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Zn superoxide dismutase from Sepiella maindroni under stress of Vibrio harveyi and Cd2+
Developmental and Comparative Immunology 47 (2014) 1–5
Contents lists available at ScienceDirect
Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci
Short communication
Identification and analysis of an intracellular Cu/Zn superoxide dismutase from Sepiella maindroni under stress of Vibrio harveyi and Cd2+ Jian-yu He, Chang-feng Chi, Hui-hui Liu ⇑ National Engineering Research Center of Marine Facilities Aquaculture, Zhejiang Ocean University, Zhoushan 316022, PR China
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Article history: Received 4 May 2014 Revised 18 June 2014 Accepted 19 June 2014 Available online 27 June 2014 Keywords: Sepiella maindroni Copper/zinc superoxide dismutase (Cu/Zn-SOD) Reactive oxygen species (ROS)
a b s t r a c t Superoxide dismutases (SODs) are ubiquitous family of metalloenzymes involved in protecting organisms from excess reactive oxygen species damage. In this paper, a novel intracellular Cu/ZnSOD from Sepiella maindroni (designated as SmSOD) was identified and characterized. The full-length cDNA sequence of SmSOD (GenBank accession No. KF908850) was 709 bp containing an open reading frame (ORF) of 459 bp, encoding 153 amino acid residues peptide with predicted pI/MW (6.02/15.75 kDa), a 131 bp50 - and 116 bp-30 - untranslated region (UTR). BLASTn analysis and phylogenetic relationship strongly suggested that the sequence shared high similarity with known Cu/Zn SODs. Several highly conserved motifs, including two typical Cu/Zn SOD family domains, two conserved Cu-/Zn-binding sites (H-47, H-49, H-64, H-120 for Cu binding, and H-64, H-72, H-81, D-84 for Zn binding) and intracellular disulfide bond (C-58 and C-146), were also identified in SmSOD. Time-dependent mRNA expression of SmSOD in hepatopancreas was recorded by quantitative real-time RT-PCR after Vibrio harveyi injection and Cd2+ exposure. The results indicated that SmSOD was an acute-phase protein involved in the immune responses against pathogens and biological indicator for metal contaminants in aquatic environment. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Marine pollution has become increasingly serious than ever before, while a lot of environmental pollutants such as chemical contaminations, salinity changes, heavy metals, lead to stimulating intracellular formation of reactive oxygen species (ROS) in organism (Cantú et al., 2009). Although small amounts of ROS are cellular requirement for their involvement in signaling pathways, regulation of variety of cellular activities and gene expression, excess ROS production increases oxidative damage in the cell, possibly by altering or inactivating proteins, lipid membranes, and DNA (Park et al., 2009). In order to limit the harmful effect of ROS production and protect their cellular components against oxidative stress, organisms have evolved to use several antioxidant enzymes like superoxide dismutases (SODs), catalases (CATs), Glutathione S-Transferases (GSTs), glutathione peroxidase (GSH-PX), etc., and a series of innate immunity responses to regulate and eliminate excessive ROS.
⇑ Corresponding author. Address: National Engineering Research Center of Marine Facilities Aquaculture, Zhejiang Ocean University, No. 1 Haida South Road, Zhoushan City, Zhejiang Province 316022, PR China. Tel./fax: +86 580 2550073. E-mail address: [email protected] (H.-h. Liu). http://dx.doi.org/10.1016/j.dci.2014.06.010 0145-305X/Ó 2014 Elsevier Ltd. All rights reserved.
Superoxide dismutases (SODs; EC 1.15.1.1) are the significant metalloenzymes, which catalyze the dismutation of superoxide radicals(O) to hydrogen peroxide (H2O2) and oxygen (O2) for protecting living cells from the cytotoxic effects of the ROS formed from superoxide anion radicals such as H2O2, hydroxyl radicals, and singlet oxygen defending against reactive oxygen species (ROS) (Park et al., 2009). Relying on the various metal contents, SODs are classified into at least three distinct groups: copper/zinc SODs (Cu/Zn-SODs), manganese SODs (Mn-SODs) and iron SODs (Fe-SODs) (Fridovich, 1975). Structurally, copper/zinc SOD was different from Mn-SODs and Fe-SODs, which contained two typical Cu/Zn SOD domains, involved in conserved the Cu-binding sites (main four His sites for Cu-binding) and Zn-binding sites (including three His sites and one Asp site for Zn-binding). Furthermore according to whether they have an N-terminal signal cleavage peptide for secretion, Cu/Zn-SODs are classified into extracellular Cu/Zn-SOD (ecCu/Zn-SOD) and intracellular Cu/Zn SOD (icCu/ Zn-SOD) (Li et al., 2011; Zelko et al., 2002). Cu/Zn-SOD had been proved to be a considerable important type in SOD superfamily for its physiological function and therapeutic potential(Ni et al., 2007), which was one of the most important free radical scavengers that respond to oxidative stress (Halliwell and Gutteridge, 1984). Until now, Cu/Zn SOD has been identified in many aquatic animals including clam (Venerupis philippinarum) (Li et al.,
