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A cytosolic glutathione s-transferase, GST-theta from freshwater prawn Macrobrachium rosenbergii: molecular and biochemical properties Jesu Arockiaraj a,⁎, Annie J. Gnanam b, Rajesh Palanisamy a, Prasanth Bhatt a, Venkatesh Kumaresan a, Mukesh Kumar Chaurasia a, Mukesh Pasupuleti c, Harikrishnan Ramaswamy d, Abirami Arasu a,e, Akila Sathyamoorthi a,f a

Division of Fisheries Biotechnology & Molecular Biology, Department of Biotechnology, Faculty of Science and Humanities, SRM University, Kattankulathur 603 203, Chennai, Tamil Nadu, India Institute for Cellular and Molecular Biology, The University of Texas at Austin, 1 University Station A4800, Austin, TX 78712, USA c Lab PCN 206, Microbiology Division, CSIR-Central Drug Research Institute, B.S. 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, Uttar Pradesh, India d PG and Research Department of Biotechnology, Bharath College of Science and Management, Thanjavur 613 005, Tamil Nadu, India e Department of Microbiology, SRM Arts & Science College, Kattankulathur 603 203, Chennai, India f Department of Biotechnology, SRM Arts & Science College, Kattankulathur 603 203, Chennai, India b

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Article history: Received 5 August 2013 Received in revised form 26 May 2014 Accepted 27 May 2014 Available online xxxx Keywords: Macrobrachium rosenbergii Glutathione S-transferase Oxidative stress Gene expression Enzyme activity

a b s t r a c t Glutathione S-transferases play an important role in cellular detoxification and may have evolved to protect cells against reactive oxygen metabolites. In this study, we report the molecular characterization of glutathione s-transferase-theta (GST-θ) from freshwater prawn Macrobrachium rosenbergii. A full length cDNA of GSTT (1417 base pairs) was isolated and characterized bioinformatically. Exposure to virus (white spot syndrome baculovirus or M. rosenbergii nodovirus), bacteria (Aeromonas hydrophila or Vibrio harveyi) or heavy metals (cadmium or lead) significantly increased the expression of GSTT (P b 0.05) in hepatopancreas. Recombinant GST-θ with monochlorobimane substrate had an optimum activity at pH 7.5 and 35 °C. Furthermore recombinant GST-θ activity was abolished by the denaturants triton X-100, Gua-HCl, Gua-thiocyanate, SDS and urea in a dose-dependent manner. Overall, the results suggest a potential role for M. rosenbergii GST-θ in detoxification and possibly conferring immune protection. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Glutathione S-transferases (GSTs) are a family of enzymes that reduce organic hydroperoxides, or catalyze the conjugation of glutathione (GSH) to a large variety of electrophilic alkylating compounds, such as hydroxyalkenals, thereby protecting the cell from the potential toxic effects of peroxides (Forman et al., 2009). Directly or indirectly GST is involved in a number of different mechanisms that are very important for the host defense and protection from oxidative damage caused by free radicals (Kim et al., 2009). In addition, it has immune-modulatory properties and is identified as a potentially promising candidate for vaccine development against different parasitic infections (Ouaissi et al., 2002).

Abbreviations: Mr, Macrobrachium rosenbergii; GST-θ, glutathione s-transferase-theta; MrGST-θ, Macrobrachium rosenbergii glutathione s-transferase-theta; GSH, glutathione; MAPEG, membrane-associated proteins in eicosanoid and glutathione metabolism; WSBV, white spot syndrome baculovirus; MrNV, Macrobrachium rosenbergii nodovirus; MCB, monochlorobimane; Cd, cadmium; Pb, lead; ORF, open reading frame; UTR, untranslated region; qRT-PCR, quantitative real time polymerase chain reaction; IPTG, isopropylβ-thiogalactopyranoside. ⁎ Corresponding author. E-mail address: [email protected] (J. Arockiaraj).

