Cloning and expression of alpha class glutathione S-transferase gene from the small hermaphroditic fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae)

Marine Pollution Bulletin 51 (2005) 776–783 www.elsevier.com/locate/marpolbul

Cloning and expression of alpha class glutathione S-transferase gene from the small hermaphroditic fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) Young-Mi Lee a, Sung Yeoul Chang b, Sang-Oun Jung a, Hee-Seok Kweon c, Jae-Seong Lee a,* a b

Department of Environmental Science, Graduate School, Hanyang University, Seoul 133-791, South Korea Laboratory for Clinical Investigation, College of Medicine, Hanyang University, Seoul 133-791, South Korea c Electron Microscopy Team, Korea Basic Science Institute, Daejeon 305-333, South Korea

Abstract In order to assess its potential as a biomarker of aquatic pollution, an alpha class glutathione S-transferase gene (GSTa gene) was cloned from the small hermaphroditic fish Rivulus marmoratus. The R. marmoratus GSTa gene spanned 1.3 kb, consisting of 6 exons encoding 221 amino acid residues. It showed high similarity to zebrafish GST. We named this R. marmoratus GSTa gene as rmGSTa. The cDNA of the rm-GSTa gene was also investigated for its phylogeny, tissue-specific and chemical-induced expression. Rm-GSTa was subcloned into a 6 · His-tagged pCRT7 TOPO TA expression vector to produce the recombinant 6 · His-tagged rm-GST protein. This will be used in future to raise an rm-GSTa antibody for use in the study of phase II metabolism involved in detoxification. We also exposed R. marmoratus to 300 lg/l of 4-nonylphenol in water, and found approximately 4-fold induction of R. marmoratus GSTa mRNA in the treated animals. In this paper, we discuss the characteristics of the R. marmoratus GSTa gene as well as its potential use in relation to environmental pollution.  2005 Elsevier Ltd. All rights reserved. Keywords: cDNA; Expression; Escherichia coli; RT-PCR

1. Introduction Glutathione S-transferases (GSTs) are a multigene family encoding enzymes involved in detoxification of electrophilic compounds during phase II metabolism by conjugation with glutathione in the liver (Leaver et al., 1997; Pham et al., 2004). Such compounds include therapeutic drugs, environmental toxins and products of oxidative stress. The GST genes are known to be highly polymorphic. These genetic variations can alter interindividual susceptibility to carcinogens and toxins as

*

Corresponding author. Tel.: +82 2 2220 0769; fax: +82 2 2299 9450. E-mail address: [email protected] (J.-S. Lee).

0025-326X/$ - see front matter  2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2005.06.016

well as affecting the toxicity and efficacy of some drugs. At present, eight distinct classes of the soluble cytoplasmic mammalian glutathione S-transferases have been identified: alpha, kappa, mu, omega, pi, sigma, theta and zeta, forming the multigene family of GST genes in mammals. Whereas in mammals, GST genes have been well characterized, less information is available on these genes in fish (Leaver et al., 1997; Henson et al., 2000; Strausberg et al., 2002; Pham et al., 2004). As the GSTs are involved in detoxification of many xenobiotics, their gene structures and expression in fish require further study in order to understand the impact of such compounds on the aquatic environment. The small hermaphroditic fish Rivulus marmoratus (R. marmoratus) is a useful species for studying the

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effects of toxic chemicals as it may be easily reared in the laboratory (Lee et al., 2000, 2002; Rotchell et al., 2001). Recently several R. marmoratus biomarker genes (e.g. vitellogenin, CYP1A, AhR, c-fos) were cloned for in vitro study as well as in vivo testing at the molecular level (Kim et al., 2004a,b; Li et al., 2004; Lee et al., 2005). A battery of biomarker genes is desirable to allow further use of this species as a molecular testing model for environmental pollution. The isolation of further potential biomarker genes, encoding GSTs and other phase I and phase II enzymes, will allow more detailed characterization of the effects of toxic stresses. In this paper, we show both the cDNA and genomic DNA sequences of a R. marmoratus GST gene, investigate its tissue-specific and chemical-induced expression, and demonstrate the production of its recombinant protein. This will facilitate the understanding of molecular events following oxidative stress and environmental endocrine-disrupting chemical exposure in this species.

