The intertidal copepod Tigriopus japonicus small heat shock protein 20 gene (Hsp20) enhances thermotolerance of transformed Escherichia coli

BBRC Biochemical and Biophysical Research Communications 340 (2006) 901–908 www.elsevier.com/locate/ybbrc

The intertidal copepod Tigriopus japonicus small heat shock protein 20 gene (Hsp20) enhances thermotolerance of transformed Escherichia coli Jung Soo Seo a, Young-Mi Lee a, Heum Gi Park b, Jae-Seong Lee

a,*

a

b

Department of Molecular and Environmental Bioscience, Graduate School, Hanyang University, Seoul 133-791, Republic of Korea Faculty of Marine Bioscience and Technology, College of Life Sciences, Kangnung National University, Gangneung 210-702, Republic of Korea Received 7 December 2005 Available online 27 December 2005

Abstract To understand the role of the Tigriopus japonicus Hsp20 gene, we isolated this gene from a whole body cDNA library and found two heat shock factor elements at the 5 0 -UTR. The transformed bacteria containing Tigriopus Hsp20 showed thermotolerance against heat shock (54
C) with different ranges of time. The Tigriopus Hsp20 gene is comprised of 174 amino acid residues and shows similarity to Caenorhabditis elegans (27% identity), silkworm (24.1% identity), moth (24.1% identity), Mexican tetra (19.5% identity), zebrafish (19.5% identity), and spiny dogfish (17.2% identity) genes, but shows more similarity in the C-terminal region that contains an a-crystallin domain. Protein motifs such as an N-glycosylation site (67–70 NKSE) and a casein kinase II phosphorylation site were found in Tigriopus Hsp20. The genomic structure of the Tigriopus Hsp20 gene did not contain introns. To characterize the biochemical characteristics of the Tigriopus Hsp20 protein, we expressed Tigriopus Hsp20 in Escherichia coli and purified the soluble protein via 6· His-tag chromatography. To analyze the gene expression of Tigriopus Hsp20 against environmental stresses (e.g., water temperature and salinity), we performed a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). On exposure to different salinities, significant change in the expression of Tigriopus Hsp20 was not observed. However, upon heat shock (30
C), Tigriopus Hsp20 expression was significantly increased, but in the case of cold shock (4 or 10
C), expression was likely downregulated. These findings provide a better understanding of cellular protection mechanisms against environmental stress such as heat shock.  2005 Elsevier Inc. All rights reserved. Keywords: Tigriopus japonicus; Hsp20; cDNA library; Copepod; Crustacea; Thermotolerance

Small heat shock proteins consist of a family of stressinducible molecular chaperones that range in size from 12 to 43 kDa and also include lens protein (e.g., a-crystallin), as they too are induced by stress and share many of the same physical and functional properties as other small heat shock proteins [1–4]. In general, the small heat shock proteins (12–43 kDa) containing aA-crystallin and aB-crystallin, heat shock protein 20 (Hsp20), and heat shock protein 27 (Hsp27) share a significant degree of sequence homology, one example being the a-crystallin domain (consisting of 80–100 amino acids). In fact, all the small heat shock proteins form high molecular weight aggregates with a molecular mass of approximately 200–800 kDa under *

Corresponding author. Fax: +82 2 2299 9450. E-mail address: [email protected] (J.-S. Lee).

0006-291X/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.12.086

conditions of cellular stress [5]. This type of multimerization is the result of interaction among small heat shock protein subunits and is believed to be crucial for chaperone activity [2,6]. Thus, under heat shock conditions, they can be refolded to their native state by other ATP-dependent molecular chaperones [2–4]. The specific physiological responses of Hsp20 in the copepod are little known. To date, previous work on Hsp20 has been carried out in mammalian, fish, Drosophila, bacterial or plant model systems [2,6–9]. In mammals, Hsp20 is associated with actin in vitro dependent on its phosphorylation state The copepod Tigriopus japonicus is a useful animal model species for toxicology, genetics, and molecular biology [10]. They inhabit rock pools of the intertidal zone of seashores in temperate and subtropical regions such as Korea,

