Cooperative binding of concanavalin A to thymocytes at 4 degrees C and micro-redistribution of concanavalin A receptors.

The mode of binding of 125I-labelled concanavalin A and succinyl-concanavalin A to rat thymocytes at 4 degrees C was investigated. Simultaneously, the free binding sites of the cell-bound lectin molecules were quantified by horseradish peroxidase binding. Concanavalin A showed cooperative binding while succinyl-concanavalin A did not. The number of molecules of concanavalin A bound to the cell surface when it was saturated was twice the number of molecules of succinyl-concanavalin A. We interpret these results as showing that the binding of native concanavalin A to thymocytes at 4 degrees C brings about a cooperative modification of the membrane which leads to appearance of new receptors. Divalent succinyl-concanavalin A has no such effect. Horseradish peroxidase binding to cell-bound lectin was shown to be related to the immobilization of membrane receptors; the more they are immobilized, the more receptor-associated lectin can bind horseradish peroxidase. This allowed us to establish that post-binding events, which we called micro-redistribution, occurred at 4 degrees C when either concanavalin A or succinyl-concanavalin A binds to cells. A cooperative restriction of the micromobility of cell receptors is produced by increasing concentrations of concanavalin A. Succinyl-concanavalin A does not restrict cell receptor mobility at any concentration tested. The results are discussed in terms of cell stimulation and cell agglutination.     

Effects of local anesthetics on membrane properties. II. Enhancement of the susceptibility of mammalian cells to agglutination by plant lectins.

Treatment of untransformed mouse and hamster cells with the tertiary amine local anesthetics dibucaine, tetracaine and procaine increases their susceptibility to agglutination by low doses of the plant lectin concanavalin A. Agglutination of anesthetic-treated untransformed cells by low doses of concanavalin A is accompanied by redistribution of concanavalin A receptors on the cell surface to form patches, similar to that occurring in spontaneous agglutination of virus-transformed cells by concanavalin A. Immunofluorescence and freeze-fracture electronmicroscopic observations indicate that local anesthetics per se do not induce this redistribution of concanavalin A receptors but modify the plasma membrane so that receptor redistribution is facilitated on binding of concanavalin A to the cell surface. Fluorescence polarization measurements on the rotational freedom of the membrane-associated probe, diphenylhexatriene, indicate that local anesthetics produce a small increase in the fluidity of membrane lipids. Spontaneous agglutination of transformed cells by low doses of concanavalin A is inhibited by colchicine and vinblastine but these alkaloids have no effect on concanavalin A agglutination of anesthetic-treated cells. Evidence is presented which suggests that local anesthetics may impair membrane peripheral proteins sensitive to colchicine (microtubules) and cytochalasin-B (microfilaments). Combined treatment of untransformed 3T3 cells with colchicine and cytochalasin B mimics the effect of local anesthetics in enhancing susceptibility to agglutination by low doses of concanavalin A. A hypothesis is presented on the respective roles of colchicine-sensitive and cytochalasin B-sensitive peripheral membrane proteins in controlling the topographical distribution of lectin receptors on the cell surface.     

Effects of local anesthetics on membrane properties II. Enhancement of the susceptibility of mammalian cells to agglutination by plant lectins

Treatment of untransformed mouse and hamster cells with the tertiary amine local anesthetics dibucaine, tetracaine and procaine increases their susceptibility to agglutination by low doses of the plant lectin concanavalin A. Agglutination of anesthetic-treated untransformed cells by low doses of concanavalin A is accompanied by redistribution of concanavalin A receptors on the cell surface to form patches, similar to that occurring in spontaneous agglutination of virus-transformed cells by concanavalin A. Immunofluorescence and freeze-fracture electronmicroscopic observations indicate that local anesthetics per se do not induce this redistribution of concanavalin A receptors but modify the plasma membrane so that receptor redistribution is facilitated on binding of concanavalin A to the cell surface. Fluorescence polarization measurements on the rotational freedom of the membrane-associated probe, diphenylhexatriene, indicate that local anesthetics produce a small increase in the fluidity of membrane lipids. Spontaneous agglutination of transformed cells by low doses of concanavalin A is inhibited by colchicine and vinblastine but these alkaloids have no effect on concanavalin A agglutination of anesthetic-treated cells. Evidence is presented which suggests that local anesthetics may impair membrane peripheral proteins sensitive to colchicine (microtubules) and cytochalasin-B (microfilaments). Combined treatment of untransformed 3T3 cells with colchicine and cytochalasin B mimics the effect of local anesthetics in enhancing susceptibility to agglutination by low doses of concanavalin A. A hypothesis is presented on the respective roles of colchicine-sensitive and cytochalasin B-sensitive peripheral membrane proteins in controlling the topographical distribution of lectin receptors on the cell surface.     

