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Cloning and expression of the Mn-SOD gene from Phascolosoma esculenta
Fish & Shellfish Immunology 29 (2010) 759e764
Contents lists available at ScienceDirect
Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Cloning and expression of the Mn-SOD gene from Phascolosoma esculenta Mengqian Wang a, Xiurong Su a, *, Ye Li a, Zhou Jun a, Taiwu Li a, b a b
Faculty of Life Science and Biotechnology, Ningbo University, Fenghua Road, Ningbo, Zhejiang Province 315211, P.R China Ningbo City College of Vocational Technology, Ningbo 315100, P. R. China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 January 2010 Received in revised form 27 June 2010 Accepted 2 July 2010 Available online 21 July 2010
Manganese superoxide dismutase (Mn-SOD; SOD2) is an important antioxidant defense enzyme. In this study, a full-length Mn-SOD cDNA was cloned from the cDNA library of Phascolosoma esculenta. The cDNA is 1385 bp in length, including an open reading frame (ORF) of 681 bp encoding 226 amino acids. The predicted protein has a calculated molecular weight of 25.2 kDa and a theoretical isoelectric point of 5.96. BLAST analysis revealed the predicted protein shared 70% identity with homologues in Caenorhabditis elegans and Gallus gallus. The SOD gene was inserted into Escherichia coli expression plasmid pET-28a (þ) to produce pET-SOD. The recombinant Mn-SOD of P. esculenta was expressed following IPTG induction, and verified by Western blot analysis using antiserum from immunized mice. Furthermore, fluorescent real-time PCR analysis revealed varying degrees of induction of SOD2 mRNA expression in the blood of P. esculenta exposed to heavy metals (Cd2þ, Cu2þ and Zn2þ) and thermal stresses (4 C and 37 C). Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Phascolosoma esculenta Mn superoxide dismutase Gene expression Western blot
1. Introduction Superoxide dismutases (SODs) are metalloenzymes capable of converting superoxide radicals to oxygen and hydrogen peroxide [1,2]. According to the metal embedded at the active site of the enzyme, SODs could be divided into iron-, manganese-, and copper zinc-containing SODs (Fe-SODs, Mn-SODs and Cu/Zn-SODs, respectively) [3e5], each with varying sensitivity to KCN, NaN3 and H2O2. Cu/Zn-SODs are mostly found in eukaryotes and rarely in bacteria [6]. In contrast, Mn-SODs and Fe-SODs are found in prokaryotes, as well as mitochondria or chloroplasts, respectively [7]. Several novel SODs have been discovered more recently, such as cambialistic SOD and nickel SOD (Ni-SOD) [8], with the former capable of using either iron or manganese at its active site [9]. Furthermore, all streptococci tested so far appeared to synthesize only a single Mn-SOD. Thus, the distribution pattern of SODs is highly specific. Mn-SOD in the mitochondria of eukaryotes, which is synthesized as a precursor protein in the cytoplasm and is then imported into the mitochondria after cleavage of its signal peptide, is a tetrameric protein with subunits of 22 kDa [10]. Phascolosoma esculenta typically live in beaches. They are highly resistant to temperature fluctuation between 17 C to 33 C [11] and heavy metal pollution. Both thermal stress and toxicants have been considered as serious factors that affect the immune response and
* Corresponding author. Tel./fax: þ86 574 87608368. E-mail address: [email protected] (X. Su). 1050-4648/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2010.07.005
survival of aquatic animals [12e15]. In this study, we report the cDNA cloning, characterization and expression analysis of the Mn-SOD gene of P. esculenta. We also describe the expression profile of Mn-SOD in response to heavy metal and thermal stresses. 2. Materials and methods 2.1. Materials P. esculenta samples were obtained form Fenghua, Zhejiang, China, and reared at room temperature. The pMD18-T vector, RNAiso Reagent, mRNA Purification Kit and cDNA Library Construction Kit were purchased from Takara Corporation of Japan. MMLV First Strand cDNA Synthesis Kit was purchased from BioBasic Corporation of Canada. Freund’s complete adjuvant and Freund’s incomplete adjuvant were purchased from Sigma Corporation of America The cDNA library of P. esculenta was previously constructed in our laboratory. 2.2. Cloning of the full-length Mn-SOD cDNA We initially obtained partial Mn-SOD cDNA sequences from the cDNA library. The 50 end of Mn-SOD was amplified by using the SOD-5 primer (50 AAGCACCACC AGACCTATGT 30 ) and the T3 primer. The PCR program was 94 C for 5 min followed by 35 cycles, at 94 C for 45 s, at 56 C for 45 s, at 72 C for 1 min and a final extension step at 72 C for 10 min. The 30 end of Mn-SOD was amplified by using the SOD-3 primer (50 ATAGT AGGCGTGTTCCCAA 30 ) and
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the T7 primer. The PCR amplification was conducted at one cycle of 95 C for 5 min followed by 35 cycles of 94 C for 45 min, 58 C for 45 min, 72 C for 1 min and a final extension step at 72 C for 10 min. The PCR products were gel-purified and sequenced.
2.3. Sequence analysis Based on the full-length Mn-SOD cDNA sequence, a putative open reading frame (ORFs) and the corresponding amino acid sequence were identified using the DNAStar program. Theoretical pI values and predicted molecular masses were calculated using Prot-Param tools (http://kr.expasy.org/tools/protparam.html). Blast search using cDNA sequences of P. esculenta Mn-SOD were performed to obtain known Mn-SOD sequences from other species.
