Interactions of lectins with CHO cell surface membranes. II. Differential effects of local anesthetics on endocytosis of CON A and WGA binding sites

Using fibroblastic CHO cells, we have examined (1) the internalization and redistribution of surface binding sites for the lectins Concanavalin A and wheat germ agglutinin and (2) the sensitivity of these processes to putative inhibitors of cytoskeletal activity. Following initial exposure to fluorescein conjugated Con A (CAF) or WGA (WGAF) at 4° C, kinetic analysis of internalization and intracellular aggregation of lectin at 37°C indicated more rapid aggregate formation in the case of WGA than in the case of Con A. Treatment with tertiary amine local anesthetics (tetracaine, dibucaine, and xylocaine) or with a lysosomatrophic amine, m-dansyl cadaverine, blocked internalization of Con A but not of WGA. Similar differential effects on redistribution of Con A and WGA were not however observed with the antimicrotubule agents colchicine and nocodazole. Simultaneous treatment of cells with WGAF and with rhodamine labeled Con A (CAR) resulted in the accumulation of both labels in a central perinuclear aggregate; a similar experiment in the presence of local anesthetic resulted in a diffuse peripheral distribution of CAR and a central aggregate of WGAF. These results suggest (1) CHO cells possess at least two distinct pathways for lectin internalization and redistribution, which can be discriminated in terms of drug sensitivity; (2) CHO cells can sort out and independently internalize different populations and lectin binding sites; and (3) different pathways may be a manifestation of biochemically distinct linkages between cytoskeletal elements and various groups of surface glycoproteins. Present findings concur with our previous results concerning the mutual independence of the surface binding sites for Con A and WGA (Emerson and Juliano, 1982).     

Concanavalin a perturbation of membrane enzymes of mammary gland

The plant lectin concanavalin A (Con A) specifically inactivates the 5′ -nucleotidase of a plasma membrane-enriched fraction from lactating mammary gland. The lectin also causes an activation of the membrane Mg⁺⁺ -ATPase, but does not affect galactosyltransferase or alkaline phosphatase. The enzyme perturbations are prevented by α-methylmannoside, an inhibitor of Con A binding, indicating that specific binding to carbohydrate structures rather than nonspecific protein-protein interaction is involved. Solubilization of the 5′ -nucleotidase in detergents (0.2% Triton X-100 or 1% deoxycholate) does not prevent Con A inactivation, indicating that incorporation into the membrane structure is not a requirement for the Con A effect. The results suggest that Con A inactivates the 5′ -nucleotidase by a direct interaction with the enzyme and that this enzyme is a Con A receptor site on the surface of mammary cells.     

Isolation of concanavalin a caps during various stages of formation and their association with actin and myosin

Regions of plasma membrane of dictyostelium discoideum amoebae that contain concanavalin A (Con A)-receptor complexes are more resistant to disruption by Triton X-100. This resistance makes possible the isolation of Con A-associated membrane fragments in sufficient quantity and homogeneity to permit the direct biochemical and ultrastructural study of receptor-cytoskeletal interactions across the cell membrane. After specific binding of Con A to the cell surface, a large amount of the cell’s actin and myosin copurifies with the plasma membrane fragments. Myosin is more loosely bound to the isolated membranes that actin and is efficiently removed by treating membranes with ATP and low ionic strength. If cells are not lysed immediately after lectin binding, all of the Con A that is bound to the cell surface is swept into a cap in a process requiring metabolic energy. When cells are lysed at different stages of cap formation, the amount of actin and myosin that copurifies with the isolated membranes remains the same. Thick and thin filaments that are attached to the protoplasmic surface of the isolated membranes underlie lectin-receptor complexes during all stages of cap formation. Once the cap is complete, the amount of actin and myosin that tightly bound to the plasma membrane is concentrated into the cap along with the Con A-receptor complexes. These results suggest that the ATP-dependent sliding of membrane-associated actin and myosin filaments is responsible for the accumulation of Con A-receptor complexes into a cap on the cell surface.     

