Regulation of concanavalin A agglutination by the extracellular matrix

The agglutinability of rat C₆ glioma cells by concanavalin A (Con A) depends upon cell density. From sparse density to near confluency agglutinability increases as cell density rises. Both the half-maximal concentration and the maximum amplitude of agglutination by Con A are functions of cell density, but are separate cell parameters differing in the extent to which they are affected by density and the point at which they become insensitive to further density increases. Both trypsin and EDTA reduce cell agglutinability. The similarity in recovery kinetics between low density cells and cells dissociated with EDTA or trypsin suggests that low density cells may lose the same surface agglutination component(s) removed by trypsin and EDTA. Density-dependent regulation of Con A agglutinability is anchorage dependent; cells grown in suspension display no such phenomenon. The cooperative cell regulation of agglutinability is mediated by the extracellular matrix, or micro-exudate. The matrix contains two activities: low density cultures produce a matrix inhibitor of Con A agglutinability, while high density cultures produce a matrix promotor.     

The role of the membrane skeleton in concanavalin A-mediated agglutination of human erythrocytes

In the erythrocyte membrane, the mobility of band 3 protein, the receptor for concanavalin A (Con A), is drastically reduced by the membrane skeleton. Yet, the vesicles free of membrane skeletal proteins, isolated from the highly agglutinable proteinase-treated cells, are found to be devoid of Con A agglutinability. The vesicles bind Con A in normal amounts, and remain agglutinable with the wheat germ and Ricinus agglutinins. Intracellular entrapment of monospecific antibodies to spectrin and 4.1 protein (two of the major skeletal components of the membrane) is also found to inhibit agglutination by 30-50%. Thus the membrane skeleton appears to play a positive role in the agglutination of the cells with Con A. The anti-ankyrin antibodies are found to be without any effect. The anti-band 3 (cytoplasmic domain) antibodies are also inhibitory to agglutination. Since Con A binding to cells alters the shape responses and deformability of the cells, and the cells resist fragmentation at 49 degrees C, the properties of the whole skeleton, especially spectrin, appear to be changed. The Con A-bound membranes also do not release the complex of spectrin-band 4.1-actin when extracted with a hypotonic medium. It appears that Con A binding leads to interaction of the cytoplasmic domain of the receptor with a skeletal component, possibly spectrin. Subsequent to this, the receptor molecules and the skeletal proteins undergo aggregation in the membrane, which is detected by their crosslinking by an 8.6-A span bifunctional reagent. The contractility believed to be associated with the membrane skeleton may be responsible for the aggregation.     

Effect of metabolic inhibitors on the agglutination of tumor cells by concanavalin A and Ricinus communis agglutinin

The agglutinations of rat ascites tumor cells by concanavalin A and by $\text{Ricinus communis}$ agglutinin were inhibited by low temperature, 2,4-dinitrophenol and cytochalasin B but not by cycloheximide. These metabolic inhibitors, however, did not inhibit the binding of the agglutinins to the cells. These results suggest that the agglutination was dependent on an active process; probably on a microfilament system responsible for cell surface movement which requires ATP, but not on protein synthesis.     

Quantitative agglutination of specific populations of sea urchin embryo cells with concanavalin A

It has been demonstrated that specific changes in carbohydrate-containing cell surface lectin receptor sites occur with differentiation and maturation of sea urchin embryo cells. In this study, evidence is presented, using a quantitative electronic particle counter assay to measure agglutination, which indicates that concanavalin A (Con A) mediated agglutination of dissociated 32/64 cell sea urchin embryos differs dramatically with respect to specific cell populations. The migratory cell type, the micromere, is significantly more agglutinable with Con A than the other cell types and colchicine treatment markedly increases sea urchin embryo cell agglutinability. The results indicate that like many malignant cells which display extensive migratory behavior, specific migratory populations of embryonic cells are agglutinable with Con A. The results are discussed with respect to the possible nature of lectin receptor sites on specific populations of embryonic cells and the possible role of colchicine-sensitive structures in controlling the display patterns of these sites.     

The effect of temperature of concanavalin A-mediated agglutination of cells with rigid receptors

The agglutination of a yeast, Candida albicans, by concanavalin A has been described. The agglutination was cell-number dependent. Prolonged incubation (60 min) was needed to reach maximum agglutination at 37° C. The rate but not the extent of agglutination was temperature dependent. The dimeric forms of concanavalin A, obtained either at low pH or after succinylation, agglutinated the yeast cells as well as the tetramer. Temperature changes affected the agglutination of yeast cells by dimers and by tetramers to the same extent.     

