Inhibition of lymphocyte 5'-nucleotidase by lectins: effects of lectin specificity and cross-linking ability

5'-Nucleotidase, an integral glycoprotein enzyme of the lymphocyte plasma membrane, is inhibited cooperatively by the lectin concanavalin A. Because divalent succinyl-concanavalin A is a poor enzyme inhibitor, both binding and lectin-induced cross-linking of 5'-nucleotidase may be necessary for inhibition. Succinyl-concanavalin A does not compete with concanavalin A for binding to the enzyme; however, maleyl-concanavalin A, another poor inhibitor, competes effectively with the parent lectin. Thus, maleyl-concanavalin A binds to the same site as concanavalin A but causes little inhibition, whereas succinyl-concanavalin A does not bind to this site. The monovalent lectin from Ricinus communis (RCA-60) is a more effective enzyme inhibitor than the related divalent lectin (RCA-120), and inactivation of the second low-affinity sugar binding site on RCA-60 does not abolish inhibition, suggesting that multivalent cross-linking is not required for 5'-nucleotidase inhibition. Peanut and wheat germ agglutinins do not inhibit the enzyme, whereas lectins from lentil, pea, soybean, Griffonia simplicifolia, and Phaseolus vulgaris inhibit 5'-nucleotidase with various degrees of effectiveness. The only lectin showing strong positive cooperativity in its interaction with 5'-nucleotidase is concanavalin A.     

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.     

Synthesis of cluster mannosides via a Ugi four-component reaction and their inhibition against the binding of yeast mannan to concanavalin A

The Ugi four-component reaction (U-4CR) was utilized to prepare divalent and trivalent cluster mannosides with different scaffolds. The glycoclusters obtained were tested for their relative inhibitory potency against the binding of yeast mannan to concanavalin A by solid-phase enzyme-linked lectin assays (ELLA) using methyl alpha-D-mannopyranoside as a standard. Among them, a divalent mannoside containing aromatic groups showed the strongest binding affinity to concanavalin A.     

Thermodynamic, kinetic, and electron microscopy studies of concanavalin A and Dioclea grandiflora lectin cross-linked with synthetic divalent carbohydrates

The jack bean lectin concanavalin A (ConA) and the Dioclea grandiflora lectin (DGL) are highly homologous Man/Glc-specific members of the Diocleinae subtribe. Both lectins bind, cross-link, and precipitate with carbohydrates possessing multiple terminal nonreducing Man residues. The present study investigates the binding and cross-linking interactions of ConA and DGL with a series of synthetic divalent carbohydrates that possess spacer groups with increasing flexibility and length between terminal alpha-mannopyranoside residues. Isothermal titration microcalorimetry was used to determine the thermodynamics of binding of the two lectins to the divalent analogs, and kinetic light scattering and electron microscopy studies were used to characterize the cross-linking interactions of the lectins with the carbohydrates. The results demonstrated that divalent analogs with flexible spacer groups between the two terminal Man residues possess higher affinities for the two lectins as compared with those with inflexible spacer groups. Furthermore, despite their high degree of homology, ConA and DGL exhibit differences in their kinetics of cross-linking and precipitation with the divalent analogs. Electron microscopy shows the loss of organized cross-linked lattices of the two lectins with analogs possessing increased distance between the terminal Man residues. The loss of lattice patterns with the analogs is distinct for each lectin. These results have important implications for the interactions of lectins with multivalent carbohydrate receptors in biological systems.     

