Localization of alpha-D-glucosyl and alpha-D-mannosyl groups of mucosubstances with concanavalin A and horseradish peroxidase.

Concanavalin A is a lectin which is known to bind specifically to alpha-D-glucosyl and alpha-D-mannosyl groups in the mucosubstances of mammalian tissues. The lectin molecule is bivalent; after its attachment to mucosubstances present in a histological specimen it can also bind horseradish peroxidase, a mannose-containing glycoprotein. The attached peroxidase may then be visualized by virtue of its histochemically demonstrable enzymatic activity. Other investigators have utilized this principle in the electron microscopic localization of cell-surface carbohydrates. A histochemical technique for light microscopy is described here, along with three control procedures which establish the specificity of the method. The technique is somewhat more sensitive than earlier ones in which fluorescent-labelled concanavalin A was used, and has the additional advantages that all the required reagents are commercially available and that sacilities for fluorescence microscopy are not needed.     

Changes in lectin-induced deoxyribonucleic acid synthesis in cultures of chick-embryo fibroblasts at various stages of development

Concanavalin A and Robinia pseudoacacia lectin decreased [(3)H]thymidine incorporation into acid-insoluble material of fibroblasts cultured from 6-10-day chick embryos. In contrast, these lectins stimulated [(3)H]thymidine incorporation in cells from 16-day embryos. These effects are due to neither [(3)H]thymidine permeability modification nor toxicity of the lectins. The specificity of lectin action was proved by blocking experiments with alpha-methyl mannopyranoside and with anti-(Robinia lectin) serum.     

Purification of a new fungal mannose-specific lectin from Penicillium chrysogenum and its aphicidal properties

Several Ascomycetes fungi are commonly used in bio-industries and provide available industrial residues for lectin extraction to be valuable. A lectin from Penicillium chrysogenum, named PeCL, was purified from a fungal culture using gel-filtration chromatography column. PeCL was found to be a mannose-specific lectin by haemagglutination activity towards rabbit erythrocyte cells and was visualised on SDS-PAGE gel. Purified PeCL fraction was delivered via artificial diet to Myzus persicae aphid and was demonstrated to be aphicidal at 0.1?% with higher toxic efficiency than a known mannose-binding lectin Concanavalin A (ConA). A fast and efficient way to purify PeCL and a potential use in pest control is described.     

Purification of Lectin from Micropropagated Roots Derived from Aseptic Seedling of <Emphasis Type="Italic">Canavalia ensiformis</Emphasis> L.

A well-organized protocol has been developed for high frequency root germination from the seed of Canavalia ensiformis on Murashige and Skoog (MS) medium. Surprisingly, the seeds that were grown on the MS medium having no growth hormone showed the best response. Roots of 30 days old aseptic seedling were homogenized and a lectin from them was purified on Sephadex G-50 affinity column. The finding that final product is a pure lectin was confirmed by specific hemagglutinating property. The final root lectin yield was 0.6% and eluted as a single peak. Root lectin specific activity was 50 times more than the seed lectin. Sugar specificity activity by hemagglutination-inhibition assay indicated that lectin belongs to glucose/mannose-specific group. Interestingly, the lectin was found to be 25 kDa, similar to molecular mass of Concanavalin A purified from seed of C. ensiformis, as revealed by SDS–PAGE. Thus, Concanavalin A from either source can be used for development of transgenic crops that are capable of expressing lectin gene and hence can efficiently perform biological nitrogen fixation by giving rise to nodules in their root. The advantage of this method is that purification of Concanavalin A in tissue culture conditions is easier, handy and is less time consuming.     

Heterogeneous glycosylation of Musca domestica arylphorin

Two distinct fractions of Musca domestica arylphorin were isolated by affinity chromatography on Concanavalin A-Sepharose column. The results show that in the hexameric arylphorin that do not bind to the lectin there is no Concanavalin A binding subunit and in the majority of the hexamers that bind to the lectin there is only one subunit with Concanavalin A binding site. The results indicate that the carbohydrate moiety of the arylphorin is not involved in its specific uptake by the fat bodies and integument.     

Influence of Concanavalin A on 3-O-methylglucose uptake in cultured chick embryo fibroblasts. Evidence for differences related to the age of embryos

Concanavalin A (Con A) was found to inhibit hexose uptake in cultured fibroblasts derived from 8-day chick embryos and to stimulate this process in those derived from 16-day embryos. Con-A effects depended on the duration of contact with cells and lectin and were inhibited by alpha-methylmannopyrannoside. Con A was shown to mask about 70% of the hexose carriers in both 8- and 16-day embryo fibroblasts. Lectin altered the hexose uptake very rapidly. Con A only modified the Vmax of the uptake system and did not alter the Km. This indicates that either the number or mobility of hexose carriers were modified by Con-A treatment. The differential effect of lectin could be due to a modification of the hexose-carrier mobility during the embryonic differentiation of fibroblasts. Secondary effects may affect cell growth.     

