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Nodavirus Infection of Sea Bass ( Dicentrarchus labrax) Induces Up-Regulation of Galectin-1 Expression with Potential Anti-Inflammatory Activity, This information is current as of October 1, 2021. Laura Poisa-Beiro, Sonia Dios, Hafiz Ahmed, Gerardo R. Vasta, Alicia Martínez-López, Amparo Estepa, Jorge Alonso-Gutiérrez, Antonio Figueras and Beatriz Novoa J Immunol published online 21 October 2009

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 21, 2009, doi:10.4049/jimmunol.0801726 The Journal of Immunology

Nodavirus Infection of Sea Bass (Dicentrarchus labrax) Induces Up-Regulation of Galectin-1 Expression with Potential Anti-Inﬂammatory Activity1,2

Laura Poisa-Beiro,* Sonia Dios,* Haﬁz Ahmed,† Gerardo R. Vasta,† Alicia Martínez-Lo´pez,‡ Amparo Estepa,‡ Jorge Alonso-Gutie´rrez,* Antonio Figueras,* and Beatriz Novoa3*

Sea bass nervous necrosis virus is the causative agent of viral nervous necrosis, a disease responsible of high economic losses in larval and juvenile stages of cultured sea bass (Dicentrarchus labrax). To identify genes potentially involved in antiviral immune defense, gene expression profiles in response to nodavirus infection were investigated in sea bass head kidney using the suppression subtractive hybridization (SSH) technique. A total of 8.7% of the expressed sequence tags found in the SSH library showed significant similarities with immune genes, of which a prototype galectin (Sbgalectin-1), two C-type lectins (SbCLA and SbCLB) from groups II and VII, respectively, and a short pentraxin (Sbpentraxin) were selected for further characterization. Results of Downloaded from SSH were validated by in vivo up-regulation of expression of Sbgalectin-1, SbCLA, and SbCLB in response to nodavirus infection. To examine the potential role(s) of Sbgalectin-1 in response to nodavirus infection in further detail, the recombinant protein (rSbgalectin-1) was produced, and selected functional assays were conducted. A dose-dependent decrease of respiratory burst was observed in sea bass head kidney leukocytes after incubation with increasing concentrations of rSbgalectin-1. A decrease in IL-1␤, TNF-␣, and Mx expression was observed in the brain of sea bass simultaneously injected with nodavirus and rSbgalectin-1 compared with those infected with nodavirus alone. Moreover, the protein was detected in the brain from infected fish, which is http://www.jimmunol.org/ the main target of the virus. These results suggest a potential anti-inflammatory, protective role of Sbgalectin-1 during viral infection. The Journal of Immunology, 2009, 183: 0000–0000.

ea bass nervous necrosis virus (SBNNV)4 is a nonenvel- the nervous system tissues (brain, retina, and spinal cord) leading oped, positive-stranded RNA virus that belongs to the Bet- to a high mortality in larval and juvenile stages of affected ﬁsh S anodavirus genus, within the Nodaviridae family, and the (1–3). The mechanism(s) of SBNNV infectivity and pathogenesis causative agent of the viral nervous necrosis. This disease, re- are largely unknown, and it remains to be determined whether the

ported in a wide range of teleost fish, is characterized by lethargy, virus evades, actively blocks, or uses the molecular recognition by guest on October 1, 2021 abnormal behavior, loss of equilibrium, and spiral swimming and, factors of the host’s immune system. histopathologically, by the development of vacuoles in cells from The innate immune response is the first line of defense against microbial pathogens and consists of a complex network of diverse *Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científi- humoral and cellular recognition and effector components, whose cas (CSIC), Vigo, Spain; †Center of Marine Biotechnology, University of Maryland tight regulation contributes to activate or deactivate specific anti- ‡ Biotechnology Institute, Baltimore, MD 21202; and Instituto de Biología Molec- microbial pathways (4). Among these components, lectins are car- ular y Celular, Universidad Miguel Hernańdez, Elche (Alicante), Spain bohydrate-binding proteins involved in pathogen recognition and Received for publication May 28, 2008. Accepted for publication September 8, 2009. neutralization that can also trigger effector mechanisms leading to The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance pathogen destruction and instruction of the adaptive immune with 18 U.S.C. Section 1734 solely to indicate this fact. mechanisms (5–8). Most animal lectins can be classified into dis- 1 This research was supported by the European Union project (SSP8-CT-2003- tinct families characterized by unique sequence motifs and struc- 501984), the project CSD2007-00002 Aquagenomics funded by the program Con- tural folds: S-type lectins (galectins), C-type lectins (including se- solider-Ingenio 2010 from the Spanish Ministerio de Educacioń y Ciencia and Xunta de Galicia (PGIDIT04PXIC40203PM). L.P.-B. and J.A.-G. were supported by For- lectins, collectins, and hyalectans), F-type lectins, I-type lectins, mación de personal universitario fellowships from Ministerio de Educacioń y Ciencia P-type lectins, pentraxins, ficolins, and cytokine lectins (Refs. 9 (Spain). Work at the Center of Marine Biotechnology, University of Maryland Bio- technology Institute, was supported by grants R01 GM070589-01 from the National and 10; see also www.imperial.ac.uk/research/animallectins). Institutes of Health, and IOS-0822257 from the National Science Foundation (to Although lectins from the various families differ greatly in the G.R.V.). domain architecture, they are all characterized by a carbohy- 2 The sequences presented in this article have been submitted to GenBank (www.ncbi. drate-recognition domain (CRD), which confers the protein its nlm.nih.gov/Genbank) under accession numbers as follows: Genomic Sbgalectin-1, 2ϩ EU660934; Sbgalectin-1, EU660937; SbCLA, EU660935; SbCLB, EU660936; Sbpen- carbohydrate-binding activity (11). Galectins are Ca -indepen- traxin, EU660933; and ESTs, from FF578829 to FF579034. dent soluble lectins, widely distributed in vertebrates, invertebrates 3 Address correspondence and reprint requests to Dr. Beatriz Novoa, Instituto de (sponges, worms, and insects), and protists (protozoa and fungi) Investigaciones Marinas, Consejo Superior de Investigaciones Científicas (CSIC), ␤ Eduardo Cabello, 6. 36208 Vigo, Spain. E-mail address: [email protected] (5, 10, 12, 13), characterized by their affinity for -galactosides (5, 12, 14). The mammalian galectins have been structurally classified 4 Abbreviations used in this paper: SBNNV, Sea bass nervous necrosis virus; CRD, carbohydrate-recognition domain; CTLD, C-type-like domain; EST, expressed se- into proto-, chimera-, and tandem-repeat types (15). The proto- quence tag; ORF, open reading frame; RLU, relative luminescence unit; SSH, sup- type galectins exhibit a single CRD per subunit and, in most cases, pression subtractive hybridization; qPCR, quantitative PCR. form noncovalently bound dimers. In addition to the CRD at the C Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 terminus, the chimera-type galectins have an N terminus region

