Nodavirus Infection of Sea Bass (Dicentrarchus labrax) Induces

Upregulation of Galectin-1 Expression in Head Kidney, with

Potential Anti-Inflammatory Activity1, 2

Laura Poisa-Beiro*, Sonia Dios*, Hafiz Ahmed†, Gerardo R. Vasta†, Alicia Martínez-

López#, Amparo Estepa#, Jorge Alonso-Gutiérrez*, Antonio Figueras*, and Beatriz

Novoa*

*Instituto de Investigaciones Marinas. Consejo Superior de Investigaciones Científicas (CSIC). Eduardo Cabello, 6. 36208 Vigo, Spain; †Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 East Pratt Street, Baltimore, MD 21202, U.S.A; #Instituto de Biología Molecular y Celular (IBMC) Universidad Miguel Hernández (UMH). Av. de la Universidad s/n. 03202 Elche (Alicante), Spain.

Running Title: Nodavirus induced galectin-1 expression and anti-inflammatory activity

Keywords: Dicentrarchus labrax, gene expression, C-type lectin, galectin, pentraxin.

Abstract

Sea bass nervous necrosis virus (SBNNV) 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 anti-

viral immune defense, gene expression profiles in response to nodavirus infection were

investigated in sea bass head kidney using the suppression subtractive hybridization

(SSH) technique. An 8.7 % of the ESTs 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 SSH were

validated by in vivo upregulation 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 carried out. 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-1β, TNF-α

and Mx expression was observed in the brain of sea bass simultaneously injected with

nodavirus and rSbgalectin-1 compared to those infected with nodavirus alone. These

results suggest a potential anti-inflammatory, protective role of Sbgalectin-1 during

viral infection.

Introduction

Sea bass nervous necrosis virus (SBNNV) is a non-enveloped, positive stranded

RNA virus that belongs to the Betanodavirus genus, within the Nodaviridae family, and

the causative agent of the viral nervous necrosis (VNN). This disease, reported in a wide

range of teleost fish, is characterized by lethargy, abnormal behavior, loss of

equilibrium and spiral swimming and, histopathologically, by the development of

vacuoles in cells from the nervous system tissues (brain, retina and spinal cord) leading

to a high mortality in larval and juvenile stages of affected fish (1-3). The mechanism(s)

of SBNNV infectivity and pathogenesis are largely unknown and it remains to be

determined whether the virus evades, actively blocks, or uses the molecular recognition

factors of the host’s immune system.

The innate immune response is the first line of defense against microbial

pathogens, and consists of a complex network of diverse humoral and cellular

recognition and effector components, whose tight regulation contributes to activate or

deactivate specific anti-microbial pathways (4). Among these components, lectins are

carbohydrate-binding proteins involved in pathogen recognition and neutralization that

can also trigger effector mechanisms leading to pathogen destruction and instruction of

the adaptive immune mechanisms (5-8). Most animal lectins can be classified into

distinct families characterized by unique sequence motifs and structural folds: S-type

lectins (galectins), C-type lectins (including selectins, collectins and hyalectans), F-type

lectins, I-type lectins, P-type lectins, pentraxins, ficolins, and cytokine lectins (9, 10; see

also www.imperial.ac.uk/research/animallectins/). Although lectins from the various

families differ greatly in the domain architecture, they are all characterized by a

carbohydrate-recognition domain (CRD), which confers the protein its carbohydrate-

binding activity (11). Galectins are Ca2+ independent soluble lectins, widely distributed

in vertebrates, invertebrates (sponges, worms, and insects), and protists (protozoa and

fungi) (5, 10, 12, 13), characterized by their affinity for β-galactosides (5, 12, 14). The

mammalian galectins have been structurally classified into proto-, chimera- and tandem-

repeat types (15). The proto-type galectins exhibit a single CRD per subunit, and in

most cases form non-covalently bound dimers. In addition to the CRD at the C-

terminus, the chimera-type galectins have an N-terminus region consisting in a short

domain followed by an intervening proline/glycine/tyrosine-rich domain with short

repetitive sequences. Tandem-repeat-type galectins consist of two homologous CRDs

tandemly arrayed in a single polypeptide, joined by a short glycine-rich region (9, 15).

Further, a novel tandem-repeat type galectin with four CRDs has been recently

described (16). In contrast, C-type lectins, a group of highly diversified soluble or

integral membrane lectins, require Ca2+ for ligand binding and their characteristic CRD

(C-type-like domain, CTLD) can be classified into those that bind mannose and fucose,

and those that bind galactose (8). Members of the pentraxin lectin family, such as C-

reactive protein and serum amyloids are characterized by subunits arranged in a cyclic

oligomeric structure. In some species, including man, pentraxins are expressed upon

stimulus by inflammatory cytokines and are considered critical components of the

acute-phase response (17). Little is known, however, about the potential role(s) of

lectins in the innate immune and tissue repair responses to viral infection in fish.

In this study, we used suppression subtractive hybridization (SSH) to examine the

sea bass (Dicentrarchus labrax) gene expression profiles in head kidney upon nodavirus

infection. A galectin, two C-type lectins, and a pentraxin were identified among the

upregulated genes, and were characterized in further detail. Moreover, evidence about

the potential anti-inflammatory and protective role of the sea bass galectin on nervous

tissue affected by viral infection was obtained.

