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Overexpression of Hsp90 from grass carp (Ctenopharyngodon idella) increases thermal protection against heat stress
Fish & Shellfish Immunology 33 (2012) 42e47
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Overexpression of Hsp90 from grass carp (Ctenopharyngodon idella) increases thermal protection against heat stress Chu-Xin Wu a,1, Feng-Yun Zhao b,1, Yuan Zhang b, Yu-Jiao Zhu b, Mei-Sheng Ma b, Hui-Ling Mao b, Cheng-Yu Hu b, c, * a b c
Nanchang Teachers College, Nanchang 330103, China Department of Bioscience, College of Life Science and Food Engineering, Nanchang University, Nanchang 330031, China Institute of Life Science, Nanchang University, Nanchang 330031, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 December 2011 Received in revised form 26 March 2012 Accepted 28 March 2012 Available online 10 April 2012
With homologous DNA probes, we had screened a grass carp heat shock protein 90 gene (CiHsp90). The full sequence of CiHsp90 cDNA was 2793 bp, which could code a 798 amino acids peptide. The phylogenetic analysis demonstrated that CiHsp90 shared the high homology with Zebrafish Grp94. Quantitative RT-PCR analysis showed that CiHsp90 was ubiquitously expressed at lower levels in all detected tissues and up-regulated after heat shock at 34 C or cold stress at 4 C. To understand the function of CiHsp90 involving in thermal protection, an expression vector containing coding region cDNA was expressed in E. coli BL21 (DE3) plysS. Upon transfer from 37 C to 42 C, these cells that accumulated CiHsp90 peptides displayed greater thermoresistance than the control cells. While incubated at 4 C for different periods, it could also improve the cell viability. After transient transfected recombinant plasmid pcDNA3.1/CiHsp90 into mouse myeloma cell line SP2/0, we found that CiHsp90 could contribute to protecting cells against both thermal and cold extremes. On the contrary, the mutant construct 6N-CiHsp90 (256e798 aa) could abolish the protection activity both in prokaryotic cells and eukaryotic cells. Additionally, both CiHsp90 and 6N-CiHsp90 peptides could reduce the level of citrate synthase aggregation at the high temperature. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Heat shock protein 90 Thermal protection Temperature stress Aggregation Grass carp
1. Introduction Heat shock proteins (Hsps) presented in all cells, both prokaryotic and eukaryotic. Many studies demonstrated that various stressors transiently would increase the production of Hsps as protection against harmful insults in cells. The exposure of organisms to elevated temperatures and other various stimuli could induce and increase the expression of Hsps [1]. According to their apparent molecular mass, these HSPs had been classified into several families: the small heat shock proteins (sHsps), the 40-kDa Hsps, the 70-kDa Hsps, the 60-kDa Hsps, the 90-kDa Hsps, and the 104-kDa Hsps [2,3]. All of these classes had important functions in fundamental cellular processes. Members of the Hsp90 family were the most abundant heat shock proteins under physiological conditions that account for
* Corresponding author. Department of Bioscience, College of Life Science and Food Engineering, Nanchang University, Nanchang 330031, China. Tel.: þ86 791 878 5566; fax: þ86 791 396 9530. E-mail address: [email protected] (C.-Y. Hu). 1 These authors contributed equally to this work. 1050-4648/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2012.03.033
1e2% of cellular proteins in most cells [4]. Hsp90 was a major chaperon in eukaryotes for it could form complexes with over 400 different proteins [5e7]. Hsp90 was up-regulated by a range of stressors such as heat or cold shock [8,9], food-deprivation [10], heavy metals [11] and diseases [12]. It had been revealed that Hsp90 would play crucial roles in protein folding, protein degradation and signal transduction [13e15]. These primary functions could help Hsp90 to exert its profoundly broad biological activities, such as cellular differentiation [16], cell proliferation and apoptosis [17], and cytoprotection [18]. In additional, studies in zebrafish demonstrated that Hsp90 was required for embryogenesis and organism development [19e21]. The importance of Hsp90 made it serve as an evolutionary capacitor of morphological changes during embryogenesis [22,23]. As aquatic vertebrates, fish were apt to suffer from a wide variety of stressors including heat shock, osmotic stress, and environmental contaminants. Hsps were key elements of the stress response system at the cellular level in fish. Although some fish Hsp90s had been cloned [24e26], limited information was available regarding Hsp90 in grass carp (Ctenopharyngodon idella) at present and its molecular mechanisms were still far from clear.
C.-X. Wu et al. / Fish & Shellfish Immunology 33 (2012) 42e47
Here we screened and identified a heat shock protein 90 gene (CiHsp90, GU258544) in the grass carp. Then, the coding region of CiHsp90 and the amino-terminal deletion mutant (6N-CiHsp90) (868e2496) were cloned into both the vector of pET-32a(þ) and pcDNA 3.1, which were transformed into E. coli and transfected into mouse myeloma cells SP2/0 for temperature treatment experiment, respectively. Moreover, the CiHsp90 and 6N-CiHsp90 (256e798 aa) peptides were expressed and purified for the citrate synthase aggregation assays. The results showed that CiHsp90 could contribute to protecting cells against both heat stress (42 C) and chilling temperature (4 C), while the mutant construct 6NCiHsp90 abolished the protection activity. In addition, both CiHsp90 and 6N-CiHsp90 could reduce the level of citrate synthase aggregation at the heat stress (42 C). 2. Materials and methods 2.1. Animals, temperature stress and sample collection The grass carp liver cDNA library was kept by our laboratory. Grass carps (about 100 g body weight) were obtained from Jiangxi Provincial Fisheries Research Institute and acclimatized to the laboratory conditions for 2 weeks in a quarantine area. For temperature stress experiments, 10 fish were put into 25 L aerated aquaria at 34 1 C or at 4 C for 2 h and then transferred back to room conditions for 20 min. Then, tissues including liver, brain, head kidney, spleen, and intestine were sampled and frozen in liquid nitrogen (we focused on the functions of Hsp90 in immunerelated tissues, so other tissues such as muscle tissue were not included in this study). Non-heat shock animals were used as a control group. Total RNA was extracted according to the manufacturer’s instruction of SV Total RNA Isolation System (Promega). Intact RNA was incubated with RNase-free DNase I to remove contaminated genomic DNA before being reverse transcribed into cDNA. 2.2. Cloning of CiHsp90 To screen CiHsp90 in the grass carp liver cDNA library, probe primers were designed based on a Danio rerio heat shock protein 90 (Hsp90b) cDNA sequences (NM_198210). The positive cDNA clone numbered YG001_F07 (pBluescript II SK/CiHsp90) was found by hybridization. Then, plasmid universal primers: T7 (50 -TAATACGACTCACTATA-30 ) and T3 (50 -ATTAACCCTCACTAAAGGGAA-30 ) were used to identify the clone. The PCR program was: 1 cycle of 94 C/5 min; 30 cycles of 94 C/30 s, 59 C/30 s, 72 C/3 min; 1 cycle of 72 C/10 min. The full length cDNA sequence was confirmed by sequencing (Beijing Genomics Institute). 2.3. Sequence analysis and phylogeny of Hsp90 The cDNA sequence was analyzed by ORF finder and the deduced amino acid sequence was analyzed with SMART and PROSITE. All homology sequences were obtained using BLAST program. Phylogenetic and molecular evolutionary analyses were conducted using PHYLIP 3.69 and optimized manually. 2.4. Tissue expression analysis of CiHsp90 First strand cDNA was synthesized using oligo(dT)18 primers with M-MLV Reverse Transcriptase (TaKaRa). Expression of CiHsp90 mRNA was assessed in different tissues using quantitative real-time RT-PCR (qRT-PCR) in a Mastercycler ep realplex (Eppendorf). CiHsp90 gene expression was detected using primers: RT-F (50 -CGTTGACGTTGACGGCACAGT-30 ) and RT-R
