- Email: [email protected]
Identification and analysis of HSP70 from Sepiella maindroni under stress of Vibrio harveyi and Cd2 +
GENE-40715; No. of pages: 7; 4C: Gene xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short communication
Identification and analysis of HSP70 from Sepiella maindroni under stress of Vibrio harveyi and Cd2 + Hui-hui Liu ⁎, Jian-yu He, Chang-feng Chi, Zhen-ming Lv 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 20 April 2015 Received in revised form 14 July 2015 Accepted 15 July 2015 Available online xxxx Keywords: Sepiella maindroni Heat shock protein 70 (HSP70) Vibrio infection Cd2 + stress
a b s t r a c t The 70-kDa heat shock proteins (HSP70) play crucial roles in protecting cells against environmental stresses, such as heat shock, heavy metals and pathogenic bacteria. The full-length HSP70 cDNA of Sepiella maindroni (designated as SmHSP70, GenBank accession no. KJ739788) was 2109 bp, including an ORF of 1950 bp encoding a polypeptide of 649 amino acids with predicted pI/MW 5.24/71.30 kDa, a 62 bp-5′-UTR and a 97 bp-3′-UTR. BLASTp analysis and phylogenetic relationship strongly suggested that the amino acid sequence was a member of HSP70 family. Multiple sequence alignment revealed that SmHSP70 and other known HSP70 were highly conserved, especially in the regions of HSP70 family signatures, the bipartite nuclear targeting sequence, ATP/GTPbinding site motif and ‘EEVD’ motif. Time-dependent mRNA expression of SmHSP70 in the liver was recorded by quantitative real-time RT-PCR after Vibrio harveyi injection and Cd2+ exposure. The results indicated that SmHSP70 played a significant role in mediating the environmental stress and immune response against pathogens. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The organisms are continually challenged by environmental conditions, which cause acute and chronic stresses. To adapt to the changes and survive from different types of injuries, eukaryotic cells have evolved networks of different responses to detect and control diverse forms of stresses (Santoro, 2000). Heat shock proteins (HSPs) are ubiquitous and highly conserved stress proteins occurring in all organisms from bacteria to humans, they have strong cytoprotective effects, and behave as molecular chaperones for other cellular proteins (Joly et al., 2010; Mjahed et al., 2012). According to their apparent molecular mass, HSPs have been classified into several families: HSP90 (85–90 kDa), HSP70 (68–73 kDa), HSP60, HSP47 and low molecular mass HSPs (16–24 kDa). Heat shock protein 70 (HSP70) is an important member of the heat shock protein superfamily and appears in almost all species except for some archaea (Tavaria et al., 1996; Krenek et al., 2013). As a kind of highly conserved protein HSP70 is associated with intracellular chaperone and extracellular immunoregulatory functions to protect cells against environmental stress, and shows tissue-/time-/ dose-dependent changes (Liu et al., 2014). Various stresses stimulate
Abbreviations: HSP70, heat shock protein 70; HSPs, heat shock proteins; SmHSP70, heat shock protein 70 of Sepiella maindroni; CST, critical solution temperature; RACE, rapid amplification of cDNA ends; ORF, open reading frame; UTRs, untranslated regions. ⁎ Corresponding author. E-mail address: [email protected] (H. Liu).
the synthesis of HSP70, which presumably acts to restore protein structure and function (Yenari et al., 1999). In recent years, marine pollution has become more and more serious than before, marine organisms have been suffering from increasing environmental stress via exposure to a plenty variety of pollutants, and even result in mutations or the death of intact organisms and their progenies. Moreover, a large portion of these contaminants can be transferred through the food chain, making them a potential threat to entire ecosystems and even human beings (Wan et al., 2008). Therefore, studies on the resistant molecules that exist in marine organisms and related applications in the assessment of environmental health are necessary (Silvia and Elena, 2005), such as HSP70s, HSP90s, superoxide dismutases (SODs), catalases (CATs), Glutathione S-Transferases (GSTs), and glutathione peroxidase (GSH-PX). As the important molecular for protein folding, multimer dissociation and association, translocation of proteins across membranes and regulation of the heat shock response, several HSP70s have been described in molluscs, including Crassostrea gigas (Isabelle et al., 2003), Ostrea edulis (Piano et al., 2005), Mytilus edulis (Luedeking and Koehler, 2004), Chlamys farreri (Wu et al., 2003), and Argopecten irradians (Song et al., 2006). Also, the studies examining the effects of environmental stressors on HSP70 gene expression have been reported in different species of molluscs and recognized the relevant physiological and ecological importance of heat shock gene expression in response of changing environments (Hamdoun et al., 2003; Piano et al., 2005; Buckley et al., 2001). However, no information is available on HSP70 expression of
http://dx.doi.org/10.1016/j.gene.2015.07.056 0378-1119/© 2015 Elsevier B.V. All rights reserved.
Please cite this article as: Liu, H., et al., Identification and analysis of HSP70 from Sepiella maindroni under stress of Vibrio harveyi and Cd2 +, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.07.056
2
H. Liu et al. / Gene xxx (2015) xxx–xxx
cephalopods at transcriptional level under aquatic pathogenic bacterial infection and environmental stress. Sepiella maindroni is an important economic cephalopod in the East Sea of China. In view of few related reports of this squid, S. maindroni was regarded as our experimental animals, and HSP70 was the candidate gene for its intracellular chaperone and extracellular immunoregulatory functions in aquatic invertebrates. The full-length HSP70 cDNA sequence was cloned from S. maindroni (designated as SmHSP70), and the mRNA expression profiles were analyzed emphatically in the liver after Vibrio harveyi infection and cadmium stress based on SYBR Green quantitative RT-PCR analysis. All of the results will contribute to better understanding of HSP70 diversity in mollusca and the biochemical resistance mechanisms used by marine consumers to cope with their allelochemically defended prey. 2. Material and methods
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 5′-RACE (5P1 and 5P2) and 3′-RACE (3P1 and 3P2, Table 1), were designed based on the known partial sequence. Full-length cDNA sequence of HSP70 was performed with the specific primers (5P, 3P as shown in Table 1) and the primers in the Smart RACE cDNA amplification kit (Clontech, USA). Both 5′-RACE and 3′-RACE were carried out according to the manufacturer's instructions. The PCR products were cloned into the PMD18-T simple vector (TaKaRa, China) and sequenced from both directions. The full-length cDNA was obtained by overlapping the forward and reverse strand sequences. The resulting sequence was verified by the amplification of the whole full length and further subjected to cluster analysis. 2.3. Sequence analysis
2.1. Experimental animals The juvenile cuttlefishes (S. maindroni, 2.0–4.0 cm in length) were collected from the Dongji aquaculture 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 2014. Seawater was changed daily. Animals were fed with microalgae during the acclimation and experimental period. No mortality was observed in either the experimental or control groups. Total RNA was isolated from the liver with Trizol reagent (TaKaRa, China) and the ratio of A 260/A280 was determined. The cDNA synthesis was carried out with M-MLV RTase cDNA Synthesis Kit (TaKaRa, China).