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0801726 2 NODAVIRUS INDUCED GALECTIN-1 IN SEA BASS

consisting of in a short domain, followed by an intervening protein sequences. Protein sequence alignments were evaluated using Clust- line/glycine/tyrosine-rich domain with short repetitive sequences. alW program (http://align.genome.jp/) and specific conserved domains Tandem-repeat-type galectins consist of two homologous CRDs were searched with several programs: Blastp from National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/blast/Blast.cgi), PROS- tandemly arrayed in a single polypeptide, joined by a short gly- ITE (www.expasy.ch/prosite) and Simple Modular Architecture Research cine-rich region (9, 15). Furthermore, a novel tandem-repeat-type Tool (http://smart.embl-heidelberg.de/); Compute pI/Mw (www.expasy. galectin with four CRDs has been described recently (16). In con- ch/tools/pi_tool.html) for Mw detection; Signal IP (www.cbs.dtu.dk/services/ trast, C-type lectins, a group of highly diversified soluble or inte- SignalP) for signal peptide detection; TMpred (www.ch.embnet. 2ϩ org/software/TMPRED_form.html),TMHMM(www.cbs.dtu.dk/services/ gral membrane lectins, require Ca for ligand binding and their TMHMM-2.0), and SOSUI (http://bp.nuap.nagoya-u.ac.jp/sosui/sosui_ characteristic CRD (C-type-like domain or CTLD) can be classi- submit.html) for transmembrane domain detection and Disulfind (http:// fied into those that bind mannose and fucose and those that bind disulfind.dsi.unifi.it) for disulfide bonds were also used. galactose (8). Members of the pentraxin lectin family, such as RACE amplification C-reactive protein and serum amyloids, are characterized by subunits arranged in a cyclic oligomeric structure. In some species, The SMART RACE cDNA Amplification Kit (BD Clontech) was used to Ј Ј including man, pentraxins are expressed upon stimulus by inflam- complete the 5 - and 3 -cDNA ends of candidate sequences (Sbgalectin-1, SbCLA, SbCLB, and Sbpentraxin), following the manufacturer’s recom- matory cytokines and are considered critical components of the mendations. PCR products were purified from 1.2% agarose gel, subcloned acute-phase response (17). Little is known, however, about the using the Original TOPO T/A Cloning Kit, and sequenced as described potential role(s) of lectins in the innate immune and tissue repair above. RACE primers are summarized in supplemental Table I.5 responses to viral infection in fish. Phylogenetic analysis In this study, we used suppression subtractive hybridization (SSH) to examine the sea bass (Dicentrarchus labrax) gene ex- Sea bass lectin protein sequences were aligned with reference ones Downloaded from obtained from GenBank using ClustalX (19). The alignment obtained pression profiles in head kidney upon nodavirus infection. A ga- was transferred and edited into MacClade (20) and then directly trans- lectin, two C-type lectins, and a pentraxin were identified among ferred to ProtTest software version 1.4 (21) as a guide to determine the the up-regulated genes and were characterized in further detail. best-fit model of protein evolution among 96 different possibilities. The Moreover, evidence about the potential anti-inflammatory and pro- relative importance and a model-averaged estimation of different evo- tective role of the sea bass galectin on nervous tissue affected by lution parameters were also calculated by this software. The best-fit model of protein evolution and parameters, calculated by ProtTest, were http://www.jimmunol.org/ viral infection was obtained. incorporated into software PHYML (22), which uses a single, fast and accurate algorithm to estimate large phylogenies by Maximum Likeli- Materials and Methods hood. Finally, the trees created by PHYML were edited using the soft- Fish and virus ware TreeViewX (23). Adult sea bass (80 g each) were obtained from a commercial fish farm. Lectin expression studies Before the experiments, fish were acclimatized to laboratory conditions for Lectin expression was assessed by real-time or quantitative PCR (qPCR) in 3 wk, maintained at 20°C, and fed daily with a commercial diet. The head kidney at 4 and 72 h postinfection. RNA was isolated as described nodavirus strain 475-9/99 isolated from diseased sea bass was provided by above, and 5 ␮g was used to obtain cDNA by the Superscript II Reverse the Istituto Zooprofilattico Sperimentale delle Venezie (Italy). Transcriptase and oligo(dT)12–18 primer (Invitrogen) following the manu- by guest on October 1, 2021 Nodavirus challenge and RNA isolation facturer’s instructions. qPCR assays were performed using the 7300 Real Time PCR System (Applied Biosystems) with specific primers (supple- Fish care and the viral challenge experiments were reviewed and approved mental Table II).5 Each primer (0.5 ␮l; 10 ␮M) and the cDNA template (1 by the Consejo Superior de Investigaciones Científicas National Commit- ␮l, pooled from three fishes) were mixed with 12.5 ␮l of SYBR green PCR tee on Bioethics. Fish were injected i.m. with 100 ␮l of nodavirus suspen- master mix (Applied Biosystems) in a final volume of 25 ␮l. The standard 8 Ϫ1 sion in MEM (10 TCID50 ml ) and placed at 25°C. Mock-infected con- cycling conditions were 95°C for 10 min, followed by 40 cycles of 95°C trol fish (n ϭ 12) were injected with medium alone and maintained under 15 s and 60°C 1 min. The comparative CT method (2Ϫ⌬⌬CT method) was the same experimental conditions. Three fish from each experimental and used to determine the expression level of analyzed genes (24). Expression control groups were sampled 4, 24, 48, and 72 h postinfection. Animals of the candidate genes was normalized using ␤-actin as the reference gene. were sacrificed by anesthetic (MS-222) overdose and dissected, and the Fold units were calculated dividing the normalized expression values of head kidney was collected. RNA from individual organs was isolated by infected tissues by the normalized expression values of the controls. homogenization with TRIzol reagent (Invitrogen) following the manufacturer’s protocol. RNA was treated with Turbo-DNA free kit (Ambion) to Genomic sequence of Sbgalectin-1 remove contaminating DNA. Total DNA was extracted from sea bass head kidney by the phenol-chlo- SSH library construction roform method. Specific primers were designed based on the Sbgalectin-1 cDNA and PCR amplification products cloned and sequenced in a 3730 The SSH technique (18) was used to identify genes in head kidney that may DNA Analyzer (Applied Biosystems). Identification of introns was con- be up-regulated upon viral infection. Because preliminary RT-PCR exper- ducted using the Wise 2 program (www.ebi.ac.uk/Wise2/advanced.html). iments had shown up-regulation of several immune genes 4 h after viral challenge (data not shown), this time point was selected for sampling spec- Expression and purification of recombinant Sbgalectin-1 imens dedicated to the SSH library construction. cDNA was synthesized (rSbgalectin-1) ␮ from 1 g of head kidney (infected and control) RNA using the SMART ϩ PCR cDNA Synthesis Kit (BD Clontech). The SSH library was constructed Expression of rSbgalectin-1 was performed using the pET-30a( ) vector using the PCR-select cDNA Subtraction Kit (BD Clontech). The PCR in the Escherichia coli (Bl21 strain) cells. Briefly, the total open reading products from the forward subtraction procedure were cloned using the frame (ORF) of Sbgalectin-1 was amplified by PCR using primers containing a NdeI (primer forward) or BamHI (primer reverse) restriction site Original TOPO T/A Cloning Kit (Invitrogen) and screened by PCR using Ј Ј specific primers. (forward, 5 -GGGGGGGGGATTCCATATGTTTAATGGTTTG-3 ; reverse, 5Ј-GGGCGCGGATCCCTATTTTATCTCAAAGC-3Ј) and cloned Sequence analysis into a pGEM-T vector (Promega). The Sbgalectin-1 insert was removed from the plasmid and ligated into the pET-30(ϩ) bacterial expression vec- Excess of primers and nucleotides was removed by enzymatic digestion tor (Novagen) using NdeI and BamHI restriction sites and transformed into using 10 and1UofExoI and shrimp alcaline phosphatase, respectively E. coli strain BL21 (DE3)-competent cells. A single colony containing the (Amersham Biosciences) at 37°C for 1 h, followed by inactivation of the transformed plasmid was used to inoculate cultures of LB medium con- enzymes at 80°C for 15 min. The PCR products were sequenced in a 3730 taining 50 ␮g/ml kanamycin, and the expression of recombinant protein DNA Analyzer (Applied Biosystems). Blastn and Blastx searching from the National Center for Biotechnology Information (www.ncbi.nlm.nih- .gov/blast/Blast.cgi) were used to identify homologous nucleotide and pro- 5 The online version of this article contains supplemental material. The Journal of Immunology 3