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2010a,b), abalone (Haliotis diversicolor supertexta) (Li et al., 2010a,b), and mussel (Mytilus edulis) (Letendre et al., 2008). However, little report has focused on molecular features and the immune mechanisms in cephalopod. Sepiella maindroni is an economically-important aquatic cephalopod in the East China Sea. The main objective of this paper is to clone the full-length cDNA of icCu/Zn-SOD (designated as SmSOD) from S. maindroni and investigate the expression profile of SmSOD after stress by Vibrio harveyi and cadmium, which would provide new insight into the function on this important, widespread and functionally diverse protein in cephalopod, and give new evidence for further discussion on the primary antioxidant enzymes in invertebrates. 2. Materials and methods 2.1. Experimental animals and SmSOD cDNA synthetizing The juvenile cuttlefishes (S. maindroni, 2.0–4.0 cm in length) were collected from the Dongji culture farm in Zhoushan, Zhejiang province, P. R. China, immediately transferred to the laboratory, and acclimated with salinity of 28–30‰ for a week at 23–25 °C before experiment in May 2013. Total RNA was isolated from the hepatopancreas with Trizol reagent (TaKaRa). The cDNA synthesis was carried out with M-MLV RTase cDNA Synthesis Kit (TaKaRa). 2.2. cDNA of SmSOD identification and full-length amplification The primers SOD-F/SOD-R (shown in Supplementary Tab. 1) were designed according to the sequence of Euprymna scolopes (AY149462), Mytilus galloprovincialis (FM177867), M. edulis (AJ581746), Haliotis discus discus (DQ530214), H. diversicolor (DQ000610), and Meretrix meretrix (GU188694). The reaction system was performed in 20 lL volume, including 2 lL 10PCR Buffer, dNTPs 0.4 lL, SOD-F 0.8 lL, SOD-R 0.8 lL, template cDNA 0.6 lL and Taq DNA polymerase (TaKaRa) 0.4 lL. The PCR amplification was conducted on a Thermal Cycler (Bio-Rad, USA), and amplification conditions were: 4 min at 94 °C, followed by 35 cycles of 60 s at 94 °C, 30 s at 53 °C, and 45 s at 72 °C, with a final extension of 10 min at 72 °C. The PCR products were gel-purified and sequenced at Shanghai Invitrogen Biological Technology Company (P.R. China). Gene specific primers for rapid-amplification of cDNA ends including 50 -RACE (5P1 and 5P2) and 30 -RACE (3P1 and 3P2, Supplementary Tab. 1), were designed based on the known partial sequence. Both 50 -RACE and 30 -RACE were carried out according to the manufacturer’s instructions in the Smart RACE cDNA amplification kit (Clontech, USA). The PCR products were cloned into the PMD18-T simple vector (TaKaRa) and sequenced from both directions. 2.3. Sequence analysis The cDNA sequence was spliced by the software of DNAstar v7.0. The homology search of nucleotide of SmSOD was conducted with BLASTn program of NCBI (http://www.ncbi.nlm.gov/BLAST/). The amino acid sequence of SmSOD was deduced by the Expert Protein Analysis System (http://www.expasy.org/). The conserved domains were predicted using SMART (http://smart.embl-heidelberg.de/) online tool. Signal peptides were predicted using the SignalP 4.0 online tool (http://www.cbs.dtu.dk/services/SignalP/) (Petersen et al., 2011). The theoretical MW and predicted pI were determined by Expasy-ProtParam online tool (http://www.expasy.org/tools/ protparam.html). Multiple sequence alignments were performed with ClustalW v1.8 (http://pbil.ibcp.fr/htm/index.php). The phylogenetic tree was constructed by Bootstrapped Neighbor-Joining
rule method from a distance matrix with the software of MEGA v4.0 software. 2.4. The temporal expression of SmSOD mRNA in hepatopancreas after V. harveyi challenged Hepatopancreas was selected as candidate tissue for investigating the temporal expression profile of SmSOD challenged by V. harveyi. The live bacteria were cultured on LB plates at 28 °C overnight, then a single colony was inoculated in 5 mL of LB broth at 28 °C for 12 h. The bacterial suspension was centrifuged at 6000g, 4 °C for 10 min to collect bacteria, and 100 lL of live V. harveyi resuspended in PBS (pH 7.4, OD 600=0.4, 1108 cfu/mL) were injected into muscles of S. maindroni. The cuttlefishes, which were injected with 100 lL PBS, acted as the control group. Total 35 infected cuttlefishes were cultured in 10 L filtered fresh well-aerated seawater, the hepatopancreas were randomly collected at 2, 4, 8, 12, 24, 48 and 72 h post-injection and frozen immediately in liquid nitrogen and stored at –70 °C. The control group was disposed as above. To analyze the temporal expression patterns of SmSOD gene at mRNA level in hepatopancreas after V. harveyi challenge, real-time PCR (qRT-PCR) was performed with SYBR PrimeScript™ RT reagent Kit (Perfect Real Time) (TaKaRa) as recommended by the manufacturer’s instructions at 7500 Real Time PCR System (Applied Biosystems, UK). Five replicates were employed for each time point in the challenge experiment. The reaction mixture of 20 lL contained qSmSOD-F 0.8 lL, qSmSOD-R 0.8 lL, 2SYBRÒ Premix Ex TaqTM II (TaKaRa) 10 lL, cDNA sample 0.8 lL, ROX II 0.4 lL, ddH2O 7.2 lL. The standard cycling conditions were: 95 °C for 1 min (initial polymerase activation), followed by 40 cycles of 10 s at 95 °C, 45 s at 59.6 °C. The PCR specificity was checked with dissociation curve analysis from 55 to 95 °C, b-actin of S. maindroni (b-Sm actin-F and b-Sm actin-R)was used as the internal standard, all the primers as shown in Tab.1. The 2DDCT method was used to analyze the mRNA expression level. All data were given in terms of relative mRNA expressed as means ± S.E. (N = 5). The data were subjected to one-way analysis of variance (one-way ANOVA) followed by an unpaired, two-tailed t-test. Differences were considered significantly at P < 0.05. 2.5. The temporal expression of SmSOD mRNA in hepatopancreas after Cd2+ exposure For Cd treatments, animals were exposed to static culture condition where cadmium chloride (CdCl25H2O) was added to the 10 L filtered seawater with dissolved Cd2+ concentration of 200 lg/L, which based on the preliminary experiment of gradient concentration. The highest concentration, which was no mortality in the heavy metals treatment during the exposure period (data not shown), was selected. Total 30 cuttlefishes were cultured in 10 L filtered well-aerated seawater with Cd2+. Seawater was changed daily and resupplied with the corresponding concentration of Cd2+. Five individuals were taken for each treatment at 24, 48, 72, 120, 168 and 264 h post-treated respectively. The methods of hepatopancreas collection, total RNA extraction, cDNA synthesis and realtime PCR analysis were performed as described above. 3. Results and discussion 3.1. cDNA sequence analysis and characterization of SmSOD full-length The full-length sequence of SmSOD was 709 bp (GenBank accession No. KF908850), which contained an open reading frame