Effect of concanavalin A on the level of sulfhydryl groups in thymocytes

Fluorescein mercury acetate (FMA), a fluorescent probe, is used for the investigation of SH-groups of thymocytes' plasma membrane. It is found that mitogenic lectin Con A decreases the amount of membrane SH-groups and increases the fluorescence polarization degree of FMA (PFMA). The value of PFMA increases also during the incubation of cells with potassium ferricyanide and H2O2 but it decreases in the presence of NADH. The analysis of the data permits a conclusion that the thymocyte activation by Con A results in the selective oxidation of certain SH-groups with the formation of disulphide cross-linking between the plasma membrane receptors bound with the lectin molecules.     

Lectins as biochemical agents for the isolation of sealed membrabe vesicles of defined polarity

The asymmetric distribution of carbohydrate on biological membranes has provided the basis for the development of lectin-affinity methodology which permits the isolation of sealed, inside-out membrane fractions from heterogeneous populations of vesicles.Optimal conditions for these separations have been assessed employing purified right-side-out and inside-out vesicles derived from the plasma membrane of human erythrocytes as a model system. In this special case, homogeneous populations of defined polarity can be produced by varying the ionic conditions during formation of the vesicles. Surface-specific enzymic markers exist also for monitoring the integrity and orientation of a given population.Multivalent lectins such as wheat germ agglutinin and soya bean agglutinin, which induce direct agglutination of erythrocyte membrane fragments containing accessible carbohydrate residues, selectively remove more than 90% of right-side-out and non-sealed membrane from mixed population, a reaction which is inhibited by GluNAc or GalNAc, respectively.Non-agglutinating lectins, e.g. concanavalin A, immobilized on an inert matrix such as Sepharose 4B, may be employed to adsorb out specifically vesicles with exposed glycopeptides on their surface. In this technique, it is necessary normally to remove the non-sealed membranes on Dextran density gradients prior to the final preparation of inside-out vesicles on Con A-Sepharose.Finally, selective immunoprecipitation of fragments with accessible sugars may also be achieved after treatment with a non-agglutinating lectin (concanavalin A) followed by incubation with anti-concanavalin A IgG which promotes rapid aggregation of membrane containing exposed receptors for the lectin.These procedures should prove generally suitable for the isolation of tightly-sealed, inside-out membrane populations in a variety of biological systems. Pure populations of vesicles, exhibiting reversed polarity, are valuable in surface-labelling studies for investigating the structure, function and transmembrane distribution of integral membrane proteins/glycoproteins.     

Schistosomula of Schistosoma mansoni clear concanavalin A from their surface by sloughing

The lectin concanavalin A (Con A) was used as a model probe to study the behavior of molecules bound to the surface of recently transformed schistosomula of Schistosoma mansoni. Con A binding was saturable (150- 180 pg/organism) and specifically competed by alpha-methyl mannoside. Both FITC-Con A and 125-I-Con A were lost from the surface of schistosomula with a halftime of 8-10 h in culture in defined medium. A comparable decrease in the binding of Con A to schistosomula cultured and then labeled with the lectin indicated that the labeling procedure itself was not inducing the observed change. Internalization of Con A was not seen by either fluorescence microscopy or electron microscope radioautography. In addition, 70-80% of the radioactivity lost from the parasite was recoverable by TCA precipitation from the culture medium as intact Con A (27,000 mol wt on SDS PAGE). Thus, the mechanism of clearance of bound Con A from the surface of cultured schistosomula is apparently by sloughing of Con A molecules intact into the culture media and not by endocytosis and degradation. Con A binding sites, visualized with hemocyanin by scanning electron microscopy, appeared homogeneously distributed over the surface of schistosomula when organisms were labeled at 4 degree C or after fixation with glutaraldehyde. However, Con A and hemocyanin formed aggregates on the surface of schistosomula when labeling was performed at 37 degrees C, which suggests that lectin binding sites have lateral mobility within the plane of the membrane. These aggregates are likely independent of metabolism by the parasite because aggregation also occurs on the surface of organisms killed with azide.     

