Inhibition of Pair Formation by Concanavalin A and Concanavalin A-Binding Glycoconjugates in Euplotes octocarinatus

ABSTRACT. Concanavalin A (≥ 50 μg/ml) inhibits pair formation in both of the two complementary mating types of Euplotes octocarinatus studied in this investigation. This effect can be reversed by methyl-α- d -mannose. Concanavalin A is accessible for methyl-α- d -mannose until pairs are formed. Methyl-α- d -mannose as well as methyl-α- d -glucose and 2-acetamido-2-deoxy- d -glucose alone do not inhibit pair formation unless applied in concentrations ≥ 60 mM. The Concanavalin A-sensitive phase of preconjugant interaction starts 2 h after cells are induced to conjugate. Based on these observations we suggest that Concanavalin A might exhibit its action by binding to carbohydrate moieties of preconjugation-specific adhesion molecules and thereby might allosterically block interactions with their counterparts. To identify preconjugation-specific alterations in number or localization of Concanavalin A-binding glycoconjugates, we probed western blots of total cell proteins or fixed cells, respectively, with digoxigenin-labeled Concanavalin A. On Concanavalin A blots 20 different Concanavalin A-binding glycoconjugates were identified in mating-competent cells. Localization of Concanavalin A-binding sites on mating-competent cells by light microscopy resulted in predominant labeling of a comma-shaped structure near the paroral membranelle. During the preconjugation period no changes in number or localization of Con A-binding glycoconjugates were detected. Possible reasons are discussed.     

Interactions of lectins with (Na+ + K+)-ATPase of eel electric organ.

Interaction of lectins with a detergent-solubilized ATPase from eel electric organ was studied. Concanavalin A, which binds to alpha-mannosides, altered the rate of enzyme migration in agar and inhibited the formation of an antigen-antibody precipitate: other lectins had no such effects. Concanavalin A similar amounts partially inhibited (Na+ + K+)-ATPase; this inhibition was reversible by alpha-methylglucoside. There was no corresponding effect of concanavalin A on the potassium p-nitrophenylphosphatase. Concanavalin A also did not interfere with ouabain binding. Thus, concanavalin A binds to an antigenic region also involved in Na+ and/or ATP binding, but does not interact with a K+ site.     

Effect of lectins on the metabolism of sulfated glycosaminolycans in cultured fibroblasts.

A short exposure of human skin fibroblasts to Concanavalin A and wheat germ agglutinin led to an intra- and extracellular accumulation of sulfated glycosaminoglycans. The intracellular accumulation was caused by an impaired degradation of sulfated glycosaminoglycans. The increase of extracellular and cell surface associated 35S-labeled proteoglycans could be ascribed to a lectin-mediated inhibition of endocytosis of these polysaccharides. Results obtained with mono- and divalent Concanavalin A derivatives were in agreement with the view that lectins inhibit endocytosis of sulfated proteoglycans by binding to the cell surface receptors specific for these polysaccharides. Proteoglycans secreted by fibroblasts formed precipitable complexes with Concanavalin A. Complex formation reduced markedly the uptake of the proteoglycan. All effects on glycosaminoglycan metabolism mediated by Concanavalin A and wheat germ agglutinin could be prevented by methyl alpha-D-mannoside and N-acetylglucoseamine, respectively.     

Analogous ultrastructure and surface properties during capping and phagocytosis in leukocytes

Ultrastructural analyses have revealed striking similarities between Concanavalin A capping and phagocytosis in leukocytes. Both processes involve extensive membrane movement to form a protuberance or pseudopods; a dense network of microfilaments is recruited into both the protuberance and the pseudopods; microtubules are disassembled either generally (capping) or in the local region of the pseudopods (phagocytosis); and cells generally depleted of microtubules by colchicine show polarized phagocytosis via the microfilament-rich protuberance rather than uniform peripheral ingestion of particles via individual pseudopods. Cap formation can thus be viewed as occurring as an exaggeration of the same ultrastructural events that mediate phagocytosis. Similar changes in cell surface topography also accompany capping and phagocytosis. Thus, in nonfixed cells, Concanavalin A-receptor complexes aggregate into the region of the protuberance in colchicine-treated leukocytes (conventional capping) or into the region of pseudopod formation in phagocytizing leukocytes. In the latter case, the movement of lectin-receptor complexes occurs from membrane overlying peripheral microtubules into filament-rich pseudopods that exclude microtubules. These data provide evidence against a role for microtubules as "anchors" for lectin receptors. Rather, they indicate a preferential movement of cell surface Concanavalin A-receptor complexes towards areas of extensive (the protuberance) or localized (pseudopods) microfilament concentration. In conventional capping, Concanavalin A must be added to the colchicine-treated cells before fixation in order to demonstrate movement of receptors from a diffuse distribution into the protuberance. However, Convanavalin A receptors are enriched in the membrane associated with phagocytic particles as compared to the remaining membrane. This particle-induced redistribution of receptors is particularly prominent in colchicine-treated cells that phagocytize and are then fixed and Concanavalin A labeled; both lectin receptors and beads are concentrated over the protuberance. Thus, the final analogy between conventionally capped and phagocytic cells is that in both cases the properties of the plasma membrane in regions of microfilament concentration are modified by Concanavalin A itself (capping) or by the phagocytized particle, to limit locally the diffusion of Concanavalin A receptors.     