J.-y. He et al. / Developmental and Comparative Immunology 47 (2014) 1–5
(ORF) of 459 bp encoding 153 amino acid (aa) residues, a 50 -UTR of 131 bp, and a 30 -UTR of 116 bp (Supplementary Fig. 1). The predicted molecular weight (MW) was 15.75 kDa and estimated pI was 6.02. BLASTn analysis suggested that the coding sequence shared high similarity and identity with known Cu/Zn SODs. Two types of Cu/Zn-SOD have been identified in most organisms, extracellular Cu/Zn-SOD (ecCu/Zn-SOD) with an N-terminal signal cleavage peptide for secretion, and intracellular Cu, Zn SOD (icCu/
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Zn-SOD) without a signal peptide (Ni et al., 2007; Halliwell and Gutteridge, 1984). The SignalP 4.0 showed that there was no signal peptide in SmSOD, suggesting that SmSOD was icCu/Zn SODs, unlike Mn-SOD or ecCu/ZnSOD, which have glycosylation of protein for conferring stability on secreted SODs (Bao et al., 2009). ClustalW analysis also revealed that the deduced amino acid sequence of SmSOD showed higher identity (70–85%) with other ten known icCu/Zn SODs (Supplementary Fig. 2). For example, it shared 82%
Fig. 1. Phylogenetic tree depicting the relationship of SmSOD with other species. All protein sequences were obtained from GenBank of NCBI, GenBank accession number was in parentheses, and phylogenetic tree was constructed using Neighbor-Joining rule method by the software of MEGA v4.0.
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identity with icCu/Zn-SOD from H. discus discus (154 aa, ABF67508), 81% identity with M. edulis (158 aa, CAE46443), 77% identity with Chlamys farreri (153 aa, ABD58974), 76% identity with Ventridens decussatus (131 aa, AAQ88163) and 71% identity with Biomphalaria glabrata (155 aa, ABD60754). The sequence size of SmSOD was 153 aa, which was identified with the average of intracellular Cu/ Zn-SOD of 154 aa (147–167 aa) and different from the extracellular Cu/Zn-SOD with 208 aa (175–251 aa) (Lin et al., 2008). 3.2. Multiple sequence alignments and phylogenetic analysis Multiple alignment revealed that several characteristic motifs and signatures of intracellular SOD were also appeared in SmSOD (Supplementary Fig. 2), for example GFHIHEFGDNT and GNAGGRLACGVI motifs that were the highest conserved region sequences of Cu/Zn-SOD family signature. Additionally, the deduced amino acids of SmSOD contained two cysteine residues C-58 and C-146, which were conserved in all Cu/ZnSODs and were predicted to form one intracellular disulfide bond (Cioni et al., 2003). Copper (H-47, H-49, H-64, H-120) and zinc (H-64, H-72, H81, D-84) binding sites were required, which were conserved in all Cu/ZnSODs, and copper and zinc ions had critical functions in stabilizing the quaternary structure to increase the probability of pathogenic mutants (Ni et al., 2007; Anju et al., 2013; Lynch and Colón, 2006). SmSOD sequence also included several fully conserved active site residues G-45, G-62, P-75 and G-83, which might be involved in maintaining the active site geometry (Li et al., 2010a,b). These features suggested that SmSOD might be a new member of the SOD protein family, and further studies were needed to seek the transcription regulating factors involved in SmSOD expression regulation. In the phylogenetic tree (Fig. 1) almost all the cytoplasmic Cu/Zn SODs (icCu/Zn SODs) clustered together as a subgroup, extracellular Cu/Zn SODs (ecCu/Zn SODs) clustered to other groups, and MnSODs obviously acted as outgroup. It was suggested that icCu/Zn SODs and ecCu/Zn SOD might be diverged from a common ancestor, consistent with some researchers’ opinions (Zelko et al., 2002). SmSOD first clustered with icCu/Zn SODs from E. scolopes, and further grouped with those of M. edulis and Ruditapes decussatus, which belonged to bivalve. The other species in the icCuZnSOD tree generally agreed with the taxonomic classification of the corresponding species. 3.3. Temporal expression of SmSOD after Vibrio challenge V. harveyi is a gram-negative bacteria in the marine organism and seawater, which often leads to high mortality in cultured bivalves, and affects the production of economic aquatic organisms in marine environment (Gómez et al., 2005). Cu/ZnSODs are considered as general stress responsive factors, whose expression at transcriptional and/or translational levels may be influenced by a variety of intracellular and environmental cues (Zelko et al., 2002). In the present study, real-time PCR (qRT-PCR) analysis indicated that SmSOD mRNA expression in hepatopancreas was upregulated significantly at the start of V. harveyi injection, reached a peak of approximate 70-fold higher than that of the control at 4 h post-injection, and then declined in the following hours. After 24 h post-injection, the level decreased to 30-fold of the blank (see Fig. 2A). Similar time-dependent expression patterns were also observed in other aquatic organisms, for example, the Cu/ ZnSOD expression was up-regulated and then recuperated in C. farreri after being injected with Candida lipolytica (Fridovich, 1975). In shrimp the activity of SOD in resistant shrimp was significantly higher at the beginning after V. harveyi injection, then recovered to normal level (Huang et al., 2013), but the expression of icCu/ZnSOD in Argopecten irradians was not affected after
Fig. 2. Expression profile of SmSOD after stress with Vibrio harveyi and Cd2+: (A) Temporal expression of the SmSOD transcripts in hepatopancreas after Vibrio harveyi infection. (B) Expression analysis of SmSOD in the hepatopancreas in response to Cd2+. Statistical analysis of differences was done by oneway analysis of variance (ANOVA) by SPSS 13.0 software. Vertical bars were mean ± SD of five technical replicates, and the asterisks above the bars represented statistically significant differences from the control samples, ‘‘*’’ at P < 0.05, ‘‘**’’ at P < 0.01.