Ultrastructural localization of lectin receptors on the surface of the rat retinal pigment epithelium. Decreased sensitivity of the avidin-biotin method due to cell surface charge

Monosaccharides on the apical processes of the retinal pigment epithelium were examined using lectin-affinity cytochemical methods. Lectin receptor sugars were localized with lectin-horseradish peroxidase (HRP) and lectin-ferritin conjugates as well as with biotinylated lectins, avidin, and biotinylated HRP. In contrast, only wheat germ agglutinin (WGA) receptors were identified with biotinylated WGA followed by avidin-ferritin or free avidin and biotinylated ferritin. Labeling with avidin-ferritin subsequent to biotinylated lectin treatment was dependent upon the source and lot of the reagent. These findings are similar to those reported for the endothelium of bone marrow sinusoids (Pino RM: Am J Anat, 169:259, 1984). Since both the retinal pigment epithelial and bone marrow sinusoidal surfaces are highly anionic (negative), we investigated the possibility that the charge of the lectin reagents and cell surfaces might affect the localization of monosaccharides on cell surfaces. Analytical isoelectric focusing revealed that biotinylated ferritin and some avidin-ferritins are highly anionic, while the other lectin reagents have more cationic (positive) components. Based on this information, a less charged biotinylated ferritin marker was made that made it possible to localize biotinylated lectins bound to the cell surface.     

Polysaccharides immobilized in polyacrylamide gel as high-capacity adsorbents for Bandeiraea simplicifolia lectin and concanavalin A.

A high-capacity adsorbent for lectins was easily prepared by immobilization of polysaccharides in a polyacrylamide gel. The properties of granulated guaran gel, used to purify by affinity chromatography the alpha-D-galactose-binding lectin from the seeds of Bandeiraea simplicifolia, were studied, as well as the binding of Concanavalin A (tetrameric) and succinylated Concanavalin A (dimeric) to a yeast mannan gel.     

Blood group B-specific lectin of Plecoglossus altivelis (Ayu fish) eggs

A lectin that agglutinates human blood group B erythrocytes but not blood group A and O erythrocytes was isolated from eggs of Ayu sweet fish (Plecoglossus altivelis). The lectin also agglutinates Ehrlich ascites carcinoma cells but not rat ascites hepatoma AH109 or rat sarcoma 150 cells tested. The lectin agglutination was most effectively inhibited by monosaccharides with the first type of configuration, i.e., L-rhamnose, L-mannose and L-lyxose at a concentration of 0.03 mM. The lectin agglutination was moderately inhibited by monosaccharides with the second type of configuration, i.e., D-galactose, D-fucose and D-galacturonic acid at a concentration of 0.4 mM. However, the agglutination was not inhibited by various other monosaccharides and oligosaccharides that have other types of configuration. The basis for an apparent B-specific hemagglutination may be due to the steric similarity of the C2 and C4 of the galactosyl series, the B-specific determinant, and the L-rhamnosyl-Sepharose column and was characterized as a homogeneous low molecular weight protein (Mr 14000) with an abundance of hydrophobic amino acids and dicarboxylic amino acid.     

Studies on plasma membranes. XXIII. Hormone-like action of concanavalin A on liver plasma membranes: inhibition of (Na+-K+)ATPase.

Concanavalin A inhibits the (Na+-K+)-ATPase activity of isolated rat-liver plasma membranes, while leaving the Mg2+-ATPase unaffected. Glucagon and cyclic AMP act supplementary to the lectin in the inhibition. The lectin effect is counteracted by insulin and L-epinephrine, and is completely abolished by the beta-adrenergic blocking agent propranolol. Results are discussed on the basis of the known interactions of concanavalin A with plasma membrane components, including its hormone-like action.     

Decrease in human lymphocyte surface glycoconjugates in leukemia as demonstrated by lectin binding

Qualitative variations in the glycoconjugates which make up the lectin receptor sites on the membranes of leukemic lymphocytes, compared with those of normal cells, have been studied by the use of three tritiated lectins: Robinia pseudoacacia lectin, Concanavalin A and Ricinus communis (var. Sanquineus) agglutinin (RCA 120). The binding specificity of these lectins has been demonstrated using specific determinants: alpha-methylmannoside and galactose for Concanavalin A and Ricinus communis agglutinin respectively. For the Robinia lectin this specificity was determined by saturation of the receptor sites with the unlabeled Robinia lectin before the addition of isotopically labeled Robinia lectin. The results show a decrease in the number of receptor sites on the leukemia cells, especially in chronic lymphoid leukemia, relative to that on normal cells. The apparent affinity constants of leukemic cells in all cases remain higher than those of normal cells.     