was induced by 1 mM isopropyl-␤-D-thiogalactopyranoside (Invitrogen). Soluble proteins were extracted using lysozyme (100 ␮g/ml), PMSF (1 mM), and 2-ME (10 mM) (Sigma-Aldrich), and the rSbgalectin-1 was isolated by affinity chromatography on lactose-Sepharose 6B column at 4°C following the protocols described previously (25). The homogeneity of the rSbgalectin-1 preparation was assessed by SDS-PAGE on 15% acrylamide:bisacrylamide gel using a MiniPROTEAN electrophoresis system (Bio-Rad). Protein determination and hemagglutination assay Protein concentration was determined on 96-well flat bottom plates with the Bio-Rad protein assay using BSA as standard following the protocols FIGURE 1. Classification of Dicentrarchus labrax head kidney ESTs described previously (25). After 30 min at room temperature, the reactions based on function or structural similarity. were read in an iEMS reader (Labsystems) at 620 nm, and the data were analyzed through the Microsoft Excell program. Hemagglutinating activity of rSbgalectin-1 was tested following the procedure reported by Nowak et al. (26) using 2-fold serial dilutions of sam- methanesulfonyl fluoride (100 ␮g/ml) and total protein concentration ples in microtiter U plates and trypsin-treated glutaraldehyde-fixed rabbit adjusted to 0.1 mg/ml using the Bradford reagent (Bio-Rad). SDS-poly- erythrocytes. To analyze the inhibitory effect of ␤-galactosides on the hem- acrylamide gels (Ready Tris-HCl 4–20% gel; Bio-Rad) were loaded with agglutinating activity of rSbgalectin-1, the saline solution was replaced by 20 ␮l of samples in buffer containing 2-ME. The rSbgalectin-1 sample of 100 mM lactose solution. The galectin-1 concentration at the highest di- 200 ng was also included in the gels. The proteins in the gel were transferred lution still showing visible agglutination was recorded. during3hat125Vin2.5mMTris, 9 mM glycine, and 20% methanol to Effects of rSbgalectin-1 on leukocyte respiratory burst and NO nitrocellulose membranes (Bio-Rad). The membranes were then blocked with Downloaded from production 2% dry milk and 0.05% Tween 20 in PBS and incubated for3hatroom temperature with a striped bass (Morone saxatilis) galectin 1 antiserum diluted Sea bass head kidney leukocytes were isolated following the method pre- 1000-fold in PBS before incubation with a peroxidase-conjugated goat anti- viously described by Chung and Secombes (27). Viability of the leukocyte rabbit Ab (Nordic). The peroxidase activity was detected by using the ECL preparation was determined by trypan blue exclusion. chemiluminescence reagents (Amersham Biosciences) and revealed by expo- To assess the potential effect of the sea bass galectin-1 on respiratory sure to x-ray films (Amersham Biosciences).

