Influence of Concanavalin A on 3-O-methylglucose uptake in cultured chick embryo fibroblasts

Abstract. Concanavalin A (Con A) was found to inhibit hexose uptake in cultured fibroblasts derived from 8-day chick embryos and to stimulate this process in those derived from 16day embryos. (1) Con-A effects depended on the duration of contact with cells and lectin and were inhibited by α-methylmannopyrannoside. (2) Con A was shown to mask about 70% of the hexose carriers in both 8- and 16-day embryo fibroblasts. Lectin altered the hexose uptake very rapidly. (3) Con A only modified the V^max of the up- take system and did not alter the K^m. This indicates that either the number or mobility of hexose carriers were modified by Con-A treatment. The differential effect of lectin could be due to a modification of the hexose-carrier mobility during the embryonic differentiation of fibroblasts. Secondary effects may affect cell growth.     

Identification of components of phospholipids metabolism in automated phosphate ester chromatography.

Transport accross the cell membrane of 3-O-methylglucose, a non-phosphorylatable glucose analogue, was measured in primary cultures of fibroblasts from 8-, 12- and 16-day chick embryos. Transport of this hexose was found to be 3.5 times and 2 times faster in fibroblasts from 16-day embryos than in fibroblasts from 8- and 12-day embryos, respectively. Compared with 8- and 12-day embryos, the rate of efflux in fibroblasts from 16-day embryos was found to be increased. 3-O-methylglucose transport in these cells did not result in an accumulation of the hexose against a concentration gradient. It was concluded that in fibroblasts from older embryos a facilitated diffusion system for hexose transport was stimulated. Embryo differentiation could be associated with a change in the plasma membrane by increasing either the number or the mobility of the glucose carriers, since the Vmax of the transport system for 3-O-methylglucose increased in fibroblasts from older embryos, while the affinity or Km of the system remained unchanged.     

Increase in the number of glucose carriers in chick fibroblasts during embryo development

Cytochalasin B (CB) has been used as a tool to ascertain whether the increase in the rate of 3-O-methylglucose (3-O-MeG) uptake between the 8th and the 16th day of development in chick embryo fibroblasts could be attributed to an increase in the number of hexose transport carriers. There was a 2—3-fold difference in glucose-specific CB binding between the 8- and the 16-day cells, a difference which is comparable to the previously reported differences in rates of 3-O-MeG uptake. We therefore suggest that glucose-specific CB binding represents binding to the 3-O-MeG carrier and that the increase in the rate of 3-O-MeG uptake from the 8th and the 16th day of development is probably due to an increase in the number of hexose carriers.     

Interaction between Dolichos biflorus lectin and chick embryonic fibroblasts at different stages of development.

The interaction between chick embryo fibroblasts and A1-specific blood group Dolichos biflorus lectin has been studied at various stages of embryo development. The site number ((0.26 plus or minus 0.03)-10-6 sites/cell) remains the same during development whereas the affinity constant apparently decreases from 8-day cells onwards. The effects of cell number, temperature and time course on the Dolichos binding to fibroblasts were not age dependent. Competitive binding experiments revealed that Dolichos receptor sites were distinct from binding sites fo Robina pseudoacacia lectin and concanavalin A, but partially related to binding sites of Ricinus lectin. Thymidine incorporation by fibroblasts in the presence of Dolichos lectin was age dependent. It was inhibited in 6-day cells and weakly stimulated in 16-day cells, but not modified in 12-day cells. Dolichos lectin effects on embryo fibroblasts were very specific because both binding to cells and effect on thymidine incorporation were blocked by N-acetylgalactosamine, the determinant of Dolichos lectin, as well as by Dolichos antiserum.     

