On the susceptibility of human platelets to aggregation by concanavalin A and the effect of this lectin on their response to ADP

Concanavalin A aggregated gel-filtered platetes in 0.9% NaCl solution signifying cross-bridging by the lectin. Aggregation of these platelets by concanavalin A was temperature dependent; it did not occur at 0–4 °C unless the platelets were previously trypsinized. The level of aggregation of trypsinized platelets by concanavalin A at 0–4°C was similar to that of untreated platelets at 37°C. It is suggested that trypsin facilitates platelet aggregation by concanavalin A at 0–4°C by causing a configurational change in membrane glycoproteins which orientates concanavalin A receptor sites into positions that favour lectin cross-bridging. Concanavalin A failed to aggregate platelets in plasma. Radioisotope studies showed that the amount of [³H]concanavalin A which combined with platelets in plasma was extremely low compared with gel-filtered platelets in saline. The aggregation of Ehrlich ascites cells by concanavalin A was considerably reduced when platelet-free plasma was added to the medium suggesting that it was due to the presence of concanavalin A-reactive components in the plasma.Concanavalin A inhibited the ADP-induced aggregation of platelets suspended in plasma or in a salts solution supplemented with calcium and fibrinogen, although the inhibitory effect was more conspicuous in the latter case. The results suggests that concanavalin A produces its inhibitory effect on ADP-induced platelet aggregation by interacting with membrane glycoproteins, and this further suggests their involvement in aggregation.     

Protection of sodium dodecyl sulfate-induced aggregation of concanavalin a by saccharide ligands

Concanavalin Å is visibly aggregated by low concentrations of sodium dodecyl sulfate, maximum aggregation being obtained at pH 4.6. Other denaturants, such as urea, guanidine hydrochloride, Triton X-100, cetyltrimethylammonium bromide, Tween 80, and Brij 35 are ineffective in promoting visible aggregation. The sodium dodecyl sulfate-induced aggregation of concanavalin Å requires the presence of an intact, saccharide-ligand binding-site. Rapid and complete reversal of the detergent effect was achieved by use of saccharides which bind to the lectin. Such compounds as tryptophan and o-nitrophenyl β-d-galactopyranoside did not inhibit the aggregation of concanavalin Å by sodium dodecyl sulfate, suggesting that the detergent does not bind the hydrophobic pocket on the surface of the protein. The results suggest that concanavalin Å may have an additional, ligand-binding site which is netal-dependent and which can be modified by the addition of a saccharide ligand.     

Interaction energies in lectin-induced erythrocyte aggregation

Two N-acetylgalactosamine-reactive lectins, Helix pomatia (HPA) and Dolichos biflorus (DBA), were used to study the energies involved in cell-cell interactions through the specific binding of these lectins to their membrane receptors on genotype AO human erythrocytes (red blood cells) (RBCs). The energy required to dissociate a unit of aggregated membrane area (gamma d) of two RBCs bridged by lectin molecules was determined from the shear force needed to dissociate two-cell aggregates in a flow channel. When HPA were used as bridging molecules, gamma d (0.4 X 10(-4) to 3.8 X 10(-4) dyn/cm) was proportional to the density (D = 175 to 1,060 molecules/micron 2) of HPA molecules bound on the RBC membrane. A similar gamma d/D ratio was also obtained for DBA. These results indicate that the number of lectin molecules bound on the interface plays an important role in determining the energy required for cell-cell dissociation. The aggregation energy per unit membrane area (gamma a) in lectin-induced aggregates was calculated from the degree of encapsulation of a lectin-bound, heat-sphered human RBC by a normal discoid RBC. A minimum of approximately 1,800 HPA molecules/micron 2 on the spheres was required to form stable aggregates with the RBC. By using spheres having a surface HPA density of 1,830 to 2,540 molecules/micron 2, or 1.1-1.5 X 10(12) combining sites/cm2, the gamma a value for HPA-induced aggregation was found to be 2.2 X 10(-3) dyn/cm. This higher value of gamma a than gamma d has been explained on the basis of several differences in aggregation and disaggregation processes. The gamma a value for DBA-induced aggregation was not obtainable by the sphere encapsulation method because of the relative low D values. A comparison of the present results with the published value of the free energy change of 5 kcal/mol for the interactions of HPA and DBA with their ligands suggests that only a small fraction of the lectin molecules bound to RBC surface participate in the bridging of adjacent cells.     

