Concanavalin A modulates a potassium channel in cultured Aplysia neurons

A novel 100 pS K(+)-selective ion channel is frequently observed in cell-attached membrane patches from cultured Aplysia neurons. The activity of this channel is moderately voltage-dependent, but channel openings are rare and brief even when the patch is strongly depolarized. However, the activity of the channel is increased dramatically by the addition of the lectin concanavalin A (Con A), to the patch pipette. The channel is also activated by Con A in the bathing medium, suggesting that the lectin's action is via an as yet unidentified intracellular second messenger. In the one single-channel patch studied, Con A had no effect on the channel mean open time; rather it decreased the average duration of the long closed times between bursts of openings. Thus Con A increases either the open probability of single channels, the number of functional channels in the patch, or both. The functional significance of the Con A-induced modulation of K+ channel activity remains to be determined.     

Separation of microvilli from Tubifex eggs upon activation: Its inhibition by concanavalin A hinders ooplasmic segregation and cleavage

The surface of mature eggs of the freshwater oligochaete Tubifex exhibits numerous microvilli. Upon activation, microvilli become narrower at their base and separated from the ooplasmic surface. Here it is shown that concanavalin A (Con A) reversibly inhibits the separation of microvilli from activated Tubifex eggs. The Con A-treated eggs undergo meioses and mitoses at a normal rate. Microvilli on these eggs change their length in a meiotic cycle-dependent manner; their core bundles of microfilaments elongate significantly during the second meiosis. The Con A-treated eggs fail to complete polar body formation, ooplasmic segregation and cleavages. Treatment with Con A of eggs that have accomplished microvillar separation does not exert any inhibitory effect on their development. Succinyl-Con A, a dimeric derivative of Con A, does not prevent microvillar separation, suggesting that the tetravalent form of Con A is essential for Con A to exert its inhibitory effect on microvillar separation.     

The effect of concanavalin A on the rat electro-olfactogram at various odorant concentrations.

We have studied the effect of concanavalin A (Con A) on the rat electro-olfactogram response to several odorants. Each odorant was applied over a range of concentrations. For hydrophobic odorants whose response was affected by Con A, the diminution in response was maximal at odorant concentrations of about 1 microM in the olfactory mucus. The (odour) concentration-dependence of the change is compatible with the idea that Con A inactivates one or more types of olfactory receptor that normally bind odorants with dissociation constants of the order of 100 nM. With hydrophilic odorants we had to apply concentrations very much higher than this to elicit any response from the system. At these high concentrations we could observe Con A-induced diminutions in response.     

Stimulation of human polymorphonuclear leukocyte phosphatidylinositol-4-phosphate kinase by concanavalin A and formyl-methionyl-leucyl-phenylalanine is calcium-independent. Correlation with maintenance of actin assembly.

Chemoattractants directly stimulate the enzyme activity that synthesizes phosphatidylinositol-4,5-bisphosphate (PIP2), phosphoinositol-4-monophosphate (PIP) kinase. The present study determined whether stimulation of this enzyme correlates with actin assembly by assessing the calcium dependence of this reaction. Incubation of neutrophils with 5 to 100 micrograms/ml Con A caused a concentration-dependent increase in PIP kinase activity ranging from 1.38- to 3.4-fold. The effective concentration which stimulated PIP kinase by 50% (17 micrograms/ml, EC50) corresponded with the EC50 for Con A-induced superoxide production (32 micrograms/ml). Like chemoattractants, the increase in PIP kinase by Con A was characterized by a 2.6-fold increase in the maximum velocity (Vmax) of the enzyme, and no change in the Km for ATP. The kinetics of FMLP- and Con A-induced filamentous actin formation preceded stimulation of PIP kinase and was sustained over the same time period that this increased enzyme activity was noted. Although transmembrane signaling by FMLP and Con A requires an increase in intracellular calcium for some polymorphonuclear leukocyte (PMN) functional responses, calcium depletion of PMN by incubation with 100 microM Quin 2 A/M and 5 mM EGTA did not prevent the stimulation of PIP kinase by FMLP or Con A. In addition, calcium depletion did not prevent the increase in filamentous actin formation by FMLP and Con A in PMN. These findings demonstrate that Con A increases PIP kinase activity in human PMN and that PIP kinase stimulation and maintenance of actin assembly are independent of calcium fluxes in these cells. Because PIP2 controls the function of the actin-regulatory proteins, profilin and gelsolin, changes in the synthetic rate of PIP2 through regulation of PIP kinase may provide a molecular basis for the prolonged stimulation of actin assembly in human PMN by agonists such as Con A and FMLP.     

