Peroxiredoxin 6 from cuttlefish (Sepiella maindroni): molecular characterisation during development and the response to thermal stress

The aim of this work was to investigate the function of the peroxiredoxin family member Peroxiredoxin 6 in Sepiella maindroni (Smprx6) during embryonic development and the response to thermal stress. The 1064 bp full-length coding sequence of Smprx6 includes an open reading frame of 660 bp encoding a polypeptide of 219 amino acids with a predicted molecular mass of 24.4 kDa. The deduced protein sequence shares significant homology with other reported Prx6 enzymes. Phylogenetic analysis classified SmPrx6 into the 1-Cys-Prx cluster, and quantitative real-time PCR (qPCR) demonstrated constitutive mRNA expression in all tested tissues and during five different embryonic developmental stages, as previously documented in other cephalopods. Thermal stress induced Smprx6 expression, which peaked at 6 h then returned to control levels. Recombinant SmPrx6 was active at different concentrations and was thiol-dependant. The results suggest that SmPrx6 plays a major role in the development of Sepiella maindroni and its response to oxidative and thermal stress.


Introduction
Cephalopods have high rates of growth and food conversion, which for aquaculture results in a short culture cycle, a high ratio of production to biomass, and high cost-effectiveness. However, at present, only small-scale culturing of a limited number of species is possible (Castellanos-Martínez and Gestal 2013). Furthermore, most reported culturing procedures rely on captured wild individuals (Vidal et al. 2014). Our research group previously developed a system for breeding Sepiella maindroni, a commercially important fishery species accounting for a large proportion of cuttlefish aquaculture in China, by creating artificial culture cycles capable of rearing at least six generations in captivity (Wei-Wei and Chun-Lin 2009). However, despite the development of an artificial rearing method, S. maindroni suffers from the predicament of a high summer mortality. Cephalopods are susceptible to environmental changes, especially water temperature fluctuations, but if they survive such changes, they readily adapt to new environmental conditions (Gestal and Castellanos-Martinez 2015). Thermal fluctuations in a marine environment can induce physiological changes through oxidative stress-related mechanisms that generate reactive oxygen species (ROS) to modulate gene expression (Bogdan et al. 2000).
Although low concentrations of ROS are essential for normal physiological signalling (Poynton and Hampton 2014), excess ROS and oxidative stress can lead to cell damage, mutations, and even death (Zheng et al. 2010). Antioxidants are known to play a critical role in regulating ROS levels by detoxifying deleterious oxygen intermediates without disrupting normal cellular functions (Wood et al. 2003b). Peroxiredoxins (Prx) are a ubiquitous family of antioxidant enzymes that prevent oxidative damage (Robinson et al. 2010) and serve as effective medicinal agents for treating ROS-related diseases (Poynton and Hampton 2014). The Prx family has six isoforms classified into two sub-groups, including five 2-Cys-Prxs (Prx1 to Prx5) and one 1-Cys Prx (Prx6). The 2-Cys Prxs are divided into 'typical' and 'atypical' classes, depending on whether the conserved cysteine residues form intermolecular or intramolecular disulfide bridges, respectively (Wood et al. 2003b).
Antioxidant enzymes operating within the oxidant system of S. maindroni are significantly affected by thermal stimulation, particularly those related to scavenging ROS, as demonstrated by cDNA microarray studies based on an EST library (in review). A better understanding of the mechanisms underpinning the responses to oxidative stress could help to improve squid rearing and cephalopod aquaculture. In this study, we identified a peroxiredoxin 6 homolog in S. maindroni (Smprx6), measured Smprx6 mRNA levels in the liver during oxidative and thermal stress, and produced and characterised the recombinant protein.