Distribution of concanavalin A in fibroblasts: direct endocytosis versus surface capping.

Indirect immunofluorescence of intact or acetone-extracted cells has allowed us to distinguish concanavalin A (Con A) which is associated with the plasma membrane of CHO cells from Con A which has been interiorized. We find that Con A is directly endocytized by these cells with no intervening stage of plasma membrane aggregation. The lectin accumulations observed by direct fluorescence are actually cytoplasmic collections of pinosomes which contain Con A. Only in a small fraction of CHO cells are true plasma membrane aggregates, or caps, found. This predominance of direct pinocytic interiorization over capping was not affected by dibutyryl cAMP or by treatments which can disrupt microtubules, including cold shock or exposure of the cells to anti-mitotic agents. Cytochalasin B, however, inhibited the uptake of Con A and at the same time promoted the formation of large surface aggregates of the lectin, or minicaps. Capping may reflect a competition between aggregation in the plane of the membrane and direct interiorization of bound lectin. Surface cap formation may be a characteristic process of cells with very low endocytic rates, such as lymphocytes.     

Synthetic glycolipids and the lectin-mediated aggregation of liposomes.

Synthetic mannose-containing glycolipids utilizing the cholesterol nucleus as a lipid anchor, and either the 6-aminohexyl- or the 6-(6-aminohexanamido)hexyl-1-thio-alpha-D-mannopyranosides as the carbohydrate ligands, have been synthesized and incorporated into small unilamellar liposomes. Incorporation of these cholesterol-mannoside derivatives at concentrations up to 14 mol% apparently does not affect the physical characteristics of the liposomes. Addition of concanavalin A to a suspension of liposomes containing the long chain cholesterol-mannose derivative causes an increase in light-scattering at 360 nm. As the increase in absorbance is completely reversed by the addition of alpha-methylmannoside, aggregation rather than fusion of the liposomes appears to be occurring. Liposomes containing 14 mol % of the short chain (6-aminohexyl-) derivative are aggregated by concanavalin A indicating that the lectin can approach to within 10 A of the lipid bilayer. Preliminary results suggest that the aggregation of vesicles containing either the long or short chain derivatives is highly dependent on the density of the sugar in the membrane.     

Enhanced concanavalin a agglutination of trypsinised erythrocytes is due to a specific class of aggregation

Using the output of a rotational viscometer as a continuous index of aggregation, we have shown previously that the concanavalin A agglutination of native human erythrocytes can be resolved into three distinct classes of aggregation, static, type I and type II. Static aggregation occurs in the absence of shear forces while both type I and II aggregations are shear-induced. We now report that the increased concanavalin A agglutination of trypsinised erythrocytes is attributable to a specific enhancement in the development of type II aggregation. While type II formation in native cell suspensions requires high concanavalin A concentrations and continual shearing, an indistinguishable type of aggregation develops in suspensions of trypsinised red cells at considerably lower lectin concentrations and in the absence of applied shear forces.     

Fibrinogen and fibrin interaction with concanavalin a dimer and its influence on coagulation

