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.     

The role of microvilli in the agglutination of cells by concanavalin A

Morphological correlates of lectin agglutinability were examined in eight cell lines of varying sensitivity to agglutination by concanavalin A (ConA). The number of microvilli on the surface of cells growing in monolayers was positively correlated with agglutinability. However, when cells were brought into suspension, they all developed numerous microvilli which persisted when the cells were treated with ConA regardless of whether or not they were agglutinated by the lectin. Treatment of cells with dibutyryl cyclic AMP (db-cAMP) and theophylline caused a parallel decrease in agglutinability and numbers of microvilli in monolayer cultures, but suspended cells from control and treated cultures were identical in appearance in the absence or presence of ConA. The surface morphology of cells agglutinated by ConA was very similar to that of cells that spontaneously agglutinated in the absence of the lectin, and surface bound ConA was rapidly withdrawn from microvilli on all cell types. Neither the morphology of cells nor the surface distribution of ConA can explain observed differences in agglutinability.     

Role of cell membrane galactosyltransferase in concanavalin A agglutination of erythrocytes.

It has been previously observed that rabbit erythrocyte cell surface galactosyltransferase appears to play a role in concanavalin A agglutination of these erythrocytes (Podolsky et al., 1974). Further, a correlation between the occurrence or level of cell surface galactosyltransferase and concanavalin A agglutinability of other cell types has also been observed. The mechanism by which rabbit erythrocyte galactosyltransferase participates in concanavalin A agglutination has now been further defined. The enzyme was solubilized and purified. Characterization of the enzyme properties has shown them to be similar to those reported for other purified galactosyltransferases. Amino acid and carbohydrate analysis showed a high asparagine content and the presence of D-mannose. Specific alpha-mannosidase treatment of the enzyme showed that some of these D-mannose residues were terminal sugars. The purified enzyme also conferred concanavalin A agglutinability to non-agglutinable human erythrocytes. However, the ability to confer concanavalin A agglutinability was unrelated to the enzyme activity per se (as measured with fetuin acceptor) but appeared to be entirely dependent on the presence of terminal alpha-linked D-mannosyl residues in the enzyme structure. These findings suggest that the presence of terminal alpha-mannosidyl residues on cell surface glycoproteins such as galactosyltransferase may be the determining factor in agglutination of cells by concanavalin A.     

Surface morphology of normal and virus-transformed cells in suspended state and their concanavalin A agglutinability

Suspended cells are heterogeneous in respect to their surface microrelief. The distribution of different microrelieves varies in different cultures. It depends on the mode of cell detachment from the substrate - by EDTA or trypsin. Oncogenic transformation is accompanied by both the increase and decrease of microvillous microrelief. There is no correlation between the surface morphology of transformed cells and their agglutinability by concanavalin A. The treatment with trypsin results in the increase of both agglutinability by concanavalin A and microvillious microrelief.     

Dynamics of changes in the surface of normal cultivated rat cells after a single exposure to the carcinogen 7,12-dimethylbenz(a)anthracene]

Changes in the cell surface after a single treatment with 7,12-dimethylbenz(a)anthracene (DMBA) of newborn rat carcass in cell culture have been studied by means of the agglutination reaction with concanavalin A. DMBA was shown to cause alterations in the cell surface. At 0.5 mkg/ml of DMBA, the difference in agglutinability of treated and untreated cells persists for 30 days. At 0.1 mkg/ml of DMBA, the agglutinability of drug-treated and control cells was similar on the 4th day after removal of carcinogen. A prolonged culturing of control cells results in an increased agglutinability of cells with concanavalin A, and in 2.5 months it becomes indistinguishable from the agglutinability level of tumor cells with concanavalin A. In 5 months, drastic karyotypic changes are registered in control cultures.     

