19 ISJ 15: 19-30, 2018 ISSN 1824-307X RESEARCH REPORT Sequence features, expression profiles and biochemical characteristics of a sigma class glutathione S-transferase gene (AiGSTσ) from bay scallop Argopecten irradians M Wang1, 3, B Wang1, M Liu1, K Jiang1, L Wang1, 2 1Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China 2Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China 3Research Platform for Marine Molecular Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China Accepted December 5, 2017 Abstract Glutathione S-transferases (GSTs) are a class of enzymes that facilitate the detoxification of xenobiotics and also play important roles in innate immunity. In the present study, a novel sigma class GST gene (designated as AiGSTσ) was cloned from the bay scallop Argopecten irradians via rapid amplification of cDNA ends (RACE) technique. The complete cDNA sequence of AiGSTσ consisted of a 5’ untranslated regions (UTR) of 48 bp, a 3’ UTR of 113 bp with a poly A tail and an open reading frame (ORF) of 618 bp. The ORF encoded a polypeptide of 205 amino acid residues with a calculated molecular mass of approximately 23.11 kDa and a theoretical isoelectric point of 5.354. The deduced amino acid sequence of AiGSTσ contained a GST_N domain and a GST_C domain, and exhibited high similarity with other reported sigma class GSTs. In the phylogenetic tree, AiGSTσ was located in the sigma class GSTs sub-branch. The AiGSTσ mRNA transcripts were constitutively expressed in the tissues of hemocytes, muscle, mantle, gill, hepatopancreas and gonad, with the highest expression level in hemocytes, and the mRNA expression levels of AiGSTσ were significantly up-regulated in hemocytes after various pathogen associated molecular patterns (PAMPs) stimulation. The purified recombinant AiGSTσ protein exhibited catalytic activity against the common substrate 1-chloro-2, 4-dinitrobenzene (CDNB) with low thermal stability and narrow optimum pH spectrum. All these results indicated that AiGSTσ was a fragile but efficient antioxidant enzyme and was potentially involved in the innate immune responses of scallop. Key Words: Argopecten irradians; glutathione S-transferase; innate immunity Introduction The innate immunity is almost the solo defence mechanism for invertebrates that protects hosts against microbial invaders (Song et al., 2015). In the innate immune defence mechanism, hemocytes can phagocytize and kill the microbial pathogens (Lu et al., 2013; Chen et al., 2014; Wang et al., 2014). When the host is attacked by microbial invaders, phagocytosis is activated with high oxygen consumption named the respiratory burst and followed by mass reactive oxygen intermediates (ROI) and reactive oxygen species (ROS) production ___________________________________________________________________________ Corresponding author: Lei W ang Key Laboratory of Experimental Marine Biology Institute of Oceanology Chinese Academy of Sciences Qingdao 266071, China E-mail: wanglei@qdio.ac.cn (Shao et al., 2017). Therefore, organisms employ the antioxidant system to maintain ROI and ROS at the normal physiological levels (Zhang et al., 2017a, b). As an essential kind of antioxidant enzymes, glutathione S-transferases (GSTs, EC 2.5.1.18) are a superfamily of multifunctional phase II enzymes primarily catalyzing reduced glutathione to both endogenous and exogenous electrophiles (Sheehan et al., 2001). GSTs have been identified from the cytosol, mitochondria and microsomes of all the prokaryotic and eukaryotic organisms that have been studied (Raza et al., 2002). Generally, based on their primary and tertiary structures, substrate and inhibitor specificity, and immunological cross reactivity, GSTs could be grouped into at least fifteen classes, which were termed as alpha (α), beta (β), delta (δ), epsilon (ε), kappa (κ), lambda (λ), mu (μ), omega (ω), phi (φ), pi (π), sigma (σ), tau (τ), theta (θ), zeta (ζ) and rho (ρ) (Wilce and Parker, 1994). 