2.4. Expression of Mn-SOD and Western blotting Mn-SOD ORF was amplified by using the primers SODBDF (50 CGGGATCC ATGCTCTCGCGGGTTGGA 30 ) and SODBDR (50 CCCAAGCTTTCACTGAGC TGCAGATGT 30 ). The PCR program was 95 C for 5 min followed by 30 cycles of 94 C for 30 s, 58 C for 30 s, 72 C for 1 min and a final extension step at 72 C for 10 min. PCR products were digested with BamH_ and HindZ and ligated into the pET-28a plasmid digested with the same enzymes. The resulting ligation was transformed and pET-SOD clones with correct insert were confirmed by enzyme digestion and sequencing. An overnight culture of a clone harboring pET-SOD was diluted into LB (1:100), and cultured for 3 more hours until log phase. IPTG (Isopropyl b-D-1-thiogalactopyranoside) was added to the final concentration of 1 mM and the culture was grown at 37 C for 1e4 h to induce the protein expression. Alternatively, 0.2 mM IPTG was added and the culture was grown at 25 C for 13e18 h. Protein samples were analyzed by 12% SDS-PAGE, and staining with Coomassie brilliant blue. Five male BALB/c mice (5-week-old) were immunized intradermally with recombinant SOD proteins mixed with equal volume of Freund’s complete adjuvant. Five mice were not immunized as the negative control. These mice were then immunized with the mixture of antigen and Freund’s incomplete adjuvant three times every week. Three days after the last immunization, blood was collected to obtain antiserum, and Western blot analysis was performed to detect protein expression.
2.5. Exposure to heavy metals P. esculenta samples (3e4 cm in shell length) were kept in a basin (5 L) and acclimated to basin conditions for 1 week prior to metal exposure. Seawater temperature was kept at 20 1.0 C throughout the experiments. P. esculenta were independently exposed to three different heavy metals (copper, cadmium and zinc) at 10 mM by adding 0.5 M stock solutions of CuCl2, CdCl2 and ZnCl2. Ten individuals were randomly removed from each replicate basin at 6, 12, 24 and 48 h post exposure. The P. esculenta was unexposed heavy metals is referred as the control group. Viability was checked daily, and dead individuals were removed immediately. When the treatments were finished, five individuals were chosen from each replicate basin, and total RNA was isolated from the blood of P. esculenta using TaKaRa RNAiso Reagent according to the manufacturer’s instructions. First strand cDNA synthesis was performed using the MMLV First Strand cDNA Synthesis Kit. cDNA samples was stored at 80 C for subsequent fluorescent real-time PCR.
2.6. Exposure to high and low temperatures Ten individuals were allocated into each of ten experimental basins containing 2 L seawater at 20 C (two replicate basins per temperature group). After a 1-week acclimation period, the water temperature of four basins was elevated to 37 C, whereas that of four others was reduced to 4 C. The remaining two tanks were kept at 20 C. Temperature treatment lasted for 6 h, 12 h, 24 h and 48 h. Five individuals were chosen from each replicate basin after each treatment period, and blood samples were removed for RNA preparation. cDNA first strand synthesis was carried out as above. 2.7. Quantitative analysis of SOD2 mRNA expression The expression of SOD2 transcript in the blood of P. esculenta after heavy metal exposure was measured by fluorescent real-time PCR. Two SOD2 gene-specific primers SOD-F (50 ATAGCACGGCAGCATGGA 30 ) and SOD-R (50 GGTCTGGTGGTGTTTTG G 30 ) were used to amplify a product of 147 bp. A constitutively expressed gene, GAPDH, was used as an internal control. Two GAPDH primers GF (50 CCAGAACATCA TCCCAGCA 30 ) and GR (50 ACGAACAGGG ACACGGAAG 30 ) were used to amplify a 104 bp fragment. PCR-grade water was used for the negative control. The fluorescent real-time PCR assay was carried out in RotorGene 6000 System (Applied Corbett Research). The amplifications were performed in a 20 ml reaction containing 10 ml of 2SYBR Premix Ex Taq II (Takala), 0.5 ml each of the forward and reverse primers (10 mM), 5 ml of 1:10 diluted cDNA, and 4 ml of PCR-grade water. The thermal profile for real-time PCR was 95 C for 10 s followed by 40 cycles of 10 s at 95 C , 15 s at 54 C and 15 s at 72 C. Fluorescence readings were taken at 72 C after each cycle. Gene of Interest and Housekeeper Gene was analyzed using a method of Comparative Delta-delta Ct. Relative fluorescence unit, calculated threshold cycle (CT), and dissociation curve were monitored by the analysis software of the system (Corbett Research). Triplicate assays per cDNA sample were carried out independently to determine the average CT values. 3. Results 3.1. Cloning, sequencing and phylogenetic analysis of cDNA The cDNA sequence of the P. esculenta Mn-SOD gene was deposited in GenBank (GenBank accession no.GQ487656). The fulllength cDNA of Mn-SOD was of 1321 bp, including a 47 bp 50 UTR, a 593 bp 30 UTR with a putative polyadenylation signal was AATAAA and a 18 bp poly(A) tail, and a 681 bp ORF encoding a polypeptide of 226 amino acids. The full-length nucleotide sequence and the deduced amino acid sequence are shown in Fig. 1. The predicted Mn-SOD proteins have four conserved residues coordinating manganese (His-56, His-100, Asp-184 and His-188); alignment positions in Fig. 1. The calculated molecular mass is 25.2 kDa, and the pI was estimated at 5.96. The protein shares the highest sequence identity of 70% with homologues from Caenorhabditis elegans and Gallus gallus respectively. Characteristic Mn-SOD signatures (DVWEHAYY) from 184 to 191 are also highly conserved. Multiple sequence alignment showed that the deduced amino acid sequence of P. esculenta Mn-SOD shares high similarity with other Mn-SOD amino acid sequences from Macrobrachium rosenbergii, Fenneropenaeus chinensis, C. elegans, G. gallus, Mus musculus and Pongo abelii (Fig. 2). Specifically, Mn-SOD of P. esculenta displays 70% similarity with homologues from C. elegans and G. gallus, and 68% similarity with homologues from M. rosenbergii and F. chinensis.