Alterations of characteristic temperatures for lectin interactions in LM cells with altered lipid composition

Alteration of the fatty acid composition of mouse LM cell lipids dramatically affected the concanavalin A binding and concanavalin A-mediated hemadsorption properties of these cells. A critical temperature for these two concanavalin A related phenomena observed at 15–19° in cells with unaltered fatty acid composition was shifted to 22–28° for cells containing a higher proportion of saturated fatty acids and lowered to 7–11° for cells containing polyunsaturated fatty acids substituted for monoenoic unsaturated fatty acids. In contrast, a second critical temperature (at 5–7°) observed for concanavalin A binding and concanavalin A-mediated hemadsorption to LM cells was essentially unchanged by alterations in cellular lipid fatty acid composition. We conclude that a change in membrane lipid freezing point is responsible for the higher critical temperature (15–19°), and factors other than lipid melting properties, perhaps cytoskeleton structure, contribute to the lower critical temperature (5–7°) for lectin interactions with the exposed surface of LM cells.     

Properties of enucleated cells. III. Changes in cytoplasmic architecture of enucleated BHK21 cells following trypsinization and replating.

At low concentrations of concanavalin A (conA), binding of the lectin to the erythrocytes appears to be the rate-limiting step in the agglutination of these cells. At higher concentrations of lectin the rate of agglutination is concentration-independent, indicating that the aggregation reaction is rate-determining. Only 5 to 7% of the 1.2 × 10⁵ receptor sites need be occupied by con A in order for agglutination to take place. Although trypsin-treated cells bind 30% less ¹²⁵I-conA, they agglutinate better than untreated cells. At high lectin concentrations, erythrocyte agglutination by wheat germ agglutinin (WGA) is more than 8 times faster than the conA-mediated reaction. Lowering of the temperature to 0 °C reduces the rate but not the extent of the agglutination by both lectins. Mechanical shear reduced the conA-mediated agglutination of native cells by more than 160-fold and that of trypsinized and neuraminidase-treated cells 6-fold and 4-fold, respectively.It is concluded that metabolic activity, receptor mobility (i.e. cluster or patch formation) and cytochalasin B-sensitive processes, all of which have been reported to be involved in the lectin-mediated agglutination of fibroblasts and other cells, do not play a role in erythrocyte agglutination. Lectin-mediated erythrocyte agglutination appears to be governed primarily by the rate and extent of binding of lectin to the cell surface, the cell surface charge (modifiable by enzyme treatments or polycations) and the shear forces in the suspension. Morphological studies confirm and amplify these conclusions.     

A correlation between concanavalin A resistance and specific alterations in growth and surface membrane-associated properties.

Mammalian cells selected for resistance to concanavalin A (ConA) cytotoxicity exhibit modifications in some fundamental cellular properties. Three independently isolated ConA-resistant hamster cell lines exhibit a complex phenotype which includes: obvious temperature-sensitive growth properties; altered cellular morphology on solid surfaces; enhanced sensitivity to membrane-active agents such as phenethyl alcohol and sodium butyrate; altered lectin agglutination properties; modified adhesiveness to substratum properties; and defective lectin-receptor mobility characteristics. Selection of a reverant cell line which showed a near wild-type sensitivity to the cytotoxic effects of ConA also showed growth and membrane-associated properties that were very similar to parental wild-type cells. Somatic cell hybrids formed through the fusion of wild-type and lectin-resistant cells exhibited the ConA-sensitive phenotype, and possessed growth and membrane-associated properties that were very similar to pseudodiploid wild-type cells and control cultures of pseudotetraploid hybrid cells. The results presented in this communication support the view that ConA is an excellent selective agent for obtaining mammalian cells with altered growth and surface membrane properties and provides convincing evidence that the altered cellular properties exhibited by the lectin-resistant cell lines are directly related to ConA resistance.     

