Basidiomycete Clitocybe nebularis is rich in lectins with insecticidal activities

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1 DOI /s APPLIED MICROBIAL AND CELL PHYSIOLOGY Basidiomycete Clitocybe nebularis is rich in lectins with insecticidal activities Jure Pohleven & Jože Brzin & Lara Vrabec & Adrijana Leonardi & Andrej Čokl & Borut Štrukelj & Janko Kos & Jerica Sabotič Received: 28 January 2011 /Revised: 3 March 2011 /Accepted: 6 March 2011 # Springer-Verlag 2011 Abstract Basidiomycete mushrooms are a rich source of unique substances, including lectins, that could potentially be useful in biotechnology or biomedical applications. Lectins are a group of carbohydrate-binding proteins with diverse biological activities and functions. Here, we demonstrate the presence of a number of lectins in the basidiomycete mushroom Clitocybe nebularis. Glucose-, galactose-, sucrose-, lactose-, and Sepharose-binding lectins were isolated from fruiting bodies using affinity chromatography on Sepharose-immobilized sugars or on Sepharose. The lectins were characterized biochemically and their binding specificities examined by agglutination and agglutination inhibition assays. In addition, insecticidal and anti-nutritional properties of the lectins were studied against a model organism, fruit fly (Drosophila melanogaster), and Colorado potato beetle (Leptinotarsa decemlineata). Of the several basidiomycete mushrooms screened, C. nebularis extract J. Pohleven (*) : J. Brzin : J. Kos : J. Sabotič Department of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia jure.pohleven@ijs.si L. Vrabec : A. Čokl Department of Entomology, National Institute of Biology, Večna pot 111, SI-1000 Ljubljana, Slovenia A. Leonardi Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia B. Štrukelj Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, SI-1000 Ljubljana, Slovenia showed the most potent insecticidal activity. Sucrose-binding lectin showed the strongest activity against D. melanogaster, followed by lactose- and galactose-binding lectins. Feeding bioassays with Colorado potato beetle revealed that C. nebularis extract exhibited high anti-nutritional activity against the insect; and of those tested, only lactose-binding lectin, named CNL showed the effect. Mushroom C. nebularis is shown to be rich in a variety of lectins with versatile biological activities, including insecticidal and antinutritional effects. C. nebularis lectins could thus have potential for use as natural insecticides. Keywords Basidiomycete. Clitocybe nebularis. Lectins. Insecticidal activity. Drosophila melanogaster. Leptinotarsa decemlineata Introduction Lectins comprise a large group of proteins with a wide variety of structures and binding specificities for carbohydrates. By recognition of and binding to distinct glycosylated cell ligands, lectins can agglutinate cells or mediate specific physiological or pathological processes (Varki et al. 2008). Their biological activity is potentially useful in biotechnological and biomedical applications. Basidiomycete fungi constitute a rich source of unique lectins (Goldstein and Winter 2007) for which various functions have been proposed. These include involvement in fruiting body development and mycorrhizal formation as well as defence against predators and parasites (Guillot and Konska 1997). Fruiting bodies of some basidiomycete species, such as those belonging to the Lepista (also classified as Clitocybe) or Cantharellus genera, are never inhabited by insects. Extracts from numerous mushroom fruiting bodies have been

