Fish & Shellfish Immunology 39 (2014) 61e68

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Isolation and characterization of tumor necrosis factor receptorassociated factor 6 (TRAF6) from grouper, Epinephelus tauvina Jingguang Wei a, Minglan Guo a, Pin Gao b, Huasong Ji b, Pengfei Li a, Yang Yan a, Qiwei Qin a, * a Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, PR China b State Key Laboratory Breeding Base for Sustainable Exploitation of Tropical Biotic Resources, College of Marine Science, Hainan University, Haikou 570228, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 January 2014 Received in revised form 2 April 2014 Accepted 23 April 2014 Available online 5 May 2014

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is one of the key adapter molecules in Tolllike receptor signal transduction that triggers downstream cascades involved in innate immunity. In the present study, a TRAF6 (named as Et-TRAF6) was identified from the marine fish grouper, Epinephelus tauvina by RACE PCR. The full-length cDNA of Et-TRAF6 comprised 1949 bp with a 1713 bp open reading frame (ORF) that encodes a putative protein of 570 amino acids. Similar to most TRAF6s, Et-TRAF6 includes one N-terminal RING domain (78aae116aa), two zinc fingers of TRAF-type (159aae210aa and 212aae269aa), one coiled-coil region (370aae394aa), and one conserved C-terminal meprin and TRAF homology (MATH) domain (401aae526aa). Quantitative real-time PCR analysis revealed that Et-TRAF6 mRNA is expressed in all tested tissues, with the predominant expression in the stomach and intestine. The expression of Et-TRAF6 was up-regulated in the liver after challenge with Lipoteichoic acid (LTA), Peptidoglycan (PGN), Zymosan, polyinosineepolycytidylic acid [Poly(I:C)] and Polydeoxyadenylic acid$Polythymidylic acid sodium salt [Poly(dA:dT)]. The expression of Et-TRAF6 was also up-regulated in the liver after infection with Vibrio alginolyticus, Singapore grouper iridovirus (SGIV) and grouper nervous necrosis virus (GNNV). Recombinant Et-TRAF6 (rEt-TRAF6) was expressed in Escherichia BL21 (DE3) and purified for mouse anti-Et-TRAF6 serum preparation. Intracellular localization revealed that EtTRAF6 is distributed in both cytoplasm and nucleus, and predominantly in the cytoplasm. These results together indicated that Et-TRAF6 might be involved in immune responses toward bacterial and virus challenges. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Epinephelus tauvina TRAF6 SGIV GNNV Intracellular localization

1. Introduction As pattern-recognition receptors (PRRs), toll-like receptors (TLRs) play a crucial role in the innate immune system by recognizing conserved pathogen-associated molecular patterns (PAMPs) and triggering the signaling pathways [1]. After the PAMPs recognition, TLRs can recruit adapter molecules Myeloid differentiation factor 88 (MyD88) and Tumor necrosis factor receptor-associated factor 6 (TRAF6) for signal transduction to activate nuclear factorkappa B (NF-kB) and mitogen-activated protein kinases (MAPKs)

* Corresponding author. Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, PR China. Tel./fax: þ86 20 89023638. E-mail address: [email protected] (Q. Qin). http://dx.doi.org/10.1016/j.fsi.2014.04.022 1050-4648/Ó 2014 Elsevier Ltd. All rights reserved.

[2]. The most likely scenario is that once recruited, MyD88 interacts with IL-1 receptor-associated kinase-4 (IRAK-4) via their respective death domains. IRAK-4 then recruits IRAK-1 to the complex, leading to its phosphorylation and activation [3]. IRAK-1 and IRAK-4 then dissociate from the complex and interact with TRAF6 in mammals [4]. TRAF6 counterparts were also identified in other vertebrates and invertebrates, such as fish [5e8], scallop [9e11], shrimp [12] and sea cucumber [13]. Their connection with pathogen challenges (bacteria or virus) have been also investigated, suggesting the existence of a Toll-signaling pathway mediated innate immunity in each species. Groupers, Epinephelus sp., are widely cultured in China and Southeast Asian countries. The emergence of bacterial and viral pathogens, including Vibrio alginolyticus, iridovirus and nervous necrosis virus, caused heavy economic losses in grouper aquaculture [14e16]. For example, the gram-negative bacterium,

