An α‐amylase is a novel receptor for Bacillus thuringiensis ssp. israelensis Cry4Ba and Cry11Aa toxins in the malaria vector mosquito Anopheles albimanus (Diptera: Culicidae)

Bacillus thuringiensis ssp. israelensis (Bti) produces four Cry toxins (Cry4Aa, Cry4Ba, Cry10Aa and Cry11Aa), and two Cyt proteins (Cyt1Aa and Cyt2Ba), toxic to mosquito‐larvae of the genus Aedes, Anopheles and Culex, important human disease vectors that transmit dengue virus, malaria and filarial parasites respectively. Previous work showed that Bti is highly toxic to Anopheles albimanus, the main vector for transmission of malaria in Mexico. In this work, we analysed the toxicity of isolated Cry proteins of Bti and identified an An. albimanus midgut protein as a putative Cry4Ba and Cry11Aa receptor molecule. Biossays showed that Cry4Ba and Cry11Aa of Bti are toxic to An. albimanus larvae. Ligand blot assays indicated that a 70 kDa glycosylphosphatidylinositol‐anchored protein present in midgut brush border membrane vesicles of An. albimanus interacts with Cry4Ba and Cry11Aa toxins. This protein was identified as an α‐amylase by mass spectrometry and enzymatic activity assays. The cDNA that codes for the α‐amylase was cloned by means of 5′‐ and 3′‐RACE experiments. Recombinant α‐amylase expressed in Escherichia coli specifically binds Cry4Ba and Cry11Aa toxins.     

Purification and Identification of a Novel Leucine Aminopeptidase from <Emphasis Type="Italic">Bacillus thuringiensis israelensis</Emphasis>

A novel leucine aminopeptidase was purified from a Bacillus thuringiensis israelensis (Bti) culture. The purification stages included heating the concentrated supernatant to 65°C for 90 min, anion-exchange chromatography by DEAE cellulose, and hydrophobic chromatography by phenyl Sepharose. The specific activity of leucine aminopeptidase after the hydrophobic chromatography increased by 215.5-fold and the yield was 16%. The molecular weight of the active enzyme was 59 kDa. Mass spectrometry analysis of the 59-kDa leucine aminopeptidase revealed that this protein has at least 41% homology with the cytosol leucine aminopeptidase produced by Bacillus cereus. Maximal leucine aminopeptidase activity occurred at 65°C, pH 10 toward leucine as the amino acid terminus. The enzyme was strongly inhibited by bestatin, dithiothreitol, and 1,10-phenanthroline, indicating that the enzyme might be considered as a metallo-aminopeptidase that has disulfide bonds at the catalytic site or at a region that influences its configuration. Examination of the purified leucine aminopeptidase’s effect on the activation of the protoxin Cyt1Aa from Bti revealed that when it acts synergistically with Bti endogenous proteases, it has only a minor role in the processing of Cyt1Aa into an active toxin.     

Assessment of protoxin composition of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains by use of polyacrylamide gel block and mass spectrometry

Assessment of protoxin composition in Bacillus thuringiensis parasporal crystals is principally hampered by the fact that protoxins in a single strain usually possess high sequence homology. Therefore, new strategies towards the identification of protoxins have been developed. Here, we established a powerful method through embedding solubilized protoxins in a polyacrylamide gel block coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of in-gel-generated peptides for protoxin identification. Our model study revealed that four protoxins (Cry1Aa, Cry1Ab, Cry1Ac and Cry2Aa) and six protoxins (Cry4Aa, Cry4Ba, Cry10Aa, Cry11Aa, Cyt1Aa, and Cyt2Ba) could be rapidly identified from B. thuringiensis subsp. kurstaki HD1 and subsp. israelensis 4Q2-72, respectively. The experimental results indicated that our method is a straightforward tool for analyzing protoxin expression profile in B. thuringiensis strains. Given its technical simplicity and sensitivity, our method might facilitate the present screening program for B. thuringiensis strains with new insecticidal properties. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Zujiao Fu and Yunjun Sun contributed equally to this work.     