Mouse 3T3 cell filtrability correlating with concanavalin A agglutinability

In a previous study of the dextran gel sphere model system, a possible correlation between cell deformability and agglutinability by concanavalin A was indicated. Cell deformability was evaluated as filtrability, using polycarbonate membrane filtration. With 25-mm diameter filters and 5-ml cell suspensions at (0.8–16) · 10⁵ cells/ml, the filtrability at a given filter pore size was highly reproducible and was not affected by variations in cell population, viability, washings of cells retained on filter, or temperature. The filtrability of EDTA-dissociated 3T3 cells through 12-μm pore size filter was 8%, and a suspension of 10⁶ cells/ml was not agglutinated by 600 μg concanavalin A. The filtrability of trypsin-dissociated 3T3 cells was 95%, and these cells were agglutinated by 200 μg of the lectin. EDTA-dissociated SV-3T3 cells had a filtrability of 73% and were also highly agglutinable. Formalin fixation reduced the high filtrability to 6%, and also abolished the agglutinability. As a further test of the correlation, trypsin-dissociated 3T3 cells were admixed with the fixed cells. The agglutinability varied with the proportions of the two cell components, and the admixtures could be separated according to filtrability into the original components with distinctly different agglutinability. Furthermore, 25% of a random population of EDTA-dissociated SV-3T3 cells retained by the filter were found to be non-agglutinable. The separated SV-3T3 cell fractions could also form admixtures of different agglutinability. It is concluded that the agglutinability of mouse 3T3 and SV-3T3 cells by concanavalin A can be correlated with the predicted by cell filtrability.     

Cell cycle dependent modulations of the surface membrane of normal and SV40 virus transformed 3T3 cells

Agglutinability with Concanavalin was studied as function of cell cycle transition in normal and SV40 virus transformed 3T3 cells. In synchronized cultures of normal cells, agglutinbility was high during mitosis and disappeared rapidly. Agglutinability of transformed cells remained high in G₁ phase but diminished gradually upon entering S phase and reached minimum in G₁ phase. Decreased agglutinability a the end of the cell cycle was also observed in synchronous SV3T3 cultures by a combined technique of haemadsorption and density gradient centrifugation. In normal 3T3 cells, similar variations in agglutin ability during interphase could not be observed.     

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.     

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.     

Mitochondrial control of cell surface characteristics in Saccharomyces cerevisiae

Defects in the inner mitochondrial membrane of petite mutants of yeast resulted not only in respiratory deficiency, but also in changes in cell surface characteristics. These were (1) concanavalin A agglutinability, (2) cell movement in a biphasic polymer system, (3) cell adhesion. Genetic analysis indicated that the control exerted by the mitochondria was on nuclear genes or on the products of these genes which were presumably specifying cell surface components. These findings ascribe a new role to mitochondria but also have implications for neoplastic transformation.     

Modulation of agglutinability by alteration of the surface topography in mouse ascites tumor cells

Factors involved in controlling agglutinability of cells with plant lectins include number, distribution, availability and mobility of cell surface lectin receptor sites. We have examined the concanavalin A (ConA)-mediated agglutination of mouse sarcoma 180 ascites tumor cells in the presence or absence of cytochalasin B (CB) using a quantitative electronic particle counter assay. These cells become substantially more agglutinable after brief treatment with low concentrations of CB. Scanning and transmission electron microscopy indicate that CB causes formation of large, broad, cell surface ruffles and loss of narrow projections that appear to be microvilli. Studies using fluorescent ConA suggest that lectin receptor sites concentrate on these ruffles and that the ruffles seem to directly mediate increased agglutinability in this system. Electron spin resonance studies suggest that CB does not alter lipid “fluidity” in these cells. The results indicate that the gross cell surface topography favoring high agglutinability is one displaying broad ruffles, not numerous narrow projections.     

Cell surface microvilli and cell agglutinability.

Detached cells of some transformed mouse fibroblast lines have a villous surface whereas similarly treated cells of other lines are relatively smooth. These differences in surface morphology of detached cells are not reflected in their agglutinability with ConA and they cannot unambigously be explained from their morphology in situ. Treatments of normal and transformed Swiss mouse fibroblasts that induce marked changes in agglutinability with ConA do not cause equivalent changes in surface morphology. It is, therefore, unlikely that agglutinability of mouse fibroblasts by ConA is determined by the number of microvilli on the cell surface.     