The cDNA sequence was spliced by the software of DNAstar v7.0. The amino acid sequence of SmHSP70 was deduced by the Expert Protein Analysis System (http://www.expasy.org/) and the homology search was conducted with BLASTp program of NCBI (http://www. ncbi.nlm.gov/BLAST/). The conserved domains were predicted using SMART (http://smart.embl-heidelberg.de/) online tool. 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 MEGA v4.0 software.
2.2. cDNA of S. maindroni HSP70 identification and full-length amplification
2.4. SmHSP70 mRNA expression in liver after V. harveyi challenge
The primers HSP70-F/HSP70-R (shown in Table 1) were designed according to the sequence of Pteria penguin (ABJ97377), Cyclina sinensis (AET13646), Perna viridis (ABJ98722), A. irradians (AAS17723), Azumapecten farreri (AAO38780), Mytilus coruscus (AGY56119), and Meretrix meretrix (ADT78478). The total RNA from the liver of S. maindroni was used as template after reverse transcription and the ratio of A260/A280 was 1.90. The reaction system was performed in 20 μL volume, including 2 μL 10 × PCR Buffer, 2 μL of MgCl 2 (25 mmol/L), dNTPs 0.4 μL (2.5 mmol/L), HSP70-F 0.8 μL (10 mol/L), HSP70-R 0.8 μL (10 μmol/L), template cDNA 0.6 μL, Taq DNA polymerase (TaKaRa, China) 0.4 μL (1 U) and 13.0 μL of PCR-Grade water. 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 56.5 °C, and 60 s at 72 °C, with a final extension
The liver was selected as a candidate tissue for investigating the temporal expression profile of SmHSP70 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 6000 ×g, 4 °C for 10 min to collect bacteria, and the resuspended live V. harveyi in 100 μL of PBS (pH 7.4, OD 600 = 0.4) were injected into muscles of S. maindroni. The cuttlefishes, which were injected with 100 μL PBS, acted as the control group. Total 35 infected cuttlefishes were cultured in 10 L filtered fresh well-aerated seawater, the livers 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 SmHSP70 gene at mRNA level in livers after V. harveyi challenge, real-time PCR (qRTPCR) was performed with SYBR PrimeScript™ RT reagent Kit (Perfect Real Time, TaKaRa, China) as recommended by the manufacturer's instructions in the 7500 Real Time PCR System (Applied Biosystems, UK). For each time point in the challenge experiment 5 biological replicate was used. The reaction mixture of 20 μL contained qSmHSP70-F 0.8 μL (10 μmol/L), qSmHSP70-R 0.8 μL (10 μmol/L), 2× SYBR® Premix Ex TaqTM II (TaKaRa, China) 10 μL, cDNA sample 0.8 μL, ROX II 0.4 μL, and ddH2O 7.2 μL. The standard cycling conditions were: 95 °C for 1 min (initial polymerase activation), followed by 40 cycles of 10 s at 95 °C and 45 s at 59.6 °C. The PCR specificity was checked with dissociation curve analysis from 55 to 95 °C, β-actin of S. maindroni (βSm actin-F and β-Sm actin-R) was used as the internal standard, and all the primers were shown in Table 1. The 2−ΔΔCT 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 significant at P b 0.05.
Table 1 PCR primer sequences for SOD cloning of S. maindroni. Degenerate primers were as follows, S: C/G; M: A/C; Y: C/T; W: A/T; R: A/G; and K: G/T. Primer For HSP70 cDNA clone HSP70-F HSP70-R For 5′-/3′-RACE Adaptor primer 5P1 5P2 3P1 3P2 For qRT-PCR qSmHSP70-F qSmHSP70-R β-Smactin-F β-Smactin-R
Sequences 5′-GTTATGACCTCGTTGATTAAGAG-3′ 5′-TTKSWSARRCGACCTTTGTC-3′ 5′-TCATTGCTCTTTCTCC-3′ 5′-CCAGGTTGGTTATCGGAGTATG-3′ 5′-AGGTCTGTGTTTGTTTTGTTGG-3′ 5′-CTTCACAACATACTCCGATAACCAACCT-3′ 5′-GAGCACTGGTAAAGAGAACAAGATCACC-3′ 5′-ACTCCGATAACCAACCTG-3′ 5′-GAGGCGACCTTTGTCATTT-3′ 5′-GCCAGTTGCTCGTTACAG-3′ 5′-GCCAACAATAGATGGGAAT-3′
Please cite this article as: Liu, H., et al., Identification and analysis of HSP70 from Sepiella maindroni under stress of Vibrio harveyi and Cd2 +, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.07.056
H. Liu et al. / Gene xxx (2015) xxx–xxx
2.5. SmHSP70 mRNA expression in liver after Cd2+ treatments For Cd treatments, mussels were maintained in static seawater in aquacultural tank with salinity of 28–30‰ at 23–25 °C. Seawater was changed daily and resupplied with the corresponding concentration of Cd2 +. Animals were exposed to cadmium chloride (CdCl2·5H2O) added to the 10 L filtered seawater with dissolved Cd2+ concentration of 200 μg/L based on the preliminary experiment of gradient concentration and 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+. Five individuals were taken for each treatment at 24, 48, 72, 120, 168 and 264 h post-treated respectively. The methods
3
of liver collection, total RNA extraction, cDNA synthesis and RT-PCR analysis were performed as described above. 3. Results and discussion 3.1. cDNA sequence analysis and characterization of SmHSP70 full-length HSP70s are responsible for the folding or assembly of native proteins and extracellular immunoregulatory functions (Reina et al., 2012). The protective mechanism of HSP70 is the induction of stress response proteins as molecular chaperones involved in heat, heavy metals, desiccation, diseases and parasites (Tukaj et al., 2011). Up to now HSP70 gene had been studied in several marine mollusca, such as scallop C. farreri
Fig. 1. The full-length cDNA and deduced amino acid sequence of HSP70 from S. maindroni. The stop codon (TAA) was indicated with an asterisk (*); the cDNA sequences had been submitted to GenBank with accession no. KJ739788. Major TRAF2-binding consensus motifs were in orange box. Caspase3 and Caspase7 cleavage site was in blue box. Tyrosine-based sorting signal was in black box. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Liu, H., et al., Identification and analysis of HSP70 from Sepiella maindroni under stress of Vibrio harveyi and Cd2 +, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.07.056
4
H. Liu et al. / Gene xxx (2015) xxx–xxx
(Wu et al., 2003), bay scallop A. irradians (Reina et al., 2012), Mytilus galloprovincialis (Anestis et al., 2007), and M. coruscus (Liu et al., 2014). In this study the complete cDNA sequence of HSP70 from S. maindroni was identified by homology cloning (GenBank accession no. KJ739788), which was 2109 bp in length, containing an ORF of 1950 bp encoding 649 amino acid residues. The 5′-UTR was 62 bp and the 3′-UTR was 97 bp (Fig. 1). The theoretical MW of SmHSP70 was 71.30 kDa and its estimated pI was 5.24. Generally, the molecular weight (MW) of HSP70 family was range from 68 to 73 kDa (Song et al., 2006). BLASTp program demonstrated that the obtained HSP70 sequence shared high homologies with known HSP70 in mollusca. Therefore it was concluded that the sequence cloned from S. maindroni belonged to HSP70 family. 3.2. Multiple sequence alignments and phylogenetic analysis The deduced amino acid sequence of SmHSP70 was aligned with the known HSP70 from other mollusca (as shown in Fig. 2), showing their high homology, especially in N terminal and C terminal. The molecular