Glutaraldehyde fixation and the mechanism of erythrocyte agglutination by concanavalin a and soybean agglutinin

To evaluate the importance of lectin receptor mobility and clustering for enhanced cell agglutinability, the effect of glutaraldehyde fixation on the agglutinability of human erythrocytes by concanavalin A and soybean agglutinin was investigated. Agglutinability was evaluated in unperturbed Microtiter^® plates. Fixation increased slightly the agglutinability of the erythrocytes by both lectins. Fixation did not alter trypsin-enhanced agglutinability. Furthermore, when fixed erythrocytes were trypsinized, their agglutinability increased to the level of unfixed, trypsinized erythrocytes.The kinetics of agglutination of fixed and unfixed erythrocytes were monitored in an electronic particle counter. The shear forces associated with the kinetic experiments diminished fixed-cell to fixed-cell agglutination, i.e., both lectins gave slower kinetics of agglutination with fixed erythrocytes than with unfixed erythrocytes. In contrast, the kinetics of concanavalin A-mediated agglutination of trypsinized-fixed erythrocytes mixed with equal numbers of trypsinized-unfixed erythrocytes were indistinguishable from the rapid kinetics of agglutination of trypsinizedunfixed erythrocytes alone. Light microscopy revealed aggregates composed of fixed and unfixed erythrocytes.We conclude that glutaraldehyde fixation does not diminish the agglutinability of human erythrocytes under low-shear conditions. Our results indicate that the enhanced agglutination of trypsinized erythrocytes is not dependent on clustering of lectin receptors. The disruption of agglutination of fixed erythrocytes by shear forces that do not disrupt agglutination of fixed erythrocytes with unfixed erythrocytes suggests that the rigidity of the fixed erythrocyte may prevent stable aggregate formation by fixed erythrocytes alone.     

Molecular changes in the membranes of mouse erythroid cells accompanying differentiation.

The development of the mouse erythroblast to a mature erythrocyte is accompanied by changes in the composition and properties of the plasma membranes of these cells. Using double fluorescence techniques, we have simultaneously determined the distribution of lectin receptors and spectrin on the membranes of these cells. The lateral mobility of the lectin receptors in the membranes decreases as differentiation proceeds, and this is accompanied by an increasing concentration of spectrin associated with the membranes. The most significant concentration of spectrin occurs, however, during the enucleation of the late erythroblast, where we observe a complete segregation of the spectrin to the incipient reticulocyte, as well as a previously observed enrichment of receptors for concanavalin A into the plasma membrane surrounding the extruding nucleus. On the basis of these and other observations, we explore the possible molecular mechanisms involved in erythroblast enucleation and the role of spectrin in the regulation of protein mobility in erythroid cell membranes.     

The effect of diabetes on hepatocyte plasma membrane fluidity and concanavalin A-induced agglutination

The techniques of fluorescence polarization and lectin-induced agglutination have been utilized to investigate the effects of the diabetic state on some of the dynamic properties of cell membranes. Hepatocyte plasma membranes from streptozotocin-induced diabetic rats exhibited a significant decrease in cholesterol and sialic acid with no alteration in phospholipid content. This membrane system also exhibited a decrease in fluorescence polarization, using the fluorescent probe, 1,6-di-phenyl-1, 3,5-hexatriene, suggesting an increase in membrane fluidity over the value observed in normal hepatocytes. When normal hepatocytes were incubated in the presence of the lectin, concanavalin A (ConA), no significant agglutination was observed. In contrast, hepatocytes from diabetic rats which exhibited a slightly decreased lectin-binding capacity underwent extensive agglutination. In addition, normal hepatocytes which were pretreated with 0.1 mM tetracaine also underwent extensive agglutination with no measurable increase in lectin-binding capacity. These results suggest that altered membrane lipid fluidity and/or cytoskeletal organization may have a profound effect on cell surface dynamics and could result in the uncoupling of the insulin receptor complex from the membrane-associated effector system(s), a defect which may play a role in the problem of insulin resistance observed in some forms of diabetes.     

