Differences in the redistribution of concanavalin A and wheat germ agglutinin binding sites on mouse neuroblastoma cells

Concanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA) bound with either ¹²⁵I, fluorescent dyes, or fluorescent polymeric microspheres were used to quantitate and visualize the distribution of lectin binding sites on mouse neuroblastoma cells. As viewed by fluorescent light and scanning electron microscopy, over 10⁷ binding sites for Con A, WGA, and RCA appeared to be distributed randomly over the surface of differentiated and undifferentiated cells. An energy-dependent redistribution of labeled sites into a central spot occurred when the cells were labeled with a saturating dose of fluorescent lectin and maintained at 37°C for 60 min. Reversible labeling using appropriate saccharide inhibitors indicated that the labeled sites had undergone endocytosis by the cell. A difference in the mode of redistribution of WGA or RCA and Con A binding sites was observed in double labeling experiments. When less than 10% of the WGA or RCA lectin binding sites were labeled, only these labeled sites appeared to be removed from the cell surface. In contrast, when less than 10% of the Con A sites were labeled, both labeled and unlabeled Con A binding sites were removed from the cell surface. Cytochalasin B uncoupled the coordinate redistribution of labeled and unlabeled Con A sites, suggesting the involvement of microfilaments. Finally, double labeling experiments employing fluorescein-tagged Con A and rhodamine-tagged WGA indicate that most Con A and WGA binding sites reside on different membrane components and redistribute independenty of each other.     

Cytochemical comparison between wheat germ agglutinin and concanavalin A bound to mouse fibroblasts in vitro.

3T3 and SV 3T3 cells, prefixed or precoated with WGA, followed by treatment with ferritin-ConA, have less label than non-pretreated cells. When the same cells were treated with ferritin-WGA, neither prefixation nor precoating with ConA had any influence. Possible explanations are discussed. ConA-ferritin distribution is clustered, and WGA-ferritin is evenly spread on both 3T3 and SV 3T3 cells. We conclude that differences in lectin-induced agglutination between these cells cannot be attributed to a difference in lectin-induced clustering.     

The affinity of concanavalin A and wheat germ agglutinin for components of the muscle spindle.

Sections through the soleus muscle of the rat were incubated with concanavalin A (Con A) or wheat germ agglutinin (WGA) conjugated to fluorescein isothiocyanate. Binding of these lectins to structures which comprise the muscle spindle was studied by fluorescence microscopy. The distribution of the lectins was heaviest in the outer capsule of the spindle and at the surface of intrafusal muscle fibres. The periaxial space in the equatorial region of spindles was unlabelled except in the immediate vicinity of the axial bundle. Binding by Con A was more extensive than by WGA, suggesting that more glucopyranoside units are accessible within the muscle spindle than are those of N-acetylglucosamine.     

Some physicochemical aspects of oligosaccharide binding to concanavalin A and wheat germ agglutinin

The binding of fluorescently labelled carbohydrates to concanavalin A and wheat germ agglutinin was studied at equilibrium and by the stopped-flow and temperature jump relaxation methods. Ligand were mainly die 4-methylumbelliferyl glycosides of α (1 → 2)-linked manno-oligosaccharides and of β (1 → 4)-linked chito oligosaccharides as limited homologous series. They offer distinct advantages, parti cularly for kinetic studies.Enthalpie and kinetic considerations suggest that concanavalin A specifically binds a single mannopyranosyl group in α (1 →2)-linked manno-oligosaccharides. This occurs preferentially at the non-reducing end. Glycosylation of a carbohydrate withe.g. an aryl group does not afect die binding kinetics and for all carbohydrates the association rate is comparable but relatively slow, which indicates that a common process is involved in the binding of all carbohydrates to concanavalin A. The affinity of a carbohydrate for concanavalin A is determined by the dissociation-rate parameter, resulting in a longer residence time for a better ligand.Interaction of chito-oligosaccharides with wheat germ agglutinin is complex. With the larger members of the 4-methylumbelliferyl chito-oligosaccharides, binding studies were only possible at low fractional saturation to avoid formation of unsoluble complexes. The binding kinetics of wheat germ agglutinin are faster than with concanavalin A and are consistent with a wheat germ agglutinin binding region composed of two adjacent subsites. For binding of the monoside as well as the bioside, two consistent kinetic models apply. They have common that for each ligand there exist two complexes with comparable population.     

