Isolation and characterization of a novel fucose-binding lectin from the gill of bighead carp (Aristichthys nobilis)

Veterinary Immunology and Immunopathology 133 (2010) 154–164

Contents lists available at ScienceDirect

Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm

Research paper

Isolation and characterization of a novel fucose-binding lectin from the gill of bighead carp (Aristichthys nobilis) Saikun Pan a,b, Jian Tang c,*, Xiaohong Gu c a

School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China School of Marine Science and Technology, Huaihai Institute of Technology, 59 Changwu Road, Lianyungang 222005, China c State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 22 April 2009 Received in revised form 19 July 2009 Accepted 27 July 2009

Lectins play an important role in many biological systems and are used increasingly in human disease therapy. To improve our understanding of fish lectins we purified and characterized a fucose-binding lectin from the gill of bighead carp (Aristichthys nobilis). The purification procedure consisted of extracting soluble proteins in 25 mM Tris–HCl buffer (pH 8.5), separation on a DEAE-Sepharose FF ion exchange column, followed by gel filtration chromatography on Sephacryl S-200 HR and Superdex 200 10/300 GL columns. The purified lectin, designated GANL, had a single protein band with an apparent molecular mass of 37 kDa when subject to SDS-PAGE under reducing conditions. GANL is a homomultimeric glycoprotein with a native molecular mass of 220 kDa and a carbohydrate content of 13.4%. The purified lectin only agglutinated rabbit native erythrocytes, and did not require Ca2+. Its activity was not inhibited by any of the mono- or disaccharides or glycoproteins tested, except for fucose. GANL contains a high proportion of Asp, Glu, Leu, Val, and Lys. The first 10 residues of the N-terminal region were determined as AGEQGGQCSA. The anti-microbial activity was assessed by measuring agglutination and inhibition of pathogen growth. Our results suggest that GANL agglutinates and inhibits the growth of Vibrio harveyi but has no antifungal activity. ß 2009 Elsevier B.V. All rights reserved.

Keywords: Lectin Fish Bighead carp (Aristichthys nobilis) Gill Isolation Antibacterial

1. Introduction Lectins are a group of diverse molecules, broadly classified as calnexin, C-, L-, P-, I-, R-, or S-type lectins. C-type lectins are further divided into various sub-groups (Angata and Linden, 2002; Suzuki et al., 2003). Two major classes of lectins, C- and S-type, are found in animals (Drickamer and Taylor, 1993). Lectins have been found in many kinds of organisms, including bacteria and higher vertebrates. It is thought that lectins play an important

* Corresponding author. Tel.: +86 510 85329016; fax: +86 510 85919611. E-mail addresses: [email protected] (S. Pan), [email protected] (J. Tang). 0165-2427/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2009.07.015

physiological role as recognition molecules within a cell, between cells, or between organisms (Liener et al., 1986). As such, lectins are involved in the control of many biological systems. For example, the L-rhamnose-binding lectin in fish eggs is involved in carbohydrate metabolism, the prevention of polyspermy, the cross-linking of carbohydrate-rich proteins in the fertilization envelope, mitogenesis, lectin-mediated cellular cytotoxicity, opsonization of pathogens, and has bactericidal activity (Ng et al., 2003). Similarly, a mannan-binding lectin from the plasma of the Atlantic salmon (Salmo salar) and a lectin from serum of blue gourami (Trichogaster trichopterus) agglutinate bacterial strains and promote phagocytosis of the bacteria (Russell and Lumsden, 2005). Because lectins have the ability to discriminate between carbohydrate structures they are particularly

S. Pan et al. / Veterinary Immunology and Immunopathology 133 (2010) 154–164

useful as biochemical and clinical reagents. For example, lectin affinity chromatography is used to isolate either soluble glycoproteins or membrane glycoproteins. Fraguas et al. (2008) developed a wheat germ agglutinin (WGA)Sepharose column which distinguishes endogenous erythropoietin (a sialoglycoprotein, EPO) from recombinant EPO and EPO analogues with high efficiency. Durham and Regnier (2006) also developed a serial lectin affinity chromatography column which is specific for the Oglycosylation sites on proteins from the human blood proteome. More importantly, Con A- or WGA-Sepharose 4B are widely used commercial affinity chromatography matrices that purify glycoproteins, polysaccharides, and IgM. Lectins are also widely used for the isolation, identification, and characterization of different cells. This characteristic is particularly useful when studying tumor cells, erythrocytes, lymphocytes, T-cells, or B-cells (Ohba et al., 2002; Bakalova and Ohba, 2003). In recent years, lectinmediated drugs have been developed to target specific cells. Furthermore, lectins have been used to trigger vesicular transport into or across epithelial cells (review by Bies et al., 2004). Lectins are also used in food safety management and disease diagnosis (Haines and Patel, 1997; Pickup et al., 2005). Fucose-specific lectins are also used in blood typing (Wu et al., 2004). There has been considerable interest in the ability of carbohydrate recognition molecules (e.g., lectins) to mediate immune function. Their ability to bind to the terminal sugars on glycoproteins and glycolipids suggests that they are important pattern recognition receptors for innate immunity (Yu and Kanost, 2003). The role of lectins as mediators of non-self recognition during the innate immune response has been well-documented in vertebrates (Epstein et al., 1996). Given that fish have a relatively weak capacity for antibody-mediated immunity, the innate immune system is thought to be of greater importance than in more developed vertebrates. The innate immune system consists of a complex of constitutively expressed components that function as the first line of defense against invading pathogens, and subsequently interact with the adaptive immune system. One element of the innate immune system is a series of C-type lectins, termed collectins, which bind to carbohydrates on the surface of invading pathogens (Epstein et al., 1996). The collectins subsequently intact with other components of the immune system, such as the complement system and cell surface receptors on phagocytic cells. C-type lectins have been cloned in common carp (Cyprinus carpio) (Savan et al., 2004), rainbow trout (Oncorhynchus mykiss) (Zhang et al., 2000), and eel (Anguilla japonica) (Tasumi et al., 2002). Inagawa et al. (2001) also cloned and characterized a tandem repeat type galectin, a b-galactoside-binding lectin, in rainbow trout. A number of lectins have also been isolated and partially characterized in the eggs of several teleosts (Tateno et al., 1998; Bazˇil and Entlicher, 1999; Hosono et al., 1999, 2005; Jung et al., 2003; Mitsuru et al., 2007; Yasuharu et al., 2008). Lectins that bind to specific sugars, such as rhamnose (Hosono et al., 1999; Okamoto et al., 2005),