5'-Nucleotidase activity of lymphocyte plasma membranes. Effect of concanavalin A]

The 5'-nucleotidase properties of isolated lymphocyte plasma membranes from young pig mesenteric nodes are described; nucleosides-5'-monophosphates are the substrates of this specific enzyme. Concanavalin A inhibits this enzyme; on the same membranes this mitogen does not affect alkaline phosphatase and activates the membrane bound (Ca2+) ATPase. The 5'-nucleotidase inhibition is due to a specific interaction of Con A with carbohydrate groups of the membrane; its high positive cooperativity suggests that the lectin promotes reorganization of the membrane bound 5'-nucleotidase. Solubilization of the 5'-nucleotidase does not prevent the effect of Con A and the solubilized enzyme is firmly bound by Con A-Sepharose 4B; these results suggest that Con A inhibits the enzyme by a direct interaction and that 5'-nucleotidase can be considered as an eventual receptor for the lectin.     

Correlation between movement of concanavalin A membrane receptors and cytolysis. A scanning electron microscopy study

The present study was undertaken to test whether cytolysis induced by Concanavalin A (Con A) requires lateral mobility of membranal lectin receptor sites into caps. Treatment of interphase murine mastocytoma cells with 10(-4) M colchicine promoted cap formation by Con A in about 30% of the cells, followed by cytolysis. Pretreatment of the cells with NaN3, low temperature, or glutaraldehyde decreased the degree of capping and, to the same extent, the degree of cytolysis. The addition of antibodies to cells bound with Con A increased the appearance of capping and cytolysis. A linear relationship with a high correlation coefficient exists between the degree of capping and cytolysis, suggesting that lateral mobility of membrane Con A receptors is required for cytolysis by the lectin. The process of cap formation by Con A up to the stage of cytolysis was followed by scanning electron microscopy.     

Concanavalin-Mediated Attachment to Membranes of a Cellular Slime Mould Cyclic AMP Phosphodiesterase

In the cellular slime mould Dictyostelium discoideum , a membrane-bound cyclic AMP phosphodi-esterase undergoes a tenfold increase in activity when amoebae reach the aggregation stage of development. Our previous studies had shown that when non-aggregating cells, which produce extracellular and intracellular forms of the enzyme, are treated with the lectin Concanavalin A (Con A), they exhibit prematurely high levels of the membrane bound enzyme. The present results indicate that this effect may be largely due not to the induction of the enzyme by Con A but rather to the binding of the intracellular form of the enzyme to membranes by Con A. This conclusion is based on the findings that: a) the enzyme activity associated with membranes from Con A treated cells can be decreased by treatment with the haptenic sugar α-methyl mannoside; b) membranes from untreated cells having only low membrane-bound phosphodiesterase activity can acquire increased activity after incubation with Con A and intracellular phosphodiesterase; c) the intracellular phosphodiesterase binds to Sepharose-Con A and is eluted with α-methyl mannoside. These results raise the possibility that some of the effects attributed to Con A in the literature may not be due directly to Con A but to glycoproteins attached to membranes by Con A.     

Concanavalin-mediated attachment to membranes of a cellular slime mould cyclic AMP phosphodiesterase.