Concanavalin A-induced translocation of part of the GTP-binding activity from the membrane to the cytosol in murine thymocytes

Concanavalin A (Con A) stimulation resulted in the rapid redistribution of part of the GTP-binding activity from the membrane to the cytosol in murine thymocytes. This change in GTP-binding activity was dependent on the Con A concentration. To investigate the relationship between this redistribution and phospholipase C (PLC) activity, the effect of GTP gamma S on the cytosol PLC activity was also examined, and it was found that GTP gamma S enhanced the phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis activity in the cytosol of Con A-stimulated thymocytes more than in that of unstimulated thymocytes. This enhancement by GTP gamma S was also dependent on the Con A concentration. The results suggest that in murine thymocytes, the GTP-binding protein (G-protein) involved in the regulation of PLC activity may be translocated from the membrane to the cytosol upon Con A stimulation. Besides, the dose dependence curve for the change in the GTP gamma S-binding activity was similar to that for inositol phosphates formation in Con A-stimulated thymocytes, suggesting that the translocation of the G-protein is closely related to PLC activation. Furthermore, the effects of cytosol fractions containing the 38-43 and 23-28 kDa GTP-binding subunits of G-proteins on the PIP2 hydrolysis activity of partially purified PLC were examined. The fraction containing the 23-28 kDa subunit evidently enhanced the PLC activity but that containing the 38-43 kDa subunit enhanced the activity to a much lower extent. Moreover, the 23-28 kDa subunit fraction of Con A-stimulated thymocytes was more effective as to enhancement of the PLC activity than that of unstimulated thymocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concanavalin A-induced increase in the membrane fluidity of chicken erythrocytes.

To determine the fluidity of the membrane lipid phase, chicken erythrocytes were labeled with a stearic acid derivative spin label. When chicken erythrocytes were treated with concanavalin A (Con A), ESR spectra showed a change in the peaks of the labels in membrane lipids, indicating an increase of membrane fluidity. The degree of the increase in fluidity of the membrane lipid phase depended on the valency of the lectin used. Tetravalent Con A induced an increase of membrane fluidity at a concentration as low as 30 micrograms/ml, while a monovalent derivative of Con A did not affect membrane fluidity. This increase in membrane fluidity was observed within 10 min after the addition of Con A. If bound Con A was removed with methyl alpha-D-mannoside later than 60 min after its addition, a complete return of the fluidity to the normal level could not be observed. However, no change was found in the composition of phospholipids or in the fatty acid compositions of phosphatidylcholine and phosphatidylethanolamine of chicken erythrocytes after the addition of Con A, indicating that this increase in membrane fluidity is not caused by a change of lipid composition. The clustering of membrane receptors of chicken erythrocytes for Con A was demonstrated when the two-dimensional distribution of ferritin-conjugated Con A on the membranes was assayed by transmission electron microscopy. Furthermore, it was shown that major receptors for Con A of chicken erythrocytes were transmembrane glycoproteins having apparent molecular weights of 100K, 45, and 33K.     

Accelerated calcium ion uptake in murine thymocytes induced by concanavalin A.

The mechanism of enhancement of Ca2+ uptake by the T cell mitogen concanavalin A (Con A) was studied in murine thymocytes. Native Con A enhanced the rate of Ca2+ uptake as much as 9-fold, an increase being observed within five minutes after Con A addition. The effect of Con A was reversed completely by alpha-methyl mannopyranoside (alpha-MM). Increased Ca2+ uptake was observed with increasing concentrations of Con A, between 2 and 400 microgram/ml, indicating that the stimulation of Ca2+ uptake is not restricted to mitogenic lectin concentrations (0.5-2 microgram/ml). Succinyl Con A exhibits only a slight effect in the same concentration ranges as native Con A. Ca2+ uptake, both in the absence and presence of Con A, is strongly dependent on energy metabolism and is carrier mediated. The augmentation of Ca2+ uptake by native Con A is due to an enhanced Vmax. Uptake of the anion, CrO42-, by thymocytes, found to be a non-saturable process, was also enhanced by Con A. The effect of Con A on CrO42- permeability appears to be independent of its effect on Ca2+ uptake.     