Interactions of concanavalin A with chick embryo fibroblasts transformed by Rous sarcoma virus. Study with an RSV mutant thermosensitive for transformation.

The interactions between concanavalin A and chick embryo fibroblasts, normal and infected with Rous sarcoma virus (RSV-BH) or its thermosensitive mutant RSV-BH-Ta, have been studied. Normal chick embryo cells and RSV-BH transformed cells showed at 4 and 25 degrees C a similar number of concanavalin A receptors per cell. Analysis of the binding data by the Scatchard relation showed that apparent changes in binding as a function of temperature are due to the thermodynamic properties of the process and not to endocytosis. The lectin receptors on the cell surface of normal and RSV-BH infected cells showed homogeneity in their binding properties. Chick cells infected with RSV-BH-Ta showed a lectin binding behavior that was dependent on the temperature at which the cells were grown. At the permissive temperature for transformation (37 degrees C), the binding process was similar to that observed for normal and RSV-BH infected cells. At the nonpermissive temperature (41 degrees C), the cells showed at least two sets of concanavalin A receptors. The new set of receptors on the cell surface had a lower lectin affinity than those observed in the same cells at 37 degrees C. Chick cells infected with RSV-BH showed an enhanced agglutinability by concanavalin A, as compared with normal cells. Cells infected with RSV-BH-Ta showed a reversal of the correlation between increased concanavalin A agglutinability and the transformed state. At the permissive temperature for transformation, the cells were not agglutinable, whereas at the nonpermissive temperature they presented agglutinability indexes as high as those observed with RSV-BH infected cells. This enhanced agglutinability observed with cells maintained at the nonpermissive temperature for transformation may be related to the new set of low affinity receptors present at 41 degrees C.     

Relationship between cytoagglutination and saturation density of cell growth

The agglutination with concanavalin A and wheat germ agglutinin of the established malignant cells, HEp 2, KB, HeLa, TDB-3, HTC and RV 3T3, and of the putatively normal cells, BHK 21, 3T3 and Wi-38 was examined as a function of their saturation densities in culture. A positive correlation between the saturation density of the cell lines and the capacity to agglutinate was demonstrated. Incubation for 15 minutes with 1.25 mg/ml of trypsin converted non-agglutinating and poorly agglutinating cells into agglutinable ones, while leaving the highly agglutinating lines largely unchanged. The magnitude of change in agglutination after trypsin treatment correlated inversely with saturation density. Although the extent of agglutination varied with the saturation density, the agglutinability of a particular line remained relatively unchanged at different cell densities.     

Change of concanavalin A induced agglutinability during preimplantation mouse development

Mouse embryos during early cleavage (zygote to eight-cell stage) were agglutinable with a low concentration (10 μg/ml) of concanavalin A (ConA). This agglutinability was reduced during the first mitotic division. Morulae were agglutinable with a slightly higher concentration (100 μg/ml), whereas blastocysts were not agglutinable even with ConA at a concentration of 5000 μg/ml; however, isolated inner cell masses agglutinated readily at 10 μg/ml of ConA. Embryos grown in vitro behaved as did those isolated directly from the genital tract. Treatment with proteolytic enzymes did not induce agglutinability of mouse blastocyst. The change in agglutinability of trophoblastic cells reflects dramatic changes in the cell surface.     

Flow microfluorometric studies of lectin binding to mammalian cells. I. General features

Flow microfluorometry has been used to quantitate cell-surface binding of fluorescein-conjugated lectins. Frequency distributions of total surface binding of Concanavalin A per cell were prepared for a variety of cultured cell populations, including established cell lines, virus-transformed lines and non-transformed parental lines. In the case of growing Chinese hamster cells (line CHO), much of the variability of Con A binding per cell could be related to variability of cell size. Experiments with cells synchronized by mitotic selection indicated that the modal surface density of binding sites was almost constant throughout the cell cycle. However, as indicated by inhibition of binding with α-methyl mannopyranoside and by the effect of trypsin, the sites on each cell were heterogeneous in chemical structure and/or exposure. Agglutinability of virus-transformed cell lines or trypsin-treated parental lines was demonstrated but could not be correlated closely with binding.     