Based on the amino acid sequence homology and structure, GSTs are grouped into three major super families' i.e., cytosolic, mitochondrial and MAPEG GSTs. Cytosolic GSTs are approximately 23–28 kDa in size and are present either as homodimers or heterodimers. The cytosolic GSTs have been further subgrouped into 13 different classes (i.e., alpha, beta, delta, epsilon, zeta, theta, mu, nu, pi, sigma, tau, phi and omega) based on their N-terminal amino acid sequences, substrate specificities, antibody cross-reactivity and sensitivities to inhibitors (Richardson et al., 2009). Cytosolic GST-θ is considered to be the most ancient group and is claimed to have a significant function in human carcinogenesis as it catalyzes the conjugation of reduced glutathione to different electrophilic and hydrophobic compounds (Hayes and Pulford, 1995). GST-θ group consists of two different types i.e., GST-θ1 and GST-θ2 which share 55% identity in their protein structure. Interestingly, given the complexity in the expression and presence of the enzymes in different locations of the cell, most GST based studies have been performed separately for each isoform. It is to be noted that the expression of GSTs in aquatic animals does not always follow the same pattern and varies with species, tissue, sex and age (Chiou et al., 1997; Hayes and Pulford, 1995). The freshwater prawn, Macrobrachium rosenbergii, is a commercially important 0378-1119/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: Arockiaraj, J., et al., A cytosolic glutathione s-transferase, GST-theta from freshwater prawn Macrobrachium rosenbergii: molecular and biochemical properties, Gene (2014),


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crustacean across Asia-Pacific. Both biotic and abiotic factors in the aquatic environment may cause oxidative stress in M. rosenbergii which ultimately may lead to untimely death. Thus, a better knowledge of GST-θ biochemistry and pattern of gene expression may help to better understand antioxidation mechanisms and disease control in this prawn. Therefore the objective of this study was to clone a GSTT cDNA from M. rosenbergii, analyze the pattern of gene expression in different tissues and in response to virus, bacteria and heavy metals, as well as to produce and characterize the recombinant GST-θ from M. rosenbergii (named as MrGST-θ). 2. Materials and methods 2.1. Isolation of GSTT cDNA A full length GSTT cDNA was isolated from the M. rosenbergii cDNA library developed from the tissue pool including the muscle, gill, hemocyte, hepatopancreas and brain (Arockiaraj et al., 2013a) and sequenced on a Genome Sequencer FLX. The identity of the GSTT cDNA was established by similarity searches using BLAST and the NCBI database (Altschul et al., 1990). 2.2. Bioinformatics analysis The open reading frame (ORF) and corresponding amino acid sequence of GSTT were obtained by using DNAssist 2.2 (Patterton and Graves, 2000). Characteristic domains or motifs were identified using the PROSITE profile database (De Castro et al., 2006). Pair-wise and multiple sequence alignments were constructed in Clustal Omega Program (Larkin et al., 2007; Thompson et al., 1994) and edited using BioEdit (Hall, 2011). The phylogenetic tree was constructed on PHYLIP 3.69v (Felsenstein, 2005) program using maximum likelihood method. GST-kappa from Xenopus laevis was set as outgroup. 2.3. Overexpression of recombinant protein The GSTT coding region was cloned into pMAL-c2X (BioLabs Inc.) expression vector according to our earlier protocol (Arockiaraj et al., 2011a, 2012a, 2013b). GSTT specific primers [F5: 5′ (GA)3 GAATTC ATG GCT GGT AAA TTA ATA CTG TAT 3′-EcoRI and R6: 5′ (GA)3 CTGCAG TTA GAG AGA CTG CCA AGC AAA TGA TGC AG 3′-PstI] used for cloning contained EcoRI and PstI restriction sites in forward and reverse respectively. Transformed Escherichia coli BL21 (DE3) cells were incubated in ampicillin (100 μg/mL) Luria broth (LB) overnight. This culture was then used to inoculate 100 mL of LB broth in 0.2% glucose-rich medium with ampicillin at 37 °C until cell density reached 0.6 at OD600. E. coli BL21 (DE3) harboring pMAL-c2x-MrGSTT was induced for over expression with 1 mM isopropyl-β-thiogalactopyranoside (IPTG) and incubated at 16 °C for 5 h. Cells were harvested by centrifugation (4000 ×g for 20 min at 4 °C) and re-suspended in column buffer (Tris–HCl, pH 7.4, 200 mM NaCl) and frozen at − 20 °C overnight. After thawing on ice, cells were disrupted by sonication. The crude MrGST-θ fusion protein followed by recombinant MrGST-θ protein was purified as previously described (Arockiaraj et al., 2012b). The concentrations of purified proteins were determined by the method of Bradford (1976) using bovine serum albumin (BSA) as standard. The purified protein was kept at −80 °C until determination of molecular functional activities. 2.4. Characterization of MrGST-θ activity The enzymatic activity (μmol/min/μg) of recombinant MrGST-θ was determined in triplicate using a GST activity assay kit (Fluorometric) with monochlorobimane (MCB) (in DMSO, dimethyl sulfoxide) as substrate as described in the manufacturer's protocol (Abcam, India, Cat No. ab65326).