155

fucose (Vitved et al., 2000), and galactose (Tasumi et al., 2002) have also been isolated and analyzed in fish. In addition, AJL-2, a C-type lectin which displays Ca2+independent activity, was isolated from the Japanese eel (A. japonica) (Suzuki et al., 2003). Thus fish appear to be an ideal source for the study of novel lectins. Because water is an ideal medium for the transmission of bacteria and parasitic microbes, fish are constantly exposed to pathogens via the skin, gills, and alimentary canal. It is generally believed that defense mechanisms against pathogens exist in the gills, and that the gill serves as a mechanical, as well as biochemical, barrier. Indeed, there is evidence that lectins can be found in gill tissue (Mistry et al., 2001; Russell and Lumsden, 2005; Savan et al., 2004; Suzuki et al., 2003; Tsutsui et al., 2005). However, there has been little effort to purify and characterize lectins from the gills of teleosts. Our objective was to purify and characterize a lectin from the gills of the bighead carp. Such information needed for further studies into the biological activity of lectins in fish or in human disease therapy. 2. Materials and methods 2.1. Fish gill collection We obtained five healthy bighead carp, Aristichthys nobilis (mean weight 2000 g), from a fish market in Lianyungang, China. The fish were selected based on their lack of external damage and signs of disease. The fish were transported to the laboratory (within 40 min) in a plastic bag filled with oxygen. The fish were then acclimated for 2 weeks in an aerated fresh water tank at 20 8C under a natural photoperiod and fed daily. Following acclimation, the fish were sacrificed and the gill tissue was removed, washed in distilled water, and stored at 80 8C until use. 2.2. Erythrocyte specificity and hemagglutination assay (HA) Rabbit and mouse red blood cells were obtained by venous puncture using vacuum blood collection tube (BD, USA) from healthy animals purchased from the Center of Laboratory Animal Sciences at Nanjing Medical University. Chicken, cow, and sheep blood were obtained by the same manner from healthy animals reared at local farm. The erythrocytes from each of these animals were placed in Alsever solution (0.42% NaCl, 0.8% sodium citrate dihydrate, 0.045% citric acid monohydrate, 2.05% D-glucose; pH 7.2) and washed five times by centrifuging at 2500 rpm for 3 min in Tris-buffered saline (TBS: 25 mM Tris–HCl, containing 0.15 M NaCl, pH 7.5) before use. The packed cells were suspended in TBS to give a 2% (V/V) suspension of native erythrocytes (Ballarin et al., 1999). 25 mL of crude lectin extract, chromatographic fractions, or the purified lectin (1 mg/mL) were serially diluted twofold with TBS in the wells of a V-bottomed microtiter plate. An equal volume of the 2% erythrocyte suspension in TBS was added to each well. The plate was gently shaken and then incubated for 1 h at room temperature. The hemagglutination titer was defined as the reciprocal of the highest dilution giving positive hemagglutination (HU/mL).

156

S. Pan et al. / Veterinary Immunology and Immunopathology 133 (2010) 154–164

Specific activity was expressed as hemagglutination units per mg of soluble protein (HU/mg) (Fock et al., 2000; Leite et al., 2005). 2.3. Purification of lectin from the gill of bighead carp DEAE-Sephrose FF and Sephacryl S-200 HR columns were purchased from Pharmacia (Peapack, NJ, USA). The Superdex 200 10/300 GL column was purchased from Amersham Biosciences (Uppsala, Sweden). We pooled the gill tissue (213 g) from all five fish. The tissue was then homogenized in a fivefold volume (W/V) of 25 mM Tris–HCl buffer (TB, pH 8.5), and stored in a refrigerator at 4 8C overnight. The homogenate was subsequently filtered using a nylon filter (four layers of 100-mesh) and the crude extract was centrifuged at 10,000  g for 30 min at 4 8C. The supernatant was further filtered through a 0.45 mm minipore nylon membrane. We injected a 50 mL aliquot of the clear filtrate to a DEAE-Sepharose FF column (1 cm  17 cm). The column was equilibrated, then eluted with 25 mM Tris–HCl (pH 8.5) at a flow-rate of 1.5 mL/min using a BioLogic DuoFlow PathfinderTM 20/80 system (Bio-Rad Laboratories, Inc., USA). Unbound protein was eluted using 25 mM Tris–HCl (buffer A, pH 8.5) (volume equivalent to 3 columns). The adsorbed protein was then eluted using a linear gradient of 0–0.5 M NaCl in buffer A. We measured the UV absorbance at 280 nm and collected fractions (2 mL) of the eluate. The hemagglutinating activity fractions were pooled and lyophilized. The lyophilized active fractions (7.5 mg) were dissolved in 1.5 mL ultra-pure water (millipore Direct-Q3, USA). The mixture was further purified by gel filtration chromatography on a Sephacryl S-200 HR column (1 cm  100 cm, Bio-Rad) and eluted with ultra-pure water at a flow-rate of 0.6 mL/min. The active fractions were pooled and lyophilized. The lyophilized fraction (2 mg) was dissolved in 1 mL phosphate buffer (50 mM) containing 0.15 M NaCl (PBS, pH 7.0), applied to a Superdex 200 10/300 GL column, and eluted using the same buffer at a flow-rate of 0.5 mL/min. 2.4. Protein concentration assay We measured the soluble protein content using the Bradford assay (Bradford, 1976), modified for use with microplates and using bovine serum albumin (Sigma Chemical Co., St. Louis, MO, USA) as a standard. Briefly, we added 20 mL of a standard BSA solution series (0–0.50 mg/mL in 0.05 mg/mL increments) to the wells of a 96-well microplate. The appropriate samples were pipetted into empty wells based on the concentration of protein (maximum 10 mg/well). The total volume of sample solution was adjusted to 20 mL per well using dd H2O. We then added 280 mL Coomassie brilliant blue G-250 solution to each well while gently shaking to avoid bubbling. Following the addition of the dye the plate was shaken for 2 min. The OD was then measured at 595 nm using a Universal Microplate Spectrophotometer (SynergyTM HT, Bio-Tek Instruments, Inc., USA) within 25 min. The measurements were conducted in triplicate.

2.5. Carbohydrate content The total neutral sugar concentration in the lectin was measured using the phenol-sulfuric acid method following the description of Masuko et al. (2005), modified for use with a microplate and using D-glucose as the standard. Briefly, we added an increasing volume of the standard solution (0–50 mL in 5 mL increments; 3 mM concentration) to 11 wells of a 96-well microplate. Distilled water was added to each well to make up the final volume of 50 mL. The plate was then shaken and the appropriate samples were added to an empty well based on the concentration of sugar (maximum 100 nmol sugar/well). The total volume of the sample solution was adjusted to 50 mL per well using dd H2O. We then added 30 mL of 5% phenol in water to each well. The plate was then shaken and 150 mL of concentrated sulfuric acid was immediately added followed by additional shaking to mix the contents. The microplate was then placed carefully (floating) in a static water bath at 90 8C for 5 min, and subsequently cooled to room temperature for 5 min in another water bath and wiped dry. The OD490 nm was measured using the Universal Microplate Spectrophotometer. 2.6. Carbohydrate-binding specificity (HI) The majority of monosaccharides, oligosaccharides and their derivatives, and glycoproteins were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The carbohydratebinding specificity was measured using the hapten assay (Hori et al., 1986; Sato et al., 2000). We measured the ability of a series of sugars (L-arabinose, D-fructose, Lfucose, D-galactose, D-raffinose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, lactose, and melibiose) and glycoproteins (transferrin and albumin egg) to inhibit the hemagglutination activity of purified lectin against native erythrocytes, obtained from a rabbit. We diluted (twofold) 25 mL of the sugar (200 mM) or glycoprotein (5 mg/mL) solution in 0.15 M NaCl. An equal volume of the lectin solution containing 4 HU was added to each well and the mixture was allowed to incubate at room temperature for 1 h. We then added 50 mL of a 2% suspension of native erythrocytes (rabbit) to each well. This mixture was subsequently incubated at room temperature for 1 h. The lowest concentration of a specific sugar or glycoprotein that completely inhibited activity of the lectin solution (minimum inhibitory concentration, MIC) was recorded and used to define the inhibitory potency. 2.7. Effects of EDTA and divalent cations on lectin hemagglutinating activity To examine the effect of divalent cations on hemagglutination activity we used native erythrocytes collected from a rabbit. Aliquots (500 mL) of the purified lectin (1 mg/mL), dissolved in 25 mM Tris–HCl (pH 7.5), were dialyzed against 100 mL of 50 mM EDTA in 20 mM Tris– HCl (pH 7.5) at 4 8C overnight. The following day the aliquots were dialyzed against 100 mL of 150 mM NaCl, 20 mM CaCl2, MgCl2, or MnCl2 in the same buffer. The

