Wheat germ agglutinin uptake by cytotoxic lymphocytes, a quantitative model of receptor-mediated endocytosis (original) (raw)
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Wheat Germ Agglutinin is Mitogenic, Non-Mitogenic and Anti-Mitogenic for Human Lymphocytes
Scandinavian Journal of Immunology, 1987
Wheat germ agglutinin (WGA) was found to stimulate DNA synthesis in human peripheral blood mononuclear cells at relatively low concentrations and to inhibit DNA synthesis at higher concentrations. Both actions were inhibited by oUgomers of N-aceiyl-D-glucosamine. Significant mitogenic activity was dependent on the use of human {as opposed to fetal calO serum to supplement the culture medium. Purified T cells responded to WGA very weakly and the incorporation of thymidinc into non-T cells in response to WGA was less than the lectin-free eontrol. The full ability of T cells to respond to WGA was restored by the addition of monocytes, but not by any other blood cells. Interleukin 2 partially restored the ability of Tceils to respond to WGA; interieukin 1 was less effective. WGA displayed a strong synergistic action with the tumour promoter, 12-O-tetradecanoyl-13-acetate (TPA), in stimulating DNA synthesis in separated T (but not non-T) cell fractions, and in unfractioned mononuclear cells. These results reconcile most of the conflicting reports in the literature concerning the interaction of WGA with human lymphocytes.
Glycoconjugate Journal, 2006
Neutrophils participate in host protection and central to this process is the regulation of oxidative mechanisms. We purified by affinity chromatography the receptor for the GlcNAc-specific WGA from CD14+ CD16+ cell lysates (WGAr). The receptor is a 141 kDa glycoprotein constituted by two subunits of 78 and 63 kDa. It is mainly composed of Ser, Asx, and Gly, and, in a minor proportion, His, Cys, and Pro. Its glycan portion contains GlcNAc, Gal, and Man; NeuAc and GalNAc were identified in a minor proportion. The amino acid sequence of the WGA receptor was predicted from tryptic peptides by MALDI-TOF, both subunits showed homology with cytokeratin type II (26 and 29% for the 78 and 63 kDa subunits, respectively); the 78 kDa subunit showed also homology with the human transferrin receptor (24%). Antibodies against WGAr induce higher oxidative burst than WGA, determined by NBT reduction; however, this effect was inhibited (p < 0.05) with GlcNAc suggesting that WGAr participates as mediator in signal transduction in neutrophils.
Fractionation of human T lymphocytes on wheat germ agglutinin-sepharose
The Journal of experimental medicine, 1976
T cells from human peripheral blood was purified by fractionation on columns charged with human immunoglobulin and rabbit anti-human immuno-globulin. When assayed with 125I- or fluorescein isothiocyanate-labeled wheat-germ agglutinin (WGA), a weakly binding and a strongly binding subpopulation could be distinguished. These T-cell subpopulations were fractionated on columns charged with WGA, convalently bound to Sepharose 6MB. The cells responding to the mitogens leukoagglutinin from Phaseolus vulgaris and concanavalin A were enriched in the strongly binding subpopulation (approximately 20% of the T cells) while they were depleted from the weakly binding subpopulation.
Production of an Antibody Specific for the Propeptide of Wheat Germ Agglutinin
PLANT PHYSIOLOGY, 1989
Wheat germ agglutinin (WGA) is synthesized as a proprotein with a glycosylated, 15 amino acid, carboxyl-terminal propeptide. This glycopeptide is cleaved from pro-WGA to produce the mature lectin during the transport of WGA to the protein bodies/vacuoles. To study the posttranslational modification of WGA, it would be useful to be able to differentiate between pro-WGA and mature WGA. Therefore, a peptide corresponding to the propeptide of WGA was synthesized (WGA-B 172-186), and an antiserum was raised in rabbits (anti-WGA-B 172-186). Anti-WGA-B 172-186 reacted with pure WGA-B 172-186 and pro-WGA in ELISA. Anti-WGA-B 172-186 was also specific for and readily differentiated between pro-WGA and mature WGA on Westem blots. This provided an assay to monitor pro-WGA on Westem blots before and after endo-fl-N-acetylglucosaminidase H digestion. Using this assay, direct evidence was obtained that the oligosaccharide of pro-WGA is of the high-mannose type.