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(50 -CCAAGGCATCGGAAGCATTAG-30 ). b-actin (primers were 50 CACTGTGCCCATCTACGA-30 and 50 -CCATCTCCTGCTCGAAGT-30 ) was utilized as an internal control for cDNA normalization. Amplifications were carried out at a final volume of 20 ml, containing 1 ml DNA sample, 10 ml SYBR Premix Ex TaqÔ (TaKaRa) and 0.5 ml of each forward and reverse primer. The PCR cycling conditions were: 1 cycle of 94 C/5 min, 40 cycles of 94 C/30 s, 59 C/30 s, 72 C/30 s, followed by dissociation curve analysis to verify the amplification of a single product. Each sample was run in triplicate. The data were subjected to one-way ANOVA followed by an unpaired, two tailed ttest. P < 0.05 was considered statistically significant. 2.5. Prokaryotic expression and purification of recombinant peptides in bacteria With pBluescript II SK/CiHsp90 as the template, the full-length coding region of the putative CiHsp90 molecule and the aminoterminal deletion mutant (256e798 aa) (6N-CiHsp90) were amplified by PCR and inserted into the Bam H I/Xho I-cleaved expression vector pET-32a(þ) (Novagen). The primers for amplification of CiHsp90 were Hsp90-F (50 -CGGGATCCATGAGGCGACTGTGGATTATC-30 ) and Hsp90-R (50 - CGCTCGAGCTACAGCTCA TCTTTGCCTGTG-30 ). And the primers for amplification of 6NCiHsp90 were F (50 -CGGGATCCTCTGACTACCTTGAGCTGGAG-30 ) and R (50 -CGCTCGAGCTACAGCTCATCTTTGCCTGTG-30 ). The recombinant expression vectors were pET-32a(þ)/CiHsp90 and pET-32a(þ)/ 6N-CiHsp90 that yielded the N-terminal 6-histidine-tagged protein, which was confirmed by PCR and sequencing. These recombinant expression vectors were transformed into E. coli BL21 (DE3) plysS (Novagen) and the recombinant proteins were prepared. The proteins were purified and then used in the thermal aggregation assays. 2.6. Bacterial cells survival assays To estimate the growth curves, cells of E. coli BL21 (DE3) plysS and those were transformed with pET-32a(þ)/CiHsp90, pET32a(þ)/6N-CiHsp90 or pET-32a(þ) (control) were grown at 37 C in LB media to an A600 of 1.5. Then, cultures were diluted 1:100 into fresh LB media supplemented with appropriate ampicillin. Continuously, optical densities (A600) of these cultures were measured every 30 min, and the means of three experiments were determined (with SD being less than 5%). A single colony of bacteria transformed with pET-32a(þ)/ CiHsp90, pET-32a(þ)/6N-CiHsp90 or control plasmids was transferred to 20 ml of LB media containing 50 mg/ml of ampicillin. Cells were grown at 37 C with vigorous shaking until they reached an OD of 0.6e0.8 at A600 nm, which were induced by the addition of IPTG to a final concentration of 1.0 mM. After 3 h of growth, the cultures were divided into three aliquots. For heat shock experiments, the first aliquot of cultures was placed at 42 C. Then 1 ml of the cultures was taken at 60, 90, 120, and 150 min, and serial dilution of 1:106 was plated onto LB agar plates containing 50 mg/ml of ampicillin. After incubation overnight at 37 C, cell viability was estimated by counting the number of colony-forming units. For cold treatments, the second aliquot of cultures was diluted 1:106 and plated onto LB agar supplemented with ampicillin. Plates were incubated at 4 C for different periods (2, 4, 6, 8 and 10 d) and then cultured overnight at 37 C. Cell viability was estimated as described above. The third aliquot of cultures was to be a control which were diluted and plated and then cultured overnight at 37 C. For both heat and cold treatments, the means of three experiments were determined (with SD being less than 5% in all cases).
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2.7. Cells culture, transfection, and survival assays The coding region of CiHsp90 or 6N-CiHsp90 (256e798 aa) was inserted into pcDNA3.1 vector (Invitrogen). Mouse myeloma cell lines (MMCL) SP2/0 were grown overnight to 90e95% confluence in 24-well plates prior to transfection. Transfections were carried out using Lipofectamine 2000 reagent (Invitrogen). For each well, 200 ng of either pcDNA3.1/CiHsp90, pcDNA3.1/6N-CiHsp90 or pcDNA3.1 was complexed with 1 ml of lipofectamine 2000 in 100 ml EMEM without serum. The transfected cells were incubated at 37 C in a CO2 incubator for 24 h. These samples were divided into three aliquots for heat (42 C) or cold (4 C) treatments (for 60 and 150 min) as above, respectively. Then, the cells were washed with PBS and digested by 1 ml 0.25% trypsin for 3e5 min at 37 C. After detached from the culture flask, cells were collected by the centrifugation (15 K, 15 s) and resuspended in 1 ml PBS. The cells were counted using a microscope counting chamber. Each experiment was repeated three times.
2.8. Thermal aggregation assays Citrate synthase (CS, Sigma) (0.1 mM) was combined with varying amounts of recombinant CiHsp90 or 6N-CiHsp90 in 50 mM HEPESeKOH buffer (pH7.5, 1 ml of total volume). Mixtures were incubated at 42 C for an hour and samples were taken every 10 min to analyze the activity of CS according to the method of Lee et al. (1995) [27]. As a control, IgG was added at the concentrations indicated.
Fig. 1. Phylogenetic analysis of Hsp90s. A neighbour-joining tree was constructed by the computer program PHYLIP. Hsp90s were derived from grass carp (Ctenopharyngodon idella), Flounder (Paralichthys olivaceus), human (Homo sapiens), Asiatic toad (Bufo gargarizans), salmon (Salmo salar), flatfishes (Saltator maximus), zebrafish (Danio rerio), pig (Sus scrofa), cow (Bos taurus) and rat (Rattus norvegicu). All sequences used for analysis were derived from GenBank and their accession numbers were shown in parentheses. The bootstrap confidence values shown at the nodes of the tree were based on 1000 bootstrap replications.
3. Results
3.3. Heat and cold stress experiments
3.1. Characterization and homology analysis of CiHsp90
As an initial step in the analysis of the protective effect of CiHsp90, the growth curves of four kinds of E. coli were evaluated. Under normal culture conditions, similar growth rates were observed for three types of recombinant cells and untransformed wild-type cells (Fig. 3A). It indicated that the plasmid of pET32a(þ), pET-32a(þ)/CiHsp90, or pET-32a(þ)/6N-CiHsp90 had little effect on the growth of E. coli at 37 C. For heat shock experiments, the aliquot of cultures were shifted to 42 C for various times. As shown in Fig. 3B, E. coli cells or those containing the vector pET-32a(þ) and pET-32a(þ)/6NCiHsp90 were unable to withstand a thermal stress of 42 C over a 150 min period. These cells died quickly with about 30% surviving after only 60 min. In contrast, 64% of E. coli cells overexpressing CiHsp90 survived at this time point. After 150 min of
With homologous DNA probes, CiHsp90 (GU258544) was screened. The full sequence of CiHsp90 cDNA was 2793 bp in length with 104 bp of 5’UTR and 292 bp of 30 UTR. The open reading frame was 2397 bp that could code a 798 amino acids peptide with an estimated molecular mass of 91,976 Da. The putative CiHsp90 protein possessed a N-terminal ATPase domain (96e255 aa), a middle region, and a C-terminal homodimerisation domain (541e709 aa). The C-terminal of CiHsp90 also has the putative ER targeting tetrapeptide KDEL (795e798 aa). To better understand the position of CiHsp90 in the evolutionary history of the Hsp90 family, an unrooted phylogenetic tree was constructed with nucleotide sequences of 12 known Hsp90 members from mammals, amphibian and fish. As shown in Fig. 1, the grass carp Hsp90 shared the high homology with zebrafish Grp94.
3.2. Tissue expression of CiHsp90 by heat shock RNA was extracted from liver, brain, head kidney, spleen, and intestine at 2 h after temperature stress, and the modulation of CiHsp90 expression was assessed in detail by qRT-PCR. As shown in Fig. 2, CiHsp90 mRNA was detected in all fish tissues tested at low levels, and was significantly up-regulated by heat or cold stress (P < 0.05), except that not significant difference was found in kidney at 4 C (P > 0.05). Expression levels of CiHsp90 mRNA differed among different tissues. In the heat shock treated group, the CiHsp90 expression in liver was most up-regulated 9.27-fold while in intestine was only 1.4-fold. In the cold stress group, the CiHsp90 expression in intestine was most upregulated 7-fold.
Fig. 2. Quantitative analysis of CiHsp90 in different tissues at 2 h after heat shock ( ) or cold stress ( ). Each bar represented the level of target mRNA relative to those in the control group, expressed as the mean SE triplicate from qRT-PCR assays for three different cDNA samples. Error bars indicated standard error.