S. Pan et al. / Veterinary Immunology and Immunopathology 133 (2010) 154–164

hemagglutination activity of the undialyzed fraction was then measured (Sato et al., 2000). 2.8. Heat stability of the lectin The heat stability of lectin was determined by incubating 500 mL aliquots of the lectin solution (2 mg/ mL) at 30, 40, 50, 60, 70, 80, and 90 8C for 30 min. The samples were then cooled at 4 8C for 30 min and assayed for hemagglutinating activity at room temperature. The agglutination value for the lectin obtained by assaying at room temperature was defined as 100% activity (Leite et al., 2005). 2.9. Effect of pH on hemagglutination The effect of pH on hemagglutination was determined according to the method of Suseelan et al. (2002) and Otta et al. (2002). Briefly, 10 mL of the lectin solution (1 mg/mL) was incubated with 90 mL of various buffer solutions in a microtiter plate for 1 h. Aliquots of the solution (50 mL) were then mixed with 50 mL of 0.25 M TBS (pH 7.5, containing 0.3 M NaCl) to adjust the pH. 25 mL aliquots of the mixture were then serially diluted (twofold) with 25 mM TBS (pH 7.5, containing 0.15 M NaCl). Last, 25 mL of native rabbit erythrocytes (2%) were added to each well and the plate was incubated at room temperature for 1 h. The agglutination value obtained by assaying the lectin with 25 mM TBS (pH 8.0) was considered to equal 100% activity. We used the following buffers: 50 mM acetate buffer (pH 4.0–5.0), 50 mM phosphate buffer (pH 6.0–7.0), 50 mM Tris–HCl buffer (pH 8.0–9.0), and 50 mM sodium bicarbonate buffer (pH 10.0–11.0) (Sato et al., 2000). 2.10. Homogeneity and molecular mass determination The electrophoresis reagents were purchased from Merck (San Diego, CA, USA). The purity and apparent molecular mass of the isolated proteins was determined by SDS-PAGE using a Mini-Protean III apparatus (Bio-Rad, Hercules, CA, USA). For visualization of protein bands, the gels were stained with Coomassie brilliant blue R-250. The gel was then analyzed with a ChemiDoc XRS system (BioRad, USA). Estimation of the molecular mass under denaturing conditions was carried out by discontinuous electrophoresis using a vertical system. The stacking gel consisted of 5% polyacrylamide in 0.5 M Tris–HCl (pH 6.8) with 0.4% (W/V) sodium dodecyl sulfate (SDS). We used a 12.5% polyacrylamide slab in 25 mM Tris–HCl, 0.2 M glycine (pH 8.8) with 0.1% (W/V) SDS. The samples and markers were dissolved in Tris–HCl buffer (pH 6.80) containing 1% (W/V) SDS and 5% (V/V) 2-mercaptoethanol. We used beta-galactosidase (E. coli) (116.0 kDa), BSA (66.2 kDa), ovalbumin (45.0 kDa), lactate dehydrogenase (35.0 kDa), restriction endozyme (25.0 kDa), beta-lactoglobulin (18.4 kDa) as protein markers. The native molecular weight of the purified lectin was determined by gel filtration on a Superdex 200 10/300 GL column. The column was equilibrated and eluted using 50 mM PBS (containing 0.15 M NaCl, pH 7.0) at a flow-rate of 0.5 mL/min. We used transferrin (80 kDa), BSA (67 kDa),

157

egg albumin (43 kDa), and RNase (13.7 kDa) as the standard proteins. The void volume (Vo) was estimated with Dextran Blue 2000 (Ambrosio et al., 2003; Leite et al., 2005). 2.11. Amino acid composition The lectin was hydrolyzed with 6 M HCl for 24 h at 110 8C. The hydrolyzate residue was then derivatized with O-phthalaldehyde (OPA) and 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl). The amino acid derivatives were analyzed by HPLC (Agilent 1100, Santa Clara, CA, USA) using a Hypersil ODS C18 (4.6 mm  150 mm) column according to the manufacturers instructions (Sato et al., 2000). 2.12. N-terminal determination The N-terminal aa sequence analysis was determined by automated Edman degradation in an Applied Biosystems (Model 491A, Foster City, CA, USA) protein sequencer equipped with a 140C microgradient system and a 785A programmable absorbance detector. 2.13. Microbial agglutination assay We measured the effect of GANL on microbial agglutination following the methods of Pang et al. (2006). We used the following target pathogens: Grampositive bacteria (Micrococcus lysodeikticus, Bacillus subtilis, and Staphylococcus aureus), Gram-negative bacteria (Vibrio harveyi and E. coli), and fungi (Saccharan cerevisiae, Candida albicans, Candida krusei, and Aspergillus niger). The microbes were cultured in nutrient broth or malt extract broth (MEB) at 37 8C (bacteria) or 30 8C (fungi) for 18–24 h then harvested. Following harvest, the microbes were washed, re-suspended in 10 mM PBS (pH 7.5), and inactivated by heating at 56 8C for 1 h. The microbial suspensions were adjusted to approximately 109 cells/mL. We prepared serial twofold dilutions of 25 mL of the GANL stock solution (1 mg/mL in 10 mM PBS, pH 7.5) in Vbottom 96-well microtiter plates using 10 mM PBS (pH 7.5). An equal volume of microbial suspensions was added to each well and the contents were completely mixed by gentle shaking for 30 s. The plates were incubated overnight at 4 8C. We evaluated agglutination by eye or under a low power microscope. The microbial agglutination titer was defined as the reciprocal of the highest dilution exhibiting visible agglutination. The negative controls consisted of equal volumes of microbial suspension and PBS (no lectin). 2.14. Microbial growth inhibition assay We evaluated bacterial growth inhibition by recording the concentration of agglutinins that reduced bacterial growth in comparison to a bacterial control, following the method described by Gaidamashvili and Staden (2002). Bacterial suspensions were cultured overnight, then diluted 1:100 with nutrient broth, and 100 mL of the diluted bacterial suspension mixed with 100 mL of