Materials and methods
Animals, embryos, acute thermal temperature challenge and sample collection S. maindroni weighing 158.2 ± 2.8 g were collected in Ningbo, China, and acclimated in a blue plastic cylinder tank (diameter 1 m, depth 0.6 m) with a salinity of 28-30% at 22 ± 2°C for 1 week before processing. Cuttlefish (120; length = 3.1 ± 0.5 cm) were randomly divided into six groups containing 20 individuals for the thermal stress experiment. After acclimation for 2 days, three groups were exposed to thermal stress conditions by gradually increasing the temperature by 1°C per h to 32°C, which is the thermal limit for survival based on breeding experiments . Additionally, three control groups were maintained at 22°C, which is the normal temperature for rearing in seawater. After treatment, five individuals were randomly sampled at 0, 0.5, 2, 6, 12, 24, 48 and 72 h, and the liver was collected and preserved in RNAlater (TaKaRa, Tokyo, Japan) for RNA extraction. Eight tissues (muscle, ink sac, liver, pancreas, ovary, testis, stomach, and gill) were collected from S. maindroni adults, and total RNA was extracted using RNAiso Plus (TaKaRa, Tokyo, Japan) according to the manufacturer's protocol.
Several clutches of fertilised eggs were collected and acclimated at 20 ± 1°C. Embryonic development of S. maindroni can be divided into 12 stages , with the early stages combined into five major developmental phases: (I) the zygote stage; (II) the pre-embryonic stage, which occurs over an extremely short period and includes cleavage, blastula, and gastrula stages; (III) the organogenesis stage, when the main body parts and organs such as the wrist, eye, heart and ink sac are formed; (IV) the morphological integrity stage, which corresponds to further ongoing development of bodily structures, with the yolk sac still present; (V) the pre-hatching stage, during which the yolk sac disappears in preparation for hatching. Embryos were collected at these five stages and used for RNA extraction.
All aspects of the experiment were conducted according to the guidelines provided by the Ethical Committee of Experimental Animal Care at Ningbo University (Ningbo, Zhejiang, China).
Identification and cloning of the full-length cDNA of SmPrx6 A squid cDNA library was constructed from the ink sac using the Creator Smart cDNA Library Construction Kit (TaKaRa, Tokyo, Japan). cDNA clones were selected randomly from the cDNA library, and single-pass sequenced using a random T3 primer on an ABI 3730 Genetic Analyzer (Applied Biosystems, Foster City, USA). A large-scale expressed sequence tag (EST) sequencing project for S. maindroni was initiated to identify and functionally annotate as many unique transcripts as possible. BLAST searches were performed to compare processed cDNA sequences against ESTs and genes available in the GenBank database (http://www. ncbi.nlm.nih.gov/blast) (Wei-Wei et al. 2012). Intriguingly, EST GT618012 (Contig 485; length = 982 bp) was homologous to Prx6 in Rattus norvegicus (RNO17295).
Amplification of flanking sequences was carried out using a 5 0 -and 3 0 -Full RACE kit (TaKaRa, Tokyo, Japan) to obtain the complete sequence of EST GT618028 following the manufacturer's instructions. Purified genes were cloned into the pMD-18T vector (Takara, Tokyo, Japan) and transformed into Escherichia coli Top10F-competent cells. Finally, M13-47 and RV-M universal primers (Table 1) were used to screen for three positive recombinants that were subsequently sequenced.

Sequence analysis, multiple sequence alignment and phylogenetic analysis
The sequence similarity of nucleotide and protein sequences was compared using BLAST (http://www.ncbi. nlm.gov/blast). Amino acid sequences were deduced by the Expert Protein Analysis System (http://www. expasy.org). Protein motif features were predicted by the Simple Modular Architecture Research Tool (http:// smart.emblheidelberg.de/) (Schultz et al. 1998). Signal peptide prediction was performed by SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP). Multiple sequence alignment of Smprx6 was performed using ClustalW (http://www.ebi.ac.uk/clustalw/). The neighbour-joining method was used to perform multiple sequence alignment of Smprx6 and its homologs in other species and construct a phylogenetic network with Transcriptional analysis of SmPrx6 by quantitative real-time RT-PCR Quantitative real-time RT-PCR was performed using an ABI 7300 (Applied Biosystems, Foster City, USA). Reactions were carried out in a final volume of 25 lL containing 12.5 lL of SYBR Green Real-time PCR Master Mix (TaKaRa, Tokyo, Japan), 9.5 lL of water, 0.5 lL of each primer (P5 and P6 for SmPrx6, and P7 and P8 for b-actin; see Table 1), and 2 lL of cDNA. Reactions were incubated at 95°C for 3 s, followed by 40 cycles at 95°C for 5 s, 56°C for 5 s, and 72°C for 20 s, and a final extension was performed at 72°C for 1 min. Reactions were performed in a 96-well plate, each sample was repeated in triplicate, and an internal control gene (b-actin) was included. Dissociation curve analysis of amplification products was performed at the end of each reaction to confirm that only one PCR product was amplified and detected. PCR data were analysed using the ABI 7300 SDS software (Applied Biosystems, Foster City, USA). To maintain consistency, the baseline was set automatically by the software. The comparative C t method was used to analyse the relative expression levels of Smprx6. C t values for the amplified target products and the internal control (bactin) were determined for each sample, and the difference in C t for the target and internal control was calculated to normalise the differences in template and the efficiency of RT-PCR. Liver from squid in the control group was used as the reference sample for calibration. DC t values for each sample were subtracted from the DC t of the calibrator to generate DDC t values. The expression level of Smprx6 was calculated using the 2 -DDCt method, and values represent the n-fold difference compared with the calibrator (Livak and Schmittgen 2001).