M. Wang et al. / Fish & Shellfish Immunology 29 (2010) 759e764
Fig. 1. Gene sequence of Mn-SOD from P. esculenta and its amino acid sequence. Note, signal.
3.2. Expression of Mn-SOD proteins and Western blotting analysis Protein expression was induced by IPTG at different concentrations followed by incubation for different times. When induced by 1 mM IPTG and cultured at 37 C for 4 h, recombinant SOD proteins accumulated to very high levels and formed inclusion bodies (Fig. 3A). On the other hand, when induced by 0.2 mM IPTG and cultured at 25 C for 18 h, the proteins were expressed both as inclusion bodies and soluble proteins. Likewise, purified Mn-SOD proteins were also resolved by SDS-PAGE and Coomassie staining revealed a single band of 29 kDa (Fig. 3B). The polyclonal antibody generated from the immunized mice was used as the primary antibody for Western blot analysis. The protein samples were obtained from E. coil BL21 with pET-28a induced by 1 mM IPTG for 4 h, uninduced E. coil BL21 with pET-SOD, E. coil BL21 with pET-SOD induced by 1 mM IPTG for 4 h (inclusion bodies), E. coil BL21 with pET-SOD induced by 0.2 mM IPTG at 25 C for 18 h (soluble protein) and whole cell lysate of P. esculenta. The
: start codon;
: manganese-binding residues;
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: Mn-SOD motif signature;
: tailing
results showed that a specific band appeared in the samples of purified recombinant protein of Mn-SOD (29 kDa) and the whole cell lysate of P. esculenta (25 kDa). In contrast, no specific band was detected in the uninduced BL-21 harboring pET-HSP or induced BL-21 with the empty vector pET-28a (Fig. 4). Furthermore, the same experiment was conducted using the serum from PBSimmunized mice as the primary antibody, and this control serum did not react with the protein and produce any band. These results suggested that the polyclonal antibody specifically recognized the Mn-SOD protein.
3.3. Mn-SOD mRNA expression in response to heavy metal exposure During metal treatments, significant mortalities were found in the groups treated with 10 mM of copper (Cu): 10% of individuals were dead within the first 48 h. There was no loss in the zinc (Zn)exposed, cadmium (Cd)-exposed and unexposed groups.
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Fig. 2. Alignment of P. esculenta Mn-SOD polypeptide sequence along with other orthologues.
Mn-SOD mRNA levels were substantially affected by the heavy metal exposures (Fig. 5). Significant differences in the Mn-SOD expression level were observed at 6 h, 12 h, 24 h and 48 h after heavy metal exposures compared with the control group (P < 0.05). Specifically, Mn-SOD mRNA level peaked at 24 h following Zn treatment (P < 0.05), 13.4-fold higher than that observed in the control group, then gradually declined to approximately the original level at 48 h. After Cd treatment, Mn-SOD mRNA level peaked at 24 h (P < 0.05), 6.4-fold higher than the control group. After Cu treatment, Mn-SOD mRNA expression was reduced and reached the lowest level at 48 h.
thermal stress on Mn-SOD mRNA expression is shown in Fig. 6. GAPDH expression showed constant levels regardless of the water temperature, whereas Mn-SOD expression was highly sensitive to both high and low water temperatures, with low water temperatures more dominant than high temperatures (P < 0.05). For example, fold increases in the 37 C group relative to the 20 C group ranged from 1.3 to 3.0 between 6 h to 48 h, with the maximum Mn-SOD mRNA levels observed at 12 h. On the other hand, fold increases in the 4 C group relative to the 20 C group ranged from 1.9 to 13.5 from 6 h to 48 h. Mn-SOD mRNA level peaked at 24 h at 4 C, then declined gradually.
3.4. Mn-SOD mRNA expression in response to temperature stress
4. Discussion
During temperature treatments, there was no loss in the 37 C -exposed, 4 C -exposed and unexposed groups. The effect of
SOD enzymes play critical roles in the antioxidant enzyme defense against ROS and superoxide anion radicals. The SOD gene
Fig. 3. Expression and purification of SOD recombinant protein. Note, (A) Lane 1: low molecular marker; lane 2: total protein of non-induced pET-28a; lane 3: total protein of pET28a induced by IPTG for 5 h; lane 4: total protein of non-induced pET-SOD; lane 5e8: total protein of pET-SOD induced by IPTG for 1 h, 2 h, 3 h, 4 h. (B) Lane 1: molecular weight marker; lane 2: purified recombinant protein of Mn-SOD.
M. Wang et al. / Fish & Shellfish Immunology 29 (2010) 759e764
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Fig. 4. Specificity of pET-SOD polyclonal antibody was determined by Western-blot. Note, Lane 1: low molecular marker, lane 2: IPTG induced E. coil BL21 with pET-28a for 4 h, lane 3: uninduced E. coil BL21 with pET-SOD, lane 4: IPTG induced E. coil BL21 with pET-SOD for 4 h, lane 5: soluble protein , lane 6: total protein of blood from P. esculenta
family is generally classified into Cu/Zn-SODs, Mn-SODs and FeSODs depending on the subcellular localization and the bound metal ion [16]. Mitochondria generate the majority of superoxide radicals, which are subsequently dismutated by Mn-SODs to produce a constant flux of H2O2. This process is known to play a vital role in controlling the redox state of the cell [17e19]. In this paper, the complete cDNA sequence of a Mn-SOD gene from P. esculenta was reported. Sequence analysis revealed conserved sequence motifs, such as the Mn-SOD signature DVWEHAYY and four conserved residues coordinating manganese interaction (His-56, His-100, Asp-184 and His-188) [14]. Homology analysis revealed phylogenetic relationships of P. esculenta with other species. The results showed that P.esculentas is most closely related to C. elegans, and to a lesser extent, to M. musculus and P. abelii. SDS-PAGE analysis showed that the Mn-SOD recombinant protein was approximately 29 kDa, and the purified Mn-SOD proteins from P. esculenta were approximately 25 kDa. Protein expression appeared to be elevated significantly with the lengthened induction time. Depending on IPTG concentration and incubation time, the proteins were partitioned into inclusion bodies or soluble protein fractions. Western blot analysis showed that the polyclonal antibody generated in mice reacted with purified Mn-SOD recombinant proteins and detected specific signals from P. esculenta whole cell lysates, but did not react with the total proteins of E. coli. These results demonstrate the specificity of this antibody for Mn-SOD from P. esculenta.