Materials and Methods

Fish and virus

Adult sea bass (80 g each) were obtained from a commercial fish farm. Prior to the

experiments, fish were acclimatized to laboratory conditions for 3 weeks, maintained at

20 ºC and fed daily with a commercial diet. The nodavirus strain 475-9/99 isolated from

diseased sea bass was provided by the Istituto Zooprofilattico Sperimentale delle

Venezie (Italy).

Nodavirus challenge and RNA isolation

Fish care and the viral challenge experiments were reviewed and approved by the

CSIC National Committee on Bioethics. Fish were injected intramuscularly with 100 µl

of nodavirus suspension in Minimum Essential Medium (MEM) (108 TCID50 ml-1) and

placed at 25 ºC. Mock-infected control fish (n = 12) were injected with medium alone,

and maintained under the same experimental conditions. Three fish from each

experimental and control groups were sampled 4, 24, 48 and 72 hours post-infection.

Animals were sacrificed by anesthetic (MS-222) overdose, dissected, and the head

kidney collected. RNA from individual organs was isolated by homogenization with

Trizol reagent (Invitrogen) following the manufacturer’s protocol. RNA was treated

with Turbo-DNA free kit (Ambion) to remove contaminating DNA.

SSH library construction

The SSH technique (18) was used to identify genes in head kidney that may be

upregulated upon viral infection. Because preliminary RT-PCR experiments had shown

up-regulation of several immune genes 4 hours after viral challenge (data not shown),

this timepoint was selected for sampling specimens dedicated to the SSH library

construction. cDNA was synthesized from 1 μg of head kidney (infected and control)

RNA using the SMART PCR cDNA Synthesis Kit (Clontech). The SSH library was

constructed using the PCR-select cDNA Subtraction Kit (Clontech). The PCR products

from the forward subtraction procedure were cloned using the Original TOPO T/A

Cloning Kit (Invitrogen), and screened by PCR using specific primers.

Sequence analysis

Excess of primers and nucleotides was removed by enzymatic digestion using 10

and 1 U of ExoI and SAP, respectively (Amershan Biosciences) at 37 ºC for 1h

followed by inactivation of the enzymes at 80 ºC for 15 min. The PCR products were

sequenced in a 3730 DNA Analyzer (Applied Biosystems). Blastn and Blastx searching

from the NCBI (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) were used to identify

homologous nucleotide and protein sequences. Protein sequence alignments were

evaluated using ClustalW program (http://align.genome.jp/) and specific conserved

domains were searched with several programs: Blastp from NCBI

(http://www.ncbi.nlm.nih.gov/blast/Blast.cgi), PROSITE

(http://www.expasy.ch/prosite/) and SMART (http://smart.embl-heidelberg.de/).

Compute pI/Mw (http://www.expasy.ch/tools/pi_tool.html) for Mw detection; Signal IP

(http://www.cbs.dtu.dk/services/SignalP) for signal peptide detection; TMpred

(http://www.ch.embnet.org/software/TMPRED_form.html), TMHMM

(http://www.cbs.dtu.dk/services/TMHMM-2.0/) and SOSUI (http://bp.nuap.nagoya-

u.ac.jp/sosui/sosui_submit.html) for transmembrane domain detection and Disulfind

(http://disulfind.dsi.unifi.it) for disulfide bonds were also used.

RACE amplification

The SMART RACE cDNA Amplification Kit (Clontech) was used to complete

the 5’ and 3’ cDNA ends of candidate sequences (Sbgalectin-1, SbCLA, SbCLB, and

Sbpentraxin), following the manufacturer’s recommendations. PCR products were

purified from 1.2 % agarose gel, subcloned using the Original TOPO T/A Cloning Kit

and sequenced as describe above. RACE primers are summarized in Supplementary

Table I.

Phylogenetic analysis

Sea bass lectin protein sequences were aligned with reference ones obtained from

GenBank using ClustalX (19). The alignment obtained was transferred and edited into

MacClade (20) and then directly transferred to ProtTest software version 1.4 (21), as a

guide to determine the best-fit model of protein evolution among 96 different

possibilities. The relative importance and a model-averaged estimation of different

evolution parameters were also calculated by this software. The best-fit model of

protein evolution and parameters, calculated by ProtTest, were incorporated into

software PHYML (22), which uses a single, fast and accurate algorithm to estimate

large phylogenies by Maximum Likelihood. Finally, the trees created by PHYML were

edited using the software TreeViewX (23).

Lectin expression studies

Lectin expression was assessed by Real-Time PCR (qPCR) in head kidney at 4

and 72 hours post-infection. RNA was isolated as described above and five µg were

used to obtain cDNA by the Superscript II Reverse Transcriptase and oligo(dT)12-18

primer (Invitrogen) following the manufacturer’s instructions. qPCR assays were

performed using the 7300 Real Time PCR System (Applied Biosystems) with specific

primers (Supplementary Table II). Each primer (0.5 μl; 10 μM) and the cDNA template

(1 μl, pooled from 3 fishes) were mixed with 12.5 μl of SYBR green PCR master mix

(Applied Biosystems) in a final volume of 25 μl. The standard cycling conditions were

95 ºC for 10 min followed by 40 cycles of 95 ºC 15 s and 60 ºC 1 min. The comparative

CT method (2- ΔΔCT method) was used to determine the expression level of analyzed

genes (24). Expression of the candidate genes was normalized using β-actin as the

reference gene. Fold units were calculated dividing the normalized expression values of

infected tissues by the normalized expression values of the controls.

Genomic sequence of Sbgalectin-1.