High-affinity calcium-stimulated, magnesium-dependent adenosine triphosphatase in Trypanosoma cruzi.

1. A high-affinity (Ca2+ + Mg2+)-ATPase and a low-affinity Mg(2+)-ATPase were identified in the 105,000 g fraction from epimastigote forms of Trypanosoma cruzi, the agent of Chagas' disease (Tulahuen strain). 2. Activities were conserved after enzyme solubilization with deoxycholate. 3. The Ca(2+)-stimulated ATPase activity was (a) lower than that of the Mg(2+)-ATPase; (b) inhibited by p-chloromercurobenzoate and orthovanadate and (c) insensitive to oligomycin. 4. Optimal stimulation by Ca2+ was observed at pH 6.5-6.8 in the presence of 1 mM MgCl2 and 0.1 M KCl. 5. The Mg(2+)-ATPase was insensitive to p-chloromercurobenzoate and orthovanadate and did not require KCl for activity. 6. Kinetic analysis of the (Ca2+ + Mg2+)-ATPase yielded a half-maximal stimulating concentration of 1.1 microM for Ca2+ and a Km of 66 microM for ATP. 7. The (Ca2+ + Mg2+)-ATPase clearly differed from the Ca(2+)- or Mg(2+)-ATPases previously characterized in the same strain of T. cruzi (Frasch et al., 1978; Comp. Biochem. Physiol. 60B, 271-275).     

Insulin-mimetic actions of wheat germ agglutinin and concanavalin A on specific mRNA levels

Insulin has pleiotropic effects on sensitive cells, including the regulation of specific mRNA accumulation initiated by the binding of insulin to its plasma membrane receptor. Lectins, such as wheat germ agglutinin (WGA) and concanavalin A (Con A), are known to be insulin mimetic. It is thought that WGA and Con A interact with the insulin receptor or associated membrane glycoproteins which, when activated, lead to insulin-mimetic responses. We attempted to determine whether WGA and Con A could induce the accumulation of a specific messenger RNA (p33-mRNA). Insulin treatment of H4IIE (H4) hepatoma cells increased the concentration of p33-mRNA within 30 min after addition, with a maximum effect of 10- to 15-fold. WGA and Con A also exhibited time- and dose-dependent stimulatory effects on p33-mRNA accumulation with maximal effects of 30- to 40-fold. The effect of insulin was maximal by 1 h and plateaued thereafter, whereas lectins had maximal effects at 2 h after addition to cell cultures. Insulin, WGA, and Con A did not significantly alter the stability (half-life) of p33-mRNA. The addition of RNA synthesis inhibitors blocked the ability of insulin, WGA, and Con A to induce the amount of p33-mRNA. These data suggest that lectins, as well as insulin, induce the synthesis of p33-mRNA in acutely treated H4 hepatoma cells.     