20 Table 1 Primers used in the present study Primer Sequence (5`-3`) Brief information adaptor primer GGCCACGCGTCGACTAGTAC Anchor primer for 3` RACE adaptor primer-oligo (dT) GGCCACGCGTCGACTAGTACT17VN Olido (dT) for cDNA synthetize Aiactin-qRT-F CAAACAGCAGCCTCCTCGTCAT Internal control for real-time PCR Aiactin-qRT-R CTGGGCACCTGAACCTTTCGTT Internal control for real-time PCR AiGSTσ-CDS-F ATGCCTTCCTACAAACTTATCTAC Gene specific primer for CDS AiGSTσ-CDS-R TTAGATCACGCTCTCGGGACGCGA Gene specific primer for CDS AiGSTσ-qRT-F CTGATCCGTCTCGCTTTCGCT Gene specific primer for real-time PCR AiGSTσ-qRT-R GCTGTTTCCCGTCCACTTCCA Gene specific primer for real-time PCR AiGSTσ-RACE-F1 CCCAAATTTGCCGAAATC Gene specific primer for RACE AiGSTσ-RACE-F2 AGTTGAACCCAGATTGTTTGAAGG Gene specific primer for RACE M13-47 CGCCAGGGTTTTCCCAGTCACGAC Vector primer for sequencing RV-M GAGCGGATAACAATTTCACACAGG Vector primer for sequencing T7 ACATCCACTTTGCCTTTCTC Vector primer for sequencing T7-ter TGCTAGTTATTGCTCAGCGG Vector primer for sequencing Among all the GSTs classes, sigma class GST (GSTσ) comprises one of the largest GST subfamilies identified from invertebrates to vertebrates, which was believed to evolve from ancestral GST genes and exhibit high levels of enzymatic activity toward the common substrate 1-chloro-2, 4-dinitrobenzene (CDNB) (Flanagan and Smythe, 2011). Recently, several sigma class GSTs were identified and investigated in marine invertebrates (Boutet et al., 2004; Lee et al., 2007; Wan et al., 2008; Ren et al., 2009; Li et al., 2012; Umasuthan et al., 2012; Yang et al., 2012; Zhang et al., 2012a, b; Wang et al., 2013a; Li et al., 2015). Among these sigma class GSTs, the abGSTsigma gene from abalone Haliotis diversicolor was significantly expressed in the hemocytes, gill, mantle and digestive gland of bacteria-challenged abalone (Ren et al., 2009). Bacterial challenge could significantly up-regulate the mRNA expression of both VpGST-1 and VpGST-2 from Manila clam Ruditapes (Venerupis) philippinarum (Li et al., 2012). The mRNA expression level of SgGST-S1 in hemocytes was significantly up-regulated after razor clam Solen grandis was stimulated by peptidoglycan (PGN) or β-1, 3-glucan (glucan) (Yang et al., 2012). While after bacterial challenge, the mRNA expression levels of sigma class GSTs in hemocytes were all significantly higher than those of the control group in mussels Mytilus galloprovincialis (Wang et al., 2013a). All these research achievements revealed that sigma class GSTs from marine invertebrates were functional diversity and might not only serve as an antioxidant enzyme involving in the detoxification but also play important roles in the modulation of innate immune responses. Bay scallop Argopecten irradians was introduced from USA in 1982 and has become one of the most important aquaculture species in China, due to its high economic value, fast growth rate and adaptation ability to different regions for aquaculture (Li et al., 2007). And A. irradians was also considered as an attractive model to study immunology because of its relatively simple innate immune system and its propensity to undergo various manipulations, which allows researchers to study the effects of both biological and non-biological factors on the innate immune responses (Matozzo, 2016). By now, several antioxidant enzyme genes have been identified and characterized in A. irradians, such as metallothionein (MT) (Wang et al., 2009), peroxiredoxin (PRX) (Li et al., 2011) and superoxide dismutase (SOD) (Bao et al., 2008, 2009a, b, 2010), however, no information about GST genes was available in bay scallop till now. To bridge this gap, the main objectives of the present study were (1) to clone the full-length cDNA of sigma class GST from A. irradians (designated as AiGSTσ), (2) to investigate the tissue distribution of AiGSTσ mRNA transcripts and their temporal expression after different pathogen associated molecular patterns (PAMPs) stimulation, and (3) to validate the activities of recombinant AiGSTσ protein under different treatments. Materials and Methods Scallops, immune stimulation and sample collection The bay scallops used in the present study were obtained from a local farm in Qingdao, China, and all the experiments were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH). The study protocol and all the experimental design were