Binding of Bacillus thuringiensis subsp. israelensis Cry4Ba to Cyt1Aa has an important role in synergism

Bacillus thuringiensis subsp. israelensis (Bti) produces at least four different crystal proteins that are specifically toxic to different mosquito species and that belong to two non-related family of toxins, Cry and Cyt named Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa. Cyt1Aa enhances the activity of Cry4Aa, Cry4Ba or Cry11Aa and overcomes resistance of Culex quinquefasciatus populations resistant to Cry11Aa, Cry4Aa or Cry4Ba. Cyt1Aa synergized Cry11Aa by their specific interaction since single point mutants on both Cyt1Aa and Cry11Aa that affected their binding interaction affected their synergistic insecticidal activity. In this work we show that Cyt1Aa loop β6-αE K198A, E204A and β7 K225A mutants affected binding and synergism with Cry4Ba. In addition, site directed mutagenesis showed that Cry4Ba domain II loop α-8 is involved in binding and in synergism with Cyt1Aa since Cry4Ba SI303-304AA double mutant showed decreased binding and synergism with Cyt1Aa. These data suggest that similarly to the synergism between Cry11Aa and Cyt1Aa toxins, the Cyt1Aa also functions as a receptor for Cry4Ba explaining the mechanism of synergism between these two Bti toxins.     

Cloning and Characterization of a Cytolytic and Mosquito Larvicidal δ-Endotoxin from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">darmstadiensis</Emphasis>

The cytolytic δ-endotoxin gene from Bacillus thuringiensis subsp. darmstadiensis was amplified from genomic DNA by PCR by using primers designed from the sequence of cyt2Aa1 gene of B. thuringiensis subsp. kyushuensis. DNA sequence analysis revealed an open reading frame translating to a 259-amino acid sequence. The cloned gene was designated cyt2Aa2. This gene was highly expressed in Escherichia coli as inclusion bodies that could be solubilized in 50 mM Na₂CO₃, pH 10.5. Activation of the solubilized protoxin by proteinase K (1% wt/wt, proteinase K/protoxin) yielded the active fragment of about 23 kDa. Cyt2Aa2 showed high hemolytic activity against sheep erythrocytes (hemolytic end- point 0.25 μg/ml) and was toxic to Culex quinquefasciatus and Aedes aegypti larvae (LC₅₀ 0.5–1.0 μg/ml). Received: 27 March 2002 / Accepted: 30 April 2002     

Cyt2Ba of Bacillus thuringiensis israelensis: activation by putative endogenous protease

The gene cyt2Ba of Bacillus thuringiensis subsp. israelensis was cloned for expression, together with p20, in an acrystalliferous strain. The large hexagonal crystals formed were composed of Cyt2Ba, which facilitated its purification. Crystal solubilization in the presence of endogenous proteases (with spores and cell debris) enabled quick and simple procedure to obtain rather pure and active toxin species by cleavage between amino acid residues 34 and 35, most likely by a camelysin-like protease that was discovered in association with activated Cyt2Ba. The product of this cleavage displayed haemolytic activity comparable to that of exogenously activated Cyt2Ba. The sequence of this putative protease shares high homology with the cell envelope-bound metalloprotease (camelysin) of the closely related species Bacillus cereus.     

Screen of Bacillus thuringiensis toxins for transgenic rice to control Sesamia inferens and Chilo suppressalis

Transgenic rice to control stem borer damage is under development in China. To assess the potential of Bacillus thuringiensis (Bt) transgenes in stem borer control, the toxicity of five Bt protoxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba and Cry1Ca) against two rice stem borers, Sesamia inferens (pink stem borer) and Chilo suppressalis (striped stem borer), was evaluated in the laboratory by feeding neonate larvae on artificial diets containing Bt protoxins. The results indicated that Cry1Ca exhibited the highest level of toxicity to both stem borers, with an LC₅₀ of 0.24 and 0.30 μg/g for C. suppressalis and S. inferens, respectively. However, S. inferens was 4-fold lower in susceptibility to Cry1Aa, and 6- and 47-fold less susceptible to Cry1Ab and Cry1Ba, respectively, compared to C. suppressalis. To evaluate interactions among Bt protoxins in stem borer larvae, toxicity assays were performed with mixtures of Cry1Aa/Cry1Ab, Cry1Aa/Cry1Ca, Cry1Ac/Cry1Ca, Cry1Ac/Cry1Ba, Cry1Ab/Cry1Ac, Cry1Ab/Cry1Ba, and Cry1Ab/Cry1Ca at 1:1 (w/w) ratios. All protoxin mixtures demonstrated significant synergistic toxicity activity against C. suppressalis, with values of 1.6- to 11-fold higher toxicity than the theoretical additive effect. Surprisingly, all but one of the Bt protoxin mixtures were antagonistic in toxicity to S. inferens. In mortality-time response experiments, S. inferens demonstrated increased tolerance to Cry1Ab and Cry1Ac compared to C. suppressalis when treated with low or high protoxin concentrations. The data indicate the utility of Cry1Ca protoxin and a Cry1Ac/Cry1Ca mixture to control both stem borer populations.     

