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. Author manuscript; available in PMC: 2015 Oct 15.
Published in final edited form as: Dev Comp Immunol. 2014 Jan 28;44(2):332–340. doi: 10.1016/j.dci.2014.01.019

A rhamnose-binding lectin from sea bass (Dicentrarchus labrax) plasma agglutinates and opsonizes pathogenic bacteria

M Cammarata 1,*, MG Parisi 1, G Benenati 1, GR Vasta 1, N Parrinello 1
PMCID: PMC4607264  NIHMSID: NIHMS727412  PMID: 24486534

Abstract

The discovery of rhamnose-binding lectins (RBLs) in teleost fish eggs led to the identification of a novel lectin family characterized by a unique sequence motif and a structural fold, and initially proposed to modulate fertilization. Further studies of the RBL tissue localization and gene organization were also suggestive of role(s) in innate immunity. Here we describe the purification, and biochemical and functional characterization of a novel RBL (DlRBL) from sea bass (Dicentrarchus labrax) serum. The purified DlRBL had electrophoretic mobilities corresponding to 24 kDa and 100 kDa under reducing and non-reducing conditions, respectively, suggesting that in plasma the DlRBL is present as a physiological homotetramer. DlRBL subunit transcripts revealed an open reading frame encoding 212 amino acid residues that included two tandemly-arrayed carbohydrate-recognition domains, and an 18-residue signal sequence at the N-terminus. The deduced size of 24.1 kDa for the mature protein was in good agreement with the subunit size of the isolated lectin. Binding activity of DlRBL for rabbit erythrocytes could be inhibited in the presence of rhamnose or galactose, did not require calcium, and was optimal at around 20°C and within the pH 6.5–8.0 range. DlRBL agglutinated Gram positive and Gram negative bacteria, and exposure of formalin-killed E. coli to DlRBL enhanced their phagocytosis by D. labrax peritoneal macrophages relative to the unexposed controls. Taken together, the results suggest that plasma DlRBL may play a role in immune recognition of microbial pathogens and facilitate their clearance by phagocytosis.

Keywords: Rhamnose binding lectin, Dicentrarchus labrax, teleost, opsonin, hemagglutinins, bacteria

1. Introduction

Lectins are carbohydrate-binding proteins widely distributed in eukaryotic taxa, and are involved in multiple biological processes, such as development and immune responses (Vasta et al., 1994; Arason, 1996; Kaltner and Stierstorfer, 1998; Kilpatrick, 2002). With regards to the latter, lectins can bind to microbial surface glycans through their CRDs (carbohydrate recognition domains), and based on their multivalent oligomeric organization and the presence of additional effector domains, not only recognize microbial pathogens but also mediate effector functions including agglutination, opsonization, and complement activation, that result in their immobilization, phagocytosis, killing and removal from the internal milieu (Gabius, 1997; Kilpatrick, 2002; Loris, 2002; Vasta et al 2004; Turner et al 1996; Sharon and Lis 2004; Fujita et al. 2004). On the basis of conserved amino acid sequence motifs in their CRDs, structural folds, and calcium requirements lectins have been classified into various families that include among others, the C-type, F-type, and rhamnose-binding lectins (RBLs), galectins and pentraxins (Drickamer 1982, Turner et al 1996; Sharon and Lis 2004; Arason 1996; Vasta et al 2004; reviewed in Vasta and Ahmed, 2008, and Vasta et al 2011).

Lectin repertoires in teleost fish are highly diversified (Vasta et al 2011; Vasta et al 2012), and include not only representatives of the lectin families described in mammals, but also members of lectin families initially identified and characterized in fish species, such as F-type lectins (Odom and Vasta, 2006; Bianchet et al, 2010) and RBLs (Tateno et al. 1998; Jimbo et al 2007; Terada et al 2007).

RBLs are Ca2+-independent lectins with specificity for rhamnose and galactosides, particularly abundant in teleosts and ascidians, and other aquatic invertebrate species, such as annelids, bivalves, and echinoderms (Ogawa et al., 2011). The sea urchin (Anthocidaris crassispina) egg dimeric lectin (SUEL) was first isolated as a D-galactoside-binding lectin and later shown to preferentially bind L-rhamnose. This is consistent with the same orientation of the hydroxyl groups at C2 and C4 in the pyranose rings of both L-rhamnose and D-galactose. The 100 amino acids-long CRD displays conserved sequence motifs, such as YGR, DPC and KYL (Terada et al., 2007), and houses eight highly conserved cysteine residues engaged in four disulfide bridges with characteristic topology that determine a unique structural fold (Jimbo et al., 2007; Terada et al., 2007).