Total DNA was extracted from sea bass head kidney by the phenol-chloroform

method. Specific primers were designed based on the Sbgalectin-1 cDNA and PCR

amplification products cloned and sequenced in a 3730 DNA Analyzer (Applied

Biosystems). Identification of introns was carried out using the Wise 2 program

(http://www.ebi.ac.uk/Wise2/advanced.html).

Expression and purification of recombinant Sbgalectin-1

Expression of recombinant Sbgalectin-1 (rSbgalectin-1) was performed using

the pET-30a(+) vector in the Escherichia coli (Bl21 strain) cells. Briefly, the total open

reading frame of Sbgalectin-1 was amplified by PCR using primers containing a NdeI

(primer forward) or BamHI (primer reverse) restriction site (F: 5´-

GGGGGGGGGATTCCATATGTTTAATGGTTTG-3´; R: 5´-

GGGCGCGGATCCCTATTTTATCTCAAAGC-3´) and cloned into a pGEM-T vector

(Promega). The Sbgalectin-1 insert was removed from the plasmid and ligated into the

pET-30(+) bacterial expression vector (Novagen) using NdeI and BamHI restriction

sites and transformed into E. coli strain BL21 (DE3) competent cells. A single colony

containing the transformed plasmid was used to inoculate cultures of LB medium

containing 50μg/ml kanamyacin, and the expression of recombinant protein was

induced by 1mM isopropyl- β-D-thiogalactopyranoside (IPTG, Invitrogen). Soluble

proteins were extracted using lysozyme (100 µg/ml), phenylmethylsulphonyl fluoride

(PMSF, 1mM) and β-mercaptoethanol (10mM) (Sigma) and the rSbgalectin-1 was

isolated by affinity chromatography on lactose-Sepharose 6B column at 4 ºC following

the protocols previously described (25). The homogeneity of the rSbgalectin-1

preparation was assessed by SDS-PAGE on 15 % acrylamide:bisacrylamide gel using a

Mini-PROTEAN 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 previously described

(25). After 30 min at room temperature, the reactions were read in an iEMS reader

(Labsystems) at 620 nm and the data were analyzed through the Excell program.

Hemagglutinating activity of rSbgalectin-1 was tested following the procedure

reported by Nowak et al. (26) using two-fold serial dilutions of samples in microtiter U

plates and trypsin-treated glutaraldehyde-fixed rabbit erythrocytes. To analyze the

inhibitory effect of β-galactosides on the hemagglutinating activity of rSbgalectin-1 the

saline solution was replaced by 100mM lactose solution. The galectin-1 concentration at

the highest dilution still showing visible agglutination was recorded.

Effects of rSbgalectin-1 on leukocyte respiratory burst and NO production

Sea bass head kidney leukocytes were isolated following the method previously

described by Chung and Secombes (27). Viability of the leukocyte preparation was

determined by Trypan blue exclusion.

To assess the potential effect of the sea bass galectin-1 on respiratory burst,

leukocyte monolayers were incubated with increasing doses of rSbgalectin-1 (0, 1.7,

3.3, 6.7 and 13.3 µg ml-1) in 96-well plates (100 µl per well) for 24 hours at 20 ºC. After

this time, the emission of relative luminescence units (RLU) was determined by

stimulating the cells with phorbol myristate acetate (PMA, Sigma) and amplified by the

addition of 5-amino-2-3-dihydro-1,4-phthalazinedione (luminol, Sigma). A stock

solution of luminol 0.1M was prepared in dimethyl sulphoxide (DMSO, Sigma) just

before use, and diluted in HBSS (Gibco) to obtain a final concentration of 0.15mM

(luminol stock solution). PMA was solubilized in the luminol stock solution to a final

concentration of 1 µg ml-1. Immediately after the addition of the solution into the wells

(100 µl per well of luminol alone or with PMA), the respiratory burst activity was

measured in a luminometer (Fluoroskan Ascent, Labsystems) at intervals of 5 minutes,

during a 30-minute time course.

To measure the potential effect(s) of rSbgalectin-1 on NO production, leukocyte

monolayers were established under the same conditions described above, increasing

doses of rSbgalectin-1 were added, and after 24 h incubation, the nitrite content of the

supernatants was measured by the Griess reaction (28). Briefly, after the incubation

period, 50 µl of leukocyte monolayer supernatants were taken to new 96-well plates. A

100 µl aliquot of 1 % sulphanilamide (Sigma) in 2.5% phosphoric acid was added to

each well, followed by 100 µl of 0.1% N-naphthyl-ethylenediamine (Sigma) in 2.5 %

phosphoric acid. ODs at 540 nm were measured in an iEMS (Labsystems). The

concentration of nitrite in the samples was determined using a standard curve generated

with solutions of sodium nitrite of known concentrations. Both assays were carried out

with three different fishes.

Effects of rSbgalectin-1 on cytokine expression

Expression levels of TNF-α, IL-1 β and Mx upon intramuscular injection of

rSbgalectin-1 and nodavirus were measured by qPCR. Four groups of fish (3 fish each)

were treated as follows: Group 1 with 100 μl of nodavirus (0.85 x 107 TCID50 ml–1);

Group 2 with 100 μl of rSbgalectin-1 (6.7 μg/ml); Group 3 with 150 μl of a mix of 50

μl of nodavirus (1.7 x 107 TCID50 ml–1) and 100 μl of rSbgalectin-1 (6.7 μg/ml); and

Group 4 with 150 μl of cell culture medium as control. Fish were sacrificed by MS-222

overdose 72 hours post challenge, and the brain and head kidney were removed

aseptically. RNA isolation, cDNA synthesis, and qPCR were carried out as described

above. Primer sequences are shown in Supplementary Table III.

