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: [email protected]
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