The role of Ia-bearing cells in T-cell proliferative response to concanavalin A

Ia-bearing cells are required for the concanavalin A (Con A)-induced T-lymphocyte proliferative response. We attempted to determine how and when the Ia-bearing cells are required in this response. As Ia-bearing cells, mitomycin C-treated nylon wool- and plastic dish-adherent cells (AACmc) were used. AACmc didn't lose their restoring capacity on treatment with anti-Thy-1, anti-Ig, but they did with anti-Ia. Using a Marbrook chamber system, it was shown that cellular contact between T cells and Ia-bearing cells is necessary for restoring the response. Results with AACmc addition after various times from Con A addition, or Ia-bearing cell elimination after various times from Con A addition indicate that the Ia-bearing cells are required for about 12 hr from the start of incubation with Con A. And it was shown that Con A-pretreated AACmc could stimulate Ia-depleted T cells, and allogenic AACmc could replace syngenic ones.     

Radiation-induced alterations in binding of concanavalin A to cells and in their susceptibility to agglutination

Cell susceptibility to agglutination mediated by a plant lectin, concanavalin A (Con A), and the binding capacity of Con A to cells following gamma-irradiation have been examined in mouse myeloid leukaemia cells cultured in suspension. Irradiation caused an immediate decrease in the amount of Con A bound to the cell surface, whereas susceptibility of irradiated cells to agglutination by Con A was unchanged when compared to that of the unirradiated cells. Post-irradiation incubation of cells at 37 degrees C resulted in a temporary, more than 1.3-fold increase in cell susceptibility to agglutination 60 min after irradiation, whereas binding capacity of cells for Con A gradually recovered following irradiation, reaching a comparable level to that of unirradiated cells 3 h after irradiation. Cell susceptibility to agglutination by Con A does not depend strongly on its binding capacity.     

The effect of temperature on concanavalin A-mediated agglutination of cells with rigid receptors.

The agglutination of a yeast, Candida albicans, by concanavalin A has been described. The agglutination was cell-number dependent. Prolonged incubation (60 min) was needed to reach maximum agglutination at 37 degrees C. The rate but not the extent of agglutination was temperature dependent. The dimeric forms of concanavalin A, obtained either at low pH or after succinylation, agglutinated the yeast cells as well as the tetramer. Temperature changes affected the agglutination of yeast cells by dimers and by tetramers to the same extent.     

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 1–220 000 mol. wt. protein is probably not a major determinant of concanavalin A agglutination.     

Inhibition of concanavalin A-induced agglutination of Novikoff tumor cells by cytochalasins and metabolic inhibitors : Role of cell-surface morphology and the distribution of concanavalin A receptors

Previous drug studies have suggested that concanavalin A (ConA)-induced cytoagglutination may be influenced by a system of contractile microfilaments. The present study was undertaken to determine the effects of microfilament-active drugs (cytochalasins B and D) (CB and CD) and inhibitors of ATP formation (NaN₃ and 2,4-dinitrophenol (DNP)) on ConA-induced agglutination of Novikoff cells and to investigate the mechanism(s) whereby these agents alter the surface properties of cells. The study described herein demonstrated that

1. 1. CB or CD inhibit cytoagglutination at concentrations above 1 or 0.1 μg/ml, respectively.

2. 2. NaN₃ and DNP both inhibit cytoagglutination, but DNP is more effective and specific.

3. 3. Combinations of metabolic inhibitors (NaN₃ or DNP) with CB lead to a greater reduction of cytoagglutination than with either agent alone.

4. 4. Maximal (>95%) cytoagglutination is still achieved in the presence of CB, CD, NaN₃, DNP, or combinations of these drugs.

5. 5. None of the drugs tested induced or allowed the redistribution of ConA receptors as measured by ferritin-ConA labeling.

6. 6. Both CB and CD induced the appearance of numerous blebs (zeioses) at the cell periphery, whereas metabolic inhibitors did not.

7. 7. The concentration of either CB or CD required to alter cell-surface morphology paralleled the concentration necessary to inhibit cytoagglutination.

These results suggest that the cytochalasins inhibit ConA-induced agglutination of Novikoff cells by their effect on cell-surface morphology, rather than by their effects on the topographical distribution of cell-surface lectin receptors, and that metabolic inhibitors reduce agglutinability by a different mechanism than the cytochalasins.     