Fig. 6. Quantitative analysis of Mn-SOD mRNA expression of P. esculenta exposed to different temperature by fluorescent real-time PCR. Note, Means with the same letters on each histogram within a detection time (4 C, 37 C) are not statistically different at P < 0.05. Standard deviation is noted by T-bar on each histogram.
Mn-SODs have been considered as general stress responsive factors whose expression might be influenced by a variety of intracellular and environmental cues at transcriptional and/or translational levels [20,21] . Previous studies have shown that heavy metal exposures and thermal stress induced expression of stress proteins [9,14,22,23]. In the present study, experimental exposure of P. esculenta either heavy metals, or low and high temperatures significantly induced the mRNA expression of Mn-SOD, albeit to different degrees. Mn-SOD expression was increasingly responsive to the treatment of Cu2þ, Cd2þ and Zn2þ, with as much 13.4-fold as control group from Zn2þ exposure. At 24 h when Mn-SOD mRNA level peaked, Zn2þ and Cd2þ treatments elicited 6- and 3-fold higher expression than that from Cu2þ, respectively. In contrast, samples treated with Cu2þ displayed a modest 2-fold increase in Mn-SOD expression at 6 h followed by a gradual decline to about 1/3 of the original level at 48 h. It is plausible that P. esculentas was more sensitive to Cu2þ; after Cu2þ treatment for 24 h, P. esculenta appeared maladjusted, and the mouth began to canker. In the present study using relatively high doses of metals (10 mM), increases in SOD expression were detectable as early as 6 h after exposure, with peak levels observed within 1 day in many instances. Mn-SOD expression appeared more sensitive to 4 C treatment than 37 C treatment, and the maximal induction from 4 C treatment was roughly 4-fold higher than that from 37 C treatment. P. esculenta live in beaches where the environmental temperature fluctuates greatly, with a tolerable temperature range of 17 C to 33 C [11]. 37 C is relatively close to the upper physiological limit, perhaps explaining why it is less effective in the induction of Mn-SOD expression than 4 C. Taken together, the increased Mn-SOD expression after heavy metal and temperature stresses suggests a role of Mn-SOD in immunopathological processes in P. esculenta.
Acknowledgements
Fig. 5. Quantitative analysis of Mn-SOD mRNA expression of P. esculenta exposed to heavy metals by fluorescent real-time PCR. Note, Means with the same letters on each histogram within a detection time (6, 12, 24 or 48 h) are not statistically different at P < 0.05. Standard deviation is noted by T-bar on each histogram.
This work was financially supported by National Natural Science Foundation (No. 40776075), and Scientific Program of Zhejiang Province (No. 2006C13089), and Project supported by the Ningbo Science Bureau, China (Grant No. 2008C50027), and Sponsored by K.C.Wong Magna Fund at Ningbo University.
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References [1] McCord JM, Fridovich I. Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein). J Biol Chem 1969;244:6049e55. [2] Bruno-Barcena JM, Anrus JM, Libby SL, Klaenhammer TR, Hassan HM. Expression of a heterologous manganese superoxide dismutase gene in intestinal Lactobacilli provides protection against hydrogen peroxide toxicity. Appl Environ Microbiol 2004;70:4702e10. [3] Gaillot O, Poyart C, Berche P, Trieu-Cuot P. Molecular characterization and expression analysis of the superoxide dismutase gene form Streptococcus agalactiae. Gene 1997;204:213e8. [4] Stevens Marla K, Hassett Daniel J, Radolf Justin D, Hansen Eric J. Cloning and sequencing of the gene encoding the Cu, Zn-superoxide dismutase of Haemophilus ducreyi. Gene 1996;183:35e40. [5] Motoshima H, Minagawa E, Tsukasaki F, Kaminogawa S. Cloning and nucleotide sequencing of genes encoding Mn-super-oxide dismutase and class II fumarase from Thermus aquaticus YT-1. J Ferm Bioeng 1998;86:21e7. [6] Hassan HM. Microbial superoxide dismutases. Adv Genet 1989;26:65e97. [7] Wang X, Yang HJ, Ruan LW, Liu X. Cloning and characterization of a thermostable superoxide dismutase from the thermophilic bacterium Rhodothermus sp. XMH10. J Ind Microbiol Biotechnol 2008;35:133e9. [8] Meile L, Fischer K, Leisinger T. Characterization of the superoxide dismutase gene and its upstream region from Methanobacterium thermoautotrophicum Marburg. FEMS Microbiol Lett 1995;128:247e53. [9] Borgstahl GE, Parge HE, Hickey MJ, Beyer Jr WF, Hallewell RA, Tainer JA. The structure of human mitochondrial manganese superoxide dismutase reveals a novel tetrameric interface of two 4-helix bundles. Cell 1992;71:107e18. [10] Prashani ME, Kang HS, Zyosa MD, Jee Y, Lee YH, Lee J. Molecular cloning and characterization of Mn-superoxide dismutase form disk abalone (Haliotis discus iscus). Comp Biochem Physiol 2006;145:318e24. [11] Zeng HX, Hong P, Ding LF, Zhu JQ. Tolerance of Phascolom a esculenta to water temperature and salinity. Fish Sci 2006;25:422e3. [12] Yeh ST, Liu CH, Chen JC. Effect of copper sulfate on the immune response and susceptibility to Vibrio alginolyticus in the white shrimp Litopenaeus vannamei. Fish Shellfish Immunol 2004;17:437e46.