Characterization of Cyt2Bc Toxin from Bacillus thuringiensis subsp. medellin

We cloned and sequenced a new cytolysin gene from Bacillus thuringiensis subsp. medellin. Three IS240-like insertion sequence elements and the previously cloned cyt1Ab and p21 genes were found in the vicinity of the cytolysin gene. The cytolysin gene encodes a protein 29.7 kDa in size that is 91.5% identical to Cyt2Ba from Bacillus thuringiensis subsp. israelensis and has been designated Cyt2Bc. Inclusions containing Cyt2Bc were purified from the crystal-negative strain SPL407 of B. thuringiensis. Cyt2Bc reacted weakly with antibodies directed against Cyt2Ba and was not recognized by an antiserum directed against the reference cytolysin Cyt1Aa. Cyt2Bc was hemolytic only upon activation with trypsin and had only one-third to one-fifth of the activity of Cyt2Ba, depending on the activation time. Cyt2Bc was also mosquitocidal against Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus, including strains resistant to the Bacillus sphaericus binary toxin. Its toxicity was half of that of Cyt2Ba on all mosquito species except resistant C. quinquefasciatus.     

Bacterial larvicides for vector control: mode of action of toxins and implications for resistance

Vector control can be an effective strategy to interrupt disease transmission and biolarvicides based on the entomopathogenic bacteria Bacillus sphaericus, and Bacillus thuringiensis serovar israelensis (Bti) have been successfully used to control species of public health relevance from the genera Aedes, Culex, Anopheles and Simulium. The most important feature of these agents is their ability to produce insecticidal proteins with selective action on the larval midgut. These protoxins are produced as crystals that, once ingested by larvae, are processed into active toxins, interact with receptors in the midgut epithelium and trigger cytopathological effects leading to larval death. B. sphaericus and Bti toxins share the initial steps of the mode of action; however, they interact with different midgut molecules. B. sphaericus presents a single larvicidal factor, the binary (Bin) toxin, whose action relies on the binding to one class of midgut receptors, while Bti crystals contain four protoxins (Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa), which display interactions with multiple midgut receptors. The mode of action of B. sphaericus displays a greater potential for resistance selection, compared to Bti, and, to date, there is no record of insect resistance to the latter, contrarily to B. sphaericus. The set of mosquitocidal toxins and their interaction with midgut target sites are described in this review, as well as the implications for the potential to select resistance amongst exposed populations. These biolarvicides have specific mode of action that rely on unique interactions and make them the most selective agents to control Diptera insects actually available.     

High-resolution crystal structure of activated Cyt2Ba monomer from Bacillus thuringiensis subsp. israelensis

The Cyt family of proteins consists of δ-endotoxins expressed during sporulation of several subspecies of Bacillus thuringiensis. Its members possess insecticidal, hemolytic, and cytolytic activities through pore formation and attract attention due to their potential use as vehicles for targeted membrane destruction. The δ-endotoxins of subsp. israelensis include three Cyt species: a major Cyt1Aa and two minor proteins, Cyt2Ba and Cyt1Ca. A cleaved Cyt protein that lacks the N- and C-terminal segments forms a toxic monomer. Here, we describe the crystal structure of Cyt2Ba, cleaved at its amino and carboxy termini by bacterial endogenous protease(s). Overall, its fold resembles that of the previously described volvatoxin A2 and the nontoxic form of Cyt2Aa. The structural similarity between these three proteins may provide information regarding the mechanism(s) of membrane-perforating toxins.     