Cellular Immunology, 1984
A lectin-resistant variant of the murine EL4 lymphocytic leukemia cell line was selected in the presence of wheat germ agglutinin for low levels of cell-surface sialic acid. H-2Kb was the major internally radiolabeled H-2b molecule on the cell-surface of WD 1, and it was not sialylated, as determined by two-dimensional gel analysis. Endo-fl-N-acetylglucosaminidase H treatment of the WDl membrane fractions suggested that the oligosaccharides on the cell-surface H-2Kb molecule were complex, but nonsialylated. Monoclonal antibody inhibition of the allogeneically primed cell-mediated cytotoxicity (CMC) reaction indicated that the T cells (BALB/c anti-EL& H-p anti-H-p) were specific only for the H-2Kb target cell antigen. These WDl variant cells were used as targets in the CMC assay using anti-H-2Kb T cells and compared with the parent EL4 in vitro line. The change in the cell-surface oligosaccharide did not affect the susceptibility to lysis by the cytotoxic T lymphocytes even though there were 2.5-fold more H-2Kb antigens on the WDl variant cell (1.5 X 10' sites/cell) than on the parent EL4 in vitro cell (5.9 X lo4 sites/cell). It was possible to isolate highly purified preparations of H-2Kb from either the EL4 or the WDl line using a monoclonal antibody affinity column. Interestingly, the variant WDl cell would no longer grow in the peritoneal cavity of the syngeneic C57BL/6 mouse.
Proceedings of the National Academy of Sciences, 1977
The binding of "25I-labeled wheat germ agglutinin (WGA) to parental and three distinct WGA-resistant Chinese hamster ovary cell lines possessing modified cell surface carbohydrate structures has been examined over a 106-fold range of WGA concentrations. The Scatchard plot for WGA binding to parental cells was complex and exhibited positively cooperative binding at the high affinity sites. One of the WGA-resistant mutants (WgaRm) was apparently not altered in its WGA-binding ability compared with parental cells. However, two of the WGA-resistant lines (WgaRI and WgaR') had distinct alterations in their WGA-binding properties specific to certain regions of the binding curve. Neither appeared to be affected in either the highest or lowest affinity regions of the binding curve. Thus, lectin-resistant cell mutants altered in specific lectin-binding sites at the cell surface provide a direct approach to analysis of the complex binding parameters that characterize the interaction of WGA with the plasma membrane.
Toxicology and Applied Pharmacology, 2009
Wheat germ agglutinin (WGA) is a plant protein that binds specifically to sugars expressed, among many others, by human gastrointestinal epithelial and immune cells. WGA is a toxic compound and an antinutritional factor, but recent works have shown that it may have potential as an anti-tumor drug and as a carrier for oral drugs. To quantitate the toxicity threshold for WGA on normal epithelial cells we previously investigated the effects of the lectin on differentiated Caco2 cells, and showed that in the micromolar range of concentrations WGA could alter the integrity of the epithelium layer and increase its permeability to both mannitol and dextran. WGA was shown to be uptaken by Caco2 cells and only ≈ 0.1% molecules were observed to cross the epithelium layer by transcytosis. Here we show that at nanomolar concentrations WGA is unexpectedly bioactive on immune cells. The supernatants of WGA-stimulated peripheral blood mononuclear cells (PBMC) can alter the integrity of the epithelium layer when administered to the basolateral side of differentiated Caco2 cells and the effects can be partially inhibited by monoclonal antibodies against IL1, IL6 and IL8. At nanomolar concentrations WGA stimulates the synthesis of proinflammatory cytokines and thus the biological activity of WGA should be reconsidered by taking into account the effects of WGA on the immune system at the gastrointestinal interface. These results shed new light onto the molecular mechanisms underlying the onset of gastrointestinal disorders observed in vivo upon dietary intake of wheat-based foods.