Effects of succinylation on thermal induced amyloid formation in Concanavalin A

We have recently shown that upon slight thermal destabilization the legume lectin Concanavalin A may undergo two different aggregation processes, leading, respectively, to amyloid fibrils at high pH and amorphous aggregates at low pH. Here we present an experimental study on the amyloid aggregation of Succinyl Concanavalin A, which is a dimeric active variant of Concanavalin. The results show that, as for the native protein, the fibrillation process appears to be favoured by alkaline pH, far from the isoelectric point of the protein. Moreover, it strongly depends on temperature and requires large conformational changes both at secondary and tertiary structure level. With respect to the native protein, the succinyl derivative forms amyloid fibrils in considerably longer times and with a minor exposure of hydrophobic regions. At physiological conditions, Concanavalin A still displays a sizeable tendency to form amyloid fibril, while the succinyl variant does not. A close correlation was observed between the progress of amyloid formation and a narrowing of the tryptophans fluorescence emission band, indicating a reduction of protein conformational heterogeneity in amyloid fibrils. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

Effect of lectin-binding to fibrinogen D and E domains on coagulation and fibrinolysis

The effect on fibrinogen coagulation and fibrinolysis of the mannose-specific lectins concanavalin A, its acetyl derivative and Lens culinaris agglutinin was studied. Concanavalin A and acetyl-concanavalin A, which bind to the four carbohydrate chains of fibrinogen, and L. culinaris agglutinin, which only binds to the carbohydrate present in fibrinogen D domains, has the same effect on the coagulation rate: an inhibition at low lectin concentrations and an increase at high concentrations. On the other hand, L. culinaris agglutinin does not alter fibrin crosslinking while acetyl-concanavalin A produces a slight inhibition of both gamma-gamma and alpha-polymer formation. However, this effect is very small when compared with the clear inhibitory effect produced by concanavalin A. Concanavalin A and acetyl-concanavalin A have an inhibitory effect on the rate of fibrin clot lysis proportional to the lectin concentration. Nearly 100% inhibition was obtained when two lectin-binding sites were occupied by either concanavalin A or acetyl-concanavalin A. However, L. culinaris agglutinin has a clearly weaker effect and more than 50% inhibition was not observed. The comparative study of the effect of the three lectins on fibrinolysis as well as on the formation of fibrinogen aggregates suggests that the inhibitory effect of concanavalin A and acetyl-concanavalin A is primarily due to their binding to the carbohydrate chains of fibrinogen E domain.     

Effect of lectins on the metabolism of sulfated glycosaminoglycans in cultured fibroblasts

A short exposure of human skin fibroblasts to Concanavallin A and wheat germ agglutinin led to an intra- and extracellular accumulation of sulfated glycosaminoglycans. The intracellular accumulation was caused by an impaired degradation of sulfated glycosaminoglycans. The increase of extracellular and cell surface associated ³⁵S-labeled proteoglycans could be ascribed to a lectin-mediated inhibition of endocytosis of these polysaccharides. Results obtained with mono- and divalent Concanavalin A derivatives were in aggreement with the view that lectins inhibit endocytosis of sulfated proteoglycans by binding to the cell surface receptors specific for these polysaccharides. Proteoglycans secreted by fibroblasts formed predipitable complexes with Concanavalin A. Complex formation reduced markedly the uptake of the proteoglycan. All effects on glycosaminoglycan metabolism mediated by Concanavalin A and wheat germ agglutin could be prevented by methyl α-D-mannoside and N-acetylglucosamine, respectively.     

