Grouper interleukin-12, linked by an ancient disulfide-bond architecture, exhibits cytokine and chemokine activities

Full length article

Grouper interleukin-12, linked by an ancient disulfide-bondarchitecture, exhibits cytokine and chemokine activities

Jui-Ling Tsai a,1, T.A. Jose Priya a,1, Kuang-Yu Hu c, Hong-Young Yan d, San-Tai Shen e,**,Yen-Ling Song a,b,*

a Institute of Zoology, National Taiwan University, Taipei City 106, TaiwanbDepartment of Life Science, National Taiwan University, Taipei City 106, TaiwancDepartment of Bioinformatics, Chung Hua University, Hsin-Chu City 300, Taiwand Laboratory of Sensory Biology, Institute of Cellular and Organismic Biology, Academic Sinica, Yi-Lan County 262, Taiwane Institute of Biological Chemistry, Academic Sinica, Taipei City 115, Taiwan

a r t i c l e i n f o

Article history:Received 4 June 2013Received in revised form3 October 2013Accepted 4 October 2013Available online 28 October 2013

Keywords:Grouper (Epinephelus coioides)IL-12 structurePBL proliferationChemotactic migrationTNF-a induction

a b s t r a c t

Interleukin-12 (IL-12) is a pleiotropic cytokine which bridges innate and adaptive immunity in defenseagainst pathogens. IL-12 proved to be an effective and successful adjuvant to enhance both the innateand adaptive immune responses and could be applicable for a rationale vaccine formulation in fishagainst pathogen infection. We have cloned the p35 and p40 cDNAs of IL-12 from orange-spottedgrouper (Epinephelus coioides). Grouper IL-12 most resembles with sea bass orthologues; moderate tolow identity with other teleost and mammalian counterparts. The structural model of grouper IL-12heterodimer revealed NC141F three amino acid patch of grouper p35, which is present in teleost p35but absent in mammalian and avian p35, and is spatially nearby the conserved cysteine residue located atA-helix of p35 to form a disulfide bond when the 14aa peptide located at loop 1 of grouper p35 wasaligned with human corresponding exon 4, instead of exon 5. The results indicated that the loss of this3aa patch during evolution was compensated by the duplication of exon 4 in mammalian p35 to gainanother cysteine residue to form a disulfide bond, evidenced by chicken p35 which does not contain NCFcorresponding 3-aa patch nor exon 4 duplication. Accordingly, the inter-chain disulfide bond of IL-12heterodimer is conserved from teleost to mammalian IL-12. A single chain grouper IL-12 (scgIL-12)construct linked by (G4S)3 was successfully expressed in baculovirus-insect cell system; its identity hasbeen confirmed by LC/MS/MS. In addition, the biological activity of recombinant scgIL-12 (rscgIL-12) aredemonstrated for its stimulation of PBL proliferation, chemotactic migration, induction of TNF-a geneexpression and a plausible adjuvant effect of prolonged protection against parasite infection in fish. Weillustrated the first time in lower vertebrate that grouper IL-12 possesses both cytokine and chemokineactivities.

! 2013 Elsevier Ltd. All rights reserved.

1. Introduction

IL-12 is a pro-inflammatory cytokine with pleiotropic effectson both innate and adaptive immune responses [1]. It is mainlyproduced by antigen presenting cells (APCs) and phagocytic cells,including monocytes and macrophages, dendritic cells, neutrophils,and B cells when host encountered infection, like other members of

IL-12 family (IL-23 and IL-27) [2,3]. The major site of actions of IL-12are on T and NK cells in cell proliferation, production of INF-g, TNF-a,GM-CSF and other cytokines, resulting in increased cytotoxic activityof these cells [4,5]. After encountering APCs, naïve CD4þ T cells candifferentiate into either type Th1 or Th2 cells, orchestrated by variouscytokines. IL-12 is the main cytokine that induces the polarization ofCD4þ T cells to the Th1 phenotype [6,7]. Therefore, IL-12 playsimportant roles in the regulation of adaptive immune response [7].

IL-12 is structurally a heterodimeric cytokine composed of a p35subunit and a p40 subunit, linked by an inter-chain disulfide bridgeto form the functional molecule. The p35 subunit resembles IL-6and GM-CSF; whereas the p40 subunit is homologous to theextracellular domains of class I cytokine receptor, such as IL-6 Ra[8]. The functions of IL-12 are mediated by IL-12R b1 and b2,

* Corresponding author. Department of Life Science, National Taiwan University,Taipei City 106, Taiwan. Tel.: þ886 2 33662455; fax: þ886 2 33669499.** Corresponding author. Current address: Genomic Research Center, AcademiaSinica, Taipei City 115, Taiwan. Tel.: +886 2 27898073; fax: +886 2 27898811.

E-mail addresses: [email protected] (S.-T. Shen), [email protected](Y.-L. Song).

1 Both authors contributed equally.

Contents lists available at ScienceDirect

Fish & Shellfish Immunology

journal homepage: www.elsevier .com/locate / fs i

1050-4648/$ e see front matter ! 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.fsi.2013.10.009

Fish & Shellfish Immunology 36 (2014) 27e37

through JAK-STAT pathway [9]. The sequence alignment ofmammalian p35 showed that the loop 1 of teleost p35, connectinghelices A and B, is shorter than that of mammalian. Among them, a14-aa peptide fragment with a Cys at 4th position of teleost p35 canbe aligned against either one of two similar 14-aa fragments ofmammalian p35 [10e12]. Those two fragments are encoded by twosimilar exons with a cysteine residue at 4th position, most likelydue to exon duplication in mammalian lineage [13]. The crystalstructure of human IL-12 showed that both the cysteine residuescontribute to form inter-chain and intra-chain disulfide bonds,respectively [14]. How the disulfide bridge arranged in non-mammalian IL-12 has not been explored?

Although expressions of p35 and p40 are regulated indepen-dently, co-expression of p35 and p40 cDNAs in target cells isrequired to generate a biologically active IL-12 heterodimer [15,16].The p40 subunit is usually produced and secreted as a free formexcess over the IL-12 heterodimer. In contrast, secretion of the p35subunit alone is hard to be detected, suggesting that p35 is unstablein the absence of p40 [17]. Meanwhile, the homodimer of p40 hasbeen shown to be a potent IL-12 antagonist [18e21]. Therefore, toexpress active IL-12 protein without p40 homodimer (p(40)2)antagonistic effect is a challenging work.

Teleosts and elasmobranchs are the most primitive vertebratesequipped with adaptive immune system similar to that of mam-mals. Due to the pivotal role of IL-12 in immune response, fish IL-12cDNA was first discovered by analysis of the pufferfish Takifugurubripes genome [11], and then cloned from several other fish, suchas carp [13], sea bass [10], zebrafish and Nile tilapia lately. It hasbeen documented that the teleost p35 mRNA is up-regulated uponviral infection [13], bacterial injection [10], LPS or Con A stimulation[12]; similar results are also found in p40 mRNA expression. Itindicated that teleost IL-12 functions as its mammalian orthologuesin immune response [22,23]. However, to our understanding, noneof teleost IL-12 has been produced as biologically active proteins tostudy their function in fish.

Groupers are potentially important and economically valuableaquaculture species in Southeast Asian countries [38]. In theaquaculture industry, grouper is widely used for intensive farming[39]. However, the intensive culture of grouper inevitably leads tothe incidences of infectious and non-infectious diseases [40,41]that result in serious economic losses [42,43] both in farms andhatcheries. For example, Cryptcaryon irritans is a ciliate protozoanparasite causing white spot disease and high mortality in marinefishes including 93 food, ornamental and wildlife fish species[27,44]. It is urgent to seek the development of prophylactic mea-sures in pathogen defense to increase survival rates, such as anadjuvant for targeted vaccines to evoke immune responses [24,25].In this study, we searched for the adequate cytokine IL-12 anadjuvant which has function both in humoral and cellular immuneresponses and also made efforts to overcome the difficulty ofmaking heterodimeric protein which was successfully expressed ininsect-baculovirus system. Moreover, we have examined theimmunological functions of gIL-12 by means of transient trans-fection of scgIL-12, p40 and p35 into grouper fin cell (GF-1),chemotactic migration of PBLs and via the induction of tumur ne-crosis factor-alpha (TNF-a). Our findings hold the potential that IL-12 could be applied to vaccine as an adjuvant to reach protectivesteps and to decrease mortality rate in the Grouper culture.

