CHARACTERIZATION,OF,TUMOR,NECROSIS,FACTOR,RECEPTOR,ASSOCIATED,FACTOR,3,(TRAF3),IN,NILE,TILAPIA:,EXPRESSION,PROFILES,AND,FUNCTIONS,IN,NF-κB,PATHWAY

XIA Hong-Li, WANG Zhi-Wen, LI Yuan, CHEN Wen-Jie, , LONG Meng, , YU Da-Peng, CHENG Jun,XIA Li-Qun, and LU Yi-Shan,

(1.Guangdong Provincial Engineering Research Center for Aquatic Animal Health Assessment, Shenzhen Public Service Platform for Evaluation of Marine Economic Animal Seedings, Shenzhen Institute of Guangdong Ocean University, Shenzhen 518120,China; 2.Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture, College of Fishery,Guangdong Ocean University, Zhanjiang 524088, China; 3.State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430000, China)

Abstract: Tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) is a highly versatile regulator in many immune pathways, including TNFR and Toll-like receptor (TLRs)/RIG-I-like receptors (RLRs) signal pathway.In this study, a TRAF3 gene was cloned from Oreochromis niloticus (named OnTRAF3), (Gen-Bank No.MN258118), which contained a RING finger, a zinc finger, a coiled-coil and MATH domain.Multiple sequence alignment showed that OnTRAF3 shares a relatively high identity with other known TRAF3 proteins.Quantitative real-time PCR (qRT-PCR) analysis showed OnTRAF3 transcripts were highly expressed in brain, skin, intestine and gill, and it could be induced by Streptococcus agalactiae.In HEK293T cells, OnTRAF3 presented both in the cytoplasm and nucleus.In addition, wide type (WT) OnTRAF3, activated NF-κB signal significantly, RING or Zinc finger was important for this activation.This study provides significant insights into the functions of TRAF3 in tilapia.

Key words: TRAF3; Immune response; NF-κB pathway; Oreochromis niloticus

The Tumor-necrosis factor receptor (TNFR) associated factors (TRAFs) are a family of adapter molecules that bind directly to the intracellular domains of the TNFR superfamily[1,2].Accumulating data showed that TRAF molecules were widely employed in both innate and adaptive immunity[1,3].Studies in mice also revealed effects varying in impaired immune function when the different TRAFs were deleted[4].Most of TRAF members shared well-defined functional domains, including a C-terminal TRAF domain that mediates TRAF dimerization and interaction with other proteins, zinc fingers as well as RING finger which known to function as E3 ubiquitin ligase[5,6].Owing to the high structural similarity, different TRAF proteins might be connect and regulate same signaling pathway, but had the overlap or opposite effect[7].Like other TRAF family members,TRAF3 functions as adapter protein and E3 ubiquitin ligase, which are important in regulating downstream signal transduction events[8—11].Additionally, TRAF3 promotes immune-related gene production, such as IL-10, TNF-α and I type IFN.On the other hand,TRAF3 negatively regulates downstream effectors in mammals, including NF-κB pathway, mitogen-activated protein kinase signal, survival of B cells and inflammatory responses[10,12,13].Therefore, TRAF3 is thought to be a distinctive member among TRAFs in that it’s multi-functional in different signal pathway.

Compared with its mammalian counterpart, the function of piscine TRAF3 was under largely un-known, TRAF3 homologues have been cloned and characterized from several species, including common carp (Cyprinus carpio)[14], yellow croaker (Larimichthys crocea)[15], humphead snapper (Lutjanus sanguineus)[16]and black carp (Mylopharyngodon piceus)[17].Notably, the surprising result showed that TRAF3 play different-even contrasting roles for NF-κB pathway in humphead snapper[16]and black carp[17].As for the exact role of OnTRAF3 in immune signal pathway has not been reported.Nile tilapia (O.niloticus) is a pivotal economic fish and widely cultured throughout the world.In recent years, it suffers serious diseases, especially caused byS.agalactiaethat resulting in large economic loss[18—20].In this study, a TRAF3 (OnTRAF3) homolog was cloned from Nile tilapia.The expression profiles of OnTRAF3 and subcellular localization were detected.Moreover, effects of OnTRAF3 (wild type or mutants) on signal transduction function were also investigated.These data will expand our knowledge about the role of TRAF3 in immune response of teleost.

1.1 Fish, challenge, and sampling

Sample of Nile tilapia [average weight of (100±10) g] were obtained from local commercial market(Shenzhen, China).Prior to experiment, the fish were acclimated in aerated sand-filtered water at 28℃ and fed for one week prior to experimental manipulation.A series of samples including gill, skin, muscle, head kidney, heart, intestine, thymus, liver, spleen and brain were collected and immediately frozen by liquid nitrogen until used for next analysis.