Differential effects of concanavalin A and succinyl concanavalin A on the macromolecular events of platelet activation

Concanavalin A is capable of activating platelets in a concentration-dependent manner as judged by [14C]serotonin secretion from prelabeled platelets. In contrast, succinyl concanavalin A does not induce platelet secretion. Concanavalin A treatment also results in a number of alterations in platelet macromolecules which are presumably associated with the process of platelet activation. These include the phosphorylation of 20 and 47 kDa platelet proteins, the increased polymerization and association of new proteins with the platelet cytoskeleton and the association of the platelet membrane glycoprotein IIb/III complex with the platelet cytoskeleton. Succinyl concanavalin A treatment results in none of these macromolecular events. This difference is observed despite the demonstration that both lectins bind to the platelet surface. Gel overlay experiments also indicate that concanavalin A and succinyl concanavalin A bind to the same receptors. These differences in the biological effects of concanavalin A and succinyl concanavalin A on platelets may be due to decreased receptor crosslinking by the succinylated derivative. The formation of multiple linked interactions between surface receptors may be an important event in the activation of platelets by concanavalin A.     

Bacteriophage-resistant mutants of Escherichia coli K12 with altered lipopolysaccharide. Studies with concanavalin A.

Three classes of mutants of Escherichia coli K12, isolated by selection for resistance to lipopolysaccharide-specific bacteriophages, were agglutinated by Concanavalin A which is presumed to interact with the lipopolysaccharide component of the outer membrane. Wheat germ and soy bean agglutinins did not agglutinate the parent or mutant strains. The adsorption of certain bacteriophages was also inhibited by Concanavalin A. The pattern of inhibition of adsorption of bacteriophages suggests that non-specific masking of receptors may occur, as well as specific masking of terminal glucose residues. Although bacteria were agglutinated by Concanavalin A, the permeability of the outer membrane seemed unaffected.     

Interactions of lectins with (Na+ + K+)-ATPase of eel electric organ

Interaction of lectins with a detergent-solubilized ATPase from eel electric organ was studied. Concanavalin A, which binds to α-mannosides, altered the rate of enzyme migration in agar and inhibited the formation of an antigen-antibody precipitate; other lectins had no such effects. Concanavalin A similar amounts partially inhibited $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$; this inhibition was reversible by α-methylglucoside. There was no corresponding effect of concanavalin A on the potassium $\text{p-}\text{nitrophenyl-phosphatase}$. Concanavalin A also did not interfere with ouabain binding. Thus, concanavalin A binds to an antigenic region also involved in Na⁺ and/or ATP binding, but does not interact with a K⁺ site.     

Effect of lectin-binding to fibrinogen D and E domains in coagulation and fibrinolysis

The effect of fibrinogen coagulation and fibrinolysis of the mannose-specific lectins concanavalin A, its acetyl derivative and Lens culinaris agglutinin was studied. Concanavalin A and acetyl-concanavalin A, which bind to the four carbohydrate chains of fibrinogen, and L. culinaris agglutinin, which only binds to the carbohydrate present in fibrinogen D domains, has the same effect on the coagulation rate: and inhibition at low lectin concentrations and an increase at high concentrations. On the other hand, L. culinaris agglutinin does not alter fibrin crosslinking while acetyl-concanavalin A produces a slight inhibition of both γ-γ and α-polymer formation. However, this effect is very small when compared with the clear inhibitory effect produced by concanavalin A. Concanavalin A and acetyl-concanavalin A have an inhibitory effect on the rate of fibrin clot lysis proportional to the lectin concentration. Near 100% inhibition was obtained when two lectin-binding sites were occupied by either concanavalin A or acetyl-concanavalin A. However, L. culinaris agglutinin has a clearly weaker effect and more than 50% inhibition was not observed. The comparative study of the effect of the three lectins on fibrinolysis as well as on the formation of fibrinogen aggregates suggests that the inhibitory effect of concanavalin A and acetyl-concanavalin A is primarily due to their binding to the carbohydrate chains of fibrinogen E domain.     