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V. alginolyticus, has frequently been identified as the pathogen responsible for the infectious disease called vibriosis. This disease is one of the major challenges facing grouper aquaculture, which has caused substantial economic losses globally [16]. Further, Singapore grouper iridovirus (SGIV) was isolated from diseased groupers, which belonged to family Iridoviridae [14,17]. SGIV caused serious systemic diseases and resulted in more than 90% mortality in grouper either in fish farm or challenge experiments. The virus infection is characterized as hemorrhage and enlargement of the spleen of infected fish [17]. The grouper nervous necrosis virus (GNNV) also caused mass mortality in grouper fry in many countries [15]. Although TLRs have been identified and characterized from grouper [18e23], whether other members like TRAF6 in the signal pathways exist remaining unknown. In the present study, the molecular characteristics of grouper TRAF6, and the tissue distributions, expression patterns after challenging with bacterial and viral pathogens were investigated. The recombinant Et-TRAF6 protein and mouse anti-Et-TRAF6 were obtained and the intracellular localization was also investigated. These present studies will help us for better understanding of its innate immune mechanisms in the anti-bacterial or anti-virus response of fishes. 2. Materials and methods 2.1. Fish Juvenile greasy grouper, Epinephelus tauvina (40e50 g) were purchased from a marin-culture farm at Honghai bay, Shanwei City, Guangdong Province, China. After maintenance in aerated flowthrough seawater for 3 days, these fish were used for the challenge experiments. 2.2. Preparation of bacterial cells and virus V. alginolyticus was cultured at 37  C for 24 h in LuriaeBertani (LB) prepared with the distilled water, then harvested by centrifugation at 3500 g for 10 min and suspended in the buffer for an appropriate concentration. Quantification was performed by plating various bacteria dilutions on agar plates. Cell lines of grouper spleen (GS) and fathead minnow (FHM) were propagated by the recommended methods with Leibovitz’s L15 and M199 culture medium with 10% fetal calf serum at 28  C [24]. Propagation of SGIV and GNNV were performed as described previously [15,17]. The viral titer of SGIV and GNNV was 105 TCID50/ ml. 2.3. Challenge experiments E. tauvina was intraperitoneal injected (i.p.) 100 ml individually with one of six groups of TLRs ligands, including Lipopolysaccharides (LPS)(10 mg/ml), Lipoteichoic acid (LTA) (5 mg/ml), Peptidoglycan (PGN) (20 mg/ml), Zymosan (100 mg/ml), polyinosinee polycytidylic acid [Poly(I:C)] (1 mg/ml) and Polydeoxyadenylic acid$Polythymidylic acid sodium salt [Poly(dA:dT)] (1 mg/ml) from SigmaeAldrich at six time points (3, 6, 12, 24, 48 and 72 h). The fish were treated with PBS as external control. For each group divided by ligands and time points, six fishes were treated and collected to eliminate individual variations. At each time point, the liver from these treated grouper was mixed and grinded in liquid nitrogen. In bacteria challenging experiment, each control and challenged sample was injected with 100 ml PBS and a live bacterial suspension (105 CFU/ml), respectively. Livers of six fishes in each group were