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Japan, and China. As these regions have different ocean currents (e.g., cold or warm currents), T. japonicus benefits from the evolution of proper cellular defense systems triggered by significant environmental stress, especially changes of water temperature or salinity. In addition, the intertidal copepod Tigriopus sp. is a particularly interesting model species for studying the phylogenetics of copepod evolution, as their populations show a wide range of genetic divergence [11]. Based on the advantages using this species for study, we previously sequenced 686 expressed sequence tags (ESTs) from T. japonicus [10] for potential use in ecotoxicogenomics. Of the 686 ESTs, we determined that the T. japonicus small heat shock protein 20 (Tigriopus Hsp20) gene may play a role in modulating cellular defense systems in response to environmental stress. In this paper, we first show the full-length cDNA sequence of Tigriopus Hsp20 with genomic DNA information, compared its amino acid residues in other species, and investigated the upand/or downregulation of the Tigriopus Hsp20 gene after environmental stresses (e.g., temperature and salinity) with extensive examination of the thermotolerance function of the Tigriopus Hsp20 recombinant protein. Materials and methods Chemicals and reagents. All chemicals and reagents used in this study were of molecular biology grade and were purchased from Sigma Chemicals, (St. Louis, Mo), Qiagen (Valencia, CA), or Invitrogen, (Carsbad, CA) unless otherwise stated. All oligonucleotide synthesis and DNA nucleotide sequencing were performed at the Bionex (Seoul, South Korea). Culture of T. japonicus and total RNA extraction. The intertidal copepod T. japonicus was maintained in the Department of Molecular and Environmental Bioscience, Hanyang University, Seoul, Korea. The identity of the species was confirmed by morphological characteristics and the sequence similarity of mitochondrial genome sequences [12]. Whole body tissues were first ground with a pestle and then homogenized in three volumes of TRIZOL with a tissue grinder. The total RNA was extracted according to the manufacturer’s suggestions, and isolated mRNA was kept at
80
C until use. cDNA and genomic DNA cloning of T. japonicus Hsp20 gene and comparison to other homologues. Expressed sequence tags (ESTs) of T. japonicus were collected by random sequencing analysis after conversion of the T. japonicus kZAPII cDNA library to pBluescript phagemid [10]. One full-length cDNA of the Tigriopus Hsp20 gene was chosen and used to search GenBank to find homologues from other species. To reveal the genomic structure of the Tigriopus Hsp20 gene, we amplified the Tigriopus Hsp20 genomic clone with two primers (TJ-Hsp20-genom-F: 5 0 -ATA AAT CGA AGA AAA AGT GCT AC-3 0 , TJ-Hsp20-stop-R: 5 0 -GGA AGA CAA TGA AGA CTA G-3 0 ) and subcloned it into a pCR2.1 TA cloning vector (Invitrogen) for sequence analysis in both directions. Thermotolerance of E. coli BL21(DE3)pLyS expressing Tigriopus Hsp20. The thermotolerance measurement was carried out, as previously reported [13], with a minor modification made by measuring the colonyforming units (cfu) after heat shock (54
C). Briefly, E. coli BL21(DE3)pLyS were transformed with either Tigriopus Hsp20 in the pCR T7 TopoN vector or the pCR T7 TopoN vector-self and inoculated at 30
C for overnight according to the above procedures. Before heat shock, 0.5 ml of culture was diluted 1:10 in fresh medium supplemented with 100 lg/ml chloramphenicol and 100 lg/ml amphicillin. Cultures were incubated at 54
C in a water bath; 100 ll samples were removed after 0, 15, 30, 45, and 60 min of heat shock, diluted in cold LB broth, and maintained on ice prior to plating in duplicate on LB agar plates. Colonies were counted after incubation for 18 h at 37
C. To verify the presence of Tigriopus