chaperones in HSP70 are the large and diverse family of allosteric two-domain proteins: the NH2-terminal ATP-binding domain (ABD) and the COOH-terminal peptide-binding domain (PBD) (Smock et al., 2010). The conserved sequences and characteristic motifs of HSP70 family also appeared in the SmHSP70, including three HSP70 family signatures, and the bipartite nuclear targeting sequence and ATP/GTPbinding site motif A. The presence of five consecutive repeats of the tetrapeptide motif GGMP in C-terminal region of SmHSP70 was another notable feature in this protein. The last four amino acids formed a motif ‘EEVD’ at the C-terminus, which is highly conserved throughout all species (Freeman et al., 1995). The function of the GGMP repeats had not been revealed, it might form a structural entity together with the helical subdomain, and the EEVD motif might mediate chaperone cofactor binding (Demand et al., 1998). To reveal the molecular phylogenetic position of SmHSP70, an unrooted phylogenetic tree was constructed with Neighbor-Joining method from a distance matrix (Fig. 3). The phylogenetic tree was composed of five major groups: molluscs, shrimp, fish, chicken, and mammalians. S. maindroni and other molluscs formed a sister group, and
Fig. 2. Multiple alignment of the deduced amino acid sequences of HSP70 from S. maindroni and other organisms. The GenBank accession no. for the sequences were as follows: Chlamys farreri (ABD58974), Mizuhopecten yessoensis (AY485262), Argopecten irradians (ACM48346), Argopecten purpuratus (ACO72585), Mytilus coruscus (KF322135), Mytilus galloprovincialis (AY861684), Pinctada fucata (ABJ97378) and Pteria penguin (EF011060).
Please cite this article as: Liu, H., et al., Identification and analysis of HSP70 from Sepiella maindroni under stress of Vibrio harveyi and Cd2 +, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.07.056
H. Liu et al. / Gene xxx (2015) xxx–xxx
5
Fig. 3. Phylogenetic tree depicting the relationship of SmHSP70 with other species. All protein sequences obtained from GenBank of the NCBI, GenBank accession number was in parentheses, and phylogenetic tree was constructed using Neighbor-Joining rule method by the software of MEGA v4.0.
then clustered to shrimps, fish, chicken and finally mammalians. The relationship was generally in agreement with the traditional taxonomy and the occurrence of HSP70 gene duplication events during evolution. Therefore, the sequence alignment, structural comparison and phylogenetic analysis revealed that SmHSP70 was a member of the HSP70 family.
The rapid induction of SmHSP70 after V. harveyi injection was in agreement with its classification as an inducible gene product, which provided an effective tool to cope with rapidly changing environmental conditions for S. maindroni. The result was corroborated by the occurrence of analogous mechanisms in other molluscan species (Encomio and Chu, 2004) and some aquatic vertebrates (Wang et al., 2009).
3.3. SmHSP70 mRNA expression in liver after V. harveyi challenge
3.4. SmHSP70 mRNA expression in liver after Cd2+ stress
V. harveyi is a gram-negative bacterium 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). The HSP70 family, one of the most abundant of various HSPs, has been confirmed maintaining cellular homeostasis and protecting organism from pathogenic stress (Encomio and Chu, 2004). In the present study, the transcriptional expression of SmHSP70 was thus investigated after challenge by V. harveyi in the liver. Not surprisingly, the transcripts showed a time dependent expression pattern, which was significantly induced to the highest level at 4 h after post-injection with 20.44-fold higher than that of the control, then decreased slowly and returned to a slightly higher than physiological level at 72 h (Fig. 4A). The result was in accordance with previous studies in other molluscs (Wang et al., 2009), and suggested that SmHSP70 was involved in the squid's response to pathogenic infection and might play an important role in the immune response. Moreover, HSP70 genes are typically intron-less (Encomio and Chu, 2004), and the absence of introns causes their rapid induction to avoid posttranscriptional editing of the related mRNA and be effectively transcribed when splicing processes may be inhibited by stressful conditions.
At present marine waters and sediments receive anthropogenic inputs, containing a multitude of chemical contaminants that are potentially toxic to aquatic organisms, and could induce a series of HSPs to protect various living organisms (Ovelgonne et al., 1995; Andrew et al., 2003). Cadmium is the major toxic metal as contaminants in marine environment to be 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). The expression of HSP70 is frequently used as a component of physiological mechanisms through which mollusca cope with environmental challenges. In Cd2+ exposure, the similar dose- and time-dependent patterns appeared in SmHSP70 mRNA expression, which sustainably increased and reached the peak approximately 159.02-fold higher than the control at 120 h (Fig. 4B), then dropped gradually to 62.15-fold at 168 h post-exposure, but as time went on appeared another peak of 92.53-fold at 264 h. The results indicated that SmHSP70 took part in the cellular immune process and undertook the important functions in protecting organisms from Cd2+ stress. Cd2+ had been also verified to induce the other HSP expression in mammalian and nonmammalian cells, such as HSP90 in C. farreri, which was more sensitive to the concentration variation of
Please cite this article as: Liu, H., et al., Identification and analysis of HSP70 from Sepiella maindroni under stress of Vibrio harveyi and Cd2 +, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.07.056
6
H. Liu et al. / Gene xxx (2015) xxx–xxx
Fig. 4. Expression profiles of SmHSP70 after stress with Vibrio harveyi and Cd2+: (A) Temporal expression of the SmHSP70 transcripts in liver after Vibrio harveyi infection; (B) expression analysis of SmHSP70 in the liver 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 b 0.05, “**” at P b 0.01.
Cd2+ (Gao et al., 2007). Therefore, the results provided us an opportunity to speculate that HSP70 gene in the liver of SmHSP70 might be a potential biomarker of heavy metals. In summary, this was the first report of full-length cloning, characterization and inducible expression of HSP70 in the liver of cephalopod S. maindroni. The analysis of phylogenetic and structural features might contribute to the understanding of adaptation and evolutionary processes of HSPs in invertebrate. The up-regulated mRNA expression of SmHSP70 in response to V. harveyi and Cd2+ indicated that SmHSP70 was inducible and involved in the immune response, and it might be the potential biomarker for marine pollution. Acknowledgments This research was supported by grants from the Natural Science Foundation of Zhejiang Province, China (LY14C190004, LY13C190001),
the Zhejiang Marine Fisheries Bureau project (2012)83, and the Ministry of Science and Technology support program (2012BAB16B02).