burst, leukocyte monolayers were incubated with increasing doses of http://www.jimmunol.org/ rSbgalectin-1 (0, 1.7, 3.3, 6.7, and 13.3 ␮g ⅐ mlϪ1) in 96-well plates (100 Results ␮ l/well) for 24 h at 20°C. After this time, the emission of relative lumi- Nodavirus infection of sea bass induces up-regulation of nescence units (RLU) was determined by stimulating the cells with PMA (Sigma-Aldrich) and amplified by the addition of 5-amino-2–3-dihydro- expression of immune-related genes including a galectin, two 1,4-phthalazinedione (luminol; Sigma-Aldrich). A stock solution of lumi- C-type lectins, and a pentraxin nol 0.1 M was prepared in DMSO (Sigma-Aldrich) just before use and diluted in HBSS (Life Technologies) to obtain a final concentration of 0.15 A SSH approach was implemented to investigate gene expression mM (luminol stock solution). PMA was solubilized in the luminol stock profiles in sea bass head kidney upon nodavirus infection and to iden- solution to a final concentration of 1 ␮g ⅐ mlϪ1. Immediately after the tify genes potentially involved in innate immunity and protective re- addition of the solution into the wells (100 ␮l/well luminol alone or with sponses against its pathogenic effects. The subtracted cDNA library by guest on October 1, 2021 PMA), the respiratory burst activity was measured in a luminometer (Fluo- was constructed from sea bass head kidneys 4 h after nodavirus in- roskan Ascent; Labsystems) at intervals of 5 min during a 30-min time course. fection. Following transformation in E. coli, 479 bacterial clones were To measure the potential effect(s) of rSbgalectin-1 on NO production, isolated and amplified by PCR, resulting in 206 reliable expressed leukocyte monolayers were established under the same conditions de- sequence tag (EST) sequences (accession nos. from FF578829 to scribed above, increasing doses of rSbgalectin-1 were added, and after 24 h FF579034; www.ncbi.nlm.nih.gov/GenBank) representing 70 single- of incubation, the nitrite content of the supernatants was measured by the Griess reaction (28). Briefly, after the incubation period, 50 ␮l of leukocyte tons potentially up-regulated in infected head kidneys. Sequence ho- monolayer supernatants were taken to new 96-well plates. A 100-␮l aliquot mology was searched using Blast taking into account Blastx entries. of 1% sulfanilamide (Sigma-Aldrich) in 2.5% phosphoric acid was added Fourteen of 206 (6.8%) showed significant similarities (e-value to each well, followed by 100 ␮l of 0.1% N-naphthyl-ethylenediamine Ͻ10Ϫ3) with structural genes from different organisms; 18 of 206 (Sigma-Aldrich) in 2.5% phosphoric acid. ODs at 540 nm were measured (8.7%) were immune or stress-related genes such as heat-shock pro- in an iEMS (Labsystems). The concentration of nitrite in the samples was ␤ ␤ ␤ determined using a standard curve generated with solutions of sodium ni- tein 90 , 2-microglobulin precursor, apcs-prov protein, -galacto- trite of known concentrations. Both assays were conducted with three dif- side-binding lectin, mannose receptor C1, Cd209e Ag, or serum lectin ferent fishes. isoform 2; 93 of 206 (45.1%) were annotated as metabolic genes; 18 Effects of rSbgalectin-1 on cytokine expression of 206 (8.7%) showed significant similarities with ribosomal genes; and 9 of 206 (4.4%) were classified as others (Fig. 1). Sequences Expression levels of TNF-␣, IL-1␤, and Mx upon i.m. injection of rSbgalectin-1 similar to hemoglobin were the most abundant in the subtraction, and nodavirus were measured by qPCR. Four groups of fish (three fish ␮ ϫ present in 42 clones. The best match was found for the phosphoglu- each) were treated as follows: group 1 with 100 l of nodavirus (0.85 Ϫ134 7 Ϫ1 ␮ ␮ conate hydrogenase (Blastx expectation value 2e ). A complete 10 TCID50 ml ); group 2 with 100 l of rSbgalectin-1 (6.7 g/ml); ␮ ␮ ϫ 7 group 3 with 150 l of a mix of 50 l of nodavirus (1.7 10 TCID50 list of the significant entries obtained through the Blastx search is Ϫ ml 1) and 100 ␮l of rSbgalectin-1 (6.7 ␮g/ml); and group 4 with 150 presented in Table 1. Besides the data described above, 25 of 206 ␮ l of cell culture medium as control. Fish were sacrificed by MS-222 (12.1%) showed nonsignificant e-values (Ն10Ϫ3) and 29 of 206 overdose 72 h postchallenge, and the brain and head kidney was removed aseptically. RNA isolation, cDNA synthesis, and qPCR were (14.1%) showed no similarity with any other sequence deposited in conducted as described above. Primer sequences are shown in supple- GenBank (Fig. 1). mental Table III.5 Among those ESTs identified from the subtracted library as Western blot analysis overexpressed upon nodavirus infection, four lectins were investigated in further detail: a galectin, two C-type lectins, and a pen- To document the up-regulation of Sbgalectin-1 after nodavirus infection, a traxin. A 400-bp clone was identified as galectin-1 (Sbgalectin-1). Western blot was conducted to provide protein expression levels in brain Ј and head kidney of nodavirus infected fish. Western blot assays were per- cDNA RACE 3 gave a 785-bp clone (GenBank accession no. formed as described previously (29). Briefly, head kidney and brain ly- EU660937; www.ncbi.nlm.nih.gov/GenBank), with an ORF (60– ophilized samples were resuspended in Milli-Q water containing phenyl- 467) encoding 135 aa with a calculated m.w. of 15244.16 and 4 NODAVIRUS INDUCED GALECTIN-1 IN SEA BASS

