Identification of the small heat shock protein,
HSP21, of shrimp Penaeus monodon and the
gene expression of HSP21 is inactivated after
white spot syndrome virus (WSSV) infection
Po-Yu Huang
a, Shih-Ting Kang
b, Wei-Yu Chen
b, Tai-Ching Hsu
c,
Chu-Fang Lo
b, Kuan-Fu Liu
d, Li-Li Chen
a,*
a
Institute of Marine Biology, National Taiwan Ocean University, No. 2, Pei-Ning Road, Keelung 20224, Taiwan, ROC
bInstitute of Zoology, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei 10617, Taiwan, ROC c
Department of Biomedical Sciences, Chung Shan Medical University, No. 110, Section 1, Chien-Kuo North Road, Taichung 40201, Taiwan, ROC
d
Tungkang Biotechnology Research Center, Fisheries Research Institute, No. 68, Feng-Yu Street, Tungkang, Pingtung 92845, Taiwan, ROC
Received 24 April 2008; revised 9 June 2008; accepted 10 June 2008 Available online 18 June 2008
KEYWORDS
White spot syndrome virus (WSSV);
Small heat shock protein (smHSP);
Penaeus monodon; Chaperone activity; Expressed sequence tag (EST)
Abstract The white spot syndrome virus (WSSV) is the causative agent of a severe disease in cultivated shrimp. The virus causes high mortality and leads to heavy stress on shrimps. In response to a variety of stresses, living organisms express particular sets of genes such as HSPs. In this study, a HSP21 gene, categorized into the small heat shock protein (smHSP) family, of shrimp Penaeus monodon was identified by annotating the EST databases established from WSSV-infected and WSSV-uninfected shrimp. The shrimp HSP21 gene was 555 bp in length. The thermal aggregation assay showed that the HSP21 had chaperone activity. The result of real-time PCR indicated that HSP21 was constitutive and inducible and was highly expressed in almost all organs such as the epithelium, gill, stomach, midgut, lymphoid organ, hepatopan-creas, nervous tissue and heart, but less expressed in haemolymph. However, HSP21 gene showed down-regulation after WSSV infection. It suggests that gene regulation of HSP21 was seriously affected by WSSV.
ª 2008 Elsevier Ltd. All rights reserved.
Introduction
Aquaculture industry is an important economic income of many countries. However, it is threatened by many path-ogens, especially viruses. Among these viruses, the white
* Corresponding author. Tel.:þ886 2 24622192x5302; fax: þ886 2
24633152.
E-mail address:[email protected](L.-L. Chen).
1050-4648/$ - see front matterª 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fsi.2008.06.002
a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m
spot syndrome virus (WSSV) is the most serious one. While the WSSV infection mechanism and the host responses induced by WSSV infection stay unclear, studies on investigating the host immune response after WSSV infection and prophylactic/therapeutic methods become pertinent[1e5]. Moreover, WSSV structural proteins such as VP19, VP28 and VP26 have been adopted to develop a prophylactic vaccine against WSSV[6,7]. In spite of this, to investigate the molecules and mechanisms relevant to the immune response is still relevant fundamental work.
To date, many approaches, such as expressed sequence tags (ESTs), RT-PCR, microarray chips and so on, have attempted to identify virus-host interactions [8]. Among these methods, ESTs have widely been applied as the foun-dation sequence of some genome-scale analyses. Such reconstructomic analyses use the EST cluster assemblies and singletons as an equivalent to a whole genome’s gene collection. EST derived cluster sequences have been widely annotated with tentative functions[9]. Recently, a series of heat shock proteins (HSPs) were identified while widely annotating the EST databases established from many crus-taceans. In response to a variety of stresses such as temper-ature extremes, xenobiotics, heavy metals, UV, oxidizing agents, or high levels of growth hormones, living organisms express particular sets of genes such as HSPs. HSPs are expressed for the stress tolerance and promote cell survival, especially by refolding proteins and preventing their denaturation[10,11], as well as participating in a vari-ety of normal cellular processes including protein traffick-ing, signal transduction, DNA replication and protein synthesis [12]. The HSP genes are highly conserved in all eukaryotes and prokaryotes, and based on the sequence homology and molecular weight of proteins, the genes have been divided into families such as hsp110, hsp100, hsp90, hsp70, hsp60, hsp40, and hsp20 [13]. These gene families consist of stress-inducible and constitutively expressed genes. Inducible genes are expressed at low levels under non-stress conditions but are expressed increasingly in response to stress. Nevertheless, basal levels of constitutive genes are high and change relatively little in response to stress[14,15].