In teleost fish, most of the RBLs described to date have been isolated from the egg cortex (Tateno et al. 1998; Lam and Ng 2002; Shiina et al.2002; Tateno et al. 2002a,b; Jimbo et al. 2007; Terada et al. 2007; Watanabe et al. 2008). Because rhamnose is absent in animal cells but widely distributed in the bacterial cell wall, the ability of trout egg RBLs to recognize and bind lipopolysaccharide and lipoteichoic acid, and agglutinate both Gram-positive and Gram-negative bacteria suggested a role in egg defense against infectious challenge (Tateno et al., 2002a). The presence of RBLs in skin mucus also supported their proposed function(s) in immune defense, and further work showed their participation in multiple antimicrobial activities such as inhibition of proliferation, cytotoxicity, and opsonization of non-self cells or particles (Hosono et al., 1999; Lam and Ng 2002; Tateno et al. 2002a; Terada et al. 2007). A putative natural ligand of fish RBL was identified as globotriasylceramide (Gb3), a glycosphingolipid that is abundant in membrane lipid rafts (Watanabe et al., 2009; Ogawa et al., 2011).

In this study we report the purification, cloning, and structural and functional characterization of an RBL present in plasma of the sea bass Dicentrarchus labrax. The protein had been observed as a secondary product in the purification of an F-type lectin (DlFBL) from sea bass plasma that we reported elsewhere (Salerno et al 2009). The purified sea bass RBL (DlRBL) showed a tetrameric structure in which each single subunit contains two tandemly-arrayed, distinct CRDs that exhibit the sequence motif typical of the teleost RBLs. Agglutinating activity for E. coli and Vibrio alginolitycus, and their opsonization for sea bass macrophages suggest that it may function in recognition of potential microbial pathogens in the blood stream.

2. Material and Methods

2.1. Chemicals and molecular biology reagents

Unless otherwise specified, chemicals and molecular biology reagents were from Sigma-Aldrich (USA), at the highest purity available.

2.2. Animals, collection of blood and tissue samples, and preparation of tissue extracts

Sea bass (Dicentrarchus labrax) (n=25; approximately 250 g each) were provided by the Ittica Trappeto fish farm (Trappeto, Palermo, Italy). Animals were anesthetized in seawater containing 0.02 % 3-aminobenzoic acid ethyl ester (MS-222 Sigma), and bled by heart puncture. The blood was allowed to clot at room temperature for 1 hr, the serum separated by centrifugation at 800 ×g (10 min, 4 °C), aliquoted, and stored at −20°C until use.

2.3. Purification of DlRBL

The DlRBL was isolated from sea bass serum by an optimized two-step chromatography procedure (Table I), following Cammarata et al. (2007). The first step consisted of affinity chromatography on fucose-agarose, with 0.2 M fucose elution. As monitored by absorbance at 280 nm, the elution profile yielded two peaks, the second of which displayed the highest hemagglutinating activity (titer: 256–512) and contained the DlFBL (Salerno et al 2009). To purify DlRBL, the pooled fractions of the first peak (20 ml) were loaded onto a galactose-agarose column (Pierce). The column was washed with 1.0 M NaCl, followed by TBS (10 volumes) at a 0.2 ml/min flow rate, and the DlRBL eluted with 20 ml of 50 mM galactose in TBS at the same flow rate, monitored by absorbance at 280 nm, and 2 ml fractions collected. The eluted fractions were tested for their hemagglutinating activity towards rabbit erythrocytes, and those that exhibited the highest activity were pooled and analyzed by SDS-PAGE electrophoresis.

Table 1.

Purification steps of DlRBP and DlFBP

Purification Stage Volume Protein Content (PC* vol) (HA) THA Specific Activity THA/PC Purification AS Stage/AS Serum Yield
ml mg HA titer AU AS %
Serum 20 160 512 10240 64 1 100
PI DlFBL (Fucose-agarose) 10 1.5 256 2560 1706 26.6
PII DlFBL* (Fucose-agarose) 9.7 0.9 1024 9932 11035 172 0.56
PII DlRBL (Galactose-agarose) 20 0.3 64 1280 4267 66.7 10 (0.18#)
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PI DlFBL: peak I of D. labrax Fucose Binding Lectin (DlFBL) after washing with TBS used for D. labrax Rhamnose Binding Lectin (DlRBL) purification source; PII DlFBL: peak II of DlFBL (Fucose) after elution with Fucose. PII DlRBL (Galactose) peak II of DlRBL after elution with Galactose. HA: hemagglutinating activity; THA: total hemagglutinating activity;

*

previously published in Salerno et al. 2009;

#

referred to serum amount.