Expression of recombinant SmPrx6
A PCR fragment encoding the mature SmPrx6 polypeptide was amplified with gene-specific primers P7 and P8 (Table 1) and cloned into the pMD18-T simple vector (TaKaRa, Tokyo, Japan), digested with restriction enzymes NdeI and XhoI (New England Biolabs, MA, USA), then subcloned into the NdeI and XhoI sites of the expression vector pET-21a(?) (Novagen, Shanghai, China). The recombinant plasmid was transformed into E. coli Origami (DE3) (Novagen, Shanghai, China) and subjected to DNA sequencing. The parent vector without the inserted fragment was used as a negative control. After sequencing to confirm in-frame insertion, positive transformants and the negative control were incubated in LB medium containing 50 lg/mL ampicillin at 37°C with shaking at 220 rpm. When the culture reached an OD 600 of 0.5-0.7, IPTG was added to a final concentration of 1 mM and culturing continued for an additional 4 h under the same conditions. Cells were harvested by centrifugation at 80009g for 2 min and suspended in 50 mM Tris containing 5 mM EDTA, 50 mM NaCl, and 5% glycerol (pH 7.9). After sonication for 60 s on ice, the rSmPrx6 and negative control samples were purified using a HisTrap Chelating Column (Amersham Biosciences, Stockholm, Sweden) according to the manufacturer's instructions. Purified recombinant proteins were analysed by 15% SDS-PAGE to check the molecular mass and purity. Assaying of the antioxidant activity was performed using the Total Antioxidant Capacity Assay Kit (Jiancheng Bioengineering Institute, Nanjing, China) using the 2,2 0 -azino-bis(3-ethyl-benzothiazoline-6-sulphonic acid) ABTS method (Miller et al. 1993). Peroxidase activity was measured based on the change in absorbance at 420 nm that accompanies the breakdown of H 2 O 2 . One unit of activity was defined as the amount of enzyme catalysing the breakdown of 1 mg of substrate by 1 g of fresh tissue in the reaction system at 37°C. Peroxidase activity was calculated using the formula supplied with the Peroxidase assay kit (Jiancheng Bioengineering Institute, Nanjing, China).

Statistical analysis
All data were expressed as the mean ± standard deviation (n = 5), and mean values were subjected to oneway analysis of variance (ANOVA). SPSS20 software was used to determine statistical significance (p \ 0.05), and Origin 8.0 was used to prepare figures.

Cloning and analysis of SmPrx6
The complete cDNA sequence of Smprx6 was obtained by overlapping EST GT618012 with amplified fragments. The sequence has been deposited in Genbank under accession number HQ662844. The full-length mRNA of Smprx6 is 1064 bp and includes an open reading frame of 660 bp encoding a polypeptide of 219 amino acids with a predicted molecular mass of 24.4 kDa and a theoretical isoelectric point of 6.17 (Fig. 1). The deduced amino acid sequence of Smprx6 is shown in Fig. 1. The protein includes a typical Prx domain (from Leu4 to Ser160) and an alkyl hydroperoxide reductase (AhpC) domain (from Leu4 to Ser140) as identified by the SMART program. A signal peptide was not predicted by SignalP. The deduced amino acid sequence of Smprx6 shares significant homology with other reported Prx6 proteins, with 71% identity with Saccostrea glomerata (ACX50969), and 70.9% identify with Haliotis discus discus (ABO26614). Multiple alignments revealed that Cys44 is conserved in all analysed Prx VI sequences. Moreover, the predicted SmPrdx6 protein has a unique cysteine residue (C44) in the peroxidase motif (42PVCTTE47) and a catalytic serine residue (S29) in the lipase motif (27NNSWA31) at the N-terminus (Fig. 2). Both of them are essential amino acid residues in the active sites that mediate peroxidase and PLA2 activity, respectively.
A phylogenetic tree was constructed based on Prx amino acid sequences (Fig. 3). Three distinct groups (1-Cys, 2-Cys, and an atypical 2-Cys subgroup) formed distinct clusters, with all Prx5 proteins part of the atypical 2-Cys subgroup, and Prx1, Prx2, Prx3, and Prx4 clustered in the 2-Cys subgroup. The Smprx6 sequence and other Prx6 sequences from invertebrates were placed in the 1-Cys subgroup.

Expression, purification and characterisation of recombinant SmPrx6
The recombinant plasmid pET-21a-SmPrx6 was transformed into E. coli Origami (DE3) as described above, and rSmPrx6 was expressed and purified under native conditions. SDS-PAGE confirmed the presence of an overexpressed protein in the whole cell lysate following IPTG induction, and the purified protein ran as a signal band with a molecular mass (*25 kDa) comparable to that predicted for rSmPrx6 (Fig. 7). The purified rSmPrx6 enzyme displayed antioxidant activity of 6.17 U/mg and POD activity of 10.17 U/mg.