collected for quantitative real-time PCR (qRT-PCR) at 3, 6, 12, 24, 48 and 72 h. In SGIV and GNNV challenging experiment, each control and challenged sample was injected 50 ml PBS and SGIV or GNNV at a concentration of 105 TCID50/ml, respectively. Livers of six fishes in each group were collected for qRT-PCR at 3, 6, 12, 24, 48, and 72 h. 2.4. RNA isolation and cDNA synthesis Total RNA was isolated from different tissues of grouper, using Trizol Reagent (Invitrogen) according to the manufacturer’s protocol. Each of the samples contained 6 independent individuals to eliminate individual differences. The RNA was treated with RQ1 RNase-Free DNase (Promega, USA) to remove contaminated DNA. The quality of total RNA was assessed by electrophoresis on 1% agarose gel. Total RNA was reverse transcribed to synthesize the first-strand cDNA by ReverTra Ace kit (TOYOBO, Japan) according to the manufacturer’s instructions. 2.5. Cloning and sequence analysis of E. tauvina TRAF6 (Et-TRAF6) 2.5.1. Degenerate primers design and initial PCR cloning Multiple-sequence alignment of TRAF6 nucleotide sequences from a variety of species was performed with the Clustal X multiple-alignment software. Degenerate primers were designed based on the conserved nucleotides of three fish TRAF6 sequences reported before, including Danio rerio (accession no. AAT37634.1), Cyprinus carpio (accession no. ADF56651.2), and Tetraodon nigroviridis (accession no. CAF99941.1). Two fragments of Et-TRAF6 gene were amplified from grouper liver cDNA by two pairs of degenerate primers DF1 (50 -CAAGGCTATGATGTAGAGTTTGACCCTCCA-30 ), DF2 (50 -ATCTCGCTTTTCGTGCACACTATGCAG-30 ), DR1 (50 TCGTTTGGCAAAGTTATCAGGGAAAAGTTG-30 ) and DR2 (50 -CACATAGCCGAATCCTTTGGGGTTGCG-30 ), using the LaTaq polymerase (Takara, Japan). PCR was performed with an initial denaturation step of 5 min at 94  C, and then 35 cycles were run as follows: 94  C 45 s, 55  C 45 s, 72  C 1.5 min, and 72  C elongation for 5 min. 2.5.2. Rapid amplification of 50 and 30 cDNA ends (RACE) of EtTRAF6 cDNA Based on the first partial sequences amplified by the degenerate primers DF1 and DR2, the 50 and 30 ends of Et-TRAF6 cDNA were obtained using the RACE approaches. The first-strand cDNA was synthesized from the total liver RNA with the SMARTÔ RACE cDNA amplification kit (Clontech) for 30 RACE and 50 RACE. Two primers F1 (50 -ACCATCCGCCTTGCCATCCTTGACC-30 ) and R1 (50 -GCAGAAACGGTGACCACAGGGTG-30 ) were designed based on the first partial sequence. PCR was performed with 10 mM F1 or R1 and 500 nM of Nested Universal Primer A (NUP, Clontech). Denaturation was performed at 94  C for 5 min, followed by 35 cycles at 94  C for 30 s, 60  C for 30 s, and 72  C for 45 s. 2.6. TA cloning, sequencing and database analysis PCR products were analyzed on 1% agarose gels, extracted with an AxyPrep DNA gel extraction kit (AxyGEN), and then ligated into pMD18-T vectors (TaKaRa) and transformed into competent Escherichia coli DH5 cells. Positive colonies were screened by PCR and at least two recombinant plasmids were sequenced. Sequences were analyzed based on nucleotide and protein databases using the BLASTN and BLASTX program (http://www.ncbi. nlm.nih.gov/BLAST/). The protein and its topology prediction were performed using software at the ExPASy Molecular Biology Server (http://expasy.pku.edu.cn). Multiple sequence alignment of the EtTRAF6 was performed with the Clustal X multiple-alignment