Stimulation and inhibition of human T cell subsets by wheat germ agglutinin

Wheat germ agglutinin (WGA), a tetravalent lectin, has both stimulatory and inhibitory effects on human T lymphocytes. It has been suggested that these actions are related and that WGA selectively stimulates a suppressive subset of T cells. We studied the ability of WGA to stimulate and inhibit subpopulations of human peripheral blood mononuclear cells (PBMC) known to have helper or suppressor activity. Fresh human PBMC were depleted of either T4+ or T8+ cells by using antibody-mediated complement lysis. The resultant cell populations were stimulated with WGA, and the proliferative response was assessed by [3H]thymidine incorporation, IL 2 receptor expression, the ability to elaborate IL 2 in culture supernatants, and the susceptibility to inhibition by the monoclonal antibody anti-Tac. Similar experiments with cells from a WGA-responsive continuous T cell culture were also performed. WGA inhibited phytohemagglutinin (PHA)-induced proliferation of PBMC depleted of either T4+ or T8+ cells. WGA also inhibited PBMC that had been depleted of adherent cells and Ia+ cells and then induced to proliferate with a combination of TPA and PHA. Our findings indicate that WGA induces IL 2-dependent proliferation in a small proportion of both T4+ and T8+ lymphocytes. We also provide evidence that the inhibitory activity of WGA is not mediated by a T4+, T8+, or Ia+ cell, suggesting that WGA acts directly on the proliferating cell rather than selectively stimulating a suppressive subpopulation.     

Effects of wheat germ agglutinin and concanavalin A on parameters of highly purified sodium-potassium adenosine triphosphatases from Squalus acanthias and Electrophorus electricus.

The effects of two lectins, wheat germ agglutinin and concanavalin A, were studied on a variety of parameters of two highly purified (Na+ + K+)-ATPases (ATP phosphohydrolase, EC 3.6.1.3), from the rectal salt gland of Squalus acanthias and from the electroplax of Electrophorus electricus. Both lectins agglutinated the rectal gland enzyme equally, but wheat germ agglutinin inhibited (Na+ + K+)-ATPase activity much more. The electroplax enzyme was only marginally agglutinated and inhibited by the lectins. Neuraminidase treatment of the rectal gland (Na+ + K+)-ATPase had no effect on germ agglutinin inhibition. The inhibition of the rectal gland (Na+ + K+)-ATPase by wheat germ agglutinin could be reversed by N,N'-diacetylchitobiose, which has a high affinity for wheat germ agglutinin. Neither ouabain inhibition nor ouabain binding to the rectal gland enzyme was affected by wheat germ agglutinin. The p-nitrophenylphosphatase activity of the rectal gland enzyme was not inhibited by wheat germ agglutinin. Na+-ATPase activity, which reflects ATP binding and phosphorylation at the substrate site was inhibited by wheat germ agglutinin and this inhibition was reversed by potassium. Evidence is cited (Pennington, J. and Hokin, L.E., in preparation) that the inhibition of the (Na+ + K+)-ATPase by wheat germ agglutinin is due to binding to the glycoprotein subunit.     

Binding of concanavalin A and wheat germ agglutinin by murine peritoneal macrophages: ultrastructural and cytophotometric studies

Summary Concanavalin A and wheat germ agglutinin were employed in conjunction with the horseradish peroxidase-diaminobenzidine method for the detection of sugar residues on the surface coat of exudate and resident murine peritoneal macrophages. Electron microscopical and cytophotometric techniques were used for the visualization and quantification of the final reaction product on the surface of cells. After incubation with concanavalin A and wheat germ agglutinin, both exudate and resident macrophages showed readily detectable final reaction product indicating the presence of numerous, easily accessible, -methyl-d-mannosyl andN-acetyl-d-glucosaminyl residues on their surface. The binding of concanavalin A was higher with resident than with exudate macrophages. With wheat germ agglutinin, a different pattern of lectin binding was observed: more electron-dense product was deposited on exudate than on resident macrophage surfaces. The binding of concanavalin A and wheat germ agglutinin to macrophages was inhibited by the competing sugars -methyl-d-mannoside andN-acetyl-d-glucosamine, respectively.     

Heterogeneity in wheat germ agglutinin binding by mouse peritoneal macrophages

Summary The binding of the lectin wheat germ agglutinin (WGA) to the cell surface of monocytes and macrophages obtained from the stimulated peritoneal cavity of mice was investigated electron microscopically, using ovomucoid-gold as an indirect marker. Resident (tissue) macrophages, identified by the presence of PO activity in the rough endoplasmic reticulum as well as in the nuclear envelope, showed low WGA binding, whereas monocytes and monocyte-derived macrophages with PO activity in the granules showed high WGA binding. Since cells devoid of PO activity showed variable WGA binding, the value of this gold-WGA-binding technique for discrimination on a quantitative basis between resident macrophages and monocytes or monocyte-derived macrophages, is discussed.     