Discussion
SmPrx6 shares common structural features with other invertebrate and vertebrate Prx6 proteins. The phylogenetic tree showed that Smprx6 falls into the 1-cys-Prx cluster, members of which contain a consensus sequence with a conserved cysteine residue (Cys 44) in the N terminus, and a AhpC domain with a peroxidase catalytic motif (PVCTT). In mammals, Cys47, His39 and Arg132 form the catalytic triad responsible for peroxidase activity, with Cys47 bound to His39 via hydrogen bonding, and electrostatically activated by Arg132 (Manevich and Fisher 2005). Interestingly, a catalytic triad (Cys44, His36 and Arg 126) is also present in squid and other mollusks (Nikapitiya et al. 2009). The catalytic motif responsible for PLA2 activity (27NNSWA31) was observed in the deduced amino acid sequence, with Ser29 the catalytic nucleophile forming part of the NNSWG consensus sequence responsible for the PLA2 hydrolase activity (Lee et al. 2015). Ser is replaced with a Met, Arg or Gly in E. sinensis (Mu et al. 2009), Xenopus laevis (Genbank accession no. NP_001084316) and Suberites domuncula (Genbank accession no. CAC38779), respectively. The effect this replacement has on the catalytic activity warrants further study.
Smprx6 was constitutively transcribed in all examined tissues, suggesting it is ubiquitously expressed in squid, and levels were highest in pancreas and liver, which is different from other aquatic animals. Ploch et al. (1999) reported that liver cells from a variety of fish species are relatively resistant to DNA modification by ROS generated by various compounds. Based on the observed high Prx6 expression levels in pancreas and liver, we propose that Prx6 in squid possesses other activities such as the PLA2 activity described above. Indeed, ubiquitous expression of EsPrx6 mRNA in different tissues has been linked to an involvement in numerous physiological functions including basal metabolism in E. sinensis (Mu et al. 2009). In channel catfish, Prx6 is expressed in liver, head, kidney, intestine, and skin, but not spleen (Yeh and Klesius 2007). However, Zheng and colleagues reported that turbot Prx6 is more highly expressed in blood and heart than liver in unchallenged fish, in contrast with rock bream and channel catfish that display tissue-specific expression profiles (Zheng et al. 2010). This apparent variation in the transcription of Prx6 in different organisms may be due to the expression of multiple isoforms of Prx family members in different organs (Huang et al. 2011).
The consumption of yolk stores during a specific period of development involves governing regulatory signals and controlling ROS by-products (Shafer et al. 2011). Most antioxidant enzymes such as catalase and glutathione peroxidase are expressed at high levels during yolk consumption. Understanding the sub-cellular management of metabolism before, during, and after yolk consumption is, therefore, an intriguing area of investigation. As observed in many other organisms, SmPrx6 mRNA was detected throughout embryonic development. Expression was highest during the pre-embryonic stage (II), which was 1358.26-fold higher than the pre-hatching stage (p \ 0.05), and expression then declined gradually from stage III to stage V. The early embryonic period can be considered a time of substantial proliferative stress because early-stage embryogenesis involves massive cellular proliferation over a very short time. Correspondingly, SmPrx6 has been implicated in complex cellular processes including cell proliferation (Wood et al. 2003a), differentiation (Rabilloud et al. 1995) and apoptosis (Zhou et al. 2000;Bernier-Villamor et al. 2004), with the highest expression in early-stage embryos. Our present results indicate that SmPrx6 plays a major role in embryonic development in S. maindroni, especially during the early stages of tissue differentiation.
Oxidative stress often accompanies thermal stress. Although low levels of ROS modulate a variety of cellular functions such as cell proliferation, high concentrations of ROS cause cell damage or apoptosis (Park et al. 2008). Among the antioxidant systems that protect organisms from excessive ROS, the Prx family of antioxidant enzymes has received much attention during the past decade. The significant upregulation of Prx transcript levels after a high-temperature stimulus indicates the induction of gene expression (Park et al. 2005). Expression of SmPrx6 increased rapidly in S. maindroni and peaked at 6 h post-stimulus (3.72-fold; p \ 0.05) before declining and eventually returning to control levels. This indicates that SmPrx6 may play a major role as an antioxidant protein to protect organisms from excessive ROS following thermal stress (Tolomeo et al. 2016).
Cephalopods are highly adaptable marine organisms, and can adjust their biology and life cycles in response to the prevailing environmental and ecosystem conditions. These organisms can presumably avoid increased levels of oxidative damage normally during short-term thermal stress, possibly by altering metabolic processes.
In conclusion, we identified and characterised Prx6 from S. maindroni, and detected Smprx6 mRNA transcripts in all tested tissues and developmental stages. The observed upregulation of Prx6 expression following a high-temperature stimulus indicates that its peroxidase activity may be important for the response to oxidative stress. Therefore, Prx6 may play a major role in maintaining proteins in a reduced state by scavenging toxic ROS.