158

S. Pan et al. / Veterinary Immunology and Immunopathology 133 (2010) 154–164

different concentrations of the agglutinin solution (62.5– 2000 mg/mL). The mixture was then incubated in a water bath at 37 8C with constant shaking. We measured the absorbance at 600 nm every hour for a period of time (5 h). We measured antifungal activity in A. niger grown for 7 days at room temperature in Petri dishes on Sabouraud dextrose agar. We prepared a fresh suspension of conidia by rinsing the surface of 7-day-old sporulated cultures with sterile water. We used Saccharan cerrvisiae, Candida krusei, and C. albicans in the yeast assay. These were grown at 30 8C for 48 h in the medium described above. The yeast cells were obtained from the 48 h culture and suspended in 0.15 M sterilized NaCl. We mixed 100 mL of the conidia suspension (2  104 cells/mL) or the yeast cells (2  104 cells/mL) with 100 mL of GANL solution (31.25–1000 mg/mL), prepared in 1% (m/v) peptone dextrose broth (pH 8.0, Sigma, St. Louis, MO) and sterilized using a 0.22 mm membrane (Millipore, USA). The mixture was then incubated in a 96-well flat culture plate (Costar, USA). Control colonies were cultivated in 1% (m/v) peptone dextrose broth in the absence of GANL. We monitored fungal growth by measuring the turbidity at 630 nm, using the Universal Microplate Spectrophotometer, between 0 and 72 h or 0 and 96 h (for A. niger). To inhibit the ability of GANL to constrain microbial growth, the assay was repeated in the presence of 0.2 M Lfucose, a sugar that fully inhibits the hemagglutinating activity of GANL. The experiments were repeated 3–4 times. We compared microbial growth rates in the presence of GANL with the control and the GANL/L-fucose cultures, following the method of Melo et al. (2005).

Table 1 Agglutination of various animal erythrocytes by a lectin from the gill of Aristichthys nobilis. Animal erythrocytesa

Hemagglutination activityb Crude extracts of gill in TB

Mouse Chicken Monopterus albus Rabbit Cow Sheep

Purified GANL

– – –

– – –

210 – –

212 – –

a Erythrocytes were collected from six different species in Alsever solution and washed five times by centrifuging at 2500 rpm for 3 min in Tris-buffer saline (pH 7.5). The packed cells were suspended in TBS to give a 2% (V/V) suspension of native erythrocytes and used to test hemagglutination activity. b Hemagglutination activity was expressed as a titer, the reciprocal of the highest twofold dilution exhibiting positive hemagglutination (HA). Dashes indicate no activity. The agglutinating assays were performed in 96-well V-bottomed microtiter plates. Samples (25 mL) were serially diluted twofold in TBS, and an equal volume of animal erythrocytes suspension was added to each well. The plates were shaken gently and then incubated at room temperature for 1 h. The data presented are from one representative experiment that was repeated three times.

3. Results First, we evaluated the specificity of the fish gill lectin by screening against the erythrocytes from six different species. Both the crude extract and the purified lectin obtained from the gill of A. nobilis displayed a high hemagglutinating titer to native erythrocytes from the rabbit. However, we did not observe agglutination against erythrocytes from the remaining animals (Table 1). Thus we used the native rabbit erythrocytes to measure hemagglutination activity during purification and characterization of the lectin. Second, we developed a procedure for purification of the lectin from A. nobilis gill tissue. In general, isolation protocols for lectins are largely dependent on the properties and source of the lectin. After several preliminary purification trials, we developed a procedure using ion exchange chromatography and gel filtration chromatography to isolate the soluble lectin from gill tissue. We extracted the lectin using 25 mM Tris–HCl buffer (pH 8.5). The crude extract was separated into five peaks on a DEAESepharose FF column. The greatest hemagglutination activity was measured in the unabsorbed fraction (peak 1, shown in Fig. 1). The active fractions obtained from the DEAE column (peak 1) were separated into five peaks (Fig. 2) when applied to the Sephacryl S-200 HR column. The active fraction was represented by peak 1. The active

Fig. 1. Ion exchange chromatography of the crude extract from gill of Aristichthys nobilis on a DEAE-Sepharose FF column. The column was equilibrated with 25 mM Tris–HCl buffer (TB, pH 8.5) at a flow rate of 1.5 mL/min. The column was first eluted with the equilibrate buffer using three column volumes, then with a linear gradient (0–0.5 M) of NaCl in TB for 15 column volumes. The eluate was monitored by absorption at 280 nm and tested for hemagglutination activity. The active fractions were pooled.

fraction obtained from the Sephacryl 200 HR column was separated into four peaks when applied to FPLC on superdex 200 column. Of these, peak 2 was the active fraction, and was designated as GANL (Fig. 3). The recovery of active protein was 3.6%. The results of the purification are summarized in Table 2. Third, we investigated the physical and biochemical properties of the lectin. The molecular mass of GANL was estimated to be 37 kDa using SDS-PAGE (Fig. 4) or 220 kDa using gel filtration on the Superdex 200 column (Fig. 5). GANL displayed a ladder-like pattern following nonreducing SDS-PAGE. In contrast, we observed a single band following the reducing SDS-PAGE. Based on these

S. Pan et al. / Veterinary Immunology and Immunopathology 133 (2010) 154–164

159

Fig. 2. Gel filtration chromatography of the active fractions obtained by DEAE ion exchange chromatography on a Sephacryl S-200 HR column. The column was eluted with ultra-pure water at a flow rate of 0.6 mL/min. The eluate was measured for absorption at 280 nm and tested for hemagglutination activity. The active fractions, denoted by the bar, were pooled.

Fig. 4. SDS-PAGE of the purified lectin (GANL). Electrophoresis was carried out on a 12.5% polyacrylamide gel in the presence or absence of 5% 2mercaptoethanol. The gel was stained with Coomassie brilliant blue R250. (M) Standard proteins; beta-galactosidase (E. coli) (116.0 kDa), BSA (66.2 kDa), ovalbumin (45.0 kDa), lactate dehydrogenase (35.0 kDa), restriction endozyme (25.0 kDa), and beta-lactoglobulin (18.4 kDa). (+ME) GANL with 2-mercaptoethanol. ( ME) GANL without 2mercaptoethanol.

that Asp, Glu, Leu, Val, and Lys were present in higher concentrations than other amino acids (Table 3). Cys-S was detected in the lectin molecule. The first 10 N-terminal amino acid sequence of GANL was determined as AGEQGGQCSA. The hemagglutination activity of GANL was not inhibited by any of the monosaccharides, disaccharides, and glycoproteins we tested, with the exception of fucose (Table 4). The hemagglutination activity of GANL incubated at 50 8C for 30 min was fourfold stronger than that at room temperature (Fig. 6). Our results suggest that the optimal temperature for GANL is close to 50 8C. We also found that 50% of the original activity was maintained even

Fig. 3. Gel filtration chromatography of the active fractions obtained by Sephacryl S-200 HR on a Superdex 200 10/300 GL column. The column was eluted with 50 mM PBS (containing 0.15 M NaCL, pH 7.0) at a flow rate of 0.5 mL/min. The eluate was measured for absorption at 280 nm and tested for hemagglutination activity. The active fractions, denoted by the bar, were pooled.

observations we conclude that GANL is a multimeric protein with six homogeneous subunits. Carbohydrate analysis using the phenol-sulfuric acid assay revealed that GANL is a glycoprotein with a carbohydrate content of 13.4%. Amino acid composition analysis of GANL revealed Table 2 Purificationa of a lectin from the gill of Aristichthys nobilis. Fraction