Western blot

In order to document the upregulation of Sbgalectin-1 after nodavirus infection, a

western blot was carried out to provide protein expression levels in brain and head

kidney of nodavirus infected fish. Western blot assays were performed as previously

described (29). Briefly, head kidney and brain lyophilized samples were resuspended in

Milli-Q water containing phenylmethanesulfonyl fluoride (100 μg/ml) and total protein

concentration adjusted to 0.1mg/ml using the Bradford reagent (BIO-RAD, Richmond,

VA, USA). Sodium dodecyl sulfate-polyacrylamide gels (Ready Tris–HCl 4–20% gel,

BIO-RAD,) were loaded with 20 μl of samples in buffer containing β-mercaptoethanol.

The rSbgalectin-1 sample of 200 ng was also included in the gels. The proteins in the

gel were transferred during 3 h at 125 V in 2.5 mM Tris, 9 mM glycine and 20%

methanol to nitrocellulose membranes (BIO-RAD). The membranes were then blocked

with 2% dry milk, 0.05% Tween-20 in PBS and incubated for 3 h at room temperature

with a striped bass (Morone saxatilis) galectin 1 antiserum diluted 1000-fold in PBS

before incubation with a peroxidase conjugated goat anti-rabbit antibody (Nordic,

Tilburg, Netherlands). The peroxidase activity was detected by using the ECL

chemiluminescence reagents (Amersham Biosciences, UK) and revealed by exposure to

X-ray films (Amersham).

Results

Nodavirus infection of sea bass induces upregulation of expression of immune related

genes including a galectin, two C-type lectins, and a pentraxin

An SSH approach was implemented to investigate gene expression profiles in

sea bass head kidney upon nodavirus infection, and identify genes potentially involved

in innate immunity and protective responses against its pathogenic effects. The

subtracted cDNA library was constructed from sea bass head kidneys 4 hours after

nodavirus infection. Following transformation in E. coli, 479 bacterial clones were

isolated and amplified by PCR resulting in 206 reliable EST sequences (accession

numbers from FF578829 to FF579034) representing 70 singletons potentially up-

regulated in infected head kidneys. Sequence homology was searched using Blast taking

into account Blastx entries. Fourteen out of 206 (6.8 %) showed significant similarities

(e-value < 10-3) with structural genes from different organisms; 18 out of 206 (8.7 %)

were immune or stress-related genes such as heat shock protein 90 beta, β-2

microglobulin precursor, apcs-prov protein, β-galactoside-binding lectin, mannose

receptor C1, Cd209e antigen or serum lectin isoform 2; 93 out of 206 (45.1 %) were

annotated as metabolic genes; 18 out of 206 (8.7 %) showed significant similarities with

ribosomal genes and 9 out of 206 (4.4 %) were classified as others (Fig. 1). Sequences

similar to hemoglobin were the most abundant in the subtraction, present in 42 clones.

The best match was found for the phosphogluconate hydrogenase (Blastx expectation

value 2e-134). A complete list of the significant entries obtained through the Blastx

search is presented in Table I. Besides the data described above, 25 out of 206 (12.1 %)

showed non-significant e-values (≥ 10-3) and 29 out of 206 (14.1 %) showed no

similarity with any other sequence deposited in GenBank (Fig. 1).

Among those ESTs identified from the subtracted library as over-expressed upon

nodavirus infection, four lectins were investigated in further detail: a galectin, two C-

type lectins, and a pentraxin. A 400 bp clone was identified as galectin-1 (Sbgalectin-1).

cDNA RACE 3’ gave a 785 bp clone (GenBank Accession No. EU660937), with an

open reading frame (60 to 467) encoding 135 amino acids with a calculated molecular

weight of 15244.16 and theoretical pI of 5.02 (Supplementary Fig. 1). The molecular

size (within the typical 14-18 kDa range of proto-type galectins) together with the

alignment results with different galectins from other species (Fig. 2A) and the presence

of the important amino acid residues for carbohydrate binding clearly confirm that the

Sbgalectin-1 is a member of the galectin family and is more closely related to the

galectin-1 group than to any other galectin family members. No secretion peptide

sequence was found in the Sbgalectin-1, in consistent with the characteristics of the

galectin family (5). At the amino acid level, this galectin presented the highest

homology with the Atlantic halibut (Hippoglossus hippoglossus) galectin (74.63%

identity). The phylogenetic analysis was concordant with the current taxonomy for all

species included, with Sbgalectin-1 clustering with the halibut galectin-1 (bootstrap

value: 99; Fig. 2B).

Characterization of the gene organization of Sbgalectin-1 (GenBank Accession

No. EU660934) revealed the presence of 3 introns in the following positions of the

ORF: 10 bp (2144 bp in length), 90 bp (879 bp in length) and 262 bp (966 bp in length)

(Fig. 2C and Supplementary Fig. 2).