Inaccessibility of certain Ricinus lectin binding sites due to the increase in hyaluronic acid during chick embryo development

Chick embryo fibroblasts constitute a useful model for investigating cell surface differentiation using Ricinus lectin as a marker. Fibroblasts from 8-day chick embryos had two classes of Ricinus lectin binding sites, whereas those from 16-day embryos displayed only one class. Hyaluronidase treatment of fibroblasts from 8-day embryos had no effect on their capacity to bind Ricinus lectin; however after this treatment, 16-day cells resembled 8-day cells since the former also exhibited two classes of lectin-binding sites. Treatment with hyaluronidase released 2-5 times more hyaluronic acid from the older cells than from the younger cells. The same hyaluronidase treatment did not change the number of 8-day cells detached by trypsin from the substrate, but increased the number of detached 16-day cells. These observations suggest (i) that the greater adhesiveness to the substrate of the 16-day cells might be due to the presence on the cell surface of a larger amount of glycosaminoglycans at 16 days than at 8 days, and (ii) that the increased accumulation of hyaluronic acid on the cell surface might be involved in an alteration in the cell membrane during differentiation.     

Changes in lectin-induced deoxyribonucleic acid synthesis in cultures of chick-embryo fibroblasts at various stages of development

Concanavalin A and Robinia pseudoacacia lectin decreased [(3)H]thymidine incorporation into acid-insoluble material of fibroblasts cultured from 6-10-day chick embryos. In contrast, these lectins stimulated [(3)H]thymidine incorporation in cells from 16-day embryos. These effects are due to neither [(3)H]thymidine permeability modification nor toxicity of the lectins. The specificity of lectin action was proved by blocking experiments with alpha-methyl mannopyranoside and with anti-(Robinia lectin) serum.     

Inaccessibility of certain Ricinus lectin binding sites due to the increase in hyaluronic acid during chick embryo development

Abstract. Chick embryo fibre-blasts constitute a useful model for investigating cell surface differentiation using Ricinus lectin as a marker. Fibroblasts from 8-day chick embryos had two classes of Ricinus lectin binding sites, whereas those from 16-day embryos displayed only one class. Hyaluronidase treatment of fibroblasts from 8-day embryos had no effect on their capacity to bind Ricinus lectin;.however after this treatment, 16-day cells resembled 8-day cells since the former also exhibited two classes of lectin-binding sites. Treatment with hyaluronidase released 2–5 times more hyaluronic acid from the older cells than from the younger cells. The same hyaluronidase treatment did not change the number of 8-day cells detached by trypsin from the substrate, but increased the number of detached 16-day cells.
These observations suggest (i) that the greater adhesiveness to the substrate of the 16-day cells might be due to the presence on the cell surface of a larger amount of glycosaminoglycans at 16 days than at 8 days, and (ii) that the increased accumulation of hyaluronic acid on the cell surface might be involved in an alteration in the cell membrane during differentiation.     

Cell growth and thymidine incorporation changes induced by Dolichos lectin in embryo fibroblasts at various stages of differentiation.

We report here the effect of Dolichos lectin on chick embryo fibroblasts from embryos between 6th and 16th day of development. There is evidence that Dolichos lectin decreases cell number and proportion of cells incorporating tritium labelled thymidine in case of chick embryo fibroblasts of 6th, 8th and 10th day of development. Dolichos lectin stimulated the proliferation of 16-day old embryo cells. No effect was noticed on 12-day embryo cells at different concentrations of Dolichos lectin used. This lectin is specifically inhibited by N-acetyl-D-galactosamine and anti-Dolichos lectin serum. The difference in response by cells during different stages of embryonic development could perhaps be explained as some regulatory changes occurring on the cell surface.     

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.     

Wheat germ agglutinin and concanavalin A inhibit the response of human fibroblasts to peptide growth factors by a post-receptor mechanism