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software. MEGA 4.0 was also used to produce the phylogenetic tree. Neighbor-joining (NJ) method implemented in MEGA 4.0 was used for phylogenetic analysis. The robustness of bifurcations was estimated with bootstrap analysis and bootstrap percentages were obtained with 1000 replicates. 2.7. Analysis of Et-TRAF6 mRNA expression profiles qRT-PCR was employed to detect the Et-TRAF6 expression profiles using b-actin as a reference gene. The qRT-PCR primers, F3 (50 CCCTATCTGCCTTATGGCTTTGA-30 )/R3 (50 -ACAGCGGACAGTTAGCGAGAGTAT-30 ) and actin-F (50 -TACGAGCTGCCTGACGGACA-30 )/actin-R (50 -GGCTGTGATCTCCTTTTGCA-30 ), were designed based on the full-length cDNA of Et-TRAF6 and b-actin. qRT-PCR was performed on Roche LightCycler 480 Real-time PCR system (Roche, Switzerland) using the 2 SYBR Green Real-time PCR Mix (TOYOBO, Japan). PCR amplification was performed in triplicate wells, using the cycling parameters: 94  C for 5 min, followed by 40 cycles of 5 s at 94  C, 10 s at 60  C and 15 s at 72  C. Relative gene expression was analyzed by the comparative Ct method (2DDCT method). Target CT values were normalized to the endogenous gene b-actin. Results for each treated sample were expressed as N-fold changes in target gene expression relative to the same gene target in the calibrator sample, both normalized to the b-actin gene. All samples were analyzed in three duplications and all data were given in term of relative mRNA expression level as means  SD, and then subjected to Student’s t-test. Differences were considered significant at p < 0.05 or p < 0.01. 2.8. Expression and purification of recombinant Et-TRAF6 and preparation of antiserum Et-TRAF6 specific primers F4 (50 -GCGCGGATCCGATGGCTTGCTTTGACAGTAATA-30 ) and R4 (50 -GCGCGTCGACG TCCCTTGAAACTGAGGCCTC-30 ) were used to amplify the DNA fragment encoding the mature peptide of Et-TRAF6. The target PCR product was digested with BamH I and Sal I (Takara), and then subcloned into the BamH I/Sal I sites of expression vector pET21b.The recombinant plasmid (pET-Et-TRAF6) was transformed into E. coli BL21 (DE3) and subjected to nucleotide sequencing. Positive clone was incubated in 200 ml LB medium (containing 100 mg/ml ampicillin) at 37  C with shaking at 220 rpm. The parent vector without an insert fragment was used as negative control. When the culture medium reached OD600 of 0.5e0.7, the cells were incubated for 4 additional hours with the induction of IPTG at the final concentration of 0.5 mmol/l. The recombinant Et-TRAF6 protein (designated as rEt-TRAF6) was purified by affinity chromatography with Ni-nitrilotriacetic acid-agarose (Qiagen, Germany) according to the manufacturer’s instruction. The resulting protein was analyzed by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized with Coomassie brilliant blue R-250. The concentration of recombinant fusion protein was determined using Bradford’s method. The polyclonal antibody against rEt-TRAF6 produced by immunizing BALB/c mice according to the conventional method [25]. The specificity of the antiserum was detected with western blot. 2.9. Intracellular localization Intracellular localization of Et-TRAF6 was performed by EGFP fusion protein expression and immunofluorescence. Et-TRAF6 specific primers F5 (50 -GCGAATTCTATGGCTTGCTTTGACAGTAATAA-30 ) and R5 (50 -GCGGATCCGTCCCTTGAAACTGAGGCCTCGTGT30 ) were used to amplify the DNA fragment encoding the mature peptide of Et-TRAF6. The target PCR product was digested with

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EcoR I and BamH I (Takara), and then subcloned into the EcoR I/ BamH I sites of expression vector pEGFP-N3. The recombinant plasmid (pEGFP-Et-TRAF6) was transformed into E. coli DH5a and subjected to nucleotide sequencing. After transfection with pEGFPEt-TRAF6 or pEGFP-N3 for 48 h, cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min, and then stained with 6-diamidino-2-pheny-lindole (DAPI) for 15 min. Finally, cells were mounted with 50% glycerol, and observed under fluorescence microscopy (Leica, Germany). For immunofluorescence localization, GS cells were seeded onto coverslips (10 mm  10 mm) in a 6-well plate. After allowing the cells to adhere for 24 h, the cells were fixed with 4% paraformaldehyde and then the coverslips were blocked using 2% bovine serum albumin (BSA) at room temperature for 30 min. Cells were incubated either with anti-Et-TRAF6 serum (1:100) or preimmune mice serum (1:100) for 1 h, rinsed with PBS three times for 10 min and then incubated with FITC-conjugated goat anti-mouse antibodies (Pierce, USA) for a further hour. Finally, cells were stained with 6-diamidino-2-pheny-lindole (DAPI) (1 mg/ml) and observed under fluorescence microscopy (Leica, Germany). 3. Results 3.1. Sequencing analysis of grouper TRAF6 cDNA The partial cDNA sequences amplified by the degenerated primers of DF1/DR1, DF2/DR2, and DF1/DR2 from E. tauvina liver cDNA were 216 bp, 195 bp, and 1322 bp in size, respectively. Sequencing and Blastx analysis of the 1322 bp fragment showed that it shares high similarity to other reported TRAF6 genes. Based on the partial cDNA sequences of TRAF6, the Et-TRAF6-specific primers of F1 and R1 were designed for the rapid amplification of 30 and 50 cDNA (RACE), and the full-length cDNA of Et-TRAF6 was amplified. The full-length cDNA of Et-TRAF6 was 1949 bp (Genbank accession no. JK000381) and contained a 1713 bp open reading frame (ORF) coding for a protein of 570 amino acid (aa), and had a 60 bp of 50 -UTR and a 176 bp of 30 -UTR including a putative polydenylation consensus signal (AATAAA) and a poly (A) tail (Fig. 1A). Similar to most TRAF6s, Et-TRAF6 includes one N-terminal RING domain (78aae116aa), two zinc fingers of TRAF-type (159aae210aa and 212aae269aa), one coiled-coil region (370aae394aa), and one conserved C-terminal meprin and TRAF homology (MATH) domain (401aae526aa) (Fig. 1B). The deduced amino acid sequence of Et-TRAF6 protein is remarkably conserved. It shared significant homology with TRAF6s from other fish species (Table 1). Multiple sequence alignments were carried out using the Clustal X multiple-alignment software. The Neighbor joining tree of TRAF6 amino acid sequences showed that the greasy grouper TRAF6 was clustered with orange-spotted grouper TRAF6, stickleback TRAF6 and fugu TRAF6. The grouping of TRAF6 proteins was well-supported by bootstrapping and in accordance with the assumed evolutionary trend of the species (Fig. 2). 3.2. Expression profiles of Et-TRAF6 To investigate the tissue expression profiles of Et-TRAF6 in healthy E. tauvina, qRT-PCR was used to analyze its expression levels in various tissues. The Et-TRAF6 expression was detected in all tested tissues, with the predominantly expression in the stomach and intestine (Fig. 3A). Liver is a unique immune organ with many resident innate and adaptive immune cells, which induce various complex and sophisticated immune responses. The liver in a fish is considered to be one important organ that has a different number of functions for