Cytotoxicity Analysis of Three Bacillus thuringiensis Subsp. israelensis δ-Endotoxins towards Insect and Mammalian Cells

Three members of the δ-endotoxin group of toxins expressed by Bacillus thuringiensis subsp. israelensis, Cyt2Ba, Cry4Aa and Cry11A, were individually expressed in recombinant acrystalliferous B. thuringiensis strains for in vitro evaluation of their toxic activities against insect and mammalian cell lines. Both Cry4Aa and Cry11A toxins, activated with either trypsin or Spodoptera frugiperda gastric juice (GJ), resulted in different cleavage patterns for the activated toxins as seen by SDS-PAGE. The GJ-processed proteins were not cytotoxic to insect cell cultures. On the other hand, the combination of the trypsin-activated Cry4Aa and Cry11A toxins yielded the highest levels of cytotoxicity to all insect cells tested. The combination of activated Cyt2Ba and Cry11A also showed higher toxic activity than that of toxins activated individually. When activated Cry4Aa, Cry11A and Cyt2Ba were used simultaneously in the same assay a decrease in toxic activity was observed in all insect cells tested. No toxic effect was observed for the trypsin-activated Cry toxins in mammalian cells, but activated Cyt2Ba was toxic to human breast cancer cells (MCF-7) when tested at 20 µg/mL.     

Cytolytic Peptide Fragments of Cyt1Aa from Bacillus thuringiensis subsp. israelensis

Cyt1Aa is the major and most active component of the parasporal crystal of the Gram-positive soil entomopathogenic bacterium Bacillus thuringiensis subsp. israelensis. The Cyt1Aa protoxin exhibits some hemolytic and cytolytic activity. However, highly active 22–25 kDa toxins are obtained after proteolysis of Cyt1Aa from both the N- and the C-termini. As shown in this study, preliminary binding of the protoxin to polylamellary liposomes or partial denaturation of Cyt1Aa and further processing by several exogenous proteases yielded short 4.9–11.5 kDa cytolytic peptide fragments of Cyt1Aa. The shortest 51 amino acid peptide was obtained after pre-incubation of Cyt1Aa with SDS and proteolysis with proteinase K. This peptide was purified, identified as the Ile87–Asp137 fragment of Cyt1Aa and was shown to exhibit more than 30 % hemolysis of rabbit erythrocytes.     

Co-Expression of the Mosquitocidal Toxins Cyt1Aa and Cry11Aa from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Subsp. <Emphasis Type="Italic">israelensis</Emphasis> in <Emphasis Type="Italic">Asticcacaulis excentricus</Emphasis>

The cyt1Aa gene from Bacillus thuringiensis subsp. israelensis (Bti), whose product synergizes other mosquitocidal toxins, and functions as a repressor of resistance developed by mosquitoes against Bacilli insecticides, was introduced into the aquatic Gram-negative bacterium Asticcacaulis excentricus alongside the cry11Aa gene. The genes were introduced as an operon, but although mRNA was detected for both genes, no Cyt1Aa toxin was detected. Both proteins were expressed using a construct in which a promoter was inserted upstream of each gene. Recombinant A. excentricus expressing both toxins was found to be approximately twice as toxic to third instar larvae of Culex quinquefasciatus as transformants expressing just Cry11Aa.     

Cloning and Expression of <Emphasis Type="Italic">cyt</Emphasis>2Ba7 Gene from a Soil-Isolated <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Bacillus thuringiensis (Bt) cyt genes coding hemolytic and cytolytic toxins constitute a gene family, which are divided into two groups: cyt1 and cyt2. A novel cyt2 gene was detected from a soil-isolated Bt strain T301, which was highly homologous to cyt2Ba1 and finally designated cyt2Ba7. Until now, Cyt2Ba has not been expressed alone in Bt or other hosts. In this study, the cyt2Ba7 gene was cloned into the vector pQE30 and expressed as a fusion protein with 6×Histidine residues in Escherichia coli. Unlike cyt1A, cyt2Ba7 was freely expressed and formed cytoplasmic inclusions without the need for a “helper” protein. The 6×His-tagged Cyt2Ba7 was purified in one step by Ni-NTA affinity chromatography, examined cytolytic activity on Sf9 cells, and developed as an antigen to obtain the antiserum against Cyt2Ba by subcutaneous injection into rabbits. This gene was also cloned into the Bt–E. coli shuttle vector pHT3101 and expressed in Bt strain 4Q7. Immunoblotting analysis revealed that the antiserum was remarkably selective and specific to Cyt2Ba. Received: 21 December 2001 / Accepted: 28 January 2002     

In vivo nanoscale analysis of the dynamic synergistic interaction of Bacillus thuringiensis Cry11Aa and Cyt1Aa toxins in Aedes aegypti