Volume (mL)

Total protein (mg)

HAb

Total HAc

Specific activityd

Purification fold

Protein recovery (%)

Crude extract DEAE–IEC Gel filtration on Sephacryl S-200 Gel filtration on Superdex 200

50 50  0.45 38  0.44 9.5  0.26

106 22.5  0.65 9.8  0.08 3.8  0.34

1024 1024 1024 4096

51200 51220  460 38952  450 38953  1060

483 2286  49.2 3978  68.5 10320  878

1 4.7  0.10 8.2  0.14 21.4  1.8

100 21.2  0.61 9.2  0.08 3.6  0.32

a The purification stages are described in Section 2. We used 50 mL aliquots of crude extract at the beginning of each purification trial. Data in the table represent the mean  S.D. of five typical purification trials. b Hemagglutination activity was expressed as a titer, the reciprocal of the highest twofold dilution exhibiting positive hemagglutination (HA). The activity was determined with rabbit native erythrocytes. c Total hemagglutination titer (HA  volume). d Hemagglutination units per mg of protein (total HA/total protein).

S. Pan et al. / Veterinary Immunology and Immunopathology 133 (2010) 154–164

160

Fig. 5. Molecular mass determination of GANL by gel filtration chromatography on a Superdex 200 10/300 GL column. The closed circles correspond to the four marker proteins: transferrin (80 kDa), BSA (67 kDa), albumin egg (45 kDa), RNase (13.7 kDa). The square corresponds to GANL (220 kDa).

after heating to 90 8C for 30 min, suggesting that this lectin protein is extremely thermostable. GANL is also stable under alkaline conditions. We found that 25% of the original activity was maintained following incubation at pH 11 for 1 h. In contrast, GANL is unstable under acidic conditions. When incubated at pH 6 for 1 h, activity declined to 12.5% of the original level. Furthermore, activity was completely abolished after incubating at pH 4 for 1 h (Fig. 7). Dialysis against EDTA and the addition of divalent cations, such as Ca2+, Mg2+, and Mn2+ did not affect the activity of GANL. These results suggest that divalent cations are not required for the hemagglutination caused by GANL. Last, we investigated the biological activity of the lectin using microbial agglutination and growth assays. The binding specificity of GANL varied significantly among the microbial targets. There was no binding activity towards fungi or the Gram-positive bacteria. In contrast, GANL bound specifically with V. harveyi. The median agglutinating titer was 16 and the mean minimum agglutinating concentration was 79.9  11.78 mg/mL (Table 5). The control solutions for both types of bacteria grew exponenTable 3 Amino acid composition of the lectin from the gill of Aristichthys nobilis. Amino acid

Mol (%)

Amino acid

Mol (%)

Asp Glu Ser His Gly Thr Arg Ala Leu

13.23 14.94 5.19 2.31 4.18 4.55 6.47 4.78 9.60

Tyr Cys-S Val Met Phe Ile Lys Pro

3.51 0.81 6.17 2.45 4.50 5.61 7.06 4.55

tially. GANL also had no effect on the growth of the fungi or Gram-positive bacteria. In contrast, the growth of V. harveyi was inhibited by incubation with GANL (Table 6 and Fig. 8). The minimal inhibitory concentration was 125 mg/mL. Among the two Gram-negative bacteria we tested, only the growth of V. harveyi was inhibited by GANL. The inhibitory effect was only observed at relatively high concentrations of GANL in comparison with those eliciting a positive aggregation response (Table 5). The growth of V. harveyi was completely restored by the addition of 0.2 M L-fucose (Fig. 8).

Table 4 Inhibition of hemagglutination by carbohydrates and glycoproteinsa. Carbohydrate and glycoprotein

Minimum inhibitory concentration (mmol/L or mg/mL)

Monosaccharides (mmol/L) D-Glucose

L-Fucose Fructose N-acetyl-D-glucosamine N-acetyl-D-galactosamine

– – – – 0.39 – – –

Disaccharides (mmol/L) Lactose melibiose

– –

Glycoproteins Transferrin Albumin egg

– –

D-Galactose D-Mannose D-Xylose

a We used rabbit native erythrocytes. Inhibitory activity is expressed as the minimum inhibitory concentration that is required to completely inhibit the hemagglutinating activity of a titer. Dashes indicate no inhibitory activity at a concentration of 100 mM for monosaccharides or disaccharides, and 2 mg/mL for the glycoproteins.

S. Pan et al. / Veterinary Immunology and Immunopathology 133 (2010) 154–164

161

Table 5 Microbial agglutinating activity of GANL. microbial

Micrococcus lysodeikticus Bacillus subtilis Staphylococcus aureus Vibrio harveyi E. coli Saccharan cerevisiae Candida albicans C. krusei Aspergillus niger

MAT of GANL mMAT

seMAT

N N N 16 N N N N N

– – – 15.1  2.47 – – – – –

seMAC (mg/mL)

– – – 79.9  11.78 – – – – –

MAT, maximum agglutinating titer; mMAT, median MAT; seMAT, mean  standard deviation (S.D.) of MAT; MAC, minimum agglutinating concentration (mg/mL); seMAC, mean  S.D. of MAC; N, no agglutinating activity. Fig. 6. Thermostability of GANL. 500 mL GANL was incubated at various temperatures for 30 min then cooled at 4 8C for 20 min. The residual hemagglutination activity was tested at room temperature. The hemagglutination activity of an untreated sample, tested at room temperature, represented 100% activity.

Fig. 7. pH stability of GANL. 10 mL sample was incubated with 90 mL buffer (pH 4–11). The titer value obtained at pH 8.0 represented 100% activity.

4. Discussion Recently, a number of lectins have been purified from the serum, mucus, and eggs of several species of fish, including grass carp (Ctenopharyngodon idellus), common carp (C. carpio) (Savan et al., 2004), blue gouramin (Trichogaster trichhopterus) (Fock et al., 2000), perch (Percafluviatilis L.) (Bazˇil and Entlicher, 1999), Atlantic salmon (S. salar) (Stratton et al., 2004), Rainbow Trout (Jensen et al., 1997), catfish (Silurus asotus) (Hosono et al., 1999), pony fish (Leiognathus nuchalis) (Okamoto et al., 2005), conger eel (Conger myriaster) (Muramoto and Kamiya, 1992), shishamo smelt (Osmerus [Spirinchus] lanceolatus) (Hosono et al., 2005), and torafugu (Takifugu rubripes) (Tsutsui et al., 2006). In addition, studies have also shown that gill tissue contains a number of novel