Table I. Up-regulated ESTs from sea bass head kidney SSH

No. of No. of e-Value Length Homology ESTs contigs (Blast X) (bp) Species (* ϭ teleost)

Ribosomal genes 40S ribosomal protein Sa (AAP20147) 1 1 4e-20 226 *Pagrus major 60S ribosomal protein L5 (CAH57700) 1 1 1e-36 260 *Platichthys ﬂesus 60S ribosomal protein L6 (AAP20201) 2 1 2e-74 508 *Pagrus major PREDICTED: similar to 60S ribosomal protein L29 (XP_517026) 2 1 5e-23 329 Pan troglodytes PREDICTED: similar to ubiquitin and ribosomal protein S27a precursor 1 1 2e-67 573 Canis familiaris (XP_531829) Ribosomal protein L7 (AAZ23151) 1 1 1e-83 583 *Oryzias latipes Ribosomal protein L9 (AAP20210) 2 1 1e-23 399 *Pagrus major Ribosomal protein L27 (AAU50549) 2 1 5e-56 410 *Fundulus heteroclitus Ribosomal protein L35 (NP_079868) 2 2 1e-20 264 Mus musculus Ribosomal protein L37 (AAP20208) 3 1 3e-04 376 *Pagrus major Ribosomal protein S7 (CAA64412) 1 1 1e-53 355 *Takifugu rubripes Structural genes Actin-related protein 2/3 complex (AAP20158) 1 1 8e-36 250 *Pagrus major Apolipoprotein H (NP_001009626) 1 1 4e-20 643 Rattus norvegicus ␤-Actin (AAR97600) 1 1 2e-81 466 *Epinephelus coioides ␤-Tubulin (BAB86853) 2 2 5e-57 351 Bombyx mori Calcium binding protein (AAA91090) 1 1 7e-14 484 Meleagris gallopavo Cytoplasmic actin type 5 (AAQ18433) 1 1 6e-29 472 Rana lessonae Gelsolin, like 1 (NP_835232) 1 1 3e-43 766 *Danio rerio Plasma retinol-binding protein II (P24775) 1 1 1e-34 461 *Oncorhynchus mykiss Downloaded from PREDICTED: similar to myosin-VIIb (XP_574117) 1 1 3e-33 301 Rattus norvegicus PREDICTED: similar to Rho-related GTP-binding protein (XP_696319) 2 1 6e-64 604 *Danio rerio Tubulin ␤-1 chain (Q9YHC3) 1 1 6e-60 352 *Gadus morhua Type I collagen ␣1 (BAD77968) 1 1 3e-19 665 *Paralichthys olivaceus Metabolic genes 3␤-Hydroxysteroid dehydrogenase (AAB31733) 3 1 8e-73 567 *Oncorhynchus mykiss Acid phosphatase 1 isoform c variant (BAD93075) 1 1 7e-29 276 Homo sapiens Acidic ribosomal phosphoprotein (AAP20211) 1 1 2e-50 368 *Pagrus major http://www.jimmunol.org/ Aldolase B (AAD11573) 1 1 3e-106 722 *Salmo salar Copper/zinc superoxide dismutase (AAW29025) 2 1 3e-78 605 *Epinephelus coioides Cystatin precursor (Q91195) 1 1 1e-20 281 *Oncorhynchus mykiss Cytochrome c oxidase subunit I (NP_008805) 5 5 1e-71 550 *Mustelus manazo Cytochrome c oxidase subunit III (NP_443365) 3 3 4e-32 392 *Crenimugil crenilabis Ferritin H chain (AAP20171) 1 1 8e-04 411 *Pagrus major Ferritin heavy subunit (AAB34575) 3 1 3e-70 420 *Salmo salar Ferritin middle subunit (AAB34576) 6 2 5e-82 518 *Salmo salar GDP-mannose pyrophosphorylase A (NP_598469) 2 1 1e-61 805 Mus musculus Glyceraldehyde 3-phosphate dehydrogenase (BAB62812) 1 1 8e-18 153 *Pagrus major Glutathione peroxidase (AAO86703) 1 1 1e-80 717 *Danio rerio Hemoglobin ␣-A subunit (Q9PVM4) 20 2 3e-63 612 *Seriola quinqueradiata Hemoglobin ␤-chain (AAZ79648) 2 2 1e-33 498 *Nibea miichthioides by guest on October 1, 2021 Hemoglobin ␤-A subunit (Q9PVM2) 15 2 1e-64 510 *Seriola quinqueradiata Hemoglobin ␤-B subunit (Q9PVM1) 5 2 3e-60 496 *Seriola quinqueradiata Nephrosin precursor (AAB62737) 4 3 1e-83 757 *Cyprinus carpio Ornithine decarboxylase antizyme large isoform (AAP82035) 2 1 9e-53 398 *Paralichthys olivaceus Phosphogluconate hydrogenase (AAH44196) 1 1 2e-134 840 *Danio rerio Phosphogluconate isomerase (CAC83778) 1 1 2e-75 459 *Mugil cephalus Predicted metal-dependent RNase (ZP_00323404) 1 1 2e-04 427 Pediococcus pentosaceus PREDICTED: similar to probable endonuclease KIAA0830 precursor 1 1 2e-07 625 *Danio rerio (XP_687673) RhAG-like protein (AAV28817) 1 1 1e-64 566 *Takifugu rubripes Serine protease I-2 (BAD91575) 3 1 1e-46 730 *Paralichthys olivaceus Spleen protein tyrosine kinase (AAK49117) 1 1 3e-64 441 *Cyprinus carpio Steroidogenic acute regulatory protein (AAW62971) 1 1 3e-23 378 *Acanthopagrus schlegelii Synaptophysin-like protein (AAQ94571) 2 1 7e-89 780 *Danio rerio Thioredoxin (AAG00612) 1 1 8e-28 395 *Ictalurus punctatus Ubiquitin-conjugating enzyme E2N-like (NP_956636) 1 1 3e-18 195 *Danio rerio Immune genes Apcs-prov protein (AAH82494) 1 1 2e-10 356 *Xenopus tropicalis ␤2 microglobulin precursor (O42197) 2 2 1e-23 572 *Ictalurus punctatus ␤-galactoside-binding lectin (A28302) 1 1 5e-34 400 *Electrophorus electricus Heat-shock protein 90 ␤ (AAP20179) 1 1 9e-14 407 *Pagrus major IgM H chain secretory form (AAL99929) 1 1 2e-69 589 *Chaenocephalus aceratus Ig L chain L2 (AAB41310) 2 1 1e-24 582 *Oncorhynchus mykiss Lymphocyte Ag 6 complex, locus D (NP_034872) 2 1 5e-05 375 Mus musculus MHC class Ib chain (BAD13366) 1 1 3e-44 538 *Paralichthys olivaceus MHC class II protein (AAA49380) 1 1 9e-11 258 *Morone saxatilis Mpx protein (AAH56287) 2 2 7e-10 440 *Danio rerio PREDICTED: similar to Cd209e Ag (XP_687353) 1 1 2e-18 605 *Danio rerio PREDICTED: similar to mannose receptor C1 (XP_686273) 1 1 7e-18 794 *Danio rerio Serum lectin isoform 2 precursor (AAO43606) 1 1 3e-23 408 *Salmo salar Tcp1 protein (AAH44397) 1 1 4e-37 403 *Danio rerio Others Differentially regulated trout protein 1 (AAG30030) 1 1 2e-07 419 *Oncorhynchus mykiss Hypothetical 18K protein-goldﬁsh mitochondrion (JC1348) 8 2 2e-26 701 *Carassius auratus