Effects of trophoblastic beta 1-glycoprotein (TBG) on the functional activity of different cell lines

The effect of TBG on the functional activity of different cell lines, spontaneous and Con A induced proliferation of PBL was studied. If concentration of TBG is higher than 50 mu kg/ml it suppresses the proliferation in many used cell lines, except choriocarcinoma and cancer of uterus. The reliable increasing of spontaneous proliferation of PBL, Jurkat and K-562 cells may be observed if concentration is more lower (0.5-15 mu kg/ml). However proliferation of other cell lines corresponds to control level, and Con A induced proliferation of PBL is inhibited. The effect was more marked at 48, as compared to 24 hours of cell incubation with TBG.     

A study of the mechanism of Con A-induced immunosuppression in vivo

The plaque-forming cell (PFC) response to sheep erythrocytes (SRBC) is suppressed in a dose-related manner when concanavalin A (Con A) is administered intravenously to mice prior to or after immunization with antigen. The magnitude of suppression as well as the duration of the Con A effect greatly depends on the concentration of antigen used for immunization. Although profound suppression of the anti-SRBC PFC response is observed in intact mice pretreated with Con A for 4-24 hr, spleen cells from these mice do not exhibit suppressive activity when transferred into normal recipients or when cotransferred with normal spleen cells into irradiated recipients. Moreover, the cells from Con A-treated mice respond as normal spleen cells to SRBC when transferred alone into irradiated hosts. Suppression of the anti-SRBC PFC is only observed when adoptive hosts of cells from Con A-treated mice are also injected with Con A within 48 hr (but not 72 hr) of cell transfer and immunization. This time course of responsiveness to the suppressive effects of Con A is similar to that observed in normal mice and in irradiated recipients of normal spleen cells. The immune response to SRBC is also suppressed in adoptive hosts of normal spleen cells that are pretreated with Con A 4-24 hr prior to irradiation and cell transfer. Although functionally inactive when transferred into adoptive hosts, spleen cells from mice pretreated with Con A for 4-24 hr can suppress a primary antibody response to SRBC in vitro. The suppressive activity, which cannot be detected in the spleens of mice when the interval between pretreatment and assay is longer than 24 hr, is present in a subpopulation that bears the Thy 1.2 and Lyt 2 phenotype. Taken together the results obtained in in vivo and in vitro functional assays suggest that a suppressor cell population is activated following in vivo treatment with Con A, but that the cells rapidly lose their state of activation when removed from a Con A environment. This phenomenon is in all probability responsible for the failure to demonstrate suppressive activity in the spleens of Con A-treated mice using in vivo functional assays.     

A comparison of the short and long term effects of insecticidal lectins on the activities of soluble and brush border enzymes of tomato moth larvae (Lacanobia oleracea)

When fed in semi-artificial diet in short- and long-term bioassays, the lectins from snowdrop (Galanthus nivalis; GNA) and jackbean (Canavalia ensiformis; Con A) affected the activities of soluble and brush border membrane (BBM) enzymes in the midgut of Lacanobia oleracea larvae. In the short term both lectins increased gut protein levels and BBM aminopeptidase activity. The lectins also increased trypsin activity, both in the gut (Con A) and in the faeces (GNA). GNA also increased the activity of alpha-glucosidase, but neither lectin had a significant effect on alkaline phosphatase activity. Trypsin mRNA levels were similar in lectin-fed and control larvae in the short term, showing that there is no direct effect on expression of the encoding genes. Larvae chronically exposed to GNA and Con A showed reductions in weight of 50-60%, and exhibited a significant reduction in alpha-glucosidase activity, but little change in other enzyme activities. Con A bound to many BBM and peritrophic matrix (PM) proteins in vitro, whereas GNA showed more specific binding, with strongest binding to a 94kDa uncharacterised BBM protein. Both lectins accumulated in gut tissues of insects after chronic exposure in vivo, but Con A was present at higher levels than GNA.     