Heterogeneity in wheat germ agglutinin binding by mouse peritoneal macrophages

The binding of the lectin wheat germ agglutinin (WGA) to the cell surface of monocytes and macrophages obtained from the stimulated peritoneal cavity of mice was investigated electron microscopically, using ovomucoid-gold as an indirect marker. Resident (tissue) macrophages, identified by the presence of PO activity in the rough endoplasmic reticulum as well as in the nuclear envelope, showed low WGA binding, whereas monocytes and monocyte-derived macrophages with PO activity in the granules showed high WGA binding. Since cells devoid of PO activity showed variable WGA binding, the value of this gold-WGA-binding technique for discrimination on a quantitative basis between resident macrophages and monocytes or monocyte-derived macrophages, is discussed.     

Electron microscope assessment of fertilization of rabbit ova treated with concanavalin A and wheat germ agglutinin.

Zonaless rabbit ova, exposed to Concanavalin A or Wheat Germ Agglutinin, then to uterine capacitated sperm produce pronuclear, 2 and 4 stage embryos that are indistinguishable from controls. Absence of cortical granules indicates that the ova were fertilized and not merely activated. Survival of lectin-bearing receptors during the period necessary for fertilization was evaluated in ova marked with ferritin-conjugated lectin.     

Inhibition of the membrane-bound adenosine triphosphatase of Escherichia coli by dicyclohexylcarbodi-imide.

The inhibition of the membrane-bound adenosine triphosphatase of Escherichia coli by DCCD (dicyclohexylcarbodi-imide) is studied under conditions of varying KCl concentration. An increase in K+ concentration and in other cations causes an increase in the DCCD sensitivity of the enzyme, as well as significant changes in the kinetic parameters.     

Rapid separation of mouse T and B lymphocytes using wheat germ agglutinin.

A separation procedure has been developed for mouse splenic T and B lymphocytes which is based on their differential agglutination by wheat germ agglutinin (WGA). In the presence of 50-100 micrograms/ml of WGA, multicellular aggregates are formed which are enriched in B cells. These aggregates can be separated from monodisperse T cells by gravity sedimentation and subsequently dissociated into single cells by treatment with N-acetylglucosamine (NAG). Immunocytochemical analyses and mitogenic assays indicate approximately 10-15% cross contamination of the resultant B and T cell fractions. The separation procedure is not only convenient and rapid but also allows the simultaneous recovery of viable T and B cells from the same spleen preparation.     

Drosophila cell fusion induced by wheat germ agglutinin.

The effects of wheat germ agglutinin on Drosophila embryonic cell lines growing on cover-glasses was examined by scanning electron microscopy. At low concentrations of the lectin (5-10 mug/ml), cells spread against the glass surface and fused to form syncytia. At high concentration, damage to the cell surface was evidenced as extensive membrane shrivelling and loss of surface microfilaments. Fusion also occurred under these conditions. There was some indication that the morphology of cells in division remains undisturbed by wheat germ agglutinin. The coalescence of cells and morphologic disotrtion induced by wheat germ agglutinin were not inhibited by N-acetylglucosamine, the hapten inhibitor of the lectin, under the conditions utilized in this study.     

Fusarium sp. growth inhibition by wheat germ agglutinin.