2. Materials and methods

2.1. Fish maintenance and cDNAs preparation

Five orange-spotted grouper (Epinephelus coioides) with anaverage body weight of 30 g and 30 cm body length were kept in

38 cm " 25 cm " 26 cm tank with recirculating fresh salt-water(30e35 ppt) at 27 # 1 $C and were fed twice daily with standardcommercial pellets. They were given a two-week acclimatizationperiod prior to treatments. Fish were water-borne infected with1.8 " 104 live theronts of Cryptocaryon irritans for 3 days to up-regulate IL-12 expression, before spleen sampling [26]. Fishspleens were collected and total RNA was extracted using TRIZOLreagent (Invitrogen Life Technologies). The first-strand cDNA wassynthesized with oligo (dT)-containing Adaptor Primer (AP; Invi-trogen, Carlsbad) as described in manufacture manual.

2.2. Cloning of p35 and p40 cDNAs of grouper IL-12 by RACEmethods

According to published fish cDNAs of IL-12 cDNAs, sequencesfrom sea bass (GenBank accession No. DQ388037 and DQ388039)and fugu (GenBank accession No.JGI7381 and JGI3377) were usedas template, and degenerate primers of p35 and p40 were designedfor PCR amplification of grouper IL-12 cDNAs. For p35 subunit,forward primer 1 and reverse primer 2 were used for PCR ampli-fication; for p40 subunit, forward primer 3 and reverse primer 4were used. The sequences of the primers are shown in Table 1. ThePCR was run for 35 cycles at 30 s at 94 $C, 30 s at 50 $C and 45 s at72 $C, and the PCR products were analyzed with 1.2% agarose gelelectrophoresis. The expected size of PCR products was subclonedinto pGEM-T Easy vector (Promega, Wisconsin) and was confirmedby nucleotide sequencing.

The complete cDNAs of p35 and p40 genes were obtained byusing 50- and 30- RACE kits. The 30-RACE of the cDNAwas amplifiedfrom the first-strand cDNA products by PCR using gene specificprimer for p35/p40 and Abridged Universal Amplification Primer(AUAP) which is complementary to AP. PCR amplification was asfollows: initial denaturation at 94 $C for 2 min, then 35 cycles of94 $C for 30 s, 55 $C for 30 s, and 72 $C for 1 min, followed by a finalelongation at 72 $C for 10 min. For 50-RACE, the first-strand cDNAwas synthesized using SMART" RACE cDNA Amplification kit(Clontech, Mountain View, USA). After diluting, 2 ml of first-strandcDNA was added into a PCR reaction containing universal PrimerMix (UPM; Clontech) and a reverse gene specific primer for p35/p40, and then a nested PCR reactionwas conducted by using nesteduniversal Primer (NUP) and gene specific nest primer. AmplifiedPCR products were subcloned into the pGEM-T Easy vector andsequenced. The primers used above are listed in Table 1.

2.3. Multiple amino acid sequences alignment and phylogenetictree analysis

The nucleotide sequences and deduced amino acid sequences ofp35 and p40 from other vertebrate species were aligned withgrouper p35 and p40 using ClustalW [28]. The secondary structure ofmultiple sequence alignments was analyzed in ESPript [29]. Thephylogenetic tree was constructed in MEGA5 [30] using theneighbor-joining method based on the multiple amino acidsequence alignment [31]. The evolutionary distanceswere computedusing the Poisson correction method and are in the units of thenumber of amino acid substitutions per site. All positions containingalignment gaps and missing data were eliminated only in pairwisesequence comparisons (Pairwise deletion option). The tree is drawnto scale with branch lengths in the same units as those of theevolutionary distances used to infer the phylogenetic tree.

2.4. Structural modeling of grouper IL-12

Structural models of grouper IL-12 were constructed by usingcrystal structures of human IL-12 complexes as templates; pdb

J.-L. Tsai et al. / Fish & Shellfish Immunology 36 (2014) 27e3728

code: 1F45 [14] and 3HMX [32]. The amino acid sequences ofgrouper p35 and p40 were aligned with human p35 and p40,respectively. Due to the similarity of exon 4 and exon 5 (eachcontaining 14-aa) of human p35, the corresponding 14-aa fragmentof grouper p35 could be aligned with either exon 4 or exon 5.Therefore, those two different models with or without cysteinedisulfide bonds restricts were constructed, respectively, by usingBuild Homology Models module of Discovery Studio 2.5 (AccelrysSoftware Inc.) with energy minimization. Structural figures weregenerated by PyMOL program (The PyMOL Molecular GraphicsSystem, Schrödinger, LLC.).

2.5. Construction of baculovirus expression vectors and transfectionof Sf21 cells

For each subunit of grouper IL-12, ORF of p35 and p40 cDNAswere amplified by PCR, including StuI restriction site at 50-end,

6" His tag and stop codons and PacI site at 30-end of cDNA,respectively. Alternatively, we constructed a cDNA vector to ex-press scgIL-12 by tethering p40 CDS followed by a (G4S)3 linkerand p35 mature peptide (Fig. 4C). It was generated by overlapPCR amplification. N-terminal half cDNA was amplified by for-ward primer 5 and reverse primer 6, and C-terminal half byforward primer 7 and reverse primer 8; and then full lengthcDNA of scgIL-12 was amplified by second run overlap PCR. ThePCR products were cloned into pABhRpX vector which containsDsRed gene for observation; the DNA sequences were confirmedby nucleotide sequencing. The expression vectors were co-transfected with BaculoGold Linearized baculovirus DNA (BDPharmigen, New Jersey) into insect Spodoptera frugiperda (Sf21)cells, respectively. To amplify its titers, monolayer Sf21 cells werecultured at 26 $C and propagated for four generations asdescribed previously. The efficiency of transfection was moni-tored by fluorescence of DsRed of the transfected cells.

Table 1Primers used in the present study.

Primers Sequence Note

1 50-GRARRASCTGTTCAGTGGAAT-30 Degenerated forward primers of p352 50-GATGMYKCGGACCTGGAAG-30 Degenerated reverse primers of p353 50-TTRGGAGGGGGCAACTACA-30 Degenerated forward primers of p404 50-AARTRAGSGGGAAGTAGGAG-3 Degenerated reverse primers of p405 5-GGGTCGGGCGGTGGGGGATCTGGGGGCGGTGGATCCGTGCCACTGATGGGTACAGGA-30 Forward primer of p35 for linker6 50-TTAATTAATTAGTGGTGGTGGTGGTGGTGGTGTGTTCGCCAGAGTTCAGGTA-30 Reverse primer of p35 for linker7 50-AGGCCTATGAAGTTGTTTATTTTCGGT-30 Forward primer of p40 for linker8 50-GCCTCCAGATCCCCCACCGCCCGACCTCCGCCACCTTTG TCTTGCTGACGTTTGC-30 Reverse primer of p40 for linker9 50-GGTGTCCTGCTGTTTGCTTG-30 Forward primer of TNF-a10 50-CTCCACTCGAGCTGGTGTTT-30 Reverse primer of TNF-a11 50-CAAAGCCAACAGGGAGAAGA-30 b-actin gene detection for real -time PCR12 50-ACCAGAGGCATACAGGGACA-30 b-actin gene detection for real-time PCR

Fig. 1. Multiple alignments of amino acid sequences of the grouper p35 subunit with vertebrate orthologues. The predicted signal peptide for each protein is shown in boldfaceletters. Identical residues in all proteins are shown on black background with white letters and similar residues (matrix Blosum 62) are shown in box. Fourteen amino acid patch ofteleost p35 corresponding to Exon4 of human and Exon5 of mammalian are in box. Unique three amino acid patch (N/KCF) and putative glycosylation site (NIT) for teleost p35 arealso shown on black background with white letters. The disulfide bonds are paired in the same number. The cysteine residue that forms an inter-chain disulfide bond with p40 ismarked with star above sequences; residues in contact with p40 within 4.0 !A with circles; Arg residues to form inter-subunit salt-bridge with triangle. The secondary structureelements are shown on the top and bottom lines for the p35 subunit of grouper IL-12 model and human IL-12 crystal structure, respectively. a-helices are depicted as helices; a-turnas T’s. aA refers to helix A.