Effects of trifluoperazine and mitogenic lectins on calcium ATPase activity and calcium transport by human lymphocyte plasma membrane vesicles

The phenothiazine, trifluoperazine, and the mitogenic lectins, phytohemagglutinin (PHA) and Concanavalin A (Con A), were tested for their effects on human lymphocyte plasma membrane Ca-activated Mg-ATPase and ATP-dependent calcium uptake. Trifluoperazine completely inhibited Ca-uptake when present from the start of the assay at concentrations of 100 microM or more. When added during measurement of calcium uptake, trifluoperazine reduced the rate of vesicular calcium accumulation but was unlike the calcium ionophore, A23187, which caused a rapid release of accumulated calcium from the vesicles. Trifluoperazine also inhibited membrane vesicle Ca-ATPase activity, but this inhibition was non-specific since the Mg-ATPase and Na,K-ATPase activities were inhibited to similar extents at the same concentration of the phenothiazine. In contrast, concentrations of PHA and Con A, which are mitogenic for lymphocytes, did not cause any change in Ca-uptake when added to suspensions of membrane vesicles. Con A had no effect and PHA had a weak inhibitory effect on Ca-ATPase activity.     

Epidermal growth factor induced membrane changes in 3T3 cells.

Epidermal growth factor (EGF) is a mitogen for Swiss 3T3 cells. Short incubation periods with physiological concentrations of EGF induced increased binding of Swiss 3T3 cells to Con A-coated nylon fibers. This effect was not induced in an EGF non-responsive 33 variant, in the transformed murine XC cells or in Swiss SV3T3 cells. The increase in Con A fiber-binding seems to be specific for EGF, since it was not observed in response to insulin, prostaglandin F2alpha or a higher serum concentration, which also initiate cell devision of confluent quiescent 3T3 cells. EGF also reduced Con A-mediated hemadsorption to 3T3, but had no effect on hemadsorption by the EFG non-responsive 3T3 variant. There was no change in the number of Con A-receptors on 3T3 cells after EGF treatment. Binding to WGA-coated fibers and WGA-mediated hemadsorption were not effected by preincubation with EGF.     

Location of amino acid and carbohydrate transport sites in the surface membrane of normal and transformed mammalian cells

Summary Amino acid and carbohydrate transport in normal and malignant transformed hamster cells was studied after binding of the protein Concanavalin A (Con. A) to the surface membrane. Experimental conditions were used so that a similar number of Con. A molecules were bound to both types of cells. The transport of amino acids was inhibited after Con. A binding in the transformed cells but not in normal cells. This was found with the metabolizable amino acidsl-leucine,l-arginine,l-glutamic acid, andl-glutamine, and with the non-metabolizable amino acids cycloleucine and -aminoisobutyric acid. Transport ofd-glucose andd-galactose was more inhibited by Con. A in transformed than in normal cells, and in both types of cellsd-glucose was inhibited more thand-galactose. The inhibition by Con. A on transport was specific, since there was no effect on the transport ofl-fucose in either normal or transformed cells. Con. A also did not effect the entry of 3-0-methyl-d-glucose.These observations can be used to locate amino acid and carbohydrate transport sites in the surface membrane in relation to the binding sites for Con. A. The results indicate that Con. A sites are associated in normal cells with transport sites ford-glucose and to a lesser extentd-galactose, and in transformed cells with transport sites for amino acids and to a greater extent than in normal cells withd-glucose andd-galactose. Malignant transformation of normal cells therefore results in a change in the location of amino acid and carbohydrate transport sites in the surface membrane in relation to the binding sites for Con. A.     

Concanavalin A increases glyoxalase enzyme activities in polymorphonuclear leukocytes and lymphocytes

Glyoxalase I converts methylglyoxal and glutathione to S-lactoylglutathione and glyoxalase II converts this compound to D-lactic acid, regenerating glutathione in the process. A recent study from my laboratory has provided evidence that S-lactoylglutathione modulates microtubule assembly in vitro whereas concanavalin A (Con A) has been shown to increase microtubule occurrence in polymorphonuclear leukocytes (PMN). The present report describes the dose-dependent activation by Con A of both glyoxalase I and II in PMN and lymphocytes. In nine experiments with PMN, Con A (100 microgram/ml) increased glyoxalase I and II activities by 19 +/- 8% and 12 +/- 10% (mean +/- S.D.). In 17 experiments with lymphocytes, activation of the two enzymes by 10 microgram/ml Con A was 30 +/- 14% and 28 +/- 8%. Changes occurred after a 1-min incubation with Con A and persisted for at least 60 min. Since both enzyme activities are increased it is not clear if S-lactoylglutathione levels are increased or decreased but presumably they change. The present findings are compatible with the hypothesis that Con A increases microtubule occurrence in PMN by affecting the glyoxalase enzymes. They also represent a newly described early biochemical change caused by Con A in lymphocytes.     