2 demonstrated to possess insecticidal properties and several of these fungi are edible, which makes them a valuable source of new candidate insecticides. The insecticidal properties of these mushrooms were attributed to proteins such as lectins or hemolysins (Mier et al. 1996; Wangetal.2002). The first protein isolated from a mushroom showing insecticidal activity was a lectin from the red cracking bolete (Xerocomus chrysenteron), named X. chrysenteron lectin (XCL; Trigueros et al. 2003). Huge yield losses are caused worldwide every year by insect pests. Since they are continuously developing resistance to routinely used biological and chemical insecticides, which also have adverse impacts on the environment, development of alternative pest control strategies is essential. In search of a new effective natural insecticide, several basidiomycete mushrooms were screened for their insecticidal activity against the model organism, fruit fly (Drosophila melanogaster). The clouded agaric mushroom (Clitocybe nebularis) showed the strongest activity, which is consistent with previous reports (Mier et al. 1996). A lectin from C. nebularis has been reported to recognize N-acetylgalactosamine (Horejsí and Kocourek 1978) and a ricin B-like lectin has been identified from the same mushroom, designated C. nebularis lectin (CNL; Pohleven et al. 2009). We have now searched for other lectins from C. nebularis fruiting bodies and characterized them biochemically. The insecticidal activities of C. nebularis lectins against D. melanogaster and a major potato pest Colorado potato beetle (Leptinotarsa decemlineata) have been examined to evaluate their potential as natural insecticides. Materials and methods Fungal material and preparation of fruiting bodies extracts Basidiocarps of 14 basidiomycete fungi covering 10 different orders from five families (Armillaria mellea, Boletus calopus, Clitocybe geotropa, C. nebularis, Cortinarius caerulescens, Hebeloma radicosum, Hebeloma sinapizans, Lactarius cilicioides, Lactarius piperatus, Lactarius scrobiculatus, Lepista nuda, Macrolepiota procera, Ramaria formosa, Sarcodon imbricatus) were collected at different locations in Slovenia and kept frozen at 70 C until use. Crude extracts were prepared using 50 mm Tris/HCl buffer, ph 7.5, containing 0.3 M NaCl (buffer A) as described (Pohleven et al. 2009). The extracts were dialyzed (3.5 kda cut-off) against water at 4 C and lyophilized. Isolation of the lectins from C. nebularis Lectins from C. nebularis fruiting bodies were isolated as described (Pohleven et al. 2009). Crude extracts were subjected to carbohydrate affinity chromatography on glucose, galactose, sucrose, or lactose immobilized on divinylsulphone-activated Sepharose 4B, and mannose on divinylsulphone-activated Sepharose 6B, prepared as described (Levi and Teichberg 1981). Non-activated Sepharose 6B or divinylsulphone-activated Sepharose 6B was used in the control for affinity chromatography. Bound proteins were eluted with 0.2 M solutions of the corresponding sugars galactose was used in the control experiment or with 0.01 M NaOH followed by immediate neutralization with 2 M Tris/HCl buffer, ph 7.5. Homogeneous lectins were obtained by serial carbohydrate affinity chromatography. After applying the extract to lactose- or glucose-sepharose, the unbound material was subjected to sucrose-sepharose and bound proteins were eluted and concentrated. Proteins bound to lactose- or glucose-sepharose in the first affinity chromatography step were eluted and then, in the second step, loaded to glucoseor lactose-sepharose, respectively. Unbound fractions were pooled and concentrated. For subsequent characterization, the lectins were additionally purified using reversed-phase HPLC as described (Pohleven et al. 2009). Biochemical characterization of the lectins Isolated lectins were characterized as described (Pohleven et al. 2009). Purity and molecular masses of the lectins were estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE; 12% gel) in the presence or absence of reducing agent, and isoelectric points by isoelectric focusing using a Pharmacia Phastsystem. Molecular masses of the lectins under non-denaturing conditions were determined by size exclusion FPLC using buffer A containing the corresponding sugar (0.2 1 M)to prevent interactions between the lectin and column matrix. Molecular masses of the lectins were determined by electrospray ionization-mass spectrometry and their N- terminal sequences by automated Edman degradation. Similarity searches