In this study, we identified a HSP21 gene, categorized into the small heat shock protein (smHSP) family, of shrimp Penaeus monodon. The results showed that the HSP21, with chaperone activity, was constitutive and heat-inducible. We also analyzed the transcriptional changes after WSSV infection. However, real-time PCR revealed that the HSP21 gene was down-regulated after WSSV infection suggesting that gene regulation of HSP21 was seriously affected by WSSV.
Materials and methods
Virus, EST database and EST-based transcription analysis
WSSV, referred to as the WSSV Taiwan-1 strain (WSSV T-1 strain), collected in 1994 in Taiwan from infected P. mono-don [16] was used to construct WSSV genomic libraries. At the same time, WSSV-infected and WSSV-uninfected ESTs, named as PmTwI and PmTwN, respectively, were
constructed using total RNA isolated from the WSSV T-1 strain infected and uninfected P. monodon postlarvae (PL20). The details of library-construction and transcription analysis of identified protein genes were described in Wang et al.[8].
Amino acid sequence comparison and phylogenetic construction
The complete coding sequence was compared with the GenBank database using the program BlastX. Amino acid sequences of nine small heat shock proteins (smHSPs) isolated from other invertebrate animals were retrieved from GenBank, and were used for sequence alignment and phylo-genetic analysis. Multiple sequence alignment was done using the program ClustalX and edited with Genedoc software. The complete coding sequences of these smHSPs were subse-quently subjected to phylogenetic analysis and were per-formed with Mega4.0 software using the Neighbor-Joining algorithm. One thousand bootstrap replicates were gener-ated to test the robustness of the trees. Sequences used in the alignment analysis and phylogenetic tree, followed by their GenBank accession number, were Nasonia vitripennis (XP_001607669.1), Locusta migratoria (ABC84493.1), Apis mellifera (XP_392405.3), Bombyx mori (NP_001036985.1), Heliconius erato (ABS57447.1), Tribolium castaneum (ABS57447.1), Aedes aegypti (XP_001657982.1), Drosophila melanogaster (NP_608326.1), and Caenorhabditis elegans (NP_001024375.1).
Experimental animals, tissue expression of HSP21 transcription and heat shock experiment
Tiger shrimp P. monodon (body length, 20 2 cm; weight, 30 5 g) were collected from the Tungkang Biotechnology Research Center located in southern Taiwan. The organs eepithelium, gill, stomach, midgut, lymphoid organ, hepa-topancreas, nervous tissue, haemolymph, and heart e were collected from P. monodon.
Shrimps were kept in a plastic tank supplied with seawater with 3% salinity. In the heat shock experiment, three shrimps were transferred from water maintained at room temperature of 24 1C to a tank containing
seawa-ter preheated to 36 1C. After 1 h at 36 1C, as the
heat shock treatment, the temperature of the water was decreased within 1 min to room temperature by draining the tank’s preheated water and adding seawater of room temperature (24C) at the same time. After this, the
tank was kept at room temperature for 0.5 h for the shrimps’ recovery. Three shrimps in the control group were transferred to another tank of water which was kept at room temperature, and after 1 h this tank was also drained of its original water while adding water of room temperature. A pleopod of three shrimps from each group was withdrawn before the heat shock as well as 0, 1, 2, 4 and 6 h after recovery and separately preserved in liquid nitrogen immediately for RNA extraction to analyze the HSP21 gene expression. Total RNA was isolated using TRIzol Reagent (Invitrogen) according to the instructions provided by the manufacturer and the modified detail was as described previously[17].
