Xerocomus chrysenteron lectin: identification of a new pesticidal protein

Xerocomus chrysenteron is an edible mushroom with insecticidal properties. In an earlier work, we found that proteins are responsible for this toxicity. Here we describe the purification of a approximately 15 kDa lectin, named XCL, from the mushroom. Its cDNA and gDNA were cloned by PCR strategies and a recombinant form was expressed in Escherichia coli. Sequence alignments and sugar specificity showed that this protein is the third member of a new saline-soluble lectin family present in fungi. This protein, either purified from mushroom or expressed in vitro in E. coli, was found to be toxic to some insects, such as the dipteran Drosophila melanogaster and the hemipteran, Acyrthosiphon pisum. The lectin possesses a high insecticidal activity compared to lectin isolated from leguminosae (Lathyrus ochrus) or from the snowdrop (Galanthus nivalis).     

Mechanisms of the insecticidal action of TEL (Talisia esculenta lectin) against Callosobruchus maculatus (Coleoptera: Bruchidae)

Plant lectins have insecticidal activity that is probably mediated through their ability to bind carbohydrates. To examine the influence of sugars on the insecticidal activity of a lectin from Talisia esculenta seeds (TEL), the lectin was mixed with mannose, glucose, or mannose plus glucose. Mannose abolished the insecticidal activity. Affinity chromatography showed that TEL bound to midgut proteins of the insect Callosobruchus maculatus. Immunoblotting showed that TEL recognized some proteins, probably glycoproteins, present in the midgut membrane of this insect. The principal proteases responsible for digestive proteolysis in fourth instar larvae of C. maculatus were purified by chromatography on activated thiol-Sepharose. These purified proteases were unable to digest TEL after a 15-h incubation. These results suggest that the insecticidal activity of TEL involves a specific carbohydrate-lectin interaction with glycoconjugates on the surface of digestive tract epithelial cells, as well as binding to assimilatory glycoproteins present in midgut extracts and resistance to enzymatic digestion by cysteine proteinases.     

Leguminous lectins as tools for studying the role of sugar residues in leukocyte recruitment

The natural physiological ligands for selectins are oligosaccharides found in glycoprotein or glycolipid molecules in cell membranes. In order to study the role of sugar residues in the in vivo lectin anti-inflammatory effect, we tested three leguminous lectins with different carbohydrate binding affinities in the peritonitis and paw oedema models induced by carrageenin in rats. L. sericeus lectin was more anti-inflammatory than D. virgata lectin, the effects being reversed by their specific binding sugars (N-acetylglucosamine and alpha-methylmannoside, respectively). However, V. macrocarpa, a galactose-specific lectin, was not anti-inflammatory. The proposed anti-inflammatory activity of lectins could be due to a blockage of neutrophil-selectin carbohydrate ligands. Thus, according to the present data, we suggest an important role for N-acetylglucosamine residue as the major ligand for selectins on rat neutrophil membranes.     

Plant‐insect interactions: what can we learn from plant lectins?

Many plant lectins have high anti‐insect potential. Although the effects of most lectins are only moderately influencing development or population growth of the insect, some lectins have strong insecticidal properties. In addition, some studies report a deterrent activity towards feeding and oviposition behavior. Transmission of plant lectins to the next trophic level has been investigated for several tritrophic interactions. Effects of lectins with different sugar specificities can vary substantially with the insect species under investigation and with the experimental setup. Lectin binding in the insect is an essential step in exerting a toxic effect. Attempts have been made to study the interactions of lectins in several insect tissues and to identify lectin‐binding receptors. Ingested lectins generally bind to parts of the insect gut. Furthermore, some lectins such as the Galanthus nivalus agglutinin (GNA) cross the gut epithelium into the hemolymph and other tissues. Recently, several candidate lectin‐binding receptors have been isolated from midgut extracts. To date little is known about the exact mechanism for insecticidal activity of plant lectins. However, insect glycobiology is an emerging research field and the recent technological advances in the analysis of lectin carbohydrate specificities and insect glycobiology will certainly lead to new insights in the interactions between plant lectins and insects, and to a better understanding of the molecular mechanisms involved. © 2010 Wiley Periodicals, Inc.     