challenge of Vibrio anguillarum. The mechanisms of Cu/Zn SOD expression after biological stimulation or suppress have been intensively investigated in mammals (Gardner et al., 2002; Akiyama et al., 2005; Peluffo et al., 2005), but not well studied in cephalopod. The significant change of SmSOD was suspected to be related to the virulence of the pathogens and the oxidative stress-induced injury. In order to response against infection, different invading microorganisms could induce different toxicity and produce different amounts, which affected SOD expression. Compared with MnSOD and ecCu/Zn-SOD, the expression of icCu/ Zn-SOD is more stable than other subtypes (Zelko et al., 2002). However, SmSOD mRNA expression increased more than 10-fold after infection than the blank, and it might be hypothesized that the juvenile of cephalopod were more sensitive than the adult in immune reaction. Therefore, as an important anti-oxidation enzyme, SmSOD is believed to play an important role in the cephalopod immune. 3.4. Temporal expression of SmSOD in hepatopancreas after Cd2+ stress Cadmium is the major toxic metals as contaminants in marine environment, being responsible for many toxic effects, including generating abnormal or denatured proteins and causing oxidative stress in a variety of living organisms (Sarkar et al., 2006). Gene
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expression frequently was used in bio-monitoring of contaminated sites based on the mRNA expression profile data, it was possible to make estimate of chemical exposure and its effects on the exposed species (Fang et al., 2010). Relying on the various metal contents, SODs could be regarded as biomarkers of contaminants pollution in aquatic environment such as cadmium pollution (Akiyama et al., 2005; Contardo and Wiegand, 2008). In Cd2+ exposure, the SmSOD mRNA expression in hepatopancreas was up-regulated at the start of experiment, but maintained low expression at the beginning hours, and appeared skyrocketing growth and reaching the peak at approximate 210-fold till 72 h post-exposure, then decreased gradually to 42-fold of blank group at 11 days postexposure (see Fig. 2B). Similar expression patterns were also observed in the clam (Mactra veneriformis), whose SOD activity significantly increased at the start of Cd2+ exposure, then decreased and finally returned to the normal level (Sarkar et al., 2006). It has been confirmed that cadmium stress can induce the ROS generation in some bivalves, and the cadmium iron would interfere with the antioxidant enzymatic defense system (Zelko et al., 2002; Geret et al., 2002). To eliminate ROS, SODs would increase, which means that cadmium stress induce the SOD gene’s transcript. These findings are consistent with the previous studies that the mRNA levels of both Cu/Zn SOD and Mn SOD were increased after metal treatments in abalones (Li et al., 2010a,b; Kim et al., 2007). Therefore, the significantly increased gene expression of SmSOD demonstrated its tolerance and defence against heavy metals contaminants to avoid the toxicity from aquatic pollutants, and it might be a potential biomarker. In conclusion, this was the first report of full-length cloning, characterization and inducible expression of icCu/Zn-SOD in hepatopancreas from cephalopod S. maindroni. The analysis of phylogenetic and structural features might contribute to the understanding of adaptation and evolutionary processes of icCu/Zn-SOD in invertebrate. The high expression level of SmSOD gene in response to V. harveyi and Cd2+ showed its crucial role in the protection of cells in case of oxidative stress of marine cephalopod, and might be potential biomarker against pollution in aquatic environment. Acknowledgements This research was supported by grants from Zhejiang Marine Fisheries Bureau Project (2012)83, the Natural Science Foundation of Zhejiang Province, China (LY14C190004), and the Hi-Tech Research and Development Program of China (863) (2012AA10 A413-5). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.dci.2014.06.010. References Akiyama, S., Inagaki, M., Tsuji, M., Gotoh, H., Gotoh, T., 2005. MRNA study on Cu/Zn superoxide dismutase induction by hemodialysis treatment. Nephron Clin. Pract. 99, c107–114. Anju, A., Jeswin, J., Thomas, P.C., Paulton, M.P., Vijayan, K.K., 2013. Molecular cloning, characterization and expression analysis of cytoplasmic Cu/Zn-
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Contents lists available at ScienceDirect
Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci
Short communication
Identification and analysis of an intracellular Cu/Zn superoxide dismutase from Sepiella maindroni under stress of Vibrio harveyi and Cd2+ Jian-yu He, Chang-feng Chi, Hui-hui Liu ⇑ National Engineering Research Center of Marine Facilities Aquaculture, Zhejiang Ocean University, Zhoushan 316022, PR China
a r t i c l e
i n f o
Article history: Received 4 May 2014 Revised 18 June 2014 Accepted 19 June 2014 Available online 27 June 2014 Keywords: Sepiella maindroni Copper/zinc superoxide dismutase (Cu/Zn-SOD) Reactive oxygen species (ROS)