A 605 bp clone was characterized as a C-type lectin (SbCLA) and RACE was

conducted to obtain a complete cDNA sequence for both 5’ and 3’ ends (GenBank

Accession No. EU660935). The complete sequence (2266 bp) presented an open

reading frame (from 187 bp to 1578 bp), which encoded 463 amino acids with a 54 kDa

predicted size protein (Supplementary Fig. 3). An alignment with CRD regions from

other species clearly confirmed that SbCLA protein is a member of the C-type lectin

family (Fig. 3A). Six cysteine residues were observed in the sequence at conserved

positions within the C-type lectin domain (CTLD). These residues are required for the

arrangement of three intramolecular disulfide bonds that are essential for CTLD fold

stability and were located at C337-C461, C348-C365 and C435-C453 positions. SbCLA

sequence had 3 out of 4 essential residues for Ca2+ ion coordination at site 1 and 3 out of

5 residues at site 2 (Fig. 3A). In addition, we observed the highly conserved “WIGL”

motif (11) and a “QPN” motif, which differed from EPN motifs (characteristic of all

mannose-binding proteins) and QPD motifs (typical of galactose-specific CTLDs).

A 408 bp clone was identified as a C-type lectin (SbCLB). RACE amplification

produced a complete cDNA sequence with 719 bp (GenBank Accession No.

EU660936), an open reading frame from 61 to 579 nucleotide position, which translated

for a 19 kDa predicted size protein with 172 amino acids (Supplementary Fig. 4). The

SbCLB protein sequence alignment with CTLDs from other species (Fig. 3B) showed

six cysteine residues at conserved positions and predicted disulfide bonds at C44-C160,

C55-C144 and C72-C168 sites. SbCLB presented 3 out of 4 essential residues for Ca2+ ion

coordination at site 1, and 1 out of 5 residues at site 2 (Fig. 3B). Moreover, we

observed a “WLGG” motif instead of the typical “WIGL” domain and the “QPD”

signature tripeptide characteristic of the galactose binding proteins.

A phylogenetic tree was constructed considering SbCLA and SbCLB CTLDs

sequences from various organisms. SbCLB clustered closer to other salmonid and non-

salmonid fish, with a boostrap value of 62. However, SbCLA was closer (79 boostrap

value) to primates and rodents (Fig. 3C).

A 356 bp clone revealed a pentraxin-like protein (Sbpentraxin). The 5’ and 3’

RACE amplification of this clone generated a 1563 bp sequence (GenBank Accession

No. EU660933) with an ORF (29 to 706) encoding for a 225 amino acid long, 26 kDa

protein (Supplementary Fig. 5). Its 857 bp 3´-UTR region resembles the unusually long

(858-1500 bp) 3´-UTR typical of the mammalian CRPs, and suggests that this protein is

a C-reactive protein (CRP). An alignment with pentraxins from other species revealed

two highly conserved cysteines, which are involved in intrachain disulfide bonds, and

the conserved pentraxin signature TWxS, where x is any amino acid (4) (Fig. 4A),

thereby confirming that Sbpentraxin protein is a bona fide member of the pentraxin

family. A search in Genbank identified the salmon (Salmo salar) pentraxin as the

protein with the highest identity (36%) at the amino acid level. A phylogenetic tree

constructed with pentraxin sequences from various organisms placed the Sbpentraxin

closer to the trout (Oncorhynchus mykiss), salmon (S. salar), and zebrafish (Danio

rerio) pentraxins with a boostrap value of 55 (Fig. 4B).

Assessment of in vivo expression of Sbgalectin-1 by qPCR revealed clear

differences between infected and control samples. Similar differences were also

observed for SbCLA and SbCLB. A significant increase in expression upon nodavirus

infection was observed after 4 hours for the three lectins, and continued beyond 72 h for

SbCLB. Expression of Sbgalectin-1 and SbCLA, however, decreased 72 h after

infection (Fig. 5).

All further studies, including in vivo expression, effects on leukocyte oxidative

burst and NO production, and cytokine expression in the brain, were focused on

Sbgalectin-1.

Sbgalectin-1 displays anti-inflammatory protective activity against viral infection

To enable functional studies on the Sbgalectin-1, the recombinant protein was

produced in a prokaryotic expression system. In SDS-PAGE, the induced bacterial

extracts showed a protein band of the expected mobility (15 kDa) (Fig. 6A). The

carbohydrate-binding activity of the rSbgalectin-1 was determined by hemagglutination,

with a 1:20 (0.5 μg rSbgalectin-1 per well, 0.325 µM) as the highest dilution at which

activity was observed. Hemagglutination by rSbgalectin-1 was carbohydrate-specific, as

lactose behaved as an effective inhibitor (Fig. 6B). A dose-dependent decrease of

respiratory burst activity was observed in leukocytes exposed to increasing rSbgalectin-

1 concentrations (0 μg/ml, 1.7 μg/ml, 3.3 μg/ml, 6.7 μg/ml and 13.3 μg/ml) (Fig. 7).

However, rSbgalectin-1 did not affect NO production by leukocytes (data not shown). A

decrease of IL-1β, TNF-α and Mx expression was observed in the brain of sea bass

simultaneously injected with nodavirus and rSbgalectin-1 with respect to those infected

with nodavirus alone, and the uninfected controls (Fig. 8). In head kidney, however,

although rSbgalectin-1 modified the expression of the analyzed genes, there was not

change comparing nodavirus alone and nodavirus plus rSbgalectin-1. Although the

expression of the inflammatory cytokines was low, the measurements were highly

reproducible.

A Western blot was carried out after extracting the proteins from head kidney

and brain of 6 nodavirus infected fish and 6 controls. A band of 15 kDa (galectin

monomer) was observed in all infected fish brain samples (Fig. 9) but not in the controls

(data not shown). Also bands of 30 and 45 kDa or 45 kDa were only observed for

rSbgalectin-1 and brain samples, respectively, which seem to be aggregates of the

proteins under the experimental conditions. No galectin bands were observed in any of

the head kidney samples (data not shown).