References Andrew, A.S., Warren, A.J., Barchowsky, A., et al., 2003. Genomic and proteomic profiling of responses to toxic metals in human lung cells. Environ. Health Perspect. 111 (6), 825–835. Anestis, A., Lazou, A., Pörtner, H.O., et al., 2007. Behavioral, metabolic, and molecular stress responses of marine bivalve Mytilus galloprovincialis during long-term acclimation at increasing ambient temperature. Am. J. Physiol. Regul. Integr. Comp. Physiol. 293 (2), R911–R921. Buckley, B.A., Owen, M., Hofmann, G.E., 2001. Adjusting the thermostat: the threshold induction temperature for the heat-shock response in intertidal mussels (genus Mytilus) changes as a function of thermal history. J. Exp. Biol. 204, 3571–3579. Demand, J., Luders, J., Hohfeld, J., 1998. The carboxy-terminal domain of Hsc70 provides binding sites for a distinct set of chaperone cofactors. Mol. Cel. Biol. 18 (4), 2023–2028.
Please cite this article as: Liu, H., et al., Identification and analysis of HSP70 from Sepiella maindroni under stress of Vibrio harveyi and Cd2 +, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.07.056
H. Liu et al. / Gene xxx (2015) xxx–xxx Encomio, V.G., Chu, F.L., 2004. Characterization of heat shock protein expression and induced thermotolerance in P. marinus parasitized eastern oysters: lab and field studies. J. Shellfish Res. 23, 289–290. Freeman, B.C., Myers, M.P., Schumacher, R., et al., 1995. Identification of a regulatory motif in Hsp70 that affects ATPase activity, substrate binding and interaction with Hdj-1. EMBO J. 14 (10), 2281–2292. Gao, Q., Song, L.S., Ni, D., Wu, L., et al., 2007. cDNA cloning and mRNA expression of heat shock protein 90 gene in the haemocytes of Zhikong scallop Chlamys farreri. Comp. Biochem. Physiol. B 147 (4), 704–715. Gómez, L.J., Villamil, L., Lemos, M.L., Novoa, B., Fiqueras, A., 2005. Isolation of Vibrio alginolyticus and Vibrio splendidus from aquacultured carpet shell clam (Ruditapes decussatus) larvae associated with mass mortalities. Appl. Environ. Microbiol. 71, 98–104. Hamdoun, A.M., Cheney, D.P., Cherr, G.N., 2003. Phenotypic plasticity of HSP70 and HSP70 gene expression in the pacific oyster (Crassostrea gigas): implications for thermal limits and induction of thermal tolerance. Biol. Bull. 205, 160–169. Isabelle, B., Arnaud, T., Sabrina, R., Michel, A., Dario, M., 2003. Molecular identification and expression of heat shock cognate 70 (hsc70) and heat shock protein 70 (hsp70) genes in the Pacific oyster Crassostrea gigas. Cell Stress Chaperones 8, 76–85. Joly, A.L., Wettstein, G., Mignot, G., et al., 2010. Dual role of heat shock proteins as regulators of apoptosis and innate immunity. J. Innate Immun. 2 (3), 238–247. Krenek, S., Schlegel, M., Berendonk, T.U., 2013. Convergent evolution of heat-inducibility during subfunctionalization of the Hsp70 gene family. BMC Evol. Biol. 13, 49. Liu, H.H., He, J.Y., Chi, C.F., Shao, J.Z., 2014. Differential HSP70 expression in Mytilus coruscus under various stressors. Gene 543, 166–173. Luedeking, A., Koehler, A., 2004. Regulation of expression of multixenobiotic resistance (MXR) genes by environmental factors in the blue mussel Mytilus edulis. Aquat. Toxicol. 69, 1–10. Mjahed, H., Girodon, F., Fontenay, M., et al., 2012. Heat shock proteins in hematopoietic malignancies. Exp. Cell Res. 318 (15), 1946–1958. Ovelgonne, J.H., Souren, J.E., Wiegant, F.A., et al., 1995. Relationship between cadmiuminduced expression of heatshock genes, inhibition of protein synthesis and cell death. Toxicology 99 (1), 19–30.
7
Piano, A., Franzellitti, S., Tinti, F., Fabbri, E., 2005. Sequencing and expression pattern of inducible heat shock gene products in the European flat oyster, Ostrea edulis. Gene 361, 119–126. Reina, C.P., Nabet, B.Y., Young, P.D., et al., 2012. Basal and stress-induced Hsp70 are modulated by ataxin-3. Cell Stress Chaperones 17 (6), 729–742. Santoro, M.G., 2000. Heat shock factors and the control of the stress response. Biochem. Pharmacol. 59 (1), 55–63. Sarkar, A., Ray, D., Shrivastava, A.N., Sarker, S., 2006. Molecular Biomarkers: their significance and application in marine pollution monitoring. Ecotoxicology 15, 333–340. Silvia, F., Elena, F., 2005. Differential HSP70 gene expression in the Mediterranean mussel exposed to various stressors. Biochem. Biophys. Res. Commun. 336, 1157–1163. Smock, R.G., Rivoire, O., Russ, W.P., et al., 2010. An interdomain sector mediating allostery in Hsp70 molecular chaperones. Mol. Syst. Biol. 6, 414. Song, L., Wu, L., Ni, D., Chang, Y., et al., 2006. The cDNA cloning and mRNA expression of heat shock protein 70 gene in the haemocytes of bay scallop (Argopecten irradians, Lamarck 1819) responding to bacteria challenge and naphthalin stress. Fish Shellfish Immunol. 21 (4), 335–345. Tavaria, M., Gabriele, T., Kola, I., et al., 1996. A hitchhiker's guide to the human Hsp70 family. Cell Stress Chaperones 1 (1), 23–28. Tukaj, S., Bisewska, J., Roeske, K., Tukaj, Z., 2011. Time- and dose-dependent induction of HSP70 in Lemna minor exposed to different environmental stressors. Bull. Environ. Contam. Toxicol. 87 (3), 226–230. Wan, Q., Whang, I., Lee, J., 2008. Molecular cloning and characterization of three sigma glutathione S-transferases from disk abalone (Haliotis discus discus). Comp. Biochem. Physiol. B 151, 257–267. Wang, Z., Wu, Z., Jian, J., Lu, Y., 2009. Cloning and expression of heat shock protein 70 gene in the haemocytes of pearl oyster (Pinctada fucata, Gould 1850) responding to bacterial challenge. Fish Shellfish Immunol. 26 (4), 639–645. Wu, L., Song, L., Xu, W., et al., 2003. Identification and cloning of heat shock protein 70 gene from scallop Chlamys farreri. High Technol. Lett. 13, 75–79. Yenari, M.A., Giffard, R.G., Sapolsky, R.M., Steinberg, G.K., 1999. The neuroprotective potential of heat shock protein 70 (HSP70). Mol. Med. Today 5, 525–531.