a b s t r a c t Superoxide dismutases (SODs) are ubiquitous family of metalloenzymes involved in protecting organisms from excess reactive oxygen species damage. In this paper, a novel intracellular Cu/ZnSOD from Sepiella maindroni (designated as SmSOD) was identified and characterized. The full-length cDNA sequence of SmSOD (GenBank accession No. KF908850) was 709 bp containing an open reading frame (ORF) of 459 bp, encoding 153 amino acid residues peptide with predicted pI/MW (6.02/15.75 kDa), a 131 bp50 - and 116 bp-30 - untranslated region (UTR). BLASTn analysis and phylogenetic relationship strongly suggested that the sequence shared high similarity with known Cu/Zn SODs. Several highly conserved motifs, including two typical Cu/Zn SOD family domains, two conserved Cu-/Zn-binding sites (H-47, H-49, H-64, H-120 for Cu binding, and H-64, H-72, H-81, D-84 for Zn binding) and intracellular disulfide bond (C-58 and C-146), were also identified in SmSOD. Time-dependent mRNA expression of SmSOD in hepatopancreas was recorded by quantitative real-time RT-PCR after Vibrio harveyi injection and Cd2+ exposure. The results indicated that SmSOD was an acute-phase protein involved in the immune responses against pathogens and biological indicator for metal contaminants in aquatic environment. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Marine pollution has become increasingly serious than ever before, while a lot of environmental pollutants such as chemical contaminations, salinity changes, heavy metals, lead to stimulating intracellular formation of reactive oxygen species (ROS) in organism (Cantú et al., 2009). Although small amounts of ROS are cellular requirement for their involvement in signaling pathways, regulation of variety of cellular activities and gene expression, excess ROS production increases oxidative damage in the cell, possibly by altering or inactivating proteins, lipid membranes, and DNA (Park et al., 2009). In order to limit the harmful effect of ROS production and protect their cellular components against oxidative stress, organisms have evolved to use several antioxidant enzymes like superoxide dismutases (SODs), catalases (CATs), Glutathione S-Transferases (GSTs), glutathione peroxidase (GSH-PX), etc., and a series of innate immunity responses to regulate and eliminate excessive ROS.
⇑ Corresponding author. Address: National Engineering Research Center of Marine Facilities Aquaculture, Zhejiang Ocean University, No. 1 Haida South Road, Zhoushan City, Zhejiang Province 316022, PR China. Tel./fax: +86 580 2550073. E-mail address: [email protected] (H.-h. Liu). http://dx.doi.org/10.1016/j.dci.2014.06.010 0145-305X/Ó 2014 Elsevier Ltd. All rights reserved.
Superoxide dismutases (SODs; EC 1.15.1.1) are the significant metalloenzymes, which catalyze the dismutation of superoxide radicals(O) to hydrogen peroxide (H2O2) and oxygen (O2) for protecting living cells from the cytotoxic effects of the ROS formed from superoxide anion radicals such as H2O2, hydroxyl radicals, and singlet oxygen defending against reactive oxygen species (ROS) (Park et al., 2009). Relying on the various metal contents, SODs are classified into at least three distinct groups: copper/zinc SODs (Cu/Zn-SODs), manganese SODs (Mn-SODs) and iron SODs (Fe-SODs) (Fridovich, 1975). Structurally, copper/zinc SOD was different from Mn-SODs and Fe-SODs, which contained two typical Cu/Zn SOD domains, involved in conserved the Cu-binding sites (main four His sites for Cu-binding) and Zn-binding sites (including three His sites and one Asp site for Zn-binding). Furthermore according to whether they have an N-terminal signal cleavage peptide for secretion, Cu/Zn-SODs are classified into extracellular Cu/Zn-SOD (ecCu/Zn-SOD) and intracellular Cu/Zn SOD (icCu/ Zn-SOD) (Li et al., 2011; Zelko et al., 2002). Cu/Zn-SOD had been proved to be a considerable important type in SOD superfamily for its physiological function and therapeutic potential(Ni et al., 2007), which was one of the most important free radical scavengers that respond to oxidative stress (Halliwell and Gutteridge, 1984). Until now, Cu/Zn SOD has been identified in many aquatic animals including clam (Venerupis philippinarum) (Li et al.,
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2010a,b), abalone (Haliotis diversicolor supertexta) (Li et al., 2010a,b), and mussel (Mytilus edulis) (Letendre et al., 2008). However, little report has focused on molecular features and the immune mechanisms in cephalopod. Sepiella maindroni is an economically-important aquatic cephalopod in the East China Sea. The main objective of this paper is to clone the full-length cDNA of icCu/Zn-SOD (designated as SmSOD) from S. maindroni and investigate the expression profile of SmSOD after stress by Vibrio harveyi and cadmium, which would provide new insight into the function on this important, widespread and functionally diverse protein in cephalopod, and give new evidence for further discussion on the primary antioxidant enzymes in invertebrates. 2. Materials and methods 2.1. Experimental animals and SmSOD cDNA synthetizing The juvenile cuttlefishes (S. maindroni, 2.0–4.0 cm in length) were collected from the Dongji culture farm in Zhoushan, Zhejiang province, P. R. China, immediately transferred to the laboratory, and acclimated with salinity of 28–30‰ for a week at 23–25 °C before experiment in May 2013. Total RNA was isolated from the hepatopancreas with Trizol reagent (TaKaRa). The cDNA synthesis was carried out with M-MLV RTase cDNA Synthesis Kit (TaKaRa). 