Thymus-independent immunogenicity in vitro of the divalent antigen DNP-polyethylene oxide

Chemically simple and physically well-defined dinitrophenyl derivatives of polyethylene oxide (DNP-PEO) can be prepared in a wide range of forms and sizes. These materials were used to investigate the molecular basis of immunogenicity and the binding of the antigens to membrane-bound receptors. Both di- and multivalent DNP-PEO activate normal murine B lymphocytes to yield primary anti-DNP antibody response in vitro. The immunogenicity is dependent on the carrier chain length but independent of T cells. Responses comparable to those induced by DNP-conjugated polymerized flagellin are induced by divalent linear materials of medium molecular weights of about 60,000. A highly multivalent material is moderately immunogenic, but at much lower antigen doses than divalent materials. The carrier PEO does not affect B-cell responses to DNP-PEO or T-cell response to succinyl concanavalin A. Moreover, it shows no polyclonal mitogenicity at concentrations as high as 1 mg/ml. Studies of antigen binding to cell surface DNP receptors show that the strongly immunogenic materials of medium molecular weights have an appreciable tendency to bind bivalently and thus potentially to crosslink receptors. The binding of smaller, less immunogenic antigen appears predominantly monovalent.     

Selective separation of microorganisms by lectins: yeast and concanavalin A as a model system

Specific aggregation and separation of microorganisms was investigated using yeasts and concanavalin A as a model system. Cells of Saccharomyces cerevisiae were specifically aggregated and so separated from those of Schizosaccharomyces pombe. Optimum aggregation with over 99% of cells aggregated was achieved by adjustment to pH value and applied agitation. Dimeric lectin structure caused a far higher degree of aggregation than did tetrameric. Degree of aggregation was also strongly influenced by the ratio of lectin/cell concentrations, optimum aggregation occurring in the middle range of ratios. A high ratio of lectin to cells inhibited aggregation, occupation of most of the available receptors preventing intercellular bonding by divalent lectins. Detachment and reuse of concanavalin A was demonstrated using switching from moderate to low pH value. Potential uses for species-specific-separation of microorganisms are discussed. (c) 1992 John Wiley & Sons, Inc.     

Influence of lectins on the binding of 125I-labeled EGF to human fibroblasts.

Lectins that interact with mannose (concanavalin A), galactose (ricin, abrin), or N-acetylglucosamine (wheat germ agglutinin) block ¹²⁵I-labeled EGF binding to the surface of cultured human fibroblasts at 37° or 5°. Lectins specific for fucose or N-acetylgalactosamine, soybean agglutinin or gorse lectin, respectively, do not interfere with growth factor binding. The inhibition of ¹²⁵I-labeled EGF binding by concanavalin A at 37° or 5° could be reversed rapidly by the addition of α-methyl mannoside. The results suggest that the fibroblast membrane receptor for EGF is, or is closely associated with, a glycoprotein or glycolipid that contains mannose, galactose and N-acetylglucosamine residues.     

Antigenic stimulation of lymphocytes I. Calorimetric exploration of metabolic responses

The rate of evolution of beat, an index of overall metabolic activity, has been measured for lymphocytes undergoing simple culture as well as in the presence of the specific antigen, DNP-BSA (dinitrophenylated bovine serum albumin), of the mitogenic lectin Con A (concanavalin A), and of inhibitors of various aspects of cell culture. Unstimulated lymphocytes evolve heat initially at a decreasing rate which stabilizes after 3 days of culture. Cells stimulated with antigen increase thermogenesis after 2 days of exposure to a maximum at 4–5 days, on a schedule that parallels increases in DNA synthesis. Cells stimulated with Con A show early increases in heat output which precede the onset of increased DNA synthesis. Inhibition of DNA synthesis alone does not significantly inhibit heat output, whereas inhibition of protein and RNA synthesis has a profound effect, as does interference with the structure of microfilaments with cytochalasin B. The completely synthetic multivalent high molecular weight antigen, PEO-DNP₈₀ (dinitrophenylated polyethylene oxide) is capable of abolishing the response of the cells to DNP-BSA and to Con A as well as of directly inhibiting the output of heat by the cells.     