The optimum temperature, pH and effect of denaturing agents triton X-100, Gua-HCL, Gua-thiocyanate, SDS and urea on recombinant MrGST-θ activity were carried out in triplicate assays as described by Hu et al. (2012).

2.5. Biotic and abiotic factor oxidative stress challenge and tissue collection Healthy prawns (15 ± 2.5 g) were obtained from Sri Sai Aqua Farm, Nellore, Andhra Pradesh, India. They were maintained in 50 L flatbottomed fiber tanks (15 prawns per tank) with aerated and filtered freshwater at 28 ± 1 °C in the laboratory. All prawns were acclimatized for one week before being injected with immune stimulants. For each challenge (see below), three experimental tanks and three control tanks were maintained. For each challenge, one animal was randomly selected from each tank (a total of 3 experimental and 3 control individuals) at each time point of analysis. The effect of various challenges on gene expression was tested: injections of white spot syndrome baculovirus (WSBV) or M. rosenbergii nodovirus virus (MrNV), infections with Aeromonas hydrophila or Vibrio harveyi (Arockiaraj et al., 2012c); and exposure to heavy metals (cadmium or lead) as described by Chen et al. (2011). For the viral challenge, PCR confirmed WSBV or MrNV infected prawn tail tissue was homogenized in sterile 2% NaCl (1:10, w/v) solution and centrifuged in a tabletop centrifuge at 3000 g for 5 min at 4 °C. The supernatant was filtered through 0.45 μm filter and used for injecting M. rosenbergii (150 μL per 15 g animal). Tissue homogenate prepared from healthy tail muscle served as control. For the bacterial infection, M. rosenbergii was injected intraperitoneally with A. hydrophila or V. harveyi (5 × 106 CFU/mL) suspended in 1× phosphate buffer saline (50 μL per 15 g prawn). Equal volume of PBS buffer injected individuals served as control. For the heavy metal challenge, prawns were placed in 50 L freshwater tanks containing cadmium (50 μg/L) or lead (40 μg/L). The heavy metal concentrations were selected based on the literature of heavy metal contamination in Indian rivers (Usha, 2013). UV treated freshwater exposure served as control. In all challenges samples were collected before (0 h), and after injection at regular intervals (3, 6, 12, 21 and 48 h). The hemolymph (0.3–0.5 mL per prawn) was collected from the heart with a sterile syringe, immediately centrifuged at 3000 g for 10 min at 4 °C and the sedimented hemocyte collected. The pleopods, walking legs, eye stalks, gills, hepatopancreas, stomach, intestine, brain and muscle were also collected. All tissues were frozen immediately at − 80 °C and stored until RNA extraction.