Hsp20, 0.5 ml of isopropyl-1-b-D-thiogalactopyranoside (IPTG) induced culture was removed prior to heat shock, cells were collected by centrifugation for 1 min at top speed in a microcentrifuge, resuspended in 50 ll of 1· sample buffer, placed in boiling water for 10 min, and frozen at
80
C before electrophoresis in SDS–PAGE, blotting to nitrocellulose, and immunodetection. Phylogenetic tree. Phylogenetic analysis was conducted using Bayesian analysis with Mr. Bayesian’s program (Ver. 3.1.1.) using the amino acid sequences of Hsp20 and crystallin from various animal species. All sequences except T. japonicus Hsp20 were retrieved from GenBank. The retrieved sequences were aligned with a Clustal X program [14] by the multiple alignment method. The gaps and missing data were completely excluded from the data analysis. The generated data matrix was converted to nexus format. This data matrix was analyzed with Mr. Bayesian’s program using the GTR model. The Markov chain Monte Carlo process was set to four chains, and 1,000,000 generations were conducted. The sampling frequency was assigned as every 100 generations. After analysis, the first 1000 trees were deleted as burn-in processes and the consensus tree was constructed. The phylogenetic trees were visualized with TreeView of PHYLIP [15]. Construction of the T. japonicus Hsp20 construct, expression and purification of recombinant T. japonicus Hsp20 in E. coli. To obtain a contiguous Tigriopus Hsp20 open-reading frame (ORF), we amplified the Tigriopus Hsp20 cDNA clone with two primers (TJ-Hsp20-start: 5 0 -ATG ACT AAG AGA TTC TCT AAA TC-3 0 , TJ-Hsp20-stop: 5 0 -CTA GTC TTC ATT GTC TTC C-3 0 ), electrophoresed the PCR product, excised the required band from the gel, and eluted it with an elution kit (Qiagen). Subsequently, we ligated the PCR product to a 6· His-tagged pCR T7 TopoN TA expression vector (Invitrogen). To overexpress the Tigriopus Hsp20 protein, E. coli strain BL21(DE3)pLyS was transformed with the Tigriopus Hsp20/pCR T7 TopoN vector. Transformed cells were grown in LB medium containing 100 lg/ml chloramphenicol and 100 lg/ml amphicillin. At a cell density (A600) of 0.7, gene expression was induced by adding IPTG at a final concentration of 1 mM. Transformed cells were harvested after 18 h incubation at 30
C and directly analyzed by SDS–PAGE. Collected cells were resuspended in ice-cold 1· homogenizing buffer (20 mM Tris, pH 7.9, 0.5 M NaCl) at 10 ml per g wet weight. Lysozyme was added at a final concentration of 200 lg/ml and the cells were incubated at 30
C for 15 min. Cells were then sonicated three times for 1 min per burst with a sonicator (Branson, USA) at a setting of 20%. The homogenate was centrifuged at 20,000g at 4
C for 20 min for separation into supernatant and pellet fractions. The supernatant and pellet fractions were used for protein purification and native structure or antibody production of active Tigriopus Hsp20, respectively. To purify the expressed Tigriopus Hsp20 protein, the His-bind system (Invitrogen, USA) was employed. Briefly, the supernatant fraction was applied to the His-bind column (1 ml settled bed volume). After column washing (30 ml) with 1· wash buffer (20 mM Tris, pH 7.9, 60 mM imidazole, and 0.5 M NaCl), recombinant Tigriopus Hsp20 was eluted in 10 fractions (10 ml) with 1· elution buffer (20 mM Tris, pH 7.9, 1 M imidazole, and 0.5 M NaCl). The pooled fractions were analyzed by SDS–PAGE and Western blotting. Protein concentration was determined with a Bio-Rad protein assay kit. Polyacrylamide gel electrophoresis and Western blotting. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (PAGE) was performed as described by Laemmli [16]. All samples were denatured in buffer containing 60 mM Tris, pH 6.8, 25% glycerol, 2% SDS, 14.4 mM 2-mercaptoethanol, and 0.1% bromophenol blue, boiled for 5 min, and separated by 12% SDS–PAGE (Bio-Rad, USA). Pre-stained molecular weight markers (Bio-Rad, USA) were run as standards on each gel. Electrophoresed proteins were transferred to a nitrocellulose membrane (Schleicher & Schuell, USA) using the method of Towbin and Gordon [17] with a Bio-Rad Mini Protean III transblotting system (Bio-Rad, USA). Following transfer of proteins to a membrane, the membrane was blocked with 3% BSA in TTBS (200 mM Tris, pH 7.0, 1.37 M NaCl, and 1% Tween 20) for 1 h at room temperature. The membrane was then incubated with anti-His G-HRP antibody (1:5000) (Invitrogen,