lectins (Mistry et al., 2001; Russell and Lumsden, 2005; Savan et al., 2004; Suzuki et al., 2003; Tsutsui et al., 2005). We successfully purified and characterized a lectin from the gill of bighead carp, an important traditional commercial freshwater fish in China, using conventional chromatographic methods. The lectin was not absorbed by weak ion exchange support, including DEAE-Sepharose and CM-Sepharose on conventional chromatography. We speculate that the relatively high carbohydrate content and complex structure may contribute to this lack of absorbtion on the ion exchange column. GANL was strongly inhibited by L-fucose. The comparison between the N-terminal amino acid sequences of GANL and other fish lectins revealed that the N-terminal sequence of GANL has low identity with lectins from other fish species, including Coho salmon (Oncorhynchus kisutch), catfish (S. asotus), rohu (Labeo rohita) and skipjack tuna (Katsuwonus pelamis). Furthermore, GANL does not share any homology with grass carp (C. idellus) lectin (Table 7). Thus we concluded that GANL is a novel lectin belonging to the L-fucose-specific lectin family. In recent decades, various fucose-specific lectins have been derived from a number of sources. The specificity of the a-linked L-fucose for the blood group H determinant is dependant on the source. Because of this property, these lectins are used as reagents in blood typing and histochemistry. There is also evidence that A. anguilla agglutinin (AAA), a fucosespecific lectin present in the serum of the fresh water eel, participates in the recognition of bacterial lipopolysaccharides by the innate immune system. This suggests that fucose-specific lectins may play a role in host defense (Wu et al., 2004). Mammalian gastrointestinal tract mucin is a glycoprotein containing 77.5% carbohydrate, comprised of N-acetyl-galactosamine, N-acetyl-glucosamine, galactose, fucose, and sialic acid at a molar ratio of 1.0:0.6:0.7:0.3:0.5 (dry weight) (Gabor et al., 2004). Fucose-binding lectins may be a candidate for intestinal lectin-mediated drug delivery. Thus the characterization of GANL provides a basis for the development of clinical applications of fish lectins. The hemagglutinating activity of GANL was not dependent on the presence of divalent cations. This property is similar to the AJL-2 and S-type lectins (Arason,

S. Pan et al. / Veterinary Immunology and Immunopathology 133 (2010) 154–164

162

Table 6 Inhibition of Vibrio harveyi growth over time by GANL. Time (min)

0 60 120 180 240 300

OD600 nm of difference concentration of GANL (mg/mL) incubated with V. harveyi Control

31.25

62.50

125

250

500

1000

0.42  0.056 0.54  0.069 1.05  0.075 1.38  0.08 1.52  0.038 1.51  0.056

0.42  0.055 0.532  0.069 1.04  0.038 1.36  0.062 1.501  0.049 1.512  0.059

0.43  0.048 0.521  0.071 0.97  0.069 1.34  0.059 1.48  0.081 1.49  0.049

0.43  0.06 0.51  0.069 0.73  0.072# 0.83  0.053# 0.92  0.064# 0.89  0.053#

0.44  0.061 0.59  0.069 0.75  0.065# 0.73  0.059# 0.85  0.054# 0.88  0.055#

0.45  0.048 0.49  0.071 0.58  0.069# 0.65  0.059# 0.75  0.081# 0.76  0.049#

0.43  0.055 0.50  0.069 0.54  0.038# 0.62  0.062# 0.73  0.049# 0.72  0.059#

Data is presented as the mean  S.D. # Significant difference from the control (P < 0.01).

1996). AJL-2 was isolated from the Japanese eel (A. japonica) (Suzuki et al., 2003), which has a highly conserved sequence of C-type lectins that are Ca2+independent. Given that fish lectins operate in an environment that has little Ca2+ such independence makes sense from an evolutionary point of view. The thermostability of various lectins appears to differ widely. Some are relatively stable while others are much less so. GANL retained half of its activity even after incubation at 90 8C for 30 min, which is unusual among the known teleost lectins. For example, two lectins, STL1 and STL2, isolated from steelhead trout (O. mykiss) (Tateno et al., 1998) are considerably less stable. The hemagglutinating activity of STL1 is completely inhibited following heating to 50 8C for 90 min. Similarly, half of the activity of STL2 is abolished under these conditions and activity is completely inhibited at 70 8C for 10 min. In addition, catfish (S. asotus) lectin activity was completely inhibited after incubation at 60 8C for 30 min and 100 8C for 5 min. A rhamnose-specific lectin, isolated from the grass carp eggs, lost its activity completely after incubation at 80 8C for 5 min (Lam and Ng, 2002). Based on the SDS-PAGE and gel filtration results we conclude that GANL is a homomeric protein with six identical subunits. A number of teleost lectins have been identified that possess a complex structure. For example, the C-type lectin, isolated from the serum and plasma of rainbow trout, is a multimeric molecule. Non-reducing

SDS-PAGE of this lectin revealed a characteristic ladder pattern (16 kDa between consecutive bands) (Jensen et al., 1997). Another C-type lectin isolated from the blood of Atlantic salmon was an oligomer with a monomeric size of 17.1 kDa (Stratton et al., 2004). Multimeric C-type lectins are thought to preferentially recognize the dense carbohydrate patterns on microbial surfaces because their conformation matches that of C-type lectin-like domains (CTLDs) in the multimer. Thus lectins with a multimeric structure are likely to play an important role in innate immunity. Because of their carbohydrate-binding specificity, lectins play an important role in pathogen recognition and clearance as part of the innate immune response. GANL specifically agglutinated V. harveyi, a fish pathogen. Conversely, we observed no agglutination activity towards

Table 7 Comparison between the N-terminal amino acid sequences of GANL and other fish lectins. Lectin

Amino acid sequence

GANL Grass carp Coho salmon SAL OLL OML1 OML2–4 STL2 (1–99) STL2 (100–195) SUEL

AGEQGGQCSA ETVVTKEGFVQRLSDDSQVIRVQQATFGRRNNNIY (35) AISITCGESDALLQCDGGKIHIKRANYGRCQHDV (34) ANMITCYGDVQKLHQETGLIIVKSXLYGR (29) VTTDIXEGQQATLNXGSSVINVVSANYGRTDRVT (34) VMTVIXEGQQETLNXGSSVVNVVSANYGRTNHVT (34) VTTDIXEGQQATLNXGSSVINVVSANYGRTDRVT (34) TRVVTCDNGENVQFLICDSGVIFIERALYGRTDGTTC —TRSITCEGSDAQLECDEGTIQIYSANYGRRDQLVC

Rohu OLABL-H OLABL-L PFL-1 PFL-2 KPL

Fig. 8. Growth curves of Vibrio harveyi in the presence of GANL (125 mg/ mL) and 0.2 M L-fucose.

ELVSEFCLKKERVCEDSSLTISCPEGEGIVIYDAIY GRKRGEVC (44) WLNGIGTQIPNKITT (15) SYPSCPSRQWTKNGQRCYL (19) SYPSCPSDWIKSGERCYLSVSQP (23) HSXEIRLEDLAXVRETAXEGXVXXLEXG (28) HSSEIRLEDLSSVRETAAXG (20) PVELCDAKCT (10)

Grass carp: lectin isolated from grass carp (Ctenopharyngodon idellus) ovaries; Coho salmon: lectin isolated from coho salmon (Oncorhynchus kisutch) eggs; SAL: catfish (Silurus asotus) egg lectin; OLL: shishamo smelt (Osmerus lanceolatus) roe lectin; OML1–4: olive rainbow smelt (Osmerus eperlanus mordax) roe lectins; STL 2: steelhead trout (Oncorhynchus mykiss) egg lectins; SUEL: sea urchin (A. crassispina) egg lectin; Rohu: rohu (Labeo rohita) plasma lectin; OLABL-H,-L: asialofetuin-binding lectin from shishamo smelt (O. lanceolatus) H-subunits and L-subunits; PFLs: lactosebinding lectin from the skin mucus of ponyfish (Leiognathus nuchalis); KPL: skipjack tuna (Katsuwonus pelamis) hard roe lectin. The number in brackets indicates the number of amino residues of each N-terminal sequence. Residues that are identical to the corresponding residues in GANL are indicated in bold face.

