotting method. As shown in Fig. 7, we found that bmGSTZ was present in the fat bodies, but there was a difference in the band intensity of bmGSTZ expressed between the strains. This result suggests that bmGSTZ is present abundantly in fat body of the R1 strain, and could contribute to permethrin resistance. Similar results were obtained in the mosquito, A. aegypti. One article described that the levels of GSTs of mosquitoes were increased in strain resistants’ to insecticides [33]. A mutation in a repressor element was proposed for the elevated expression of the GSTs. It is of interest to analyze the promoter region of the bmGSTZ.

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Here, we provided the first evidence that a novel GST (Zeta-class GST) is present in the silkmoth, B. mori (bmGSTZ). Taking into account our observations, we suggest that bmGSTZ fulfills functions in response to detoxification of xenobiotics containing chloride. We have characterized bmGSTD, bmGSTS, and bmGSTZ (the currently cloned enzyme) [11–13]. The existence of other GSTs in B. mori must be examined to understand their correlation to the insecticide detoxification system in this species. It may be of importance to compare detailed properties, such as expression rates, activities, substrate specificity, and resistance spectrums among GSTs as well as related enzymes of B. mori. Investigation along these lines is now underway in our laboratories. Acknowledgments This work was partially supported by KAKENHI (19780042), and Kyushu University Interdisciplinary Programs in Education and Projects in Research Development. References [1] I. Listowsky, M. Abramovitz, H. Homma, Y. Niitsu, Intracellular binding and transport of hormones and xenobiotics by glutathione S-transferase, Drug Metab. Rev. 19 (1988) 305–318. [2] R.N. Armstrong, Structure, catalytic mechanism, and evolution of the glutathione transferases, Chem. Res. Toxicol. 10 (1997) 2–18. [3] B. Mannervik, P.G. Board, J.D. Hayes, I. Listowsky, W.R. Pearson, Nomenclature for mammalian soluble glutathione transferases, Methods Enzymol. 401 (2005) 1–8. [4] C.P. Tu, B. Akgül, Drosophila glutathione S-transferases, Methods Enzymol. 401 (2005) 204–226. [5] H. Ranson, J. Hemingway, Mosquito glutathione transferases, Methods Enzymol. 401 (2005) 226–241. [6] X. Li, M.A. Schuler, M.R. Berenbaum, Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics, Annu. Rev. Entomol. 52 (2007) 231–253. [7] A.A. Enayati, H. Ranson, J. Hemingway, Insect glutathione transferases and insecticide resistance, Insect Mol. Biol. 14 (2005) 3–8. [8] R. Sawicki, S.P. Singh, A.K. Mondal, H. Benesc, P. Zimniak, Cloning, expression and biochemical characterization of one Epsilon-class (GST-3) and ten Deltaclass (GST-1) glutathione S-transferases from Drosophila melanogaster, and identification of additional nine members of the epsilon class, Biochem. J. 370 (2003) 661–669. [9] A.G. Clark, N.A. Shamaan, Evidence that DDT-dehydrochlorinase from the housefly is a glutathione S-transferase, Pest. Biochem. Physiol. 22 (1984) 249–261. [10] J.G. Vontas, G.J. Small, J. Hemingway, Glutathuone S-transferases as antioxidant defense agents confer pyrethroid resistance in Nilaparvata lugens, Biochem. J. 357 (2001) 65–72. [11] K. Yamamoto, P. Zhang, F. Miake, N. Kashige, Y. Aso, Y. Banno, H. Fujii, Cloning, expression, and characterization of theta-class glutathione S-transferase from the silkworm, Bombyx mori, Comp. Biochem. Physiol. 141B (2005) 340–346. [12] K. Yamamoto, P. Zhang, Y. Banno, H. Fujii, Identification of a sigma-class glutathione S-transferase from the silkworm, Bombyx mori, J. Appl. Entomol. 130 (2006) 515–522. [13] K. Yamamoto, H. Fujii, Y. Aso, Y. Banno, K. Koga, Expression and Characterization of a sigma-class glutathione S-transferase of the fall webworm, Hyphantria cunea, Biosci. Biotechnol. Biochem. 71 (2007) 553–560.

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