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Fig. 1. Sequences and domain topology of Et-TRAF6. (A) The nucleotide (upper row) and deduced amino acid (lower row) sequences of the ORF and UTR regions are shown and numbered on the left. The RING domain (amino acids 78e116) and two TRAF domains (amino acids 159e210 and 212e269) were marked by the single and double underline. The coiled-coil region (amino acids 370e394) was boxed, and the MATH domain (amino acids 401e526) was shaded. The bold letters indicated the polyadenylation signal sequence (AATAAA). (B) Schematic representation of the domain topology of Et-TRAF6.

J. Wei et al. / Fish & Shellfish Immunology 39 (2014) 61e68 Table 1 Comparisons of the deduced amino acid sequence of Et-TRAF6 with TRAF6s from other known fish species. Fish species

Accession number

Identity (%)

Epinephelus coioides TRAF6 Gasterosteus aculeatus TRAF6 Pundamilia nyererei TRAF6 Oreochromis niloticus TRAF6 Takifugu rubripes TRAF6 Tetraodon nigroviridis TRAF6 Cyprinus carpio TRAF6 Danio rerio TRAF6

AGQ45557.1 ABJ15863.1 XP_005749680.1 XP_003450846.1 XP_003969671.1 CAF99941.1 ADF56651.2 AAT37634.1

98 87 86 86 81 79 65 65

the fish. To determine whether Et-TRAF6 was involved in the host immune responses, RT-qPCR was also employed to examine the expression profiles of Et-TRAF6 in the liver after challenged with different viral/bacterial agents. The expression was able to be induced by LTA, PGN, Zymosan, Poly(I:C) and Poly(dA:dT), but not for LPS. The time-course analysis revealed 18.0, 85.4, 16.7 and 41.0fold induction of Et-TRAF6 expression after 48 h injection of LTA, PGN, Zymosan, Poly(I:C) and Poly(dA:dT) compared with PBS, and then decreased to 4.93, 4.23, 7.13, 1.2 and 8.97-fold at 72 h (Fig. 3B). Further, RT-qPCR revealed also an induction of Et-TRAF6 expression after bacterial and virus challenge by V. alginolyticus, SGIV and GNNV infection (Fig. 3C). The time-course analysis revealed 326-fold induction of Et-TRAF6 expression after 48 h injection of V. alginolyticus compared with PBS, and then decreased to 11.2-fold at 72 h (Fig. 3C). The expression profiles increased up to 7.44 and 25.4-fold induction after 72 h injection of SGIV and GNNV compared with PBS (Fig. 3C).