De novo prediction of three-dimensional structures for major protein families

The family 10 xylanase from Streptomyces olivaceoviridis E-86 contains a (beta/alpha)(8)-barrel as a catalytic domain, a family 13 carbohydrate binding module (CBM) as a xylan binding domain (XBD) and a Gly/Pro-rich linker between them. The crystal structure of this enzyme showed that XBD has three similar subdomains, as indicated by the presence of a triple-repeated sequence, forming a galactose binding lectin fold similar to that found in the ricin toxin B-chain. Comparison with the structure of ricin/lactose complex suggests three potential sugar binding sites in XBD. In order to understand how XBD binds to the xylan chain, we analyzed the sugar-complex structure by the soaking experiment method using the xylooligosaccharides and other sugars. In the catalytic cleft, bound sugars were observed in the xylobiose and xylotriose complex structures. In the XBD, bound sugars were identified in subdomains alpha and gamma in all of the complexes with xylose, xylobiose, xylotriose, glucose, galactose and lactose. XBD binds xylose or xylooligosaccharides at the same sugar binding sites as in the case of the ricin/lactose complex but its binding manner for xylose and xylooligosaccharides is different from the galactose binding mode in ricin, even though XBD binds galactose in the same manner as in the ricin/galactose complex. These different binding modes are utilized efficiently and differently to bind the long substrate to xylanase and ricin-type lectin. XBD can bind any xylose in the xylan backbone, whereas ricin-type lectin recognizes the terminal galactose to sandwich the large sugar chain, even though the two domains have the same family 13 CBM structure. Family 13 CBM has rather loose and broad sugar specificities and is used by some kinds of proteins to bind their target sugars. In such enzyme, XBD binds xylan, and the catalytic domain may assume a flexible position with respect to the XBD/xylan complex, inasmuch as the linker region is unstructured.     

Mechanism of entomotoxicity of the plant lectin from Hippeastrum hybrid (Amaryllis) in Spodoptera littoralis larvae

Plant lectins have received a lot of attention because of their insecticidal properties. When orally administered in artificial diet or in transgenic plants, lectins provoke a wide range of detrimental effects, including alteration of the digestive enzyme machinery, fecundity drop, reduced feeding, changes in oviposition behavior, growth and development inhibition and mortality. Although many studies reported the entomotoxicity of lectins, only a few of them investigated the mode of action by which lectins exert toxicity. In the present paper we have studied for the first time the insecticidal potential of the plant lectin from Hippeastrum hybrid (Amaryllis) (HHA) bulbs against the larvae of the cotton leafworm (Spodoptera littoralis). Bioassays on neonate larvae showed that this mannose-specific lectin affected larval growth, causing a development retardation and larval weight decrease. Using primary cell cultures from S. littoralis midguts and confocal microscopy we have elucidated FITC-HHA binding and internalization mechanisms. We found that HHA did not exert a toxic effect on S. littoralis midgut cells, but HHA interaction with the brush border of midgut cells interfered with normal nutrient absorption in the S. littoralis midgut, thereby affecting normal larval growth in vivo. This study thus confirms the potential of mannose-specific lectins as pest control agents and sheds light on the mechanism underlying lectin entomotoxicity.     

Facile and rapid direct gold surface immobilization with controlled orientation for carbohydrates

Effective surface immobilization is a prerequisite for numerous carbohydrate-related studies including carbohydrate-biomolecule interactions. In the present work, we report a simple and rapid modification technique for diverse carbohydrate types in which direct oriented immobilization onto a gold surface is accomplished by coupling the amine group of a thiol group-bearing aminophenyl disulfide as a new coupling reagent with an aldehyde group of the terminal reducing sugar in the carbohydrate. To demonstrate the generality of this proposed reductive amination method, we examined its use for three types of carbohydrates: glucose (monosaccharide), lactose (disaccharide), and GM1 pentasaccharide. Through successful mass identifications of the modified carbohydrates, direct binding assays on gold surface using surface plasmon resonance and electrochemical methods, and a terminal galactose-binding lectin assay using atomic force microscopy, we confirmed several advantages including direct and rapid one-step immobilization onto a gold surface and exposure of functional carbohydrate moieties through oriented modification of the terminal reducing sugar. Therefore, this facile modification and immobilization method can be successfully used for diverse biomimetic studies of carbohydrates, including carbohydrate-biomolecule interactions and carbohydrate sensor or array development for diagnosis and screening.     