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different temperatures treatment for 60 min and 150 min. As shown in Fig. 4A, the survival rate of the cells transfected and nontransfected with plasmid pcDNA3.1/CiHsp90 were similar at 37 C. As the temperature was elevated at 42 C, although all the samples viability were lower, the pET-32a(þ)/CiHsp90 cells exhibited a higher survival rate (9.6%) than the pET-32a(þ) cells (2.7%) and the pET-32a(þ)/6N-CiHsp90 cells (3.5%) after 150 min (Fig. 4B). Exposure to a temperature of 4 C for different periods, the survival rate of the pET-32a(þ)/CiHsp90 cells was also higher than others (with about 30% increases after 150 min) (Fig. 4C).
Fig. 3. Effect of recombinant CiHsp90 on cell viability upon cold or heat stress in vivo. (A) Growth of wild-type (B), transformed pET-32a(þ) (:), pET-32a(þ)/CiHsp90 (A) and pET-32a(þ)/6N-CiHsp90 (﹡) E. coli at 37 C. (B and C) Viability of E. coli subjected to 42 C or 4 C treatments. At the times indicated after temperature downshift, plates were transferred to 37 C and cell viability was estimated. Means of three independent experiments are shown (SD was less than 5%). ,: wild-type E. coli, : transformed pET-32a(þ), : pET-32a(þ)/CiHsp90, : pET-32a(þ)/6N-CiHsp90.
thermal stress approximately 18% of E. coli cells overexpressing CiHsp90 were still viable, while that of the others cells were less than 5e7%. To test whether CiHsp90 might be relevant for cell viability at chilling temperatures, these aliquots from IPTG-induced cultures were plated and kept at 4 C for different times. As shown in Fig. 3C, whether the pET-32a(þ) cells, pET-32a(þ)/6N-CiHsp90 cells or pET-32a(þ)/CiHsp90 cells lost viability upon storage in the cold, although at significantly different rates. After 8 d at 4 C, for example, the survival rate of the control pET-32a(þ) cells was 5.16% whereas that of pET-32a(þ)/CiHsp90 cells was 16.67%. Meanwhile, the effect of grass carp Hsp90 over-expression on cells protection was analyzed in mouse myeloma cells after
Fig. 4. The viability of the noninduced and induced SP2/0 cells. The cells were transfected with plasmid pcDNA3.1/CiHsp90 ( ), pcDNA3.1/6N-CiHsp90 ( ) or pcDNA3.1 ( ). The non-transfected cells were the negtive controls (,). (A) Cells were incubated at 37 C. (B and C) Cells were heated at 42 C or cooled at 4 C and returned to 37 C for 24 h. The cells survival rates were measured from three independent experiments. Error bars indicate SD (n ¼ 3).
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C.-X. Wu et al. / Fish & Shellfish Immunology 33 (2012) 42e47
3.4. CiHsp90 inhibits the aggregation of heat-induced citrate synthase In an attempt to understand the functional role of CiHsp90 as a molecular chaperone, a citrate synthase (CS) aggregation assay was utilized. At 42 C, CS began to form insoluble aggregates rapidly that could be detected by light scattering in either the presence or absence of IgG, and with approximately 100% aggregation occurring after 60 min. However, the aggregation could be reduced in the presence of CiHsp90 or 6N-CiHsp90. Incubation with CiHsp90 or 6N-CiHsp90 at a peptides-to-CS ratio of 1:1 resulted in about 40e43% CS aggregation after 60 min. 2:1 ratio of peptides: CS almost completely inhibited CS aggregation (Fig. 5). 4. Discussion Hsp90s were ubiquitous, highly conserved proteins that were found in most organisms [4]. In the present study, the grass carp full-length cDNA of Hsp90 gene (CiHsp90) had been successfully cloned. The molecular characteristics of putative CiHsp90 protein indicated that it might play multiple functions in grass carp just like those reported in other organisms. On the other hand, the existence of C-terminal KDEL endoplasmic reticulum (ER) retention signal in CiHsp90 suggested that it might be a member of Grp94 (Hsp90 beta) [28]. Meantime, the phylogenetic analysis based on nucleotide data also demonstrated that these sequences of Hsp90 were clustered into two major groups, in which the CiHsp90 shared the highest homology with zebrafish Grp94 (Fig. 1). Grp94 was a ubiquitously expressed chaperone, especially with high levels in secretory tissues and played an important role in cellular immune responses. Its upregulation was often used as a hallmark of responses to ER stress [29]. When cells were exposed to a range of stressful conditions, such as heat, cold, and osmotic stress, the over-expression of Hsp90 could be immediately induced to recover cells from stress and guard cells from subsequent insults. Wang and Edens (1993)
demonstrated that the expression of Hsp90 was temperaturedependent in turkey leukocytes [30]. The latest study also indicated that a heat shock treatment could rapidly activate the transcription of Hsp90, and potentially involved in the immune responses against bacteria challenge [31,32]. In this study, we found that CiHsp90 expression were up-regulated in all detected tissues after 34 C heat shock or 4 C cold stress. Also, the up-regulated CiHsp90 expression levels varied from tissue to tissue. The tissuespecific expression of the chaperone might be attributed to differences in the rates of tissue protein synthesis [33]. It suggested that Hsp90 could play a pivotal role in the response to temperature stress in various tissues. It was well established that small heat shock proteins (sHsps) were able to protect cells against thermal extremes. Overexpression of CsHSP17.5 from Castanea sativa in E. coli was correlated with maintenance of viability both under heat-stress conditions and at low temperatures [34]. Likewise, Xenopus 30C in bacteria conferred resistance against thermal challenge [35]. To explore the underlying function of CiHsp90, the coding region cDNA was cloned into a vector and overexpressed in bacteria and MMCL SP2/0 cells. We found these cells were more resistant to cold challenge of 4 C and heat shock of 42 C than the control cells. The results suggested that CiHsp90 could adopt similar strategies to protect cells under different extreme environmental conditions. On the contrary, the amino-terminal deletion mutant (6N-CiHsp90) abolished the protective effect, which demonstrated that the full-length protein was required for CiHsp90’s protection activity. The previous studies demonstrated that Hsp90 could suppress the aggregation of many unstable proteins, such as protein kinase CK-II at low ionic strength [14,36]. Based on kinetic studies, Jakob et al. (1995) proposed that Hsp90 could bind to early unfolding intermediates, preventing their irreversible aggregation [37]. In aerobic organisms, the tricarboxylic acid cycle (TCA cycle) was part of a metabolic pathway involved in the chemical conversion of carbohydrates, fats and proteins into carbon dioxide and water to generate ATP. Citrate synthase (CS), a key pace-making enzyme in the first step of the TCA cycle, catalyzed the condensation reaction of acetyl coenzyme A and oxaloacetate to form citrate. CS was very sensitive to thermal inactivation and aggregates rapidly, which had been used as a well established model substrate protein to analyze the chaperone function of Hsp [37]. For example, some small Hsps such as Xenopus 30C could act as a molecular chaperone and prevent heat-induced CS aggregation by binding to the target protein and maintaining it in solution [35]. Here, we found that both CiHsp90 and 6N-CiHsp90 were very efficient in suppressing the thermal aggregation of CS. It indicated that there was little correlation between the prevention of protein aggregation and the cell protective activity [38]. Thus, the cellular protective mechanism of CiHsp90 under stress conditions was complex that to be further explored. As Hsp90s could form complexes with a wide range of protein kinases, mechanisms for Hsp90 involved in the cellular process might be very complicated. In mammals, Hsp90 participated in the regulation of protein synthesis by interacting with heme-regulated inhibitor (HRI), resulting in apoptosis [39]. Therefore, the exact biochemical functions of CiHsp90 in fish cells remained to be determined.
Acknowledgements Fig. 5. CiHsp90 protect CS from heat-induced aggregation. CS (0.1 mM) were incubated at 42 C in the absence (B) or presence of CiHsp90 (at a 1:1 (:) or 2:1 (6) molar ratio) and 6N-CiHsp90 (at a 1:1 (,) or 2:1 (,) molar ratio) as indicated, respectively. As the control, IgG (C) was added to CS at concentration of 0.5 mM in the absence of CiHsp90. The data are representative of 4 trials and calculated as a percentage of the maximum aggregation of CS after 60 min and was expressed as the mean SD.