Discussion

In the present study we analyzed the effect of nodavirus infection on the sea bass

head kidney transcriptome using a SSH approach. The percentages of the significant

ESTs obtained were higher than for cDNA or subtracted libraries after experimental

infection or immunestimulation of Japanese flounder (Paralichthys olivaceus) (30), carp

(Cyprinus carpio) (31), salmon (S. salar) (32) and gilthead 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 critical

components in the mammalian immune response, such as galectins (34, 35); Cd209e

(35), C-type lectins (31, 32, 34, 36, 37), pentraxins (32, 38), MHC class II and heat

shock protein 90 (30, 32, 35), β-2 microglobulin, immunoglobulin heavy and light

chains (30, 32), and T-complex protein (Tcp) (32). This considerable number of

common upregulated transcripts supports the notion that multiple components of both

innate and adaptative immune responses of fish have been conserved in the vertebrate

lineages leading to the mammals.

Among the immune genes found differentially expressed in the library we focused

on lectins as in mammals they play important roles in innate immunity and regulation of

adaptive responses (10, 12, 39-45), and are also involved in numerous cellular processes

such as targeting of proteins within cells and cell-to-cell and cell-to-matrix adhesion

(44, 46-49). Therefore, we characterized the complete cDNA sequences of a galectin

(Sbgalectin-1), two C-type lectins (SbCLA and SbCLB), and a pentraxin (Sbpentraxin)

not reported so far in sea bass. Because in mammals, galectins have been demonstrated

to mediate important roles in host responses to viral infection (34, 50-53; recently

reviewed in 54), this study was further centred on Sbgalectin-1.

Within the proto-type galectins, Sbgalectin-1 is closely related to the mammalian

galectin-1 than to any other subgroups. Sbgalectin-1 exhibits all the essential amino

acids (H44, N46, R48, H52, D54, N61, W68, E71, and R73, numbering based on bovine

galectin-1 sequence, 55) that are responsible for galectin-1 carbohydrate-binding

activities (56). Based on its molecular weight and agglutinating activity, Sbgalectin-1 is

most likely present as a dimer. The phylogenetic analysis grouped Sbgalectin-1 in the

same cluster with halibut (H. hippoglossus) galectin-1 with a bootstrap value of 99 (Fig.

2B), suggesting a conservative evolution of this family of proteins. Like other galectin-1

genes, Sbgalectin-1 gene contains four exons, of which exon 3 houses all the amino acid

residues that bind the carbohydrate ligand. Despite some differences in intron sizes,

Sbgalectin-1 gene showed similarity to that of the mammalian galectin-1 genes

including exon-intron boundaries (57-59).

Comparison of SbCLA and SbCLB with other sequences already published

suggested that they are clearly members of the C-type lectin family, characterized by the

CTLD (Fig. 3). Although most CTLD-containing proteins reported so far are of

mammalian origin, a few C-type lectins have been already identified in teleost species

(36, 60-67). SbCLA can be classified 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 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 C-type lectins. It was also conserved

in SbCLA (WIGL; Fig. 3A), but in SbCLB the motif had substitutions in the second

[leucine; as in carp (C. carpio)] and fourth [glycine; as described for salmon (S. salar)]

positions (WLGG; Fig. 3B). Another interesting difference between SbCLA and SbCLB

resided in the characteristic EPN (mannose/fucose-binding) or QPD (galactose-binding)

motifs of C-type lectins (8, 69). SbCLB had a QPD motif suggesting recognition of

galactose and related ligands (Fig. 3B). However, SbCLA displayed a QPN motif (Fig.

3A), which is intermediate between the two typical EPN and QPD CTLD sequence

motifs previously identified (11, 69). This QPN tripeptide has asparagine instead of

aspartate at the third position, which although it may not affect the Ca2+ ion

coordination at site 2, it may influence its carbohydrate specificity (65). A phylogenetic

tree was constructed based on the SbCLA and SbCLB CTLDs and those from various

organisms. SbCLB was closer to other salmonid and non-salmonid fish with a boostrap

value of 62. However, SbCLA was much closer (79 boostrap value) to primates and

rodents (Fig. 3C), suggesting that despite bearing the unique QPN carbohydrate-binding

motif, this CTLD has been strongly conserved from the fish to the mammalian taxa.

The results of upregulation of expression of Sbgalectin-1 upon experimental

nodavirus infection as revealed by the SSH approach were verified by in vivo

expression studies using qPCR. The results showed a clear difference between infected

and control samples with a significant increase in Sbgalectin-1 expression from 4h post-

infection, followed by a significant decrease at 72h post-infection (Fig. 5). SbCLA and

SbCLB, also included for comparison in the in vivo expression studies, showed an

increase in expression at 4h post-infection. However, although after that timepoint

SbCLA followed an expression profile similar to Sbgalectin-1, for SbCLB expression

continued to significantly increase at 72 h post-infection (Fig. 5). Similar results were

described previously in mammalian galectins by Zuñiga et al. (70), who reported

galectin-1 expression was higher in Trypanosoma cruzi infected mice macrophages than

uninfected controls. Soanes et al. (65) detected a higher expression three C-type lectin

receptors in liver of Atlantic salmon in response to infection by Aeromonas salmonicida

than in the controls. In mammals, lectins mediate recognition of a wide range of

pathogens including Gram-positive and Gram-negative bacteria, yeast, parasites and

viruses and can trigger effectors mechanisms such as activation of the complement

system, increasing opsonization, phagocytosis, and apoptosis, which leads to the

destruction of the infectious agent (71-74). Lectins can also function as regulators of

adaptive immunity, leading to apoptosis of selected T cells subsets, or enhancing the

production of cytokines in non-activated and activated T cells, among others (75-77).