[13] Cheng W, Hsiao IS, Hsu CH, Chen JC. Change in water temperature on the immune response of Taiwan abalone Haliotis diversicolor supertexta and its susceptibility to Vibrio parahaemolyticus. Fish Shellfish Immunol 2004;17:235e43. [14] Kim KY, Lee SY, Cho YS, Bang IC, Kim KH, Kim DS, et al. Molecular characterization and mRNA expression during metal exposure and thermal stress of copper/zinc- and manganesesuperoxide dismutases in disk abalone, Haliotis discus discus. Fish Shellfish Immunol 2007;23:1043e59. [15] Viola A, Pregnolato G, Albergoni V. Effect of in vitro cadmium exposure on natural killer (NK) cells of catfish, Ictalurus melas. Fish Shellfish Immunol 1996;6:167e72. [16] Mruk DD, Silvestrini B, Mo M, Chenga CY. Antioxidant superoxide dismutaseda review: its function, regulation in the testis, and role in male fertility. Contraception 2002;65:305e11. [17] Boveris A, Chance B. The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen. Biochem J 1973; 134:707e16. [18] Cadenas E, Davies KJ. Mitochondrial free radical generation, oxidative stress, and aging. Free Radic Biol Med 2000;29:222e30. [19] Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev 1979;59:527e605. [20] Zelko IN, Mariani TJ, Folz RJ. Superoxide dismutase multigene family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and ECSOD (SOD3) gene structures, evolution, and expression. Free Radic Biol Med 2002; 33:337e49. [21] Cho YS, Choi BN, Kim KH, Kim SK, Kim DS, Bang IC, et al. Differential expression of Cu/Zn superoxide dismutase mRNA during exposures to heavy metals in rockbream (Oplegnathus fasciatus). Aquaculture 2006; 253:667e79. [22] Gao Q, Song LS, Ni DJ, Wu LT, Zhang H, Chang YQ. cDNA cloning and mRNA expression of heat shock protein 90 gene in the haemocytes of Zhikong scallop Chlamys farreri. Comp Biochem Physiol B 2007;147:704e15. [23] Farcy E, Serpentini A, Fiévet B, Lebel JM. Identification of cDNAs encoding HSP70 and HSP90 in the abalone Haliotis tuberculata: transcriptional induction in response to thermal stress in hemocyte primary culture. Comp Biochem Physiol B 2007;146:540e50.
Contents lists available at ScienceDirect
Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Cloning and expression of the Mn-SOD gene from Phascolosoma esculenta Mengqian Wang a, Xiurong Su a, *, Ye Li a, Zhou Jun a, Taiwu Li a, b a b
Faculty of Life Science and Biotechnology, Ningbo University, Fenghua Road, Ningbo, Zhejiang Province 315211, P.R China Ningbo City College of Vocational Technology, Ningbo 315100, P. R. China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 January 2010 Received in revised form 27 June 2010 Accepted 2 July 2010 Available online 21 July 2010
Manganese superoxide dismutase (Mn-SOD; SOD2) is an important antioxidant defense enzyme. In this study, a full-length Mn-SOD cDNA was cloned from the cDNA library of Phascolosoma esculenta. The cDNA is 1385 bp in length, including an open reading frame (ORF) of 681 bp encoding 226 amino acids. The predicted protein has a calculated molecular weight of 25.2 kDa and a theoretical isoelectric point of 5.96. BLAST analysis revealed the predicted protein shared 70% identity with homologues in Caenorhabditis elegans and Gallus gallus. The SOD gene was inserted into Escherichia coli expression plasmid pET-28a (þ) to produce pET-SOD. The recombinant Mn-SOD of P. esculenta was expressed following IPTG induction, and verified by Western blot analysis using antiserum from immunized mice. Furthermore, fluorescent real-time PCR analysis revealed varying degrees of induction of SOD2 mRNA expression in the blood of P. esculenta exposed to heavy metals (Cd2þ, Cu2þ and Zn2þ) and thermal stresses (4 C and 37 C). Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Phascolosoma esculenta Mn superoxide dismutase Gene expression Western blot
1. Introduction Superoxide dismutases (SODs) are metalloenzymes capable of converting superoxide radicals to oxygen and hydrogen peroxide [1,2]. According to the metal embedded at the active site of the enzyme, SODs could be divided into iron-, manganese-, and copper zinc-containing SODs (Fe-SODs, Mn-SODs and Cu/Zn-SODs, respectively) [3e5], each with varying sensitivity to KCN, NaN3 and H2O2. Cu/Zn-SODs are mostly found in eukaryotes and rarely in bacteria [6]. In contrast, Mn-SODs and Fe-SODs are found in prokaryotes, as well as mitochondria or chloroplasts, respectively [7]. Several novel SODs have been discovered more recently, such as cambialistic SOD and nickel SOD (Ni-SOD) [8], with the former capable of using either iron or manganese at its active site [9]. Furthermore, all streptococci tested so far appeared to synthesize only a single Mn-SOD. Thus, the distribution pattern of SODs is highly specific. Mn-SOD in the mitochondria of eukaryotes, which is synthesized as a precursor protein in the cytoplasm and is then imported into the mitochondria after cleavage of its signal peptide, is a tetrameric protein with subunits of 22 kDa [10]. Phascolosoma esculenta typically live in beaches. They are highly resistant to temperature fluctuation between 17 C to 33 C [11] and heavy metal pollution. Both thermal stress and toxicants have been considered as serious factors that affect the immune response and
* Corresponding author. Tel./fax: þ86 574 87608368. E-mail address: [email protected] (X. Su). 1050-4648/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2010.07.005
survival of aquatic animals [12e15]. In this study, we report the cDNA cloning, characterization and expression analysis of the Mn-SOD gene of P. esculenta. We also describe the expression profile of Mn-SOD in response to heavy metal and thermal stresses. 2. Materials and methods 2.1. Materials P. esculenta samples were obtained form Fenghua, Zhejiang, China, and reared at room temperature. The pMD18-T vector, RNAiso Reagent, mRNA Purification Kit and cDNA Library Construction Kit were purchased from Takara Corporation of Japan. MMLV First Strand cDNA Synthesis Kit was purchased from BioBasic Corporation of Canada. Freund’s complete adjuvant and Freund’s incomplete adjuvant were purchased from Sigma Corporation of America The cDNA library of P. esculenta was previously constructed in our laboratory. 2.2. Cloning of the full-length Mn-SOD cDNA We initially obtained partial Mn-SOD cDNA sequences from the cDNA library. The 50 end of Mn-SOD was amplified by using the SOD-5 primer (50 AAGCACCACC AGACCTATGT 30 ) and the T3 primer. The PCR program was 94 C for 5 min followed by 35 cycles, at 94 C for 45 s, at 56 C for 45 s, at 72 C for 1 min and a final extension step at 72 C for 10 min. The 30 end of Mn-SOD was amplified by using the SOD-3 primer (50 ATAGT AGGCGTGTTCCCAA 30 ) and
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the T7 primer. The PCR amplification was conducted at one cycle of 95 C for 5 min followed by 35 cycles of 94 C for 45 min, 58 C for 45 min, 72 C for 1 min and a final extension step at 72 C for 10 min. The PCR products were gel-purified and sequenced.