2.2. cDNA of SmSOD identification and full-length amplification The primers SOD-F/SOD-R (shown in Supplementary Tab. 1) were designed according to the sequence of Euprymna scolopes (AY149462), Mytilus galloprovincialis (FM177867), M. edulis (AJ581746), Haliotis discus discus (DQ530214), H. diversicolor (DQ000610), and Meretrix meretrix (GU188694). The reaction system was performed in 20 lL volume, including 2 lL 10PCR Buffer, dNTPs 0.4 lL, SOD-F 0.8 lL, SOD-R 0.8 lL, template cDNA 0.6 lL and Taq DNA polymerase (TaKaRa) 0.4 lL. The PCR amplification was conducted on a Thermal Cycler (Bio-Rad, USA), and amplification conditions were: 4 min at 94 °C, followed by 35 cycles of 60 s at 94 °C, 30 s at 53 °C, and 45 s at 72 °C, with a final extension of 10 min at 72 °C. The PCR products were gel-purified and sequenced at Shanghai Invitrogen Biological Technology Company (P.R. China). Gene specific primers for rapid-amplification of cDNA ends including 50 -RACE (5P1 and 5P2) and 30 -RACE (3P1 and 3P2, Supplementary Tab. 1), were designed based on the known partial sequence. Both 50 -RACE and 30 -RACE were carried out according to the manufacturer’s instructions in the Smart RACE cDNA amplification kit (Clontech, USA). The PCR products were cloned into the PMD18-T simple vector (TaKaRa) and sequenced from both directions. 2.3. Sequence analysis The cDNA sequence was spliced by the software of DNAstar v7.0. The homology search of nucleotide of SmSOD was conducted with BLASTn program of NCBI (http://www.ncbi.nlm.gov/BLAST/). The amino acid sequence of SmSOD was deduced by the Expert Protein Analysis System (http://www.expasy.org/). The conserved domains were predicted using SMART (http://smart.embl-heidelberg.de/) online tool. Signal peptides were predicted using the SignalP 4.0 online tool (http://www.cbs.dtu.dk/services/SignalP/) (Petersen et al., 2011). The theoretical MW and predicted pI were determined by Expasy-ProtParam online tool (http://www.expasy.org/tools/ protparam.html). Multiple sequence alignments were performed with ClustalW v1.8 (http://pbil.ibcp.fr/htm/index.php). The phylogenetic tree was constructed by Bootstrapped Neighbor-Joining
rule method from a distance matrix with the software of MEGA v4.0 software. 2.4. The temporal expression of SmSOD mRNA in hepatopancreas after V. harveyi challenged Hepatopancreas was selected as candidate tissue for investigating the temporal expression profile of SmSOD challenged by V. harveyi. The live bacteria were cultured on LB plates at 28 °C overnight, then a single colony was inoculated in 5 mL of LB broth at 28 °C for 12 h. The bacterial suspension was centrifuged at 6000g, 4 °C for 10 min to collect bacteria, and 100 lL of live V. harveyi resuspended in PBS (pH 7.4, OD 600=0.4, 1108 cfu/mL) were injected into muscles of S. maindroni. The cuttlefishes, which were injected with 100 lL PBS, acted as the control group. Total 35 infected cuttlefishes were cultured in 10 L filtered fresh well-aerated seawater, the hepatopancreas were randomly collected at 2, 4, 8, 12, 24, 48 and 72 h post-injection and frozen immediately in liquid nitrogen and stored at –70 °C. The control group was disposed as above. To analyze the temporal expression patterns of SmSOD gene at mRNA level in hepatopancreas after V. harveyi challenge, real-time PCR (qRT-PCR) was performed with SYBR PrimeScript™ RT reagent Kit (Perfect Real Time) (TaKaRa) as recommended by the manufacturer’s instructions at 7500 Real Time PCR System (Applied Biosystems, UK). Five replicates were employed for each time point in the challenge experiment. The reaction mixture of 20 lL contained qSmSOD-F 0.8 lL, qSmSOD-R 0.8 lL, 2SYBRÒ Premix Ex TaqTM II (TaKaRa) 10 lL, cDNA sample 0.8 lL, ROX II 0.4 lL, ddH2O 7.2 lL. The standard cycling conditions were: 95 °C for 1 min (initial polymerase activation), followed by 40 cycles of 10 s at 95 °C, 45 s at 59.6 °C. The PCR specificity was checked with dissociation curve analysis from 55 to 95 °C, b-actin of S. maindroni (b-Sm actin-F and b-Sm actin-R)was used as the internal standard, all the primers as shown in Tab.1. The 2DDCT method was used to analyze the mRNA expression level. All data were given in terms of relative mRNA expressed as means ± S.E. (N = 5). The data were subjected to one-way analysis of variance (one-way ANOVA) followed by an unpaired, two-tailed t-test. Differences were considered significantly at P < 0.05. 2.5. The temporal expression of SmSOD mRNA in hepatopancreas after Cd2+ exposure For Cd treatments, animals were exposed to static culture condition where cadmium chloride (CdCl25H2O) was added to the 10 L filtered seawater with dissolved Cd2+ concentration of 200 lg/L, which based on the preliminary experiment of gradient concentration. The highest concentration, which was no mortality in the heavy metals treatment during the exposure period (data not shown), was selected. Total 30 cuttlefishes were cultured in 10 L filtered well-aerated seawater with Cd2+. Seawater was changed daily and resupplied with the corresponding concentration of Cd2+. Five individuals were taken for each treatment at 24, 48, 72, 120, 168 and 264 h post-treated respectively. The methods of hepatopancreas collection, total RNA extraction, cDNA synthesis and realtime PCR analysis were performed as described above. 3. Results and discussion 3.1. cDNA sequence analysis and characterization of SmSOD full-length The full-length sequence of SmSOD was 709 bp (GenBank accession No. KF908850), which contained an open reading frame
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(ORF) of 459 bp encoding 153 amino acid (aa) residues, a 50 -UTR of 131 bp, and a 30 -UTR of 116 bp (Supplementary Fig. 1). The predicted molecular weight (MW) was 15.75 kDa and estimated pI was 6.02. BLASTn analysis suggested that the coding sequence shared high similarity and identity with known Cu/Zn SODs. Two types of Cu/Zn-SOD have been identified in most organisms, extracellular Cu/Zn-SOD (ecCu/Zn-SOD) with an N-terminal signal cleavage peptide for secretion, and intracellular Cu, Zn SOD (icCu/