Effect of concanavalin A on the activity of membrane-bound and detergent-solubilized Mg2+ -ATPase

Plasma membranes were isolated after binding liver and hepatoma cells to polylysine-coated polyacrylamide beads, and the effect of concanavalin A on the membrane-bound Mg2+ -ATPase and the Mg2+ -ATPase solubilized by octaethylene glycol monododecyl ether (C12E8) was studied. In the experiment of membrane-bound Mg2+ -ATPase, plasma membranes were pretreated with Concanavalin A and the activity was assayed. Concanavalin A stimulated the activity of both liver and hepatoma enzymes assayed above 20 degrees C. Concanavalin A abolished the negative temperature dependency characteristic of liver plasma membrane Mg2+ -ATPase. On the other hand, Concanavalin A prevented the rapid inactivation due to storage at -20 degrees C, which was characteristic of hepatoma plasma membrane Mg2+ -ATPase. With solubilized Mg2+ -ATPase from liver plasma membranes, the negative temperature dependency was not observed. Concanavalin A, which was added to the assay medium, stimulated the activity of the enzyme solubilized in C12E8 at a high ionic strength. However, Concanavalin A failed to show any effect on the enzyme solubilized in C12E8 at a low ionic strength. With solubilized Mg2+ -ATPase from hepatoma plasma membranes, Concanavalin A could not prevent the inactivation of the enzyme during incubation at -20 degrees C.     

Suppression of a thymus dependent humoral response in mice by concanavalin A in vivo

Mice treated with Concanavalin A prior to immunization with sheep erthyrocytes exhibit a markedly reduced plaque forming spleen cell response. This immunosuppressive effect could be reversed by using higher doses of antigen or priming the animals with nonimmunizing doses of antigen prior to Concanavalin A injection designed to either by-pass or enhance thymus derived lymphocyte functions. It was also demonstrated that Concanavalin A in vivo activated the thymus derived lymphocyte subpopulation in the spleen, and this activation was dose dependent and correlated with the immunosuppression observed. Animals injected with Concanavalin A at various times prior to whole body lethal irradiation would not support the plaque forming cell response of adoptively transferred normal syngeneic spleen cells. This effect was shown to be time and dose of Concanavalin A dependent. It was also shown that the route of injection of Concanavalin A prior to irradiation determined the results observed, in that the intravenous route resulted in the suppression of transferred cells, while the intraperitoneal route showed no effect. It is suggested that Concanavalin A induced immunosuppression of the humoral, thymus dependent immune response in mice results for the activation of a subpopulation of thymus derived suppressors cells, and that the effect is short lived, radiation resistant, and dose of Concanavalin A and antigen dependent.     

Role of the membrane concanavalin A binding site in platelet-fibrin interactions

Concanavalin A was employed to study the role of platelet membrane glycoproteins in platelet-fibrin interactions during clot formation. A rheological technique was used to study the interactions, measuring the clot rigidity and platelet contractile force simultaneously during the formation of network structure. Concanavalin A lowered the clot rigidity and contractile force of a platelet-rich plasma clot by a small extent. Plasma glycoproteins probably compete with platelet membranes for concanavalin A binding in platelet-rich plasma. Both native concanavalin A (tetrameric) and succinyl concanavalin A (dimeric) lowered the clot rigidity and contractile force of a washed platelet-fibrin clot dramatically, almost down to those values found for fibrin clots. Inhibition studies with alpha-methyl-D-mannoside indicated that the concanavalin A effects were specific for the concanavalin A binding capacity to platelets. The effects of native concanavalin A on platelet-fibrin clots were only partially reversible, while the succinyl concanavalin A effects were completely reversible. The observed concanavalin A effects are probably mainly due to concanavalin A binding to platelet membrane glycoproteins. The concanavalin A binding site appears to play an important role in the fibrin binding to platelets.     

Fluctuation methods to study protein aggregation in live cells: concanavalin A oligomers formation

Prefibrillar oligomers of proteins are suspected to be the primary pathogenic agents in several neurodegenerative diseases. A key approach for elucidating the pathogenic mechanisms is to probe the existence of oligomers directly in living cells. In this work, we were able to monitor the process of aggregation of Concanavalin A in live cells. We used number and brightness analysis, two-color cross number and brightness analysis, and Raster image correlation spectroscopy to obtain the number of molecules, aggregation state, and diffusion coefficient as a function of time and cell location. We observed that binding of Concanavalin A to the membrane and the formation of small aggregates paralleled cell morphology changes, indicating progressive cell compaction and death. Upon protein aggregation, we observed increased membrane water penetration as reported by Laurdan generalized polarization imaging.     