This work was supported by grants from the Science and Technology Project of Jiangxi Province, China (No. 20111BBF60020) and from the Research Foundation of Education Bureau of Jiangxi Province, China (No. GJJ12662).
C.-X. Wu et al. / Fish & Shellfish Immunology 33 (2012) 42e47
References [1] Ritossa F. A new puffing pattern induced by temperature shock and DNP in Drosophila. Cell Mol Life Sci 1962;18(12):571e3. [2] Buchner J. Supervising the fold: functional principles of molecular chaperones. FASEB J 1996;10:10e9. [3] Dong CW, Zhang YB, Zhang QY, Gui JF. Differential expression of three Paralichthys olivaceus Hsp40 genes in responses to virus infection and heat shock. Fish Shellfish Immunol 2006;21(2):146e58. [4] Csermely P, Schnaider T, Soti C, Prohászka Z, Nardai G. The 90-kDa molecular chaperone family: structure, function, and clinical applications. A comprehensive review. Pharmacol Ther 1998;79(2):129e68. [5] Buchner J. Hsp90 & Co. e a holding for folding. Trends Biochem Sci 1999;24: 136e41. [6] Young JC, Moarefi I, Hartl FU. Hsp90: a specialized but essential proteinfolding tool. J Cell Biol 2001;154:267e73. [7] Zhao R, Davey M, Hsu YC, Kaplanek P, Tong A, Parsons AB, et al. Navigating the chaperone network: an integrative map of physical and genetic interactions mediated by the hsp90 chaperone. Cell 2005;120:715e27. [8] Palmisano AN, Winton JR, Dickhoff WW. Sequence features and phylogenetic analysis of the stress protein hsp90alpha in chinook salmon (Oncorhynchus tshawytscha), a poikilothermic vertebrate. Biochem Biophys Res Commun 1999;258:784e91. [9] Hermesz E, Abraham M, Nemcsok J. Identification of two hsp90 genes in carp. Comp Biochem Physiol C 2001;129:397e407. [10] Cara JB, Aluru N, Moyano FJ, Vijayan MM. Food-deprivation induces HSP70 and HSP90 protein expression in larval gilthead sea bream and rainbow trout. Comp Biochem Phys 2005;142:426e31. [11] Ali A, Krone PH, Pearson DS, Heikkila JJ. Evaluation of stress-inducible hsp90 gene expression as a potential molecular biomarker in Xenopus laevis. Cell Stress Chaperones 1996;1:62e9. [12] Wang B, Li F, Dong B, Zhang X, Zhang C, Xiang J. Discovery of the genes in response to white spot syndrome virus (WSSV) infection in Fenneropenaeus chinensis through cDNA microarray. Mar Biotechnol 2006;8:491e500. [13] Freeman BC, Morimoto RI. The human cytosolic molecular chaperones hsp90, hsp70 (hsc70) and hdj-1 have distinct roles in recognition of a non-native protein and protein refolding. EMBO J 1996;15:2969e79. [14] Miyata Y, Yahara I. The 90-kDa heat shock protein, HSP90, binds and protects casein kinase II from self-aggregation and enhances its kinase activity. J Biol Chem 1992;267:7042e7. [15] Picard D. Heat-shock protein 90, a chaperone for folding and regulation. Cell Mol Life Sci 2002;59:1640e8. [16] Lele Z, Hartson SD, Martin CC, Whitesell L, Matts RL, Krone PH. Disruption of zebrafish somite development by pharmacologic inhibition of Hsp90. Dev Biol 1999;210(1):56e70. [17] Sreedhar AS, Kalmar E, Csermely P, Shen YF. Hsp90 isoforms: functions, expression and clinical importance. FEBS Lett 2004;562:11e5. [18] Sreedhar AS, Csermely P. Heat shock proteins in the regulation of apoptosis: new strategies in tumor therapy: a comprehensive review. Pharmacol Ther 2004;101:227e57. [19] Sass JB, Weinberg ES, Krone PH. Specific localization of zebrafish hsp90 alpha mRNA to myoD-expressing cells suggests a role for hsp90 alpha during normal muscle development. Mech Dev 1996;54:195e204. [20] Sumanas S, Larson JD, Miller Bever M. Zebrafish chaperone protein GP96 is required for otolith formation during ear development. Dev Biol 2003;261: 443e55.
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[21] Du SJ, Li H, Bian Y, Zhong Y. Heat-shock protein 90alpha1 is required for organized myofibril assembly in skeletal muscles of zebrafish embryos. Proc Natl Acad Sci USA 2008;105:554e9. [22] Queitsch C, Sangster TA, Lindquist S. Hsp90 as a capacitor of phenotypic variation. Nature 2002;417:618e24. [23] Sollars V, Lu X, Xiao L, Wang X, Garfinkel MD, Ruden DM. Evidence for an epigenetic mechanism by which Hsp90 acts as a capacitor for morphological evolution. Nat Genet 2002;33:70e4. [24] Roberts RJ, Agius C, Saliba C, Bossier P, Sung YY. Heat shock proteins (chaperones) in fish and shellfish and their potential role in relation to fish health: a review. J Fish Dis 2010;33:789e801. [25] Zhang XZ, Dai LP, Wu ZH, Jian JC, Lu YS. Expression pattern of heat shock protein 90 gene of humphead snapper Lutjanus sanguineus during pathogenic Vibrio harveyi stress. J Fish Biol 2011;79:178e93. [26] Chen YM, Kuo CE, Wang TY, Shie PS, Wang WC, Huang SL, et al. Cloning of an orange-spotted grouper Epinephelus coioides heat shock protein 90AB (HSP90AB) and characterization of its expression in response to nodavirus. Fish Shellfish Immunol 2010;28:895e904. [27] Lee GJ, Pokala N, Vierling E. Structure and in vitro molecular chaperone activity of cytosolic small heat shock proteins from pea. J Biol Chem 1995;270: 10432e8. [28] Marzec M, Eletto D, Argon Y. GRP94: an HSP90-like protein specialized for protein folding and quality control in the endoplasmic reticulum. Biochim Biophys Acta 2012;1823(3):774e87. [29] Eletto D, Dersh D, Argon Y. GRP94 in ER quality control and stress responses. Semin Cell Dev Biol 2010;21:479e85. [30] Wang SY, Edens FW. Stress-induced heat-shock protein synthesis in peripheral leukocytes of turkeys, Meleagris gallopavo. Comp Biochem Physiol 1993; 106:621e8. [31] Gao Q, Zhao J, Song L, Qiu L, Yu Y, Zhang H, et al. Molecular cloning, characterization and expression of heat shock protein 90 gene in the haemocytes of bay scallop Argopecten irradians. Fish Shellfish Immunol 2008;24:379e85. [32] Manchado M, Salas-Leiton E, Infante C, Ponce M, Asensio E, Crespo A, et al. Molecular characterization, gene expression and transcriptional regulation of cytosolic HSP90 genes in the flatfish Senegalese sole (Solea senegalensis Kaup). Gene 2008;416:77e84. [33] Kawabe S, Yokoyama Y. cDNA cloning and expression of grp94 in the Pacific oyster Crassostrea gigas. Comp Biochem Physiol B 2009;154(3):290e7. [34] Soto A, Allona I, Collada C, Guevara MA, Casado R, Rodriguez-Cerezo E, et al. Heterologous expression of a plant small heat-shock protein enhances Escherichia coli viability under heat and cold stress. Plant Physiol 1999;120: 521e8. [35] Fernando P, Heikkila JJ. Functional characterization of Xenopus small heat shock protein, Hsp30C: the carboxyl end is required for stability and chaperone activity. Cell Stress Chaperones 2005;5:148e59. [36] Miyata Y, Yahara I. Interaction between casein kinase II and the 90-kDa stress protein, HSP90. Biochemistry 1995;34:8123e9. [37] Jakob U, Lilie H, Meyer I, Buchner J. Transient interaction of Hsp90 with early unfolding intermediates of citrate synthase. Implications for heat shock in vivo. J Biol Chem 1995;270:7288e94. [38] Johnson BD, Chadli A, Felts SJ, Bouhouche I, Catelli MG, Toft DO. Hsp90 chaperone activity requires the full-length protein and interaction among its multiple domains. J Biol Chem 2000;275:32499e507. [39] Pal JK. Hsp90 regulates protein synthesis by activating the heme-regulated eukaryotic initiation factor 2a (eIF-2a) kinase in rabbit reticulocyte lysates. J Biosci 1998;23:353e60.