Factors affecting hemagglutination by concanavalin A and soybean agglutinin

While investigating the effect of temperature on hemagglutination by concanavalin A, we noted three factors that seriously interfere with the usual microscopic agglutination assay and produce misleading or ambiguous results. (1) Adherence of concanavalin A-treated erythrocytes to surfaces of plastic Petri dishes, especially at (2) commonly used cell densities, effectively prevents determination of agglutination. (3) In addition, incubation times usually used may be insufficient to demonstrate agglutination. Failure to account for these factors may explain the previously reported temperature-sensitive, concanavalin A-mediated agglutination of trypsinized erythrocytes and transformed cells (Vlodavsky, I., Inbar, M. and Sachs, L., (1972) Biochim. Biophys. Acta 274, 364–369). By controlling these factors, we demonstrated that concanavalin A does agglutinate trypsinized, human erythrocytes equally well at 24 and 4 °C.Investigation of the kinetics of erythrocyte agglutination by lectins revealed that the rate of agglutination by concanavalin A is markedly slower at lower temperatures while soybean agglutinin-mediated agglutination is faster at lower temperatures. Ultracentrifugation data indicate that at low temperature concanavalin A exists partially as a dimer (mol. wt 50 000) and at warmer temperatures exists mainly as a tetramer (mol. wt 100 000). The correlation of the effect of temperature on molecular weight with the agglutinating activity of concanavalin A suggests that temperature-dependent forms of concanavalin A may determine the rate of cell agglutination by this lectin. No temperature-dependent change in molecular form was observed with soybean agglutinin.     

Adriamycin-induced changes in the surface membrane of sarcoma 180 ascites cells

Adriamycin increases (a) the rate of agglutination of Sarcoma 180 cells by concanavalin A after brief exposure of 2–3 h and (b) membrane fluidity as measured by ESR within 30 min of exposure at concentrations of the anthracycline of 10^?7–10^?5 M. The effect of adriamycin on agglutination is not due to an increase in the number of surface receptors for concanavalin A, since the extent of binding of the lectin is not altered by adriamycin and no change occurs in the rate of occupancy of the concanavalin A binding sites by the lectin in cells treated with the antibiotic. The order parameter, a measurement of membrane fluidity, decreases in cells exposed to adriamycin and is dose-related. The results indicate that adriamycin can induce changes in the surface membrane of Sarcoma 180 cells within a brief period of exposure to a low but cytotoxic level of this agent.     

Effects of cholesterol and lipoproteins on endocytosis by a monocyte-like cell line

The human monocyte/macrophage-like cell line U937 is a cholesterol auxotroph. Incubation of these cells in the growth medium in which delipidated fetal calf serum has been substituted for fetal calf serum depletes cellular cholesterol and inhibits growth. The cholesterol requirement of these cells for growth can be satisfied by human low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL), but not by high-density lipoprotein (HDL). U937 cells can bind and degrade LDL via a high-affinity site and this recognition is altered by acetylation of LDL. This indicates that these cells express relatively high LDL receptor activity and low levels of the acetyl-LDL receptor. The cells were used to study the role of cholesterol in lectin-mediated and fluid-phase endocytosis. Growth of the cells in the medium containing delipidated fetal calf serum results in impairment of both concanavalin A-mediated endocytosis of horseradish peroxidase and concanavalin A-independent endocytosis of Lucifer Yellow. Supplementation of the medium with cholesterol prevents cellular cholesterol depletion, supports growth and stimulates Lucifer Yellow endocytosis but fails to restore horseradish peroxidase endocytosis. However, if the cells are incubated in the presence of no less than 40 μg LDL protein/ml to maintain normal cell cholesterol levels, concanavalin A-mediated endocytosis of horseradish peroxidase is activated. The effect of LDL is specific since neither VLDL nor HDL₃ at the same protein concentration activates horseradish peroxidase uptake by the cells. Furthermore, the activation of endocytosis by LDL is not inhibited by the inclusion of heparin or acetylation of the LDL indicating that binding of LDL to the LDL receptor is not required for these effects. The mediation of activation of horseradish peroxidase endocytosis by the lectin is presumed to involve binding of LDL to concanavalin A associated with the cell surface which in turn stimulates horseradish peroxidase binding and uptake by adsorptive endocytosis. The rate of fluid endocytosis and endosome formation seems to depend on cellular cholesterol content presumably because cholesterol is involved in maintaining the appropriate plasma membrane structure and fluidity.     