Concanavalin A dimer interacts with fibrinogen and soluble fibrin at pH 5.2 Analysis of the binding data shows that there are in both cases four binding sites per molecule and that the dissociation constant does not change by removal of fibrinopeptides A and B. Ultracentrifugal studies shows that no aggregates of fibrinogen or fibrin are formed through concanavalin A binding and that up to four molecules of concanavalin A dimer can be bind to one molecule of fibrinogen or fibrin. These results imply that the four carbohydrate chains in the molecule are accessible to concanavalin A dimer. There is a diminution in the coagulation of fibrinogen by thrombin at low relative lectin concentrations and an increase at high concentrations. However, the lectin always favours the aggregation of fibrin monomers and does not have any inhibitory effect on the release of fibrinopeptides. We conclude that the electric charge in the neighbourhood of the carbohydrate in both chains, Bβ and γ plays an important role in the attraction between monomeric fibrin and fibrinogen-monomeric fibrin. The different effect of concanavalin A on the coagulation, depending on the relative concentration of the lectin, would be the result of the screening of this electric charge favouring either the interaction of fibrinogen-monomeric fibrin or the polymerization of monomeric fibrin.     

Neo-mannosylated liposomes: synthesis and interaction with mouse Kupffer cells and resident peritoneal macrophages

In order to target liposomes to cells expressing at their surface mannose receptors, e.g. mouse Kupffer cells and peritoneal macrophages, we have developed a new synthetic strategy which allows a chemically well defined preparation of neo-mannosylated vesicles. alpha-D-Thiomannopyranoside residues, substituted with a hydrophilic spacer arm and functionalized with a sulfhydryl group, were covalently coupled to preformed large unilamellar vesicles containing 4-(p-maleimidophenyl)butyryl phosphatidylethanolamine. Liposomes, containing 15 mol% of mannosyl residues, were specifically aggregated with concanavalin A; this aggregation could be reversed by an excess of free methyl alpha-D-mannopyranoside indicating that the surface ligands were freely accessible to the lectin. The neo-mannosylated liposomes presented in vitro an increased binding to cells possessing alpha-D-mannose specific binding sites. At 37 degrees C a specific binding, up to 9-fold compared to control vesicles, was observed. These neo-mannosylated vesicles represent attractive tools for targeting bio-active molecules to macrophage-associated diseases.     

CD23 molecule acts as a galactose-binding lectin in the cell aggregation of EBV-transformed human B-cell lines

Epstein-Barr virus (EBV)-transformed human B-cell lines, L-KT9and DH3 cells express CD23 antigen, and grow in a mixture ofsingle and aggregated cells. The CD23 molecule has high aminoacid sequence homology with C-type lectin and recently we haveshown that the solubilized CD23 molecule can really interactwith galactose residues on glycoproteins. In this study, therefore,we tested whether CD23 antigen on the cell surface really actsas a galactose-binding lectin in the aggregation of these cells.The EBV-transformed cells (L-KT9) were separated into an aggregated-cell-richfraction and a single-cell-rich fraction. Aggregated cells disaggregatedafter removal of galactose by ß-galactosidase treatment,whereas single cells made large aggregation on sialidase treatment,and this aggregation was inhibited in the presence of asialo-fetuin.On the other hand, naturally aggregated cells become singlecells with anti-CD23 monoclonal antibody (mAB) as well as thesoluble form of CD23, but not with anti-CD21 mAB. In addition,L-KT9 and DH3 cells bound to asialo-fetuin-coupled Sepharose(ASF-Sepharose) and this binding was significantly inhibitedby pre-treatment of cells with anti-CD23, but not with anti-CD21or other antiadhesion molecules. From these results, we concludethat the naturally aggregated state of EBV-transformed cellsoccurs mainly through the interaction of CD23 as a lectin moleculeand galactose residues as its ligand. CD23 molecule cell aggregation EBV-transformed B cells glycosidase treatment low-affinity IgE receptor     

Reactivity with lectins of the saccharide components of rhodopsin in reconstituted membranes. Orientation of the carbohydrates