S.agalactiaeZQ0910 was cultured according to the method described by Wang,et al.[21].The experimental fish were randomly divided into three groups(36 fish per group).For challenged group, fish were injected intraperitoneally with 100 μL of formalin-inactivatedS.agalactiaesuspension (1×107cells/mL).Meanwhile, the control group fish were injected with 100 μL 1×PBS.Each sample contained three independent individuals respectively.At time points of 0,12h, 24h, 48h, 72h and 96h post-injection, sample from the gill, skin, head kidney, spleen, intestine and thymus were collected aseptically from the control and infected groups, and immediately frozen by liquid nitrogen, followed by storage at –80℃ for quantitative real-time PCR analysis.

1.2 RNA isolation and cDNA synthesis

Total RNA was isolated from the sample of Nile tilapia using TransZol Up (Trans, China) according to the manufacturer’s instructions.The quality of total RNA was detected by Qubit3.0.The first-strand cDNA synthesis was carried out through EasyScript One-step gDNA Removal and cDNA Synthesis SuperMix (Trans, China).

1.3 Cloning of cDNA sequence

The coding sequence of TRAF3 obtained from whole-genome sequencing data ofO.niloticus.PCR was conducted using a Thermal Cycler 2720 (Thermo fisher) by specific primers, under the following amplification conditions: 94℃ for 5min, followed by 35 cycles at 94℃ for 30s, 65℃ for 30s, and 72℃ for 2min, with a final extension at 72℃ for 10min.

1.4 Bioinformatics

The open reading frame (ORF) was analyzed with the ORF Finder program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html).The nucleotide and predicted amino acid sequences of OnTRAF3 were analyzed using Genetyx 7.0 software.The protein analysis was conducted with ExPASy tools (http://expasy.org/tools/).Multiple-sequence alignment of the reported TRAF3 amino acid sequences was performed using ClustalX2.0 and a phylogenetic tree was constructed by MEGA 5.0 software.

1.5 Real-Time PCR

qRT-PCR was performed by IQ5 Real-time PCR System (Bio-Rad laboratories) with SYBR Green Master mix (TOYOBO).All of specific primers used were listed in the Tab.1.Reactions were performed in a final volume of 10 μL, comprising of 0.3 μL cDNA sample, 5 μL SYBR Green Master mix, 0.5 μL (10 μmol/L) each primer and 3.7 μL PCR-grade water.The PCR conditions were as follows: 94℃ for 3min, followed by 40 cycles of 94℃ for 30s, 55℃ for 30s and 72℃ for 30s.Each sample was assayed in triplicate.The qRT-PCR data was analyzed by 2–∆∆Ctmethod.The results were expressed as mean±SD and statistical analysis was performed using SPSS10.0 software.

1.6 Construction of expression plasmids

The entire ORF of TRAF3 was amplified with primers pDsRed-3F/3R containingNheI andHindⅢrestriction enzyme sites (Tab.1) and inserted into the corresponding sites of the pDsRed-Monomer-N1 plasmid respectively.Similarly, the recombine plasmid of pcDNA-TRAF3 also constructed withKpnI andEcoRI restriction enzyme sites (Tab.1).Moreover, four truncated forms of OnTRAF3, including∆RING (deletion of residues 76－114), ∆Zinc finger(deletion of residues 159—273), ∆coiled-coil (deletion of residues 278－389) and ∆MATH (deletion of residues 443—556) were amplified by recombinant PCR with primers TRAF3T1/T2/T3/T4-F, TRAF3T1-R1, TRAF3T1-F2, TRAF3T2-R1, TRAF3T2-F2,TRAF3T3-R1, TRAF3T3-F2, TRAF3T4-R1,TRAF3T4-F2 and TRAF3T1/T2/T3/T4-R (Tab.1),then subcloned into the pcDNA3.1 vector.All the recombinant plasmids were transferred intoEscherichia coliand sequenced.The recombinant plasmids were extracted using an E.Z.N.A.Endo-free Plasmid Mini Kit (Promega) according to the manufacturer’s instructions, which were used for further analysis.