The effects of Phaseolus vulgaris erythro- and leucoagglutinating isolectins (PHA-E and PHA-L) delivered via artificial diet and transgenic plants on the growth and development of tomato moth (Lacanobia oleracea) larvae; lectin binding to gut glycoproteins in vitro and in vivo

Red kidney bean, Phaseolus vulgaris, contains a lectin phytohemagglutinin (PHA) with toxicity towards higher animals. PHA exists in the isoforms PHA-E and PHA-L, which agglutinate erythrocytes and lymphocytes, respectively. Lacanobia oleracea larvae were reared from hatch on artificial diets containing PHA-E or PHA-L at 2% (w/w) dietary protein, and on transgenic Arabidopsis plants expressing either lectin at 0.4-0.6% of total soluble proteins. In artificial diet bioassays neither lectin affected larval survival, development, growth nor consumption. In transgenic plant bioassays both PHA-E and PHA-L promoted larval growth and development. This effect was greatest for PHA-E. Mean larval biomass of insects fed on plants expressing PHA-E was significantly greater (up to two-fold) than controls during the final two instars and the insects developed at a significantly greater rate so that after 26 days 83% of PHA-E exposed insects were in the final instar compared to 44% for control insects. PHA-E and PHA-L were detected by Western blotting in haemolymph, sampled from insects fed diets or plant material containing the lectins. However, despite the demonstrated potential for both isolectins to bind to gut glycopolypeptides in vitro neither was found to accumulate in vivo in the guts of exposed insects. Since lectin binding to gut polypeptides is thought to be necessary for insecticidal activity the failure of PHA-E and PHA-L to bind in vivo may account for their lack of toxicity to L. oleracea.     

Oligomerisation and carbohydrate binding in an Atlantic salmon serum C-type lectin consistent with non-self recognition

A C-type lectin was previously isolated from the blood of healthy Atlantic salmon (Salmo salar) and this salmon serum lectin (SSL) was found to opsonise bacteria. Selective binding to bacteria in vivo requires that the lectin be able to recognise a carbohydrate pattern on the bacterial surface distinguishable from that of the host. In order to investigate this selectivity in the lectin, a phage-display antibody was prepared and then used for detection of lectin by Western blotting. A carbohydrate binding-inhibition assay with Western blot detection of the lectin showed mannose to be the primary ligand and related sugars including glucose, N-acetylglucosamine and methyl alpha-D-mannopyranoside to be additional ligands of this lectin. The SSL in serum detected by Western blotting was shown to form a complex oligomer. These results show that the salmon serum lectin is oligomeric in blood and that it recognizes a broad spectrum of carbohydrates with optimal binding to mannose. The lectin might therefore be an ideal opsonin for multiple salmon pathogens with carbohydrate arrays on their surfaces. No similar lectins were identified in the sera of other fish by Western blotting using the phage-display antibody. Molecular analysis will be required in order to determine whether homologous lectins are expressed in related fish species. It is anticipated that similar lectins might have related pathogen recognition roles in divergent fish species.     

Lectins of oil-seed flax plants exposed to abiotic stress

The seedlings of six cultivars of oil-seed flax (Linum humile Mill.) differing in the extent of adaptation to abiotic stresses (hypo- and hyperthermia, osmotic stress, and salinity) were used to assess hemag-glutination activity and carbohydrate specificity of total lectin preparations extracted from various cell compartments. In the course of adaptation of plants resistant to hyperthermia, osmotic stress and salinity, we observed a considerable rise in the coefficient of activity of membrane lectins, whereas the adaptation to hypothermia elevated the coefficient of activity of cell wall lectins. As to total soluble lectins, the adaptation of flax plants was associated with the changes in the range of their carbohydrate specificity. For instance, following the adaptation to hyperthermia, they were found to bind glucose and glucosamine, to osmotic stress—mannose and xylose, to salinity—galactose, glucose, and glucosamine; after cold resistance was developed, total soluble lectins were found to recognize lactose and fructose. It was concluded that lectins may participate in specific adaptation of flax plants to various abiotic stress factors.     