In the cellular slime mould Dictyostelium, a membrane-bound cyclic AMP phosphodiesterase undergoes a tenfold increase in activity when amoebae reach the aggregation stage of development. Our previous studies had shown that when non-aggregating cells, which produce extracellular and intracellular forms of the enzyme, are treated with the lectin Concanavalin A (Con A), they exhibit prematurely high levels of the membrane bound enzyme. The present results indicate that this effect may be largely due not to the induction of the enzyme by Con A but rather to the binding of the intracellular form of the enzyme to membranes by Con A. This conclusion is based on the findings that: a) the enzyme activity associated with membranes from Con A treated cells can be decreased by treatment with the haptenic sugar alpha-methyl mannoside: b) mambranes from untreated cells having only low membrane-bound phosphodiesterase activity can acquire increased activity after incubation with Con A and intracellular phosphodiesterase; c) the intracellular phosphodiesterase binds to Sepharose-Con A and is eluted with alpha-methyl mannoside. These results raise the possibility that some of the effects attributed to Con A in the literature may not be due directly to Con A but to glycoproteins attached to membranes by Con A.     

Cell contact mediated differentiation in dictyostelium.

Major biochemical and ultrastructural changes occur in Dictyostelium discoideum plasma membranes following aggregation of the amoebae. The effects of cyclic AMP, Concanavalin A (Con A), and disruption of cell contacts on membrane particle synthesis and the subsequent differentiation of prespores and mature spores were determined. The results indicated that prespore cell differentiation always failed under conditions in which large particle formation was inhibited or cells bearing particles were restricted in their contacts. Although prespore cells exposed to Con A formed mature spores devoid of prespore vacuoles, the cell walls were defective. The research suggests that the interactions between membrane particles of apposing amoebae may initiate differentiation of prespores and mature spores.     

Lectin-induced inhibition of plasma membrane 5′-nucleotidase: Sensitivity of purified enzyme

To determine whether the lectin-induced inhibition of plasma membrane 5′-nucleotidase resulted from direct interaction of the lectin with the enzyme or indirectly from a membranous change due to lectin binding to other membrane glycoproteins, the enzyme was purified and its sensitivity tested in the absence of other membrane components. A 5000 fold purification was achieved by solubilization in Lubrol PX followed by gel filtration (Sephadex G-100), anion exchange (DEAE-Biogel A) and selective adsorption (hydroxylapatite) chromatography. The purified enzyme was even more sensitive to inhibition by high concentrations of concanavalin A, wheat germ agglutinin or Rincinus communis agglutinin than was the membrane-bound enzyme indicating that inhibition is due to direct binding of the lectins to the glycoprotein enzyme itself. Divalent succinyl Con A inhibited neither form of the enzyme suggesting the need for crosslinking for inhibition by the native lectin. The purified enzyme could not be activated by low concentrations of lectins which stimulated the membrane bound enzyme.     

Distribution and mobility of lectin receptors on synaptic membranes of identified neurons in the central nervous system

The distribution and mobility of concanavalin A (Con A) and Ricinus communis agglutinin (RCA) receptors (binding sites) on the external surfaces of Purkinje, hippocampal pyramidal, and granule cells and their attached boutons were studied using ferritin-lectin conjugates. Dendritic fields of these cells were isolated by microdissection and gently homogenized. Cell fragments and pre- and postsynaptic membranes were labeled with the ferritin-lectin conjugates at a variety of temperatures, and the distribution of lectin receptors was determined by electron microscopy. Both classes of these lectin receptors were concentrated at nearly all open and partially open postsynaptic junctional membranes of asymmetric-type synapses on all three neuron types. Con A receptors were most concentrated at the junctional membrane region, indicating that the mature neuron has a specialized nonrandom organization of carbohydrates on its outer surface. Lectin receptors located on postsynaptic junctional membranes appeared to be restricted in their mobility compared to similar classes of receptors on extrajunctional membrane regions. Labeling with ferritin-RCA and - Con A at 37 degrees C produced clustering of lectin receptors on nonjunctional surfaces; however, Con A and RCA receptors retained their nonrandom topographic distribution on the postsynaptic junctional surface. The restricted mobility of lectin receptors was an inherent property of the postsynaptic membrane since the presynaptic membrane was absent. It is proposed that structures in the postsynaptic density may be transmembrane-linked to postsynaptic receptors and thereby determine topographic distribution and limit diffusion of specialized synaptic molecules. Speicalized receptor displays may play an important role in the formation and maintenance of specific synaptic contacts.     