Quantification of HSP21 mRNA expression by real-time PCR
Real-time PCR of shrimp smHSP mRNA was performed using gene-specific primers. b-actin mRNA was used as the normalization control. PCR primers for gene-specific primer sets were designed using Primer Express V.2.0 software (Applied Biosystems Inc.). The sense and antisense primers for HSP21 were 50- CACGAGGAGAAGTCTGACAAC-30 and
50-GAGAGCGAGGACTTGATGAG-30, respectively. The
for-ward and reverse primers for b-actin were 50-CYGAGCGYG
GYTACWCCTT-30 and 50
-TAGCAMAGYTTCTCCTTGATGTCA-30, respectively. Real-time PCR was performed in a
two-step process. In the first two-step, sample RNA was reverse tran-scribed with 50 ng random hexamers in a volume of 20 ml using 200 U of Superscript III Reverse Transcriptase (Invitro-gen) and 40 U of RNaseOUT Recombinant RNase Inhibitor (Invitrogen). In the second step, real-time PCR was carried out in a MicroAmp Optical 96-well plate using power SYBR Green PCR Master Mix (Applied Biosystems Inc.), with 2 ml cDNA in each well. PCR reactions were monitored in real time using the ABI PRISM 7300 Real-time PCR system (Applied Biosystems Inc.). The thermal cycling conditions for real-time PCR were 50C for 2 min, then 95C for
10 min, and 40 cycles of denature (95C, 15 s) and
anneal-ing/extension (60C, 1 min). The real time PCR was
con-ducted in triplicate with different shrimp samples. The data obtained from real-time PCR analysis were analyzed using the comparative Ct method according to the user
bulletin of ABI PRISM 7300 system and then subjected to paired sample t-test. Differences were considered signifi-cant at p < 0.05.
Recombinant HSP21 over-expression in Escherichia coli and antiserum production
The DNA fragment encoding the full-length HSP21 was amplified from shrimp cDNA by PCR with the S-F/S-R primer set (50-TACGGATCCAATGGAGGGCTTCAAGCAC-30/
50-ATCCTCGAGTCAGTTCT GTGCGATGGG-30; the underlined
bases indicate, respectively, the BamHI and XolI restriction sites that were used for subsequent cloning) and ligated to pGEM-T Easy (Promega) plasmid. After confirming the sequence, the resultant plasmid, pGEM-T-HSP21 was cleaved with BamHI and XolI, and the amplified fragment was then cloned to pER28bþ (Novagen) at BamHI and XolI sites. The resultant recombinant plasmid, pET28-HSP21 was transformed into E. coli BL21 (DE3) strain. E. coli BL21 (DE3) cells were cultured in LB medium with 25 mg/ ml kanamycin and the protein was induced with 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG). The
recombi-nant HSP21 proteins tagged with 6 consecutive histidines (6$His-tagged HSP21) were purified by using QIAexpression-ist nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography (Qiagen) according to the manufacturer’s recommendations. The resins were washed with buffer (pH 8.0) containing 25 mM Tris and 8 M urea and eluted with the buffer containing 500 mM imidazole. The eluted protein was then concentrated using Amicon Ultra-15 centrifugal filters (Millipore) in PBS buffer and stored at20C for
fur-ther antiserum production and fur-thermal aggregation assay.
New Zealand white rabbits were hyperimmunized by injection with 250 mg proteins emulsified in complete Freund’s adjuvant. Subsequent booster injections were car-ried out with 250 mg protein emulsified in incomplete Freund’s adjuvant. The antiserum was collected after the antibody titer had peaked.