Please cite this article as: Liu, H., et al., Identification and analysis of HSP70 from Sepiella maindroni under stress of Vibrio harveyi and Cd2 +, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.07.056
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short communication
Identification and analysis of HSP70 from Sepiella maindroni under stress of Vibrio harveyi and Cd2 + Hui-hui Liu ⁎, Jian-yu He, Chang-feng Chi, Zhen-ming Lv 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 20 April 2015 Received in revised form 14 July 2015 Accepted 15 July 2015 Available online xxxx Keywords: Sepiella maindroni Heat shock protein 70 (HSP70) Vibrio infection Cd2 + stress
a b s t r a c t The 70-kDa heat shock proteins (HSP70) play crucial roles in protecting cells against environmental stresses, such as heat shock, heavy metals and pathogenic bacteria. The full-length HSP70 cDNA of Sepiella maindroni (designated as SmHSP70, GenBank accession no. KJ739788) was 2109 bp, including an ORF of 1950 bp encoding a polypeptide of 649 amino acids with predicted pI/MW 5.24/71.30 kDa, a 62 bp-5′-UTR and a 97 bp-3′-UTR. BLASTp analysis and phylogenetic relationship strongly suggested that the amino acid sequence was a member of HSP70 family. Multiple sequence alignment revealed that SmHSP70 and other known HSP70 were highly conserved, especially in the regions of HSP70 family signatures, the bipartite nuclear targeting sequence, ATP/GTPbinding site motif and ‘EEVD’ motif. Time-dependent mRNA expression of SmHSP70 in the liver was recorded by quantitative real-time RT-PCR after Vibrio harveyi injection and Cd2+ exposure. The results indicated that SmHSP70 played a significant role in mediating the environmental stress and immune response against pathogens. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The organisms are continually challenged by environmental conditions, which cause acute and chronic stresses. To adapt to the changes and survive from different types of injuries, eukaryotic cells have evolved networks of different responses to detect and control diverse forms of stresses (Santoro, 2000). Heat shock proteins (HSPs) are ubiquitous and highly conserved stress proteins occurring in all organisms from bacteria to humans, they have strong cytoprotective effects, and behave as molecular chaperones for other cellular proteins (Joly et al., 2010; Mjahed et al., 2012). According to their apparent molecular mass, HSPs have been classified into several families: HSP90 (85–90 kDa), HSP70 (68–73 kDa), HSP60, HSP47 and low molecular mass HSPs (16–24 kDa). Heat shock protein 70 (HSP70) is an important member of the heat shock protein superfamily and appears in almost all species except for some archaea (Tavaria et al., 1996; Krenek et al., 2013). As a kind of highly conserved protein HSP70 is associated with intracellular chaperone and extracellular immunoregulatory functions to protect cells against environmental stress, and shows tissue-/time-/ dose-dependent changes (Liu et al., 2014). Various stresses stimulate
Abbreviations: HSP70, heat shock protein 70; HSPs, heat shock proteins; SmHSP70, heat shock protein 70 of Sepiella maindroni; CST, critical solution temperature; RACE, rapid amplification of cDNA ends; ORF, open reading frame; UTRs, untranslated regions. ⁎ Corresponding author. E-mail address: [email protected] (H. Liu).
the synthesis of HSP70, which presumably acts to restore protein structure and function (Yenari et al., 1999). In recent years, marine pollution has become more and more serious than before, marine organisms have been suffering from increasing environmental stress via exposure to a plenty variety of pollutants, and even result in mutations or the death of intact organisms and their progenies. Moreover, a large portion of these contaminants can be transferred through the food chain, making them a potential threat to entire ecosystems and even human beings (Wan et al., 2008). Therefore, studies on the resistant molecules that exist in marine organisms and related applications in the assessment of environmental health are necessary (Silvia and Elena, 2005), such as HSP70s, HSP90s, superoxide dismutases (SODs), catalases (CATs), Glutathione S-Transferases (GSTs), and glutathione peroxidase (GSH-PX). As the important molecular for protein folding, multimer dissociation and association, translocation of proteins across membranes and regulation of the heat shock response, several HSP70s have been described in molluscs, including Crassostrea gigas (Isabelle et al., 2003), Ostrea edulis (Piano et al., 2005), Mytilus edulis (Luedeking and Koehler, 2004), Chlamys farreri (Wu et al., 2003), and Argopecten irradians (Song et al., 2006). Also, the studies examining the effects of environmental stressors on HSP70 gene expression have been reported in different species of molluscs and recognized the relevant physiological and ecological importance of heat shock gene expression in response of changing environments (Hamdoun et al., 2003; Piano et al., 2005; Buckley et al., 2001). However, no information is available on HSP70 expression of
http://dx.doi.org/10.1016/j.gene.2015.07.056 0378-1119/© 2015 Elsevier B.V. All rights reserved.
Please cite this article as: Liu, H., et al., Identification and analysis of HSP70 from Sepiella maindroni under stress of Vibrio harveyi and Cd2 +, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.07.056
2
H. Liu et al. / Gene xxx (2015) xxx–xxx
cephalopods at transcriptional level under aquatic pathogenic bacterial infection and environmental stress. Sepiella maindroni is an important economic cephalopod in the East Sea of China. In view of few related reports of this squid, S. maindroni was regarded as our experimental animals, and HSP70 was the candidate gene for its intracellular chaperone and extracellular immunoregulatory functions in aquatic invertebrates. The full-length HSP70 cDNA sequence was cloned from S. maindroni (designated as SmHSP70), and the mRNA expression profiles were analyzed emphatically in the liver after Vibrio harveyi infection and cadmium stress based on SYBR Green quantitative RT-PCR analysis. All of the results will contribute to better understanding of HSP70 diversity in mollusca and the biochemical resistance mechanisms used by marine consumers to cope with their allelochemically defended prey. 2. Material and methods
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 5′-RACE (5P1 and 5P2) and 3′-RACE (3P1 and 3P2, Table 1), were designed based on the known partial sequence. Full-length cDNA sequence of HSP70 was performed with the specific primers (5P, 3P as shown in Table 1) and the primers in the Smart RACE cDNA amplification kit (Clontech, USA). Both 5′-RACE and 3′-RACE were carried out according to the manufacturer's instructions. The PCR products were cloned into the PMD18-T simple vector (TaKaRa, China) and sequenced from both directions. The full-length cDNA was obtained by overlapping the forward and reverse strand sequences. The resulting sequence was verified by the amplification of the whole full length and further subjected to cluster analysis. 2.3. Sequence analysis
2.1. Experimental animals The juvenile cuttlefishes (S. maindroni, 2.0–4.0 cm in length) were collected from the Dongji aquaculture 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 2014. Seawater was changed daily. Animals were fed with microalgae during the acclimation and experimental period. No mortality was observed in either the experimental or control groups. Total RNA was isolated from the liver with Trizol reagent (TaKaRa, China) and the ratio of A 260/A280 was determined. The cDNA synthesis was carried out with M-MLV RTase cDNA Synthesis Kit (TaKaRa, China).