Tab.1 Sequences of primers used in this study

1.7 Subcellular localization analysis of OnTRAF3

HEK-293T cells were cultured at 37℃ in highglucose Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (Gibco, USA).The HEK-293T cells were seeded onto coverslips (10 mm×10 mm) in a 24-well plate.After the cell adhering for 18h, the HEK-293T cells were transiently transfected with empty pDsRed-Monomer-N1 and recombinant pDsRed-TRAF3, using Lipofectamine™ 3000 Reagent (Invitrogen, USA) according to the manufacture’s protocol.After transfection for 24h, HEK-293T cells were washed with PBS (pH 7.4) and fixed with 4% paraformaldehyde for 30min, and then stained with 4’, 6- diamidino-2-pheny-lindole (DAPI) (1 μg/mL) for 15min.Finally, the cells were rinsed with PBS, mounted with 50% glycerol, and observed using fluorescence microscopy (Leica, Germany).

1.8 Cell transfection and luciferase reporter assay

HEK-293T (1×106cells/well) were cultured at 37℃ in MEM containing 10% (v/v) fetal bovine serum.For transfection, cells were seeded into 48-well plates and cultured for 24h at 37℃, then 254 ng plasmids containing 125 ng recombinant or pcDNA3.1 plasmids, 125 ng NF-κB reporter plasmids (Promega) and 4 ng pRL-TK Renilla (Renilla reniformis) luciferase plasmids were co-transfected using LipofectamineTM3000 (Invitrogen, USA).Each sample was run in triplicate.At 48h post-transfection, cells were harvested with passive lysis buffer (Promega, USA).The firefly (Photinus pyralis) and Renilla luciferase activities were determined with a Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions.

2.1 Bioinformatic analysis of OnTRAF3

The complete coding sequence of TRAF3 was identified fromO.niloticus, (GenBank No.MN258118),which encoded a putative protein of 591 amino acids.The deduced protein of OnTRAF3 contained a RING finger (76—114), a TRAF-type zinc finger (159—273),coiled-coil (278—389) and a MATH domain (443—566)at the C-terminus.These functional domains are conserved from mammals to fish, especially the C-terminal MATH domain.The deduced amino acid of OnTRAF3shared highest identity (99%) toMaylandia zebraTRAF3.Additionally, a phylogenetic tree was constructed using MEGA5.0.The phylogenetic analysis showed that OnTRAF3 was clustered tightly withM.zebraTRAF3 (Fig.1).

Fig.1 Phylogenetic analysis of OnTRAF3

2.2 Tissue distribution of OnTRAF3

Quantitative RT-PCR analysis revealed that On-TRAF3 was ubiquitously expressed in all the tissues tested.The highest level of OnTRAF3 transcript was detected in the brain, which was about 41-fold the level detected in the liver, the tissue with the lowest level of OnTRAF3 transcript.Moderately expression level was observed in skin, intestine, gill, spleen, head kidney and spleen (Fig.2).

Fig.2 Constitutive expression of OnTRAF3 in healthy tilapia tissues determine with qRT-PCR

2.3 Expression of OnTRAF3 in different organs of Nile tilapia after S.agalactiae challenge

The temporal expression of OnTRAF3 after challenged byS.agalactiaewas analyzed by qRTPCR.Compared with the control group, OnTRAF3 expression in the gill was initially down regulated,then reaching a maximum level at 12h following the challenge withS.agalactiae, after which it subsequently declined and remained at a low level until 96h.A similar expression pattern was detected in the skin, head kidney and spleen, the highest expression was observed respectively at 96h, 4h and 48h.In intestine, OnTRAF3 mRNA level was stable (even decreased) and then slightly up-regulated, subsequently,there was a secondly raise and reached the peak at 72h.As shown in Fig.3, the similar phenomenon was found in thymus, in which double raise was detected,but the maximum expression increased to 5.9-fold at 48h.

Fig.3 Expression analyses of OnTRAF3 in different tissues after S.agalatiae stimulation

2.4 Subcellular location of OnTRAF3

The subcellular localization of OnTRAF3 was determined by pDsRed-TRAF3 fusion protein expression in HEK-293T cells.The red fluorescence in pDsRed-TRAF3 fusion protein transfected HEK-293T cells were distributed in the cytoplasm and nucleus of HEK-293T (Fig.4).

Fig.4 Subcellular localization of OnTRAF3 in HEK-293T cells by fluorescence microscopy.The cells are transfected with pDsRed-Monomer-N1 or pDsRed- TRAF3 and the nucleus is stained with 4’,6-dimaidino-2-pheny-lindole (DAPI)

2.5 The effect of OnTRAF3 in NF-κB activity

As shown in Fig.5, over expression of WT On-TRAF3 efficiently induced the activation of NF-κB signaling.Subsequently, we tested all of the mutants for their ability to induce NF-κB signaling, four types of domain-missing mutant of OnTRAF3 were constructed and transfected into 293T cells.The results showed that the deletion of coiled-coil and MATH domain did not affect the activation, but no significant increase activity was observed in ∆RING or∆zinc finger mutant compared to WT OnTRAF3.