The antifungal role of wheat germ agglutinin (WGA) isolated from a Romanian dihaploid variety of wheat against two pathogenic fungal species of Fusarium, F. graminearum and F. oxysporum, is demonstrated. WGA was prepared from unprocessed wheat germs by a new purification procedure using chitin and fetuin-Sepharose as affinity chromatography supports. SDS-PAGE and chitinase assay showed that the WGA preparation migrated as a single protein band and was devoid of any contaminating enzyme chitinase, well known for its antifungal effects. Based on its affinity for N-acetylglucosamine residues, WGA binding to the chitin-containing walls of the fungi was detected by fluorescence microscopy using WGA coupled with fluorescein isothiocyanate (FITC). In vitro testing of WGA action on early developmental stages of both fungal strains resulted in various modifications of the germ tubes, visualised by light microscopy: swelling, vacuolation of the cellular content and lysis of cell walls. Viability tests performed on potato tuber slices showed that the microbial infection was prevented from spreading by pretreatment of the fungal suspension with WGA.     

Changes in the binding of concanavalin A and wheat germ agglutinin to human lymphocytes during in vitro transformation

Concanavalin A binds to human circulating lymphocytes in a complex manner suggesting the presence of multiple binding sites. Saturation of one or more of these binding sites is observed at concentrations of concanavalin A which induce blast transformation in lymphocytes. In contrast, only one saturable binding site is observed for wheat germ agglutinin. During $\text{in vitro}$ transformation, the amount of concanavalin A which can be bound by lymphocytes increases, whereas the amount of wheat germ agglutinin which can be bound remains unchanged. Since the size increases during transformation, there must be a fall in the density of surface receptors for wheat germ agglutinin whereas the density of concanavalin A receptors remains unchanged.     

Morphological changes of rat small intestine after short-time exposure to concanavalin A or wheat germ agglutinin

Dietary lectins of gluten origin have been suggested to play an important role in the mechanisms leading to the characteristic morphology of the intestine found in patients with celiac disease. To further explore this issue we have used Wheat Germ Agglutinin (WGA) or Concanavalin A (Con A) to challenge rat small intestine and study the ultrastructural changes of such a treatment. Both lectins affected the enterocytes at the base of the villi more than those at the top. The morphological findings included disarrangement of the cytoskeleton, increased endocytosis and shortening of the microvilli. The interrelationship between the observed changes, and their relevance for similar morphological alterations found in patients with celiac disease are discussed. In conclusion, the morphological findings in our rat model resemble early changes in patients with celiac disease, thus supporting the idea that lectins or lectin-like substances are involved in the pathogenesis of this disease.     

Expression and secretion of wheat germ agglutinin by Saccharomyces cerevisiae.

Genes encoding pre-protein and prepro-protein of wheat germ agglutinin isolectin 2 (WGA2) were chemically synthesized and expressed in the yeast Saccharomyces cerevisiae under the control of the ENO1 promoter. Yeast harboring either a pre-WGA2 or a prepro-WGA2 gene expression plasmid secreted a mature form of WGA2 into the culture medium. The amount of WGA2 secreted by the strain KS58-2Ddel, which has a ssl1 mutation causing a supersecretion of human lysozyme [Suzuki, K., Ichikawa, K. & Jigami, Y. (1989) Mol. Gen. Genet. 219, 58-64], was 20-fold greater than that secreted by the wild-type strain KK4. The recombinant WGA2 from the cells containing the prepro-WGA2 gene expression plasmid was purified to homogeneity by a three-step ion-exchange chromatography scheme. As in wheat, the N-terminal signal peptide of recombinant WGA2 purified from yeast culture was processed to form an N-terminal 5-oxoprolyl (pyroglutamyl) residue. Likewise, we found that the C-terminal pro-region of recombinant WGA2 had also been processed in yeast. Using electrospray ionization mass spectrometry, we found the processed C-terminus to be heterogeneous in both recombinant WGA2 purified from yeast and in authentic WGA2. The major component of the recombinant WGA2 contained two additional amino acids at its C-terminus compared to that of authentic WGA2. In spite of this difference in the C-terminus, the recombinant WGA2 exhibited a sugar binding activity that was indistinguishable from that of authentic WGA2.