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.     

Effect of wheat germ agglutinin on T lymphocyte activation

Wheat germ agglutinin (WGA) is low mitogenic or nonmitogenic for human T lymphocytes and inhibits phytohemagglutinin (PHA)-induced mitotic response of the lymphocytes. In this study, the effect of WGA was analyzed in terms of interleukin 2 (IL2) production, expression of IL2 receptor, and IL2 responsiveness of the T lymphocytes. WGA as well as PHA could induce IL2 mRNA and IL2 production and also elevate cytoplasmic free Ca2+ concentration. The IL2 production was reduced by inhibitors of calmodulin and protein kinase C. The IL2 receptor (Tac) expression was induced at about 20% of the lymphocytes by WGA and the expression induced by PHA was not blocked by the addition of WGA. The lymphocytes precultured with WGA for 3 days could proliferate by the addition of IL2 after removal of WGA. The IL2-dependent proliferation of PHA-blasts was blocked by the addition of WGA. These results indicate that WGA inhibits T lymphocyte proliferation by inhibiting the responsiveness of the lymphocytes to IL2 but not by interfering with IL2 production and IL2 receptor expression.     

T3-T cell receptor (Ti) complex-independent activation of T cells by wheat germ agglutinin

The T3-Ti complex appears to play a central role in the activation of T cells by antigens and mitogens. Wheat germ agglutinin (WGA) is a unique lectin which inhibits T cell proliferation induced by mitogens, but it also induces marked IL 2 production by peripheral blood T cells. The pattern of responses induced by WGA suggests that this lectin may use a different mechanism of T cell activation other than the mechanism employed by the common T cell stimulants. We first investigated the production of IL 2 by Jurkat cells (E6-1) stimulated with WGA, before and after modulation of the surface T3-Ti complex. IL 2 production was markedly reduced after modulation of the T3 antigen from the cell surface when these cells were stimulated with PHA. In contrast, little change was observed in WGA-induced IL 2 production after modulation. Furthermore, we examined the effect of WGA on a T3-mutant of E6-1 cells (T3.1) which does not produce IL 2 in response to PHA or PHA plus PMA. WGA-stimulated T3.1 cells produced a significant amount of IL 2 with or without added PMA. In addition, a small but consistent rise in intracytoplasmic free calcium was observed when these cells were stimulated with WGA. These results demonstrate the presence of an alternative mechanism of T cell activation independent of the T3-Ti complex.     

Effect of wheat germ agglutinin on the interleukin pathway of human T lymphocyte activation

Wheat germ agglutinin (WGA) inhibits proliferation of human peripheral blood mononuclear cells (PBMC) induced by mitogens and antigens. We investigated the mechanism by which WGA inhibits PHA-induced human lymphocyte proliferation with regard to the interleukin pathway. Our data revealed that although PBMC-proliferation was markedly suppressed by WGA, levels of IL 2 activity in WGA-inhibited cultures were not reduced, but instead were increased, suggesting failure to utilize IL 2. Furthermore, the addition of exogenous IL 2 failed to overcome the suppression. Consistent with these observations, culturing PBMC with PHA plus WGA markedly decreased the number of high-affinity IL 2 receptor per cell, as determined by binding of purified [3H]IL 2, relative to cultures containing PHA alone. WGA immobilized on support beads bound detergent-solubilized IL 2 receptors from PHA-activated T cells, but did not bind human IL 2. However, WGA did not competitively block the binding of [3H]IL 2 to PHA-induced lymphoblasts. These results suggest that WGA inhibits lymphocyte proliferation by binding to and decreasing the number of high-affinity IL 2 receptors displayed on T cells, without impairing IL 2 production.     

Purification of wheat germ agglutinin by affinity chromatography

Wheat germ agglutinin was isolated in pure form and in high yield from an extract of wheat germ by affinity chromatography of 6-amino-1-hexyl-2-deoxy-β-d-glucopyranoside-Sepharose 4B. The purified agglutinin behaved as a single species electrophoretically and, as judged by its migration on sodium dodecyl sulfate-polyacrylamide gels, has an apparent molecular weight of 17,000. The amino acid composition of the isolated agglutinin was in good agreement with that previously reported.     