theoretical isoelectric point of 5.02 (supplemental Fig. 1).5 The tins from other species (Fig. 2A), and the presence of the important molecular size (within the typical 14–18 kDa range of prototype amino acid residues for carbohydrate binding clearly conﬁrm that galectins) together with the alignment results with different galec- the Sbgalectin-1 is a member of the galectin family and is more The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

FIGURE 2. Molecular characterization of Sbgalectin-1. A, ClustalW alignment comparing the predicted Sbgalectin-1 sequence with other similar indicates the conserved amino acids. The critical amino acid residues are highlighted in gray. B, Sbgalectin-1 phylogenetic tree ء .galectin sequences obtained with Phyml software following the model of protein evolution recommended by ProtTest (WAGϩIϩG and p-invar: 0.02). Numbers beside branches are bootstrap values (percentages), based on an analysis of 1000 maximum likelihood bootstrap replicates. Galectin sequences from different groups of Eukarya were used to classify our query. Those belonging to Fungi were used as outgroup. C, Genomic structure of Sbgalectin-1. Size of exons (u) and introns (Ⅺ) are shown in base pair. The number of exons is indicated in the top. by guest on October 1, 2021 closely related to the galectin-1 group than to any other galectin of five residues at site 2 (Fig. 3A). In addition, we observed the family members. No secretion peptide sequence was found in the highly conserved “WIGL” motif (11) and a “QPN” motif, which Sbgalectin-1, in accordance with the characteristics of the galectin differed from EPN motifs (characteristic of all mannose-binding family (5). At the amino acid level, this galectin presented the proteins) and QPD motifs (typical of galactose-specific CTLDs). highest homology with the Atlantic halibut (Hippoglossus hippo- A 408 bp clone was identified as a C-type lectin (SbCLB). glossus) galectin (74.63% identity). The phylogenetic analysis was RACE amplification produced a complete cDNA sequence with concordant with the current taxonomy for all species included, 719 bp (GenBank accession no. EU660936; www.ncbi.nlm.nih. with Sbgalectin-1 clustering with the halibut galectin-1 (bootstrap gov/GenBank), an ORF from 61 to 579 nt position, which trans- value: 99; Fig. 2B). lated for a 19-kDa predicted size protein with 172 aa (supplemen- Characterization of the gene organization of Sbgalectin-1 (Gen- tal Fig. 4).5 The SbCLB protein sequence alignment with CTLDs Bank accession no. EU660934; www.ncbi.nlm.nih.gov/GenBank) from other species (Fig. 3B) showed six cysteine residues at con- revealed the presence of three introns in the following positions of served positions and predicted disulfide bonds at C44-C160,C55- the ORF: 10 bp (2144 bp in length), 90 bp (879 bp in length), and C144, and C72-C168 sites. SbCLB presented three of four essential 262 bp (966 bp in length) (Fig. 2C and supplemental Fig. 2).5 residues for Ca2ϩ ion coordination at site 1 and one of five residues A 605-bp clone was characterized as a C-type lectin (SbCLA), at site 2 (Fig. 3B). Moreover, we observed a “WLGG” motif in- and RACE was conducted to obtain a complete cDNA sequence stead of the typical “WIGL” domain and the “QPD” signature for both 5Ј- and 3Ј-ends (GenBank accession no. EU660935; www. tripeptide characteristic of the galactose-binding proteins. ncbi.nlm.nih.gov/GenBank). The complete sequence (2266 bp) A phylogenetic tree was constructed considering SbCLA and presented an ORF (from 187 to 1578 bp), which encoded 463 aa SbCLB CTLDs sequences from various organisms. SbCLB clus- with a 54-kDa predicted size protein (supplemental Fig. 3).5 An tered closer to other salmonid and non-salmonid fish, with a boos- alignment with CRD regions from other species clearly confirmed trap value of 62. However, SbCLA was closer (79 boostrap value) that SbCLA protein is a member of the C-type lectin family (Fig. to primates and rodents (Fig. 3C). 3A). Six cysteine residues were observed in the sequence at con- A 356-bp clone revealed a pentraxin-like protein (Sbpentraxin). served positions within the CTLD. These residues are required for The 5Ј- and 3Ј-RACE amplification of this clone generated a the arrangement of three intramolecular disulfide bonds that are 1563-bp sequence (GenBank accession no. EU660933; www. essential for CTLD fold stability and were located at C337-C461, ncbi.nlm.nih.gov/GenBank) with an ORF (29–706) encoding for a C348-C365, and C435-C453 positions. SbCLA sequence had three of 225 aa long, 26 kDa protein (supplemental Fig. 5).5 Its 857-bp four essential residues for Ca2ϩ ion coordination at site 1 and three 3Ј-untranslated region resembles the unusually long (858–1500 6 NODAVIRUS INDUCED GALECTIN-1 IN SEA BASS Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. Molecular characterization of SbCLA and SbCLB. A, ClustalW alignment comparing the predicted SbCLA CTLD sequence with other indicates the conserved amino acids. Conserved cysteines and other important amino acids are highlighted in light gray. The ء .similar CTLD sequences highly conserved “WIGL” motif is highlighted in dark gray. EPN/QPN signatures are shown in bold. Numbers 1 and 2 indicate the residues for Ca2ϩ indicates the conserved amino ء .coordination. B, ClustalW alignment comparing the predicted SbCLB CTLD sequence with other similar CTLD sequences acids. Conserved cysteines and other important amino acids are highlighted in light gray, and the highly conserved “WIGL” motif is highlighted in dark gray. EPN/QPD signatures are shown in bold. Numbers 1 and 2 indicate the residues for Ca2ϩ coordination. C, SbCLA and SbCLB phylogenetic tree obtained with Phyml software following the model of protein evolution recommended by ProtTest (WAGϩIϩG and p-invar: 0.04). Numbers beside branches are bootstrap values (percentages), based on an analysis of 1000 maximum likelihood bootstrap replicates. C-Lectin sequences from different groups of Eukarya were used to classify our query. Those belonging to Crustracea were used as outgroup. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 4. Molecular characterization of Sbpentraxin. A, ClustalW alignment comparing the predicted Sbpentraxin sequence with other similar pen- indicates the conserved amino acids. Conserved cysteines are highlighted in light gray and the pentraxin signature TWxS, where x is ء .traxin sequences any amino acid, is highlighted in dark gray. B, Sbpentraxin phylogenetic tree obtained with Phyml software following the model of protein evolution recommended by ProtTest (JTTϩG and p-invar: 0.02). Numbers beside branches are bootstrap values (percentages), based on an analysis of 1000 maximum likelihood bootstrap replicates. Pentraxin sequences from different groups of Eukarya were used to classify our query. Those belonging to Mammalia were by guest on October 1, 2021 used as outgroup. bp) 3Ј-untranslated region typical of the mammalian C-reactive pro- In SDS-PAGE, the induced bacterial extracts showed a protein teins and suggests that this protein is a C-reactive protein. An align- band of the expected mobility (15 kDa) (Fig. 6A). The carbo- ment with pentraxins from other species revealed two highly con- hydrate-binding activity of the rSbgalectin-1 was determined by served cysteines, which are involved in intrachain disulfide bonds, hemagglutination, with a 1:20 (0.5 ␮g rSbgalectin-1/well, 0.325 and the conserved pentraxin signature TWxS, where x is any amino ␮M) as the highest dilution at which activity was observed. acid (4) (Fig. 4A), thereby confirming that Sbpentraxin protein is a Hemagglutination by rSbgalectin-1 was carbohydrate specific, be- bona fide member of the pentraxin family. A search in GenBank iden- cause lactose behaved as an effective inhibitor (Fig. 6B). A dose- tified the salmon (Salmo salar) pentraxin as the protein with the high- dependent decrease of respiratory burst activity was observed in est identity (36%) at the amino acid level. A phylogenetic tree con- leukocytes exposed to increasing rSbgalectin-1 concentrations (0, structed with pentraxin sequences from various organisms placed the 1.7, 3.3, 6.7, and 13.3 ␮g/ml) (Fig. 7). However, rSbgalectin-1 did Sbpentraxin closer to the trout (Oncorhynchus mykiss), salmon (S. not affect NO production by leukocytes (data not shown). A de- salar), and zebrafish (Danio rerio) pentraxins with a boostrap value of crease of IL-1␤, TNF-␣, and Mx expression was observed in the B 55 (Fig. 4 ). brain of sea bass simultaneously injected with nodavirus and Assessment of in vivo expression of Sbgalectin-1 by qPCR re- rSbgalectin-1 with respect to those infected with nodavirus alone vealed clear differences between infected and control samples. and the uninfected controls (Fig. 8). In head kidney, however, Similar differences were also observed for SbCLA and SbCLB. A although rSbgalectin-1 modified the expression of the analyzed significant increase in expression upon nodavirus infection was genes, there was not change comparing nodavirus alone and nodavi- observed after 4 h for the three lectins and continued beyond 72 h rus plus rSbgalectin-1. Although the expression of the inflammatory for SbCLB. Expression of Sbgalectin-1 and SbCLA, however, de- creased 72 h after infection (Fig. 5). cytokines was low, the measurements were highly reproducible. All further studies, including in vivo expression, effects on leu- Western blot analysis was conducted on extracts from head kidney kocyte oxidative burst and NO production, and cytokine expres- and brain of six nodavirus-infected fish and six controls. A band of 15 sion in the brain, were focused on Sbgalectin-1. kDa (galectin monomer) was observed in all brain samples from the infected fish, but not in the controls (Fig. 9), shows the results of three Sbgalectin-1 displays anti-inflammatory protective activity infected and three control brain samples). Additional bands of 30 and against viral infection 45 kDa or 45 kDa were only observed for rSbgalectin-1 and brain To enable functional studies on the Sbgalectin-1, the recombi- samples, respectively, presumably the oligomers (dimers and trimers) nant protein was produced in a prokaryotic expression system. of the 15-kDa galectin, which assemble under the experimental 8 NODAVIRUS INDUCED GALECTIN-1 IN SEA BASS Downloaded from http://www.jimmunol.org/