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Zn-SOD) without a signal peptide (Ni et al., 2007; Halliwell and Gutteridge, 1984). The SignalP 4.0 showed that there was no signal peptide in SmSOD, suggesting that SmSOD was icCu/Zn SODs, unlike Mn-SOD or ecCu/ZnSOD, which have glycosylation of protein for conferring stability on secreted SODs (Bao et al., 2009). ClustalW analysis also revealed that the deduced amino acid sequence of SmSOD showed higher identity (70–85%) with other ten known icCu/Zn SODs (Supplementary Fig. 2). For example, it shared 82%
Fig. 1. Phylogenetic tree depicting the relationship of SmSOD with other species. All protein sequences were obtained from GenBank of NCBI, GenBank accession number was in parentheses, and phylogenetic tree was constructed using Neighbor-Joining rule method by the software of MEGA v4.0.
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identity with icCu/Zn-SOD from H. discus discus (154 aa, ABF67508), 81% identity with M. edulis (158 aa, CAE46443), 77% identity with Chlamys farreri (153 aa, ABD58974), 76% identity with Ventridens decussatus (131 aa, AAQ88163) and 71% identity with Biomphalaria glabrata (155 aa, ABD60754). The sequence size of SmSOD was 153 aa, which was identified with the average of intracellular Cu/ Zn-SOD of 154 aa (147–167 aa) and different from the extracellular Cu/Zn-SOD with 208 aa (175–251 aa) (Lin et al., 2008). 3.2. Multiple sequence alignments and phylogenetic analysis Multiple alignment revealed that several characteristic motifs and signatures of intracellular SOD were also appeared in SmSOD (Supplementary Fig. 2), for example GFHIHEFGDNT and GNAGGRLACGVI motifs that were the highest conserved region sequences of Cu/Zn-SOD family signature. Additionally, the deduced amino acids of SmSOD contained two cysteine residues C-58 and C-146, which were conserved in all Cu/ZnSODs and were predicted to form one intracellular disulfide bond (Cioni et al., 2003). Copper (H-47, H-49, H-64, H-120) and zinc (H-64, H-72, H81, D-84) binding sites were required, which were conserved in all Cu/ZnSODs, and copper and zinc ions had critical functions in stabilizing the quaternary structure to increase the probability of pathogenic mutants (Ni et al., 2007; Anju et al., 2013; Lynch and Colón, 2006). SmSOD sequence also included several fully conserved active site residues G-45, G-62, P-75 and G-83, which might be involved in maintaining the active site geometry (Li et al., 2010a,b). These features suggested that SmSOD might be a new member of the SOD protein family, and further studies were needed to seek the transcription regulating factors involved in SmSOD expression regulation. In the phylogenetic tree (Fig. 1) almost all the cytoplasmic Cu/Zn SODs (icCu/Zn SODs) clustered together as a subgroup, extracellular Cu/Zn SODs (ecCu/Zn SODs) clustered to other groups, and MnSODs obviously acted as outgroup. It was suggested that icCu/Zn SODs and ecCu/Zn SOD might be diverged from a common ancestor, consistent with some researchers’ opinions (Zelko et al., 2002). SmSOD first clustered with icCu/Zn SODs from E. scolopes, and further grouped with those of M. edulis and Ruditapes decussatus, which belonged to bivalve. The other species in the icCuZnSOD tree generally agreed with the taxonomic classification of the corresponding species. 3.3. Temporal expression of SmSOD after Vibrio challenge V. harveyi is a gram-negative bacteria in the marine organism and seawater, which often leads to high mortality in cultured bivalves, and affects the production of economic aquatic organisms in marine environment (Gómez et al., 2005). Cu/ZnSODs are considered as general stress responsive factors, whose expression at transcriptional and/or translational levels may be influenced by a variety of intracellular and environmental cues (Zelko et al., 2002). In the present study, real-time PCR (qRT-PCR) analysis indicated that SmSOD mRNA expression in hepatopancreas was upregulated significantly at the start of V. harveyi injection, reached a peak of approximate 70-fold higher than that of the control at 4 h post-injection, and then declined in the following hours. After 24 h post-injection, the level decreased to 30-fold of the blank (see Fig. 2A). Similar time-dependent expression patterns were also observed in other aquatic organisms, for example, the Cu/ ZnSOD expression was up-regulated and then recuperated in C. farreri after being injected with Candida lipolytica (Fridovich, 1975). In shrimp the activity of SOD in resistant shrimp was significantly higher at the beginning after V. harveyi injection, then recovered to normal level (Huang et al., 2013), but the expression of icCu/ZnSOD in Argopecten irradians was not affected after
Fig. 2. Expression profile of SmSOD after stress with Vibrio harveyi and Cd2+: (A) Temporal expression of the SmSOD transcripts in hepatopancreas after Vibrio harveyi infection. (B) Expression analysis of SmSOD in the hepatopancreas in response to Cd2+. Statistical analysis of differences was done by oneway analysis of variance (ANOVA) by SPSS 13.0 software. Vertical bars were mean ± SD of five technical replicates, and the asterisks above the bars represented statistically significant differences from the control samples, ‘‘*’’ at P < 0.05, ‘‘**’’ at P < 0.01.