The insecticidal Cry11Aa and Cyt1Aa proteins are produced by Bacillus thuringiensis as crystal inclusions. They work synergistically inducing high toxicity against mosquito larvae. It was proposed that these crystal inclusions are rapidly solubilized and activated in the gut lumen, followed by pore formation in midgut cells killing the larvae. In addition, Cyt1Aa functions as a Cry11Aa binding receptor, inducing Cry11Aa oligomerization and membrane insertion. Here, we used fluorescent labeled crystals, protoxins or activated toxins for in vivo localization at nano-scale resolution. We show that after larvae were fed solubilized proteins, these proteins were not accumulated inside the gut and larvae were not killed. In contrast, if larvae were fed soluble non-toxic mutant proteins, these proteins were found inside the gut bound to gut-microvilli. Only feeding with crystal inclusions resulted in high larval mortality, suggesting that they have a role for an optimal intoxication process. At the macroscopic level, Cry11Aa completely degraded the gastric caeca structure and, in the presence of Cyt1Aa, this effect was observed at lower toxin-concentrations and at shorter periods. The labeled Cry11Aa crystal protein, after midgut processing, binds to the gastric caeca and posterior midgut regions, and also to anterior and medium regions where it is internalized in ordered “net like” structures, leading finally to cell break down. During synergism both Cry11Aa and Cyt1Aa toxins showed a dynamic layered array at the surface of apical microvilli, where Cry11Aa is localized in the lower layer closer to the cell cytoplasm, and Cyt1Aa is layered over Cry11Aa. This array depends on the pore formation activity of Cry11Aa, since the non-toxic mutant Cry11Aa-E97A, which is unable to oligomerize, inverted this array. Internalization of Cry11Aa was also observed during synergism. These data indicate that the mechanism of action of Cry11Aa is more complex than previously anticipated, and may involve additional steps besides pore-formation activity.     

Alanine-162 of Bacillus thuringiensis Cyt2Aa2 toxin is essential for membrane binding and oligomerisation

Cyt2Aa2 is a cytolytic toxin produced by Bacillus thuringiensis subsp. darmstadiensis. It is specifically toxic to dipteran larvae in vivo and is also active against several cell types, such as erythrocytes. The active toxin is proposed to bind to the cell membrane, and membrane pore formation by toxin oligomerisation leads to cell lysis. This study aimed to characterise the role of residues (I139, S159, L160, S161, A162, D209 and V215) potentially involved in the membrane binding of Cyt2Aa2. All mutants, except I139A and V215A, showed similar characteristics to the wild-type toxin after proteinase K cleavage. Three mutants, S159A, L160A and S161A, showed high haemolytic activity but low toxicity against Aedes aegypti. Membrane interaction assays showed that these mutants could bind to rat red blood cells (rRBCs) and oligomerise. The mutant D209N had no haemolytic activity but was still mildly toxic to A. aegypti. The mutant A162V could not lyse rRBCs, even at high concentrations, and showed no toxicity against A. aegypti. Our data suggest that alanine 162 of the Cyt2Aa2 toxin is involved in membrane binding and oligomerisation. Substitution of this amino acid altered the conformation of the toxin and affected its biological activity.     

Evidence of the Importance of the Met115 for Bacillus thuringiensis subsp. israelensis Cyt1Aa Protein Cytolytic Activity in Escherichia coli

Cyt1Aa is a cytolytic toxin, found together with the delta-endotoxins in Bacillus thuringiensis subsp. israelensis parasporal insecticidal crystals. The latter are used as an environmental friendly insecticide against mosquitoes and black flies. Contrary to Cry delta-endotoxin, the mode of action of Cyt1Aa is not completely understood. In the absence of direct structural data, a novel mutated cyt1Aa gene was used to obtain indirect informations on Cyt1Aa conformation changes in the lipid membrane environment. A mutated cyt1Aa gene named cyt1A97 has been isolated from a B. thuringiensis israelensis strain named BUPM97. The nucleotide sequence predicted a protein of 249 amino acids residues with a calculated molecular mass of 27 kDa. Both nucleotide and amino acid sequences similarity analysis revealed that cyt1A97 presents one amino acid different from the native cyt1Aa gene. This mutation was located in the helix α C corresponding to a substitution of Met¹¹⁵ by a Thr. The heterologous expression of the cyt1A97 and another cyt1Aa-type gene called cyt1A98, not affected by such mutation used as control, was performed in Escherichia coli. It revealed that the mutated Cyt1A97 protein was over produced as inclusion bodies showing a very weak toxicity to E. coli contrarily to Cyt1A98 that stopped E. coli growth. Hence, hydrophobic residue Met at position 115 of Cyt1Aa should play a very important role for the maintenance of the structure and cytolytic functions of Cyt1Aa.     