Western blot analysis
For Western blotting analysis, proteins that had been separated in SDS-PAGE were transferred onto a polyvinyli-dene difluoride (PVDF) membrane (Micron Separations) by semi-dry blotting. Membranes were blocked in 5% skim milk (Difco Laboratories) in TBS (0.2 M NaCl and 50 mM TriseHCl, pH 7.4). Immunodetection was performed by incubation of the blot in a polyclonal rabbit anti-HSP21 serum diluted 1:5000 in TBS with 5% skim milk for 1 h at room tempera-ture. Subsequently, anti-rabbit IgG antibody conjugated with horseradish peroxidase (Sigma) was used at a concen-tration of 1:5000 and detection was performed with a West-ern Blot Chemiluminescence Reagent (NEN Life Sciences). Thermal aggregation assay
Thermal aggregation of citrate synthase (from porcine heart, Sigma) was monitored in HEPES buffer (50 mM HEPES, pH 8.0, 25 mM NaCl, 0.5 mM DTT) at 45C. The buffer containing recombinant HSP21 at different concentrations was preincubated for 10 min at 45C before the addition
of citrate synthase to a final concentration of 20 mg/ml.
Figure 1 The nucleotide sequence and deduced amino acid sequence of the Penaeus monodon small heat shock protein gene.
Reactions were continuously monitored at 320 nm in a Ultro-spec 3100 pro (Amersham Pharmacia, UV/Visible spectro-photometer). The absorbance of citrate synthase alone at 60 min of heating was defined as 100% aggregation. Immune challenge
In order to reveal whether the transcription of HSP21 was affected by WSSV infection, shrimps were injected with WSSV and tissues were collected after infection using pro-cedures described in Chen et al.[17]. The control shrimps were injected with 0.9% NaCl solution. A pleopod of three shrimps from each group was withdrawn at 0, 2, 4, 6, 8, 12, 18, 24, 36 and 72 h post-injection and separately pre-served in liquid nitrogen immediately for RNA extraction.
The expression level of HSP21 mRNA in the WSSV challenged group and control group were also analyzed by real-time PCR.
Results
Sequence analysis of shrimp small heat shock protein (smHSP)
According to the EST database, an unpublished sequence with an opening reading frame (ORF) was identified (Fig. 1). This ORF encodes a protein of 184 amino acids with a theo-retical size of about 21 kDa (and a predicted isoelectric point of 5.38). The nucleotide sequence surrounding the
Figure 2 Comparisons between members of the smHSP family from invertebrate organisms including shrimp are shown in multiple sequence alignment and phylogenetic analysis.
methionine start codon (AAAATGG) of the predicted protein conformed to the Kozak rule of an efficient context for eukaryotic translation initiation. A polyadenylation signal (AATAA) was located 181 bp downstream of the transla-tional stop codon.
The deduced amino acid sequence of the ORF was homologous with known small heat shock protein (smHSP) (Fig. 2). Nine smHSPs from diverse genera were selected for analysis. The sequence of shrimp smHSP identified to a high degree with those of similar genes in eight insect species and one roundworm.
A phylogenetic tree was constructed by analyzing the amino acid sequences of P. monodon smHSP and the nine similar smHSPs (Fig. 3). The result indicated that P mono-don smHSP shared higher identity with insects than with roundworm.
Tissue expression pattern of the HSP21
The expression of HSP21 mRNA in different tissues was as-sessed by real-time PCR. The result showed that HSP21 was highly expressed in almost all organs such as epithe-lium, gill, stomach, midgut, lymphoid organ, hepatopan-creas, nervous tissue and heart, but poorly in haemolymph (Fig. 4).
To determine whether P. monodon HSP21 was inducible by heat shock, three shrimps were also subjected to heat shock treatment. Pleopods were used in this analysis. As shown inFig. 5, heat shock treatment increased the amount of HSP21 mRNA transcript in P. monodon. The transcripts were found before treatment as well as 0, 1, 2, 4, and 6 h after recovery. HSP21 expression reached its greatest peaks during the first hour after heat shock treatment and recov-ery. It gradually decreased to the pretreatment level after 6 h. The significant difference (p < 0.05) in HSP21 expres-sion was found between the heat shock treated and the control group at 0, 1, 2 and 4 h after recovery. It indicated that HSP21 was expressed constitutively and inducible im-mediately by heat shock.