Surface glycoprotein of human natural killer cells recognized by wheat germ agglutinin

We analyzed surface glycoproteins of human natural killer (NK) cells by utilizing lectins. Among the lectins tested, wheat germ agglutinin (WGA) was found to bind preferentially to CD16(Leu11)-positive lymphocytes as determined by two-colour flow cytometry. Analysis of glycoproteins in the lysate prepared from NK cells with sodium dodecyl sulfate (SDS) gel electrophoresis followed by Western blotting and¹²⁵I labeled WGA staining revealed that a glycoprotein with anM _r of 65 kDa was strongly bound to the lectin, but no corresponding glycoprotein was detected in the lysate of T lymphocytes. This glycoprotein (GP65) gave several spots in the pI range 4.1–4.6 on 2-dimensional gel electrophoresis. Sialidase treatment of GP65 resulted in a single spot on the 2-dimensional gel, suggesting that GP65 is heterogeneous in the degree of sialylation. GP65 was shown to be exposed on the cell surface, since it was radiolabeled with¹²⁵I by the lactoperoxidase-catalyzed method. We next isolated GP65 from human peripheral blood lymphocytes by a combination of chromatography on a cation-exchange column and a WGA-agarose column and preparative SDS gel electrophoresis. It is suggested that GP65 is a novel surface glycoprotein on human NK cells.     

Immunocytochemical localization of wheat germ agglutinin in wheat

Immunocytological techniques were developed to localize the plant lectin, wheat germ agglutinin (WGA), in the tissues and cells of wheat plants. In a previous study we demonstrated with a radioimmunoassay that the lectin is present in wheat embryos and adult plants both in the roots and at the base of the stem. We have now found, using rhodamine, peroxidase, and ferritin-labeled secondary antibodies, that WGA is located in cells and tissues that establish direct contact with the soil during germination and growth of the plant In the embryo, WGA is found in the surface layer of the radicle, the first adventitious roots, the coleoptile, and the scutellum. Although found throughout the coleorhiza and epiblast, it is at its highest levels within the cells at the surface of these organs. In adult plants, WGA is located only in the caps and tips of adventitious roots. Reaction product for WGA was not visualized in embryonic or adult leaves or in other tissues of adult plants. At the subcellular level, WGA is located at the periphery of protein bodies, within electron-translucent regions of the cytoplasm, and at the cell wall-protoplast interface. Since WGA is found at potential infection sites and is known to have fungicidal properties, it may function in the defense against fungal pathogens.     

The platelet activation induced by wheat germ agglutinin

In human platelets, wheat germ agglutinin (WGA) induced serotonin release without cell agglutination. WGA induced the phosphorylation of both 40-kDa and 20-kDa proteins in a parallel manner, and at least, the phosphorylation of 40-kDa protein was preceded by transient formation of endogenous diacylglycerol (DG) accompanied by a decrease in phosphatidylinositol (PI). Both phosphorylation of these two proteins and serotonin release were inhibited by prior treatment of platelets with dibutyryl cyclic AMP, W-7, or TMB-8. These results suggest that both phosphatidylinositol turnover and Ca2+ mobilization play an essential role in WGA-induced platelet activation.     

Wheat germ agglutinin regulates cell division in wheat seedling roots

The mitogenic activity of wheat germ agglutinin (WGA) has been studied in roots of 4-day-old wheat seedlings. WGA had a more pronounced stimulating effect on cell division than the known mitogens concanavalin A and phytohemagglutinin whereas gliadin had no effect. Treatment of wheat seedling roots with exogenous WGA led to the accumulation of indoleacetic acid and cytokinins, hormones that play an important role in the activation of plant cell growth. The data on the combined effect of 24-epibrassinolide and WGA on cell division and accumulation of phytohormones in seedling roots support a possible link between the endogenous WGA level and hormonal regulation of cell division in the root meristem of wheat plants.     

Binding of wheat germ agglutinin to extracellular network produced by cultured human fibroblasts

The occurrence of diverse carbohydrate moieties on the cell surface and in the extracellular matrix, makes lectins the suitable probes to study the distribution of appropriate determinants produced in cell culture. Biotin-labelled wheat germ agglutinin (WGA) was used in microscopic and photometric detection of lectin binding to monolayer of human skin fibroblasts. The incubation of confluent fibroblast monolayer with labelled WGA reveals two principal patterns of binding of this lectin: to cell surface structures and, predominantly, to extracellular fibres; the alignment and density of extracellular network are not uniform. After binding of WGA to confluent culture, light microscopic analysis revealed the ubiquitous fibrillar network between and over cells, with some regions of increased compactness and altered orientation of fibrils. Binding to cell surfaces (manifested as specks) was predominant for the fibroblasts at the logarithmic phase of growth. N-acetylglucosamine (0.2 M) and native lectin (100 microg/ml) had a partial inhibitory effect on WGA binding to the extracellular network. Treatment with neuraminidase (0.1 unit/ml) of untreated or prefixed monolayers resulted in a significant decrease in WGA binding to fibrils (and increase in PNA binding), indicating that terminal sialic acid residues are mainly involved in the network-WGA interaction. Mild trypsinization (10 microg/ml) removed the target sites, which retained the ability to bind WGA, being spotted on hydrophobic Immobilon P paper; biotinylated lectin, bound to adsorbed glycopeptides, could be eluted and quantified in solid-phase inhibition assay.     