Production of monoclonal antibodies against a cell surface concanavalin a binding glycoprotein

Concanavalin A-binding (Con A)-binding cell surface glycoproteins were isolated, via Con A-affinity chromatography, from Triton X-100-solubilized Chinese hamster ovary (CHO) cell plasma membranes. The Con A binding glycoproteins isolated in this manner displayed a significantly different profile on sodium dodecyl sulfate-polyacrylamide gels than did the Tritonsoluble surface components, which were not retarded by the Con A-Sepharose column. [¹²⁵I]-Con A overlays of the pooled column fractions displayed on sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) demonstrated that there were virtually no Con A receptors associated with the unretarded peak released by the Con A-Sepharose column, whereas the material which was bound and specifically eluted from the Con A-Sepharose column with the sugar hapten α-methyl-D-mannopyranoside contained at least 15 prominent bands which bound [¹²⁵I]-Con A. In order to produce monoclonal antibodies against various cell surface Con A receptors, Balb/c mice were immunized with the pooled Con A receptor fraction. Following immunization spleens were excised from the animals and single spleen cell suspensions were fused with mouse myeloma P3/X63-Ag8 cells. Numerous hybridoma clones were subsequently picked on the basis of their ability to secrete antibody which could bind to both live and glutaraldehyde-fixed CHO cells as well as to the Triton-soluble fraction isolated from the CHO plasma membrane fraction. Antibody from two of these clones was able to precipitate a single [¹²⁵I]-labeled CHO surface component of ～265,000 daltons.     

Plasma Membrane Glycoproteins of Ciona intestinalis Spermatozoa that Interact with the Egg1

In the ascidian Ciona intestinalis the species-specific interaction between the spermatozoon and the egg occurs between the vitelline coat (VC) of the egg and the plasma membrane of the apical part of the head of the spermatozoa. Concanavalin A (Con A)-binding sites are present on this area of the sperm surface. We used Con A to identify and isolate the spermatozoon plasma membrane components that may be involved in the interaction with the VC. These glycoproteins have been identified on SDS-PAGE of a sperm membrane fraction (SMF) enriched with the extermal proteins, after incubation of the gel with ³H-Con A. Affinity chromatography on Con A-agarose has been used for the purification of sperm plasma membrane proteins with and affinity for the lectin. The biological activity of the Con A-retained fraction was determined with binding and fertilization assays.     

Phagocytosis of collagen fibrils by periosteal fibroblasts in long bone explants. Effect of concanavalin A

In an attempt to determine whether phagocytosis of collagen by fibroblasts involves binding of the fibril to the plasma membrane, the effect of the lectin concanavalin A (Con A) was studied in an in vitro model system. Metacarpal bone rudiments from 19-day-old mouse fetuses were incubated with varying concentrations of the lectin. Quantitative electron microscopic analysis indicated that Con A caused a dose-related increase in the amount of phagocytosed collagen fibrils in periosteal fibroblasts, suggesting either an enhanced uptake or a decreased intracellular breakdown of fibrils. Since a Con A-inducible increase was not seen in the combined presence of both the lectin and the proteinase inhibitor leupeptin, which is known to inhibit the intracellular digestion of phagocytosed fibrillar collagen, it is unlikely that Con A stimulated phagocytosis. Based on the finding that Con A interfered with the digestion of a synthetic substrate by the collagenolytic lysosomal enzyme cathepsin B it is suggested that the augmentation of intracellular fibrillar collagen under the influence of the lectin was due to a decreased intracellular digestion. Since Con A did not inhibit the uptake of collagen fibrils by the fibroblasts it is concluded that Con A-inhibitable binding sites for collagen molecules are unlikely to be involved in phagocytosis of collagen fibrils by fibroblasts.     