2.4. Hemagglutination assay

Rabbit and sheep erythrocytes (RBC; supplied by Istituto Zooprofilattico della Sicilia) were washed three times with PBS, centrifuged at 500 ×g for 10 min at 4°C and suspended at 1% in TBS containing 0.1% (w/v) pig gelatin. A volume (25 μl) of sea bass serum (1:10) or 25 μl of the purified DlRBL preparation (250 μg/ml) dialyzed in TBS were serially (2-fold) diluted with TBS-gelatin in 96-well round-bottom microtiter plates (Nunc, Denmark), and mixed with an equal volume of RBC suspension. The hemagglutinating titer (HT) was measured after a 1 h incubation at 37°C and expressed as the reciprocal of the highest dilution showing clear agglutination.

2.5. Protein content estimation

Protein content was estimated according to the method of Bradford (1979) using bovine serum albumin (BSA) as a standard.

2.6. Polyacrylamide gel electrophoresis

SDS-PAGE (10%) was carried out following the method of Laemmli (1970) under reducing conditions (5% mercaptoethanol). To assess the apparent molecular size of DlRBL, the polyacrylamide gels were calibrated with low range standard proteins (Bio-Rad, Richmond, CA). Proteins were stained with Coomassie brillant Blue R250.

2.7. N-terminal sequencing

The purified lectin from SDS-PAGE gels run under reducing conditions was electrophoretically transferred (4 μg/well) to Immobilon polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA), at 160 mA for 1 h. The membrane was stained with an aqueous solution of Coomassie blue R-250, extensively rinsed with distilled water, and the band corresponding to the isolated protein was excised and applied to a protein sequencer (Procise, Perkin-Elmer, Switzerland). The sequence was determined by automated Edman degradation using a protein sequencer at the Institute of Protein Biochemistry (CNR Naples).

2.8. Phylogenetic analysis

The DlRBL protein sequence identified in a D. labrax EST library as Rhamnose binding protein (Appendix 3, Contig_1715; Sarropoulou et al 2009) was aligned with RBLs sequences from several species [(Channa argus (gi|189096288|gb|ACD76075.1); Lutjanus sanguineus (gi|322423476|gb|ADX01347.1); Tetraodon nigroviridis (gi|47222797|emb|CAG01764.1); Oncorhynchus mykiss STL2 (gi|185134480|ref|NP_001117668.1|); Ciona intestinalis (gi|198412427|ref|XP_002131218.1|); Nematostella vectensis (gi|156358465|ref|XP_001624539.1|predicted protein); Strongylocentrotus purpuratus SAL (gi|72110389|ref|XP_788214.1|)] and a phylogenetic tree was constructed by the Neighbor-Joining method (NJ) after 1000 bootstrap iterations by using CLC workbench 6.4. Calculations of theoretical protein characteristics from the deduced peptide sequence were performed with ProtParam (www.expasy.ch) and signal peptide cleavage site have been predicted by the SignalP algorithm (Ito et al 1991). The putative tertiary structure of each CRD from DlFBL was developed with SWISS-MODEL and the Swiss-PdbViewer (Guex and Peitsch, 1997; Arnold et al., 2006; Schwede et al., 2003) by using of the RBL CSL3 structure as the template (Protein Data Bank accession code 2zx2A; 1.80 Å).

2.9. Carbohydrate specificity

The carbohydrate specificity of DlRBL was assessed by inhibition of its agglutinating activity for rabbit RBC by saccharides. Inhibition experiments were carried out using decreasing concentrations (starting from 100 mM, in TBS pH 7.4; 5mM CaCl2; pH 7.4) of monosaccharides (L-fucose, L-rhamnose, D-galactose, D-glucose, D-mannose, N-Acetylgalactosamine, and N-Acetylglucosamine), oligosaccharides (maltose, lactose, lactulose), and mannan (from Saccharomyces cerevisae).