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The recombinant Et-TRAF6 protein (designated as rEt-TRAF6) was purified by affinity chromatography with Ni-nitrilotriacetic acidagarose (Qiagen, Germany) according to the manufacturer’s instruction (Fig. S1 lane 3). To identify the biological properties of Et-TRAF6, the recombinant protein was used to immunize mouse to prepare immune serum. The specificity of anti-TRAF6 serum was examined by western blot. The rEt-TRAF6 protein (5 mg) was specifically recognized by polyclonal antibody against Et-TRAF6 (Fig. S1 lane 4), while no bands were found detected by negative control serum (Fig. S1 lane 5), indicating that the anti-TRAF6 antibody specifically recognized the purified Et-TRAF6 protein. 3.4. Intracellular localization of Et-TRAF6 Intracellular localization of Et-TRAF6 was determined by pEGFPEt-TRAF6 fusion protein expression and immunofluorescence assay with anti-Et-TRAF6 serum. Firstly, a recombinant plasmid, pEGFPEt-TRAF6, was constructed and transfected into FHM cells. The green fluorescence signal in pEGFP-Et-TRAF6 transfected cells was distributed in both the cytoplasm and nucleus, and predominantly in the cytoplasm, whereas the signal was detected in both the cytoplasm and nucleus in the control cells (Fig. 4A). Secondly, the intracellular localization of Et-TRAF6 in GS cells was determined by immunofluorescence assay. In GS cells, the FITC green fluorescence was distributed in both the cytoplasm and nucleus, and predominantly in the cytoplasm, while no green fluorescence signal was detected in samples detected by the preimmune mice serum (Fig. 4B). 4. Discussion

3.3. Expression, purification, and antibody preparation of recombinant Et-TRAF6 The parent vector pET-21b and the plasmid pET-Et-TRAF6 were transformed into E. coli BL21 (DE3), respectively. After IPTG induction, cell lysates were obtained and boiled for 10 min, then subjected to SDS-PAGE analysis. One band at 66 kDa (Fig. S1 lane 1e 2) was visualized by Coomassie brilliant blue staining, which matched to the expected molecular mass of Et-TRAF6 based on the predicted molecular mass and the deduced amino acid sequence.

Fig. 2. Neighbor joining analysis of TRAF6 proteins. NJ tree showing the relationship between Et-TRAF6 and other known TRAF proteins. The full-length amino acid sequences of TRAFs from different organisms were aligned using the Clustal X 2.0 program (http://www.ebi.ac.uk/tools/clustalw2). The phylogenetic tree was constructed using MEGA software 4.0 (http://www.megasoftware.net/index.html). The relationships among the various components were analyzed by the Neighbor-joining (NJ) method. Numbers on the branches indicate percent bootstrap confidence values from 1000 replicates. Greasy grouper (Accession No. JK000381), orange-spotted grouper (Accession No. AGQ45557.1); fugu (Accession No. XP_003969671.1); stickleback (Accession No. ABJ15863.1); human (Accession No. NP_665802.1); mouse (Accession No. BAA12705.1); zebrafish (Accession No. AAT37634.1); chicken (Accession No. XP_004941605.1).