2. Materials and methods The small hermaphroditic fish R. marmoratus were raised in an aquarium at the Department of Environmental Science, Graduate School, Hanyang University, Seoul in South Korea. Approximately 5 g wet weight of R. marmoratus was used for RNA isolation to make an R. marmoratus cDNA library. Total RNA was extracted from R. marmoratus samples frozen in liquid nitrogen. The whole body tissues were ground with a pestle and then homogenized in three volumes of TRIZOL with a tissue grinder. Total RNA was extracted according to the manufacturerÕs protocol. We then purified mRNA using a mRNA purification kit (Invitrogen, USA) according to the manufacturerÕs protocol and stored the isolated mRNA at
70 C until used. To make the unidirectional cDNA library, we used a kZAPII cDNA library packaging kit (Stratagene) with the help of oligo(dT) according to the manufacturerÕs protocol. The linker and adaptor with EcoRI and XhoI restriction enzyme sites were used to make unidirectional cDNA clones. The packaging and titration of primary R. marmoratus k recombinant phages was performed according to the manufacturersÕ protocol. To analyze EST clones of R. marmoratus, a small quantity (3–4 ll) of R. marmoratus kZAPII cDNA library was taken and subjected to in vitro conversion to pBluescript phagemid DNA using helper phage and Escherichia coli SOLR strain. Of colonies rescued, some 50–100 were randomly picked to identify the inserts. After cutting the inserts with EcoRI, the recombinant pBluescript phagemid DNAs (over 1.0 kb) were subjected to sequence analysis with an ABI 377 automated sequencer. The R. marmoratus EST sequences were com-

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pared with the GenBank database to identify the closest homologues, and were submitted to the R. marmoratus EST database (to date, not released to the public domain). One full-length cDNA of R. marmoratus GST gene identified by random sequencing analysis was chosen, and the database was searched to find homologues from other species. To reveal the genomic structure of R. marmoratus GST gene, we amplified the R. marmoratus GST genomic clone with two primers (Rm-GST-F 5 0 ATG TCT GGA AAA GTT ACT CTG AC-3 0 , RmGST-R 5 0 -TTA CAG TGA CGT CAT GTT GAA CAC-3 0 ) and subcloned to pCR2.1 TA cloning vector (Invitrogen) for sequence analysis in both directions. We compared the R. marmoratus GST sequence to the GenBank database to find similar clones from other species. To construct a phylogenetic tree for the R. marmoratus GST cDNA, we retrieved GST homologues from several species including zebrafish, plaice, largemouth bass, rabbit, chicken, rat and human, then used an Expansion of ClustalW by DDBJ (http://www.ddbj. nig.ac.jp/E-mail/homology.html) with a Kimura-2 parameter, and visualized the phylogenetic tree with TreeView of PHYLIP. To analyze tissue-specific expression of R. marmoratus GST gene, we extracted total RNAs from several tissues (brain, eye, gonad, intestine, liver, muscle, skin) of R. marmoratus, and stored them at
80 C prior to use. We used two RT-PCR primers (Rm-GST-F 5 0 -ATG TCT GGA AAA GTT ACT CTG AC-3 0 , Rm-GST-R 5 0 -TTA CAG TGA CGT CAT GTT GAA CAC-3 0 ; expected product 668 bp). Internal control primers (RmGAPDH-F 5 0 -CGG TAA GCT GTG GAG GGA CGG CCG CG-3 0 , Rm-GAPDH-R 5 0 -TGG TGC TCG GTG TAT CCC AGA ATG CC-3 0 ; expected product 265 bp) were used with Rm-GST primers in the same reaction to control for the amount of template first-strand cDNAs. We followed the conventional method of RT-PCR with the reaction mixture (1 ll of first-strand cDNA, 5 ll of 10 · LA buffer, 8 ll of 2.5 mM dNTPs, 5 ll of 25 mM MgCl2, 100 pM each primer, 0.5 ll of LA Taq polymerase (TaKaRa)) subjected to amplification (1 cycle, 95 C, 5 min; 40 cycles, 98 C, 25 s, 50 C, 1 min, 72 C, 2 min; 1 cycle 72 C, 10 min) using an iCycler (Bio-Rad). We carried out this experiment in triplicate, and applied the StudentÕs t-test for statistical analysis of the data. To analyze the expression of GST upon 4-nonylphenol exposure, we treated R. marmoratus adults with 300 lg/l of 4-nonylphenol in water for 96 h, and sampled liver and ovary tissues. Total RNA was extracted from tissues stored at
80 C prior to use. We used two RT-PCR primers as mentioned above. Internal control primers (Rm-GAPDH-F 5 0 -CGG TAA GCT GTG GAG GGA CGG CCG CG-3 0 , Rm-GAPDH-R 5 0 TGG TGC TCG GTG TAT CCC AGA ATG CC-3 0 ;