conducted with approval from Experimental Animal Ethics Committee of Institute of Oceanology, Chinese Academy of Sciences. Approximately 200 scallops with an average 50 mm in shell length were employed for the PAMPs stimulation treatment. The scallops were randomly divided into 6 groups and each group contained about 30 - 40 individuals. The scallops were received an injection of 50 μL phosphate buffered saline (PBS, 0.14 mol L-1 sodium chloride, 3 mmol L-1 potassium chloride, 8 mmol L-1 disodium hydrogen phosphate dodecahydrate, 1.5 mmol L-1 21 Table 2 Information of GST proteins used in phylogenetic analysis Class Species Accession Number omega Chlamys farreri ADF32018 Craassostrea gigas XP_011429380 Danio rerio NP_001002621 Haliotis discus discus ABO26600 Haliotis madaka ALU63761 Perna viridis AGN03944 sigma Argopecten irradians ANG56313 Chlamys farreri ACF25904 Chlamys farreri ADF32019 Hyriopsis cumingii AGU68336 Pinctada fucata JAS04242 Ruditapes philippinarum AEW46325 rho Chlamys farreri ACF25903 Cyprinus carpio BAS29983 Ruditapes philippinarum AEW46331 Sebastes schlegelii ANW83217 Siniperca chuatsi ACI32418 Solea senegalensis BAG12568 zeta Chlamys farreri ADD82544 Cyprinus carpio BAS29981 Oplegnathus fasciatus ADY80028 Xenopus laevis XP_018084636 microsomal Chlamys farreri ADF45336 Gallus gallus NP_001129022 Microtus ochrogaster XP_005364596 Osmerus mordax ACO10098 Sinonovacula constricta ALC77324 Xenopus tropicalis NP_001011245 potassium phosphate monobasic, pH 7.4), lipopolysaccharides from Escherichia coli 0111:B4 (LPS, L2630, Sigma-Aldrich, USA, 0.5 mg mL-1 in PBS), PGN from Staphylococcus aureus (77140, Sigma-Aldrich, USA, 0.5 mg mL-1 in PBS), glucan from baker’s yeast Saccharomyces cerevisiae (G5011, Sigma-Aldrich, USA, 0.5 mg mL-1 in PBS) or polyinosinic-polycytidylic acid (poly IC, P1530, Sigma-Aldrich, USA, 0.5 mg mL-1 in PBS), respectively. The injected scallops were returned to seawater tanks immediately and five individuals were randomly sampled from each stimulated and unstimulated group at 3, 6, 12, 24 and 48 h post injection. The hemolymphs were collected and centrifuged at 800 g, 4 °C for 10 min to harvest the hemocytes for RNA preparation. Hemocytes, muscle, mantle, gill, hepatopancreas and gonad from five untreated scallops were collected to determine mRNA transcripts of AiGSTσ. RNA isolation and cDNA synthesis Total RNA was isolated from the hemocytes of scallops with RNAiso plus reagent (9108, Takara, Japan). The first-strand synthesis was carried out using the DNaseⅠ (RQ1, M6101, Promega, USA) treated raw RNA as template and adaptor primer-oligo (dT) as primer (Table 1). The reaction were performed at 42 °C for 1 h, terminated by heating at 95 °C for 5 min, and then stored at -80 °C till use. EST analysis and cloning of full-length AiGSTσ cDNA An EST (Ai_F00346) from bay scallop cDNA library in National Center for Biotechnology Information (NCBI) homologous to previously identified sigma class GST genes was selected for further cloning the cDNA of AiGSTσ. Two gene-specific primers, AiGSTσ-RACE-F1/F2 (Table 22 1), were designed to clone the 3’ sequence of AiGSTσ cDNA by rapid amplification of cDNA ends (RACE) technique. And the coding sequence (CDS) of AiGSTσ was amplified and confirmed using another two gene-specific primers, AiGSTσ-CDS-F/R. All PCR amplification was performed in an A300 Fast Thermal Cycler (LongGene, China), and the PCR products were purified using Monarch DNA Gel Extraction Kit (T1020S, NEB, USA) and cloned into the pMD18-T simple vector (D103A, Takara, Japan). After being transformed into the competent cells Escherichia coli strain DH5α (CB101, Tiangen, China), the positive recombinants were identified via anti-ampicillin selection and verified by PCR screening using M13-47 and RV-M primers (Table 1). Three of the positive clones were sequenced using a PRISM 3730XL automated sequencer (Thermo Fisher Scientific, USA). Bioinformatical analysis of cDNA and protein sequences The protein sequences information for homologous and phylogenetic analysis was listed in Table 2. The search for protein sequence similarity was conducted with blastp 2.6.0. The deduced