Rhodopsin-containing liposomes may provide a model for investigating the interaction of intrinsic membrane glycoproteins in biological systems. As part of the characterization of this preparation, the surface orientation of the carbohydrates of rhodopsin, assembled from purified bovine rhodopsin and egg phosphatidylcholine was examined, and is the topic of this report. The major tool used in these studies was the interaction with the carbohydrate-specific reagents, plant lectins. Two techniques were used: lectin-mediated aggregation of the liposomes, as measured by light scattering; the binding of 125I-labeled succinylated concanavalin A, and Scatchard analysis as a measure of affinity. The preparation most extensively examined had a mole ratio of rhodopsin:phospholipid of 1:100. Among a variety of lectins which were examined, only concanavalin A, succinylated concanavalin A, and wheat germ agglutinin were able to mediate the aggregation of rhodopsin-containing liposomes, as expected. The aggregation with concanavalin A was prevented by the presence of sugars having the alpha-D-glucopyranosyl configuration, and that brought about with wheat germ agglutinin, by N-acetylglucosamine (GlcNAc). In addition, the aggregation with concanavalin A was reversed with methyl alpha-D-mannoside, and with wheat germ agglutinin, by GlcNAc, suggesting that membrane fusion did not take place. On a molar basis, wheat germ agglutinin brought about a greatly reduced extent of aggregation as compared to concanavalin A, suggesting the relative inaccessibility of GlcNAc residues in the liposomes as compared to mannose. The initial rate of the aggregation, however, were similar with either lectin. The first-order rate constants were unaffected by wide variation in the concentrations of concanavalin A and wheat germ agglutinin, and by variation in the mole ratios of rhodopsin in the liposomes from 0.2 to 19 moles per 100 moles of egg lecithin. Rhodopsin-liposomes were also prepared from a total lipid extract of rod outer segments instead of egg lecithin. Similar kinetic properties were exhibited by this preparation as were obtained with the liposome prepared with the purified phospholipid. Scatchard analysis of the binding of 125I-labeled succinylated concanavalin A by rhodopsin liposomes indicated the presence of a single class of binding site as the preferred fit, with an apparent Kd of 2.8 X 10(-7) M. The binding was destroyed or extensively interfered with by trypsinization and by periodate treatment.     

Binding of human fibroblast interferon to concanavalin A-agarose. Involvement of carbohydrate recognition and hydrophobic interaction.

Human fibroblast interferon binds to a concanavalin A-agarose (Con A-Sepharose) equilibrated with methyl alpha-D-mannopyranoside, or levan; in contrast, it is only partially retarded on a similar column equilibrated with ethylene glycol. Interferon does not bind, however, to a lectin column equilibrated with both methyl alpha-D-mannopyranoside and ethylene glycol. Thus, a hydrophobic interaction between fibroblast interferon and the immobilized lectin seems to account for a large portion of the binding forces involved. Other hydrophobic solutes, such as dioxane, 1, 2-propanediol, and tetraethylammonium chloride, were found equally or more efficient than ethylene glycol in displacing interferon from the lectin column. The elution pattern of interferon from a concanavalin A-agarose (Con A-Sepharose) column, at a constant ehtylene glycol concentration and with an increasing mannoside concentration, reveals the existence of four distinct interferon components. The selective adsorption to and elution from a concanavalin A-agarose (Con A-Sepharose) column resulted in about a 3000-fold purification of human fibroblast interferon and complete recovery of activity. The specific activity of the partially purified interferon preparation is about 5 X 10(7) units per mg of protein. The chromatographic behavior of human leukocyte interferon is remarkable in that it does not bind to concanavalin A-agarose at all indicating the absence of carbohydrate moieties recognizable by the lectin, or if present, their masked status. When concanavalin A was coupled to an agarose matrix (cyanogen bromide activated) at pH 8.0 and 6.0 human fibroblast interferon bound to both lectin-agarose adsorbents and could be recovered with methyl alpha-D-mannopyranoside. Concanavalin A, immobilized directly on agarose matrix at pH 8.0 and 6.0, thus displays only carbohydrate recognition toward interferon. By contrast, unless a hydrophobic solute was included in the solvent containing methyl mannoside, human fibroblast interferon could not be recovered from concanavalin A-agarose coupled at pH 9.0. When concanavalin A was immobilized via molecular arms, in tetrameric as well as dimeric forms, the binding of interferon again occurred exclusively through carbohydrate recognition. Thus, the hydrophobic interaction can be eliminated by appropriate immobilization of the lectin, and then adsorbed glycoproteins, as exemplified here by interferon, can be recovered readily with methyl mannoside alone.     