J.-L. Tsai et al. / Fish & Shellfish Immunology 36 (2014) 27e37 29

2.6. Purification of recombinant proteins and western blotting

For protein expression, monolayer Sf21 cells in T-flask wereinfected by recombinant virus at a multiplicity of infection (MOI)between 3 and 6. After three days of infection, culture medium

were collected and mixed with equal volume of binding buffer(50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 7.4) withprotease inhibitor cocktail (Roche) and incubated at 4 $C for 10min.The supernatant was centrifuged at 10,000 " g at 4 $C for 10 minand the supernatant was mixed with Ni-NTA agarose slurry

Fig. 2. Multiple alignments of amino acid sequences of the grouper p40 subunit with vertebrate orthologues. Sequences are presented as in Supplemental Fig. 1. Cysteine residuesunique for teleost and unique for tetrapod are shown in black background with white letters. The disulfide bonds are paired in the same number. The cysteine residue that forms aninter-chain disulfide bond with p35 is denoted with star; residues in contact with p35 within 4.0!A with circles. The secondary structure elements are shown on the top and bottomlines for the p40 subunit of grouper IL-12 model and human IL-12 crystal structure, respectively. a-helices, and a-strands are depicted as helices and arrows; a-turn as T’s. aD1Arefers to strand A of domain 1.

J.-L. Tsai et al. / Fish & Shellfish Immunology 36 (2014) 27e3730

(Bioman Scientific, Taiwan) at 4 $C for 1 h. The mixture was sub-sequently loaded into a column and allowed to flow continuouslywith washing buffer (50 mM NaH2PO4, 300 mM NaCl, 30 mMimidazole). The recombinant protein was eluted by using theelution buffer (50mMNaH2PO4, 300mMNaCl, 250mM imidazole).Protein concentration was determined by using the Bradfordmethod with BSA as standard.

The recombinant protein was analyzed by electrophoresis in 12%SDS-PAGE and visualized by SYPRO RUBY staining and Westernblotting. For Western blotting, the SDS-PAGE gel was transferred toPVDF membrane and blotted with rabbit anti-His antibody (1:4000;Bioman Scientific, Taiwan) and alkaline phosphatase-conjugatedgoat Ig against rabbit IgG (1:4000; Abcam, USA). 4-Nitro bluetetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate(NBT/BCIP, Roche) in Tris buffer were used for color development.

2.7. LC/MS/MS analysis

To identify the purified recombinant scgIL-12 (rscgIL-12), in-gel digestion was performed. In brief, the protein band in 12%

SDS-PAGE was manually excised from the gel and sliced intopieces. The gel pieces were alkylated with iodoacetamide anddigested with sequencing grade trypsin (Promega, USA) ac-cording to standard protocols. The dried digested peptides werereconstituted in 0.1% FA in H2O and analyzed on Waters SynaptG2 HDMS (Waters, Milford, MA). Samples were injected onto a2 cm " 180 mm capillary trap column and separated on a75 mm " 25 cm nanoACQUITY 1.7 mm BEH C18 column using ananoACQUITY Ultra-Performance LC system (Waters, Milford,MA). The MS was operated in ESI positive V mode with aresolving power of 10,000 and calibrated. Data acquisition wasperformed using data directed analysis (DDA) method. The DDAmethod included one full MS scan (m/z 350e1600, 1 s/scan) andthree MS/MS scans (m/z 100e2000, 1 s/scan) performedsequentially on the three most intense ions presented in the fullscan mass spectrum. Analysis of all MS/MS samples wassearched over in-house scgIL-12 database by using Mascotprogram (Matrix Science; version 2.4.0) with a fragmention mass tolerance of 0.05 Da and a parent ion tolerance of25 ppm.

Fig. 3. Structural model analysis of grouper IL-12. (A) Stereo view of grouper IL-12 model. Helices A-D are represented as cartoon in magenta, lime, cyan, and blue color,respectively; the same color coding is used throughout this report unless specified. Side chains of Loop1 (in orange) are shown in thin sticks; for those corresponding to mammalianexon4 are in thick sticks. Disulfide bonds are shown in spheres; their appearing order and residue numbers are indicated and inter-chain disulfide bond denoted with asterisk.Atoms are colored by element: N in blue, O in red, S in yellow, and C in the same with cartoon presentation. (B) Stereo view of grouper IL-12 model focus on inter-chain salt-bridgerelay. Salt-bridge (D108of helix A and R178of helix D) unique for p35 and E211 of p40 subunit are presented in sticks. Side chains of helices constituted the inner hydrophobic core areshown in spheres. (C) Overall structure of heterodimeric grouper IL-12, viewed from the back in figure A. The conserved salt-bridge interlocks two subunits are shown in spheresand C atoms in yellow. Conserved residues including WSEWS motif and several hydrophobic residues of E and F strands of D3 are presented in sticks. Resides of p40 subunit aredenoted in brown; p35 in blue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

J.-L. Tsai et al. / Fish & Shellfish Immunology 36 (2014) 27e37 31

2.8. Preparation of fresh PBLs

Five to 10 milliliters of whole blood from adult grouper fish (BWw600 g, n ¼ 3) were withdrawn from the caudal vein with 1/4volumes of anticoagulant (0.8% EDTA in 0.9% NaCl). To this, fourfolddilution of AL-mediummix (Aim V serum-free lymphocyte medium,Leibovitz’s L-15 medium containing 50 mM 2-MT and 7.5% NaHCO3)was added and mixed gently. Then, 5 mL of the diluted blood wascarefully layered over 4 mL lymphoprep (Axis Shield) in a 15 ml tubeand centrifuged at 350"g for 20 min at room temperature. Aftercentrifugation, the PBL cells formed as visually distinct phase at thesample/medium interface were transferred into a clean 50 mL tubeusing amicropipette. Then, the harvested PBL fractionwaswashed in40 mL AL-medium and centrifuged for 10 min at 600"g. The PBLpellet was dissolved in 1mL of AL-medium. The number of cells wascounted by a Hemocytometer, and the viability of the PBLs waschecked by Tryptan blue staining test.

2.9. Preparation of Con A-stimulated PBLs and cell proliferationassay

The biologic activities of rscgIL-12 and rp40 were examinedusing cell proliferation assay. A total of 5 " 106 PBL cells/ml of AL-medium supplemented with 5% Fetal bovine serum were stimu-lated with 2.5 mg of Concavaline A (Con A). Following incubation for72 h at 25 $C, Con A-stimulated PBLs were harvested, and the ac-tivity of residual Con Awas inhibited by incubation in the presenceof 0.1 M methyl a-D-mannopyranoside (a-MM) (Sigma, St. Louis,MO) for 30 min. Cells were then washed once in AL-medium, re-suspended and seeded in a 24well plate at a density of 5"105 cells/mL of AL-medium. The cells were then supplemented with variousconcentrations of rscgIL-12 (2.5 rM, 25 rM, 125 rM, 375 rM and625 rM) and rp40 (2.5 rM, 25 rM, 125 rM, 375 rM and 625 rM),the concentrations of which were estimated by Bradford’s proteinconcentration measurement test. Cells were cultured in a CO2incubator at 25 $C for 4 days. The cells were then harvested bycentrifugation at 2800 rpm for 10 min, and the cells were addedwith 5:1 ratio of 0.4% Trypan Blue Stain for 5 min at room tem-perature. The viable cells were quantified using a hemocytometer.