Synthesis of Guaiacol-α-D-glucoside and Curcumin-bis-α-D-glucoside by an Amyloglucosidase from Rhizopus

Guaiacol (2-methoxyphenol) and curcumin [1E,6E-1,7-di(4-hydroxy-3-methoxy-phenyl)-1,6-heptadiene-3,5-dione] were converted into their corresponding glucosides using glucose and an amyloglucosidase from Rhizopus. Guaiacol-α-D-glucoside yields ranged from 3 to 52% with the highest at pH 7.0. Curcumin-bis-α-D-glucoside yields ranged from 3 to 48% with the highest at pH 4.0 with 50% (w/w D-glucose) of enzyme. The phenolic hydroxyl group of guaiacol and both phenolic hydroxyl groups of curcumin were glucosylated at the C1 carbon of α-D-glucose indicating that the enzymatic reaction is stereospecific. Both guaiacol-α-D-glucoside and curcumin-bis-α-D-glucosides had antioxidant activities.     

Ion Channels Induced in Planar Lipid Bilayers by the Bacillus thuringiensis Toxin Cry1Aa in the Presence of Gypsy Moth (Lymantria dispar) Brush Border Membrane

The apical brush border membrane, the main target site of Bacillus thuringiensis toxins, was isolated from gypsy moth (Lymantria dispar) larval midguts and fused to artificial planar lipid bilayer membranes. Under asymmetrical N-methyl-d-glucamine-HCl conditions (450 mm cis/150 mm trans, pH 9.0), which significantly reduce endogenous channel activity, trypsin-activated Cry1Aa, a B. thuringiensis insecticidal protein active against the gypsy moth in vivo, induced a large increase in bilayer membrane conductance at much lower concentrations (1.1–2.15 nm) than in receptor-free bilayer membranes. At least 5 main single-channel transitions with conductances ranging from 85 to 420 pS were resolved. These Cry1Aa channels share similar ionic selectivity with P _Cl/P _NMDG permeability ratios ranging from 4 to 8. They show no evidence of current rectification. Analysis of the macroscopic current flowing through the composite bilayer suggested voltage-dependence of several channels. In comparison, the conductance of the pores formed by 100–500 nm Cry1Aa in receptor-free bilayer membranes was significantly smaller (about 8-fold) and their P _Cl/P _NMDG permeability ratios were also reduced (2- to 4-fold). This study provides a detailed demonstration that the target insect midgut brush border membrane material promotes considerably pore formation by a B. thuringiensis Cry toxin and that this interaction results in altered channel properties. Received: 23 February 2001/Revised: 15 June 2001     

A mannose-specific tetrameric lectin with mitogenic and antibacterial activities from the ovary of a teleost,the cobia (<Emphasis Type="Italic">Rachycentron canadum</Emphasis>)

A tetrameric lectin, with hemagglutinating activity toward rabbit erythrocytes and with specificity toward d-mannosamine and d(+)-mannose, was isolated from the ovaries of a teleost, the cobia Rachycentron canadum. The isolation protocol comprised ion exchange chromatography on CM-cellulose and Q-Sepharose, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono Q, and finally gel filtration by FPLC on Superose 12. The lectin was adsorbed on all ion exchangers used. It exhibited a molecular mass of 180 kDa in gel filtration on Superose 12 and a single 45-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it is a tetrameric protein. The hemagglutinating activity of the lectin was stable up to 40°C and between pH 4 and pH 10. All hemagglutinating activity disappeared at 60°C and at pH 1 and pH 13. The hemagglutinating activity was doubled in the presence of 0.1 μM FeCl₃. The lectin exerted antibacterial activity against Escherichia coli with 50% inhibition at 250 μg. There was no antifungal activity toward Coprinus comatus, Fusarium oxysporum, Mycosphaerella arachidicola, and Rhizoctonia solani at a dose of 300 μg. The lectin exhibited maximal mitogenic response from mouse splenocytes at a concentration of 14 μM.