TRAF6 is one of the crucial factors in the TLR signaling pathway, the ubiquitinated TRAF6 activates TAK1, which subsequently phosphorylates IKK and MAPK, leading to NF-kB and AP-1 activation, respectively [26,27]. Previous studies have shown that TRAF6 contains four conserved domains, including a RING domain, a series of zinc fingers, a coiled-coil region and a MATH domain [5,7,28]. In mammals, the N-terminal RING domain and zinc fingers coordinate TRAF6 auto-ubiquitination and its interaction with Ubc13 [29,30]. The coiled-coil region is essential for auto-ubiquitination and activation of the NF-kB signaling pathway [31]. Furthermore, the conserved MATH domain links TRAF6 to upstream molecules [32]. Similar to the TRAF6s of mammals and other species, Et-TRAF6 also contains the classical domains, including one RING domain, two zinc fingers, one coiled-coil region, and one MATH domain. The neighbor joining tree showed that the evolutionary trend of TRAF6 was in accordance with that of the species. High similarity of gene sequence and structural domains suggests that Et-TRAF6 may have a function similar to that of other mammalian and teleost TRAF6s. In E. tauvina, Et-TRAF6 was expressed in all examined tissues, indicating an ubiquitous and constitutive expression of Et-TRAF6, which is similar to expression pattern of teleost fish TRAF6 genes. TRAF6 has been reported in other teleost fish, such as D. rerio, C. carpio, Cirrhinus mrigala and Ctenopharyngodon idella. TRAF6 was found to be predominantly expressed in the gill of D. rerio [7], liver of C. carpio [5], kidney of C. mrigala [33], and in the main immunerelated tissues (head kidney, posterior kidney, and spleen) of C. idella [8]. In our study, Et-TRAF6 expression was high in the main immune-related tissues (stomach, intestine, brain, and spleen) and low in the liver, skin, and muscle. The discrepancies among species in expression patterns might be a result of species variation and differences in developmental stage. Once pathogen invading, the host could trigger the immune response through TRAF6 via the TLR signaling pathway. In mammals, TRAF6 has been shown to

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Fig. 3. Expression of Et-TRAF6 mRNA in healthy and immune-challenged groupers. (A) Tissue distribution of Et-TRAF6 in healthy groupers by qRT-PCR analysis. The expression of Et-TRAF6 in muscle was set to 1.0. (B) Quantification of mRNA expression upon TLRs ligand induction. Six groups of TLRs ligands LPS (10 mg/ml), LTA (5 mg/ml), PGN (20 mg/ml), Zymosan (100 mg/ml), Poly(I:C) (1 mg/ml) and Poly(dA:dT) (1 mg/ml) were i.p. injected into adult groupers at six time points (3, 6, 12, 24, 48 and 72 h). Total RNA was extracted by Trizol and reverse-transcripted into cDNA. qRT-PCR was conducted using the primers. Graphs of mRNA induction fold were presented. (C) Temporal expression of Et-TRAF6 in liver after V. alginolyticus, SGIV and GNNV challenge. Expression values were normalized to those of b-actin, using relative standard curve method. qRT-PCR was carried out three replicates per sample. Data are expressed as the mean fold change (means  S.E., n ¼ 3) from the PBS challenged group. Statistical significance was calculated by Student’s t-test. Bars with “a” at p < 0.05 or “b” at p < 0.01 indicate statistical differences.

participate in activation of the TLR signaling pathways in response to some bacterial and viruses. For example, grass carp TRAF6 (CiTRAF6) was significantly up-regulated during Ichthyophthirius multifiliis infection. In zebrafish embryos and adults, both SHRV and Edwardsiella tarda induced zfTRAF6 upregulation. Orange-spotted grouper TRAF6 (EcTRAF6) increased after infection with Cryptocaryon irritans. We detected that the expression Et-TRAF6 can be induced by bacterial and virus infection. In the liver of grouper, EtTRAF6 was significantly up-regulated not only after challenging with PGN, Zymosan, Poly(I:C) and Poly(dA:dT), but also after V.

alginolyticus, SGIV and GNNV infections. Taken together, our work will help to elucidate the functions of TRAF6 for better understanding of grouper innate immunity and ultimately development of disease control strategies. Localizations of the components in TLR signaling pathways are very important for our understanding of the protein function and molecular organization [35]. In this study, Et-TRAF6 was located in the cytoplasm and nuclear in GS cell, which is similar with Litopenaeus vannamei TRAF6 (LvTRAF6) in S2 cells. The cytoplasmic localization of TRAF6 is consistent with its putative function in