2.3. Sequence analysis Based on the full-length Mn-SOD cDNA sequence, a putative open reading frame (ORFs) and the corresponding amino acid sequence were identified using the DNAStar program. Theoretical pI values and predicted molecular masses were calculated using Prot-Param tools (http://kr.expasy.org/tools/protparam.html). Blast search using cDNA sequences of P. esculenta Mn-SOD were performed to obtain known Mn-SOD sequences from other species.
2.4. Expression of Mn-SOD and Western blotting Mn-SOD ORF was amplified by using the primers SODBDF (50 CGGGATCC ATGCTCTCGCGGGTTGGA 30 ) and SODBDR (50 CCCAAGCTTTCACTGAGC TGCAGATGT 30 ). The PCR program was 95 C for 5 min followed by 30 cycles of 94 C for 30 s, 58 C for 30 s, 72 C for 1 min and a final extension step at 72 C for 10 min. PCR products were digested with BamH_ and HindZ and ligated into the pET-28a plasmid digested with the same enzymes. The resulting ligation was transformed and pET-SOD clones with correct insert were confirmed by enzyme digestion and sequencing. An overnight culture of a clone harboring pET-SOD was diluted into LB (1:100), and cultured for 3 more hours until log phase. IPTG (Isopropyl b-D-1-thiogalactopyranoside) was added to the final concentration of 1 mM and the culture was grown at 37 C for 1e4 h to induce the protein expression. Alternatively, 0.2 mM IPTG was added and the culture was grown at 25 C for 13e18 h. Protein samples were analyzed by 12% SDS-PAGE, and staining with Coomassie brilliant blue. Five male BALB/c mice (5-week-old) were immunized intradermally with recombinant SOD proteins mixed with equal volume of Freund’s complete adjuvant. Five mice were not immunized as the negative control. These mice were then immunized with the mixture of antigen and Freund’s incomplete adjuvant three times every week. Three days after the last immunization, blood was collected to obtain antiserum, and Western blot analysis was performed to detect protein expression.
2.5. Exposure to heavy metals P. esculenta samples (3e4 cm in shell length) were kept in a basin (5 L) and acclimated to basin conditions for 1 week prior to metal exposure. Seawater temperature was kept at 20 1.0 C throughout the experiments. P. esculenta were independently exposed to three different heavy metals (copper, cadmium and zinc) at 10 mM by adding 0.5 M stock solutions of CuCl2, CdCl2 and ZnCl2. Ten individuals were randomly removed from each replicate basin at 6, 12, 24 and 48 h post exposure. The P. esculenta was unexposed heavy metals is referred as the control group. Viability was checked daily, and dead individuals were removed immediately. When the treatments were finished, five individuals were chosen from each replicate basin, and total RNA was isolated from the blood of P. esculenta using TaKaRa RNAiso Reagent according to the manufacturer’s instructions. First strand cDNA synthesis was performed using the MMLV First Strand cDNA Synthesis Kit. cDNA samples was stored at 80 C for subsequent fluorescent real-time PCR.