Purification of recombinant HSP21 and detection HSP21 in P. monodon
The HSP21 gene was expressed in E. coli as a 6$His-tagged fusion protein. After induction with IPTG at 37C, the
SDS-PAGE result showed that a band (about 23 kDa) corre-sponding to the HSP21-6$His fusion protein was observed in the induced bacterium (Fig. 6A, lane 2). No protein was found at the same position in the non-induced control (Fig. 6A, lane 1). The data indicated that the HSP21 gene was expressed and a purified HSP21-6$His fusion protein was obtained (Fig. 6A, lane 3). Western blotting was
Penaeus Nasonia Locusta Apis Bombyx Heliconius Tribolium Aedes Drosophila Caenorhabditis 56 53 100 97 34 63 20 0.05
Figure 3 Phylogenetic tree was constructed by the Neighbor-Joining method and shows relatedness of nine smHSPs from invertebrate organisms. Robustness was tested by 1000 boot-strap replications and the indicated distance given the value of 0.1 means the 10% differences in amino acid residues between compared sequences. Bootstrap values are given in percentages.
Different tissues ofP. monodom
1 2 3 4 5 6 7 8 9
Relative mRNA level of
HSP21 0 1 2 3 4 5
Figure 4 Detection of HSP21 transcripts by real-time PCR. Lanes; (1), epithelium; (2), gills; (3), stomach; (4), midgut; (5), lymphoid organ; (6), hepatopancreas; (7), nervous tissue; (8), haemolymph and (9), heart.
Time after heat shock treatment
p 0 1 2 4 6
Relative mRNA level of
HSP21 0 1 2 3 4
Heat shock treated group Control group
Figure 5 Detection of the HSP21 transcripts before and after heat shock treatment by real-time PCR. P, pretreated; 0, 1, 2, 4, and 6 show the hours after heat shock treatment. The signif-icant difference (p < 0.05) of HSP21 expression between the heat shock treated and the control group are indicated with asterisks.
performed to detect the HSP21 in P. monodon. Epithelium tissue was selected to be tested owing to the result that HSP21 was highly expressed in the tissue and it was easily extracted in buffer. As shown in Fig. 6B, an obvious band about 21 kDa was detected (lane 1). As compared with purified HSP21-6$His fusion protein (lane 2), the molecular weight of HSP21 detected in tissue is less because of the lack of the His tag (and some amino acids co-expressed by expression vector pET28bþ). It indicated that the real molecular weight of HSP21 in shrimp tissue was 21 kDa. Chaperone function of HSP21
HSP21 was active in a standard chaperone assay, which measures the heat-induced aggregation of citrate synthase [18]. At an HSP21/citrate synthase ratio of 1:1 (w/w), HSP offers protection to the extent of only 20%. The extent of protection increases as the concentration of HSP21 increased, and at a ratio 8:1 (chaperone/target) approxi-mately 85% protection is observed (Fig. 7). Thus, recombi-nant HSP21 is an active chaperone in this assay, comparable in specific activity to other HSP21s that have been studied.
Transcription analysis of HSP21 in WSSV-infected shrimps
Real-time PCR analysis was used to detect HSP21-specific transcripts from shrimp specimens before injection (0 h) and at 2, 4, 6, 8, 12, 18, 24, 36 and 72 h after intramuscular injection of WSSV. The result showed that the HSP21 mRNA level was significantly decreased after WSSV infection (Fig. 8). The expression level of HSP21 mRNA was reduced
to about 50% after 2 h post-infection. At 6 h post-infection, the HSP21 mRNA level even reduced to 10%. The significant difference (p < 0.05) of HSP21 expression between the WSSV challenged and the control group appeared at all sampling times after WSSV injection. It indicated that the expression mechanism of HSP21 was highly affected by WSSV.