The cDNA sequence was spliced by the software of DNAstar v7.0. The amino acid sequence of SmHSP70 was deduced by the Expert Protein Analysis System (http://www.expasy.org/) and the homology search was conducted with BLASTp program of NCBI (http://www. ncbi.nlm.gov/BLAST/). The conserved domains were predicted using SMART (http://smart.embl-heidelberg.de/) online tool. 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 MEGA v4.0 software.
2.2. cDNA of S. maindroni HSP70 identification and full-length amplification
2.4. SmHSP70 mRNA expression in liver after V. harveyi challenge
The primers HSP70-F/HSP70-R (shown in Table 1) were designed according to the sequence of Pteria penguin (ABJ97377), Cyclina sinensis (AET13646), Perna viridis (ABJ98722), A. irradians (AAS17723), Azumapecten farreri (AAO38780), Mytilus coruscus (AGY56119), and Meretrix meretrix (ADT78478). The total RNA from the liver of S. maindroni was used as template after reverse transcription and the ratio of A260/A280 was 1.90. The reaction system was performed in 20 μL volume, including 2 μL 10 × PCR Buffer, 2 μL of MgCl 2 (25 mmol/L), dNTPs 0.4 μL (2.5 mmol/L), HSP70-F 0.8 μL (10 mol/L), HSP70-R 0.8 μL (10 μmol/L), template cDNA 0.6 μL, Taq DNA polymerase (TaKaRa, China) 0.4 μL (1 U) and 13.0 μL of PCR-Grade water. 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 56.5 °C, and 60 s at 72 °C, with a final extension
The liver was selected as a candidate tissue for investigating the temporal expression profile of SmHSP70 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 6000 ×g, 4 °C for 10 min to collect bacteria, and the resuspended live V. harveyi in 100 μL of PBS (pH 7.4, OD 600 = 0.4) were injected into muscles of S. maindroni. The cuttlefishes, which were injected with 100 μL PBS, acted as the control group. Total 35 infected cuttlefishes were cultured in 10 L filtered fresh well-aerated seawater, the livers 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 SmHSP70 gene at mRNA level in livers after V. harveyi challenge, real-time PCR (qRTPCR) was performed with SYBR PrimeScript™ RT reagent Kit (Perfect Real Time, TaKaRa, China) as recommended by the manufacturer's instructions in the 7500 Real Time PCR System (Applied Biosystems, UK). For each time point in the challenge experiment 5 biological replicate was used. The reaction mixture of 20 μL contained qSmHSP70-F 0.8 μL (10 μmol/L), qSmHSP70-R 0.8 μL (10 μmol/L), 2× SYBR® Premix Ex TaqTM II (TaKaRa, China) 10 μL, cDNA sample 0.8 μL, ROX II 0.4 μL, and ddH2O 7.2 μL. The standard cycling conditions were: 95 °C for 1 min (initial polymerase activation), followed by 40 cycles of 10 s at 95 °C and 45 s at 59.6 °C. The PCR specificity was checked with dissociation curve analysis from 55 to 95 °C, β-actin of S. maindroni (βSm actin-F and β-Sm actin-R) was used as the internal standard, and all the primers were shown in Table 1. The 2−ΔΔCT 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 significant at P b 0.05.
Table 1 PCR primer sequences for SOD cloning of S. maindroni. Degenerate primers were as follows, S: C/G; M: A/C; Y: C/T; W: A/T; R: A/G; and K: G/T. Primer For HSP70 cDNA clone HSP70-F HSP70-R For 5′-/3′-RACE Adaptor primer 5P1 5P2 3P1 3P2 For qRT-PCR qSmHSP70-F qSmHSP70-R β-Smactin-F β-Smactin-R
Sequences 5′-GTTATGACCTCGTTGATTAAGAG-3′ 5′-TTKSWSARRCGACCTTTGTC-3′ 5′-TCATTGCTCTTTCTCC-3′ 5′-CCAGGTTGGTTATCGGAGTATG-3′ 5′-AGGTCTGTGTTTGTTTTGTTGG-3′ 5′-CTTCACAACATACTCCGATAACCAACCT-3′ 5′-GAGCACTGGTAAAGAGAACAAGATCACC-3′ 5′-ACTCCGATAACCAACCTG-3′ 5′-GAGGCGACCTTTGTCATTT-3′ 5′-GCCAGTTGCTCGTTACAG-3′ 5′-GCCAACAATAGATGGGAAT-3′
Please cite this article as: Liu, H., et al., Identification and analysis of HSP70 from Sepiella maindroni under stress of Vibrio harveyi and Cd2 +, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.07.056
H. Liu et al. / Gene xxx (2015) xxx–xxx
2.5. SmHSP70 mRNA expression in liver after Cd2+ treatments For Cd treatments, mussels were maintained in static seawater in aquacultural tank with salinity of 28–30‰ at 23–25 °C. Seawater was changed daily and resupplied with the corresponding concentration of Cd2 +. Animals were exposed to cadmium chloride (CdCl2·5H2O) added to the 10 L filtered seawater with dissolved Cd2+ concentration of 200 μg/L based on the preliminary experiment of gradient concentration and 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+. Five individuals were taken for each treatment at 24, 48, 72, 120, 168 and 264 h post-treated respectively. The methods
3
of liver collection, total RNA extraction, cDNA synthesis and RT-PCR analysis were performed as described above. 3. Results and discussion 3.1. cDNA sequence analysis and characterization of SmHSP70 full-length HSP70s are responsible for the folding or assembly of native proteins and extracellular immunoregulatory functions (Reina et al., 2012). The protective mechanism of HSP70 is the induction of stress response proteins as molecular chaperones involved in heat, heavy metals, desiccation, diseases and parasites (Tukaj et al., 2011). Up to now HSP70 gene had been studied in several marine mollusca, such as scallop C. farreri
Fig. 1. The full-length cDNA and deduced amino acid sequence of HSP70 from S. maindroni. The stop codon (TAA) was indicated with an asterisk (*); the cDNA sequences had been submitted to GenBank with accession no. KJ739788. Major TRAF2-binding consensus motifs were in orange box. Caspase3 and Caspase7 cleavage site was in blue box. Tyrosine-based sorting signal was in black box. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Liu, H., et al., Identification and analysis of HSP70 from Sepiella maindroni under stress of Vibrio harveyi and Cd2 +, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.07.056
4
H. Liu et al. / Gene xxx (2015) xxx–xxx
(Wu et al., 2003), bay scallop A. irradians (Reina et al., 2012), Mytilus galloprovincialis (Anestis et al., 2007), and M. coruscus (Liu et al., 2014). In this study the complete cDNA sequence of HSP70 from S. maindroni was identified by homology cloning (GenBank accession no. KJ739788), which was 2109 bp in length, containing an ORF of 1950 bp encoding 649 amino acid residues. The 5′-UTR was 62 bp and the 3′-UTR was 97 bp (Fig. 1). The theoretical MW of SmHSP70 was 71.30 kDa and its estimated pI was 5.24. Generally, the molecular weight (MW) of HSP70 family was range from 68 to 73 kDa (Song et al., 2006). BLASTp program demonstrated that the obtained HSP70 sequence shared high homologies with known HSP70 in mollusca. Therefore it was concluded that the sequence cloned from S. maindroni belonged to HSP70 family. 3.2. Multiple sequence alignments and phylogenetic analysis The deduced amino acid sequence of SmHSP70 was aligned with the known HSP70 from other mollusca (as shown in Fig. 2), showing their high homology, especially in N terminal and C terminal. The molecular