Approach to the mechanism of Zajdela's hepatoma cell growth inhibition induced by concanavalin A and phytohaemagglutinin

Cell growth of tumour ascites cells was inhibited by concanavalin A, phytohaemagglutinin and Ricinus lectin at 2-100 micrograms/ml. As expected, the Ricinus lectin inhibited the protein synthesis estimated by leucine incorporation and decreased thymidine incorporation, whereas concanavalin A and phytohaemagglutinin stimulate the uptake and the incorporation of both leucine and thymidine, and thus, synthesis of protein and DNA. These results suggest that different mechanisms are involved in the hepatoma cell growth inhibition by the lectins. This difference was not related to the kinetic characteristics of the lectin interactions with the cells which represent a first and necessary step. It was showed that concanavalin A and phytohaemagglutinin as well as chloroquine inhibited the 14C-labelled asialofetuin degradation. We can conclude that Ricinus lectin present a toxic effect whereas both concanavalin A and phytohaemagglutinin show an anti-protease activity.     

The interaction of N-acetylhexosaminidase with insolubilized concanavalin A.

The specific interaction between human N-acetylhexosaminidase and concanavalin A was evaluated with respect to temperature, time, pH and concentration of specific ligand in incubation mixtures containing the enzyme and insolubilized lectin. Elution of the enzyme from insolubilized concanavalin A is dependent on both temperature and concentration of alpha-methyl mannoside. Conditions for a high yield of the enzyme from chromatography on insolubilized concanavalin A are described.     

<Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp7 produces an outer-membrane lectin that specifically binds to surface-exposed extracellular polysaccharide produced by the bacterium

Azospirillum sp promotes the growth of many important crop plants. We demonstrated lectin binding activity in outer-membrane protein extracts of A. brasilense Sp7 by hemagglutination assays. The lectin specifically recognised the exopolysaccharide (EPS) produced by aggregated cells. Affinity chromatography using EPS-Sepharose was used to identify a 67 kDa outer-membrane lectin (OML) that recognised a binding region in the extracellular polysaccharide. Results show the specific recognition and binding between EPS and OML. The potential relationship between cell-to-cell aggregation and the OML–EPS interaction is discussed. Paola Mora and Federico Rosconi contributed equally to this study. Laura Franco Fraguas and Susana Castro-Sowinski equally supervised this study.     

Immobilization of glycoenzymes using crude concanavalin A and glutaraldehyde

Insoluble concanavalin A complexes of glucose oxidase and amyloglucosidase have been prepared by mixing the glycoenzyme solutions with either pure concanavalin A or a buffer extract of jackbean meal. The complexes obtained using excess lectin exhibited relatively low specific activity compared with those obtained with low lectin concentration. The crosslinked complexes, however, exhibited relatively higher thermostability. In addition, glutaraldehyde treated concanavalin A-glucose oxidase complexes, especially those prepared using excess lectin, exhibited significantly broader pH-activity profiles compared to the soluble enzyme. Electrophoresis of the concanavalin A-glucose oxidase and concanavalin A-amyloglucosidase from pure lectin and from crude extract showed similar protein composition. The concanavalin A-amyloglucosidase complex was crosslinked to obtain relatively large (0.15–0.17 mm diameter) and homogeneous particles by treatment with 2% glutaraldehyde followed by homogenization. Glutaraldehyde treatment, however, caused a marked decrease in the retained activity of the particles, especially on starch. The crosslinked concanavalin A-amyloglucosidase preparations were, however, only slightly more stable against thermal denaturation than the soluble enzyme.     