The effect of concanavalin A-stimulated mononuclear cells on low density lipoprotein receptor activity of cultured fibroblasts

Secretory products of freshly isolated human circulating blood cells such as platelets, monocytes, and B lymphocytes, but not T lymphocytes, have previously been shown to enhance low density lipoprotein (LDL) metabolism by arterial wall cells. This study was undertaken to evaluate how secretory factor(s) from mononuclear cells that had been stimulated by concanavalin A (Con A) alters LDL receptor activity by cultured human skin fibroblasts. Conditioned medium from Con A-stimulated mononuclear cells produced an increase of 125I-LDL degradation accompanied by increased thymidine incorporation into DNA. The effect of conditioned medium from the Con A-stimulated mononuclear cells was mediated by the LDL receptor pathway. Degradation of HDL and methylated LDL, neither of which is taken up by the classical LDL receptor pathway, was not affected. The conditioned medium from these Con A-stimulated cells also failed to stimulate fluid pinocytosis, as measured by the uptake of [14C]sucrose. Some strains of fibroblasts, deficient in LDL receptors, responded to the conditioned medium from the Con A-stimulated mononuclear cells by increasing the very small amounts of LDL degraded by these cells. Fibroblasts from other homozygous familial hypercholesterolemic cell strains were unresponsive, however. The effect on LDL receptors was characterized by an increase in LDL receptor number without a change in the affinity of LDL for its receptor. Thus stimulated mononuclear cells secrete mitogens that also stimulate LDL receptor activity in human skin fibroblasts.     

Effect of concanavalin A on the killing of tumor cells by antibody and complement.

Concanavalin A (Con A) was found to inhibit the killing of antibody-sensitized line-1 tumor cells (TA) by guinea pig complement (GPC) but not by human complement (HuC). Other plant lectins (wheat germ, leucoagglutinin, and pokeweed mitogen) were also tested but Con A was the only lectin found to inhibit antibody-GPC-mediated killing. The inhibitory effect of Con A was observed when the GPC was mixed with Con A or when the antibody-sensitized cells were pretreated with Con A (TA-Con A) before the addition of GPC. The effect could be reversed by treatment of such cells with alpha-D-methylglucopyranoside or by incubation at 37 degrees C for approximately 2 hr. Con A appeared to act by preventing the binding of the first component of GPC (GPC1) to antibody-sensitized tumor cells. Differences in the binding of the first component of HuC (HuC1) and GPC1 to TA-Con A suggested that a difference in the binding site for HuC1 and GPC1 might exist. There was no difference in the number of GPC1 molecules fixed to antibody-sensitized sheep erythrocytes (EA) or EA treated with Con A in experiments using the same antibody as used with the tumor cells and the same Con A preparation. It would consequently appear that the inhibitory effect of Con A on the binding of GPC1 to TA is not due solely to an interaction of Con A with the antibody.     

Bestatin,a new specific inhibitor of aminopeptidases,enhances activation of small lymphocytes by concanavalin A

Bestatin, a new competitive aminopeptidase-inhibitor of dipeptide nature, was shown to enhance markedly activation of peripheral blood lymphocytes by concanavalin A (Con A). More than 40 percent stimulation over the control (the culture with Con A only) was observed at 50 μg/ml of bestatin, and this stimulatory effect was most predominant at an early stage of lymphocyte blasto-genesis by Con A: bestatin had most effect when added to the culture simultaneously with Con A and no appreciable effect when added 44 h after Con A. The effect of bestatin on T lymphocyte activation in vitro is discussed in relation to its in vivo enhancing effect on cell-mediated immunity and a role in lymphocyte blastogenesis of some proteolytic activities possibly located at the cell surface is emphasized.     