chaperones in HSP70 are the large and diverse family of allosteric two-domain proteins: the NH2-terminal ATP-binding domain (ABD) and the COOH-terminal peptide-binding domain (PBD) (Smock et al., 2010). The conserved sequences and characteristic motifs of HSP70 family also appeared in the SmHSP70, including three HSP70 family signatures, and the bipartite nuclear targeting sequence and ATP/GTPbinding site motif A. The presence of five consecutive repeats of the tetrapeptide motif GGMP in C-terminal region of SmHSP70 was another notable feature in this protein. The last four amino acids formed a motif ‘EEVD’ at the C-terminus, which is highly conserved throughout all species (Freeman et al., 1995). The function of the GGMP repeats had not been revealed, it might form a structural entity together with the helical subdomain, and the EEVD motif might mediate chaperone cofactor binding (Demand et al., 1998). To reveal the molecular phylogenetic position of SmHSP70, an unrooted phylogenetic tree was constructed with Neighbor-Joining method from a distance matrix (Fig. 3). The phylogenetic tree was composed of five major groups: molluscs, shrimp, fish, chicken, and mammalians. S. maindroni and other molluscs formed a sister group, and
Fig. 2. Multiple alignment of the deduced amino acid sequences of HSP70 from S. maindroni and other organisms. The GenBank accession no. for the sequences were as follows: Chlamys farreri (ABD58974), Mizuhopecten yessoensis (AY485262), Argopecten irradians (ACM48346), Argopecten purpuratus (ACO72585), Mytilus coruscus (KF322135), Mytilus galloprovincialis (AY861684), Pinctada fucata (ABJ97378) and Pteria penguin (EF011060).
Please cite this article as: Liu, H., et al., Identification and analysis of HSP70 from Sepiella maindroni under stress of Vibrio harveyi and Cd2 +, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.07.056
H. Liu et al. / Gene xxx (2015) xxx–xxx
5
Fig. 3. Phylogenetic tree depicting the relationship of SmHSP70 with other species. All protein sequences obtained from GenBank of the NCBI, GenBank accession number was in parentheses, and phylogenetic tree was constructed using Neighbor-Joining rule method by the software of MEGA v4.0.
then clustered to shrimps, fish, chicken and finally mammalians. The relationship was generally in agreement with the traditional taxonomy and the occurrence of HSP70 gene duplication events during evolution. Therefore, the sequence alignment, structural comparison and phylogenetic analysis revealed that SmHSP70 was a member of the HSP70 family.
The rapid induction of SmHSP70 after V. harveyi injection was in agreement with its classification as an inducible gene product, which provided an effective tool to cope with rapidly changing environmental conditions for S. maindroni. The result was corroborated by the occurrence of analogous mechanisms in other molluscan species (Encomio and Chu, 2004) and some aquatic vertebrates (Wang et al., 2009).
3.3. SmHSP70 mRNA expression in liver after V. harveyi challenge
3.4. SmHSP70 mRNA expression in liver after Cd2+ stress
V. harveyi is a gram-negative bacterium 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). The HSP70 family, one of the most abundant of various HSPs, has been confirmed maintaining cellular homeostasis and protecting organism from pathogenic stress (Encomio and Chu, 2004). In the present study, the transcriptional expression of SmHSP70 was thus investigated after challenge by V. harveyi in the liver. Not surprisingly, the transcripts showed a time dependent expression pattern, which was significantly induced to the highest level at 4 h after post-injection with 20.44-fold higher than that of the control, then decreased slowly and returned to a slightly higher than physiological level at 72 h (Fig. 4A). The result was in accordance with previous studies in other molluscs (Wang et al., 2009), and suggested that SmHSP70 was involved in the squid's response to pathogenic infection and might play an important role in the immune response. Moreover, HSP70 genes are typically intron-less (Encomio and Chu, 2004), and the absence of introns causes their rapid induction to avoid posttranscriptional editing of the related mRNA and be effectively transcribed when splicing processes may be inhibited by stressful conditions.
At present marine waters and sediments receive anthropogenic inputs, containing a multitude of chemical contaminants that are potentially toxic to aquatic organisms, and could induce a series of HSPs to protect various living organisms (Ovelgonne et al., 1995; Andrew et al., 2003). Cadmium is the major toxic metal as contaminants in marine environment to be 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). The expression of HSP70 is frequently used as a component of physiological mechanisms through which mollusca cope with environmental challenges. In Cd2+ exposure, the similar dose- and time-dependent patterns appeared in SmHSP70 mRNA expression, which sustainably increased and reached the peak approximately 159.02-fold higher than the control at 120 h (Fig. 4B), then dropped gradually to 62.15-fold at 168 h post-exposure, but as time went on appeared another peak of 92.53-fold at 264 h. The results indicated that SmHSP70 took part in the cellular immune process and undertook the important functions in protecting organisms from Cd2+ stress. Cd2+ had been also verified to induce the other HSP expression in mammalian and nonmammalian cells, such as HSP90 in C. farreri, which was more sensitive to the concentration variation of
Please cite this article as: Liu, H., et al., Identification and analysis of HSP70 from Sepiella maindroni under stress of Vibrio harveyi and Cd2 +, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.07.056
6
H. Liu et al. / Gene xxx (2015) xxx–xxx
Fig. 4. Expression profiles of SmHSP70 after stress with Vibrio harveyi and Cd2+: (A) Temporal expression of the SmHSP70 transcripts in liver after Vibrio harveyi infection; (B) expression analysis of SmHSP70 in the liver 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 b 0.05, “**” at P b 0.01.
Cd2+ (Gao et al., 2007). Therefore, the results provided us an opportunity to speculate that HSP70 gene in the liver of SmHSP70 might be a potential biomarker of heavy metals. In summary, this was the first report of full-length cloning, characterization and inducible expression of HSP70 in the liver of cephalopod S. maindroni. The analysis of phylogenetic and structural features might contribute to the understanding of adaptation and evolutionary processes of HSPs in invertebrate. The up-regulated mRNA expression of SmHSP70 in response to V. harveyi and Cd2+ indicated that SmHSP70 was inducible and involved in the immune response, and it might be the potential biomarker for marine pollution. Acknowledgments This research was supported by grants from the Natural Science Foundation of Zhejiang Province, China (LY14C190004, LY13C190001),
the Zhejiang Marine Fisheries Bureau project (2012)83, and the Ministry of Science and Technology support program (2012BAB16B02).