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Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Overexpression of Hsp90 from grass carp (Ctenopharyngodon idella) increases thermal protection against heat stress Chu-Xin Wu a,1, Feng-Yun Zhao b,1, Yuan Zhang b, Yu-Jiao Zhu b, Mei-Sheng Ma b, Hui-Ling Mao b, Cheng-Yu Hu b, c, * a b c
Nanchang Teachers College, Nanchang 330103, China Department of Bioscience, College of Life Science and Food Engineering, Nanchang University, Nanchang 330031, China Institute of Life Science, Nanchang University, Nanchang 330031, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 December 2011 Received in revised form 26 March 2012 Accepted 28 March 2012 Available online 10 April 2012
With homologous DNA probes, we had screened a grass carp heat shock protein 90 gene (CiHsp90). The full sequence of CiHsp90 cDNA was 2793 bp, which could code a 798 amino acids peptide. The phylogenetic analysis demonstrated that CiHsp90 shared the high homology with Zebrafish Grp94. Quantitative RT-PCR analysis showed that CiHsp90 was ubiquitously expressed at lower levels in all detected tissues and up-regulated after heat shock at 34 C or cold stress at 4 C. To understand the function of CiHsp90 involving in thermal protection, an expression vector containing coding region cDNA was expressed in E. coli BL21 (DE3) plysS. Upon transfer from 37 C to 42 C, these cells that accumulated CiHsp90 peptides displayed greater thermoresistance than the control cells. While incubated at 4 C for different periods, it could also improve the cell viability. After transient transfected recombinant plasmid pcDNA3.1/CiHsp90 into mouse myeloma cell line SP2/0, we found that CiHsp90 could contribute to protecting cells against both thermal and cold extremes. On the contrary, the mutant construct 6N-CiHsp90 (256e798 aa) could abolish the protection activity both in prokaryotic cells and eukaryotic cells. Additionally, both CiHsp90 and 6N-CiHsp90 peptides could reduce the level of citrate synthase aggregation at the high temperature. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Heat shock protein 90 Thermal protection Temperature stress Aggregation Grass carp
1. Introduction Heat shock proteins (Hsps) presented in all cells, both prokaryotic and eukaryotic. Many studies demonstrated that various stressors transiently would increase the production of Hsps as protection against harmful insults in cells. The exposure of organisms to elevated temperatures and other various stimuli could induce and increase the expression of Hsps [1]. According to their apparent molecular mass, these HSPs had been classified into several families: the small heat shock proteins (sHsps), the 40-kDa Hsps, the 70-kDa Hsps, the 60-kDa Hsps, the 90-kDa Hsps, and the 104-kDa Hsps [2,3]. All of these classes had important functions in fundamental cellular processes. Members of the Hsp90 family were the most abundant heat shock proteins under physiological conditions that account for
* Corresponding author. Department of Bioscience, College of Life Science and Food Engineering, Nanchang University, Nanchang 330031, China. Tel.: þ86 791 878 5566; fax: þ86 791 396 9530. E-mail address: [email protected] (C.-Y. Hu). 1 These authors contributed equally to this work. 1050-4648/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2012.03.033
1e2% of cellular proteins in most cells [4]. Hsp90 was a major chaperon in eukaryotes for it could form complexes with over 400 different proteins [5e7]. Hsp90 was up-regulated by a range of stressors such as heat or cold shock [8,9], food-deprivation [10], heavy metals [11] and diseases [12]. It had been revealed that Hsp90 would play crucial roles in protein folding, protein degradation and signal transduction [13e15]. These primary functions could help Hsp90 to exert its profoundly broad biological activities, such as cellular differentiation [16], cell proliferation and apoptosis [17], and cytoprotection [18]. In additional, studies in zebrafish demonstrated that Hsp90 was required for embryogenesis and organism development [19e21]. The importance of Hsp90 made it serve as an evolutionary capacitor of morphological changes during embryogenesis [22,23]. As aquatic vertebrates, fish were apt to suffer from a wide variety of stressors including heat shock, osmotic stress, and environmental contaminants. Hsps were key elements of the stress response system at the cellular level in fish. Although some fish Hsp90s had been cloned [24e26], limited information was available regarding Hsp90 in grass carp (Ctenopharyngodon idella) at present and its molecular mechanisms were still far from clear.
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Here we screened and identified a heat shock protein 90 gene (CiHsp90, GU258544) in the grass carp. Then, the coding region of CiHsp90 and the amino-terminal deletion mutant (6N-CiHsp90) (868e2496) were cloned into both the vector of pET-32a(þ) and pcDNA 3.1, which were transformed into E. coli and transfected into mouse myeloma cells SP2/0 for temperature treatment experiment, respectively. Moreover, the CiHsp90 and 6N-CiHsp90 (256e798 aa) peptides were expressed and purified for the citrate synthase aggregation assays. The results showed that CiHsp90 could contribute to protecting cells against both heat stress (42 C) and chilling temperature (4 C), while the mutant construct 6NCiHsp90 abolished the protection activity. In addition, both CiHsp90 and 6N-CiHsp90 could reduce the level of citrate synthase aggregation at the heat stress (42 C). 2. Materials and methods 2.1. Animals, temperature stress and sample collection The grass carp liver cDNA library was kept by our laboratory. Grass carps (about 100 g body weight) were obtained from Jiangxi Provincial Fisheries Research Institute and acclimatized to the laboratory conditions for 2 weeks in a quarantine area. For temperature stress experiments, 10 fish were put into 25 L aerated aquaria at 34 1 C or at 4 C for 2 h and then transferred back to room conditions for 20 min. Then, tissues including liver, brain, head kidney, spleen, and intestine were sampled and frozen in liquid nitrogen (we focused on the functions of Hsp90 in immunerelated tissues, so other tissues such as muscle tissue were not included in this study). Non-heat shock animals were used as a control group. Total RNA was extracted according to the manufacturer’s instruction of SV Total RNA Isolation System (Promega). Intact RNA was incubated with RNase-free DNase I to remove contaminated genomic DNA before being reverse transcribed into cDNA. 2.2. Cloning of CiHsp90 To screen CiHsp90 in the grass carp liver cDNA library, probe primers were designed based on a Danio rerio heat shock protein 90 (Hsp90b) cDNA sequences (NM_198210). The positive cDNA clone numbered YG001_F07 (pBluescript II SK/CiHsp90) was found by hybridization. Then, plasmid universal primers: T7 (50 -TAATACGACTCACTATA-30 ) and T3 (50 -ATTAACCCTCACTAAAGGGAA-30 ) were used to identify the clone. The PCR program was: 1 cycle of 94 C/5 min; 30 cycles of 94 C/30 s, 59 C/30 s, 72 C/3 min; 1 cycle of 72 C/10 min. The full length cDNA sequence was confirmed by sequencing (Beijing Genomics Institute). 2.3. Sequence analysis and phylogeny of Hsp90 The cDNA sequence was analyzed by ORF finder and the deduced amino acid sequence was analyzed with SMART and PROSITE. All homology sequences were obtained using BLAST program. Phylogenetic and molecular evolutionary analyses were conducted using PHYLIP 3.69 and optimized manually. 2.4. Tissue expression analysis of CiHsp90 First strand cDNA was synthesized using oligo(dT)18 primers with M-MLV Reverse Transcriptase (TaKaRa). Expression of CiHsp90 mRNA was assessed in different tissues using quantitative real-time RT-PCR (qRT-PCR) in a Mastercycler ep realplex (Eppendorf). CiHsp90 gene expression was detected using primers: RT-F (50 -CGTTGACGTTGACGGCACAGT-30 ) and RT-R