Antimetabolic effect of phytohemagglutinin to the grain aphid Sitobion avenae fabricius

The insecticidal activity of plant lectins against a wide range of insect species have been intensively studied. Understanding the mechanism of the toxicity of lectins is one of the studied aspects. In the present research, the first step was determine the effect of phytohemagglutinin (PHA) on the development, fecundity and mortality of grain aphid. Next, the effect of PHA lectin on the activity of such enzymes as: α- and β-glucosidases, alkaline (AkP) and acid (AcP) phosphatases, aminopeptidase N and cathepsin L involved in the metabolism of sugar, phosphorus and proteins of an adult apterae aphids was investigated. The PHA lectin added into the liquid diet increased the pre-reproductive period, mortality of Sitobion avenae, the time of generation development and decreased its fecundity and the intrinsic rate of natural increase. In addition, activity of α-glucosidase, alkaline phosphatase and aminopeptidase N of adult apterae exposed to PHA were reduced. The results indicate that the insecticidal activity of PHA on S. avenae may involve changes in activity of the enzymes in the midgut and it may be part of its toxicity.     

A new lectin family with structure similarity to actinoporins revealed by the crystal structure of Xerocomus chrysenteron lectin XCL

A newly defined family of fungal lectins displays no significant sequence similarity to any protein in the databases. These proteins, made of about 140 amino acid residues, have sequence identities ranging from 38% to 65% and share binding specificity to N-acetyl galactosamine. One member of this family, the lectin XCL from Xerocomus chrysenteron, induces drastic changes in the actin cytoskeleton after sugar binding at the cell surface and internalization, and has potent insecticidal activity. The crystal structure of XCL to 1.4 A resolution reveals the architecture of this new lectin family. The fold of the protein is not related to any of the several lectin folds documented so far. Unexpectedly, the structure similarity is significant with actinoporins, a family of pore-forming toxins. The specific structural features and sequence signatures in each protein family suggest a potential sugar binding site in XCL and a possible evolutionary relationship between these proteins. Finally, the tetrameric assembly of XCL reveals a complex network of protomer-protomer interfaces and generates a large, hydrated cavity of 1000 A3, which may become accessible to larger solutes after a small conformational change of the protein.     

Assay and characterization of carbohydrate binding by the lectin discoidin I immobilized on nitrocellulose

Discoidin I is the most abundant galactose binding lectin produced by the cellular slime mold Dictyostelium discoideum and has been implicated in cell-substratum adhesion. We have developed an assay of carbohydrate binding activity utilizing binding of 125I-asialofetuin to discoidin I, or to other lectins, immobilized on nitrocellulose. Among the proteins examined, only lectins exhibited the ability to bind asialofetuin. Specificity of asialofetuin binding was demonstrated by competition with monosaccharides, which inhibited binding consistent with the known sugar specificity of the lectins examined. Experiments with fetuin and derivatives differing in their oligosaccharide structure indicated a requirement for terminal galactosyl residues for probe binding to discoidin I. We have used this assay to characterize the carbohydrate binding behavior of discoidin I. The extent of asialofetuin binding to discoidin I was dependent on the concentrations of both lectin and ligand. Interpretation of equilibrium binding data suggested that, under saturating conditions, 1 mol of oligosaccharide was bound per mole discoidin I monomer. Furthermore, discoidin I in solution and discoidin I on nitrocellulose were equally effective at competing for soluble asialofetuin, suggesting that immobilization had no effect on the carbohydrate binding behavior of discoidin I. Binding was strongly inhibited by ethylenediaminetetraacetic acid; both Ca2+ and Mn2+ could overcome that inhibition, but Mg2+ could not. Preincubation of discoidin I at 60 degrees C stimulated asialofetuin binding 2-fold by increasing the affinity, while preincubation at higher temperatures resulted in a complete loss of activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of lectin concentrations in different Rhizoctonia solani strains