Taking these roles into account, the identification of the galectin and C-type lectins in

our subtracted library indicates these molecules could have important defense roles in

fish immunity. Moreover, their upregulation in response to the early stages of in vivo

nodavirus infection validates the results from the SSH approach.

Functional in vitro assays carried out with the recombinant Sbgalectin-1 provided

further insight into its potential anti-inflammatory activity. A dose-dependent decrease

of respiratory burst was observed in head kidney total leukocytes after incubation with

different Sbgalectin-1 concentrations. A parallel study carried out in turbot (Psetta

maxima) with rSbgalectin-1 yielded similar results (data not shown). Similarly, a

decrease in expression of proinflammatory cytokines, IL-1β and TNF-α, was observed

in the brain of sea bass, which is the target of the infection, simultaneously injected with

nodavirus and rSbgalectin-1 respect to the ones infected with nodavirus alone, which

suggests a potential anti-inflammatory role for the recombinant galectin-1, such as

previously proposed in mammals (78). Although, exposure of nodavirus to rSbgalectin-

1 in the inoculum could decrease its virulence by direct binding, this remains to be

demonstrated rigorously. Moreover, a reduction in Mx protein expression was observed

in fish simultaneously injected with nodavirus and rSbgalectin-1 with respect to the

nodavirus infected ones, which could indicate that rSbgalectin-1 decreases nodavirus

pathogenicity. Also, the results provided by the in vivo Western Blot assays seem to

reveal an induced Sbgalectin-1 expression in brain after nodavirus infection, as no

galectin bands were observed neither in brain of control fish nor in head kidney samples

suggesting again its possible role on the target tissue for the virus. Although it was

reported above that Sbgalectin-1 is most likely present as a dimer (30 kDa), different

molecular size bands were observed in Western blots probably due to the experimental

conditions. These bands included monomers (15 kDa) and trimers (45 kDa), making

impossible to elucidate from this results the molecular size of the active protein in

tissues. Nodavirus causes vacuolization in the central nervous system and it has been

reported that in mammals, the oxidized galectin-1 enhances axonal regeneration in

peripheral and central nerves, even at relatively low concentrations (78). Studies by

O’Farrell et al. (34) in rainbow trout demonstrated upregulation of galectin-9 in

leukocytes upon rhabdovirus infection. It has been well established that galectins play a

role in the initiation, activation and resolution phases of innate and adaptive immune

responses by promoting oxidative burst (79, 80), inhibition of nitric oxide production

(81), anti-inflammatory or pro-inflammatory effects (45), and cytokine production (82).

Furthermore, a given galectin (for example, galectin-1) may exert pro- or anti-

inflammatory effects depending on multiple factors, such as the concentration reached

at the inflammatory foci, the extracellular microenvironment, and the target cells (83).

In addition to their multiple immunoregulatory activities, galectins may bind to the

envelope glycoproteins and inhibit viral fusion to the host cell membrane, thereby

exerting direct anti-viral effects. Galectin-1 exhibits anti-viral properties against Nipah

virus, possibly by interaction with specific N-glycans on the viral envelope (50, 52).

Although our results suggest that Sbgalectin-1 could have similar activity, and explain

the observed differential Mx protein expression, the direct binding and/or neutralization

of nodavirus by Sbgalectin-1 remains to be demonstrated. Further studies aimed at

elucidating the detailed mechanisms of Sbgalectin-1 in sea bass immunity and

protection against viral pathogenesis are ongoing.

Acknowledgements

We wish to thank Dr. G. Bovo for kindly providing the nodavirus strain

used in these studies and Dr. V. Mulero for generously providing fish tissues.

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Footnotes:

1 Corresponding author: Beatriz Novoa. Telephone: 34 986 214463. Fax: 34 986

292762. E-mail: virus@iim.csic.es

2 This research was supported by EU project (SSP8-CT-2003-501984), the

project CSD2007-00002 Aquagenomics funded by the program Consolider-Ingenio

2010 from the Spanish Ministerio de Educación y Ciencia (MEC) and Xunta de Galicia

(PGIDIT04PXIC40203PM). L. P-B and J. A-G were supported by FPU fellowships

from Ministerio de Educación y Ciencia, Spain. Work at the Center of Marine

Biotechnology, UMBI, was supported by grants RO1 GM070589-01 from the National

Institutes of Health, and IOS-0822257 from the National Science Foundation, USA, to

G.R.V. 3 The sequences presented in this article have been submitted to GenBank under

accession numbers as follows: Genomic Sbgalectin-1, EU660934; Sbgalectin-1,

EU660937; SbCLA, EU660935; SbCLB, EU660936; Sbpentraxin, EU660933; ESTs,

from FF578829 to FF579034.

Figure legends

FIGURE 1. Classification of Dicentrarchus labrax head kidney ESTs based on

function or structural similarity.

FIGURE 2. Molecular characterization of Sbgalectin-1.

A, ClustalW alignment comparing the predicted Sbgalectin-1 sequence with other

similar galectin sequences. * indicates the conserved amino acids. The critical amino

acid residues are highlighted in grey.