2.10. Physical and chemical treatments

To examine the divalent cation requirements for lectin binding, either CaCl2 or MgCl2 was added to the HA medium to reach a 5–10 mM final concentration. EDTA (10mM) or EGTA (10mM) was used to examine the effect of Ca2+/Mg2+ depletion on the lectin activity. To examine the thermal stability of the protein, the purified lectin (250 μg/ml) was incubated at 18°C, 37°C, 50°C, 60°C, 70°C and 90°C for 20 min and cooled down for 10 min on ice before the HA. Susceptibility of the lectin to freeze-thaw was examined by carrying out the HA on samples maintained at −20°C for 2 months and thawed at room temperature. The pH dependence of the DlRBL agglutinating activity was examined by Sorensen phosphate buffer (SPB; 0,15 M KH2PO4, 0,15 M Na2HPO4) with pH values ranging from 3 to 11 by addition of either acetic acid (down to pH 3) or sodium carbonate (up to pH 11). The lectin solution (250 μg/ml) was incubated the various buffer solutions in a microtiter plate for 30 min before the HA assay.

2.11. Bacterial suspensions and agglutination

Bacteria (Escherichia coli; Aeromonas hidrophyla, Staphylococcus aureus and Vibrio alginolyticus) (Table 3)were grown to log phase in tryptic soy broth (TSB) containing 3% NaCl at 25°C, with continuous shaking (120 rpm) in a Gallenkamp incubator. Cell numbers were estimated by absorbance at 600 nm, as previously correlated to plate counts. Bacteria were fixed by adding formaldehyde to the bacterial stock suspension to a 2% final concentration, and the mixture shaken (120 rpm) overnight at 21°C. After centrifugation at 6,000 ×g for 15 min (4°C), the formalin-killed bacteria were washed three times with sterile PBS, suspended in PBS containing 0.1% (w/v) gelatin to obtain 1× 109 cells/ml, and stored at 4°C until use.

Table 3.

Hemagglutinating (titer-1) against various arythrocyte types and bacteria by a Rhamnose (DlRBL) and Fucosea (DlFBL) binding lectins from Dicentrarchus labrax activity of purified lectin (50 μg/ml).

Erythrocytesb Agglutination activityc
Purified RBL Purified FBL
Human A 16 64
Human B 16 2
Human AB 16 2
Human 0 16 64
Rabbit 32–128 256–512
Bacteria
E. coli 128 –
V. alginolyticus 8 –
A. hidrophyla 16 –
S. aureus 16 –
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a

Salerno et al 2010

b

Erythrocytes were collected in Alsever solution and washed five times by centrifuging at 800 g for 5 min in Tris-buffer saline (pH 7.5). The packed cells were suspended in TBS to give a 1% (V/V) suspension of native erythrocytes and used to test hemagglutination activity.

c

Hemagglutination activity was expressed as a titer, the reciprocal of the highest twofold dilution exhibiting hemagglutination (HA). Dashes indicate no activity. The agglutinating assays were performed in 96-well V-bottomed microtiter plates. Samples (25mL) were serially diluted twofold in TBS, and an equal volume of erythrocyte suspension was added to each well. The plates were shaken gently and then incubated at 37°C for 1 h (bacteria at room temperature for 24 hours). The data presented are from one representative experiment that was repeated three times.

2.12. Opsonic activity for peritoneal macrophages

For the phagocytosis assay, the formalin-killed bacteria Escherichia coli were washed three times with sterile PBS, suspended 1×109/ml in carbonate buffer (0.1M Na2CO3, 0.1M NaHCO3, pH 9.5) containing 0.1 mg/ml FITC and incubated 30 min at 37°C with gentle shaking. FITC-treated formalin-killed bacteria were washed three times in NaCl 0.9% and twice in PBS containing 2 mM CaCl2 (PBS-Ca), and suspended (1×108/ml) in PBS-Ca. FITC-treated formalin-killed bacteria (1×108/ml) were mixed in a microtube with purified F-lectin (5, 10, 25 μg/ml) in PBS-Ca, incubated at 18°C for 60 min, and washed twice with the same buffer. In the controls the purified lectin was replaced with PBS-Ca. The opsonized bacteria (100 μl) were incubated with an equal volume of Peritoneal cavity cells (PCC) (1×107/ml) for 30 min at 18°C. Fluorescence of the non-phagocytosed bacteria, was quenched by adding trypan blue (2 mg/ml) in 0.02 M citrate buffer pH 4.4, containing 0.15 M NaCl and 2 mg/ml crystal violet in PBS [32]. The phagocytosed fluorescent bacteria were observed under a UV light microscope equipped with Nomarsky differential interference contrast optic (Diaplan, Leica, Wetzlar, Germany). The phagocytic rate (PR) was determined as the percent of cells showing internalized bacteria, and the phagocytic index (PI) as the average of ingested bacteria relative to the total phagocyte number.