The effect of plant lectins on amino acid uptake and DNA synthesis in cultured human skin fibroblasts stimulated by various peptide mitogens was studied. Wheat germ agglutinin (WGA), at a concentration of 5 micrograms/ml, which by itself had little effect on 3H-aminoisobutyric acid (AIB) uptake, markedly inhibited stimulation of 3H-AIB uptake by somatomedin-C, insulin, epidermal growth factor (EGF) and platelet-derived growth factor. This inhibition could not be overcome by increasing the concentration of peptide added. Neither WGA nor concanavalin A (Con A) significantly affected basal 3H-thymidine incorporation. However both lectins, at concentrations of 5-20 micrograms/ml, decreased EGF- and insulin-stimulated DNA synthesis while succinyl Con A, a divalent lectin derivative, did not. The inhibitory effects of lectins on mitogenic stimulation were reversed by alpha-methyl mannose (Con A) or N-acetylglucosamine (WGA), and were not due to a reduction in the binding of growth factors to their receptors. It is concluded that certain lectins noncompetitively inhibit the response of human fibroblasts to multiple peptide mitogens at the post-receptor level, possibly by interfering with lateral mobility and aggregation of mitogen-receptor complexes.     

Cell surface glycosaminoglycans (GAGs) of cultured chick fibroblasts : Modifications in relation to the stage of embryo development

We have investigated the changes in glycosaminoglycan (GAG) composition between cultured fibroblasts derived from 8- and 16-day chick embryos. GAG composition has been studied after [3H]glucosamine and [35S]sulfate labeling. Both the 8- and 16-day embryo fibroblasts were found to contain hyaluronic acid (HA), dermatan sulfate (DS), heparan sulfate (HS) and chondroitin sulfates (CS), the latter being the major component in 8- and 16-day cells. These four GAGs were quantified after their separation using cellulose acetate electrophoresis. The amounts of HA and CS were respectively shown to increase 2-fold and 4-fold between the 8th and 16th day of development, whereas the amounts of HS and DS resp. diminished 2.5-fold and 1.2-fold. These results show that the relative proportions of the different GAGs alter during embryo development. The fibroblasts from 8-day-old embryos detached more rapidly from the culture dishes than the cells from 16-day-old embryos when treated with trypsin. However, this difference was not directly related to the different GAG content.     

Interaction between Dolichos biflorus lectin and chick embryonic fibroblasts at different stages of development

The interaction between chick embryo fibroblasts and A1-specific blood group Dolichos biflorus lectin has been studied at various stages of embryo development. The site number (($\text{0.26±0.03) · 10}{}^6\text{sites/cell}$) remains the same during development whereas the affinity constant apparently decreases from 8-day cells onwards. The effects of cell number, temperature and time course on the Dolichos binding to fibroblasts were not age dependent. Competitive binding experiments revealed that Dolichos receptor sites were distinct from binding sites of Robina pseudoacacia lectin and concanavalin A, but partially related to binding sites of Ricinus lectin. Thymidine incorporation by fibroblasts in the presence of Dolichos lectin was age dependent. It was inhibited in 6-day cells and weakly stimulated in 16-day cells, but not modified in 12-day cells. Dolichos lectin effects on embryo fibroblasts were very specific because both binding to cells and effect on thymidine incorporation were blocked by $\text{N-}\text{acetylgalactosamine}$, the determinant of Dolichos lectin, as well as by Dolichos antiserum.     

Effects of valinomycin on hexose transport and cellular ATP pools in mouse fibroblasts

The K+ ionophore valinomycin at concentrations of 1 X 10(-8) M and over, stimulated 2-deoxy-D-glucose (2DG) and 3-O-methylglucose (3OMG) uptake in Swiss 3T3 fibroblasts. The rate-limiting step of 2DG uptake was transport rather than phosphorylation, in the control or valinomycin-treated cells. Kinetic analysis showed that valinomycin increased the Vmax for 2DG uptake without change of the Km. The valinomycin-stimulated 2DG uptake was insensitive to 10 micrograms/ml cycloheximide, and extracellular K+ concentrations between 0.1 and 50 mM. On the other hand, valinomycin at the concentration of 1 X 10(-8) M and over, induced a rapid decrease in cellular ATP content, followed by stimulation of 2DG uptake and recovery of the ATP content. A similar relationship between the reduction of cellular ATP content and the subsequent stimulation of 2DG uptake was observed when the cells were treated not only with 2,4-dinitrophenol and iodoacetic acid, but also with other monovalent cation ionophores or inhibitors of oxidative phosphorylation. These results suggest that valinomycin may posttranslationally stimulate hexose transport by increasing the number of functional carriers of hexose or changing their mobility, and the rapid decrease in cellular ATP pools by valinomycin may be a trigger of the stimulation of the hexose transport in Swiss 3T3 fibroblasts.     