A study on concanavalin a binding to human erythrocytes

Interactions of concanavalin A with human erythrocytes were studied using ¹²⁵I-labelled concanavalin A and a centrifugal technique with dibutyl phthalate which permitted complete separation of bound and free concanavalin A. Binding of ¹²⁵I-labelled concanavalin A to human erythrocytes was dependent on cell concentration, pH and temperature. Specificity of binding was confirmed by inhibition and dissociation studies with sugars and native concanavalin A. Positive cooperative binding of concanavalin A to human erythrocytes was observed at low concanavalin A concentrations (less than 1 μ/ml) in both buffers studied. Positive cooperativity at higher concanavalin A concentrations (more than 100 μ/ml) was seen in Tris-Hepes buffer but not in phosphate-buffered saline. Consistent with this cooperative effect was the observation that although dissociation of ¹²⁵I-labelled concanavalin A from the erythrocytes was complete in the presence of 1 mg/ml of the native lectin, release was inhibited by low concentrations (1 μ/ml). A comparison of concanavalin A binding with hemagglutination studies suggest that the amount of concanavalin A bound determines the rate of erythrocyte agglutination and the size of the aggregates formed.     

Amyloid fibrils formation and amorphous aggregation in concanavalin A

We here report an experimental study on the thermal aggregation process of concanavalin A, a protein belonging to the legume lectins family. The aggregation process and the involved conformational changes of the protein molecules were followed by means of fluorescence techniques, light scattering, circular dichroism, zeta potential measurements and atomic force microscopy. Our results show that the aggregation process of concanavalin A may evolve through two distinct pathways leading, respectively, to the formation of amyloids or amorphous aggregates. The relative extent of the two pathways is determined by pH, as amyloid aggregation is favored at high pH values ( approximately 9), while the formation of amorphous aggregates is favored at low pH ( approximately 5). At difference from amorphous aggregation, the formation of amyloid fibrils requires significant conformational changes on the protein, both at secondary and tertiary structural level. To our knowledge, this is the first observation of amyloid fibrils from concanavalin A.     

Studies on oxalate oxidase from beet stems upon immobilization on concanavalin A.

Oxalate oxidase (EC 1.2.3.4) was purified from beet stems and immobilized on concanavalin A. The bound enzyme showed a high resistance of denaturation and increased the storage stability at 4 degrees C. The immobilized oxidase showed a broad optimum at pH 3.5-5, compared to the free enzyme with a sharp optimum at pH 4.5. There was a 3-fold increase in the apparent Km value on immobilization. The lectin interaction also eliminated the inhibitory effect produced on the enzyme by azide, nitrate and glycollate. The stimulatory effect on the enzyme activity by the flavins was not seen with the bound enzyme. The interaction of oxidase on concanavalin A-Sepharose 4B column and its reversal with methyl alpha-D-mannoside, indicated the presence of polysaccharides. The glycoprotein nature was further confirmed by periodic acid-sciff staining procedure of the enzyme after gel electrophoresis.     

Interactions between chondroitin sulfate and concanavalin A

Chondroitin sulfate, the major extracellular matrix glycosaminoglycan, formed an insoluble complex with concanavalin A at pH 5.4 or below. Concanavalin A (500 μg/ml) reacted only with a relatively narrow concentration range of chondroitin sulfate (optimally between 5 and 50 μg/ml) at pH 5.4 in 0.05 M buffer. Similar precipitin-like interactions were seen between concanavalin A and hyaluronic acid or heparin. No precipitating complexes formed between concanavalin A and the glycosaminoglycans at these concentrations in physiological salt solutions (approx. 0.15 M) unless the pH was below 4.5. Precipitating self-aggregates of concanavalin A appeared to be promoted by chondroitin sulfate at pH 7.3, but no significant precipitation occurred between the reactants at this pH even at very high concentrations, nor did soluble complexes form as determined by affinity chromatography on Sephadex G-200 or fractionation on Bio-Gel P-200. Thus, binding between the lectin and glycosaminoglycans appeared to depend upon reversible non-specific electrostatic interactions observed only at low pH and low ionic strength. Stable interactions were not seen in experiments using physiologically balanced salts at near neutral pH.