References Andrew, A.S., Warren, A.J., Barchowsky, A., et al., 2003. Genomic and proteomic profiling of responses to toxic metals in human lung cells. Environ. Health Perspect. 111 (6), 825–835. Anestis, A., Lazou, A., Pörtner, H.O., et al., 2007. Behavioral, metabolic, and molecular stress responses of marine bivalve Mytilus galloprovincialis during long-term acclimation at increasing ambient temperature. Am. J. Physiol. Regul. Integr. Comp. Physiol. 293 (2), R911–R921. Buckley, B.A., Owen, M., Hofmann, G.E., 2001. Adjusting the thermostat: the threshold induction temperature for the heat-shock response in intertidal mussels (genus Mytilus) changes as a function of thermal history. J. Exp. Biol. 204, 3571–3579. Demand, J., Luders, J., Hohfeld, J., 1998. The carboxy-terminal domain of Hsc70 provides binding sites for a distinct set of chaperone cofactors. Mol. Cel. Biol. 18 (4), 2023–2028.
Please cite this article as: Liu, H., et al., Identification and analysis of HSP70 from Sepiella maindroni under stress of Vibrio harveyi and Cd2 +, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.07.056
H. Liu et al. / Gene xxx (2015) xxx–xxx Encomio, V.G., Chu, F.L., 2004. Characterization of heat shock protein expression and induced thermotolerance in P. marinus parasitized eastern oysters: lab and field studies. J. Shellfish Res. 23, 289–290. Freeman, B.C., Myers, M.P., Schumacher, R., et al., 1995. Identification of a regulatory motif in Hsp70 that affects ATPase activity, substrate binding and interaction with Hdj-1. EMBO J. 14 (10), 2281–2292. Gao, Q., Song, L.S., Ni, D., Wu, L., et al., 2007. cDNA cloning and mRNA expression of heat shock protein 90 gene in the haemocytes of Zhikong scallop Chlamys farreri. Comp. Biochem. Physiol. B 147 (4), 704–715. Gómez, L.J., Villamil, L., Lemos, M.L., Novoa, B., Fiqueras, A., 2005. Isolation of Vibrio alginolyticus and Vibrio splendidus from aquacultured carpet shell clam (Ruditapes decussatus) larvae associated with mass mortalities. Appl. Environ. Microbiol. 71, 98–104. Hamdoun, A.M., Cheney, D.P., Cherr, G.N., 2003. Phenotypic plasticity of HSP70 and HSP70 gene expression in the pacific oyster (Crassostrea gigas): implications for thermal limits and induction of thermal tolerance. Biol. Bull. 205, 160–169. Isabelle, B., Arnaud, T., Sabrina, R., Michel, A., Dario, M., 2003. Molecular identification and expression of heat shock cognate 70 (hsc70) and heat shock protein 70 (hsp70) genes in the Pacific oyster Crassostrea gigas. Cell Stress Chaperones 8, 76–85. Joly, A.L., Wettstein, G., Mignot, G., et al., 2010. Dual role of heat shock proteins as regulators of apoptosis and innate immunity. J. Innate Immun. 2 (3), 238–247. Krenek, S., Schlegel, M., Berendonk, T.U., 2013. Convergent evolution of heat-inducibility during subfunctionalization of the Hsp70 gene family. BMC Evol. Biol. 13, 49. Liu, H.H., He, J.Y., Chi, C.F., Shao, J.Z., 2014. Differential HSP70 expression in Mytilus coruscus under various stressors. Gene 543, 166–173. Luedeking, A., Koehler, A., 2004. Regulation of expression of multixenobiotic resistance (MXR) genes by environmental factors in the blue mussel Mytilus edulis. Aquat. Toxicol. 69, 1–10. Mjahed, H., Girodon, F., Fontenay, M., et al., 2012. Heat shock proteins in hematopoietic malignancies. Exp. Cell Res. 318 (15), 1946–1958. Ovelgonne, J.H., Souren, J.E., Wiegant, F.A., et al., 1995. Relationship between cadmiuminduced expression of heatshock genes, inhibition of protein synthesis and cell death. Toxicology 99 (1), 19–30.
7
Piano, A., Franzellitti, S., Tinti, F., Fabbri, E., 2005. Sequencing and expression pattern of inducible heat shock gene products in the European flat oyster, Ostrea edulis. Gene 361, 119–126. Reina, C.P., Nabet, B.Y., Young, P.D., et al., 2012. Basal and stress-induced Hsp70 are modulated by ataxin-3. Cell Stress Chaperones 17 (6), 729–742. Santoro, M.G., 2000. Heat shock factors and the control of the stress response. Biochem. Pharmacol. 59 (1), 55–63. Sarkar, A., Ray, D., Shrivastava, A.N., Sarker, S., 2006. Molecular Biomarkers: their significance and application in marine pollution monitoring. Ecotoxicology 15, 333–340. Silvia, F., Elena, F., 2005. Differential HSP70 gene expression in the Mediterranean mussel exposed to various stressors. Biochem. Biophys. Res. Commun. 336, 1157–1163. Smock, R.G., Rivoire, O., Russ, W.P., et al., 2010. An interdomain sector mediating allostery in Hsp70 molecular chaperones. Mol. Syst. Biol. 6, 414. Song, L., Wu, L., Ni, D., Chang, Y., et al., 2006. The cDNA cloning and mRNA expression of heat shock protein 70 gene in the haemocytes of bay scallop (Argopecten irradians, Lamarck 1819) responding to bacteria challenge and naphthalin stress. Fish Shellfish Immunol. 21 (4), 335–345. Tavaria, M., Gabriele, T., Kola, I., et al., 1996. A hitchhiker's guide to the human Hsp70 family. Cell Stress Chaperones 1 (1), 23–28. Tukaj, S., Bisewska, J., Roeske, K., Tukaj, Z., 2011. Time- and dose-dependent induction of HSP70 in Lemna minor exposed to different environmental stressors. Bull. Environ. Contam. Toxicol. 87 (3), 226–230. Wan, Q., Whang, I., Lee, J., 2008. Molecular cloning and characterization of three sigma glutathione S-transferases from disk abalone (Haliotis discus discus). Comp. Biochem. Physiol. B 151, 257–267. Wang, Z., Wu, Z., Jian, J., Lu, Y., 2009. Cloning and expression of heat shock protein 70 gene in the haemocytes of pearl oyster (Pinctada fucata, Gould 1850) responding to bacterial challenge. Fish Shellfish Immunol. 26 (4), 639–645. Wu, L., Song, L., Xu, W., et al., 2003. Identification and cloning of heat shock protein 70 gene from scallop Chlamys farreri. High Technol. Lett. 13, 75–79. Yenari, M.A., Giffard, R.G., Sapolsky, R.M., Steinberg, G.K., 1999. The neuroprotective potential of heat shock protein 70 (HSP70). Mol. Med. Today 5, 525–531.
Please cite this article as: Liu, H., et al., Identification and analysis of HSP70 from Sepiella maindroni under stress of Vibrio harveyi and Cd2 +, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.07.056