S. Pan et al. / Veterinary Immunology and Immunopathology 133 (2010) 154–164

the fungi and Gram-positive bacteria we tested. The ability of GANL to inhibit bacterial growth and agglutinate specific pathogens suggests that GANL may play an important role in immobilizing invading bacteria. However, GANL did not agglutinate all the Gram-negative bacteria. This suggests that the agglutinating activity of GANL is not dependant on recognition of the bacterial cell wall LPS. The growth of V. harveyi was fully restored by L-fucose (Fig. 8). Therefore, we hypothesize that the L-fucose carbohydrate-binding site of the lectin is involved in mediating the inhibition of bacterial growth. Unlike AAA, a fucose-binding lectin mentioned above, GANL exhibited a high selective aggregating activity to certain bacteria. Our study revealed that this selective aggregating active involved in L-fucosebinding site of the lectin. The agglutinating properties of GANL supports the concept that every novel lectin has its own binding characteristics (Wu et al., 2004). Although Wu et al. (2004) made great effort to elucidate the binding profile of AAA, the exact mechanism of fucose-binding lectin agglutinate bacteria is not yet known. In this respect, extensive agglutinating activity of GANL to various bacteria species needed further test to support our observations and the exact mechanism of the interaction of GANL with bacteria needed to further investigate. Furthermore, the selective aggregating capacities of GANL could also lead to new uses for diagnostic purpose.

Acknowledgments Technical support was provided by staff at the Jingsu Key Laboratory of Marine Biotechnology. This study was supported financially by the Jiangsu key subject-Marine Biology.

References Ambrosio, A.L., Sanz, L., Sa´nchez, E.I., Wolfenstein-Todel, C., Calvete, J.J., 2003. Isolation of two novel mannan- and L-fucose-binding lectins from the green alga Enteromorpha prolifera: biochemical characterization of EPL-2. Arch. Biochem. Biophys. 415, 245–250. Angata, T., Linden, E.C.M.B.-V.d., 2002. I-type lectins. Biochim. Biophys. Acta 1572, 294–316. Arason, G.J., 1996. Lectins as defence molecules in vertebrates and invertebrates. Fish Shellfish Immun. 6, 277–289. Bakalova, R., Ohba, H., 2003. Purifiation of normal lymphocytes from leukemic T-cells by lectin-affinity adsorbents—correlation with lectin-cell binding. Cancer Lett. 192, 59–65. Ballarin, L., Tonello, C., Guidolin, L., Sabbadin, A., 1999. Purification and characterization of a humoral opsonin, with specificity for D-galactose, in the colonial ascidian Botryllus schlosseri. Comp. Biochem. Phys. B 123, 115–123. Bazˇil, V., Entlicher, G., 1999. Complexity of lectins from the hard roe of perch (Percafluviatilis L.). Int. J. Biochem. Cell Biol. 31, 431–442. Bies, C., Lehr, C.-M., Woodley, J.F., 2004. Lectin-mediated drug targeting: history and applications. Adv. Drug Deliver. Rev. 56, 425–435. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Drickamer, K., Taylor, M.E., 1993. Biology of animal lectins. Annu. Rev. Cell Biol. 9, 237–264. Durham, M., Regnier, F.E., 2006. Targeted glycoproteomics: serial lectin affinity chromatography in the selection of O-glycosylation sites on proteins from the human blood proteome. J. Chromatogr. A 1132, 165–173. Epstein, J., Eichbaum, Q., Sheriff, S., Ezekowitz, R.A.B., 1996. The collectins in innate immunity. Curr. Opin. Immunol. 8, 29–35.

163

Fock, W.L., Chen, C.L., Lam, T.J., Sin, Y.M., 2000. Isolation and characterisation of a serum lectin from blue gourami, Trichogaster trichopterus (Pallus). Fish Shellfish Immun. 10, 489–504. Fraguas, L.F., Carlsson, J., Lo¨nnberg, M., 2008. Lectin affinity chromatography as a tool to differentiate endogenous and recombinant erythropoietins. J. Chromatogr. A 1212, 82–88. Gabor, F., Bogner, E., Weissenboeck, A., Wirth, M., 2004. The lectin–cell interaction and its implications to intestinal lectin-mediated drug delivery. Adv. Drug Deliver. Rev. 56, 459–480. Gaidamashvili, M., Staden, J.V., 2002. Interaction of lectin-like proteins of South African medicinal plants with Staphylococcus aureus and Bacillus subtilis. J. Ethnopharmacol. 80, 131–135. Haines, J., Patel, P.D., 1997. Antibody- and lectin-based assays for the rapid analysis of food-grade gums and thickeners. Trends Food Sci. Technol. 8, 395–400. Hori, K., Miyazawa, K., Ito, K., 1986. Preliminary characterization of agglutinins from seven marine algal species. Bull. Jpn. Soc. Sci. Fish 52, 323–331. Hosono, M., Ishikawa, K., Mineki, R., Murayama, K., Numata, C., Ogawa, Y., Takayanagi, Y., Nitta, K., 1999. Tandem repeat structure of rhamnosebinding lectin from catfish (Silurus asotus) eggs. Biochim. Biophys. Acta 1472, 668–675. Hosono, M., Sugawara, S., Ogawa, Y., Kohno, T., Takayanagi, M., Nitta, K., 2005. Purification, characterization, cDNA cloning, and expression of asialofetuin-binding C-type lectin from eggs of shishamo smelt (Osmerus [Spirinchus] lanceolatus). Biochim. Biophys. Acta 1725, 160–173. Inagawa, H., Kuroda, A., Nishizawa, T., Honda, T., Ototake, M., Yokomizo, U., Nakanishi, T., Soma, G., 2001. Cloning and characterisation of tandem-repeat type galectin in rainbow trout (Oncorhynchus mykiss). Fish Shellfish Immun. 11, 217–281. Jensen, L.E., Thiel, S., Petersen, T.E., Jensenius, J.C., 1997. A rainbow trout lectin with multimeric structure. Comp. Biochem. Physiol. 116B, 385–390. Jung, W.K., Park, P.J., Kim, S.K., 2003. Purification and characterization of a new lectin from the hard roe of skipjack tuna, Katasuwonus pelamis. Int. J. Biochem. Cell B 35, 255–256. Lam, Y.W., Ng, T.B., 2002. Purification and characterization of a rhamnosebinding lectin with immunoenhancing activity from grass carp (Ctenopharyngodon idellus) ovaries. Protein Expr. Purif. 26, 378–385. Leite, Y.F.M.M., Silva, L.M.C.M., Amorim, R.C.D.N., Freire, E.A., Jorge, D.M.D.M., Grangeiro, T.B., Benevides, N.M.B., 2005. Purification of a lectin from the marine red alga Gracilaria ornata and its effect on the development of the cowpea weevil Callosobruchus maculatus (Coleoptera: Bruchidae). Biochim. Biophys. Acta 1724, 137–145. Liener, I.E., Sharon, N., Goldstein, I.J. (Eds.), 1986. The Lectins: Properties, Functions, and Applications in Biology and Medicine. Academic Press, Orlando, pp. 1–600. Masuko, T., Minamib, A., Iwasakib, N., Tokifumi, M., Nishimurac, S.-I., Leea, Y.C., 2005. Carbohydrate analysis by a phenol-sulfuric acid method in microplate format. Anal. Biochem. 339, 69–72. Melo, V.M.M., Vasconcelos, I.M., Gomes, V.M., Cunha, M.D., Soares, A.A., Oliveira, J.T.A., 2005. A cotyledonary agglutinin from Luetzelburgia auriculata inhibits the fungal growth of Colletotrichum lindemuthianum, Fusarium solani and Aspergillus niger and impairs glucose-stimulated acidification of the incubation medium by Saccharomyces cerevisiae cells. Plant Sci. 169, 629–639. Mistry, A.C., Honda, S., Hirose, S., 2001. Structure, properties and enhanced expression of galactose-binding C-type lectins in mucous cells of gills from freshwater Japanese eels (Anguilla japonica). Biochemistry 360, 107–115. Mitsuru, J., Rika, U., Ryuichi, S., Koji, M., Hisao, K., 2007. Purification, cloning and characterization of egg lectins from the teleost Tribolodon brandti. Comp. Biochem. Physiol. B 147, 164–171. Muramoto, K., Kamiya, H., 1992. The amino-acid sequence of a lectin from conger eel, Conger myriaster, skin mucus. Biochim. Biophys. Acta 1116, 129–136. Ng, T.B., Lam, Y.W., Woo, N.Y.S., 2003. The immunostimulatory activity and stability of grass carp (Ctenopharyngodon idellus) roe lectin. Vet. Immunol. Immunopathol. 94, 105–112. Ohba, H., Bakalova, R., Moriwaki, S., Nakamura, O., 2002. Fractionation of normal and leukemic T-cells by lectin-affinity column chromatography. Cancer Lett. 184, 207–214. Okamoto, M., Tsutsui, S., Tasumi, S., Suetake, H., Kikuchi, K., Suzuki, Y., 2005. Tandem repeat L-rhamnose-binding lectin from the skin mucus of ponyfish, Leiognathus nuchalis. Biochem. Biophys. Res. Commun. 333, 463–469. Otta, Y., Amano, K., Nishiyama, K., Ando, A., Ogawa, S., Nagata, Y., 2002. Purification and properties of a lectin from ascomycete mushroom, Ciborinia camelliae. Phytochemistry 60, 103–107.