Lectin-associated proteins from the seeds of Leguminosae

The seeds of Pisum sativum (pea), Canavalia ensiformis, Vicia faba, Vicia sativa, and Ricinus communis were shown to contain proteins which are associated to the respective lectins (lectin binders). The lectin binders from Pisum sativum and Canavalia ensiformis were studied more closely. Both are single proteins not resembling the variety of membrane glycoproteins found in animals and plants which bind to lectins. The pea lectin binder is a tetrameric glycoprotein composed of identical subunits of the Mr 51 000. Its interaction with the lectin is abolished by acidic buffers or by glucose. The Concanavalin A binder, which does not contain sugar, is composed of one kind of subunit, Mr of 35 000. As in the case of the pea lectin binder, glucose and acid dissociate the lectin-lectin binder complex, but in contrast to the pea lectin binder low NaCl concentrations also cause this effect. During germination and growth, the Concanavalin A binder appears in the roots.     

Purification, characterization, and molecular cloning of lectin from winter buds of Lysichiton camtschatcensis (L.) Schott

A novel lectin was purified to homogeneity from winter buds of Lysichiton camtschatcensis (L.) Schott of the Araceae family. It was a tetramer composed of two non-covalently associated polypeptides with small subunits (11 kDa) and large subunits (12 kDa). Sequencing of both subunits yielded unique N-terminal sequences. A cDNA encoding the lectin was cloned. The isolated cDNA contained an open reading frame that encoded 267 amino acids. It encoded both subunits, indicating that the lectin is synthesized as a single precursor protein that is post-translationally processed into two different subunits with 45% sequence identity. Each subunit contained a mannose-binding motif known to be conserved in monocot mannose-binding lectins, but its activity was not inhibited by monosaccharides, including methyl α-mannoside. Asialofetuin and yeast invertase were potent inhibitors. Lectin activity was detected in the buds formed during the winter season but not in the expanded leaves.     

Conformational stability of legume lectins reflect their different modes of quaternary association: solvent denaturation studies on concanavalin A and winged bean acidic agglutinin

Thermodynamic parameters associated with the unfolding of the legume lectin, WBA II, were determined by isothermal denaturation. The analysis of isothermal denaturation data provided values for conformational stability and heat capacity for WBA II unfolding. To explore the role of intersubunit contact in stability, we carried out similar studies under identical conditions on Concanavalin A, a legume lectin of nearly similar size, buried hydrophobic surface area and tertiary structure to that of WBA II but with a different oligomerization pattern. Both proteins showed a reversible two-state unfolding with guanidine hydrochloride. As expected, the change in heat capacity upon unfolding was similar for both proteins at 3.5 and 3.7 kcal mol(-1) K(-1) for Concanavalin A and WBA II, respectively. Although the deltaG(H20) at the maximum stability of both proteins is around 16 kcal/mol, Concanavalin A exhibits greater stability at higher temperatures. The T(g) obtained for Concanavalin A and WBA II were 21 degrees C apart at 87.2 and 66.6 degrees C, respectively. The higher conformational stability at higher temperatures and the T(g) of Concanavalin A as compared to that of WBA II are largely due to substantial differences in the degree of subunit contact in these dimeric proteins. Ionic interactions and hydrogen bonding between the monomers of the two proteins also seem to play a significant role in the observed stability differences between these two proteins.     

Embryo Cell Surfaces—Lectin Binding and Cell Proliferation

The interaction between chick embryo fibroblasts and various lectins has been studied at different stages of embryo development. There is evidence that Robinia lectin, Dolichos lectin, and Conca navalin A decrease cell number and proportion of cells incorporating [³H] thymidine in case of 8and 10day-old chick embryo fibroblasts, whereas they stimulated the proliferation of 16-dayold embryo cells. No effect was noticed in 12-day cells.
These results suggest that some cell surface changes occur during embryo development. The site number of Dolichos lectin remains the same during embryo development, and the affinity constant decreases. The site number of Robinia lectin and Concanavalin A decreases from the 8^th to the 12^th day of development, and slowly increases on the 16–day cells, the affinity constant remaining rather constant.
The results indicate that the age–dependent effect of lectin on embryo cells could not be directly related to the number of lectin–binding sites. Competitive binding experiments revealed that Dolichos receptor sites were distincts from binding sites of Robinia lectin and Concanavalin A, and Robina receptor sites distinct from those of Concanavalin A.
Lectin effects on embryo fibroblasts were very specific as determined by inhibitory assays.     