protein sequences were analyzed by the EditSeq module in Lasergene program suite 14.0.0.88. The function domains were predicted using Simple Modular Architecture Research Tool (SMART) 7.0. Multiple sequence alignments were performed with Clustal Omega 1.2.4 and visualized by multiple alignment show module in Sequence Manipulation Suite 2.0. A Neighbor-Joining (NJ) phylogenic tree was constructed with MEGA 7.0.26. To derive confidence value for the phylogeny analysis, bootstrap trials were replicated 1,000 times. Expression patterns analysis via quantitative real-time PCR The mRNA transcripts of AiGSTσ in different tissues or their temporal expression patterns in hemocytes of scallops stimulated with various PAMPs were investigated by quantitative real-time PCR (qRT-PCR). All qRT-PCR reactions were performed with the SYBR premix ExTaq (Tli RNaseH plus) (RR420, Takara, Japan) using 100 ng cDNA template in a LineGene K FQD-48A (A4) Fluorescence Quantitative PCR Detection System (Bioer, China). All the primers using in qRT-PCR were listed in Table 1. The mRNA expression levels of AiGSTσ were normalized to those of β-actin for each sample. The relative mRNA expression levels of AiGSTσ were generated using comparative CT method (2-ΔΔCt method) (Schmittgen and Livak, 2008). All the data were subjected to one-way analysis of variance (ANOVA) followed by a multiple comparison using CoStat 6.400, and the p values less than 0.05 were considered statistically significant. Recombinant and purification of AiGSTσ in E. coli The CDS of AiGSTσ was amplified using two gene-specific primers, AiGSTσ-CDS-F/R (Table 1), and ligated to the expression vector pEASY-Blunt E1 (CE111, Transgen, China). The recombinant plasmid, pEASY-Blunt E1/AiGSTσ, was isolated by Monarch Plasmid Miniprep Kit (T1010S, NEB, USA) and then transformed into E. coli strain BL21 (DE3) (CD601, Transgen, China). The positive transformants, E. coli BL21 (DE3)/pEASY-Blunt E1/AiGSTσ, were incubated in ArtMedia Protein Expression auto-inducing medium (CP101, Transgen, China) containing 100 mg L-1 ampicillin (GG101, Transgen, China) at 28 °C with shaking at 220 rpm for 24 h. The recombinant protein (designated as rAiGSTσ) was purified using a His-tag Protein Purification Kit (P2226, Beyotime, China) under natural condition. The resultant protein was separated by 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and visualized with Protein Stains H (C510041, Sangon, China). Analysis of enzymatic activity of rAiGSTσ The specific activities of rAiGSTσ were measured as described in previous reports (Habig et al., 1974 Wan et al., 2008; Umasuthan et al., 2012). Briefly, the reaction was carried out in a 1 mL mixture containing 100 mM PBS, 10 mM GSH (S0073, Beyotime, China), and an appropriate amount of rAiGSTσ. The enzyme mixture was incubated at 25 °C for 5 min before the reaction was initiated by adding 1 mM CDNB (703318, Cayman Chemical, USA) and absorbance was monitored for 5 min at 340 nm while the reaction was maintained at 25 °C. The changes in absorbance per minute were converted into amounts of substrate conjugated per min per mg enzyme by using the molar extinction coefficient for CDNB ε340 = 9.6 mM-1 cm-1. To characterize the rAiGSTσ, enzymatic activity was evaluated at different temperature and pH. To determine the optimal temperature, protein samples were treated at 10 °C intervals between 10 °C and 90 °C for 1 h. To investigate the optimal pH, protein samples were treated between pH 3.5 and 10.5 at 1.0 pH intervals using different buffers for 1 h. Acetate, phosphate and glycine-NaOH buffers were used to obtain the pH ranges of 3.5-5.5, 6.5-7.5 and 8.5-10.5, respectively, according to previously reports (Wang et al., 2013b, 2015). Results Sequence features of AiGSTσ A sigma class GST gene, AiGSTσ, was identified from the bay scallop EST database, and its full-length cDNA sequence was obtained via RACE technique and deposited into GenBank under the accession number KU301768. The full-length cDNA sequence of AiGSTσ comprised 779 bp, containing a 5’ untranslated regions (UTR) of 48 bp, a 3’ UTR of 113 bp with a poly A tail and an open reading frame (ORF) of 618 bp. The ORF encoded a polypeptide of 205 amino acid residues with a calculated molecular mass of approximately 23.11 kDa and a theoretical isoelectric point of 5.354. No signal peptide was revealed in the deduced amino acid sequence of AiGSTσ by SignalP program. A GST_N domain (from Y4 to R73) and a GST_C domain (from I92 to N190) were found in the deduced amino acid sequence of AiGSTσ (Fig. 1). 