Chronic ethanol administration alters hepatic surface membranes as evidenced by decreased concanavalin A binding

The effects of chronic ethanol administration on the hepatic surface membrane were examined. The binding of the lectin, concanavalin A (Con A), to isolated hepatocytes was used to ascertain changes in the hepatic plasma membrane, especially in regard to glycoprotein composition, due to chronic ethanol feeding. Hepatocytes, isolated from rats fed ethanol for 5 to 7 weeks, had a decreased ability to bind Con A when compared to hepatocytes from either the pair-fed controls or ad libitum chow-fed rats. Since decreased Con A binding was more apparent at high Con A concentrations, reduced lectin binding likely reflected changes in the composition of surface membrane glycoproteins in the livers of the ethanol-fed rats. When ethanol (50 mM) was added to the incubation medium containing hepatocytes from ethanol-fed rats, pair-fed controls, or chow-fed rats, no effects on Con A binding were observed. These results indicate that chronic ethanol administration induces changes in the oligosaccharide chains of plasma membrane glycoproteins in the liver. Such alterations may play a role in the pathogenesis of alcoholic liver disease.     

Concanavalin A increases spontaneous beat rate of embryonic chick heart cell aggregates

The plant lectin concanavalin A (Con A), at concentrations of 5–200 μg/ml, induced a twofold to fivefold increase in spontaneous beat rate of cultured aggregates of ventricular cells from seven-day chick embryos. This response was time, dose, and temperature dependent and was accompanied by a decrease in transmembrane potential. It could be blocked or reversed by α-methyl-D-mannoside but was not reversed by dilution alone. Binding of the lectin occurred in the cold, but a temperature-dependent process was also necessary to produce the response. Divalent (succinyl) Con A did not cause a beat rate increase. Whole heart aggregates responded similarly but less intensely than ventricular aggregates. Atrial aggregates, and whole heart aggregates treated with 5 μg/ml of Con A, produced a biphasic chronotropic response, first decreasing then increasing their beat rates. These results suggest that saccharide-bearing macromolecules on the heart cell surface play a role in regulating spontaneous beat rate.     

Concanavalin a and wheat germ agglutinin receptors on dictyostelium: Their visualization by scanning electron microscopy with microspheres

The cellular slime mold, Dictyostelium discoideum, is a convenient model for studying cellular interactions during development. Evidence that specific cell surface components are involved in cellular interactions during its development has been obtained by Gerisch and co-workers (1, 2) using immunological techniques. Smart and Hynes (3) have shown that a cell surface protein can be iodinated on cells in aggregation phase, but not in vegetative phase, by the lactoperoxidase procedure. Recently, McMahon et al. (4), and Hoffman and McMahon have demonstrated, by SDS gel electrophoresis, considerable differences in cell surface proteins and glycoproteins of plasma membranes isolated from cells at different stages of development. Plant lectins have also been used to monitor changes in cell surface properties of D. discoideum cells during development. Weeks and co-workers (5, 6) have detected differences in the binding and agglutination of cells by concanavalin A (Con A). Gillette and Filosa (7) have shown that Con A inhibits cell aggregation and prematurely induces cyclic AMP phosphodiesterase. Capping of Con A receptors has also been reported (8). Reitherman et al. (9) have recently reported that agglutination of cells by several plant lectins and the slime mold agglutination, discoidin, changes during development. Such studies indicate that differences in surface properties exist for cells at various stages of development. However, owing to the uncertainties in the factors which contribute to lectin-induced cell agglutination (10), the molecular basis for these observations remain to be determined. In this study, we have used microspheres (11-14) coupled to either Con A or wheat germ agglutinin (WGA) as visual markers to study by scanning electron microscopy the topographical distribution of lectin receptors on D. discoideum cells fixed at different stages of development. We also describe the effect of labeling on the distribution of lectin receptors and on the morphology of the cell surface.