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(50 -CCAAGGCATCGGAAGCATTAG-30 ). b-actin (primers were 50 CACTGTGCCCATCTACGA-30 and 50 -CCATCTCCTGCTCGAAGT-30 ) was utilized as an internal control for cDNA normalization. Amplifications were carried out at a final volume of 20 ml, containing 1 ml DNA sample, 10 ml SYBR Premix Ex TaqÔ (TaKaRa) and 0.5 ml of each forward and reverse primer. The PCR cycling conditions were: 1 cycle of 94 C/5 min, 40 cycles of 94 C/30 s, 59 C/30 s, 72 C/30 s, followed by dissociation curve analysis to verify the amplification of a single product. Each sample was run in triplicate. The data were subjected to one-way ANOVA followed by an unpaired, two tailed ttest. P < 0.05 was considered statistically significant. 2.5. Prokaryotic expression and purification of recombinant peptides in bacteria With pBluescript II SK/CiHsp90 as the template, the full-length coding region of the putative CiHsp90 molecule and the aminoterminal deletion mutant (256e798 aa) (6N-CiHsp90) were amplified by PCR and inserted into the Bam H I/Xho I-cleaved expression vector pET-32a(þ) (Novagen). The primers for amplification of CiHsp90 were Hsp90-F (50 -CGGGATCCATGAGGCGACTGTGGATTATC-30 ) and Hsp90-R (50 - CGCTCGAGCTACAGCTCA TCTTTGCCTGTG-30 ). And the primers for amplification of 6NCiHsp90 were F (50 -CGGGATCCTCTGACTACCTTGAGCTGGAG-30 ) and R (50 -CGCTCGAGCTACAGCTCATCTTTGCCTGTG-30 ). The recombinant expression vectors were pET-32a(þ)/CiHsp90 and pET-32a(þ)/ 6N-CiHsp90 that yielded the N-terminal 6-histidine-tagged protein, which was confirmed by PCR and sequencing. These recombinant expression vectors were transformed into E. coli BL21 (DE3) plysS (Novagen) and the recombinant proteins were prepared. The proteins were purified and then used in the thermal aggregation assays. 2.6. Bacterial cells survival assays To estimate the growth curves, cells of E. coli BL21 (DE3) plysS and those were transformed with pET-32a(þ)/CiHsp90, pET32a(þ)/6N-CiHsp90 or pET-32a(þ) (control) were grown at 37 C in LB media to an A600 of 1.5. Then, cultures were diluted 1:100 into fresh LB media supplemented with appropriate ampicillin. Continuously, optical densities (A600) of these cultures were measured every 30 min, and the means of three experiments were determined (with SD being less than 5%). A single colony of bacteria transformed with pET-32a(þ)/ CiHsp90, pET-32a(þ)/6N-CiHsp90 or control plasmids was transferred to 20 ml of LB media containing 50 mg/ml of ampicillin. Cells were grown at 37 C with vigorous shaking until they reached an OD of 0.6e0.8 at A600 nm, which were induced by the addition of IPTG to a final concentration of 1.0 mM. After 3 h of growth, the cultures were divided into three aliquots. For heat shock experiments, the first aliquot of cultures was placed at 42 C. Then 1 ml of the cultures was taken at 60, 90, 120, and 150 min, and serial dilution of 1:106 was plated onto LB agar plates containing 50 mg/ml of ampicillin. After incubation overnight at 37 C, cell viability was estimated by counting the number of colony-forming units. For cold treatments, the second aliquot of cultures was diluted 1:106 and plated onto LB agar supplemented with ampicillin. Plates were incubated at 4 C for different periods (2, 4, 6, 8 and 10 d) and then cultured overnight at 37 C. Cell viability was estimated as described above. The third aliquot of cultures was to be a control which were diluted and plated and then cultured overnight at 37 C. For both heat and cold treatments, the means of three experiments were determined (with SD being less than 5% in all cases).
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2.7. Cells culture, transfection, and survival assays The coding region of CiHsp90 or 6N-CiHsp90 (256e798 aa) was inserted into pcDNA3.1 vector (Invitrogen). Mouse myeloma cell lines (MMCL) SP2/0 were grown overnight to 90e95% confluence in 24-well plates prior to transfection. Transfections were carried out using Lipofectamine 2000 reagent (Invitrogen). For each well, 200 ng of either pcDNA3.1/CiHsp90, pcDNA3.1/6N-CiHsp90 or pcDNA3.1 was complexed with 1 ml of lipofectamine 2000 in 100 ml EMEM without serum. The transfected cells were incubated at 37 C in a CO2 incubator for 24 h. These samples were divided into three aliquots for heat (42 C) or cold (4 C) treatments (for 60 and 150 min) as above, respectively. Then, the cells were washed with PBS and digested by 1 ml 0.25% trypsin for 3e5 min at 37 C. After detached from the culture flask, cells were collected by the centrifugation (15 K, 15 s) and resuspended in 1 ml PBS. The cells were counted using a microscope counting chamber. Each experiment was repeated three times.
2.8. Thermal aggregation assays Citrate synthase (CS, Sigma) (0.1 mM) was combined with varying amounts of recombinant CiHsp90 or 6N-CiHsp90 in 50 mM HEPESeKOH buffer (pH7.5, 1 ml of total volume). Mixtures were incubated at 42 C for an hour and samples were taken every 10 min to analyze the activity of CS according to the method of Lee et al. (1995) [27]. As a control, IgG was added at the concentrations indicated.
Fig. 1. Phylogenetic analysis of Hsp90s. A neighbour-joining tree was constructed by the computer program PHYLIP. Hsp90s were derived from grass carp (Ctenopharyngodon idella), Flounder (Paralichthys olivaceus), human (Homo sapiens), Asiatic toad (Bufo gargarizans), salmon (Salmo salar), flatfishes (Saltator maximus), zebrafish (Danio rerio), pig (Sus scrofa), cow (Bos taurus) and rat (Rattus norvegicu). All sequences used for analysis were derived from GenBank and their accession numbers were shown in parentheses. The bootstrap confidence values shown at the nodes of the tree were based on 1000 bootstrap replications.
3. Results
3.3. Heat and cold stress experiments
3.1. Characterization and homology analysis of CiHsp90
As an initial step in the analysis of the protective effect of CiHsp90, the growth curves of four kinds of E. coli were evaluated. Under normal culture conditions, similar growth rates were observed for three types of recombinant cells and untransformed wild-type cells (Fig. 3A). It indicated that the plasmid of pET32a(þ), pET-32a(þ)/CiHsp90, or pET-32a(þ)/6N-CiHsp90 had little effect on the growth of E. coli at 37 C. For heat shock experiments, the aliquot of cultures were shifted to 42 C for various times. As shown in Fig. 3B, E. coli cells or those containing the vector pET-32a(þ) and pET-32a(þ)/6NCiHsp90 were unable to withstand a thermal stress of 42 C over a 150 min period. These cells died quickly with about 30% surviving after only 60 min. In contrast, 64% of E. coli cells overexpressing CiHsp90 survived at this time point. After 150 min of
With homologous DNA probes, CiHsp90 (GU258544) was screened. The full sequence of CiHsp90 cDNA was 2793 bp in length with 104 bp of 5’UTR and 292 bp of 30 UTR. The open reading frame was 2397 bp that could code a 798 amino acids peptide with an estimated molecular mass of 91,976 Da. The putative CiHsp90 protein possessed a N-terminal ATPase domain (96e255 aa), a middle region, and a C-terminal homodimerisation domain (541e709 aa). The C-terminal of CiHsp90 also has the putative ER targeting tetrapeptide KDEL (795e798 aa). To better understand the position of CiHsp90 in the evolutionary history of the Hsp90 family, an unrooted phylogenetic tree was constructed with nucleotide sequences of 12 known Hsp90 members from mammals, amphibian and fish. As shown in Fig. 1, the grass carp Hsp90 shared the high homology with zebrafish Grp94.
3.2. Tissue expression of CiHsp90 by heat shock RNA was extracted from liver, brain, head kidney, spleen, and intestine at 2 h after temperature stress, and the modulation of CiHsp90 expression was assessed in detail by qRT-PCR. As shown in Fig. 2, CiHsp90 mRNA was detected in all fish tissues tested at low levels, and was significantly up-regulated by heat or cold stress (P < 0.05), except that not significant difference was found in kidney at 4 C (P > 0.05). Expression levels of CiHsp90 mRNA differed among different tissues. In the heat shock treated group, the CiHsp90 expression in liver was most up-regulated 9.27-fold while in intestine was only 1.4-fold. In the cold stress group, the CiHsp90 expression in intestine was most upregulated 7-fold.
Fig. 2. Quantitative analysis of CiHsp90 in different tissues at 2 h after heat shock ( ) or cold stress ( ). Each bar represented the level of target mRNA relative to those in the control group, expressed as the mean SE triplicate from qRT-PCR assays for three different cDNA samples. Error bars indicated standard error.