were performed using blastp and tblastn algorithms against different databases at the NCBI ( Haemagglutination and haemagglutination inhibition assay Human groups A, AB, B, O, and bovine erythrocytes were used in a haemagglutination assay. Group A erythrocytes were used in a haemagglutination inhibition assay with a glycoprotein asialofetuin and sugars (glucose, galactose, sucrose, lactose, fructose, and mannose). The assays were performed in triplicate as previously described (Pohleven et al. 2009). Agglutination was examined visually after 1-h incubation at room temperature and the haemagglutination titre (reciprocal of the lowest lectin concentration

3 exhibiting agglutination) as well as the haemagglutination inhibition titre (reciprocal of the lowest inhibitor concentration giving complete inhibition of agglutination) were determined. Toxicological tests on D. melanogaster The fruit fly (D. melanogaster, wild-type strain Canton S) was used as a model organism for assessing the insecticidal activity of mushroom extracts and the isolated lectins. The feeding bioassay was carried out using a rearing medium containing 1.2% agar, 8.5% yeast extract, 1.1% Nipagin, 0.5% propionic acid, together with the addition of 5% cornmeal and 3.2% sucrose in the experiments with mushroom extracts. Lyophilized extracts of 14 mushroom species in dry weight concentrations of 60, 20, 10, 5, 2.5, and 1.25 mg/ml, and selected lectins in concentrations of 600, 200, 60, 20, 6, and 2 μg/ml were added to the medium before it was poured into cm tubes. As a reference assay, 500 μg/ml of bovine serum albumin was added to the medium. Corresponding sugars (0.2 M) were added to the rearing medium with the lectins for the control assays. Ten eggs of D. melanogaster were deposited on the medium and the tubes were maintained at 25 C for 14 days, when the number of fully developed flies was recorded. Each test was performed with at least five replicates. Relative mortality (RM) with standard deviation was determined as 1 (average number of fully developed test flies/average number of fully developed reference flies). Insecticidal activity was expressed as the median lethal concentration (LC 50 ) value, determined by nonlinear sigmoid curve fitting of logarithms of concentrations versus RM using Prism (GraphPad Software). Toxicological tests on L. decemlineata larvae Anti-nutritional properties of the lectins and of the crude extract of C. nebularis on Colorado potato beetle (L. decemlineata) larvae were evaluated in a feeding bioassay. Groups of six 2-day-old larvae hatched from eggs collected in the field were put on potato (Solanum tuberosum) leaves soaked in lectin solutions containing lactose-binding C. nebularis lectin (1.5 and 2.2 mg/ml), glucose-binding lectin (1.5 and 2.0 mg/ml), sucrose-binding lectin (1.5 and 3.3 mg/ml), or crude C. nebularis extract, then drained. Leaves soaked in buffer containing bovine serum albumin (1.5 or 3.0 mg/ml) were used in control tests. Leaves were replaced every 24 h, when the number of surviving larvae and their weights were recorded. The amount of added protein in ingested food was estimated to be 0.015% (w/w, for 1.5 mg/ml lectin solution), 0.018% (w/w, for 2 mg/ml lectin solution), or 0.030% (w/w, for 3.3 mg/ml lectin solution) of the leaf weight. Results C. nebularis lectins Several lectins were isolated from C. nebularis fruiting bodies by carbohydrate affinity chromatography using glucose, galactose, sucrose, or lactose immobilized to Sepharose or using Sepharose alone (Fig. 1, Table 1). Galactose-Sepharose-bound lectins (CnGalLs) migrated as three bands with molecular masses of 19, 17.5, and 15.5 kda, as estimated from SDS-PAGE (Fig. 1, lane 2). Lactose-Sepharose-bound lectins (CnLacLs) gave a similar band pattern with lactose-sepharose (Fig. 1, lane 3), whereas glucose-sepharose-bound lectins (CnGlcLs) migrated as three proteins with apparent masses of 31, 17.5, and 15.5 kda (Fig. 1, lane 5). In the second step of serial affinity chromatography, glucose- or lactose- Sepharose-bound