3. Results

3.1. D. labrax RBL purification and characterization

Fractions from the first peak eluted from the fucose-agarose column revealed hemagglutinating activity (1:32 – 1:256) that could only be inhibited by L-rhamnose or D-galactose, but not by concentrations up to 200 mM of L-fucose, glucose, lactose, GalNAc, GlcNAc or other sugars tested (Table 1). Accordingly, the protein contained in the first peak was further purified by affinity chromatography on a D-galactose–agarose column (1.6 × 5 cm), eluted with D-galactose. A typical affinity chromatography purification profile is shown in Fig. 1. The eluted protein represented approximately 0.1–0.5 % of the total serum protein loaded onto the column. Hemagglutination titers (HT) of the eluted fractions (n=28–36, 0.3–0.5 mg/ml; Fig.1) for rabbit RBC ranged from 1:16 to 1:64, and the activity was completely abolished by a pre-incubation with 4.2 mM L-rhamnose. As shown in Table 2, the DlRBL pool had 60- to 70-fold higher activity (specific activity: 4,267) than that of whole serum. Fractions 6–12 lacked hemagglutinating activity. The L-rhamnose-eluted fractions, examined by SDS-PAGE under non-reducing conditions, revealed a single 102.0 ± 3.2 kDa component, while under reducing conditions the apparent mass was 24.1 ± 1.6 kDa (Fig.2).

Fig. 1. Affinity chromatography of D.labrax Rhamnose binding protein.

Fig. 1

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The pooled fractions of the first peak peak derived from a Fucose agarose column were loaded onto a galactose-agarose column. Absorbance at 280 nm (□); hemagglutinating activity (magenta bar).

Table 2.

Inhibition of hemagglutination activity of the Dicentrarchus labrax isolated lectin against RE by various Carbohydrates

Carbohydrate Minimum inhibitory concentration (mM)
Monosaccharides
D-Glucose –
D-Galactose 70.0
D-Mannose –
L-Fucose –
L-Rahmnose 4.2
N-acetyl-D-glucosamine –
N-acetyl-D-galactosamine –
Disaccharides
Lactose –
Lactulose –
Maltose –
Polysaccharides Mannan –
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We used rabbit erythrocytes. Inhibitory activity is expressed as the minimum inhibitory concentration that is required to completely inhibit the hemagglutinating activity of a titer. Dashes indicate non inhibitory activity at a concentration of 200mM for monosaccharides or disaccharides.

Fig. 2. SDS-PAGE of D. labrax purified lectins.

Fig. 2

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SDS-PAGE analysis of DlRBL (2.5 μg) in the absence (lane 1) or presence (lane 2) of reducing agent (NR) (2-mercaptoethanol) on a 12.5% acrylamide gel stained with Coomassie Blue R-250.

3.2. N terminal sequence of DlRBL

The selected protein identified by SDS-PAGE under non-reducing conditions was transferred to PVDF membrane and sequenced. The N-terminal sequence was GVPTETVTTCEGNHVHRL. A GenBank search revealed identities at several amino acid positions with RBL-type lectin family members, and a 100% of identity was obtained with a complete open reading frame transcript identified in a D. labrax EST library as Rhamnose binding protein (Sarropoulou et al 2009).

3.3. DlRBL sequence analysis

The open reading frame of the DlRBL encoding 221 amino acids (Supplemental Fig. 1). The nucleotide sequence revealed a 71-nucleotide 5′-UTR that precedes the putative translation start site. The deduced protein sequence contains a signal peptide at the N-terminal with a cleavage site that resides between A19 and G20 (Fig.3A) as predicted by the SignalP algorithm. This sequence contains the already identified 18 amino acid sequence identified at the N-terminal. The calculated molecular mass of the DlRBL was 24.1 kDa (ProtParam; www.expasy.ch), with a theoretical isoelectric point of 5.84.

Fig. 3. Structural analysis of DlRBL.