Interactions of concanavalin A with chick embryo fibroblasts transformed by rous sarcoma virus Study with an RSV mutant thermosensitive for transformation

The interactions between concanvalin A and chick embryo fibroblasts, normal and infected with Rous sarcoma virus (RSV-BH) or its thermosensitive mutant RSV-BH-Ta, have been studied. Normal chick embryo cells and RSV-BH transformed cells showed at 4 and 25 °C a similar number of concanavalin A receptors per cell. Analysis of the binding data by the Scatchard relation showed that apparent changes in binding as a function of temperature are due to the thermodynamic properties of the process and and not to endocytosis. The lectin receptors on the cell surface of normal and RSV-BH infected cells showed homogeneity in their binding properties. Chick cells infected with RSV-BH-Ta showed a lectin binding behavior that was dependent on the temperature at which the cells were grown. At the permissive temperature for transformation (37 °C), the binding process was similar to that observed for normal and RSV-BH infected cells. At the nonpermissive temperature (41 °C), the cells showed at least two sets of concanavalin A receptors. The new set of receptors on the cell surface had a lower lectin affinity than those observed in the same cells at 37 °C.Chick cells infected with RSV-BH showed an enhanced agglutinability by concanavalin A, as compared with normal cells. Cells infected with RSV-BH-Ta showed a reversal of the correlation between increased concanavalin A agglutinability and the transformed state. At the permissive temperature for transformation, the cells were not agglutinable, whereas at the nonpermissive temperature they presented agglutinability indexes as high as those observed with RSV-BH infected cells. This enhanced agglutinability observed with cells maintained at the nonpermissive temperature for transformation may be related to the new set of low affinity receptors present at 41 °C.     

Inhibitors of protein synthesis increase the agglutinability mediated by concanavalin A of the unfertilized mouse oocyte

The agglutinability mediated by concanavalin A has been studied in the zona-free mouse oocyte before and after fertilization or treatment with inhibitors of protein synthesis. At lectin concentrations of 10 μg/ml ot higher the zygote is clearly more agglutinable than the unfertilized oocyte. Treatment with 10 μg/ml of cycloheximide or puromycin, which causes parthenogenetic activation, greatly increases the agglutinability of the unfertilized oocyte; the agglutinability of the zygote and of the early blastocyst is not increased by such treatment. The possibility is discussed that the repression of cell division requires the synthesis of unstable protein molecules of the cell surface which are also involved in lectinmediated agglutinability.     

Reaction of lectins with human erythrocytes : III. Surface charge density and agglutination

A number of commercially available enzymes were used to modify the cell surface of human erythrocytes to varying degrees. In protease-treated erythrocytes the decrease in surface charge (determined by cell electrophoresis or analysis of sialic acid content) correlates with an increase in agglutinability with concanavalin A (ConA) and wheat germ agglutinin (WGA). On the other hand, treatment with neuraminidase leads to very large decrease in surface charge with only an intermediate increase in agglutinability with both lectins. Subsequent protease treatment of these cells enhances their agglutinability appreciably without further altering their surface charge. It is concluded that the increased agglutinability following protease treatment is due both to a decrease in the net negative charge and a removal of peptides and glycopeptides from the cell surface that may sterically hinder the agglutination reaction.     

Cell surface properties of L1210 leukaemia cells.

The surface properties of vincristine-colchicine sensitive and resistant L1210 leukaemic cells have been studied using concanavalin A mediated agglutination assay as well as electron microscopic visualization of concanavalin A receptors. 3H-colchicine uptake by the sensitive and resistant lines has also been compared. The resistant L1210 leukaemic cells proved less agglutinable than the sensitive ones at the same concanavalin A concentration. Previous treatments with either colchicine, vincristine or chlorpromazine caused a marked decrease in the agglutinability of the sensitive L1210 leukaemic cells, while agglutination of the resistant ones was lowered slightly by the same treatments. The 3H-colchicine uptake of the sensitive cells was three times higher than that of the resistant ones.