2.6. Analysis of GSTT gene expression Total RNA from the control and infected M. rosenbergii was isolated using Tri Reagent™ (Life Technologies), according to the manufacturer's protocol with slight modifications (Arockiaraj et al., 2012d, 2013c). Using 2.5 μg of RNA, first strand cDNA was synthesized by SuperScript® VILO™ cDNA Synthesis Kit (Life Technologies). The resulting cDNA solution was stored at −20 °C for further analysis. The expression level of GSTT gene was measured in triplicate by quantitative reverse-transcription polymerase chain reaction (qRTPCR) (BIO-RAD CFX384 Touch Real-Time PCR Detection System) as previously described (Arockiaraj et al., 2011b). The following GSTT primers were used: MrGST-θ F1: GTT GTG CAG CAT TGA GGT TTA T and MrGST-θ R2: GTA TCC TAC ACC ATG TGC TCT G. β-Actin (GenBank accession number AY651918) was used as reference with primer gene β-actin F3: ACC ACC GA ATT GCT CCA TCC TCT and β-actin R: ACT GCC ACT TSGT TCAS CCA TC CGA TTF. The comparative CT method (2−δδCT method) was used for quantification (Livak and Schmittgenm, 2001).

Please cite this article as: Arockiaraj, J., et al., A cytosolic glutathione s-transferase, GST-theta from freshwater prawn Macrobrachium rosenbergii: molecular and biochemical properties, Gene (2014),

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2.7. Statistics Relative mRNA expression and enzyme activities were compared by one-way ANOVA followed by Tukey's multiple range test using SPSS 11.5 at the 5% significance level. 3. Results 3.1. Bioinformatics analysis of GSTT The full length GSTT cDNA (accession number HF570114) was 1417 base pair (bp) nucleotides long with a 189 bp 5′ untranslated region (UTR), an open reading frame (ORF) of 663 bp and a 565 bp 3′ UTR (Supplementary material). The ORF encodes a polypeptide of 221 amino acids with an estimated molecular weight of 25 kDa and a predicted theoretical isoelectric point (pI) of 8.77. Two putative polyadenylation sites were also observed in the 3′ UTR portion at 1387–1392 and 1403–1408 respectively. The translated MrGST-θ polypeptide sequence neither has a signal peptide nor transmembrane regions and contains two soluble GST domains at N (3–84) and C (90–221) terminal respectively. Moreover, MrGST-θ possesses 20 substrate binding sites out of which 10 are present at N-terminal domain (Tyr8, Val12, Arg17, Gln31, Leu37, Lys39, Pro57, Glu68, Ser69 and Asp81) and others at C-terminal (His105, Arg109, Ala110, Val113, Gly114, Phe116, Arg117, Leu121, Glu175 and Gln178). Further, motif analysis showed that the MrGST-θ consists of 4 casein kinase II phosphorylation


sites (78–81, 128–131, 166–169 and 211–214), 1 tyrosine kinase phosphorylation site (144–152) and 2 protein kinase C phosphorylation sites (211–213 and 215–217). MrGST-θ amino acid sequences showed more similarities at the N terminal region when aligned with six other homologous sequences (Supplementary material). Furthermore, MrGST-θ had a 37% sequence identity (similarity = 61%) with GSTT from Locusta migratoria (Supplementary material). The evolutionary distance of MrGST-θ was determined using 24 homologous sequences by constructing a phylogenetic tree. The phylogenetic tree showed two distinct clades, i.e., vertebrates and invertebrate GSTT whereas GST kappa remained as outgroup. As expected, MrGST-θ clustered together with the arthropods (Fig. 1).

3.2. MrGST-θ recombinant protein expression and activity analysis Recombinant MrGST-θ protein together with the fusion protein, maltose binding protein (MBP) appeared as a single band with a size of 67.5 kDa (42.5 kDa for MBP and 25 kDa for MrGST-θ) on SDS-PAGE. After MBP cleavage, the MrGST-θ recombinant protein was further purified by DEAE-Sepharose ion exchange chromatography producing a single band with a molecular weight of 25 kDa on SDS-PAGE (Fig. 2). Optimum enzyme activity was determined to be at 35 ± 2 °C and pH 7.5 and amounted to 8.258 ± 0.81 μmol/min/μg protein. All the tested denaturants inhibited the activity in a concentration dependent manner.