2.6. Exposure to high and low temperatures Ten individuals were allocated into each of ten experimental basins containing 2 L seawater at 20 C (two replicate basins per temperature group). After a 1-week acclimation period, the water temperature of four basins was elevated to 37 C, whereas that of four others was reduced to 4 C. The remaining two tanks were kept at 20 C. Temperature treatment lasted for 6 h, 12 h, 24 h and 48 h. Five individuals were chosen from each replicate basin after each treatment period, and blood samples were removed for RNA preparation. cDNA first strand synthesis was carried out as above. 2.7. Quantitative analysis of SOD2 mRNA expression The expression of SOD2 transcript in the blood of P. esculenta after heavy metal exposure was measured by fluorescent real-time PCR. Two SOD2 gene-specific primers SOD-F (50 ATAGCACGGCAGCATGGA 30 ) and SOD-R (50 GGTCTGGTGGTGTTTTG G 30 ) were used to amplify a product of 147 bp. A constitutively expressed gene, GAPDH, was used as an internal control. Two GAPDH primers GF (50 CCAGAACATCA TCCCAGCA 30 ) and GR (50 ACGAACAGGG ACACGGAAG 30 ) were used to amplify a 104 bp fragment. PCR-grade water was used for the negative control. The fluorescent real-time PCR assay was carried out in RotorGene 6000 System (Applied Corbett Research). The amplifications were performed in a 20 ml reaction containing 10 ml of 2SYBR Premix Ex Taq II (Takala), 0.5 ml each of the forward and reverse primers (10 mM), 5 ml of 1:10 diluted cDNA, and 4 ml of PCR-grade water. The thermal profile for real-time PCR was 95 C for 10 s followed by 40 cycles of 10 s at 95 C , 15 s at 54 C and 15 s at 72 C. Fluorescence readings were taken at 72 C after each cycle. Gene of Interest and Housekeeper Gene was analyzed using a method of Comparative Delta-delta Ct. Relative fluorescence unit, calculated threshold cycle (CT), and dissociation curve were monitored by the analysis software of the system (Corbett Research). Triplicate assays per cDNA sample were carried out independently to determine the average CT values. 3. Results 3.1. Cloning, sequencing and phylogenetic analysis of cDNA The cDNA sequence of the P. esculenta Mn-SOD gene was deposited in GenBank (GenBank accession no.GQ487656). The fulllength cDNA of Mn-SOD was of 1321 bp, including a 47 bp 50 UTR, a 593 bp 30 UTR with a putative polyadenylation signal was AATAAA and a 18 bp poly(A) tail, and a 681 bp ORF encoding a polypeptide of 226 amino acids. The full-length nucleotide sequence and the deduced amino acid sequence are shown in Fig. 1. The predicted Mn-SOD proteins have four conserved residues coordinating manganese (His-56, His-100, Asp-184 and His-188); alignment positions in Fig. 1. The calculated molecular mass is 25.2 kDa, and the pI was estimated at 5.96. The protein shares the highest sequence identity of 70% with homologues from Caenorhabditis elegans and Gallus gallus respectively. Characteristic Mn-SOD signatures (DVWEHAYY) from 184 to 191 are also highly conserved. Multiple sequence alignment showed that the deduced amino acid sequence of P. esculenta Mn-SOD shares high similarity with other Mn-SOD amino acid sequences from Macrobrachium rosenbergii, Fenneropenaeus chinensis, C. elegans, G. gallus, Mus musculus and Pongo abelii (Fig. 2). Specifically, Mn-SOD of P. esculenta displays 70% similarity with homologues from C. elegans and G. gallus, and 68% similarity with homologues from M. rosenbergii and F. chinensis.
M. Wang et al. / Fish & Shellfish Immunology 29 (2010) 759e764
Fig. 1. Gene sequence of Mn-SOD from P. esculenta and its amino acid sequence. Note, signal.
3.2. Expression of Mn-SOD proteins and Western blotting analysis Protein expression was induced by IPTG at different concentrations followed by incubation for different times. When induced by 1 mM IPTG and cultured at 37 C for 4 h, recombinant SOD proteins accumulated to very high levels and formed inclusion bodies (Fig. 3A). On the other hand, when induced by 0.2 mM IPTG and cultured at 25 C for 18 h, the proteins were expressed both as inclusion bodies and soluble proteins. Likewise, purified Mn-SOD proteins were also resolved by SDS-PAGE and Coomassie staining revealed a single band of 29 kDa (Fig. 3B). The polyclonal antibody generated from the immunized mice was used as the primary antibody for Western blot analysis. The protein samples were obtained from E. coil BL21 with pET-28a induced by 1 mM IPTG for 4 h, uninduced E. coil BL21 with pET-SOD, E. coil BL21 with pET-SOD induced by 1 mM IPTG for 4 h (inclusion bodies), E. coil BL21 with pET-SOD induced by 0.2 mM IPTG at 25 C for 18 h (soluble protein) and whole cell lysate of P. esculenta. The
: start codon;
: manganese-binding residues;
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: Mn-SOD motif signature;
: tailing
results showed that a specific band appeared in the samples of purified recombinant protein of Mn-SOD (29 kDa) and the whole cell lysate of P. esculenta (25 kDa). In contrast, no specific band was detected in the uninduced BL-21 harboring pET-HSP or induced BL-21 with the empty vector pET-28a (Fig. 4). Furthermore, the same experiment was conducted using the serum from PBSimmunized mice as the primary antibody, and this control serum did not react with the protein and produce any band. These results suggested that the polyclonal antibody specifically recognized the Mn-SOD protein.
3.3. Mn-SOD mRNA expression in response to heavy metal exposure During metal treatments, significant mortalities were found in the groups treated with 10 mM of copper (Cu): 10% of individuals were dead within the first 48 h. There was no loss in the zinc (Zn)exposed, cadmium (Cd)-exposed and unexposed groups.
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Fig. 2. Alignment of P. esculenta Mn-SOD polypeptide sequence along with other orthologues.
Mn-SOD mRNA levels were substantially affected by the heavy metal exposures (Fig. 5). Significant differences in the Mn-SOD expression level were observed at 6 h, 12 h, 24 h and 48 h after heavy metal exposures compared with the control group (P < 0.05). Specifically, Mn-SOD mRNA level peaked at 24 h following Zn treatment (P < 0.05), 13.4-fold higher than that observed in the control group, then gradually declined to approximately the original level at 48 h. After Cd treatment, Mn-SOD mRNA level peaked at 24 h (P < 0.05), 6.4-fold higher than the control group. After Cu treatment, Mn-SOD mRNA expression was reduced and reached the lowest level at 48 h.
thermal stress on Mn-SOD mRNA expression is shown in Fig. 6. GAPDH expression showed constant levels regardless of the water temperature, whereas Mn-SOD expression was highly sensitive to both high and low water temperatures, with low water temperatures more dominant than high temperatures (P < 0.05). For example, fold increases in the 37 C group relative to the 20 C group ranged from 1.3 to 3.0 between 6 h to 48 h, with the maximum Mn-SOD mRNA levels observed at 12 h. On the other hand, fold increases in the 4 C group relative to the 20 C group ranged from 1.9 to 13.5 from 6 h to 48 h. Mn-SOD mRNA level peaked at 24 h at 4 C, then declined gradually.