B, Sbgalectin-1 phylogenetic tree obtained with Phyml software following the model of

protein evolution recommended by ProtTest (WAG+I+G 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 (grey boxes) and introns (open

boxes) are shown in bp. The number of exons is indicated in the top.

FIGURE 3. Molecular characterization of SbCLA and SbCLB.

A, ClustalW alignment comparing the predicted SbCLA CTLD sequence with other

similar CTLD sequences. * indicates the conserved amino acids. Conserved cysteines

and other important amino acids are highlighted in light grey. The highly conserved

“WIGL” motif is highlighted in dark grey. EPN/QPN signatures are shown in bold.

Numbers 1 and 2 indicate the residues for Ca2+ coordination.

B, ClustalW alignment comparing the predicted SbCLB CTLD sequence with other

similar CTLD sequences. * indicates the conserved amino acids. Conserved cysteines

and other important amino acids are highlighted in light grey and the highly conserved

“WIGL” motif is highlighted in dark grey. EPN/QPD signatures are shown in bold.

Numbers 1 and 2 indicate the residues for Ca2+ coordination.

C, SbCLA and SbCLB phylogenetic tree obtained with Phyml software following the

model of protein evolution recommended by ProtTest (WAG+I+G 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.

FIGURE 4. Molecular characterization of Sbpentraxin.

A, ClustalW alignment comparing the predicted Sbpentraxin sequence with other

similar pentraxin sequences. * indicates the conserved amino acids. Conserved

cysteines are highlighted in light grey and the pentraxin signature TWxS, where x is any

amino acid, is highlighted in dark grey.

B, Sbpentraxin phylogenetic tree obtained with Phyml software following the model of

protein evolution recommended by ProtTest (JTT+G 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 used as

outgroup.

FIGURE 5. Real-Time PCR results for SbCLA (a), SbCLB (b) and Sbgalectin-1 (c)

expression level upon controls in the head kidney of intramuscular 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 β-actin expression.

n=3 in each experimental group.*: indicates expression values significantly different

from controls.

FIGURE 6. Biochemical characterization of recombinant Sbgalectin-1.

A, Recombinant Sbgalectin-1 SDS-PAGE, a: sample before passing to the

chromatography column; b: sample after passing to the chromatography column

(purified rSbgalectin-1). The induced bacterial extracts showed a protein band of the

expected mobility (15 kDa)

B, Hemagglutination assay of recombinant Sbgalectin-1. The carbohydrate-binding

activity of the rSbgalectin-1 was determined by hemagglutination, with a 1:20 (0.5 μg

rSbgalectin-1 per well, 0.325 µM) as the highest dilution at which activity was

observed. Hemagglutination by rSbgalectin-1 was carbohydrate-specific, as lactose

behaved as an effective inhibitor.

FIGURE 7. Respiratory burst activity of sea bass leukocytes incubated with increasing

rSbgalectin-1 concentrations (0 μg/ml, 1.7 μg/ml, 3.3 μg/ml, 6.7 μg/ml and 13.3 μg/ml)

along time in an individual fish (A), or as maximum well activity, mean of three fish

(B). Respiratory burst activity is expressed as relative luminescence units (RLU). *:

indicates RLU units significantly different from controls.

FIGURE 8. Real-Time PCR results for IL-1β, TNF-α and Mx expression level in the

brain (A, B, C) or in the head kidney (D, E, F) of intramuscular infected sea bass. n=3.

*: indicates expression values significantly different from control group. #: indicates

expression values significantly different from nodavirus group.

FIGURE 9. Western blot of Sbgalectin-1 from brain of 3 nodavirus infected fish (1-3:

infected samples), rGal corresponds to the rSbgalectin-1.

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

HOMOLOGY Nº of ESTs Nº of contigs e-value (Blast X) Length (bp) Species (* =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 (XP_531829) 1 1 2e-67 573 Canis familiaris

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

Beta actin (AAR97600) 1 1 2e-81 466 *Epinephelus coioides

Beta-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

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 beta-1 chain (Q9YHC3) 1 1 6e-60 352 *Gadus morhua

Type I collagen alpha 1 (BAD77968) 1 1 3e-19 665 *Paralichthys olivaceus

METABOLIC GENES

3 beta-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

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 heavy 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 alpha-A subunit (Q9PVM4) 20 2 3e-63 612 *Seriola quinqueradiata

Hemoglobin beta chain (AAZ79648) 2 2 1e-33 498 *Nibea miichthioides

Hemoglobin beta-A subunit (Q9PVM2) 15 2 1e-64 510 *Seriola quinqueradiata

Hemoglobin beta-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 (XP_687673) 1 1 2e-07 625 *Danio rerio

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

Beta-2 microglobulin precursor (O42197) 2 2 1e-23 572 *Ictalurus punctatus

Beta-galactoside-binding lectin (A28302) 1 1 5e-34 400 *Electrophorus electricus

Heat shock protein 90 beta (AAP20179) 1 1 9e-14 407 *Pagrus major

IgM heavy chain secretory form (AAL99929) 1 1 2e-69 589 *Chaenocephalus aceratus

Immunoglobulin light chain L2 (AAB41310) 2 1 1e-24 582 *Oncorhynchus mykiss

Lymphocyte antigen 6 complex, locus D (NP_034872) 2 1 5e-05 375 Mus musculus

Major histocompatibility complex 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 antigen (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