2.10. TNF-a gene induction and chemotactic migration of PBLs

We have used 24 well plates with polycarbonate Transwell"permeable supports (5 mm pore size; Corning Inc. Life Sciences)

for further assays to avoid the possibilities of recombinant pro-tein degradation during and after the extraction process. To beginwith, the capabilities of rscgIL-12, rp40 and rp35 on the induc-tion of grouper tumor necrosis factor-a (gTNF-a) gene expressionwere evaluated. In brief, 2 " 105 Grouper fin cells (GF-1) weresuspended in 100 mL of L-15 Medium (Leibovitz) supplementedwith 5% FBS, 100 IU penicillin/ml, 100 mg of streptomycin/ml and2.5 mg amphotericin/ml and plated onto Transwell" inserts keptupon a 24 well plate. The cells were incubated overnight at 25 $Cto permit attachment on the transwell. The medium level waschecked periodically and fresh medium was added as required.GF-1 cells were then transfected transiently with 3 mg each of theplasmid constructs of scIL-12, p40 and scIL-12 þ p40 cDNAs inpcDNA3.1þ using 3 mg/well of Cellfectin II Reagent (Invitrogen).The empty vector (pcDNA3.1þ alone) was transfected for themock control. At 5 h post-transfection, the medium was changedto fresh AL-medium, and the lower compartment of the Trans-well, was added with 2 " 105 freshly prepared PBL cells in 600 mLof AL-medium. The upper Traswell containing the transfected GF-1 cells and the lower compartment holding the PBL were co-cultured at 25 $C for 36 h. Afterward, the PBL in the lowerchamber was harvested by centrifugation for 10 min at 600"g.Total RNA from the above PBL pellet was extracted using TRIzolreagent (Life Technologies, USA) and cDNAwas prepared by usingSuperscript III Reverse Transcriptase. A PCR was performed for thePBL cDNA template to confirm the presence of positive bands of208 bp using primers 9 and 10 (Table 1) corresponding to nu-cleotides 265e473 bp of gTNFa (GenBank accession No.FJ009049). The PCR condition was as follows: 94 $C for 4 min,followed by 30 cycles of 94 $C for 30 s, 58 $C for 30 s and 72 $C for45 s. The resultant 208 bp PCR product was sequenced andconfirmed its 100% identity with grouper TNF-a (GenBank:FJ009049) and 83% identity with stripped beakperch Oplegnathusfasciatus TNF-a (GenBank: ACM69339). Then, real-time PCR wasperformed using primers 9 and 10 and 2" KAPA" SYBR# qPCRMaster Mix (KAPA Biosystems) in an ABI 7500 Q-PCR systemusing the standard program. The grouper b-actin gene served asinternal control which was performed with primer 11 and 12.The gTNFa mRNA expression in the PBL was calculated relative togrouper b-actin expression. The relative expression was repre-sented by the equation: (gTNFa expression level in PBL)/(b-actinexpression level). The data were analyzed using analysis ofvariance (ANOVA) and Duncan’s multiple range test (Duncan’sMRT) to determine the significant difference between test

Fig. 4. Grouper IL-12 recombinant protein expression in Sf21 cells. Proteins purified from culture supernatant of Sf21 cells which were transfected with baculovirus carrying eitherp40 subunit, both p35 and p40 subunits, or scgIL-12 were analyzed by 12% SDS-PAGE and Western blotting with anti-His tag antibody. Lane 1: arrows from up to bottom representw80 kDa p(40)2, w60 kDa p70, w40 kDa p40 monomer and w30 kDa p40 monomer, respectively; Lane 2: arrows from up to bottom represent w80 kDa p(40)2, w40 kDa p40monomer and w30 kDa p40 monomer, respectively; Lane 3: arrow represents w130 kDa p(70)2; Lane 4: upper arrow represents w40 kDa p40 monomer and the lower onerepresents w30 kDa p40 monomer; Lane 5: upper arrow represents w40 kDa p40 monomer and the lower one represents w30 kDa p40 monomer; Lane 6: arrow indicatesw65 kDa p70 molecule; Lane 7: un-transfected cell culture supernatant and Lane 8: crude protein.

J.-L. Tsai et al. / Fish & Shellfish Immunology 36 (2014) 27e3732

samples. The specificity of real-time PCR products was confirmedby melting curve analysis.

Subsequently, the chemotaxis function of rp40, rscgIL-12, andrscgIL-12 þ rp40 was further assayed by using the above Trans-well" permeable system, but with a reverse approach. For thisassay, the transfected GF-1 cells were plated onto the lower 24 wellplate chamber and the freshly prepared PBL was added into theupper Transwell" permeable insert at 36 h post-GF-1 transfection.The cell number and the transfection procedure were all the sameas above mentioned. The PBL was allowed to transmigrate from theupper side of the Transwell" polycarbonate permeable membraneinto the lower side for about 90 min. At this time point, the poly-carbonate permeable membranes were gently pulled out from theTranswell" stained with May-Grunwald and Giema stains. Briefly,the membrane was first fixed in methanol for 5 min and immersedin May-Grunwald solution diluted 1:1 with Sorenson’s buffer(0.2 M Na2HPO4$12H2O and 0.2 M NaH2PO4$2H2O) for 15 min.Following washing in tap water, the membrane was immersed inGiema (1:9 in Sorenson’s buffer) for 10min. After repeated washingin tap water and soaking in Sorenson’s buffer for 4 min, themembranes were slightly dried and mount onto a microscopicslide. The migrated cells were observed microscopically and thenumber of cells was quantified by Stereo Investigator (MBFBioscience) using optical fractionator approach. The differenceswere analyzed using ANOVA and Duncan’s new multiple rangetests. P < 0.05 was considered statistically significant.

To test in vivo immunogenic effect, grouper fingerlings with anaverage BW of 2 g and a body length of 4 cmwere divided into twogroups and held in separate tanks. Each group contains three rep-licates with 16 fish each. One group was kept as untreated and thesecond group of fish were orally intubated with chitosan nano-encapsulated gscIL-12 at a concentration of 20 mg/g fish BW. At20 days post IL-12 administration, fish from both untreated andimmunized group were challenged by C. irritans parasite theronts[46]. The appearance of white spots on the body surface from 2 to 3days after challengewas considered as specific infection. Number ofdead fish was recorded daily. Data analysis was performedwith SASsoftware (SAS Institute, 1989).

3. Results

3.1. Cloning of p35 and p40 cDNA of grouper IL-12

The cloned p35 cDNA (GenBank accession No. HQ154062)(926 bp) contains 591 bp of ORF and 330 bp of 30-untranslated re-gionwhich contain seven rapid mRNA degradation motifs (AUUUA)and a canonical polyadenylation site at 15 bp upstream of polyA tail(Supplemental Fig. S1). The ORF encodes a 196-aa protein with aputative 25-aa signal peptide. Meanwhile, the cloned p40 cDNA(HQ154061) (1563 bp) contains a 313 bp of 50-UTR, 1071 bp of ORF,and 179 bp of 30-UTR (Supplemental Fig. S2). The deduced proteinconsisted of 356-aa residues, including a putative 18-aa signalpeptide.

3.2. Characterization of p35 and p40 subunits of grouper IL-12

The multiple amino acid sequence alignments of p35 and p40subunits of grouper IL-12 with other vertebrate orthologues wereshown in Figs. 1 and 2, respectively. Grouper p35 showed highestidentity with sea bass orthologues (78.4%); moderate identity withfreshwater fish p35 (from 32 to 51% identity with carp, zebrafish,pufferfish, and fugu); and least identity with mammalian p35 (20e25%). The secondary structure prediction indicated that grouperp35 contains four a-helices as other orthologues. Despite of lowidentity with mammalian p35, residues for important structural

features are highly conserved among vertebrates, including hy-drophobic residues of four a helices, residues located in the inter-face of IL-12 complex (denoted above the sequence alignment inFig. 1), and six of seven cysteine residues. However, the followingtwo features are conserved in teleost p35 but different frommammalian counterpart. The loop 1 connecting helix A and B ofgrouper p35 is 23-aa shorter than that of human, and three extraresidues including a cysteine residue (NCF) was found in the end ofhelix C.