lectins were loaded to lactose- or glucose-sepharose, respectively. Thereby we obtained a homogeneous unbound glucose-binding lectin (CnGlcL; Fig. 1, lane 6) and a previously reported lactose-binding lectin, CNL (Fig. 1, lane 4; Pohleven et al. 2009). A 20- kda sucrose-binding lectin, designated CnSucL (Fig. 1, lane 7), was also obtained using serial affinity chromatography after the lactose- or glucose-sepharose unbound extract material was subjected to sucrose-sepharose. Control affinity chromatography using Sepharose or divinylsulphoneactivated Sepharose yielded two proteins showing band patterns which corresponded exactly to the 17.5 and 15.5 kda-bands (Fig. 1, lane 8). The two Sepharose-binding lectins (CnSepLs) were designated CnSepL-1 and CnSepL-2. No mannose-binding lectins were obtained, since only the same bands as with Sepharose were obtained (not shown). The same migration of C. nebularis lectins was observed under reducing conditions, indicating that they are not present as covalent associations. Fig. 1 SDS-PAGE analysis of the lectins isolated from C. nebularis. 1 Molecular weight standard proteins; 2 galactose-sepharose-bound lectins (CnGalLs), 3 lactose-sepharose-bound lectins (CnLacLs), 4 lactose-binding C. nebularis lectin (CNL; Pohleven et al. 2009), 5 glucose-sepharose-bound lectins (CnGlcLs), 6 glucose-binding lectin (CnGlcL), 7 sucrose-binding lectin (CnSucL), 8 Sepharose-binding lectins (CnSepLs)

4 Table 1 Biochemical properties of the lectins from C. nebularis Lectin name SDS-PAGE MM (kda) ESI-MS MM (Da) No. of subunits pi a N-terminal amino acid sequence CnSepL , NSAFGNSVIDLTGNDPAENTPXIXV CnSepL , NSAFGN CnGlcL 31 29, TTGTPINHNGDVNKKLGXFN CnGalLs 19, 17.5, blocked CnSucL 21 20,293 1 >9.3 VNPNLPGPNDVFVGFRGTNN CNL 19 15, MSITPGTYNITNVAYTNRLIDLTGSNP b a Isoelectric point b Pohleven et al. (2009); Fig. 2 Isoelectric focusing revealed that the isolated lectins are acidic proteins with isoelectric points (pi) between 4 and 5, except CnSucL with a pi above ph 9.3 (Table 1). The N- terminal amino acid sequences of the lectins were determined, except for CnGalLs which were blocked to Edman degradation suggesting modification of their amino termini (Table 1). Similarity searches of N-terminal amino acid sequences revealed no significant similarities of either CnGlcL or CnSucL to other proteins, whereas CnSepL-1 and -2 sequences were similar to that reported for CNL which contains the ricin B lectin domain (Fig. 2; Pohleven et al. 2009). Specificities of C. nebularis lectins for different types of erythrocytes and sugars Specificities of the lectins for human and bovine erythrocytes were determined by haemagglutination assay (Fig. 3). The strongest agglutinating activity was observed for CNL on human blood group A erythrocytes and the lectin also agglutinated AB, B, O, and bovine erythrocytes (Pohleven et al. 2009). On the other hand, besides agglutinating group A erythrocytes, CnSepLs were more specific for groups B and O. CnGalLs also agglutinated all types of erythrocyte tested, with a preference for type A erythrocytes, while CnGlcL and CnSucL exhibited negligible agglutinating activity (Fig. 3). The binding specificities of CNL, CnGalLs, and CnSepLs for sugars and a glycoprotein asialofetuin were determined by haemagglutination inhibition assay using human blood group A erythrocytes. CnGlcL and CnSucL were not used because of their negligible agglutinating activity. CnSepLs and Fig. 2 N-terminal amino acid sequence alignment of CNL and CnSepL-1. Identical amino acid residues are shaded in dark gray and similar residues in light gray. Complete amino acid sequence of CNL was previously reported (Pohleven et al. 2009) CnGalLs gave similar patterns of inhibition; both were strongly inhibited by asialofetuin and lactose and less so by other sugars (Table 2). On the other hand, CNL was inhibited only by asialofetuin, and less so by lactose (Pohleven et al. 2009), and the inhibition was less potent than the inhibition of CnSepLs and CnGalLs (Table 2). Insecticidal activity of mushroom