23 Fig. 1 Nucleotide and deduced amino acid sequences of AiGSTσ. The nucleotides and amino acids were numbered along the left margin. The function domain was in shade. The asterisks indicated the stop codon. Two single typical polyadenylation signal sequences (AATAAA AATAAA) was underlined. Phylogenetic ananlysis of AiGSTσ The deduced protein sequence of AiGSTσ exhibited high similarity with other previously identified sigma class GSTs, such as 78 % identity with that of sigma class GST 2 from Chlamys farreri (ADF32019). The NJ phylogenetic tree based on protein sequences from multiple GST genes was positioned separately into five main branches, and AiGSTσ were clustered with sigma class GST 2 from C. farreri and located in the sigma class GSTs sub-branch (Fig. 2). Tissue distribution of AiGSTσ mRNA transcripts The qRT-PCR technique was employed to detect the distribution of AiGSTσ mRNA transcripts in different tissues with β-actin gene as internal control (Fig. 3). The highest mRNA expression level of AiGSTσ was found in hemocytes, which was 21.30-fold (p < 0.05) of that in muscle, while that in hepatopancreas was 13.28-fold (p < 0.05) of that in muscle. Expression profiles of AiGSTσ mRNA transcripts The temporal mRNA expression profiles of AiGSTσ in hemocytes after various PAMPs stimulation were also examined via qRT-PCR (Fig. 4A-D). The mRNA transcripts of AiGSTσ all increased for the first time at 3-6 h and reached the peak at 12 h post different PAMPs stimulation. The mRNA transcripts of AiGSTσ significantly increased at 3 h after LPS stimulation (2.79-fold compared with the origin level, p < 0.05, Fig. 4A), with the highest level observed at 12 h (17.74-fold, p < 0.05, Fig. 4A). The mRNA expression level of AiGSTσ was up-regulated at 6 h post PGN stimulation (3.13-fold, p < 0.05, Fig. 4B) and then up-regulated to the highest level at 12 h (6.82-fold, p < 0.05, Fig. 4B), and finally down-regulated to the normal level at 48 h. In the glucan stimulation group, after a significant increase at 3 h post stimulation (3.18-fold, p < 0.05, Fig. 4C), the mRNA transcripts of AiGSTσ increased to the peak at 12 h (12.15-fold, p < 0.05, Fig. 4C), and finally decreased to the original level at 48 h. 24 Fig. 2 Consensus neighbor-joining phylogenetic tree based on the amino acid sequences of GSTs from different organisms. The evolutionary history was inferred using the Neighbor-Joining method. The bootstrap consensus tree inferred from 1,000 replicates was taken to represent the evolutionary history of the taxa analyzed. All positions containing gaps and missing data were eliminated. The numbers at the forks indicated the bootstrap value. The sequence information has been listed in Table 2. The mRNA transcripts of AiGSTσ significantly increased at 6 h post poly IC stimulation (6.71-fold, p < 0.05, Fig. 4D), reached the peak at 12 h (9.07-fold, p < 0.05, Fig. 4D), and then decreased to the normal level at 24 h. In the normal group, no significant change of AiGSTσ mRNA expression level was observed during the whole experiment, while after PBS injection, a slight but significant increase was observed at 6 h (2.93-fold, p < 0.05, Fig. 4A-D). Purification of recombinant AiGSTσ protein To investigate the potential activities of AiGSTσ, the recombinant plasmid pEASY- Blunt E1/AiGSTσ was transformed into E. coli strain BL21 (DE3). After auto-induction, the whole-cell lysate was separated by SDS-PAGE, and a distinct band of rAiGSTσ was revealed (Fig. 5). Biochemical characteristics of recombinant AiGSTσ protein According to the method previously described, the activity of rAiGSTσ was measured for five times, and rAiGSTσ exhibited detectable activity towards CDNB, which was 3.28 ± 0.03 μmol min-1 mg-1. To investigate the stability of AiGSTσ, the enzymatic activities of rAiGSTσ were measured at different pH and temperature. For the optimal pH assay, rAiGSTσ could maintain more than 50 % of its activity at a pH range from 7.5 to 9.5, but lost more than 60 % of its activity when the pH was lower than 6.5 or at 10.5 (Fig. 6A). While when the temperature increased from 10 °C to 20 °C, rAiGSTσ exhibited stable enzymatic activities, but lost more than 60 % of its enzymatic activity over 30 °C and almost devitalized at 50 °C (Fig. 6B). 