challenge of Vibrio anguillarum. The mechanisms of Cu/Zn SOD expression after biological stimulation or suppress have been intensively investigated in mammals (Gardner et al., 2002; Akiyama et al., 2005; Peluffo et al., 2005), but not well studied in cephalopod. The significant change of SmSOD was suspected to be related to the virulence of the pathogens and the oxidative stress-induced injury. In order to response against infection, different invading microorganisms could induce different toxicity and produce different amounts, which affected SOD expression. Compared with MnSOD and ecCu/Zn-SOD, the expression of icCu/ Zn-SOD is more stable than other subtypes (Zelko et al., 2002). However, SmSOD mRNA expression increased more than 10-fold after infection than the blank, and it might be hypothesized that the juvenile of cephalopod were more sensitive than the adult in immune reaction. Therefore, as an important anti-oxidation enzyme, SmSOD is believed to play an important role in the cephalopod immune. 3.4. Temporal expression of SmSOD in hepatopancreas after Cd2+ stress Cadmium is the major toxic metals as contaminants in marine environment, being responsible for many toxic effects, including generating abnormal or denatured proteins and causing oxidative stress in a variety of living organisms (Sarkar et al., 2006). Gene
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expression frequently was used in bio-monitoring of contaminated sites based on the mRNA expression profile data, it was possible to make estimate of chemical exposure and its effects on the exposed species (Fang et al., 2010). Relying on the various metal contents, SODs could be regarded as biomarkers of contaminants pollution in aquatic environment such as cadmium pollution (Akiyama et al., 2005; Contardo and Wiegand, 2008). In Cd2+ exposure, the SmSOD mRNA expression in hepatopancreas was up-regulated at the start of experiment, but maintained low expression at the beginning hours, and appeared skyrocketing growth and reaching the peak at approximate 210-fold till 72 h post-exposure, then decreased gradually to 42-fold of blank group at 11 days postexposure (see Fig. 2B). Similar expression patterns were also observed in the clam (Mactra veneriformis), whose SOD activity significantly increased at the start of Cd2+ exposure, then decreased and finally returned to the normal level (Sarkar et al., 2006). It has been confirmed that cadmium stress can induce the ROS generation in some bivalves, and the cadmium iron would interfere with the antioxidant enzymatic defense system (Zelko et al., 2002; Geret et al., 2002). To eliminate ROS, SODs would increase, which means that cadmium stress induce the SOD gene’s transcript. These findings are consistent with the previous studies that the mRNA levels of both Cu/Zn SOD and Mn SOD were increased after metal treatments in abalones (Li et al., 2010a,b; Kim et al., 2007). Therefore, the significantly increased gene expression of SmSOD demonstrated its tolerance and defence against heavy metals contaminants to avoid the toxicity from aquatic pollutants, and it might be a potential biomarker. In conclusion, this was the first report of full-length cloning, characterization and inducible expression of icCu/Zn-SOD in hepatopancreas from cephalopod S. maindroni. The analysis of phylogenetic and structural features might contribute to the understanding of adaptation and evolutionary processes of icCu/Zn-SOD in invertebrate. The high expression level of SmSOD gene in response to V. harveyi and Cd2+ showed its crucial role in the protection of cells in case of oxidative stress of marine cephalopod, and might be potential biomarker against pollution in aquatic environment. Acknowledgements This research was supported by grants from Zhejiang Marine Fisheries Bureau Project (2012)83, the Natural Science Foundation of Zhejiang Province, China (LY14C190004), and the Hi-Tech Research and Development Program of China (863) (2012AA10 A413-5). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.dci.2014.06.010. References Akiyama, S., Inagaki, M., Tsuji, M., Gotoh, H., Gotoh, T., 2005. MRNA study on Cu/Zn superoxide dismutase induction by hemodialysis treatment. Nephron Clin. Pract. 99, c107–114. Anju, A., Jeswin, J., Thomas, P.C., Paulton, M.P., Vijayan, K.K., 2013. Molecular cloning, characterization and expression analysis of cytoplasmic Cu/Zn-
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