Characterization of an SV40-transformed 3T3 cell line expressing an unusual phenotype.

A transformed variant derived as a clone from normal 3T3 cells infected with simian virus 40 (SV40) has been found to possess a phenotype intermediate between that of normal cells and that characteristic of the transformed state, yet cells of the variant still test positively for the SV40-specific nuclear T-antigen. The variant exercises growth control, although not as stringently as do normal cells. Its cell size more closely resembles that of normal cells than of transformed cells. The variant also exhibits levels of spontaneous agglutination that are in line with those characteristic of the normal cells from which it was derived, and far higher than corresponding values for cells exhibiting the fully transformed phenotype. Plasma membranes of variant cells more closely resemble those of transformed cells than of normal cells as estimated by polyacrylamide gel electrophoresis. Perhaps the most distinguishing characteristic of the transformed variant is its complete immunity to agglutination by concanavalin A (Con A), even at concentrations of the lectin as high as 500 mug/ml. Moreover, trypsinization does not render variant cells as agglutinable in the presence of Con A as are untreated fully transformed cells. By contrast the variant displays a low tolerance of Con A toxicity, as monitored by ability to grow after treatment with the lectin, and on this count resembles transformed cells. Moreover a survey of several normal cell lines has revealed that even they do not consistently show resistance to Con A toxicity. These observations indicate that Con A-mediated agglutination and inability to grow after treatment with Con A are quite independent and do not bear a cause and effect relationship.     

Studies on the iodinated surface membrane proteins and concanavalin A agglutination of transformed Syrian hamster cells.

Chemically transformed Syrian hamster cells exhibit marked agglutination in the presence of the plant lectin, concanavalin A. In this report, we describe conditions which can alter this concanavalin A agglutinability, and compare the surface proteins from transformed cells which express different degrees of agglutinability. Lactoperoxidase-catalyzed iodination of tertiary Syrian hamster cells reveals the major iodinatable protein to be approximately 220 000 daltons. The transformed Syrian hamster cells do not contain this protein in an iodinatable form. Analyses of the transformed cells grown under conditions which decrease the concanavalin A agglutinability do not demonstrate any iodination of the 220 000 mol. wt. protein. These results depict the effects of growth and dibutyryl cyclic AMP on the iodinatable cell surface proteins of transformed cells and indicate that the absence of the I-220 000 mol. wt. protein is probably not a major determinant of concanavalin A agglutination.     

Effect of a phorbol ester on basic surface properties of trichomonads.

The effect of nanomolar concentrations of 12-O-tetradecanoilphorbol-13-acetate (TPA) on the cell surface of the urogenital parasitic protozoa Trichomonas vaginalis and Tritrichomonas foetus was evaluated by means of measurements of the parasites' surface tension, electrokinesis, lectin agglutination tests, and adhesion to inert substrates. TPA-treated parasites had their adhesion increased to both plastic and glass substrates. This was accompanied by increases in the parasites' net negative surface charge and also by changes in their surface tension. The lectin agglutination assays suggest that the increase in surface negativeness may be related in some extent to alterations in the oligosaccharide composition. Successive treatment of the microorganisms with TPA and sphingosine, a well-known competitive inhibitor of the phorbol ester active site, depressed the tendency of trichomonads to exhibit a phenotype of activated cells.     