FIGURE 5. Real-time PCR results for SbCLA (A), SbCLB (B), and Sbgalectin-1 (C) expression level upon controls in the head kidney of i.m. infected sea bass. Fold units were calculated dividing the expression values of infected tissues by the expression values of the controls, once normalized regarding to the ␤-actin expression. n ϭ 3 in each experimental group. .indicates expression values significantly different from controls ء by guest on October 1, 2021 conditions. No galectin bands were observed in any of the head kidney samples (data not shown). FIGURE 6. Biochemical characterization of recombinant Sbgalectin-1. A, rSbgalectin-1 SDS-PAGE. a, Sample before passing to the chroma- Discussion tography column; b, sample after passing to the chromatography col- In this study, we analyzed the effect of nodavirus infection on the umn (purified rSbgalectin-1). The induced bacterial extracts showed a sea bass head kidney transcriptome using a SSH approach. The protein band of the expected mobility (15 kDa). B, Hemagglutination percentages of the significant ESTs obtained were higher than for assay of rSbgalectin-1. The carbohydrate-binding activity of the rSbgalectin-1 was determined by hemagglutination, with a 1:20 (0.5 ␮g rSbgalectin-1/well, cDNA or subtracted libraries after experimental infection or im- 0.325 ␮M) as the highest dilution at which activity was observed. Hemagglu- mune stimulation of Japanese flounder (Paralichthys olivaceus) tination by rSbgalectin-1 was carbohydrate specific, as lactose behaved as an (30), carp (Cyprinus carpio) (31), salmon (S. salar) (32), and gilt- effective inhibitor. head seabream (Sparus aurata) (33). Within the genes related to the immune system category, however, we identified genes that had already been described in those fish libraries (Table I) and are not reported so far in sea bass. Because in mammals, galectins critical components in the mammalian immune response such as have been demonstrated to mediate important roles in host re- galectins (34, 35), Cd209e (35), C-type lectins (31, 32, 34, 36, 37), sponses to viral infection (Refs. 34, 50–53; recently reviewed in pentraxins (32, 38), MHC class II and heat-shock protein 90 (30, Ref. 54), this study was further centered on Sbgalectin-1. ␤ 32, 35), 2-microglobulin, Ig H and L chains (30, 32), and T- Within the proto-type galectins, Sbgalectin-1 is closely related to complex protein (32). This considerable number of common up- the mammalian galectin-1 than to any other subgroups. Sbgalectin-1 regulated transcripts supports the notion that multiple components exhibits all the essential amino acids (H44,N46,R48,H52,D54,N61, of both innate and adaptative immune responses of fish have been W68,E71, and R73, numbering based on bovine galectin-1 se- conserved in the vertebrate lineages leading to the mammals. quence; Ref. 55) that are responsible for galectin-1 carbohydrate- Among the immune genes found differentially expressed in the binding activities (56). On the basis of its m.w. and agglutinating library, we focused on lectins as in mammals they play important activity, Sbgalectin-1 is most likely present as a dimer. The phy- roles in innate immunity and regulation of adaptive responses (10, logenetic analysis grouped Sbgalectin-1 in the same cluster with 12, 39–45) and are also involved in numerous cellular processes halibut (H. hippoglossus) galectin-1 with a bootstrap value of 99 such as targeting of proteins within cells and cell-to-cell and cell- (Fig. 2B), suggesting a conservative evolution of this family of to-matrix adhesion (44, 46–49). Therefore, we characterized the proteins. Like other galectin-1 genes, Sbgalectin-1 gene contains complete cDNA sequences of a galectin (Sbgalectin-1), two C- four exons, of which exon 3 houses all the amino acid residues that type lectins (SbCLA and SbCLB), and a pentraxin (Sbpentraxin) bind the carbohydrate ligand. Despite some differences in intron The Journal of Immunology 9

FIGURE 9. Western blot of Sbgalectin-1 of brain from three control ﬁsh (1–3) and from three nodavirus-infected ﬁsh (4–6: infected samples), rGal corresponds to the rSbgalectin-1. Arrows show the mobility corresponding to the Sbgalectin-1 oligomers (15, 30, and 45 kDa).

within the group II of integral membrane proteins characterized by an N-terminal transmembrane domain and C-terminal CTLD (11, 68). In contrast, SbCLB belongs to group VII, which consists of a unique CRD with no other associated N- or C-terminal domains

(68) and a signal peptide for extracellular secretion. Both SbCLA Downloaded from and SbCLB sequences displayed the highly conserved six cysteine residues required for CTLD fold stability. It is noteworthy that they differed, however, in the conserved WIGL motif present in FIGURE 7. Respiratory burst activity of sea bass leukocytes incubated C-type lectins. It was also conserved in SbCLA (WIGL; Fig. 3A), with increasing rSbgalectin-1 concentrations (0, 1.7, 3.3, 6.7, and 13.3 but in SbCLB the motif had substitutions in the second [leucine; as ␮ g/ml) along time in an individual fish (A), or as maximum well activity, in carp (C. carpio)] and fourth [glycine; as described for salmon http://www.jimmunol.org/ mean of three fish (B). Respiratory burst activity is expressed as relative (S. salar)] positions (WLGG; Fig. 3B). Another interesting differ- indicates RLU units significantly different ء .(luminescence units (RLU ence between SbCLA and SbCLB resided in the characteristic from controls. EPN (mannose/fucose-binding) or QPD (galactose-binding) motifs of C-type lectins (8, 69). SbCLB had a QPD motif suggesting sizes, Sbgalectin-1 gene showed similarity to that of the mam- recognition of galactose and related ligands (Fig. 3B). However, malian galectin-1 genes including exon-intron boundaries SbCLA displayed a QPN motif (Fig. 3A), which is intermediate (57–59). between the two typical EPN and QPD CTLD sequence motifs Comparison of SbCLA and SbCLB with other sequences al- previously identified (11, 69). This QPN tripeptide has asparagine

ready published suggested that they are clearly members of the instead of aspartate at the third position, which although it may not by guest on October 1, 2021 C-type lectin family, characterized by the CTLD (Fig. 3). Al- affect the Ca2ϩ ion coordination at site 2, it may influence its though most CTLD-containing proteins reported so far are of carbohydrate specificity (65). A phylogenetic tree was constructed mammalian origin, a few C-type lectins have been already iden- based on the SbCLA and SbCLB CTLDs and those from various tified in teleost species (36, 60–67). SbCLA can be classified organisms. SbCLB was closer to other salmonid and nonsalmonid