Concanavalin A-induced impairment of fibroblast spreading on laminin but not on fibronectin

The effect of concanavalin A (Con A) on the adhesion of 8-day-old chick embryo fibroblasts (CEFs) to fibronectin (FN) and laminin (LM) was studied. Con A was shown to inhibit the spreading of CEF on a LM substrate. In contrast, no inhibition of CEF spreading on the FN substrate could be detected when the quantity of FN coated varied from 0.5 to 4 pmoles. The effect induced by Con A was specific, since it was abolished by 100 mM alpha-methylmannopyranoside. The inhibition of CEF spreading was only observed when the lectin was added during the 20 min following cell plating. In addition, the effect of Con A on CEF spreading on the LM substrate was shown to be dependent upon its presence at the cell surface, since under conditions which accelerate the uptake of the lectin, the effect on cell spreading is no longer detectable. Furthermore, the number of CEFs attached to LM was not modified by the lectin. The molecular weight of the isolated Con A binding sites revealed glycoproteins ranging from 30,000 to 72,000. On the other hand, these Con A binding sites did not interact with LM-Sepharose. Only a protein with a molecular weight of 68,000 which did not express affinity for Con A bound tightly to the LM-Sepharose. These data suggested that cell surface Con A binding sites do not interfere with the initial step of CEF adhesion to LM but play a key role during their spreading on this glycoprotein.     

Modification of the N-linked oligosaccharides in cell surface glycoproteins during chick embryo development. A using lectin affinity and a high resolution chromatography study

Important differences in asparagine-linked glycopeptides were observed in vitro cultured fibroblasts derived from chick embryo at different stages of development. Cells from 8-day and 16-day embryos were labeled metabolically with [3H]mannose. Cell surface glycopeptides obtained after mild trypsin treatment were extensively digested with pronase and then chromatographed on concanavalin-A-Sepharose and other immobilized lectins. The most important changes concerned the complex type chains. The ratio between triantennary plus tetraantennary and biantennary chains increased about 2.5-fold from the 8th to the 16th day of development. In the same way, complex chains with bisecting N-acetylglucosamine increased from 8-day to 16-day cells as shown by Phaseolus-vulgaris-erythroagglutinin--agarose chromatography. In 16-day cells, the majority of triantennary chains (60%) with alpha-linked mannose substituted at C2 and C6 positions and biantennary chains (50%) were shown to contain fucosyl (alpha 1----6)N-acetylglucosaminyl structure in the core region by their ability to bind to a lentil lectin affinity column. Similarly, in 8-day cells, triantennary chains (50%) were more fucosylated than biantennary chains (35%). Thus, complex structures exhibited an increased fucosylation of their invariable core from the 8th to the 16th day of development, except for fucosylated triantennary chains which were retained on Phaseolus vulgaris Leucoagglutin and on lentil lectin. These latter structures were present at the surface of 8-day cells and absent at the surface of 16-day cells. After chromatography on Bio-Gel P6 and treatment with endo-beta-N-acetylglucosaminidase H, the [3H]-mannose-labeled glycopeptides were separated by high resolution chromatography into glycopeptides with complex chains and glycopeptides with high-mannose chains. Analysis of the high-mannose oligosaccharides released after endo-beta-N-acetylglucosaminidase H treatment by chromatography on Bio-Gel P4 indicated that the same type of high-mannose chains were present at the surface of 8-day and 16-day cells. Quantification of mannose, galactose and sialic acid residues using gas liquid chromatography was consistent with a decrease of the relative amount of oligomannose chains and an increase of the relative amount of complex type chains in 16-day cells compared to 8-day cells. Thus N-linked oligosaccharides derived from cell surface glycoproteins undergo changes during embryo development resulting in greater complexity of carbohydrate chains.     