Fig. 3

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A: Sequence analysis of DlRBL: Signal P-NN program predicted a signal peptide located in the N-terminal region, and provided of the cutting site between Ala19 and Gly20. B: Homology modeling: The N and C-CRD of DlRBL were modeled on to the Oncorhynchus mykiss rhamnose binding lectin CSL3 structure (accession code 2zx2A). The differences in the structure are highlighted in red and indicated by arrows

A comparative sequence analysis (Supplemental Fig.1) revealed the presence of two CRDs spanning from Thr27 to Cys118 (N-CRD) and from Val125 to Cys214 (C-CRD) connected by a six amino acid linker peptide. The Fig. 4A show a multiple alignment of CRD from different species, conservation of the canonical RBL sequence motif is shown by a grey tone gradient; the four cysteine bridges are shown by blue lines; the blue arrows indicate the EYGK residues involved in carbohydrate binding in mouse RBL and present in D. labrax RBL.

Fig. 4. Phylogenetic analysis of RBL-type lectins.

Fig. 4

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A: Alignment of RBL CRDs from various species: The multiple alignment of CRD conservation of the canonical RBL sequence motif is shown by a grey tone gradient; cysteine bridges are shown by blue lines; the blue arrows indicate the residues involved in carbohydrate binding in mouse RBL. B: Phylogenetic analysis of RBL sequences: The phylogram was created from neighbor-joining analysis using CLC workbench 6.4. Distances were corrected for multiple substitutions and gap positions were excluded. Bootstrap values are percentages from 1000 iterations. The scale bar measures substitutions per site. RBL abbreviated names: N or C letter indicated the N-terminal or C terminal CRD followed by the species name. Cargus = Channa argus (gi|189096288);L_sanguineus = Lutjanus sanguineus (gi|322423476); T_nigroviridis = Tetraodon nigroviridis (gi|47222797); O_mykiss = Oncorhynchus mykiss STL2 (gi|185134480); C_intestinalis = Ciona intestinalis (gi|198412427); N_vectensis = Nematostella vectensis (gi|156358465); S_purpuratus = Strongylocentrotus purpuratus SAL (gi|72110389).

3.4. Phylogenetic analysis, structure and CRD comparison

BLAST analysis revealed that the DlRBL deduced amino acid sequence presents close homologies with RBLs from invertebrates and fish depending on their CRD organization. In the phylogenetic tree, DlRBL clustered with the other binary teleost RBLs, and homologies with CRDs of vertebrate and invertebrate RBLs, although the teleost binary RBL cluster that includes DlRBL was clearly distinguishable from the invertebrate RBLs. The detailed phylogenetic analysis of the CRDs in RBLs showed highly conserved sequences in their N-CRDs or C-CRDs indicating a probably ancient CRDs duplication (Fig. 4B red and blue box). In contrast, the N- and C-CRD from the echinoderm Strongylocentrotus purpuratus and the urochordate Ciona intestinalis clustered together indicating a closer similarity between their binary C- and N-CRDs and a more recent origin of this duplication (Fig. 4B green box). The homology model of DlRBL based on the Oncorhynchus mykiss CSL3 RBL structure (40.31%, of identity E value 0.00e-1) showed substantial structural overlap (Figure 3B).

3.5. Biochemical and binding properties of DlRBL

The hemagglutinating activity of the purified DlRBL stored at −20 °C was stable for more than 2 months. The activity was reduced up to 70% by incubating the sample for 30 min at 60°C, almost abolished after incubation at 90°C (Figure 5A), and reduced in 50–60% when tested in a medium at pH lower or higher than 7.0–7.4 (Figure 5B). The hemagglutinating activity of DlRBL did not appear to require Ca2+ or Mg2+, since CaCl2 or MgCl2 (5mM) or EDTA or EGTA (10 mM, either in the presence or absence of CaCl2 or MgCl2) added to the eluted fractions (HA ranging from 64 to 512) did not significantly affect the activity. The DlRBL eluted fractions (0.3–0.4 mg/ml protein) agglutinated both erythrocytes and bacteria (Supplemental Fig. 2, Table 3). Rabbit erythrocytes were agglutinated at the highest titers, whereas sheep erythrocytes were not agglutinated at all. With regards the bacteria, the purified DlRBL strongly bound to E. coli (HT=128), and to lesser extent to A. hidrophyla, S. aureus and V. alginolyticus, (Table 3).

Fig. 5. Thermal stability and optimal pH range of DlRBL binding activity.

Fig. 5

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DlRBL was incubated at various temperatures for 30 min then cooled on ice for 10 min. The residual hemagglutination activity was tested at room temperature. The hemagglutination activity of an untreated sample, tested at room temperature, represented 100% activity sample was incubated with buffer ranging from pH 3–11. The titer value obtained at pH 8.0 represented 100% activity.