3.4. Mn-SOD mRNA expression in response to temperature stress
4. Discussion
During temperature treatments, there was no loss in the 37 C -exposed, 4 C -exposed and unexposed groups. The effect of
SOD enzymes play critical roles in the antioxidant enzyme defense against ROS and superoxide anion radicals. The SOD gene
Fig. 3. Expression and purification of SOD recombinant protein. Note, (A) Lane 1: low molecular marker; lane 2: total protein of non-induced pET-28a; lane 3: total protein of pET28a induced by IPTG for 5 h; lane 4: total protein of non-induced pET-SOD; lane 5e8: total protein of pET-SOD induced by IPTG for 1 h, 2 h, 3 h, 4 h. (B) Lane 1: molecular weight marker; lane 2: purified recombinant protein of Mn-SOD.
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Fig. 4. Specificity of pET-SOD polyclonal antibody was determined by Western-blot. Note, Lane 1: low molecular marker, lane 2: IPTG induced E. coil BL21 with pET-28a for 4 h, lane 3: uninduced E. coil BL21 with pET-SOD, lane 4: IPTG induced E. coil BL21 with pET-SOD for 4 h, lane 5: soluble protein , lane 6: total protein of blood from P. esculenta
family is generally classified into Cu/Zn-SODs, Mn-SODs and FeSODs depending on the subcellular localization and the bound metal ion [16]. Mitochondria generate the majority of superoxide radicals, which are subsequently dismutated by Mn-SODs to produce a constant flux of H2O2. This process is known to play a vital role in controlling the redox state of the cell [17e19]. In this paper, the complete cDNA sequence of a Mn-SOD gene from P. esculenta was reported. Sequence analysis revealed conserved sequence motifs, such as the Mn-SOD signature DVWEHAYY and four conserved residues coordinating manganese interaction (His-56, His-100, Asp-184 and His-188) [14]. Homology analysis revealed phylogenetic relationships of P. esculenta with other species. The results showed that P.esculentas is most closely related to C. elegans, and to a lesser extent, to M. musculus and P. abelii. SDS-PAGE analysis showed that the Mn-SOD recombinant protein was approximately 29 kDa, and the purified Mn-SOD proteins from P. esculenta were approximately 25 kDa. Protein expression appeared to be elevated significantly with the lengthened induction time. Depending on IPTG concentration and incubation time, the proteins were partitioned into inclusion bodies or soluble protein fractions. Western blot analysis showed that the polyclonal antibody generated in mice reacted with purified Mn-SOD recombinant proteins and detected specific signals from P. esculenta whole cell lysates, but did not react with the total proteins of E. coli. These results demonstrate the specificity of this antibody for Mn-SOD from P. esculenta.
Fig. 6. Quantitative analysis of Mn-SOD mRNA expression of P. esculenta exposed to different temperature by fluorescent real-time PCR. Note, Means with the same letters on each histogram within a detection time (4 C, 37 C) are not statistically different at P < 0.05. Standard deviation is noted by T-bar on each histogram.
Mn-SODs have been considered as general stress responsive factors whose expression might be influenced by a variety of intracellular and environmental cues at transcriptional and/or translational levels [20,21] . Previous studies have shown that heavy metal exposures and thermal stress induced expression of stress proteins [9,14,22,23]. In the present study, experimental exposure of P. esculenta either heavy metals, or low and high temperatures significantly induced the mRNA expression of Mn-SOD, albeit to different degrees. Mn-SOD expression was increasingly responsive to the treatment of Cu2þ, Cd2þ and Zn2þ, with as much 13.4-fold as control group from Zn2þ exposure. At 24 h when Mn-SOD mRNA level peaked, Zn2þ and Cd2þ treatments elicited 6- and 3-fold higher expression than that from Cu2þ, respectively. In contrast, samples treated with Cu2þ displayed a modest 2-fold increase in Mn-SOD expression at 6 h followed by a gradual decline to about 1/3 of the original level at 48 h. It is plausible that P. esculentas was more sensitive to Cu2þ; after Cu2þ treatment for 24 h, P. esculenta appeared maladjusted, and the mouth began to canker. In the present study using relatively high doses of metals (10 mM), increases in SOD expression were detectable as early as 6 h after exposure, with peak levels observed within 1 day in many instances. Mn-SOD expression appeared more sensitive to 4 C treatment than 37 C treatment, and the maximal induction from 4 C treatment was roughly 4-fold higher than that from 37 C treatment. P. esculenta live in beaches where the environmental temperature fluctuates greatly, with a tolerable temperature range of 17 C to 33 C [11]. 37 C is relatively close to the upper physiological limit, perhaps explaining why it is less effective in the induction of Mn-SOD expression than 4 C. Taken together, the increased Mn-SOD expression after heavy metal and temperature stresses suggests a role of Mn-SOD in immunopathological processes in P. esculenta.
Acknowledgements
Fig. 5. Quantitative analysis of Mn-SOD mRNA expression of P. esculenta exposed to heavy metals by fluorescent real-time PCR. Note, Means with the same letters on each histogram within a detection time (6, 12, 24 or 48 h) are not statistically different at P < 0.05. Standard deviation is noted by T-bar on each histogram.
This work was financially supported by National Natural Science Foundation (No. 40776075), and Scientific Program of Zhejiang Province (No. 2006C13089), and Project supported by the Ningbo Science Bureau, China (Grant No. 2008C50027), and Sponsored by K.C.Wong Magna Fund at Ningbo University.
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