Receptor mobility and the binding of cells to lectin-coated fibers

The ability of cells to bind to nylon fibers coated with lectin molecules interspaced with varying numbers of albumin molecules has been analyzed. The cells used were lymphoma cells, normal lymphocytes, myeloid leukemia cells, and normal and transformed fibroblasts, and the fibers were coated with different densities of concanavalin A or the lectins from soybean or wheat germ. Cells fixed with glutaraldehyde did not bind to lectin-coated fibers. The number of cells bound to fibers could be increased by increasing the density of lectin molecules on the fiber, the density of specific receptors on the cell, or the mobility of the receptors. It is suggested that binding of cells to fibers involves alignment and binding of specific cell surface receptors with lectin molecules immobilized on the fibers, and that this alignment requires short-range rapid lateral mobility (RLM) of the receptors. The titration of cell binding to fibers coated with different densities of lectin and albumin has been used to measure the relative RLM of unoccupied cell surface receptors for the lectin. The results indicate a relationship of RLM to lectin-induced cell-to-cell binding. The RLM or receptors for concanavalin A (Con A) was generally found to be higher than that of receptors for the lectins from wheat germ or soybean. Receptor RLM could be decreased by use of metabolic inhibitors or by lowering the temperature. Receptors for Con A had a lower RLM on normal fibroblasts than on SV40-transformed fibroblasts, and trypsinization of normal fibroblasts increased Con A receptor RLM. Normal lymphocytes, lymphoma cells, and lines of myeloid leukemia cells that can be induced to differentiate had a high receptor RLM, whereas lines of myeloid leukemia cells that could not be induced to differentiate had a low receptor RLM. These results suggest that the RLM of Con A receptors is related to the transformation of fibroblasts and the ability of myeloid leukemia cells to undergo differentiation     

Embryo Cell Surfaces—Lectin Binding and Cell Proliferation

The interaction between chick embryo fibroblasts and various lectins has been studied at different stages of embryo development. There is evidence that Robinia lectin, Dolichos lectin, and Conca navalin A decrease cell number and proportion of cells incorporating [³H] thymidine in case of 8and 10day-old chick embryo fibroblasts, whereas they stimulated the proliferation of 16-dayold embryo cells. No effect was noticed in 12-day cells.
These results suggest that some cell surface changes occur during embryo development. The site number of Dolichos lectin remains the same during embryo development, and the affinity constant decreases. The site number of Robinia lectin and Concanavalin A decreases from the 8^th to the 12^th day of development, and slowly increases on the 16–day cells, the affinity constant remaining rather constant.
The results indicate that the age–dependent effect of lectin on embryo cells could not be directly related to the number of lectin–binding sites. Competitive binding experiments revealed that Dolichos receptor sites were distincts from binding sites of Robinia lectin and Concanavalin A, and Robina receptor sites distinct from those of Concanavalin A.
Lectin effects on embryo fibroblasts were very specific as determined by inhibitory assays.     

Hexose and amino acid transport by chicken embryo fibroblasts infected with temperature-sensitive mutant of Rous sarcoma virus. Comparison of transport properties of whole cells and membrane vesicles

The effect of transformation on hexose and amino acid transport has been studied using whole cells and membrane vesicles of chicken embryo fibroblasts infected with the temperature-sensitive mutant of the Rous sarcoma virus, TS-68. In whole cells, TS-68-infected chicken embryo fibroblasts cultured at the permissive temperature (37 degrees C) had a 2-fold higher rate of 2-deoxy-D-glucose uptake than the same cells cultured at the non-permissive temperature (41 degrees C). However, both the non-transformed and transformed cells had comparable rates of alpha-aminoisobutyric acid transport. Membrane vesicles, isolated from TS-68-infected chicken embryo fibroblasts cultured at 41 degrees C or 37 degrees C, displayed carrier-mediated, intravesicular uptake of D-glucose and alpha-aminoisobutyric acid. Membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 37 degrees C had an approx. 50% greater initial rate of stereospecific hexose uptake than the membrane vesicles from fibroblasts cultured at 41 degrees C. The two types of membrane vesicle had similar uptake rates of alpha-aminoisobutyric acid. The results of hexose and amino acid uptake by the membrane vesicles correlated well with those observed with the whole cells. Km values for stereospecific D-glucose uptake by the membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 41 and 37 degrees C were similar, but the V value was greater for the membrane vesicles from TS-68-infected cells cultured at 37 degrees C. Cytochalasin B competitively inhibited stereospecific hexose uptake in both types of membrane vesicle. These findings suggest that the membrane vesicles retained many of the features of hexose and amino acid transport observed in whole cells, and that the increased rate of hexose transport seen in the virally-transformed chicken embryo fibroblasts was due to an increase in the number or availability of hexose carriers.     