J.S. Seo et al. / Biochemical and Biophysical Research Communications 340 (2006) 901–908 USA) at room temperature for 3 h and rinsed three times with TTBS for 60 min at room temperature. Detection was performed with an ECL plus Western Blotting kit (Amersham, USA). Non-denaturing PAGE was performed on a 12% polyacrylamide gel according to Anderson et al. [18]. Effects on gene expression pattern after environmental stress. To analyze expression pattern of the Tigriopus Hsp20 gene upon exposure to environmental stresses such as salinity or temperature change, different concentrations of saline solutions (e.g., 0&, 10&, 30&, and 40&) were used to treat T. japonicus adults (approximately 100 individuals) for 24 days and 48 days. To see the effects of temperature, we exposed T. japonicus adults (approximately 100 individuals) to different temperatures of water (4, 10, and 30
C) for 5, 10, 20, 30, 60, 90, 120, and 180 min. After exposure, the exposed adult T. japonicus were maintained at 18
C with a 12 h light/12 h dark photoperiod and 20& salinity. After environmental stress exposure, total RNA was extracted from T. japonicus and stored at
80
C prior to use. To determine the level of gene expression, we used two RT-PCR primers as mentioned above. Internal control primers (TJ-GAPDH-F, 5 0 -GAT CTG GAC AGA ACA TCA TC-3 0 ; TJ-GAPDH-R, 5 0 -GAA TAC CCC AAG TAT CCC TTC-3 0 ; expected product 250 bp) were used with TJ-Hsp20 primers (TJ-Hsp20-F, 5 0 -AAT CGG AAT ACA AAG ATG GAA CA-3 0 ; TJ-Hsp20-R, 5 0 -CTT CAC ATC CTT CAT TTG ACA ATT-3 0 ; expected product 350 bp) in the same reaction to normalize the input amount of template first-strand cDNAs. We followed the conventional method of RT-PCR with the reaction mixture (1 ll of firststrand cDNA, 5 ll of 10· PCR reaction buffer, 1 ll of 10 mM dNTPs, 10 pM each primer, 0.5 ll of NeoTherm Taq polymerase (GeneCraft, Germany)) subjected to amplification (1 cycle, 95
C, 5 min; 30 cycles,

A

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94
C, 30 s, 55
C, 30 s, and 72
C, 30 s; 1 cycle, 72
C, 7 min) using an iCycler (Bio-Rad). The relative level of gene expression was quantified after we checked the intensity of the ethidium bromide-stained bands from T. japonicus Hsp20 and T. japonicus GAPDH genes. These experiments were carried out in at least triplicate. For statistical analysis, we employed Student’s t test.

Results and discussion Cloning of Tigriopus Hsp20 gene Of the 686 ESTs sequenced, we isolated one clone that showed high similarity to the nematode Nippostrongylus brasiliensis Hsp20 gene, which shares a similar a-crystallin domain. The T. japonicus Hsp20 gene consists of 174 amino acid residues with a 1014 bp transcript (Fig. 1). This gene was registered with GenBank under AY522572. In the 3 0 untranslated region of this gene, a poly (A) signal sequence (AATAAA) was found 10 bp upstream of the poly (A) tail (Fig. 1). This is usually observed in vertebrate genes but in Tigriopus genes, two kinds of poly (A) signal sequences such as AATAAA and AATTTA were observed. Most cDNA transcripts had usual poly (A) signal sequences (AATAAA), but 15–30% of cDNAs had another type of poly (A) signal sequence such as AATTTA. This would B