3.6 Effect of DlFBL on phagocytic activity of peritoneal macrophages

The phagocytic rate of peritoneal macrophages increased significantly after E. coli were opsonized with 10 μg/ml the isolated DlRBL (from 6.9 ± 2.9% to 34.5 ± 4. 2 p<0.001; 600 cells were counted in three distinct assays) while the phagocytic index increased 2.4-fold (from 1.7±0.8 to 4.1±1.6 p< 0.005) (Figure 6).

Fig. 6. Phagocytosis of DlRBL-exposed bacteria.

Fig. 6

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Opsonic effect of DlRBL on sea bass peritoneal cavity leukocyte phagocytosis against Escherichia coli. A,C; Peritoneal cavity phagocytes exposed to DlRBL-opsonized FITC stained bacteria. B,D Peritoneal cavity phagocytes exposed to PBS-treated FITC stained bacteria. A, B; under fluorescence microscope. C, D ; under light microscope. IB: ingested bacteria; FB: free bacteria. Bar 20 μm.

4. Discussion

Lectins play important roles in the immune responses of invertebrates and vertebrates either by recognizing exposed glycans on potential pathogens or by their immunoregulatory roles through the binding to carbohydrates on the surfaces of immunocompetent cells (Kuhlman, et al. 1989; Cooper, et al. 1994; Tino and Wright 1996; Matsushita et al. 1996; Vasta and Ahmed, 2008; Vasta, 2009). It has been proposed that the RBLs are involved in the regulation of carbohydrate metabolism, control of fertilization and cytotoxicity (Tateno et al 2002a; Watanabe et al 2009). In addition, an enhancing effect on cell proliferation has been reported for RBL of human dermal fibroblasts (Faury et al., 2008). Immune challenge in fish upregulates RBLs expression, leading to increased opsonization and phagocytosis of the microbes, and inducing the synthesis and release of pro-inflammatory cytokines (Lam and Ng 2002; Tateno et al. 2002a; Terada et al. 2007; Jia et al., 2009; Watanabe et al., 2009; Ogawa et al., 2011).

In a previous report we characterized a 34 kDa fucose-binding lectin from Dicentrarchus labrax serum (DlFBL) isolated through Sepharose 6BCL affinity chromatography (Cammarata et al 2001). In subsequent studies (Cammarata et al 2007; Salerno et al 2009; Cammarata et al 2012) we established that DlFBL and SauFBL, a lectin from the gilt head sea bream (Sparus aurata) of similar binding properties, were both members of the F-type lectin family,and are also present in eggs (Parisi et al 2010). During affinity chromatography of D. labrax serum on L-fucose-agarose a protein peak with hemagglutinating activity that was susceptible to inhibition by L-rhamnose and D-galactose but not by L-fucose was identified, suggesting that this was a serum lectin distinct from DlFBL. In the present study we optimized the purification procedure for this lectin, which we designated DlRBL, and characterized its molecular, structural, and biological properties.

For the purification of DlRBL we developed a two-step affinity chromatography procedure in which the pooled active fractions from the first peak of the L-fucose-agarose column were loaded onto a galactose-agarose affinity column, and eluted with 50 mM galactose. Based on the electrophoretic mobility in polyacrylamide gels, the purified DlRBL is constituted by 24 kDa subunits. Cloning of full length DlRBL transcript revealed that it consists of an open reading frame encoding 212 aminoacid residues including 18 residue signal sequence at the N-terminal. This was fully supported by a transcript (Accession number: NP_001117668, BAA92256; Sarropoulou et al 2009) identified in a D. labrax EST library. The deduced size of 24.1 kDa for the mature protein is in good agreement with subunit size of the isolated lectin (24 kDa).

Comparative sequence analysis revealed that DlRBL is a binary tandem domain RBL-type lectin with the N- and C-CRDs connected by a six-amino acid peptide linker.

The RBL CRD can be characterized by highly conserved 4 disulfide bridges at homologous positions as well as the conserved motif.

BLAST analysis of DlRBL disclosed sequence homologies with multiple RBLs from invertebrate and vertebrate species RBL CRDs. In the phylogenetic tree, DlRBL included in a cluster of teleost binary RBL-type lectins, clearly distinguishable from those invertebrate RBL with a single CRD. Both N-CRD and C-CRD from fish showed highly conserved sequences indicating a probably ancient origin of CRDs duplication. The N- and C-CRDs from Nematostella vectensis, Strongilocentrotus purpuratus and Ciona intestinalis clustered together, indicating a more recent CRD duplication.