Parallel to p35 subunit, grouper p40 protein subunit of IL-12showed highest identity (73.9%) with sea bass ortholog; moderateidentity (34.6e43.3%) with freshwater fish; least identity (22e27%)with mammalian (Fig. 2). Resembling class I cytokine receptor,grouper p40 contains one Ig domain and two fibronectin type IIIdomains. Conserved residues are mainly located on the b strandswhich are usually important for structural integrity and stability,including a canonical W333SEWS signature in D3F strand. In addi-tion, two loops connecting between F and G strands of D2 domainand between A and B strands of D3 domain are highly conservedwhich are located on the interface of heterodimeric IL-12.

3.3. Structural models of grouper IL-12

The multiple sequence alignment of p35 showed that a 14-aafragment between Helix A and B of grouper and other teleost p35can be aligned with either one of two similar exons (exon 4 and 5,each encoding 14-aa with a cysteine residue at the fourth position).To address this issue, structural models of grouper p35 subunitalone or in complex with p40 to form heterodimeric IL-12 wereconstructed according to their sequence alignment on exon 4 orexon 5 of human p35, respectively. When aligned grouper andhuman p35 based on exon 4, the structural model of grouper IL-12is an energy favor structure (Fig. 3A). The major core structure ofgrouper p35 is constituted by four a helices bundle in upeupedownedown topology. The loop 1, connecting helices A and B,tethers with helices B and D of p35 and p40 subunit through threedisulfide bonds, including Cys64 with Cys169 of helix D, Cys81 withCys101 of helix B, and Cys88 with Cys207 of p40 (Fig. 3A). In thecrystal structure of human IL-12, the exon 5 cysteine residue of p35loop 1 provides an extra disulfide bridge with helix A (C37eC110;data not shown) which is absent in teleost p35. Whereas, our ho-mology model of grouper IL-12 showed that the NC141F, unique forteleost p35, is spatially nearby the conserved cysteine residue ofhelix A (Cys38) and to form disulfide bond.

By contrast, when aligned the 14-aa fragment of grouper 35with exon 5 of human p35 and constrain C88eC38of grouper p35 toform a disulfide bond, the inter-chain disulfide bond is absent andstructure in an energy unfavorable (data not shown). It is unlikelybecause the cysteine residue of p40 (Cys207 in grouper and Cys199 inhuman) to form inter-chain disulfide bond is actually highlyconserved (Fig. 2). Therefore, it is more reasonable to align teleostp35 with mammalian p35 on exon 4 instead of exon5 as shown inFig. 1. When comparing the human IL-12 structure with grouper IL-12 model, we found that peptides of exon 4 and exon 5 of humanp35 need to be packed underneath helices bundle crowdedly, dueto extra 14 -aa residues in human IL-12 (data not shown).

As hypothesized, the structural model of grouper IL-12 showedthat the inner core of p35 four helices is mainly constituted ofhydrophobic residues, particularly those with bulky side chains(Fig. 3B). Meanwhile, the intra-helix salt-bridge between helix B(D108 for grouper p35) and helix D (R178) are conserved in thegrouper IL-12 and form a hydrogen bond/salt-bridge relay with T77

of p35 and E211 of p40 (Fig. 3B). All the above four residues arehighly conserved in vertebrate IL-12 as shown in multiple sequencealignment (Figs. 1 and 2). This inter-helix and inter-subunit salt-

J.-L. Tsai et al. / Fish & Shellfish Immunology 36 (2014) 27e37 33

bridge relay is unique for IL-12, but not found in other IL-12 familymembers (IL-23, IL-27) or IL-6 (data not shown).

In addition to the aforementioned inter-chain disulfide bondand salt-bridge relay, the interface of grouper IL-12 heterodimer ismainly composed of the interactions of D helix of p35 subunit withhinge region of p40 D2/D3 domains in a “key-in-hole” feature asshown in Fig. 3C. Arg184 residue, protruded from D helix of p35,formed a salt-bridge with Asp326 of D3E strand of p40 subunit,surrounded by several hydrophobic residues from D helix of p35(I180 and Y188), and D2 domain (Y136) and D3 domain (W265, Y271,F272, P273, L274, and F328) of p40 subunit as shown in Fig. 3C (someresidues data not shown). Those residues are highly conserved invertebrate IL-12 as shown in multiple sequence alignment (Figs. 1and 2) and play an important role in IL-12 heterodimer confor-mation. Mutation of either Arg or Asp of this inter-subunit salt-bridge abrogates the formation of heterodimer IL-12 (2). This salt-bridge of grouper p40 was further enforced by a p-cation stackingmotif, formed by positively charged side chains (R323 and K325) witharomatic residues of canonical W333SEWS motif from the adjacentstrand (Fig. 3C).

3.4. Phylogenetic analysis of IL-12

To illustrate the relationship of the cloned p35 and p40 ofgrouper IL-12 with other related molecules, two phylogenetic treesbased on their multiple amino acid sequence alignments wereconstructed as shown in Supplemental Figs. S3 and S4. These twotrees were both divided into two major groups, Actinopterygii andMammalia. And, both the grouper IL-12 p35 and p40 subunits wereclosely related with homologs in sea bass. The topologies of thesetwo trees were quite consistent with the NCBI taxonomy.

3.5. Over-expression of single chain grouperIL-12 recombinantprotein

For functional studies, we first tried to produce p40 subunit ofgrouper IL-12 in insect-baculovirus expression system. The Ni-NTApurified proteins revealed four protein bands in non-reducing PAGEaround 30, 40, and 80 kDa molecular weights, respectively (lane 2of Fig. 4). Under reducing condition, only 30 and 40 kDa bandsappeared (lane 5 of Fig. 4A). In order to obtain the biological activeheterodimeric IL-12, we tried to co-express both p35 and p40subunits of grouper IL-12 in baculovirus expression system. West-ern blotting of recombinant proteins showed two protein bandswith molecular weight around 60 and 80 kDa in non-reducingPAGE (Lane 1, Fig. 4). Since, these 60 and 80 kDa bands are sus-pected for whether it is p(40)2 or IL-12, we have constructed scgIL-12 (GenBank accession No. KC662465) by combining p40 subunitwith p35 subunit using (G4S)3 linker (Supplementary Fig. S5). ThescgIL-12 was also expressed in baculovirus system and the re-combinant protein was purified. Western blot analysis showed thatrscgIL-12 appeared as a single band (Lane 6, Fig. 4) at molecularweight at around 65 kDa under reducing SDS-PAGE. Without b-ME,this band was shifted above130 kDa (Lane 3, Fig. 4)

3.6. LC/mass/mass Identification of single chain grouper IL-12recombinant protein

Six trypsin-digested peptide fragments were identified andsequenced which covered both p35 and p40 subunits (Fig. 5A). Theapparent mass-to-charge ratio (m/z) corresponded amino acid se-quences of six identified peptides (Fig. 5B). Two representativefragmented mass spectra were shown to confirm their amino acidsequences (Fig. 5C and D). The LC/MS/MS data clearly showed thatthe purified protein is our rscgIL-12.