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Hsp20 Hsp20

C

ATAAATCGAAGAAAAAGTGCTACAAACGACACGAAAAATGGCTCTTTTGTTTCCCATGAGCTCTGAACGTGACTTTTTCC ATCCTCATTCCTTACTGAGTGAGGATATCTTCGGATTCCCTTGGGTGTCATCCCGAAAGCGGAGATTAAATCGACCTAAA CCCATAACCAGATCCAAAAGGCCCCTTCCATCCGAGTGGAAAGATTGTCGGGATCACTTCGAAGCAAAACTTGCCGTAAA AGGGTTCAATGCTGACGATTTCCATTTGGGCCTCAACAACTCTTATACAAGATTAACCATTGTGGCCAATGCTGAAGAAC AAGGCGAGGATGGAGTGGTTATGACTAAGAGATTCTCTAAATCCTTGGATCTCCCCGAGGATTGCCTCCCCGATGATCTC M T K R F S K S L D L P E D C L P D D L GAATCGGAATACAAAGATGGAACACTCCATTTGACAGTTCCTAAATATGTTCCTGAGAAGAGGCTTCGGAAGAACGAGGA E S E Y K D G T L H L T V P K Y V P E K R L R K N E D TGAGGATTCTCCCTTCGAAATCATTCCCAAACTCATGTCAGGGGAATTTTTGGATTCCAATAAGAGCGAAATTAAGGATT E D S P F E I I P K L M S G E F L D S N K S E I K D S CGAATGAGGCATTCCAATTGAATATGGATGTCTCTGGGTTCAAACCGGAAAATCTTCAAGTGGAACTCACTCCTAATGGT N E A F Q L N M D V S G F K P E N L Q V E L T P N G GTGATCAACATCTCTGGACATTTCGAGGATAAGAGTGAAGGTCGACACATTTCGCGACAAGTCCACAAATCGTTCACTTT V I N I S G H F E D K S E G R H I S R Q V H K S F T L GCCCAAGAATTGTCAAATGAAGGATGTGAAGTCTCGCTTGGACAACCAAGGGAAACTAACTATTACTGCACCCCAAGACC P K N C Q M K D V K S R L D N Q G K L T I T A P Q D P CAAATAAGGCTATTGGTAACGAACCGCGCAAATTGCCCATTAATTTGGAAGACAATGAAGACTAGAACGGGTCATGAGGT N K A I G N E P R K L P I N L E D N E D * GATTTGTAAATATGTATTGCTGGTCTCGAATTTTGTTGATGTTATTGTGAATATGACTTGAAATGTAAATAAACTTTGAA TAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (1014 bp)

Fig. 1. Nucleotide sequence of cDNA of T. japonicus Hsp20 gene and its deduced amino acid residues. (A) PCR amplification of T. japonicus Hsp20 cDNA gene. (B) PCR amplification of the T. japonicus Hsp20 genomic gene. (C) Nucleotide sequence of T. japonicus Hsp20 gene. The underlined sequences at the 3 0 -UTR indicate the poly(A) signal sequence. M, k/HindIII size marker (A) or 1 kb ladder (B).

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be an alternative to transcription of their cDNA information. Through the previous studies, small Hsps (heat shock proteins) have one or two introns in the coding region in higher vertebrates but not in lower chordates or nematodes [19]. As shown in Fig. 1B, genomic DNA PCR provided evidence that the Tigriopus Hsp20 gene is intronless. To analyze the 5 0 -untranslated region of the T. japonicus Hsp20 gene, we used the motif search site (motif.genome.jp) and found two matched sites for heat shock factors from Drosophila and yeast, as shown in Fig. 1C (underlined at the 5 0 -UTR) [20]. This may provide the thermotolerance potential of this gene in Tigriopus.

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Heat shock (min) Fig. 2. Thermotolerance of transformed T. japonicus Hsp20. Transformed E. coli BL 21(DE3)pLyS, grown as described in Materials and methods, were incubated at 54
C for the times indicated, plated on LB plate and incubated at 37
C for 18 h. Colonies were counted and the log10 values of colony-forming units (cfu) per milliliter were plotted against the length of heat shock in min. These experiments were performed in triplicate (means ± SD).

Tigriopus C.elegans Silkworm Moth Mexican tetra Zebrafish Spiny dogfish

To determine whether Tigriopus Hsp20 is able to defend against heat stress in E. coli, we tested the potential role of Tigriopus Hsp20 on thermotolerance after transforming E. coli BL21(DE3)pLyS with an expression vector containing the Tigriopus Hsp20 gene. As a control, a pCR/T7 Topo vector lacking a Tigriopus Hsp20 cDNA insert was used. After exposure to heat shock (54
C), the transformed bacteria containing Tigriopus Hsp20 showed significantly more resistance to heat shock, indicating that