Different effects of concanavalin A and its succinylated derivative on superoxide release in peritoneal macrophages

The effects of tetravalent concanavalin A and its succinylated derivative on the intracellular production of superoxide anion (O₂^?) and its release into cell exterior of peritoneal macrophages were observed. Both tetravalent concanavalin A and its succinylated derivative induced marked enhancement of intracellular reduction of nitroblue tetrazolium, which could be inhibited by α-methyl-D-glucoside. The extent of activation of nitroblue tetrazolium reduction induced by both types of the lectin paralleled the activation ratio of oxygen consumption.There was littele difference in the extent of intracellular O₂^? production induced by two types of the lectin. Nitroblue tetrazolium reduction was not affected significantly by pretreatment with colchicine, rotenone or malonate, inhibitors of the cytoskeletal system and of the electron transport system. In contrast, tetravalent concanavalin A induced a higher rate of superoxide release than did succinylated divalent concanavalin A, which lacks the cross-linking activity of surface glycoproteins.These results indicate that superoxide production following oxygen consumption and superoxide release into cell exterior are controlled independently by a separate membrane mechanism and that superoxide production system is not essentially dependent on the involvement of the cytoskeletal system.     

Concanavalin a binding and Ca2+ fluxes in rat spleen cells

Addition of the mitogenic lectin concanavalin A to rat spleen cells results in a small increase in the steady-state Ca²⁺ content of the cells. ⁴⁵Ca²⁺ fluxes were measured under conditions where artifacts due to Ca²⁺ binding to concanavalin A could be excluded. Both ⁴⁵Ca²⁺ influx into and efflux from these cells are significantly activated by the lectin. If ⁴⁵Ca²⁺ is added 30 min after concanavalin A the rate of influx is further enhanced. The increase in ⁴⁵Ca²⁺ influx correlates well with binding of concanavalin A to the cells. At low concentrations (optimal mitogenic) of the lectin (1 and 3 μg/ml) no significant increase in ⁴⁵Ca²⁺ influx occurs but an increase in ⁴⁵Ca²⁺ efflux is still observed. The results suggest that concanavalin A binding to the cell surface causes an increase in Ca²⁺ influx into the cells and that activation of Ca²⁺ efflux occurs as a response to an increase in the cytosolic Ca²⁺ activity. Thus, Ca²⁺ may well play a role in triggering lymphocyte activation.     

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.     

Alteration of quaternary structure and biological activity of concanavalin A

A major molecular species of concanavalin A (Con A), a mitogenic lectin from jack bean seeds, has a quaternary structure composed of four homologous subunits and a tetravalent sugar-binding ability. We show that the tetrameric Con A can be converted into a monovalent monomeric form by either photochemical alkylation or hydrogen peroxide/dioxane oxidation of about two tryptophan residues. A divalent dimeric derivative of Con A is also prepared by sulfomethylamidation of about four carboxyl groups. Chemical properties and mitogenic and hemagglutinating activities of these new Con A derivates are compared with those of the tetravalent Con A, as well as of the Con A derivatives that have appeared in the literature on cell biological studies. The significance of the lectin valences in lymphocyte activation and hemagglutination is also discussed.     

Schistosoma mansoni: lectin-dependent cytotoxicity of hemocytes from susceptible host snails, Biomphalaria glabrata

Normally benign hemocytes from a strain (M-line) of the snail, Biomphalaria glabrata, susceptible to Schistosoma mansoni, became cytotoxic toward the sporocyst stage if the parasite was first treated with the lectin, concanavalin A. Concanavalin A binding was inhibitable with alpha-methyl mannoside and killing was dose-dependent. Maximal levels of concanavalin A-induced cytotoxicity were comparable with levels observed when hemocytes from a resistant snail strain (13-16-R1) encountered untreated sporocysts. Induction of the cytotoxic response did not occur if hemocytes alone were pretreated with the lectin. A unique method incorporating ultraviolet microscopy and the vital fluorescent dye, eosin Y, was used for discriminating between live and dead sporocysts. This model may prove useful in understanding mechanisms used by invertebrate effector cells in recognition and killing of invading organisms.     