Hexose and amino acid transport by chicken embryo fibroblasts infected with temperature-sensitive mutant of rous sarcoma virus: Comparison of transport properties of whole cells and membrane vesicles

The effect of transformation on hexose and amino acid transport has been studied using whole cells and membrane vesicles of chicken embryo fibroblasts infected with the temperature-sensitive mutant of the Rous sarcoma virus, TS-68. In whole cells, TS-68-infected chicken embryo fibroblasts cultured at the permissive temperature (37°C) had a 2-fold higher rate of 2-deoxy-d-glucose uptake than the same cells cultured at the non-permissive temperature (41°C). However, both the non-transformed and transformed cells had comparable rates of α-aminoisobutyric acid transport. Membrane vesicles, isolated from TS-68-infected chicken embryo fibroblasts cultured at 41°C or 37°C, displayed carrier-mediated, intravesicular uptake of d-glucose and α-aminoisobutyric acid. Membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 37°C had an approx. 50% greater initial rate of stereospecific hexose uptake than the membrane vesicles from fibroblasts cultured at 41°C. The two types of membrane vesicle had similar uptake rates of α-aminoisobutyric acid. The results of hexose and amino acid uptake by the membrane vesicles correlated well with those observed with the whole cells. K_m values for stereospecific d-glucose uptake by the membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 41 and 37°C were similar, but the V value was greater for the membrane vesicles from TS-68-infected cells cultured at 37°C. Cytochalasin B competitively inhibited stereospecific hexose uptake in both types of membrane vesicle. These findings suggest that the membrane vesicles retained many of the features of hexose and amino acid transport observed in whole cells, and that the increased rate of hexose transport seen in the virallytransformed chicken embryo fibroblasts was due to an increase in the number or availability of hexose carriers.     

Cell-to-cell binding induced by different lectins

The cell-to-cell binding induced by concanavalin A (Con A) and the lectins from wheatgerm, soybean, and waxbean has been analyzed by measuring the ability of single cells to bind to lectin-coated cells immobilized on nylon fibers. The cells used were lymphoma, myeloid leukemia, and normal fibroblast cells. With all lectins, cell-to-cell binding was inhibited if both cells were prefixed with glutaraldehyde. However, in most cases cell-to-cell binding was enhanced when only the lectin-coated cell was prefixed. With normal fibroblasts, treatment of either one or both cells with trypsin enhanced the cell-to-cell binding induced by Con A and the wheatgerm lectin. Neuraminidase, which increases the number of receptors for soybean agglutinin, increased cell-to-cell binding only if both cells were treated. Although cell-to- cell binding induced by the lectins from soybean and wheatgerm could be partially reversed by the appropriate competitive saccharide inhibitor, binding induced by Con A could not be reversed. The experiments indicate that cell-to-cell binding induced by a lectin can be prevented by an insufficient density of receptors for the lectin, insufficient receptor mobility, or induced clustering of receptors. These effects can explain the differences in cell-to-cell binding and agglutination observed with different cell types and lectins. They also suggest that cell-to-cell binding induced by different lectins with a variety of cell types is initiated by a mechanism involving the alignment of complementary receptors on the colliding cells for the formation of multiple cell-to-lectin-to-cell bridges.