Discussion
Diseases caused by viruses are the greatest challenge to world wide shrimp aquaculture. However, up to this date, there is no effective method to control viruses, especially white spot syndrome virus (WSSV). A better understanding of shrimp immune responses will be very helpful for disease control. For this purpose, a gene named small heat shock protein (smHSP) was firstly identified by annotating the EST 72 55 40 33 24 17 100 130 170 kDa M 1 2 3 1 2
A
B
11Figure 6 Recombinant expression in E. coli purification and western blot of shrimp HSP21. (A), SDS-PAGE of expressed and purified of protein. Lanes M, pre-stained molecular weight marker; (1), non-induced; (2), induced; (3), purified HSP21-6$His fusion protein. (B), Western blotting of HSP21. Lanes 1, epithelium tissue lysate; 2, purified HSP21-6$His fusion protein. 100% 80% 70% 60% 50% 40% 30% 20% 10% 0% 90% 0 5 10 15 20 25 30 35 40 45 50 55 60 Time (min)
Figure 7 Chaperone activity of HSP21. Thermally induced aggregation of citrate synthase (20 mg/ml) alone (A); and in the presence of HSP21 at 1:1 (-); 2:1 (:); 4:1 (,); and 8:1 (6) ratio of HSP21/citrate synthase (w/w). The light scattering of the solution was monitored at 320 nm as described under ‘‘Materials and Methods’’.
Time after WSSV challenge
2
0 4 6 8 12 18 24 36 72
Relative mRNA level of HSP21
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 WSSV challenged group Control group
Figure 8 Analysis of HSP21 expression in the WSSV chal-lenged group and control group by real-time PCR at 0, 2, 4, 6, 8, 12, 18, 24, 36 and 72 h post-injection. The significant difference (p < 0.05) of HSP21 expression between the WSSV challenged and the control group are indicated with asterisks.
databases and was compared with the GenBank database. The subunit molecular weights of smHSPs range from approximately 12 to 43 kDa, although most fall within from 17 to 30 kDa, and generally exist as large multimeric assemblies insolution. In this study, we identified a smHSP of shrimp P. monodon with a molecular weight of 21 kDa. A thermal aggregation assay indicated that smHSP and HSP21 had chaperone activity.
The real-time PCR result showed that HSP21 was expressed less in haemolymph. In this tissue, HSP21 was also expressed less after heat shock (data not shown). This might be because the main function of the haemo-lymph tissue was not to express the proteins with chaper-one activity.
Research on HSPs indicated that the heat shock response is a basic and highly conserved process that is essential for surviving environmental stresses. That is, HSPs are induced in expression against environmental stress [10,11,19]. Therefore, the speculation that the expression of smHSP should be induced after heat shock and after WSSV infection was reasonable. Actually, HSP21 was inducible after heat shock. However, contrary to expectation, the expression of HSP21 was repressed after WSSV infection. We speculate that this might be in relation with the host’s apoptosis. Referring to HSP27, it was discovered to have the function in blocking apoptosis[20,21]. That is, reducing the expres-sion of small HSP could finally lead to a cells’ apoptosis. Pre-vious studies have indeed found signs of increasing numbers of cells showing DNA fragmentation, an obvious hallmark of apoptosis, by TUNEL assay in P. monodon infected with WSSV[22]. Thus, we speculate that the apoptosis induced by WSSV infection might be in connection with the down-regulation of HSP21. But, the next question is, what turned off HSP21? WSSV or the host itself?
Besides apoptosis, the studies of smHSP, Mj HSP16.5, of Methanococcus jannaschii provided a reasonable construc-tion. Kim et al. observed the crystal structure of Mj HSP16.5 and then proposed the chaperone mechanism of smHSP. Mj HSP16.5 contained a ‘‘windows’’ structure in which denatured proteins caused by stress can be attracted to [23]. This can prevent proteins aggregating and miss-folding. As the stress was removed, the proteins renature and might fall off from the ‘‘windows’’ because of the re-naturation process or disassembly of the smHSP complex [24]thus providing a mechanism for the host to protect pro-teins from miss-folding. However, during a viral infection, the host’s resources are turned to produce the components of viruses. As the result shown in Fig. 8, the quantity of HSP21 mRNA was reduced to about 10% after 6 h post-infec-tion. We speculate that WSSV occupied host resources so that the host could not express smHSP or WSSV might turn off HSP21 to stop the chaperone activity in order to in-crease viral protein expression. However, further research is required to confirm these speculations.
Acknowledgments
This work was supported by the National Council Grant. We are indebted to Mr. Vince J. Genis and Miss Angela Wang for their helpful criticism of the manuscript.
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