Surface membrane redistribution and stabilization of concanavalin A-specific receptors following Yaba tumor poxvirus infection

Monkey kidney cells productively infected with Yaba tumor poxvirus clearly exhibit plasma membrane alterations when treated with both fluorescein-labeled and unlabeled concanavalin A. The convanavalin A-mediated cytoagglutination reaction for Yaba-infected Jinet and CV-1 cells increased linearly from 12 to 16 h post-infection, reaching a maximum by 24-28 h. Treatment of either Yaba-infected CVC-1 or Jinet cells with methyl-D-glucopyranoside before or after addition of concanavalin A completely blocked or reversed the cytoaglutination response. Trypsin treatment of uninfected CV-1 or Jinet cells enhanced concanavalin A-mediated cytoagglutination properties. Conversely, trypsin treatment of Yaba-infected Jinet cells resulted in a reduced cytoagglutination response. Increasing temperature and lectin concentration enhance concanavalin A-mediated cytoagglutination for uninfected, trypsin-treated and Yaba-infected CV-1 cells. Cytosine arabinoside has little or no effect on the Yaba-induced cell cytoagglutination reaction while cycloheximide blocks the cytoagglutinatin response if added prior to 12 h post-infection. Fluorescein-labeled concanavalin A binding studies have revealed that at 4 degrees C, Yaba-infected CV-1 cells display a predominantly 'patchy' pattern of topological fluorescence, while trypsin-treated and uninfected CV-1 cells at 4 degrees C display a uniform pattern of fluorescence binding. Patchy fluorescence, indicative of concanavalin A-suspeptible, receptor-site clustering on the surface membrane, was reduced 50% if Yaba-infected CV-1 cells were treated with glutaraldehyde (2.5%) before addition of fluorescein-labeled concanavalin A at 4 degrees C. Similar pre-fixatin of trypsin-treated CV-1 cells resulted in uniform, fluorescent labelling patterns at all assay temperatures.     

Changes in structural organization of surface membrane during erythrocyte maturation

The effect of concanavalin A and its succinylated derivative on cell agglutination and potassium compartmentation of mature and immature erythrocytes was observed. The binding of tetravalent concanavalin A to the surface glycoproteins of rabbit erythrocytes leads to a change in the properties of the surface membrane, which results in an induction of cell agglutination and concomitant release of potassium from the cells. Both of the phenomena induced by concanavalin A are temperature dependent, and observed at above 15°C.Divalent succinylated concanavalin A, lacking the inducing activity of surface glycoprotein cross-linking into patches and caps, caused neither cell agglutination nor change in the potassium compartmentation of erythrocytes and reticulocytes.In the case of immature reticulocytes, however, remarkable agglutination of the cells was induced without a change in the potassium compartmentation after treatment with tetravalent concanavalin A.It is suggested that changes in the molecular organization of the surface membrane occur in which potassium compartmentation of the reticulocytes becomes more susceptible to surface glycoprotein cross-linking during cellular maturation.     

Similarities in the membrane fluidity of 3T3 and SV101-3T3 cells and its relation to concanavalin A- and wheat germ agglutinin-induced agglutination.

Intact, viable ultransformed 3T3 and transformed SV101-3T3 cells were labeled with fatty acid spin labels and with 2,2,6,6-tetramethylpiperidine-1-oxyl in order to measure the fluidity properties of membrane lipids. Both cell types were grown in regular calf serum and in a lipid-depleted serum supplemented with either oleate or elaidate. The temperature dependence of the spectra obtained revealed inflections that correlate with the temperature below which agglutination with concanavalin A is inhibited, and another inflection that correlates with the temperature below which agglutination with wheat germ agglutinin is inhibited, suggesting that (a) the lipid phase(s) in the vicinity of the receptor(s) for these two lectins differ, and (b) a fluid membrane in the vicinity of the lectin receptor(s) is necessary for agglutination with either concanavalin A or wheat germ agglutinin. Studies with a partially characterized plasma membrane fraction suggest that the plasma membrane fluidity parameters closely resemble those of the intact cell. 3T3 and SV101-3T3 cells show virtually identical fluidity profiles by all of the tests we have applied.     

Lectin-induced cell agglutination and membrane cholesterol levels

The membranes of human and guinea pig erythrocytes were enriched with, or depleted of cholesterol. Ehrlich ascites carcinoma cells were also enriched with cholesterol and the extra slerol shown to be present in the plasma membrane. Enrichment of the cells with sterol made them less susceptible to agglutination by concanavalin A (ConA) or phytohemagglutinin (PHA), while removal of sterol from the erythrocytes increased their susceptibilily to agglutination. It is suggested that following changes in surface membrane sterol levels there are changes both in short-range movement of individual receptor molecules and in cell shape and deformability which control the agglutinability of the cells.