Membrane lipid fluidity as rate limiting in the concanavalin A-mediated agglutination of pyBHK cells

The initial rate of concanavalin A-mediated agglutination of polyoma transformed Baby Hamster Kidney (pyBHK) cells follows Arrhenius kinetics. There is a smooth decrease in the agglutination rate from 37°C to 22°C with an activation energy of $\text{11.8 ± 0.2}\text{kcal/mol}$ in this region. There is a sharp decrease in agglutination rate below 22°C. The addition of 0.1 mM 1,3-di-tert-2-hydroxyl-5-methylbenzene, a lipid perturber, increases the agglutination rate by a factor of two and increases the membrane lipid fluidity as determined by the spin label method. The rotational correlation time of the spin label 2N14 $\text{(2,2-}\text{dimethyl-5-dodecyl-5-methyloxazolidine}\text{-N-}\text{oxide}$) was measured. The sum of the enthalpy of activation of rotational diffusion and the enthalpy of activation of translational diffusion is very nearly equal to the enthalpy of activation of agglutination. This is consistent with the rate limiting step of agglutination being receptor diffusion, which is probably limited in pyBHK cells by membrane lipid fluidity.     

Cellular energy dependent agglutination of rat ascites tumor cells mediated by concanavalin A and Ricinus communis agglutinin.

Effect of various metabolic inhibitors on the agglutination of rat ascites tumor cells mediated by concanavalin A and Ricinus communis agglutinin was studied using a quantitative assay method for agglutination in which turbidity of cell suspension is measured. Cell agglutination was inhibited by low temperature, cytochalasin B and inhibitors of energy generating systems without affecting lectin binding, and agglutination was not affected by hydroxyurea, actinomycin D or cycloheximide. The inhibitors of energy generating systems decreased the cellular ATP level and inhibited macromolecular synthesis under the conditions where they inhibited the agglutinations. In contrast, cytochalasin B did not depress the cellular ATP level nor inhibit RNA and protein syntheses. These results suggest that the agglutination is associated with cellular energy dependent processes other than macromolecular synthesis; probably with some cellular surface movements participated by microfilament activity.     

Effect of metabolic state on agglutination of human erythrocytes by concanavalin A.

Intact freshly drawn or stored human erythrocytes, which show little agglutination by concanavalin A, become agglutinable by this lectin in the presence of adenosine. alpha-Methylglucose (10 mM) completely inhibits this agglutination. The concanavalin A agglutination shows no sensitivity to vinblastine or cytochalasin B. Resealed membranes preparaed with ATP in lysing and resealing medium give modest agglutinability, while the presence of adenosine in both the lysing and the resealing medium results in a substantial agglutinability of the resealed membranes. Mild trypsin treatment of the erythrocytes causes an enhanced sensitivity to adenosine activation of the concanavalin A agglutination, while extensive trypsin treatment produced highly agglutinable erythrocytes that shown no response to the presence of adenosine in the lectin solution. The extensively treated erythrocytes also show concanavalin A agglutination at temperatures below 37 degrees C, under conditions in which intact or moderately treated erythrocytes do not agglutinate, with or without adenosine present. Results suggest that the adenosine activation of concanavalin A agglutination of intact human erythrocytes is mediated through a metabolic conversion of adenosine to a rapidly turned over metabolite which participates directly in the activation of agglutination. The agglutinability does not appear to depend on whole cell ATP levels, but may involve a particular pool of ATP. The effect of variation of cellular metabolic state and the response of particular systems involved in lectin-mediated agglutinability to cellular metabolism seem to be worth consideration in explaining the frequently large differences in agglutinability of und in cells in different biological states, such as those encountered in normal and transformed cells.     

Measurement of concanavalin A induced agglutination of follicular, luteal and atretic rat granulosa cells

To determine changes in the surface membrane of granulosa cells related to follicular atresia and luteinization, their agglutination behavior with Concanavalin A has been studied by quantitative spectrophotometry. As compared to the granulosa cells of normal Graafian follicles, the agglutination rate (delta A546/5 min) and final level of agglutination significantly increased in atretic and decreased in luteinized cells indicating the increase of Con A binding sites with atresia and decrease with luteinization. Treatments of granulosa cells with EDTA and trypsin enhanced agglutination of follicular and luteal cells but had no effect on atretic granulosa cells.