Electrophoresis of DlRBL in polyacrylamide gels under non-reducing conditions indicated that the 24 kDa subunits are organized as a 100 kDa tetramer. The presence of two CRDs in each polypeptide subunit, and therefore, the physiological DlRBL tetramer displays 8 CRDs, suggesting that this multivalency is responsible for the observed agglutinating properties for erythrocytes and bacteria. A similar subunit size and quaternary organization has been described for other teleost RBLs such as the CSL1 isolated from chum salmon eggs, that is also organized as a homotetramer, and therefore displays 8 CRDs (Watanabe et al 2009). In contrast, the RBL subunits of CSL2 and CSL3, isolated from the same species, can also form homooctadecamers and homodimers, respectively, and therefore display 36 and 4 CRDs.

DlRBL actively agglutinates rabbit and human erythrocytes but does not require divalent cations for binding to the carbohydrate ligands, as all cations and chelators tested had no effect on the DlRBL hemagglutinating activity. Similar observations have been made in other fish RBLs, including the steelhead trout (Oncorhynchus mykiss) egg lectin, grass carp (Ctenopharyngodon idellus) roe lectin, rudd (Scardinius erythropthalmus) roe lectin, and olive rainbow smelt (Osmerus eperlanus mordax) roe lectin (Lam and Ng 2002; Hosono et al 1993).

In addition to the hemagglutinating activity, DlRBL bind both Gram-negative and Gram-positive bacteria. The ability to recognize and bind lipopolysaccharides and lipoteichoic acid and agglutinate both Gram-positive and Gram-negative bacteria has been described in trout RBLs, suggesting an antibacterial activity (Tateno et al., 2002a). We show clearly the presence of DlRBL in the serum. Other authors have demonstrated thet RBLs are mainly localized in the tissues related to the immune system, such as mucous cells of gills, goblet cells of intestine, spleen, thrombocytes, lymphocytes, monocytes and neutrophils (Watanabe et al 2008; Tateno et al 2002c).

In addition, RBLs have also been found in the cortex of teleost eggs as well as in the skin mucus, further confirming their protective role. A putative natural ligand of fish RBL is the glycosphingolipid globotriasylceramide (Gb3), abundant in membrane lipid rafts (Watanabe et al., 2009; Ogawa et al., 2011).

The exposure of formalin-killed E. coli to DlRBL enhanced their phagocytosis by peritoneal macrophages. The opsonic activity of DlRBL, together with its capacity of bacterial agglutination observed support the hypothesis that the plasma DlRBL mediates not only agglutination and immobilization of potentially pathogenic microorganisms, but also promotes their phagocytosis and clearance from circulation, thereby playing a key role in host defense against infectious challenge. Finally, our results confirmed a model for innate immunity in fish proposed by Watanabe et al (2009) in which RBLs recognize various kinds of pathogens in inflammatory site thought the blood circulation and enhance their phagocytosis by binding on the leukocyte surface.

Supplementary Material

Supp1

Supplemental Fig. 1 The nucleotidic and deduced protein sequence of DlRBL. The sequence of the N-terminal peptide is underlined with a single line. The in-frame stop codon is marked with an asterisk. The polyadenylation motif is double underlined. The two CRDs are highlighted in grey.

Supplemental Fig. 2 Bacterial agglutination by purified DlRBL. (A) Escherichia coli; (B) E. coli + DlRBL (10 μg/ml); (C) Vibrio alginolyticus; (D) V. alginolyticus + DlRBL (10 μg/ml) Bar 20 μm.

Supp2

Acknowledgments

We are grateful to Mario Guarcello for expert fish maintenance. This work was supported by grants from MIUR, and MC RITMARE project (CNR and CONISMA) and by grant 5R01GM070589-06 from the National Institutes of Health to GRV.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp1

Supplemental Fig. 1 The nucleotidic and deduced protein sequence of DlRBL. The sequence of the N-terminal peptide is underlined with a single line. The in-frame stop codon is marked with an asterisk. The polyadenylation motif is double underlined. The two CRDs are highlighted in grey.

Supplemental Fig. 2 Bacterial agglutination by purified DlRBL. (A) Escherichia coli; (B) E. coli + DlRBL (10 μg/ml); (C) Vibrio alginolyticus; (D) V. alginolyticus + DlRBL (10 μg/ml) Bar 20 μm.

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