25 Fig. 3 Tissue distribution of AiGSTσ mRNA transcripts detected by qRT-PCR. The β-actin gene was used as an internal control to calibrate the cDNA template for each sample. The mRNA expression level of AiGSTσ in hemocytes, muscle, mantle, gill, hepatopancreas and gonad of five adult scallops was normalized to that of muscle. Vertical bars represented mean ± SD (n = 5), and bars with different characters indicated significantly different (p < 0.05). Discussion Sigma class GSTs are a large sub-family of GSTs (Flanagan and Smythe, 2011), and accumulating research achievements revealed that sigma class GSTs from marine invertebrates were functional diversity and might not only serve as an antioxidant enzyme involving in the detoxification but also play important roles in the modulation of innate immune responses (Boutet et al., 2004; Lee et al., 2007; Wan et al., 2008; Ren et al., 2009; Li et al., 2012; Yang et al., 2012; Umasuthan et al., 2012; Zhang et al., 2012a, b; Wang et al., 2013a; Li et al., 2015). In the present study, the full-length cDNA sequence of AiGSTσ was obtained from bay scallop A. irradians. The deduced polypeptide of AiGSTσ consisted of 205 amino acids, and its calculated molecular weight was 23.11 kDa, which was very close to GSTs of vertebrate and invertebrate. The amino acid sequence of AiGSTσ shared as high as 78 % identity with the previously identified sigma class GST 2 from C. farreri. In the phylogenetic tree, AiGSTσ was located in the sigma class GSTs sub-branch. Its sequence characteristics, high similarity with other known sigma class GSTs and the phylogenetic relationship collectively suggested that AiGSTσ is a novel member of invertebrate sigma class GST family and may have similar function with sigma class GSTs from other marine invertebrates. Sigma class GST acts as the principal scavenger of xenobiotics (Flanagan and Smythe, 2011), and it was reported to be ubiquitously distributed in multiple tissues in marine invertebrates (Boutet et al., 2004; Lee et al., 2007; Wan et al., 2008; Ren et al., 2009; Li et al., 2012; Yang et al., 2012; Umasuthan et al., 2012; Zhang et al., 2012a, b; Wang et al., 2013a; Li et al., 2015). In the present study, the tissue distribution of AiGSTσ mRNA transcripts was detected by qRT-PCR to investigate its possible function, and the ubiquity of AiGSTσ transcripts indicated that it could be involved in many important physiological processes of scallops. Similar to the observation in sigma class GSTs from M. galloprovincialis, S. grandis and V. philippinarum (Yang et al., 2012; Zhang et al., 2012a; Wang et al., 2013a), the highest mRNA expression level of AiGSTσ was observed in hemocytes, followed by hepatopancreas. The variable tissue distribution of AiGSTσ mRNA transcripts was speculated to be related with tissue dependent oxidative load. The hemocytes have been considered to play pivotal roles 26 Fig. 4 Temporal mRNA expression profiles of AiGSTσ detected by qRT-PCR in hemocytes at 3, 6, 12, 24 and 48 h post different PAMPs stimulation (A: LPS, B: PGN, C: glucan, D: poly IC). The β-actin gene was used as an internal control to calibrate the cDNA template for each sample. Each values was shown as mean ± SD (n = 5), and bars with different characters indicated significantly different (p < 0.05). in the innate immune response in invertebrates mainly via phagocytosis, which was usually companied with oxidative stress, while the hepatopancreas is regarded as the main organ where multiple oxidative reactions and antioxidant defenses occur with high metabolic activity (Song et al., 2015). Additionally, hemocytes and hepatopancreas