Fig. 4. Intracellular localization of Et-TRAF6. (A) Subcellular localization of Et-TRAF6 by fluorescence microscopy. FHM cells were transfected with pEGFP-N3 (upper row) or pEGFPEt-TRAF6 (lower row). The location of the nucleus was shown by DAPI staining. (B) Expression of Et-TRAF6 in GS cells. After allowing the cells to adhere for 48 h in 6-well plates, indirect immunofluorescence staining of Et-TRAF6 was performed using mice anti-Et-TRAF6 serum and Goat anti-mouse FITC secondary antibody (lower row). Preimmune mouse serum was used as control (upper row). Blue images show the location of the nucleus, stained by DAPI. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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forming a receptor complex when the TLR signaling pathway is activated by PAMPs [36e38]. The nuclear localization of TRAF6 has been reported in human [39,40]. It has been found that TRAF6 negatively regulated c-Myb in the nuclei of B-lymphocytes that maintained cell homeostasis and immune surveillance [40]. In conclusion, the full-length cDNA encoding TRAF6 was isolated from greasy grouper, E. tauvina. It was expressed in all tested tissues, and the expression in the liver was not only up-regulated after challenge with viral/bacterial agents, but also after infections with bacteria and virus. These results indicated that EtTRAF6 might be involved in immune response toward bacterial and virus challenge. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (31202022), the National High Technology Development Program of China (863) (2012AA092201), the National Sparking Plan Project of China (2013GA780001), the Knowledge Innovation Program of the Chinese Academy of Sciences (SQ201203), and the Science and Technology Program of Guangdong (2012A020603018). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fsi.2014.04.022. References [1] Medzhitov R. Toll-like receptors and innate immunity. Nat Rev Immunol 2001;1:135e45. [2] Janssens S, Beyaert R. A universal role for MyD88 in TLR/IL-1R-mediated signaling. Trends Biochem Sci 2002;27:474e82. [3] Suzuki N, Suzuki S, Duncan GS, Millar DG, Wada T, Mirtsos C, et al. Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4. Nature 2002;416:750e4. [4] Ninomiya-Tsuji J, Kishimoto K, Hiyama A, Inoue J, Cao ZD, Matsumoto K. The kinase TAK1 can activate the NIK-I kappa B as well as the MAP kinase cascade in the IL-1 signalling pathway. Nature 1999;398:252e6. [5] Kongchum P, Hallerman EM, Hulata G, David L, Palti Y. Molecular cloning, characterization and expression analysis of TLR9, MyD88 and TRAF6 genes in common carp (Cyprinus carpio). Fish Shellfish Immunol 2011;30:361e71. [6] Stockhammer OW, Rauwerda H, Wittink FR, Breit TM, Meijer AH, Spaink HP. Transcriptome analysis of Traf6 function in the innate immune response of zebrafish embryos. Mol Immunol 2010;48:179e90. [7] Phelan PE, Mellon MT, Kim CH. Functional characterization of full-length TLR3, IRAK-4, and TRAF6 in zebrafish (Danio rerio). Mol Immunol 2005;42:1057e71. [8] Zhao F, Li YW, Pan HJ, Wu SQ, Shi CB, Luo XC, et al. Grass carp (Ctenopharyngodon idella) TRAF6 and TAK1: molecular cloning and expression analysis after Ichthyophthirius multifiliis infection. Fish Shellfish Immunol 2013;34: 1514e23. [9] Qiu L, Song L, Yu Y, Zhao J, Wang L, Zhang Q. Identification and expression of TRAF6 (TNF receptor-associated factor 6) gene in Zhikong scallop Chlamys farreri. Fish Shellfish Immunol 2009;26:359e67. [10] Chen HM, Yang HH, Guangxu S, Yan XM, Campbell R, Berenson JR. The Cdomain of TRAF6 is a potential new target for inhibition of multiple myeloma. Blood 2003;102:931A. [11] He CB, Wang Y, Liu WD, Gao XG, Chen PH, Li YF, et al. Cloning, promoter analysis and expression of the tumor necrosis factor receptor-associated factor 6 (TRAF6) in Japanese scallop (Mizuhopecten yessoensis). Mol Biol Rep 2013;40:4769e79. [12] Wang PH, Wan DH, Gu ZH, Deng XX, Weng SP, Yu XQ, et al. Litopenaeus vannamei tumor necrosis factor receptor-associated factor 6 (TRAF6) responds to Vibrio alginolyticus and white spot syndrome virus (WSSV) infection and activates antimicrobial peptide genes. Dev Comp Immunol 2011;35:105e14. [13] Lu Y, Li C, Zhang P, Shao Y, Su X, Li Y, et al. Two adaptor molecules of MyD88 and TRAF6 in Apostichopus japonicus Toll signaling cascade: molecular cloning and expression analysis. Dev Comp Immunol 2013;41:498e504.

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