Fig. 1. A phylogenetic tree of MrGST-θ with 24 other homologous glutathione s-transferase with GST kappa of Xenopus laevis as outgroup from vertebrates and invertebrates was reconstructed by maximum likelihood method. The tree is based on an alignment corresponding to full-length amino acid sequences. The numbers shown at the branches denote the bootstrap majority consensus values of 1000 replicates. The GenBank accession number is given in the parentheses.

Please cite this article as: Arockiaraj, J., et al., A cytosolic glutathione s-transferase, GST-theta from freshwater prawn Macrobrachium rosenbergii: molecular and biochemical properties, Gene (2014),


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Fig. 2. Expression and purification of the recombinant MrGST-θ protein. Protein samples were separated by SDS-PAGE and stained with Coomassie brilliant blue. Lane 1, protein marker; lane 2, before induction with IPTG; lane 3, after IPTG induction; lane 7, purified fusion protein and lane 10, purified recombinant protein.

3.3. Gene expression analysis of MrGST-θ The highest GSTT expression was observed in hepatopancreas followed by hematocyte (P b 0.05) (Fig. 3A). Hence, the hepatopancreas was analyzed for the gene expression response to biotic and abiotic challenges. In WSBV injected M. rosenbergii species, GSTT gene expression increased 5.5-fold at 12 h post-injection (p.i.) compared to the control group (P b 0.05; Fig. 3B). MrNV also caused a significant (P b 0.05) 19-fold increase in gene expression at 24 h p.i. (Fig. 3B). The GSTT gene expression response to A. hydrophila infection peaked at 5-fold (P b 0.05) at 12 h p.i. (Fig. 3B). V. harveyi also caused increased gene expression (P b 0.05) at 12 h p.i. in comparison to control (Fig. 3B). Cadmium and lead caused a 20-fold upregulation of GSTT in comparison to control (P b 0.05) at 24 h p.i. (Fig. 3B).

4. Discussion The putative protein product of the cloned M. rosenbergii GSTT possessed the characteristic features of cytosolic GST such as the absence of signal sequence at N terminal and presence of two domains at N and C terminals respectively. Dirr et al. (1994) reported that most cystolic GSTs have two binding sites i.e. G-site and H-site, at N and C terminals respectively. It is predicted that G-site is highly specific for glutathione binding and H-site for electrophilic substrates. In this study, we observed that these binding sites were also present at N-terminal (G-site) and C-terminal (H-site). Interestingly, the structure, numbers and position of the amino acid of the substrate binding sites varied in different species and GST isomer forms (Hu et al., 2012; Ji et al., 1997). It is reported by Hu et al. (2012) that these changes or modification in amino acid of the substrate binding sites could lead to a decreased affinity and an increased flexibility for GST. Our enzyme activity studies showed that the recombinant protein produced was functional suggesting that the putative binding sites are active. It has been suggested that GST-θ is an ancient class among the other GST group and include a large number of subclasses and isoforms in various species. The multiple sequence alignment of the putative MrGST-θ amino acid indicates that the N terminal was conserved in comparison to the C