3.7. Functional assays of recombinant proteins

The rscgIL-12 enhanced the proliferation of Con A-stimulatedPBL significantly at a concentration of 125 rM. And, the prolifera-tion was maintained at a constant level for higher concentrations(above 125 rM). Meanwhile, the treatment of rp40 enhanced theproliferation of Con A-stimulated PBL significantly at concentra-tions of 25 and 125 rM. Nevertheless, there was no change inresponse for lower concentration (below 125 rM) and showed le-thal effect at concentrations above 125 rM (Fig. 6A). Further, scgIL-12 exerted significantly high induction of gTNFa mRNA expressionin PBL, as measured by real-time PCR. Apparently, p40 also inducedgTNF-a transcript prominently. But when p40 was co-transfectedwith scgIL-12, the level of gTNF-a gene expression significantlyreduced from that of scgIL-12 alone (Fig. 6B). In the chemotaxisassay, the results showed that both scgIL-12 and p40 ensuredchemotactic effect which was demonstrated by the significantlyincrease in number of PBL that were migrated from upper to thelower side of the polycarbonate membrane. However, co-transfection of p40 with scgIL-12 significantly reduced thechemotactic effect of scgIL-12 (Fig. 6C). The in vivo effect of scIL-12showed that the survival of parasite challenged fish was prolongedwhen scIL-12 was given orally (Fig. 6D). The fish from untreatedgroup presented a lethal effect and 100% fish were found deadbetween D3 and D5 post-challenge, while 83% of scIL-12 adminis-tered fish were survived until 7 day post-challenge.

4. Discussion

It has been shown that AUUUAmotifs of mRNA contribute to theinstability of transcripts encoded by important regulatory genes,such as cytokines, lymphokines and proto-oncogenes in mammal.From the analysis of 30-UTR of vertebrates p35 mRNA, we foundthat four to six of AUUUAmotifs prevalently occur in the p35mRNAof vertebrates, from mammal to piscine (data not shown). Unlikep35 subunit, there is only one AUUUA motif in the 30-UTR of p40mRNA. It has been reported that almost no free p35 subunit of IL-12can be detected which associates with low expression of stablemRNA; by contrast excess p40 in monomer or dimer usually foundconcomitant with relative higher level of mRNA [19]. The differ-ential mRNA stability of p35 and p40 may contribute to controlfunctional heterodimeric IL-12 level.

The WSXWS signature of p40 is an important structural featureof IL-12 in efficient receptor folding and secretion for hemopoietinreceptor family [33] and conserved in grouper p40 subunit. We alsonoticed that the C-terminal tail after the WSEWS signature ofgrouper p40 is mainly composed of positive charge and polar res-idues, including 10 Lys/Arg and 6 polar residues among 19aa. It is aunique feature for teleost p40 which is not found in other verte-brate p40. There is no discussion regarding the function of thispositive charge patch so far. We hypothesize that the electroposi-tive patch of piscine p40 subunit may help teleost IL-12 interactwith membrane surface molecules such as receptors, heparin sul-fate, or glycoproteins to facilitate the binding with its cognate re-ceptors and the signaling transduction. Actually, the p40 subunit isa soluble version of membrane-bound class 1 cytokine receptor.Without transmembrane segment, therefore, this positive chargepatch of teleost IL-12 may provide interaction sites withmembrane.

Even though the secondary structure prediction showed thatp35 and p40 of grouper IL-12 possesses their characterizingstructural features, respectively, their identities with mammalianorthologues are low (below 25%). Particularly for the p35 loop 1which connects helix A and B, there are 45-aa for grouper p35 but68-aa for humanp35. Due to the recent duplication of exon4/exon 5

J.-L. Tsai et al. / Fish & Shellfish Immunology 36 (2014) 27e3734

in mammalian p35 [13], corresponding 14-aa fragment in loop 1 ofpiscine p35 could be aligned with either exon 4 or exon 5 ofmammalian orthologues equally. Our structural models of grouperIL-12 strongly support the notion that the 14-aa fragment of teleostp35 should be aligned against with exon 4 of mammalian p35.When aligned with mammalian exon 4, the inter-chain disulfidebridge of piscine p35 is conserved and the extra three amino acidresidues (NC121F) of grouper p35 are spatially nearby the conservedcysteine residue (C38 for grouper p35) of helix A to form a disulfidebond. Although the inter-chain disulfide bond is not absolutelyrequired for IL-12 heterodimer formation, significantly reducedamount of IL-12 are observed when IL-12 lack these cysteine resi-dues [14]. Our structural model indicates that the inter-chain di-sulfide bond is a common feature for vertebrate IL-12, from fish tomammal. Perhaps, site-directed mutagenesis at Cys88 position ofgrouper p35 can prove whether teleost and mammal are reallyusing the same region exon 4 to form inter-chain disulfide bondwith p40. It needs further studies to clarify this issue.

Interestingly, chicken p35 does not contain NCF fragment orexon4 duplication to pair with cysteine residue on helix A (Fig. 1).From the evolutional viewpoint, the above three residues (N/KCF)of piscine p35 are lost during evolution, resulting in the conservedcysteine residue of helix A unpaired as found in chicken. Theduplication of exon 5 in mammalian p35, which provides a cysteineresidue to form a disulfide bond with helix A, seems to be the resultof evolution pressure to compensate the loss of NCF fragment.

Western blot of rp40 (Fig. 4) indicated that the protein band atmolecular weight around 80 kDa would be mediated by disulfidebond because it was disappeared in reducing condition. The other

protein bands at molecular mass around 30 and 40 kDa are mostlikely p40, due to heterogenous glycosylation, because there arefive putative Asn glycosylation sites in p40 peptide of grouper IL-12.Similar results were found for p40 of murine IL-12 expressed byinsect-baculovirus system due to the heterogeneity of glycosylation[19].Western blot of co-expressed p35 and p40 proteins (Fig. 4) alsorevealed similar protein patterns of p40 expression. Co-expressedp35 and p40 showed two proteins bands around 60 and 80 kDa,of which the 60 kDa is most likely the IL-12 heterodimer and the80 kDa would be the p40 homodimer. It has been reported thatboth p40 and p35 subunits have to be produced within the samecell to obtain the biologically active heterodimeric IL-12 [17].However, we could not identify p35 subunit from the purifiedprotein by mass spectrometry analysis. It has been reported thatonly low levels of IL-12 heterodimer and an excess of free p40 andno p35 subunit alone was secreted in free form in EpsteineBarrvirus (EBV)-transformed human B lymphoblastoid cell lines[20,34]. Up regulation of p35/p40 mRNA ratio would balance theprotein expression level and improve the heterodimeric IL-12expression level [35]. Our results imply that co-transfection ofp35 and p40 into insect cell by baculovirus still could not providebalanced expression for two subunits to form intact grouper IL-12.Henceforth, scgIL-12 was constructed by joining both the p40 andp35 subunits, incorporating a linker sequence in between the twoentities. Grouper IL-12 was promptly biosynthesized as a singlechain IL-12 (scIL-12) protein, which was confirmed by Westernblotting and also by LC/Mass/Mass. However, western blot of rscgIL-12 (Fig. 4), a clear band of IL-12 was observed at molecular weightaround 65 kDa. Without b-ME, it was shifted above 130 kDa and

Fig. 5. LC/MS/MS analysis of recombinant scgIL-12. (A). Amino acid sequence of rscgIL-12. Six peptide fragments identified by selected LC/MS peak were underlined. Predictedsignal peptide of p40 were denoted by dash underline and (G4S)3 linker in italics. (B). The apparent mass-to-charge ratio (m/z), experimental data of mass derived from observedm/z, calculated mass of peptide, and corresponding amino acid sequences of six identified peptides. Modified amino acid C (carbamidomethylation) and M (oxidation) wereunderlined. (C) and (D) are two representative spectra of peptides identified in the recombinant proteins with monoisotropic mass of neutral peptide 702.40 and 1776.89,respectively. Identification of the peptides was conducted by comparison of the fragmentation patterns of individual peptides with those predicted from locally generated grouperIL-12 databases.

J.-L. Tsai et al. / Fish & Shellfish Immunology 36 (2014) 27e37 35

above, implying that the dimerization is mediated by disulfidebridges (Lane: 3, Fig. 4).