164

S. Pan et al. / Veterinary Immunology and Immunopathology 133 (2010) 154–164

Pang, Q., Zhang, S., Liu, X., Wu, D., 2006. Humoral immune responses of amphioxus Branchiostoma belcheri to challenge with Escherichia coli. Fish Shellfish Immun. 21, 139–145. Pickup, J.C., Hussain, F., Evans, N.D., Rolinski, O.J., Birch, D.J.S., 2005. Fluorescence-based glucose sensors. Biosens. Bioelectron. 20, 2555–2565. Russell, S., Lumsden, J.S., 2005. Function and heterogeneity of fish lectins. Vet. Immunol. Immunopathol. 108, 111–120. Sato, Y., Murakami, M., Miyazawa, K., Hori, K., 2000. Purification and characterization of a novel lectin from a freshwater cyanobacterium, Oscillatoria agardhii. Comp. Biochem. Phys. B 125, 169–177. Savan, R., Endo, M., Sakai, M., 2004. Characterization of a new C-type lectin from common carp Cyprinus carpio. Mol. Immunol. 41, 891– 899. Stratton, L., Wu, S., Richards, R.C., Ewart, K.V., 2004. Oligomerisation and carbohydrate binding in an Atlantic salmon serum C-type lectin consistent with non-self recognition. Fish Shellfish Immun. 17, 315–323. Suseelan, K.N., Mitra, R., Pandey, R., Sainis, K.B., Krishna, T.G., 2002. Purification and characterization of a lectin from wild sunflower (Helianthus tuberosus L.) tubers. Arch. Biochem. Biophys. 407, 241–247. Suzuki, Y., Tasumi, S., Tsutsui, S., Okamoto, M., Suetake, H., 2003. Molecular diversity of skin mucus lectins in fish. Comp. Biochem. Phys. B 136, 723–730. Tasumi, S., Ohira, T., Kawazoe, I., Suetake, H., Suzuki, Y., Aida, K., 2002. Primary structure and characteristics of a lectin from skin mucus of the Japanese eel Anguilla japonica. J. Biol. Chem. 277, 27305–27311. Tateno, H., Saneyoshi, A., Ogawa, T., Muramoto, K., Kamiya, H., Saneyoshi, M., 1998. Isolation and characterization of rhamnose-binding lectins

from eggs of steelhead trout (Oncorhynchus mykiss) homologous to low density lipoprotein receptor superfamily. Biol. Chem. 273, 19190–19197. Tsutsui, S., Okamoto, M., Tasumi, S., Suetake, H., Kikuchi, K., Suzuki, Y., 2006. Novel mannose-specific lectins found in torafugu, Takifugu rubripes: a review. Comp. Biochem. Physiol. D 1, 122–127. Tsutsui, S., Tasumi, S., Suetake, H., Kikuchi, K., Suzuki, Y., 2005. Demonstration of the mucosal lectins in the epithelial cells of internal and external body surface tissues in pufferfish (Fugu rubripes). Dev. Comp. Immunol. 29, 243–253. Vitved, L., Holmskov, U., Koch, C., Teisner, B., Hansen, S., Skjodt, K., 2000. The homologue of mannose-binding lectin in the carp family Cyprinidae is expressed at high level in spleen, and the deduced primary structure predicts affinity for galactose. Immunogenetics 51, 955– 964. Wu, A.M., Wu, J.H., Singh, T., Liu, J.-H., Herp, A., 2004. Lectinochemical studies on the affinity of Anguilla anguilla agglutinin for mammalian glycotopes. Life Sci. 75, 1085–1103. Yasuharu, W., Nobuyuki, S., Fuminori, S., Hiroshi, Y., Junko, K., Sachiko, N.T., Jun, H., Kazuhiro, S., Hisao, K., Hiroki, M., Tomohisa, O., Koji, M., 2008. Isolation and characterization of L-rhamnose-binding lectin, which binds to microsporidian Glugea plecoglossi, from ayu (Plecoglossus altivelis) eggs. Dev. Comp. Immunol. 32, 487–499. Yu, X.Q., Kanost, M.R., 2003. Manduca sexta lipopolysaccharide-specific immulectin-2 protects larvae from bacterial infection. Dev. Comp. Immunol. 27, 189–196. Zhang, H., Robison, B., Thorgaard, G.H., Ristow, S.S., 2000. Cloning, mapping and genomic organization of a fish C-type lectin gene from homozygous clones of rainbow trout (Oncorhynchus mykiss). Biochim. Biophys. Acta 494, 14–22.