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Fig. 1. Size fractionation of Rivulus marmoratus cDNA. M1, k/HindIII size marker; M2, 200 bp ladder. The cDNAs were electrophoresed on 1% TBE gel. The 1 kb band is indicated by a dotted line and an arrow.

expected product 265 bp) were used with Rm-GST primers in the same reaction to normalize the input amount of template first-strand cDNAs. We carried out RT-PCR as above. The relative level of gene expression was quantified after we checked the intensity of the ethidium bromide-stained bands from R. marmoratus GST and GAPDH genes. These experiments were carried out in triplicate and we applied the StudentÕs t-test for statistical analysis.

To produce recombinant protein from the amplified RT-PCR product of R. marmoratus GST cDNA, we electrophoresed the RT-PCR product, excised the required band from the gel, and eluted by elution kit (Qiagen). Subsequently, we ligated the RT-PCR product to the 6 · His-tagged pCRT7 TOPO TA expression vector (Invitrogen) and transformed into E. coli BL21(DE3)pLysS competent cells. After the isolation of R. marmoratus GST cDNA inserts, we sequenced them to select the clones containing full-length R. marmoratus GST cDNA. To confirm the expression of recombinant Rivulus GST protein, we induced the recombinant protein with 1 mM IPTG during the log phase of growth. We sampled the treated E. coli for 24 h after induction, and immunoblotted against antiHisG-HRP (Invitrogen) after polyacrylamide gel electrophoresis. The film was developed by Amersham ECL-plus Western blotting detection system.

3. Results and discussion We constructed a R. marmoratus whole body cDNA library, and titrated it according to the standard protocol (Kim et al., 2004b) in order to study global gene

Fig. 2. cDNA nucleotide sequence and deduced amino acid residues of Rivulus marmoratus GSTa gene. Poly(A) signal sequence is underlined at the 3 0 -untranslated region.

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Fig. 3. (A) Agarose (1%) gel electrophoresis of Rivulus marmoratus GSTa gene PCR product from R. marmoratus. M, k/HindIII molecular size marker, Lane 1, the amplified GSTa gene product. (B) Representative DNA sequence of the R. marmoratus GSTa gene (GenBank accession no. AY626242).

expression in this fish. After we fractionated R. marmoratus cDNAs as shown in Fig. 1, we pooled fractions (over 1 kb) and constructed the unidirectional R. marmoratus cDNA library using kZAPII/EcoRI and XhoI, which will facilitate the sequencing of 5 0 -ends. We randomly picked colonies after conversion of R. marmora-

tus kZAPII cDNA library to pBluescript with the aid of helper phage, and sequenced them from the 5 0 -end with T7 primer. This approach has been taken by several research groups to study global gene expression patterns in specific tissues or developmental stages of fish (Ju et al., 2000). The resulting clones will be useful to

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Fig. 4. Relative comparison of genomic structure of Rivulus marmoratus GSTa gene to other species (rat and human). Exon 1 was used as a base for alignment.

construct a species-specific cDNA chip for toxicogenomic studies with Rivulus marmoratus and may be used to isolate new biomarker genes and to confirm existing findings from known biomarker genes such as vitello-

genin, CYP1A, and AhR. The GST gene was selected from these sequences (1577 ESTs from R. marmoratus whole body tissues) for further analysis due to its importance in the response to oxidative stress in aquatic organisms (Fig. 2). In mammals, there are several GST family genes such as alpha (a), mu (l), pi (u), theta (h) and sigma (r). From the high similarity of our clone to the alpha (a) GST family genes, we named it ÔrmGSTaÕ. The rm-GSTa gene had a 961 bp transcript, including 5 0 -untranslated, open reading frame (ORF) and 3 0 -untranslated regions. It has one poly(A) signal sequence, AATAAA at 16 bp before the polyadenylation site (as underlined in Fig. 2). This cDNA sequence has been submitted to GenBank (Accession number AY626242).