Here we firstly demonstrate that the rscgIL-12 can stimulate theproliferation of Con A-stimulated PBLs, in a dose dependentmanner. The rscgIL-12 significantly stimulated cell proliferationsuggesting that the bio-produced rscgIL-12 successfully retainedthe biological activity. Also, rp40 showed significant cell prolifera-tion effect at a low dose (25 pM), however, it is lethal to the cells athigher concentrations (Fig. 6A). This indicates that grouper rp40bioactivity is consistent with those of human and mouse p40subunit [18e20]. Further studies are required to determinewhether piscine rp40 and rp(40)2 function is similar to mammalianones.

Chemokines are involved in fighting against various pathogeninfections and in shaping the immune response. One of the well-known defense mechanisms of chemokines is mediated throughcompetitive binding. For IL-12, p40 could be the competitor proteincombat for IL-12Rb1. To assess the competitive receptor bindingfunction of gIL-12 and gp40, TNF-a gene induction and chemotacticeffect assay were performed. Also, IL-12 can up-regulate the tran-scriptional expression of TNF-a which has been demonstrated forrepairing and regenerating potential of damaged nervous system,frequently caused by nervous necrotic virus (NNV) infection ingrouper. The results showed that, the effect of scgIL-12 on theexpression of TNF-awas significantly up-regulated. In addition, p40itself is able to induce TNF-a to a significant level, but p35 aloneshowed no effect. Reports showed that incubation of mouse spleencells when cultured with IL-12 p40 can aid in TNF-a gene inductionas measured by ELISA [45]. Addition of p40 significantly inhibited

the effect of scgIL-12 as anticipated. Our data thus demonstrate thatthe production of p(40)2 mediates the induction of TNF-awhich onthe other hand competes with IL-12 to IL-12Rb1 antagonistically[37]. Besides without suspicion, addition of p35 did not alter theeffect of scgIL-12 based on the data of statistical analysis.

The results from chemotactic activity of rscgIL-12 were consis-tent with the TNF-a induction assay. The rscgIL-12 was found toexhibit chemotaxis which was revealed by the significantly greaternumber of PBL migrated toward the rscgIL-12 protein ought to beextant in the GF-1 transfected culture medium. In the same way,rp40 alone showed chemotaxis effect and moreover, addition ofrp40 to rsgIL-12 significantly suppressed the effect. Our data onceagain suggest that the production of p(40)2 likely mediates themigration of PBL via IL-12Rb1 and compete with IL-12 for bindingto IL-12Rb1 thus inhibit IL-12-induced biologic activity, demon-strating that p(40)2 is an antagonist of IL-12 [20,36]. Further, weassumed that as cytokines, IL-12 should be involved in immuneactivation which supports the development of both cell-mediatedand humoral immunity. Thus, we made a preliminary test for itsin vivo adjuvant effect by oral administration of scIL-12 DNA intofish. Subsequent challenge with a parasite C. irritans, showed pro-longed pathogen resistance in fish (Fig. 6D), one of the character-istics of an adjuvant. This prolonged protection in turn depicts thatscIL-12 could be a candidate protein for adjuvant in fish againstpathogen.

In conclusion, this study was to clone, express and characterizeboth p35 and p40 subunits of IL-12 from orange-spotted grouper.The first time results obtained from the functional assays of IL-12such as cell proliferation, TNF-a induction and chemotactic

Fig. 6. (A) Cell proliferation of Con A-stimulated PBLs treated with recombinant proteins. A total of 5 " 105 Con A-stimulated PBLs were treated with rp40 or rscgIL-12 (2.5 rM,25 rM, 125 rM, 375 rM and 625 rM). The viable cells were quantified by Tryptan blue staining (ANOVA and Duncan’s new multiple range tests, p < 0.05, n ¼ 5). (B) Relativetranscriptional expression of gTNF-a. PBL was induced by GF-1 cells transfected with either scgIL-12, p40 or scgIL-12 þ p40, respectively. The b-actin was used as the normalizationcontrol. Different alphabets above the column show significant difference statistically (Duncan’s multiple T-test, p < 0.05, n ¼ 3). (C) Chemotactic migration of PBL toward GF-1 cellstransfected with p40, scgIL-12 and scgIL-12 þ p40, as analyzed in Transwell" permeable inserts. Un-transfected and mock transfected GF-1 cells were taken as control. (Duncan’smultiple T-test p < 0.05, n ¼ 3). (D) Total of 16 fish with three replicates was orally intubated with chitosan encapsulated gscIL-12 at a concentration of 20 mg/g fish BW. At 20 dayspost-gscIL-12 administration, fish were challenged by Cryptocaryon irritans parasite theronts. The data showed the mean survival of fish.

J.-L. Tsai et al. / Fish & Shellfish Immunology 36 (2014) 27e3736

migration are very encouraging. The increasing economic lossescaused by diseases in aquaculture indicate the need to develop newprophylactic strategies. The results showed that in teleost IL-12functions as mammalian orthologues and may serve as a goodcandidate element for pathogenic defense in fish aquaculture.

Acknowledgments

We greatly appreciate Dr. Fan-Hua Nan, Department of Aqua-culture, NTOU for providing healthy groupers, Dr. Ying-Chou Lee,Institute of Fisheries Science, NTU, for helping statistical analysisand Dr. Chia-Chi Ku, Institute of Immunology, College of Medicine,NTU for her instructions regarding the chemotactic assay. Greatappreciation also extends to Dr. Yu-May Lee, Institute of BiologicalChemistry, Academia Sinica, and Dr. Shih-Hu Ho, Taiwan OceanResearch Institute, National Applied Research Laboratory, forvaluable suggestions and revision of the manuscript. We thank BCTof NTU for protein preparation for mass analysis. LC-MS/MS datawere acquired at the Common Mass Spectrometry Facilities ofAcademia Sinica. The work was supported financially by NationalScience Council, R.O.C with Grant No. NSC101-2321-B-002-055 toYen-Ling Song.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.fsi.2013.10.009.

References

[1] Watford WT, Moriguchi MA, Morinobu, O’Shea JJ. The biology of IL-12: coor-dinating innate and adaptive immune responses. Cytokine Growth Factor Rev2003;14:361e8.

[2] Vignali DA, Kuchroo VK. IL-12 family cytokines: immunological playmakers.Nat Immunol 2012;13:722e8.

[3] Trinchieri G. Interleukin-12 and the regulation of innate resistance andadaptive immunity. Nat Rev Immunol 2003;3:133e46.

[4] Kobayashi ML, Fitz MR, Hewick RM, Clark SC, Chan S, Loudon R, et al. Iden-tification and purification of natural killer cell stimulatory factor (NKSF), acytokine with multiple biologic effects on human lymphocytes. J Exp Med1989;170:827e45.

[5] Stern AS, Podlaski FJ, Hulmes JD, Pan YC, Quinn PM, Wolitzky AG, et al. Pu-rification to homogeneity and partial characterization of cytotoxic lymphocytematuration factor from human B-lymphoblastoid cells. Proc Natl Acad Sci U SA 1990;87:6808e12.

[6] Buchanan JM, Vogel LA, Van Cleave VH, Metzger DW. Interleukin 12 alters theisotype-restricted antibody response of mice to hen eggwhite lysozyme. IntImmunol 1995;7:1519e28.

[7] Trinchieri G. Interleukin-12: a cytokine produced by antigen-presenting cellswith immunoregulatory functions in the generation of T-helper cells type 1and cytotoxic lymphocytes. Blood 1994;84:4008e27.

[8] Jones LL, Vignali DA. Molecular interactions within the IL-6/IL-12 cytokine/receptor superfamily. Immunol Res 2011;51:5e14.

[9] Stark GR, Darnell JE. The JAK-STAT pathway at twenty. Immunity 2012;36:503e14.

[10] Nascimento DS, do Vale A, Tomas AM, Zou J, Secombes CJ, dos Santos NM.Cloning, promoter analysis and expression in response to bacterial exposureof sea bass (Dicentrarchus labrax L.) interleukin-12 p40 and p35 subunits. MolImmunol 2007;44:2277e91.