Modulation of lymphocyte proliferative responses by a canine Lyme disease vaccine of recombinant outer surface protein A (OspA)

The modulation of human lymphocyte proliferative responses was demonstrated with a recombinant outer surface protein A (OspA) vaccine preparation for the prevention of Borrelia burgdorferi infection. After exposure to either the unaltered vaccine preparation or OspA prepared in saline, normal lymphocyte responses to the mitogens concanavalin A, phytohemagglutinin-M or pokeweed mitogen, or the antigen BCG were consistently reduced. Whole cell extracts of B. burgdorferi also modulated immune responses but required a much greater quantity of protein than needed for the OspA preparation. The magnitude of modulation was directly dependent on the quantity of OspA. OspA interferes with the response of lymphocytes to proliferative stimuli including a blocking of cell cycle phase progression. Future studies designed to delete the particular region or component of the OspA molecule responsible for this effect may lead to improved vaccine preparations.     

Interaction of Concanavalin A and a Divalent Derivative with Lymphocytes and Reconstituted Lymphocyte Membrane Glycoproteins

Both concanavalin A (con A) and its divalent derivative, succinyl-concanavalin A (S-con A) are mitogenic for porcine lymph node lymphocytes. We have compared the binding of these two lectins to intact porcine lymphocytes and phospholipid vesicles containing reconstituted lymphocyte membrane glycoproteins. Both con A and S-con A showed high- and low-affinity binding to intact cells, as indicated by LIGAND analysis of Scatchard plots of binding data. Despite the apparently identical saccharide specificities of the two lectins, high-affinity binding sites for S-con A were only one-third as numerous as high-affinity sites for the parent lectin. Large numbers of low-affinity binding sites existed for con A, while many fewer were present for S-con A. It is suggested that these sites result from hydrophobic association. Con A bound to lymphocytes in a positively cooperative fashion, while S-con A showed non-cooperative behavior. Lectin binding to large unilamellar phospholipid vesicles containing reconstituted lymphocyte membrane glycoproteins was measured using a rapid filtration assay, and was linear with the glycoprotein content of the vesicles. Almost all of the outward-facing glycoprotein was functional in terms of lectin binding. Reconstituted glycoproteins showed only a single class of high-affinity binding sites for both con A and S-con A, with association constants similar to those measured for intact cells. Con A, but not S-con A, showed positively cooperative binding to reconstituted vesicles. Cooperativity was observed in both gel phase and liquid crystalline phase lipid, and was thus not dependent on long-range lateral rearrangement of glycoprotein receptors. Results suggested that con A induces a microre-distribution of receptors on the lymphocyte membrane surface, leading to the exposure of glycoproteins that were previously inaccessible to the lectin. S-Con A does not cause glycoprotein redistribution, and a large fraction of the receptors remain cryptic.     

Suppression of DNA synthesis in mitogen stimulated leukocytes with partial trypsin digests of Staphylococcus aureus protein A

Staphylococcus aureus protein A (SPA) acts as a mitogen stimulating DNA synthesis and is not antagonistic to the mitogenic activity of other mitogens. The mitogenicity of SPA is due to the protein's multivalent Fc antibody receptors. Partial trypsin digestion of SPA yields monovalent Fc antibody receptors which are not mitogenic. Using a thymidine incorporation assay, results indicated that tryptic digests of SPA added to mouse splenic leukocytes 12 hours after stimulation with either concanavalin A, lipopolysaccaride or SPA suppressed the DNA synthesis normally elicited by these mitogens. SPA was digested with 1% trypsin at pH 8 for varying periods of time. As the digestion time increased from 0 to 60 minutes the mitogenicity of the SPA-trypsin digests decreased indicating cleavage from multivalent to monovalent SPA Fc antibody receptors. The decrease in mitogenicity of the SPA digests was directly associated with increased ability to inhibit DNA synthesis in mitogen stimulated leukocytes. A proposed mechanism for the inhibition of DNA synthesis suggests that SPA-monovalent Fc antibody receptors mimic cellular Fc receptor function thereby influencing cellular gene expression.     