terminal. In addition, the H-site shows a high degree of homology in amino acid sequences and is important for xenobiotic compound binding, specificity and activity of GSTs (Armstrong, 1997; Hayes and Pulford, 1995). GSTT has two polyadenylation sites in the 3′ UTR at 1387–1392 and 1403–1408 which suggest the possibility of having multiple functions (Caizzi et al., 1990). Similar findings were observed in GST-pi from Crassostrea gigas (Boutet et al., 2004) and GST-rho from Antarctic bivalve (Park et al., 2009). The MrGST-θ enzyme had an optimum activity at 7.5 pH and 35 °C. The optimum pH and temperature for the GST activity also varied among GST isomers from different species as discussed by Hu et al. (2012). GST is widely used as a tag for the recombinant expression of proteins in heterologous systems. Usually the fusion proteins (GST-protein interest) lie inside the cell, which needs to be broken down with detergents. It is generally assumed that breaking the cells with detergents does not affect the purification strategy or the function of the protein of interest. Our results show that the activity of MrGST-θ enzyme decreased with the increase in the denaturant concentrations. Our results are consistent with Seroude and Cribbs (1994) where in they have shown that detergents can inhibit the human GST activity but not the DNA binding properties. According to the available literature, the pattern of GST expression among tissues and species varies (Kim et al., 2009; Rhee et al., 2008). Our results are in agreement with these and other studies in fish and shellfish which have found GSTT expression in the gills, hepatopancreas, hemocytes, mantle, kidney, liver, intestine, muscle, gonad, excretory system and digestive gland (Leaver et al., 1993; Meyer et al., 1991). It has been shown by various researchers that GST gene expression is influenced by heavy metals (Chen et al., 2011; Li et al., 2012), thermal stress (Kim et al., 2009), organic pollutants (Park et al., 2009; Xu et al., 2010), bacteria (Hu et al., 2012; Wang et al., 2013), virus (Duan et al., 2013) and microorganism glycan (Yang et al., 2012). In the present study, challenges with pathogenic bacteria and virus promoted a strong upregulation of GSTT. It may be assumed that after infection changes in the GSTT gene influence the respiratory bursts in prawn and activate the reactive oxygen metabolite production which in turn destroy pathogen and protect the host cell (Hunaiti and Soud, 2000). Similarly,

Please cite this article as: Arockiaraj, J., et al., A cytosolic glutathione s-transferase, GST-theta from freshwater prawn Macrobrachium rosenbergii: molecular and biochemical properties, Gene (2014),

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Fig. 3. Gene transcript patterns of M. rosenbergii GSTT determined by real time PCR. 3A: tissue distribution of GSTT from three individuals. Data are expressed as a ratio to pleopod GSTT. The different letters indicate statistically significance at P b 0.05 level by one-way ANOVA and Tukey's multiple range test. 3B: The time course of GSTT mRNA expression in hepatopancreas at 0, 3, 6, 12, 24, and 48 h post challenge with WSBV, MrNV, A. hydrophilla, V. harveyi, cadmium and lead. Data are expressed as a ratio to GSTT from three individuals relative to three control individuals. Statistical difference between the challenged and the control group is indicated with asterisks.

upregulation was observed with exposure to metals Cd and Pb which is in agreement with the results obtained in human as well as in rats (Wright et al., 1998). GST has the ability to provide protection from diverse range of chemical molecules indicating that individual GST genes are regulated in a distinct fashion and each encodes a protein with unique catalytic activity. It is believed that knowledge of the activities of individual isoenzymes from different species occurring in diverse sources will allow a rational understanding of the biological consequences of GST over expression and under expression.

Acknowledgments This research is supported by SERB Young Scientist Fellowship Scheme (SERC/LS-437/2011), Science and Engineering Research Board (SERB), Department of Science and Technology, Ministry of Science and Technology, Government of India, New Delhi. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.

5. Conclusions References So far, many GSTs from aquatic organism have been characterized at molecular level, but only a very few GSTs reported for crustacea. MrGSTθ has sequence similarity to other GST and the present study suggests that also in crustaceans GSTs are likely to play a variety of functions such as in immune responses and detoxification. Conflict of interest The authors declare that the article do not have any conflict of interest.

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Please cite this article as: Arockiaraj, J., et al., A cytosolic glutathione s-transferase, GST-theta from freshwater prawn Macrobrachium rosenbergii: molecular and biochemical properties, Gene (2014),

A cytosolic glutathione s-transferase, GST-theta from freshwater prawn Macrobrachium rosenbergii: molecular and biochemical properties.

Glutathione S-transferases play an important role in cellular detoxification and may have evolved to protect cells against reactive oxygen metabolites...
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