Activity and specificity of covalently immobolized wheat germ agglutinin toward cell surfaces

Wheat germ agglutinin protein, which is able to agglutinate tumor cells better than normal cells, was covalently bound to polyacrylamide gel beads. The specific binding activity of the protein was preserved on these beads and was expressed heterogeneously by the binding of mouse leukemia cells (L1210) to the protein coupled gels. The selective activity of the immobilized protein was maximal when the number of sites available to covalently couple the protein was lowest. The application of this observation to the general field of covalent immobilization of proteins and enzymes may be of considerable utility.     

Wheat germ agglutinin activates human T lymphocytes by stimulation of phosphoinositide hydrolysis

Recently, it has become evident that stimulated phosphoinositide (PI) hydrolysis plays a crucial role in early T lymphocyte activation. We have investigated the effects of the nonmitogenic lectin wheat germ agglutinin (WGA) on several parameters associated with PI hydrolysis in human T cells. It was found that WGA was as effective as anti-T3 antibody and PHA in producing a rise in cytosolic free Ca++ ((Ca++)i) in blood T cells and in cells of the T cell line CCRF-CEM. It was inferred that identical cells within the blood T cell preparation responded to each of the three agents, refuting the contention that WGA only stimulated a subfraction of circulating mature T lymphocytes. WGA-induced, but not PHA-induced rises in (Ca++)i could be blocked completely by N-acetyl-D-glucosamine, demonstrating that the sugar-binding characteristics of the lectin dictate its action on T lymphocytes. Anti-T3 antibody, PHA, and WGA all initiated inositol phosphate formation in blood T cells, indicating that each of the agents stimulated PI hydrolysis. The combination of WGA with nonmitogenic amounts of phorbol-12-myristate-13-acetate resulted in strong mitogenicity. It is concluded that WGA, like anti-T3 antibody and PHA, is a pan-T activator of PI hydrolysis.     

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.     

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.     

Inhibition of Vorticella microstoma stalk formation by wheat germ agglutinin

Fluorescently labeled conjugates of wheat germ agglutinin and concanavalin A stained the contractile stalk but not the cell body of Vorticella microstoma trophonts. Binding of the fluorescent conjugants did not noticeably alter the activity of the trophonts. However, unconjugated wheat germ agglutinin prevented free swimming telotrochs from adhering to a glass surface and deploying a contractile stalk during differentiation into trophonts. These observations indicated that the stalk, the material that binds the stalk to surfaces, and the precursors for these components have saccharide residues in common.     

Stimulation of the membrane-bound, magnesium-dependent adenosine triphosphatase of mouse neuroblastoma by concanavalin A and wheat germ agglutinin.

The effect of the plant lectins concanavalin A and wheat germ agglutinin on the membrane-bound Mg⁺²-dependent ATPase of an adrenergic clone of mouse neuroblastoma was examines. When cell membranes were treated with concanavalin A or wheat germ agglutinin, a dose-related increase in ATPase-specific activity was observed. Maximal stimulation was greater with wheat germ agglutinin than with concanavalin A; half-maximal and maximal stimulation occurred at similar lectin concentrations. Concanavalin A-dependent stimulation was blocked by α-methylmannoside but not by N-acetylglucosammine. Conversely, stimulation with wheat germ agglutinin was prevented by N-acetylglucosamine but not by α-methylmannoside. The combined effects of concanavalin A and wheat germ agglutinin were greater than the individual effects of either, but were not additive. The results suggest that these lectins interact specifically with membrane glycoproteins or glycolipids, resulting in enhancement of Mg⁺²-dependent ATPase activity.     

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.     

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.     

Evidence for multiple mechanisms of interaction between wheat germ agglutinin and human platelets

Wheat germ agglutinin induced aggregation and secretion of serotonin from human platelets in plasma. This aggregation of platelets was blocked by ethylenediaminetetraacetate, azide or prostaglandin E₁. The secretion of serotonin was not affected by ethylenediaminetetraacetate but was inhibited by progstaglin E₁. Thus, wheat germ agglutinin acts on platelets in plasma as a true aggregating agent.Washed platelets showed increased light transmission when treated with the lectin which was not blocked by ethylenediaminetetraacetate or prostaglandin E₁. The capacity to inhibit platelet clumping by the above agents was restored if plasma was added back to the cell suspension. Washed platelets did not release serotonin under the conditions of cell clumping. Thus, in contrast to platelets in plasma, washed platelets are agglutinated by the lection.Platelets fixed in formaldehyde were not agglutinated by the lectin in the aggregometer but agglutination was observed in the microtiter plate. This agglutination may be mediated by interplatelet bridging. These results show that the same agent may act on platelets by different mechanisms depending on the state of the cell and its environment.