extracts on D. melanogaster The insecticidal effect of different dialysed mushroom extracts was first determined by adding them to the rearing medium at a high concentration (60 mg/ml). Those that caused an RM above 80% (Table 3) were then tested at a lower concentration (20 mg/ml). Several mushroom extracts exhibited potent activities. Among them C. nebularis, L. piperatus, B. calopus, L. nuda, and H. radicosum showed highest RMs (>90%) and were further tested at lower concentrations of 10, 5, 2.5, and 1.25 mg/ml to determine the LC 50 values. The strongest insecticidal activity in extracts was observed with C. nebularis, followed by L. piperatus, B. calopus, L. nuda, and H. radicosum (Table 3). Insecticidal activities of C. nebularis lectins on D. melanogaster The extract of C. nebularis exhibited the most potent insecticidal activity on D. melanogaster, so the individual lectins isolated from this mushroom were tested to evaluate their contribution to insecticidal activity. The strongest activity was observed with CnSucL (LC 50 of 5.5±0.6 μg/ml), followed by CNL (47.9±3.1 μg/ml), CnLacLs (234.4±49.3 μg/ml), and CnGalLs (603.2±39.3 μg/ml). CnGlcL exhibited negligible activity (4% RM at a concentration of 200 μg/ml). The insecticidal activities of the lectins were abolished when the corresponding sugars (0.2 M) were added to the medium, except at high concentrations of CnSucL at which the sugar did not inhibit the effect (not shown).

5 Appl Microbiol Biotechnol Fig. 3 Specificities of C. nebularis lectins for different types of erythrocytes. Differently shaded bars correspond to agglutination of different types of erythrocytes. The lowest concentrations of the lectins in micromolar that exhibited agglutination of erythrocytes are indicated above each bar, and each is the mean of three independent experiments with standard deviations shown. N.D. not determined; > denotes the highest concentration of lectin at which agglutination was not elicited. Specificity of the lectin for erythrocytes is expressed as relative haemagglutination titre the agglutination titre for each type of erythrocyte, relative to the titre obtained by CNL for blood group A erythrocytes (Pohleven et al. 2009), taken as 100% Anti-nutritional properties of C. nebularis lectins on L. decemlineata larvae basidiomycete C. nebularis. Such a variety of lectins has not been shown for any other mushroom species, though available homobasidiomycete genome sequences anticipate such diversity. In addition, their insecticidal activity was demonstrated, suggesting that these lectins constitute an array of proteins that enable a synergistic defensive action by recognition of various glycans, conveying higher toxicity against a wide range of predatory organisms. Furthermore, other functions of individual lectins, such as developmental or regulatory roles, cannot be excluded. The isolated C. nebularis lectins are mainly small acidic proteins that form homodimeric structures by noncovalent interaction. CnGalLs, CNL, and CnSepLs are probably isolectins, since they display similar biochemical properties and have comparable sugar-binding specificities, all being potently inhibited by asialofetuin and lactose. This is consistent with the methods of isolation, in which galactose, lactose or Sepharose (galactose-anhydrogalactose copolymer) were used. The similarity of CNL and CnSepLs is also evident from their N-terminal amino acid sequences The crude extract of C. nebularis showed a high antinutritional effect on Colorado potato beetle larvae, which attained only 10 20% of the weight gained in control group (Fig. 4) and none proceeded to pupate. A preliminary feeding assay with mixtures of lactose-binding lectins isolated from C. nebularis indicated that they are responsible for the antinutritional effect of C. nebularis extract. Only CNL of the purified lectins from C. nebularis showed significant antinutritional effect and this was amount-dependent (Fig. 4). On the other hand, CnGlcL and CnSucL had no effect, even at the higher concentration used