Fig. 5. Similarity of Rivulus marmoratus GSTa to other species. The symbol ‘‘ ’’, ‘‘:’’ and ‘‘.’’ indicate identical residue, strongly conserved substitution and weakly conserved substitution, respectively. R. marmoratus, Mangrove rivulus; Danio rerio, zebrafish; Pleuronectes platessa, plaice; Micropterus salmoides, largemouth bass; Oryctolagus cuniculus, rabbit; Gallus gallus, chicken; Rattus norvegicus, rat; Homo sapiens, human.

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We determined the genomic structure of the rmGSTa gene, by designing two primers to amplify the corresponding genomic clone (Fig. 3A), sequenced it in both directions (Fig. 3B) and submitted this genomic DNA sequence to the GenBank database (Accession number AY626240). The rm-GSTa gene consisted of 6 exons and conformed to the GT-AG rule at all exon– intron junctions. The cDNA sequence and that derived from the genomic sequence were identical, indicating that both were derived from the same gene. In fish, to date, the only reported GST genomic sequences have been for GSTA in plaice and largemouth bass (Leaver et al., 1997; Pham et al., 2004) and zebrafish (unpublished result). These fish GSTA genes consist of five or seven exons. Therefore, the rm-GSTa is likely to represent a different gene if it is assumed that the number of exons is conserved between species. In other species, there is a large number of GST family genes, indicating that further R. marmoratus GSTs, potentially important for detoxification, remain to be discovered. We compared the rm-GSTa gene with other known GSTa genes from rat and human. In Fig. 4, we show that the rm-GSTa gene is very compact (1.3 kb) in comparison to those of rat (12.1 kb) and human (7.4 kb). Fish genes tend to possess small introns compared with those of the corresponding mammalian homologues (Lee et al., 1995). We compared the R. marmoratus GST gene to GSTs of other species (e.g. zebrafish, plaice, largemouth bass, chicken, rabbit, rat, human) as shown in Fig. 5. The R. marmoratus GST gene showed higher similarity to those of zebrafish (64.3%) and rabbit (52.1%) than those of rat and human, which would be expected considering the phylogenetic relationships of these organisms. Conserved regions at the N-terminal, middle and C-terminal of the amino acid sequence imply a conservation of function between rm-GSTa and these GSTs from diverse species. To reveal the molecular phylogenetic position of rmGSTa cDNA, we retrieved the alpha family of GST from GenBank to reveal the phylogenetic position of R. marmoratus GST gene, and constructed a phylogenetic tree with aid of the NJ method after we used an Expansion of ClustalW by DDBJ (http://www.ddbj. nig.ac.jp/E-mail/homology.html), and visualized with TreeView of PHYLIP as shown in Fig. 6A. In this tree, the R. marmoratus GST gene had the same clade with zebrafish with a low value of bootstrap (Fig. 6A box), indicating that this phylogenetic relationship was not highly supported. Also the relationship with other taxa (largemouth bass and plaice) was not greatly supported as shown in Fig. 6B. These kinds of molecular topology were supported by other methods such as maximum parsimony and the Bayesian method (data not shown). This phylogenetic tree may indicate which R. marmoratus GSTa antibody would be not cross-

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reacted well between taxa which were shown in different clade. Of them, chicken has two different GSTa genes. To determine whether this duplication of GSTa genes occurs in R. marmoratus as well, we attempted to amplify the genomic clone of further rm-GSTa genes with the same PCR primers, based on the assumption that an

Fig. 6. Phylogenetic tree of Rivulus marmoratus GSTa with those of other species. The tree was constructed by NJ method with a Kimura 2-parameter after ClustalW analysis. The used species were as follow; R. marmoratus (AY626242) (in this study), zebrafish (BC060914) (Strausberg et al., 2002), plaice (X95200) (Leaver et al., 1997), largemouth bass (AY335905) (Henson et al., 2000), rabbit (M74529) (Gardik et al., 1991), chicken (L15387) (Liu et al., 1993), rat (XM_217195) (Vargo et al., 2004) and human (NM_145740) (Zhan and Rule, 2004).