Fig.5 Over-expression of OnTRAF3 efficiently activate the activity of NF-κB in HEK293 Cells

TRAFs have been proposed to be molecular bridge that transduces signals from members of TN-FR superfamily, adaptive and innate immune receptors, as well as cytokine receptors[6,22—25].In mammals, TRAF3 participates in many key immune signaling pathways, as a membrane associated, intracellular, and nuclear protein[26].In the current study, the whole coding sequence OnTRAF3 was cloned, moreover,some of molecular biological characteristics were characterized.Multiple sequence alignment and phylogenetic analysis revealed that OnTRAF3 shared very high identify with other known teleost TRAF3 proteins.Structural analysis revealed that OnTRAF3 posed typical functional domains of TRAFs, with a RING a Zinc finger, coiled-coil and MATH domain.Interestingly, unlike humphead snapper[16]and blackcarp[17]TRAF3 with two zinc fingers, just only on zinc finger was found in Nile tilapia.Whether reduction in the number of zinc finger had an impact on the function of OnTRAF3 need further study.

qRT-PCR analysis revealed that OnTRAF3 transcript was observed in all examined tissues at different expression levels, which was similar to TRAF3 from mammals and other teleost fish.This suggest that diverse roles of TRAF3 in many physiological processes.

AfterS.agalactiaestimulation, the expression of OnTRAF3 increased at different times in gill, skin,head kidney and spleen.Those results revealed that OnTRAF3 participated in innate immune response of piscine fish.Furthermore, the intestine and thymus differed in this regard, there were double up-regulation at different time points afterS.agalactiaechallenge, even the secondly raise was more noticeable(5.4-fold and 5.9-fold).Taken together, OnTRAF3 was involved in both innate and adaptive immune response for the Gram-positive bacterial infection.Increasing human and mouse evidence indicates that alterations in TRAF3 expression contribute to the pathogenesis of systemic inflammation[27,28].Moreover,the up-regulation of TRAF3 serves as a feedback mechanism to suppress inflammation of mammals[29].The evidences above implied that TRAF3 was important for inflammation response.We speculate that OnTRAF3 may also be related to inflammation which caused by pathogen invasion, but further research is needed to confirm this hypothesis.

Subcellular localization analysis suggested that OnTRAF3 presented both in cytoplasm and nucleus of HEK-293T.According to the reports, most of TRAFs, including TRAF3, are generally considered to be cytoplasmic proteins, and have interactions with other proteins in the cytoplasm of cells[30].Moreover,nucleus TRAF3 is important for B cell survival by regulates stability of the CREB (cAMP response element binding protein)[31].Those hinted that OnTRAF3 not only participated in immune signal transduction,but also had function in survival and proliferation of immune cells.Nuclear factor κB is a family of inducible transcription factors that regulate cell survival,proliferation and differentiation, immune and inflammatory responses[6,32,33].So far, activation of NF-kB signaling was commonly classified as two distinct routes: canonical or noncanonical pathways[33].Accumulating evidence suggested that TRAF3 is a critical suppressor of the noncanonical NF-κB pathway in mammals, and our previous study revealed thatLutjanus sanguineusTRAF3 efficiently inhibits NF-κB activity in FHM cells[6,13,16,33—35].However, in this study, OnTRAF3 play a contrasting role in NF-κB pathway.Over expression of OnTRAF3 significantly activated NF-κB activity, which in line with black carp TRAF3[17].Furthermore, the activation effect of OnTRAF3 was partly reduced when the RING or zinc finger domain deleted, implying the pivotal role of RING and zinc finger domain in TRAF3 for modulating NF-κB activity.Meanwhile, in pigeon, Zinc finger and Coiled coil domains were essential for the ac-tivation of NF-κB[36].Those implies that the role of TRAF3 regulating in NF-κB activity, and the domain responsible, vary between species.

In the present study, we cloned the whole coding sequence of TRAF3 from Nile tilapia.The putative amino acid sequence of OnTRAF3 shared similar functional domains, including RING, zinc finger,coiled-coil and MATH domain, which conserved in almost TRAF proteins.OnTRAF3 was ubiquitously expressed in all detected tissues and its expression was up-regulated significantly after S.agalactiaechallenge.The RING and zinc finger of OnTRAF3 were pivotal for activating NF-κB pathway.We concluded that OnTRAF3 might be involvement in teleost antimicrobial defense at different approach,which provided a new thought into versatile regulator of TRAF3.