in the assay. Discussion Multiple lectins, displaying diverse biochemical and sugarbinding properties, have been shown to be present in Table 2 Binding-specificities of the lectins from C. nebularis for sugars and asialofetuin a The lowest concentration of sugar or a glycoprotein giving complete inhibition of agglutination, presented as the mean of three independent experiments b c Pohleven et al. (2009) At or below this concentration no inhibition of agglutination was observed Sugar/glycoprotein inhibitor Asialofetuin Lactose Galactose Glucose Mannose Sucrose Fructose Minimum inhibitory concentration of inhibitor (mm)a CnSepLs CnGalLs CNLb >476.19c >476.19c

6 Table 3 Insecticidal activity of mushroom extracts on D. melanogaster Mushroom species Relative mortality (%) of mushroom extract at 60 mg/ml Relative mortality (%) of mushroom extract at 20 mg/ml LC 50 (mg/ml) C. nebularis <1.25 L. piperatus ±0.1 B. calopus 97.3± ±0.1 L. nuda ±0.2 H. radicosum ± ±0.5 M. procera ±11.6 N.D. H. sinapizans ±12.4 N.D. R. formosa ±10.0 N.D. A. mellea 86.5± ±10.0 N.D. L. cilicioides 67.6±7.8 N.D. N.D. S. imbricatus 64.9±14.0 N.D. N.D. C. caerulescens 32.4±7.3 N.D. N.D. L. scrobiculatus 27.0±10.6 N.D. N.D. C. geotropa 16.2±10.6 N.D. N.D. N.D. not determined (Fig. 2). Moreover, the sequences of CnSepLs contain a motif of the lactose-binding site determined by crystallographic analysis of CNL (unpublished observation). However, in the agglutination assay using different types of erythrocytes, distinct specificities of these isolectins were observed. CNL and CnGalLs displayed higher specificity for human blood group A than for blood groups B and O erythrocytes, while CnSepLs show preference for human types B and O over type A erythrocytes. This suggests that CNL and CnGalLs have higher affinity for α-n-acetylgalactosamine- and CnSepLs for α-galactose-containing carbohydrates. In the haemagglutination inhibition assay using human blood group A erythrocytes, a strong inhibition of CnSepLs and CnGalLs, greater than that of CNL, was observed. This was probably because CNL is highly specific for human blood group A determinantcontaining carbohydrates (Pohleven et al. 2009), so the inhibitors could not compete strongly for carbohydratebinding sites of the lectin, as is the case for CnSepLs and CnGalLs. Finally, in order to screen for a potentially effective natural insecticide candidate, the insecticidal and antinutritional properties of basidiomycete fungi, and in particular of C. nebularis lectins, were studied against a lepidopteran model organism D. melanogaster and an economically important pest, coleopteran L. decemlineata. Experiments on D. melanogaster using fruiting body extracts from a number of basidiomycetes revealed that several mushrooms have relatively potent activities, the strongest being from C. nebularis, followed by L. piperatus, B. calopus, L. nuda, and H. radicosum. The insecticidal potential of an individual basidiomycete appears to be species-specific, since different species of the same order (e.g., Clitocybe, Lactarius) displayed incongruous insecticidal activities. These activities belong to macromolecules, presumably proteins, since small molecules like secondary metabolites were removed from the extracts by dialysis. Similarly, in the study of Mier et al. (1996), one of the strongest toxicities has been noted with C. nebularis extracts and another set of experiments suggested that the insecticidal effect of C. nebularis was caused by proteins such as lectins or hemolysins (Wang et al. 2002). Toxicological tests of C. nebularis lectins against D. melanogaster revealed that some possess higher insecticidal activities than the mushroom extracts and the cysteine protease inhibitor clitocypin and serine protease inhibitors CnSPIs from C. nebularis (Avanzo et al. 2009; Renko et al. 2010). XCL, a lectin isolated from mushroom X. chrysenteron, was the first fungal lectin shown to possess insecticidal properties. Recombinant XCL, known Fig. 4 Anti-nutritional effects of CNL and C. nebularis extract on Colorado potato beetle larvae. Bars represent means of larval weight with standard deviations shown. Mean weights of larvae treated with CNL were compared with those treated with BSA (control) for each day, using the two-sample t test. Statistically significant differences are indicated by two (P<0.01) or three (P<0.001) asterisks above bars that represent compared groups. The amount of the lectin or BSA in ingested food is presented as the percentage of the leaf weight