Purification of the glycoprotein lectin from the broad bean (Vicia faba) and a comparison of its properties with lectins of similar specificity.

1. The lectin from the broad bean (Vicia faba) was purified by affinity chromatography by using 3-O-methylglucosamine covalently attached through the amino group to CH-Sepharose (an omega-hexanoic acid derivative of agarose). Its composition and the nature of its subunits were compared with concanavalin A and the lectins from pea and lentil. 2. Unlike the other three lectins, broad-bean lectin is a glycoprotein; a glycopeptide containing glucosamine and mannose was isolated from a proteolytic digest. 3. The mol.wt. is about 47500; the glycoprotein consists of two apprently identical subunits, held together by non-covalent forces. Fragments of the subunits, similar to those found in concanavalin A and soya-bean agglutinin, were found in active preparations. 4. Broad-bean lectin was compared with concanavalin A and the lectins from pea and lentil in an investigation of the inhibition of their action by a number of monosaccharides, methyl ethers of monosaccharides, disaccharides and glycopeptides. The most striking differences concern 3-O-substituted monosaccharides, which are strong inhibitors of the action of broad-bean, pea and lentil lectins but not of the action of concanavalin A. There is, however, no strong inhibition of the action of these lectins by 3-Olinked disaccharides.     

Blood group B-specific lectin of Plecoglossus altivelis (Ayu fish) eggs

A lectin that agglutinates human blood group B erythrocytes but not blood group A and O erythrocytes was isolated from eggs of Ayu sweet fish (Plecoglossus altivelis). The lectin also agglutinates Ehrlich ascites carcinoma cells but not rat ascites hepatoma AH109 or rat sarcoma 150 cells tested. The lectin agglutination was most effectively inhibited by monosaccharides with the first type of configuration, i.e., L-rhamnose, L-mannose and L-lyxose at a concentration of 0.03 mM. The lectin agglutination was moderately inhibited by monosaccharides with the second type of configuration, i.e., D-galactose, D-fucose and D-galacturonic acid at a concentration of 0.4 mM. However, the agglutination was not inhibited by various other monosaccharides and oligosaccharides that have other types of configuration. The basis for an apparent B-specific hemagglutination may be due to the steric similarity of the C₂ and C₄ of the galactosyl series, the B-specific determinant, and the L-rhamnosyl series, which are the best inhibitors of the lectin activity. The lectin was affinity purified on an L-rhamnosyl-Sepharose column and was characterized as a homogeneous low molecular weight protein (M_r 14 000) with an abundance of hydrophobic amino acids and dicarboxylic amino acid.     

Influence of Concanavalin A on 3-O-methylglucose uptake in cultured chick embryo fibroblasts

Abstract. Concanavalin A (Con A) was found to inhibit hexose uptake in cultured fibroblasts derived from 8-day chick embryos and to stimulate this process in those derived from 16day embryos. (1) Con-A effects depended on the duration of contact with cells and lectin and were inhibited by α-methylmannopyrannoside. (2) Con A was shown to mask about 70% of the hexose carriers in both 8- and 16-day embryo fibroblasts. Lectin altered the hexose uptake very rapidly. (3) Con A only modified the V^max of the up- take system and did not alter the K^m. This indicates that either the number or mobility of hexose carriers were modified by Con-A treatment. The differential effect of lectin could be due to a modification of the hexose-carrier mobility during the embryonic differentiation of fibroblasts. Secondary effects may affect cell growth.     

Alteration of macrophage surface in the course of immunological activation: decay of metabolic response to concanavalin A

“Activated” macrophages, isolated from lungs of BCG-infected rabbits, exhibited a greater perturbability of oxidative metabolism than their normal counterpart, when exposed in vitro to unrelated microorganisms or treated with phospholipase C. They were much less metabolically responsive when incubated with Concanavalin A. The metabolic effect of this lectin gradually decayed as time elapsed from the day of infection.These results are interpreted to show a modification of the surface properties of the macrophage in the course of the immunological activation. The possible mechanisms of generation of these surface alterations are discussed.     

Novel peptide surface for reversible immobilization of concanavalin A

Concanavalin A (Con A) was spontaneously adsorbed on polymyxin B surface. This peptide-lectin interaction was strong, K(D)=1.9 x 10(-10), based predominantly on creation of hydrophobic bonds, and was completely reversible. Concanavalin A on polymyxin B (PmB) retained higher binding capacity for yeast mannan, compared with covalently immobilized lectin. Kinetics of mannan-concanavalin A interaction were significantly different in dependence on type of concanavalin A immobilization.