FIGURE 8. Real-time PCR results for IL-1␤, TNF-␣, and Mx expression level in the brain (A, B, and C)orin the head kidney (D, E, and F) of i.m. indicates ء .infected sea bass, n ϭ 3 expression values signiﬁcantly different from control group. # indicates expression values signiﬁcantly different from nodavirus group. 10 NODAVIRUS INDUCED GALECTIN-1 IN SEA BASS

fish with a boostrap value of 62. However, SbCLA was much associated glycan ligands (80). Recognition of the latter can lead to closer (79 boostrap value) to primates and rodents (Fig. 3C), sug- the formation of galectin-glycoprotein lattices in the control of gesting that despite bearing the unique QPN carbohydrate-binding biological processes, including host-pathogen interactions, and im- motif, this CTLD has been strongly conserved from the fish to the mune cell activation and homeostasis. The observation that in mammalian taxa. Western blot analysis Sbgalectin-1 appears as a 15-kDa monomer The results of up-regulation of expression of Sbgalectin-1 upon is not surprising, because it would be expected that the dimers experimental nodavirus infection as revealed by the SSH approach dissociate under the denaturing electrophoresis conditions. The were verified by in vivo expression studies using qPCR. The re- presence of abundant 30- and 45-kDa oligomers in the rSbgalectin-1 sults showed a clear difference between infected and control sam- control, however, suggests that strong hydrophobic interactions ples with a significant increase in Sbgalectin-1 expression from 4h take place among its subunits and opens the possibility that the postinfection, followed by a significant decrease at 72 h postin- monomers and oligomers (15, 30, and 45 kDa) are potentially fection (Fig. 5). SbCLA and SbCLB, also included for comparison functional protein species in the infected tissues. This may reflect in the in vivo expression studies, showed an increase in expression structural differences in the Sbgalectin-1 folding relative to that at 4 h postinfection. However, although after that time point reported for the mammalian prototype galectins (55, 56) and is SbCLA followed an expression profile similar to Sbgalectin-1, for consistent with the 45-kDa species observed in the infected ani- SbCLB expression continued to significantly increase at 72 h mals, Moreover, the lack of the 30-kDa dimer in the latter suggests postinfection (Fig. 5). Similar results were described previously in that the Sbgalectin-1 trimerization may be specific to the nodavirus mammalian galectins by Zun˜iga et al. (70), who reported galec- infection in brain, an intriguing observation that warrants further tin-1 expression was higher in Trypanosoma cruzi-infected mice investigation. macrophages than uninfected controls. Soanes et al. (65) detected Nodavirus causes vacuolization in the CNS, and it has been Downloaded from a higher expression three C-type lectin receptors in liver of Atlan- reported that in mammals, the oxidized galectin-1 enhances axonal tic salmon in response to infection by Aeromonas salmonicida than regeneration in peripheral and central nerves, even at relatively in the controls. In mammals, lectins mediate recognition of a wide low concentrations (78). Studies by O’Farrell et al. (34) in rainbow range of pathogens including Gram-positive and Gram-negative trout demonstrated up-regulation of galectin-9 in leukocytes upon bacteria, yeast, parasites, and viruses and can trigger effectors rhabdovirus infection. It has been well established that galectins mechanisms such as activation of the complement system, increas- play a role in the initiation, activation, and resolution phases of http://www.jimmunol.org/ ing opsonization, phagocytosis, and apoptosis, which leads to the innate and adaptive immune responses by promoting oxidative destruction of the infectious agent (71–74). Lectins can also func- burst (81, 82), inhibition of NO production (83), anti-inflammatory tion as regulators of adaptive immunity, leading to apoptosis of or proinflammatory effects (45), and cytokine production (84). Fur- selected T cells subsets or enhancing the production of cytokines thermore, a given galectin (e.g., galectin-1) may exert pro- or anti- in nonactivated and activated T cells, among others (75–77). Tak- inflammatory effects depending on multiple factors, such as the ing these roles into account, the identification of the galectin and concentration reached at the inflammatory foci, the extracellular C-type lectins in our subtracted library indicates these molecules microenvironment, and the target cells (85). In addition to their could have important defense roles in fish immunity. Moreover, multiple immunoregulatory activities, galectins may bind to the by guest on October 1, 2021 their up-regulation in response to the early stages of in vivo no- envelope glycoproteins and inhibit viral fusion to the host cell davirus infection validates the results from the SSH approach. membrane, thereby exerting direct antiviral effects. Galectin-1 ex- Functional in vitro assays conducted with the recombinant hibits antiviral properties against Nipah virus, possibly by inter- Sbgalectin-1 provided further insight into its potential anti- action with specific N-glycans on the viral envelope (50, 52). Al- inflammatory activity. A dose-dependent decrease of respiratory though our results suggest that Sbgalectin-1 could have similar burst was observed in head kidney total leukocytes after incu- activity, and explain the observed differential Mx protein expres- bation with different Sbgalectin-1 concentrations. A parallel study sion, the direct binding and/or neutralization of nodavirus by conducted in turbot (Psetta maxima) with rSbgalectin-1 yielded Sbgalectin-1 remains to be demonstrated. Further studies aimed at similar results (data not shown). Similarly, a decrease in ex- elucidating the detailed mechanisms of Sbgalectin-1 in sea bass pression of proinflammatory cytokines, IL-1␤ and TNF-␣, was immunity and protection against viral pathogenesis are ongoing. observed in the brain of sea bass, which is the target of the infection, simultaneously injected with nodavirus and rSbgalectin-1 re- Acknowledgments spect to the ones infected with nodavirus alone, which suggests a We thank Dr. G. Bovo (Istituto Zooprofilattico Sperimentale delle Venezie, potential anti-inflammatory role for the recombinant galectin-1, Italy) for providing the nodavirus strain used in these studies and Dr. V. such as previously proposed in mammals (78). Although, exposure Mulero (Universidad de Murcia, Spain) for generously providing fish of nodavirus to rSbgalectin-1 in the inoculum could decrease its tissues. virulence by direct binding, this remains to be demonstrated rigorously. Moreover, a reduction in Mx protein expression was Disclosures observed in fish simultaneously injected with nodavirus and The authors have no financial conflict of interest. rSbgalectin-1 with respect to the nodavirus-infected ones, which could indicate that rSbgalectin-1 decreases nodavirus pathogenic- References ity. Also, the results provided by the in vivo Western Blot assays 1. Renault, T., P. H. Haffner, F. Baudin Laurencin, G. Breuil, and J. R. Bonami. revealed a tissue-specific induction of Sbgalectin-1 expression in 1991. Mass mortalities in hatchery-reared sea bass (Lates calcarifer) larvae associated with the presence in the brain and retina of virus-like particles. Bull. Eur. brain after nodavirus infection, because no galectin bands were Ass. Fish Pathol. 11: 68–73. observed either in brain of control fish or in head kidney samples, 2. Frerichs, G. N., H. D. Rodger, and Z. Peric. 1996. Cell culture isolation of piscine neuropathy nodavirus from juvenile sea bass, Dicentrarchus labrax. J. Gen. Vi- suggesting again its possible role on the target tissue for the virus. rol. 77: 2067–2071. Most mammalian prototype galectins form noncovalently associ- 3. Munday, B. L., J. Kwang, and N. Moody. 2002. Betanodavirus infections of ated dimers, although under certain conditions higher order aggre- teleost fish: a review. J. Fish Dis. 25: 127–142. 4. Garlanda, C., B. Bottazzi, A. Bastone, and A. Mantovani. 2005. Pentraxins at the gates can assemble (79). The galectin oligomerization process can crossroads between innate immunity, inflammation, matrix deposition and female be driven further by binding to both soluble and cell membrane- fertility. Annu. Rev. Immunol. 23: 337–366. The Journal of Immunology 11

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