Lectins are carbohydrate-binding proteins that contain at least one carbohydrate binding domain which can bind to a specific mono- or oligosaccharide. These proteins are widely distributed in plants. However, over the last decade evidence is accumulating that lectins occur also in numerous fungi belonging to both the Ascomycota and Basiodiomycota. Rhizoctonia solani is known to be an important pathogen to a wide range of host plants. In this study, isolates of R. solani from different anastomosis groups have been screened for the presence of lectin using agglutination assays to detect and quantitate lectin activity. The evaluation included determination of the lectin content in mycelium as well as in sclerotia. The amount of lectin in the sclerotia was higher than in the mycelium of the same strains. The R. solani strains with the highest amounts of lectin have been selected for cultivation, extraction and purification of the lectin.     

Carbohydrate binding properties of banana (Musa acuminata) lectin II. Binding of laminaribiose oligosaccharides and beta-glucans containing beta1,6-glucosyl end groups.

This paper extends our knowledge of the rather bizarre carbohydrate binding poperties of the banana lectin (Musa acuminata). Although a glucose/mannose binding protein which recognizes alpha-linked gluco-and manno-pyranosyl groups of polysaccharide chain ends, the banana lectin was shown to bind to internal 3-O-alpha-D-glucopyranosyl units. Now we report that this lectin also binds to the reducing glucosyl groups of beta-1,3-linked glucosyl oligosaccharides (e.g. laminaribiose oligomers). Additionally, banana lectin also recognizes beta1,6-linked glucosyl end groups (gentiobiosyl groups) as occur in many fungal beta1,3/1,6-linked polysaccharides. This behavior clearly distinguishes the banana lectin from other mannose/glucose binding lectins, such as concanavalin A and the pea, lentil and Calystegia sepium lectins.     

Isolation and characterization of a galactose binding lectin with insulinomimetic activities. From the seeds of the bitter gourd Momordica charantia (Family Cucurbitaceae)

A galactose binding lectin was isolated from the seeds of the bitter gourd Momordica charantia by delipidation with petroleum ether, extraction with phosphate buffered saline, ammonium sulfate precipitation and affinity chromatography on lactogel. The lectin had a molecular weight of 124,000 and approximately 5% carbohydrate content. The molecular weights of the individual subunits were 37,000, 35,000 and 33,000. The lectin exhibited potent hemagglutinating activity. In addition, it demonstrated antilipolytic and lipogenic activities in isolated rat adipocytes although it did not possess intrinsic lipolytic activity. The antilipolytic activity was susceptible to destruction by heat, trypsin, chymotrypsin, glutathione and galactose, indicating that the integrity of the protein moiety, the disulfide linkages, and galactose, which is the sugar specifically bound by the lectin, all play an important role in interaction with the adipocyte leading to an expression of this insulin-like activity.     

Physicochemical Properties and Oxidative Inactivation of Soluble Lectin from Water Buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) Brain

Lectins are carbohydrate-binding proteins present in a wide variety of plants and animals, which serve various important physiological functions. A soluble β-galactoside binding lectin has been isolated and purified to homogeneity from buffalo brain using ammonium sulphate precipitation (40–70%) and gel permeation chromatography on Sephadex G_50–80 column. The molecular weight of buffalo brain lectin (BBL) as determined by SDS-PAGE under reducing and non-reducing conditions was 14.2 kDa, however, with gel filtration it was 28.5 kDa, revealing the dimeric form of protein. The neutral sugar content of the soluble lectin was estimated to be 3.3%. The BBL showed highest affinity for lactose and other sugar moieties in glycosidic form, suggesting it to be a β-galactoside binding lectin. The association constant for lactose binding as evidenced by Scatchard analysis was 6.6 × 10³ M⁻¹ showing two carbohydrate binding sites per lectin molecule. A total inhibition of lectin activity was observed by denaturants like guanidine HCl, thiourea and urea at 6 M concentration. The treatment of BBL with oxidizing agent destroyed its agglutination activity, abolished its fluorescence, and shifted its UV absorption maxima from 282 to 250 nm. The effect of H₂O₂ was greatly prevented by lactose indicating that BBL is more stable in the presence of its specific ligand. The purified lectin was investigated for its brain cell aggregation properties by testing its ability to agglutinate cells isolated from buffalo and goat brains. Rate of aggregation of buffalo brain cells by purified protein was more than the goat brain cells. The data from above study suggests that the isolated lectin may belong to the galectin-1 family but is glycosylated unlike those purified till date.     