-MTKRFSKSLDLPEDCLPDDLESEYKDGTLHLTVPKYVPEKRLRKNEDEDSPFEIIPKLM ----------------------MLMLRSPFSDSN--VLDHFLDEITGSVQF.YWRNADHN MSLLP.VLG-.W.RVRHNH-WP.RLV.QDFG.AL--TPNDMLAAVACPVL.EDYFR.WRQ MSLLP.VFGYES.RYYHHS-WP.RLI.QNFG.AL--TPD.MLTAVACPLL.TDYYR.WRQ ---MDIAIQHPWFRRA.G--YP.RLF.QFFGEGL--FDYDLFPYATSTVSPYYRYSLFRN ---MDIAIQHPWFRRT.G--YPTRLF.QFFGEGL—FDYDLFPFTTSTVSPYYRHSLFRN ---MDLAIQYPWFRRS.GSFYP.RLF.QFFGEGL--FDYDLFPFFSSTISPYYRQSVFRN : .

N-glycosylation site Tigriopus C.elegans Silkworm Moth Mexican tetra Zebrafish Spiny dogfish

SGEFLDSNKSEIKDSNEAFQLNMDVSGFKPENLQVELTPNGVINISGHFEDKSEGRHISR .FN.S.NIGEQ.VNDESK.SVQL...H....D.KI..DGRELKIEGIQ-.K...HGYSK. LAAASRDLG.S..ADKDK..V.L..QH.S..EIS.KTADGYIVVEGK.E.K.D.HGY... LAAAARDIG.N..ADKDKL.I.L..QH.S..EIS.KTADGF.VVEGK.E.K.D.HGY... FLDSSN.GM..VRSDRDK.MVYL..KH.S..E.N.KVAEDY.EIQGK.G.RQDDHGY... ILDSSN.GV..VRSDR.K.TVYL..KH.S.DE.S.KV.DDY.EIQGK.G.RQDDHGY... FLDS---GI..VRSEKDR.MI.LN.KH.S..E.S.KIVDDY.EIHGK.A.RQEDQGRV.. ..: ... : : ::*. *.*:::.:: : . : * :.: .*

Tigriopus C.elegans Silkworm Moth Mexican tetra Zebrafish Spiny dogfish

QVHKSFTLPKNCQMKDVKSRLDNQGKLTITAPQDP-NKAIGNEPRKLPINLEDNED---SFS.MIL..EDVDLTS...AIS.E...Q.E..KKT------.SS.SI...FVAKH----.FVRRYA..EGAAPET.E...SSD.V......RKVPDAVK.ERKVPIAQTGPVRKEIKDQ .FVRRYA..EGAASET.E...SSG.V......LKVPDAVK.ERKVPIAQTGPVRKEIKDQ EF.RRYR..S.VDQSAITCT.SAD.Q...CG.KSG-GSES.RGD.SI.VTRD.KTNSTPS EF.RRYR..S.VDQSAITCT.SAD.L..LCG.KTS-GIDA.RGD.TI.VTR..KSNSGSS EF.RTYH..S.LNESAIACS.S.E.L..LCC.KTRPGDDSNWQD.PI.VSR.EKQGTQPE .. : **.. : . :. * * : * :. . .

Tigriopus C.elegans Silkworm Moth Mexican tetra Zebrafish Spiny dogfish

------------------SEGTQDAENK SGKEKGNK-S--------S--------IRADP-----

Casein kinase II phosphorylation site

174 144 186 185 173 173 177

Fig. 3. Similarity of T. japonicus Hsp20 to other species. The symbol ‘‘*’’, ‘‘:’’, and ‘‘.’’ indicate identical residues, strongly positive residues, and weakly positive residues, respectively. N-glycosylation sites (67–70 NKSE) and casein kinase II phosphorylation sites (138–141 SRLD) are indicated by an overbar.