were also considered as the main immune related organs in scallops (Song et al., 2015), the high mRNA expression level of AiGSTσ in these two organs indicated that it could be involved in the innate immunity of scallop. It has been reported that sigma class GSTs could rapidly respond to various foreign particles or invading microbes in mRNA levels. For examples, a sigma class GST gene from H. diversicolor could be significantly induced in the hemocytes, gill, mantle and digestive gland of bacteria-challenged abalone (Ren et al., 2009). Bacterial challenge could significantly induce the mRNA expression of two sigma class GSTs from V. philippinarum (Li et al., 2012). The mRNA expression of a sigma class GST in hemocytes was significantly up-regulated after razor clam was stimulated by PGN or glucan (Yang et al., 2012). While after bacterial challenge, the mRNA expression levels of sigma class GSTs in hemocytes were all significantly up-regulated in M. galloprovincialis (Wang et al., 2013a). In the present study, the mRNA transcripts of AiGSTσ could be significantly induced by the stimulation of four typical PAMPs, confirming the hypothesis that it could be involved in the innate immune response of scallops. Additionally, a slight but significant increase of AiGSTσ mRNA transcripts was also observed at 6 h after PBS injection, indicating AiGSTσ might be also involved in the responses to injury in scallop. To further investigate the potential role of AiGSTσ in bay scallop, the catalytic activity of its recombinant protein was determined in vitro using 27 Fig. 5 SDS-PAGE analysis of the rAiGSTσ protein in E. coli strain BL21 (DE3). Line M was the unstained protein marker (26610 Thermo Fisher Scientific, USA). Line U was the supernatant of non-induced bacteria lysate. Line I was the supernatant of auto-induced bacteria lysate. Line P was the purified recombinant protein. CDNB as substrate. In a previous research, the recombinant HdGSTS1 and HdGSTS2 proteins in H. discus discus exhibited catalytic activities of 0.17 ± 0.01μmol min-1 mg-1 and 1.06 ± 0.02 μmol min-1 mg-1, respectively, with relatively broad optimum pH spectrum and temperature range (Wan et al., 2008). While rRpGSTσ from R. philippinarum demonstrated a high catalytic ability toward CDNB of 4.64 ± 0.17 μmol-1 min-1 mg-1, but xhibited narrow optimal pH spectrum and temperature range (Umasuthan et al., 2012). Similarly, in the present study, AiGSTσ exhibited a high enzymatic activity of 3.28 ± 0.03 μmol min-1 mg-1, but lost more than 60% of its activity when the pH was lower than 6.5 or when the temperature was over 30 °C. It has been reported that both sea surface temperature rise and ocean acidification affect survival and reproduction of marine organisms negatively, including scallop (Zhang et al., 2014; Lagos et al., 2016). So, the lower active stability of rAiGSTσ, especially susceptible to low pH or high temperature, might provide valuable insights into a possible mechanism of large scale mortalities of cultured bay scallops in summer. In conclusion, the full-length cDNA encoding a sigma class GST was identified from bay scallop A. irradians. It was constitutively expressed in all the tested tissues, including hemocytes, muscle, mantle, gill, hepatopancreas and gonad, and the mRNA expression levels of AiGSTσ were all up-regulated in hemocytes after various PAMPs stimulaton. The purified rAiGSTσ protein exhibited relatively high catalytic activity against CDNB with low thermal stability and narrow optimum spectrum of pH. All these results indicated that it was a fragile but efficient antioxidant enzyme and was potentially involved in the innate immune responses of scallop. This study would enrich the understanding of the scallop innate immunity. 28 Fig. 6 The enzymatic activities of rAiGSTσ under different treatment (A: pH, B: temperature). Each values was shown as mean ± SD (n = 5), and bars with different characters were significantly different (p < 0.05). 29 Acknowledgement This research was supported by the Key Research Program of the Chinese Academy of Sciences (KFZD-SW-106). 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