Glutaraldehyde cross-linking of lectins to marker enzymes: protection of binding site by specific sugars

The role of bound specific sugars in protecting the sugar binding activity of several galactose binding proteins during their covalent conjugation to horse radish peroxidase by glutaraldehyde-mediated cross-linking was examined by: a) affinity matrix binding of the conjugate, b) enzyme linked lectin assay and c) hemagglutination assay. During conjugation using 1% glutaraldehyde, protection of jack fruit (Artocarpus integrifolia) lectin (jacalin) activity depended on concentration of specific sugar present during conjugation; optimum protection was offered by 50 mM galactose. This indicated the presence of one or more primary groups at the binding site of jacalin, which is (are) essential for sugar binding. On the other hand, such essential amino group(s) was not indicated at the sugar binding site of the peanut lectin, bovine heart galectin or of the human serum anti alpha-galactoside antibody, since exclusion of sugar during their conjugation to HRP did not diminish sugar binding activity. The differential behavior is discussed in the light of reported differences in sugar specificities. Results indicated that sugar mediated blocking of active site may be used in characterization of the latter in lectins.     

The staining of sciatic nerve glycoproteins on polyacrylamide gels with concanavalin A-peroxidase.

The plant lectin concanavalin A (Con A) possesses a remarkably specific capacity to bind primarily α-d-mannose or α-d-glucose sugar residues on macromolecules (cf. 1). The multivalent Con A will bind to carbohydrates on cell surfaces, and free binding sites on the attached Con A will bind to horseradish peroxidase which is a glycoprotein (2). Since peroxidase may be visualized by reaction with diaminobenzidine (3), it has been possible using this method to specifically “stain” carbohydrate residues on cell surface macromolecules (2, 4). The same principles for staining cell surfaces should apply to “staining” glycoproteins separated by polyacrylamide electrophoresis. In this paper, we examine the staining of glycoproteins in sciatic nerve by a Con A-peroxidase labeling technique. The method is more sensitive for mannose or glucose containing glycoproteins than the periodic acid-Schiff's (PAS) method commonly used.     

Purification and Characterization of Two Major Lectins from Araucaria brasiliensis syn. Araucaria angustifolia Seeds (Pinhão)

Two major lectins (lectin I and lectin II) were purified to homogeneity from the seeds of Araucaria brasiliensis (Gymnospermae). The purity of the lectins was confirmed by polyacrylamide gel electrophoresis, isoelectric focusing, and high performance liquid chromatography. They are glycoproteins in nature containing 6.3 and 2.9%, respectively, of neutral sugar and have absorption coefficients of 3.8 and 4.7, respectively, at 280 nanometers. The molecular weights of both lectins obtained by gel filtration on Sephacryl S-400 were equal: 200,000. After dissociation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, molecular weights were 20,000 and 34,000, respectively, for lectin I and lectin II, suggesting they are decameric and hexameric in nature. The amino acid composition of both lectins showed little difference, but both had high amounts of acidic amino acids and lacked methionine in their molecule. The carbohydrate binding specificity of lectins was directed towards mannose, glucose, and their oligomers. High inhibitory activity was also found with thyroglobulin. The erythroagglutinating activity of the lectins was enhanced in the presence of high-molecular-weight substances both at 37 and 4°C. Divalent cations do not appear to be essential for activity. They maintained their agglutinating activity over a broad but different range of pH: 5.5 to 7.5 and 6.5 to 7.5, respectively. Both lectins agglutinated erythrocytes of human ABO blood types equally well.