Facilitation of adenylate cyclase stimulation in macrophages by lectins.

Preincubation of guinea pig peritoneal macrophages with concanavalin A (Con A) markedly enhanced the accumulation of 3′,5′-cyclic-adenosine monophosphate (cAMP) in response to the adenylate cyclase (AC) stimulators prostaglandin E₁ (PGE₁) and isoproterenol (IP). Basal cAMP levels were not altered. Maximal enhancement of cAMP accumulation was induced by preincubation with 50–100 μg/ml Con A for 10 min at 37 °C. Con A-induced facilitation of macrophage responsiveness was prevented by α-methyl-d-mannoside (αMM). No facilitation was induced by the divalent derivative, succinyl-Con A or by Con A immobilized on Sepharose beads. Con A-induced facilitation developed normally in macrophages treated with the microfilament blocking agent, cytochalasin B. The responsiveness of macrophages to PGE₁ and IP was also augmented by phytohemagglutinin (PHA) but wheat germ agglutinin (WGA), soy bean agglutinin (SBA), pokeweed mitogen (PWM), and Lotus tetragonolobus lectin (LL) showed no enhancing effect. The effect of Con A on cAMP levels was the result of augmented cAMP synthesis and not of reduced degradation or a block in cAMP egress from the cells. Lectin-induced facilitation of AC stimulation could be mediated via one of the following mechanisms: (i) induction of receptor clustering; (ii) causing a conformational change in the receptors; (iii) inhibition of negative cooperativity; (iv) causing an increase in membrane fluidity; (v) disruption of microtubules by acting as a Ca²⁺ ionophore; or (vi) inactivation of a sugar-containing inhibitor of AC.     

The N-terminal of a heparin-binding sperm membrane mitogen possess lectin-like sequence

Glycosaminoglycans like heparin and heparin sulfate in follicular fluid induce changes in the intracellular environment during the spermatozoal functional maturation. We previously reported the isolation, purification and partial characterization of a heparin binding sperm membrane protein (HBSM). In the present study, the amino acids analysis provided evidence of a single sequence, which suggest the homogeneity of the purified HBSM. Fourteen amino acids--(1)A D T I V A V E L D T Y P N(14)--correspond to the amino terminal sequence of Concanavalin A (Con A) and contain 45.2% carbohydrate by weight. HBSM possess mitogenic property on lymphocytes with comparable magnitude to the well-known mitogen; Con A, inducing 83% radiolabel thymidine incorporation in growing lymphocytes. Unlike Con A, there was no agglutination of cell by HBSM upto 5 ng/ml concentration. Interestingly, we found that heparin and chondroitin sulfate-conjugated HBSM inhibit the proliferative activity. Similar effect was also found with an in-house isolate sulfated glycans; G-I (28% sulfate). In contrast, there was no inhibition by the desulfated form; G-ID. Altogether, our data suggest that the mechanism of cell proliferative pathway may be different for HBSM and Con A.     

Inhibition of intercellular adhesion by concanavalin A is associated with concanavalin A-mediated redistribution of surface receptors

The inhibition of adhesion between aggregates and layers of embryonic retinal cells by concanavalin A (Con A) and Con A-mediated rearrangements of Con A receptors on retinal cells were studied. A short incubation of aggregates and layers with 10 micrograms/ml Con A substantially reduced aggregate-to-layer adhesion in a subsequent assay without soluble lectin present. This effect of Con A was dose-dependent, temperature-sensitive, involved events subsequent to Con A binding, and was reduced by cytochalasin B. The inhibition produced by succinylated Con A was substantially increased by incubation with antibody to Con A. Visualization of ConA- receptor complexes by fluorescence microscopy revealed that binding of Con A induced clearing of Con A receptors from filopodia, flattened regions of growth cones, and the edges of axons. This clearing reaction was prevented by the same agents that reduced Con A's inhibition of cell adhesion: low temperature, succinylation of Con A, or cytochalasin B. Aggregate-layer adhesion was restored by releasing Con A at 37 degrees C. Inhibitors of protein and ATP synthesis did not prevent recovery of ability to make adhesions. However, release of Con A at lowered temperatures did not prevent recovery. The results suggest that intercellular adhesion is inhibited by events associated with redistribution of Con A-receptor complexes on retinal cells.