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Tigriopus Hsp20 provides thermotolerance to the transformed E. coli as shown in Fig. 2. To date, the mechanism of sHsp chaperone activity is poorly characterized but is hypothesized to involve temperature-induced rearrangement of the sHsp oligomer. Similarly, the small heat shock proteins, Hsp16-2 from Caenorhabditis elegans [6], p26 from Artemia [13], and human a-B crystallin [21], also enhance heat resistance of E. coli. Therefore, sHsp would protect a cell system during environmental stress as well as in prevention of irreversible aggregation of denaturing proteins [3,4,22]. In addition, the ATP-independent chaperone, which enhanced stress tolerance in a cell system, contained a specific a-crystallin structure [3,4,22]. Sequence analysis of Tigriopus Hsp20 Based on the translated Tigriopus Hsp20 gene, it was determined that the putative pI and molecular weight of Tigriopus Hsp20 were 5.12 and 19.84 kDa, respectively, as expected. To date, a few species are available to compare sequence similarity to the Hsp20 gene. Through PROSITE analysis [23], we also found that Tigriopus Hsp20 has several secondary modification motifs, such as an N-glycosylation site (67–70 NKSE) and a casein kinase II phosphorylation site (138– 141 SRLD) (Fig. 3). These biochemical characteristics would support its potential role in phosphorylation of Tigriopus Hsp20 in cells or tissues. As shown in Fig. 3, Tigriopus

Hsp20 showed more similarity to other species in the C-terminal region, compared to the N-terminal region. Phylogenetic tree To uncover the relationship of T. japonicus Hsp20 with other related genes, such as crystallin genes, we conducted a phylogenetic analysis using genes from different species as shown in Fig. 4. To do this analysis, we collected amino acid sequences from various kinds of Hsps and crystallins, resulting in a phylogenetical dissection of their relationship. As shown in Fig. 4, the tree was separated to three clades. One is a Hsp20 gene family and the two others were crystallin and Hsp27 families. The Hsp20 gene family showed a closer relationship to crystallin, indicating that Hsp20 would be derived from a-crystallin-type heat shock protein. The Hsp20 gene family clade was separated into two small clades: one containing a silkworm Bombyx mori Hsp20 and another containing the T. japonicus Hsp20 and nematode Hsp20. To obtain a more informative phylogenetic tree using these genes, it would be necessary to use more sequence information from diverse species. Expression and purification of recombinant Tigriopus Hsp20 To check the biochemical characteristics of Tigriopus Hsp20, we constructed a recombinant Tigriopus Hsp20

Bombyx mori hsp19.9 99 96

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Anopheles gambiae str. PEST Plodia interpunctella crystallin Bombyx mori hsp20.1

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Nipponstronglus brasiliensis hsp20 Caenorhabditis briggsae Hypothetical protein Artemia francisana crystallin Human crystallin

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Zebrafish hsp25 Topminnow hsp27

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Fig. 4. Phylogenetic tree (unrooted) of T. japonicus Hsp20 with the relevant genes of other species constructed by the Bayesian method.

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subunits. Kato et al. [24] reported that Hsp20 shows aggregated and dissociated forms with apparent molecular masses of 200–300 and 67 kDa, respectively. van de Klundert et al. [8] also detected two forms of mammalian Hsp20 with 470 and 43 kDa using size-exclusion chromatography that may convert to either form depending on protein concentration.

protein using an E. coli expression system. The recombinant Tigriopus Hsp20 was induced by adding IPTG to the culture of E. coli BL21(DE3)pLyS transformed with the pCR/T7 Topo vector containing Tigriopus Hsp20 gene. Eighteen hours after culture, the cells were harvested, disrupted, and separated into pellet and supernatant fractions. To purify the recombinant Tigriopus Hsp20, the Hisbind column was applied after the supernatant fraction of the recombinant Tigriopus Hsp20-expressing E. coli was separated. The recombinant Tigriopus Hsp20 was eluted from the column with buffer containing a high concentration of imidazole (1.0 M). Proteins in each fraction were analyzed to see the expression level of the recombinant Tigriopus Hsp20 protein by SDS–PAGE and Western blotting. A distinct protein band of approximately 24 kDa, similar to the size (19.84 kDa) predicted from its amino acid sequence, was detected in transformed but not in untransformed E. coli by SDS–PAGE (Fig. 5A). To further verify that the protein expressed would be His-tagged Tigriopus Hsp20, Western blot analysis was performed using a His-tag monoclonal antibody (Invitrogen, CA). This His-tag antibody was cross-reacted to a recombinant Tigriopus Hsp20 protein that corresponds in size to the expressed protein seen by protein staining (Fig. 5B). These results indicate that the expressed recombinant Tigriopus Hsp20 was purified with apparent homogeneity. To determine whether Tigriopus Hsp20 may form a homomultimer, native-PAGE was used for separation and revealed a hetero-oligomer complex of Tigriopus Hsp20 of 52 kDa (Fig. 5C). In relation to this result, many reports have previously detailed investigation of the biochemical properties of eukaryotic Hsp20 expressed from E. coli. Under the E. coli expression system, Hsp20 showed various homo- or hetero-oligomeric complexes of 2–40 12% SDS-PAGE