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different temperatures treatment for 60 min and 150 min. As shown in Fig. 4A, the survival rate of the cells transfected and nontransfected with plasmid pcDNA3.1/CiHsp90 were similar at 37 C. As the temperature was elevated at 42 C, although all the samples viability were lower, the pET-32a(þ)/CiHsp90 cells exhibited a higher survival rate (9.6%) than the pET-32a(þ) cells (2.7%) and the pET-32a(þ)/6N-CiHsp90 cells (3.5%) after 150 min (Fig. 4B). Exposure to a temperature of 4 C for different periods, the survival rate of the pET-32a(þ)/CiHsp90 cells was also higher than others (with about 30% increases after 150 min) (Fig. 4C).
Fig. 3. Effect of recombinant CiHsp90 on cell viability upon cold or heat stress in vivo. (A) Growth of wild-type (B), transformed pET-32a(þ) (:), pET-32a(þ)/CiHsp90 (A) and pET-32a(þ)/6N-CiHsp90 (﹡) E. coli at 37 C. (B and C) Viability of E. coli subjected to 42 C or 4 C treatments. At the times indicated after temperature downshift, plates were transferred to 37 C and cell viability was estimated. Means of three independent experiments are shown (SD was less than 5%). ,: wild-type E. coli, : transformed pET-32a(þ), : pET-32a(þ)/CiHsp90, : pET-32a(þ)/6N-CiHsp90.
thermal stress approximately 18% of E. coli cells overexpressing CiHsp90 were still viable, while that of the others cells were less than 5e7%. To test whether CiHsp90 might be relevant for cell viability at chilling temperatures, these aliquots from IPTG-induced cultures were plated and kept at 4 C for different times. As shown in Fig. 3C, whether the pET-32a(þ) cells, pET-32a(þ)/6N-CiHsp90 cells or pET-32a(þ)/CiHsp90 cells lost viability upon storage in the cold, although at significantly different rates. After 8 d at 4 C, for example, the survival rate of the control pET-32a(þ) cells was 5.16% whereas that of pET-32a(þ)/CiHsp90 cells was 16.67%. Meanwhile, the effect of grass carp Hsp90 over-expression on cells protection was analyzed in mouse myeloma cells after
Fig. 4. The viability of the noninduced and induced SP2/0 cells. The cells were transfected with plasmid pcDNA3.1/CiHsp90 ( ), pcDNA3.1/6N-CiHsp90 ( ) or pcDNA3.1 ( ). The non-transfected cells were the negtive controls (,). (A) Cells were incubated at 37 C. (B and C) Cells were heated at 42 C or cooled at 4 C and returned to 37 C for 24 h. The cells survival rates were measured from three independent experiments. Error bars indicate SD (n ¼ 3).
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3.4. CiHsp90 inhibits the aggregation of heat-induced citrate synthase In an attempt to understand the functional role of CiHsp90 as a molecular chaperone, a citrate synthase (CS) aggregation assay was utilized. At 42 C, CS began to form insoluble aggregates rapidly that could be detected by light scattering in either the presence or absence of IgG, and with approximately 100% aggregation occurring after 60 min. However, the aggregation could be reduced in the presence of CiHsp90 or 6N-CiHsp90. Incubation with CiHsp90 or 6N-CiHsp90 at a peptides-to-CS ratio of 1:1 resulted in about 40e43% CS aggregation after 60 min. 2:1 ratio of peptides: CS almost completely inhibited CS aggregation (Fig. 5). 4. Discussion Hsp90s were ubiquitous, highly conserved proteins that were found in most organisms [4]. In the present study, the grass carp full-length cDNA of Hsp90 gene (CiHsp90) had been successfully cloned. The molecular characteristics of putative CiHsp90 protein indicated that it might play multiple functions in grass carp just like those reported in other organisms. On the other hand, the existence of C-terminal KDEL endoplasmic reticulum (ER) retention signal in CiHsp90 suggested that it might be a member of Grp94 (Hsp90 beta) [28]. Meantime, the phylogenetic analysis based on nucleotide data also demonstrated that these sequences of Hsp90 were clustered into two major groups, in which the CiHsp90 shared the highest homology with zebrafish Grp94 (Fig. 1). Grp94 was a ubiquitously expressed chaperone, especially with high levels in secretory tissues and played an important role in cellular immune responses. Its upregulation was often used as a hallmark of responses to ER stress [29]. When cells were exposed to a range of stressful conditions, such as heat, cold, and osmotic stress, the over-expression of Hsp90 could be immediately induced to recover cells from stress and guard cells from subsequent insults. Wang and Edens (1993)
demonstrated that the expression of Hsp90 was temperaturedependent in turkey leukocytes [30]. The latest study also indicated that a heat shock treatment could rapidly activate the transcription of Hsp90, and potentially involved in the immune responses against bacteria challenge [31,32]. In this study, we found that CiHsp90 expression were up-regulated in all detected tissues after 34 C heat shock or 4 C cold stress. Also, the up-regulated CiHsp90 expression levels varied from tissue to tissue. The tissuespecific expression of the chaperone might be attributed to differences in the rates of tissue protein synthesis [33]. It suggested that Hsp90 could play a pivotal role in the response to temperature stress in various tissues. It was well established that small heat shock proteins (sHsps) were able to protect cells against thermal extremes. Overexpression of CsHSP17.5 from Castanea sativa in E. coli was correlated with maintenance of viability both under heat-stress conditions and at low temperatures [34]. Likewise, Xenopus 30C in bacteria conferred resistance against thermal challenge [35]. To explore the underlying function of CiHsp90, the coding region cDNA was cloned into a vector and overexpressed in bacteria and MMCL SP2/0 cells. We found these cells were more resistant to cold challenge of 4 C and heat shock of 42 C than the control cells. The results suggested that CiHsp90 could adopt similar strategies to protect cells under different extreme environmental conditions. On the contrary, the amino-terminal deletion mutant (6N-CiHsp90) abolished the protective effect, which demonstrated that the full-length protein was required for CiHsp90’s protection activity. The previous studies demonstrated that Hsp90 could suppress the aggregation of many unstable proteins, such as protein kinase CK-II at low ionic strength [14,36]. Based on kinetic studies, Jakob et al. (1995) proposed that Hsp90 could bind to early unfolding intermediates, preventing their irreversible aggregation [37]. In aerobic organisms, the tricarboxylic acid cycle (TCA cycle) was part of a metabolic pathway involved in the chemical conversion of carbohydrates, fats and proteins into carbon dioxide and water to generate ATP. Citrate synthase (CS), a key pace-making enzyme in the first step of the TCA cycle, catalyzed the condensation reaction of acetyl coenzyme A and oxaloacetate to form citrate. CS was very sensitive to thermal inactivation and aggregates rapidly, which had been used as a well established model substrate protein to analyze the chaperone function of Hsp [37]. For example, some small Hsps such as Xenopus 30C could act as a molecular chaperone and prevent heat-induced CS aggregation by binding to the target protein and maintaining it in solution [35]. Here, we found that both CiHsp90 and 6N-CiHsp90 were very efficient in suppressing the thermal aggregation of CS. It indicated that there was little correlation between the prevention of protein aggregation and the cell protective activity [38]. Thus, the cellular protective mechanism of CiHsp90 under stress conditions was complex that to be further explored. As Hsp90s could form complexes with a wide range of protein kinases, mechanisms for Hsp90 involved in the cellular process might be very complicated. In mammals, Hsp90 participated in the regulation of protein synthesis by interacting with heme-regulated inhibitor (HRI), resulting in apoptosis [39]. Therefore, the exact biochemical functions of CiHsp90 in fish cells remained to be determined.
Acknowledgements Fig. 5. CiHsp90 protect CS from heat-induced aggregation. CS (0.1 mM) were incubated at 42 C in the absence (B) or presence of CiHsp90 (at a 1:1 (:) or 2:1 (6) molar ratio) and 6N-CiHsp90 (at a 1:1 (,) or 2:1 (,) molar ratio) as indicated, respectively. As the control, IgG (C) was added to CS at concentration of 0.5 mM in the absence of CiHsp90. The data are representative of 4 trials and calculated as a percentage of the maximum aggregation of CS after 60 min and was expressed as the mean SD.
This work was supported by grants from the Science and Technology Project of Jiangxi Province, China (No. 20111BBF60020) and from the Research Foundation of Education Bureau of Jiangxi Province, China (No. GJJ12662).