7 to be a very potent insecticidal lectin (LC 50 68±20 μg/ml; Trigueros et al. 2003), shows an activity against D. melanogaster comparable to that of CNL (LC ± 3.1 μg/ml) and both lectins recognize N-acetylgalactosamine-containing carbohydrates (Trigueros et al. 2003; Damian et al. 2005; Pohleven et al. 2009). However, CnSucL proved to be an even more potent insecticide, showing approximately 10 times stronger insecticidal activity (LC ±0.6 μg/ml). Feeding bioassays with Colorado potato beetle larvae revealed a high antinutritional activity of C. nebularis extract against the insect. Of the isolated lectins tested, however, only CNL showed an anti-nutritional effect. A plant lectin, designated Gleheda, which was isolated from ground ivy (Glechoma hederacea) also showed insecticidal activity against Colorado potato beetle larvae (Wang et al. 2003a). Similarly to CNL, this legume lectin is specific for N-acetylgalactosamine and also shows preference for type A over type B human erythrocytes (Wang et al. 2003b). Anti-nutritional activity of Gleheda against Colorado potato beetle larvae was demonstrated when leaves were dipped in 20 mg/ml solution of the lectin, whereas CNL exerted the effect at approximately tenfold lower concentration of 2.2 mg/ml. The insecticidal activity of C. nebularis lectins appear to be elicited by specific lectin carbohydrate interactions since, in tests with D. melanogaster, the effect was abolished when the corresponding sugars were added to the rearing medium. In addition, it has been shown that the lectins are not toxic to several human cell lines (Pohleven et al. 2009), and we therefore conclude that the insecticidal activity is not due to a general cytotoxic action of the lectins. Different activities of the individual C. nebularis lectins on the two insect species tested are probably the result of the recognition and binding to specific glycosylated target receptors, presumably in the digestive tract of the insects, which leads to disruption of the digestive system. For example, galectin CGL2 from fungus Coprinopsis cinerea was demonstrated to mediate its toxic effect towards the nematode Caenorhabditis elegans by binding to a specific glycoconjugate Galβ1,4Fucα1,6 (Butschi et al. 2010). In general, plant lectins specific for mannose and glucose are mainly toxic to insects belonging to hemiptera order, while those exhibiting insecticidal activity against coleopteran and lepidopteran insects generally display specificity for N-acetyl-D-glucosamine, galactose and N- acetyl-d-galactosamine (Vasconcelos and Oliveira 2004), in agreement with the results of our assays. The insecticidal activity of the C. nebularis lectins, together with their resistance to proteolytic degradation as demonstrated for CNL (Pohleven et al. 2009), makes them candidates for natural insecticides. In conclusion, we have established further evidence that basidiomycetes are a valuable source of a variety of lectins and have demonstrated that mushroom C. nebularis is rich in a variety of lectins with versatile biological activities including insecticidal and anti-nutritional effects. These findings suggest their potential defensive role against predators in the mushroom. Furthermore, C. nebularisderived lectins could have a potential for use as natural insecticidal pest control agents. Acknowledgments The authors thank Dr. Špela Schrader for help with tests involving Drosophila, Dr. Bogdan Kralj for mass spectrometric analysis and Prof. Roger Pain for critical review of the manuscript. 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