Fig. 7. Expression of Rivulus marmoratus GSTa gene in different tissues (eye, brain, liver, intestine, ovary, skin, muscle). R. marmoratus GSTa expression was divided by R. marmoratus GAPDH expression to determine relative expression. These experiments were performed in triplicate.

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Fig. 8. Expression of GSTa gene from ovary and liver tissues of Rivulus marmoratus exposed to 300 lg/l of 4-nonylphenol. R. marmoratus GSTa expression was divided by R. marmoratus GAPDH (GenBank accession number AY864771) expression to determine relative expression. These experiments were performed in triplicate, and were applied StudentÕs t-test for statistical analysis.

additional rm-GSTa gene would have nearly identical sequences at the PCR primer binding sites at the 5 0 -end and 3 0 -end of its open reading frame (ORF). However, no additional GSTa genes were detected, indicating either that R. marmoratus has only one GSTa gene or that the sequences at these primer binding sites are not conserved. Thus, further studies are necessary to determine the number of GSTa genes in this species. To analyze the tissue-specific expression of the rmGSTa gene, we extracted several tissues (brain, eye, gonad, intestine, liver, muscle, skin) from adult R. marmoratus. We used R. marmoratus GAPDH gene as internal control of expression. After RT-PCR amplification of rm-GSTa cDNA, we found that the rm-GSTa cDNA was expressed ubiquitously in the examined tissues (Fig. 7), however, there were dramatic differences in the level of expression between tissues. Gonad, intestine

and liver showed high expression, while the neural tissues (eye and brain) and muscle showed lower expression. Singhal et al. (1994) reported that the human GST was also different regarding activity toward 4hydroxynonenal–GSH conjugation in liver and testis. Also there is little information on tissue-specific expression of this gene in other species. In R. marmoratus, we expect that these tissues (gonad, intestine and liver) would show greater metabolic activity, resulting in the production of more reactive oxygen species. Potentially, rm-GSTa would play a role in detoxification, thereby protecting the cells from reactive oxygen species and the products of peroxidation. To analyze 4-nonylphenol-induced expression of the R. marmoratus CYP1A gene in liver, we treated R. marmoratus adults with 300 lg/l of 4-nonylphenol in water, and carried out RT-PCR. As shown in Fig. 8, there was over-expression of rm-GSTa in the treated group in liver rather than that of ovary. However, in the case of the R. marmoratus CYP1A gene, there was not a statistically significant induction (Lee et al., 2005), reflecting nonylphenol as a weak endocrine-disrupting chemical (Ishibashi et al., 2004). Thus, this finding would suggest application as a new biomarker for weak endocrinedisrupting chemicals, although other weak endocrinedisrupting chemicals have yet to be tested. A recombinant clone of the rm-GSTa gene was constructed in pCR T7 Topo TA expression vector. First, we amplified the cDNA of rm-GSTa (Fig. 9A) and subcloned it into the above vector. After checking the sequence identity, we expressed it in E. coli BL21(DE3)pLys as shown in Fig. 9B. As shown in Fig. 9C, expression was seen in the E. coli system, resulting in cross-reactivity to anti-His antibody. In Fig. 9C, we found there were two bands after IPTG induction at 29 kDa (rm-GST) and at 38 kDa (additional band). The band at 38 kDa is assumed to be due to non-specific hybridization, as control also showed the faint band at the same position. In fact, we did not find the hybridized

Fig. 9. (A) RT-PCR product of Rivulus marmoratus GSTa gene. M, k/HindIII size marker; Lane 1, RT-PCR product of R. marmoratus GSTa gene. (B) Induction of R. marmoratus GSTa after transformation into E. coli BL21(DE3)pLys. R. marmoratus GSTa was induced by 1 mM IPTG for 4 h after it reached a log phase of growth. The induced R. marmoratus GSTa recombinant proteins are marked by arrows. (C) Western blot of R. marmoratus GSTa against anti-His antibody. The R. marmoratus GSTa protein is marked by arrow.

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band at 38 kDa when we used mouse anti-GST antibody (data not shown).

Acknowledgements We thank anonymous reviewers for their constructive comments to improve the earlier version, and also thank Prof. J.K. Chipman (School of Biosciences, University of Birmingham, UK) for his comments on the revised manuscript. This work was funded by the KRF-ABRL grant (H00007) to Jae-Seong Lee.

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