Interferon abrogates the arrest of DNA synthesis in heterologous thymocytes treated with lectins

Mouse or rat thymocytes are triggered to undergo blastogenesis by mitogenic doses of concanavalin A and lentil lectin, when these plant mitogens are applied to the freshly cultured thymocytes at time 0. However, these mitogens did not elicit a mitogenic response if added to mouse or rat thymocytes that were preincubated in culture medium for 24 hours. Incubation of the thymocytes with heterologous preparations of interferon during the period of 24 hours before application of the mitogens, brought about an enhanced incorporation of tritiated thymidine. The data presented suggest that heterologous interferons could significantly abrogate the block in DNA synthesis in thymocytes that were preincubated for 24 hours in culture medium prior to addition of the mitogens.     

Acridinium ester conjugated to lectin as chemiluminescent histochemistry marker

Cell differentiation/dedifferentiation includes changes in oligosaccharide composition and distribution in the cell surface glycoconjugates. Lectins have been used as auxiliary tools in histopathological diagnosis of mammary, uterus and brain pathologies. Acridinium ester (AE) conjugated to biomolecules has been employed in chemiluminescent analytical applications. This work aimed to use a lectin, concanavalin A (Con A), conjugated to AE as a chemiluminescent histochemistry tool. Biopsies of normal and infiltrating duct carcinoma (IDC) of mammary tissues were treated by a Con A-AE derivative. Photon emission, observed during the breakage of the chemical bound between Con A and AE, was quantified, expressed in relative light units (RLU) and correlated to the labelling of the normal and transformed tissues. The results demonstrated that RLU presented a linear relationship with the labelled tissue area in the range 0.125-1.0 cm² (r=0.98). Furthermore, RLU was much higher for the IDC (1283.920×10³±220.621×10³) than the normal tissue (2.565×10³±0.247×10³), namely, about 500 times higher. The Con A-AE conjugation efficiency, differential staining of normal and IDC tissues, and quantification of results contribute to a decrease in the subjectivity in routine histopathological diagnoses and indicate that acrydinum ester can join other lectin marker to be used in histochemistry.     

Acridinium ester conjugated to lectin as chemiluminescent histochemistry marker

Abstract

Cell differentiation/dedifferentiation includes changes in oligosaccharide composition and distribution in the cell surface glycoconjugates. Lectins have been used as auxiliary tools in histopathological diagnosis of mammary, uterus and brain pathologies. Acridinium ester (AE) conjugated to biomolecules has been employed in chemiluminescent analytical applications. This work aimed to use a lectin, concanavalin A (Con A), conjugated to AE as a chemiluminescent histochemistry tool. Biopsies of normal and infiltrating duct carcinoma (IDC) of mammary tissues were treated by a Con A–AE derivative. Photon emission, observed during the breakage of the chemical bound between Con A and AE, was quantified, expressed in relative light units (RLU) and correlated to the labelling of the normal and transformed tissues. The results demonstrated that RLU presented a linear relationship with the labelled tissue area in the range 0.125–1.0?cm² (r=0.98). Furthermore, RLU was much higher for the IDC (1283.920×10³±220.621×10³) than the normal tissue (2.565×10³±0.247×10³), namely, about 500 times higher. The Con A–AE conjugation efficiency, differential staining of normal and IDC tissues, and quantification of results contribute to a decrease in the subjectivity in routine histopathological diagnoses and indicate that acrydinum ester can join other lectin marker to be used in histochemistry.     

Lymphocyte stimulation by concanavalin a studied by the fluorescent probe acridine orange

Summary Viable mouse thymocytes or spleen leucocytes stained with acridine orange (AO) were divided into one part used for stimulation, and the other part for control. Analysis of cellular green-fluorescence emission enabled physicochemical changes in lymphocytes to be detected after 30 min stimulation with the mitogens concanavalin A (Con A) and pokeweed mitogen (PWM). No change in fluorescence was observed with the nonmitogenic reagent wheat germ lectin (WGL) or with allogeneic cell stimulation (MLR). When green fluorescence intensity of individual cells was monitored by microfluorimetry, 30 min stimulation with Con A induced an increase, whereas PWM induced a decrease. When analysed by fluorescence spectrophotometry, Con A induced a 2 nm blue shift in emission maximum and a decrease in polarization values.Supported by grants from the Anti-Cancer Council of Victoria. We are grateful to Dr. H.A. Ward for helpful discussion