Interaction of Concanavalin A with the Cell Wall of Bacillus subtilis

Interactions between concanavalin A and cell wall digests of Bacillus subtilis 168 resulted in insoluble complexes as observed by double gel diffusion, turbidity, and analysis of the precipitate. The macromolecular constituent of the cell walls complexing with concanavalin A was the polyglucosylglycerol phosphate teichoic acid. The complex exhibited two pH optima: 3.1 and 7.4. The complex could be dissociated by saccharides which bind to concanavalin A. In contrast to concanavalin A-neutral polysaccharide complexes, formation of the concanavalin A-wall complex was inhibited by salts. It was subsequently shown that salts induce conformational changes in cell wall digests. The data suggested that for complex formation to occur a rigid rod conformation in the glucosylated teichoic acid is probably necessary. Concanavalin A can be used as a probe to study structural features of bacterial cell walls.     

Cell surface differences of Naegleria fowleri and Naegleria lovaniensis exposed with surface markers

Differences in the distribution of diverse cell surface coat markers were found between Naegleria fowleri and Naegleria lovaniensis. The presence of carbohydrate-containing components in the cell coat of the two species was detected by selective staining with ruthenium red and alcian blue. Using both markers, N. fowleri presented a thicker deposit than N. lovaniensis. The existence of exposed mannose or glucose residues was revealed by discriminatory agglutination with the plant lectin Concanavalin A. These sugar residues were also visualized at the cell surface of these parasites either by transmission electron microscopy or by fluorescein-tagged Concanavalin A. Using this lectin cap formation was induced only in N. fowleri. The anionic sites on the cell surface detected by means of cationized ferritin were more apparent in N. fowleri. Biotinylation assays confirmed that even though the two amoebae species have some analogous plasma membrane proteins, there is a clear difference in their composition.     

Plant lectins detect age and region specific differences in cell surface carbohydrates and cell reassociation behavior of embryonic mouse cerebellar cells

When plated at high cell density in a microwell culture system, freshly dissociated embryonic mouse cerebellar cells assemble into reproducible, 3-dimensional patterns. The addition of the dimeric lectin Succinyl Concanavalin A blocks reversibly the formation of the microwell pattern, suggesting that cell surface carbohydrates affect the reassociation behavior of embryonic mouse cerebellar cells. Agglutination studes of dissociated cell populations harvested from different regions of the embryonic brain reveal that different lectins agglutinate cell populations from different embryonic brain regions. Cells from E13 cerebellum are agglutinated with Concanavalin A, wheat germ agglutinin, Ricinus communis agglutinin, mol wt 60,000, Ricinus communis agglutinin, mol wt 120,000, and Lens culinaris, but not by soybean agglutinin or a fucose-binding protein. Cells from the midbrain are agglutinated only with Concanavalin A, Ricinus communis agglutinin, mol wt 60,000 and Ricinus communis agglutinin, mol wt 120,000; those from the cerebral cortex are agglutinated only with Lens culinaris; and those from the medulla are agglutinated only with Ricinus communis agglutinin, mol wt 60,000, and Ricinus communis agglutinin, mol wt 120,000. In addition, agglutination of cerebellar cells with Concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinin is diminished over the course of development from embryonic day 13 to postnatal day 7. These studies suggest regional differences in the cell surfaces of the developling brain that are further modulated during the differentiation of the tissues. On a poly(D-lysine) treated substrate in microwell cultures, cell migration is unique to the cerebellum of the 4 brain regions studied. Surfaces treated with carbohydrate-derivatized poly(D-lysine) are currently being tested for their efficacy as substrates for differential cell migration.     

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.     

Partial characterization of an allergenic glycoprotein from peanut (Arachis hypogaea L.)

A concanavalin A-reactive glycoprotein allergen has been isolated from peanut (Arachis hypogaea). The allergen was separated by affinity chromatography and purified by gel permeation and ion-exchange chromatography. The monomeric molecular weight is 65,000 and the pI is 4.6. The presence of one cysteine residue per molecule results in some dimer formation. Concanavalin A-reactive glycoprotein is a potent allergen for peanut-sensitive patients in both in vivo and in vitro tests. It is allergenically stable, on in vitro examination, at temperatures of up to 100 degrees C and over the pH range 2.8-10. Removal of the carbohydrate moiety failed to eliminate the allergenicity. Concanavalin A-reactive glycoprotein is identified in the crossed immunoelectrophoretic pattern as a major antigen of peanut protein extract but its structural characteristics indicate that it is probably not a component of the major storage-protein complex, arachin.