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Salinity (‰) Fig. 6. Gene expression of the T. japonicus Hsp20 gene after exposure to different salinities between 0&, 10&, 30& or 40&. Control was maintained in the condition of 20& salinity and a 20
C water temperature for the entire experiment. The relative expression of Tigriopus Hsp20 was obtained by dividing GAPDH expression. These experiments were performed in triplicate (mean ± SD), and Student’s t test was applied for statistical analysis.

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20 Fig. 5. Expression and purification of recombinant T. japonicus Hsp20. (A) Protein samples were separated by SDS–PAGE (12%) and visualized by Coomassie R-250 blue staining. (B) Western blot analysis using anti-His-tag monoclonal antibody. Antibodies were used in dilutions of 1:5000. Lane M, standard size marker; lane 1, pCR-T7TOPO vector-expressing E. coli induced with 1 mM IPTG for 18 h at 30
C; lane 2, whole cell lysate of Tigriopus Hsp20-expressing E. coli induced with 1 mM IPTG for 18 h at 30
C; lane 3, soluble fraction; lane 4, His-bind column purified fraction (3 lg of protein). (C) His-tag purified soluble Tigriopus Hsp20 was separated by native-PAGE (12%) and visualized by Coomassie R-250 blue staining. The positions of standard size markers (M) are shown on the left.

J.S. Seo et al. / Biochemical and Biophysical Research Communications 340 (2006) 901–908

Analysis of gene expression pattern after environmental stress To investigate whether different types of environmental stressors such as temperature and salinity may affect the A

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gene expression pattern of Tigriopus Hsp20, the organisms were exposed to different ranges of salinity (0&–40&). The Tigriopus Hsp20 gene was amplified by semi-quantitative RT-PCR but there were no differences between experimental groups (Fig. 6). Some information is available on the relationship of gene expression and salinity stress; for example, Burton and Feldman [25] and Willet and Burton [26] reported that proline biosynthesis genes or glutamatepyruvate transaminase plays a role in regulating cellular organic osmolytes during environmental salinity stress in the copepod Tigriopus californicus. Also, Henry et al. [27] reported that salinity-mediated carbonic anhydrase induction in crustacea plays a role in modulating cellular organic osmolytes during environmental salinity stress. When we exposed the copepod T. japonicus to a different spectrum of temperatures, the expression pattern of Tigriopus Hsp20 increased in a time-dependent manner after a non-lethal incubation at 30
C (Fig. 7) under various experiment temperatures (4–30
C). However, the expression pattern of the Tigriopus Hsp20 gene was decreased time-independently at 4 and 10
C (Fig. 7). These results suggest that Tigriopus Hsp20 shows increased mRNA expression upon water temperature stress but not upon salinity. Arrigo and Landry [5] and other researchers reported that expression of the heat shock protein family increased thermal tolerance upon heat shock [28,29]. However, Liang and MacRae [30] reported that synthesis of small heat shock protein 26 (p26) in Artemia was not induced by heat under the experimental conditions. This suggests that the Tigriopus Hsp20 gene may have other functional characteristics compared to those of p26 from the brine shrimp Artemia.

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This work was supported by a Korea Research Foundation grant (2004; Grant No. C00013) funded to Jae-Seong Lee. We also thank Dr. Sang-Oun Jung for his excellent assistance to make the phylogenetic tree, and Mr. Tae-Jin Park for his help to maintain the T. japonicus. References

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Fig. 7. Gene expression of T. japonicus Hsp20 gene after exposure to different temperatures between 4, 10 or 30
C. Control was maintained in the condition of 20& salinity and 20
C water temperature for the entire experiment. The relative expression of Tigriopus Hsp20 was obtained by dividing GAPDH expression. These experiments were performed in triplicate (mean ± SD), and Student’s t test was applied for statistical analysis. Con, control group. *Significantly different from control (p < 0.05).

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