C.-X. Wu et al. / Fish & Shellfish Immunology 33 (2012) 42e47
References [1] Ritossa F. A new puffing pattern induced by temperature shock and DNP in Drosophila. Cell Mol Life Sci 1962;18(12):571e3. [2] Buchner J. Supervising the fold: functional principles of molecular chaperones. FASEB J 1996;10:10e9. [3] Dong CW, Zhang YB, Zhang QY, Gui JF. Differential expression of three Paralichthys olivaceus Hsp40 genes in responses to virus infection and heat shock. Fish Shellfish Immunol 2006;21(2):146e58. [4] Csermely P, Schnaider T, Soti C, Prohászka Z, Nardai G. The 90-kDa molecular chaperone family: structure, function, and clinical applications. A comprehensive review. Pharmacol Ther 1998;79(2):129e68. [5] Buchner J. Hsp90 & Co. e a holding for folding. Trends Biochem Sci 1999;24: 136e41. [6] Young JC, Moarefi I, Hartl FU. Hsp90: a specialized but essential proteinfolding tool. J Cell Biol 2001;154:267e73. [7] Zhao R, Davey M, Hsu YC, Kaplanek P, Tong A, Parsons AB, et al. Navigating the chaperone network: an integrative map of physical and genetic interactions mediated by the hsp90 chaperone. Cell 2005;120:715e27. [8] Palmisano AN, Winton JR, Dickhoff WW. Sequence features and phylogenetic analysis of the stress protein hsp90alpha in chinook salmon (Oncorhynchus tshawytscha), a poikilothermic vertebrate. Biochem Biophys Res Commun 1999;258:784e91. [9] Hermesz E, Abraham M, Nemcsok J. Identification of two hsp90 genes in carp. Comp Biochem Physiol C 2001;129:397e407. [10] Cara JB, Aluru N, Moyano FJ, Vijayan MM. Food-deprivation induces HSP70 and HSP90 protein expression in larval gilthead sea bream and rainbow trout. Comp Biochem Phys 2005;142:426e31. [11] Ali A, Krone PH, Pearson DS, Heikkila JJ. Evaluation of stress-inducible hsp90 gene expression as a potential molecular biomarker in Xenopus laevis. Cell Stress Chaperones 1996;1:62e9. [12] Wang B, Li F, Dong B, Zhang X, Zhang C, Xiang J. Discovery of the genes in response to white spot syndrome virus (WSSV) infection in Fenneropenaeus chinensis through cDNA microarray. Mar Biotechnol 2006;8:491e500. [13] Freeman BC, Morimoto RI. The human cytosolic molecular chaperones hsp90, hsp70 (hsc70) and hdj-1 have distinct roles in recognition of a non-native protein and protein refolding. EMBO J 1996;15:2969e79. [14] Miyata Y, Yahara I. The 90-kDa heat shock protein, HSP90, binds and protects casein kinase II from self-aggregation and enhances its kinase activity. J Biol Chem 1992;267:7042e7. [15] Picard D. Heat-shock protein 90, a chaperone for folding and regulation. Cell Mol Life Sci 2002;59:1640e8. [16] Lele Z, Hartson SD, Martin CC, Whitesell L, Matts RL, Krone PH. Disruption of zebrafish somite development by pharmacologic inhibition of Hsp90. Dev Biol 1999;210(1):56e70. [17] Sreedhar AS, Kalmar E, Csermely P, Shen YF. Hsp90 isoforms: functions, expression and clinical importance. FEBS Lett 2004;562:11e5. [18] Sreedhar AS, Csermely P. Heat shock proteins in the regulation of apoptosis: new strategies in tumor therapy: a comprehensive review. Pharmacol Ther 2004;101:227e57. [19] Sass JB, Weinberg ES, Krone PH. Specific localization of zebrafish hsp90 alpha mRNA to myoD-expressing cells suggests a role for hsp90 alpha during normal muscle development. Mech Dev 1996;54:195e204. [20] Sumanas S, Larson JD, Miller Bever M. Zebrafish chaperone protein GP96 is required for otolith formation during ear development. Dev Biol 2003;261: 443e55.
47
[21] Du SJ, Li H, Bian Y, Zhong Y. Heat-shock protein 90alpha1 is required for organized myofibril assembly in skeletal muscles of zebrafish embryos. Proc Natl Acad Sci USA 2008;105:554e9. [22] Queitsch C, Sangster TA, Lindquist S. Hsp90 as a capacitor of phenotypic variation. Nature 2002;417:618e24. [23] Sollars V, Lu X, Xiao L, Wang X, Garfinkel MD, Ruden DM. Evidence for an epigenetic mechanism by which Hsp90 acts as a capacitor for morphological evolution. Nat Genet 2002;33:70e4. [24] Roberts RJ, Agius C, Saliba C, Bossier P, Sung YY. Heat shock proteins (chaperones) in fish and shellfish and their potential role in relation to fish health: a review. J Fish Dis 2010;33:789e801. [25] Zhang XZ, Dai LP, Wu ZH, Jian JC, Lu YS. Expression pattern of heat shock protein 90 gene of humphead snapper Lutjanus sanguineus during pathogenic Vibrio harveyi stress. J Fish Biol 2011;79:178e93. [26] Chen YM, Kuo CE, Wang TY, Shie PS, Wang WC, Huang SL, et al. Cloning of an orange-spotted grouper Epinephelus coioides heat shock protein 90AB (HSP90AB) and characterization of its expression in response to nodavirus. Fish Shellfish Immunol 2010;28:895e904. [27] Lee GJ, Pokala N, Vierling E. Structure and in vitro molecular chaperone activity of cytosolic small heat shock proteins from pea. J Biol Chem 1995;270: 10432e8. [28] Marzec M, Eletto D, Argon Y. GRP94: an HSP90-like protein specialized for protein folding and quality control in the endoplasmic reticulum. Biochim Biophys Acta 2012;1823(3):774e87. [29] Eletto D, Dersh D, Argon Y. GRP94 in ER quality control and stress responses. Semin Cell Dev Biol 2010;21:479e85. [30] Wang SY, Edens FW. Stress-induced heat-shock protein synthesis in peripheral leukocytes of turkeys, Meleagris gallopavo. Comp Biochem Physiol 1993; 106:621e8. [31] Gao Q, Zhao J, Song L, Qiu L, Yu Y, Zhang H, et al. Molecular cloning, characterization and expression of heat shock protein 90 gene in the haemocytes of bay scallop Argopecten irradians. Fish Shellfish Immunol 2008;24:379e85. [32] Manchado M, Salas-Leiton E, Infante C, Ponce M, Asensio E, Crespo A, et al. Molecular characterization, gene expression and transcriptional regulation of cytosolic HSP90 genes in the flatfish Senegalese sole (Solea senegalensis Kaup). Gene 2008;416:77e84. [33] Kawabe S, Yokoyama Y. cDNA cloning and expression of grp94 in the Pacific oyster Crassostrea gigas. Comp Biochem Physiol B 2009;154(3):290e7. [34] Soto A, Allona I, Collada C, Guevara MA, Casado R, Rodriguez-Cerezo E, et al. Heterologous expression of a plant small heat-shock protein enhances Escherichia coli viability under heat and cold stress. Plant Physiol 1999;120: 521e8. [35] Fernando P, Heikkila JJ. Functional characterization of Xenopus small heat shock protein, Hsp30C: the carboxyl end is required for stability and chaperone activity. Cell Stress Chaperones 2005;5:148e59. [36] Miyata Y, Yahara I. Interaction between casein kinase II and the 90-kDa stress protein, HSP90. Biochemistry 1995;34:8123e9. [37] Jakob U, Lilie H, Meyer I, Buchner J. Transient interaction of Hsp90 with early unfolding intermediates of citrate synthase. Implications for heat shock in vivo. J Biol Chem 1995;270:7288e94. [38] Johnson BD, Chadli A, Felts SJ, Bouhouche I, Catelli MG, Toft DO. Hsp90 chaperone activity requires the full-length protein and interaction among its multiple domains. J Biol Chem 2000;275:32499e507. [39] Pal JK. Hsp90 regulates protein synthesis by activating the heme-regulated eukaryotic initiation factor 2a (eIF-2a) kinase in rabbit reticulocyte lysates. J Biosci 1998;23:353e60.