[11] Yoshiura Y, Kiryu I, Fujiwara A, Suetake H, Suzuki Y, Nakanishi T, et al.Identification and characterization of Fugu orthologues of mammalianinterleukin-12 subunits. Immunogenetics 2003;55:296e306.

[12] Arts JA, Tijhaar EJ, Chadzinska M, Savelkoul HF, Verburg-van Kemenade BM.Functional analysis of carp interferon-gamma: evolutionary conservation ofclassical phagocyte activation. Fish Shellfish Immunol 2010;29:793e802.

[13] Huising MO, van Schijndel JE, Kruiswijk CP, Nabuurs SB, Savelkoul HF, Flik G,et al. The presence of multiple and differentially regulated interleukin-12p40genes in bony fishes signifies an expansion of the vertebrate heterodimericcytokine family. Mol Immunol 2006;43:1519e33.

[14] Yoon CS, Johnston C, Tang J, Stahl M, Tobin JF, Somers WS. Charged residuesdominate a unique interlocking topography in the heterodimeric cytokineinterleukin-12. EMBO J 2000;19:3530e41.

[15] Gubler UA, Chua O, Schoenhaut DS, Dwyer CM, McComas W, Nabavi N, et al.Coexpression of two distinct genes is required to generate secreted bioactive

cytotoxic lymphocyte maturation factor. Proc Natl Acad Sci U S A 1991;88:4143e7.

[16] Schoenhaut DS, Chua AO, Wolitzky AG, Quinn PM, Dwyer CM, McComas W,et al. Cloning and expression of murine IL-12. J Immunol 1992;148:3433e40.

[17] D’Andrea AM, Rengaraju NM, Valiante J, Chehimi MK, Aste M, Chan SH, et al.Production of natural killer cell stimulatory factor (interleukin 12) by pe-ripheral blood mononuclear cells. J Exp Med 1992;176:1387e98.

[18] Gillessen SD, Carvajal PL, Podlaski FJ, Stremlo DL, Familletti PC, Gubler U, et al.Mouse interleukin-12 (IL-12) p40 homodimer: a potent IL-12 antagonist. Eur JImmunol 1995;25:200e6.

[19] Heinzel FP, Hujer AM, Ahmed FN, Rerko RM. In vivo production and functionof IL-12 p40 homodimers. J Immunol 1997;158:4381e8.

[20] Ling P, Gately MK, Gubler U, Stern AS, Lin P, Hollfelder K, et al. Human IL-12p40 homodimer binds to the IL-12 receptor but does not mediate biologicactivity. J Immunol 1995;154:116e27.

[21] Mattner F, Fischer S, Guckes S, Jin S, Kaulen H, Schmitt E, et al. The interleukin-12 subunit p40 specifically inhibits effects of the interleukin-12 heterodimer.Eur J Immunol 1993;23:2202e8.

[22] Secombes CJ, Wang T, Bird S. The interleukins of fish. Dev Comp Immunol2011;35:1336e45.

[23] Overgard AC, Nepstad I, Nerland AH, Patel S. Characterisation and expressionanalysis of the Atlantic halibut (Hippoglossus hippoglossus L.) cytokines: IL-1beta, IL-6, IL-11, IL-12beta and IFNgamma. Mol Biol Rep 2012;39:2201e13.

[24] Metzger DW. Interleukin-12 as an adjuvant for induction of protective anti-body responses. Cytokine 2010;52:102e7.

[25] Metzger DW, Buchanan JW, Collins JT, Lester TL, Murray KS, Van Cleave VH,et al. Enhancement of humoral immunity by interleukin-12. Ann N Y Acad Sci1996;795:100e15.

[26] Schade B, Fischer HG. Toxoplasma gondii induction of interleukin-12 is asso-ciated with acute virulence in mice and depends on the host genotype. VetParasitol 2001;100:63e74.

[27] Colorni A, Diamant A. Ultrastructural features of Cryptocaryon irritans, a ciliateparasite of marine fish. Eur J Protistol 1993;29:425e34.

[28] Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H,et al. Clustal W and Clustal X version 2.0. Bioinformatics 2007;23:2947e8.

[29] Gouet PEC, Stuart DI, Metoz F. ESPript: analysis of multiple sequence align-ments in postscript. Bioinformatics 1999;15:305e8.

[30] Tamura KDP, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecularevolutionary genetics analysis using maximum likelihood, evolutionary dis-tance, and maximum parsimony methods. Mol Biol Evol 2011;28:2731e9.

[31] Saitou N, Nei M. The neighbor-joining method: a new method for recon-structing phylogenetic trees. Mol Biol Evol 1987;4:406e25.

[32] Luo J, Wu SJ, Lacy ER, Orlovsky Y, Baker A, Teplyakov A, et al. Structural basisfor the dual recognition of IL-12 and IL-23 by ustekinumab. J Mol Biol2010;402:797e812.

[33] de Vos AM, Ultsch M, Kossiakoff AA. Human growth hormone and extracel-lular domain of its receptor: crystal structure of the complex. Science1992;255:306e12.

[34] Boulanger MJ, Chow DC, Brevnova EE, Garcia KC. Hexameric structure andassembly of the interleukin-6/IL-6 alpha-receptor/gp130 complex. Science2003;300:2101e4.

[35] Lupardus PJ, Garcia KC. The structure of interleukin-23 reveals the molecularbasis of p40 subunit sharing with interleukin-12. J Mol Biol 2008;382:931e41.

[36] Slight SR, Lin Y, Messmer M, Khader SA. Francisella tularensis LVS-inducedInterleukin-12 p40 cytokine production mediates dendritic cell migrationthrough IL-12 receptor beta1. Cytokine 2011;55:372e9.

[37] Carra GFG, Trinchieri G. Biosynthesis and posttranslational regulation of hu-man IL-12. J Immunol 2000;164:4752e61.

[38] Harikrishnan R, Balasundaram C, Heo MH. Molecular studies, disease statusand prophylactic measures in grouper aquaculture: economic importance,disease and immunology. Aquaculture 2010;309:1e14.

[39] Boonyaratpalin M. Nutritional requirements of marine food fish cultured inSoutheast Asia. Aquaculture 1997;151:283e313.

[40] Chua FHC, Loo JJ, Wee JY. Investigation of outbreaks of a novel disease, “SleepyGrouper Disease”, affecting the brown spotted grouper, Epinephelus tauvinaForskal. J Fish Dis 1994;17:417e27.

[41] Fukuda Y, Nguyem HD, Furuhashi M, Nakai T. Mass mortality of culturedsevenband grouper, Epinephelus septemfasticatus, associated with viral ner-vous necrosis. Fish Pathol 1999;31:165e70.

[42] Lee KK. Pathogensis studies on Vibrio alginolyticus in the grouper, Epinephelusmalabaricus Bloch et Schneider. Microb Pathog 1995;19:39e48.

[43] Yeh SP, Chang CA, Liu CY, Cheng W. Dietary sodium alginate administrationaffects fingerling growth and resistance to Streptococcus sp. and iridovirus,and juvenile non-specific immune responses of the oranges spotted grouper,Epinephelus coioides. Fish Shellfish Immunol 2008;25:19e27.

[44] Colorni A, Burgess P. Cryptocaryon irritans Brown 1951, the cause of whitespot disease in marine fish: an update. Aquarium Sci Conservation 1997;1:217e38.

[45] Davis Craig, Turner Joanne. Role of IL-12 in T cell activation in old mice. ASenior Honors Thesis. Ohio State University; 2008.

[46] Jose Priya TA, Lin YH, Wang YC, Yang CS, Chang PS, Song YL. Codon changedimmobilization antigen (iAg), a potent DNA vaccine in fish against Crypto-caryon irritans infection. Vaccine 2012;30(5):893e903.

J.-L. Tsai et al. / Fish & Shellfish Immunology 36 (2014) 27e37 37