Interaction Of Wheat Germ Agglutinin Research Articles

Synthetic assembly of α-O-linked-type GlcNAc using polymer chemistry affords sugar clusters, which effectively bind to lectins

Fischer’s glycoside synthesis was applied to linker precursor alcohols of two different lengths having appropriate alkane chains to obtain the corresponding α-glycoside and it was found to be applicable with moderate yields. Water-soluble glycomonomers were systematically prepared from N-acetyl-d-glucosamine (GlcNAc) by introducing two kinds of alcohols having different methylene lengths. Typical radical polymerizations of the glycomonomers with acrylamide as a modulator for control of the distance between carbohydrate residues in water in the presence of ammonium persulfate (APS)-N,N,N’,N’-tetramethylethylenediamine (TEMED) gave a series of glycopolymers with various α-glycoside-type GlcNAc residue densities. Fluorometric analysis of the interaction of wheat germ agglutinin (WGA) with the glycopolymers was performed and the results showed unique binding specificities based on structural differences.

Rapid Detection of Listeria monocytogenes in Milk by Surface Plasmon Resonance Using Wheat Germ Agglutinin

The current study made an attempt to develop a novel surface plasmon resonance (SPR) sensor using wheat germ agglutinin as a biomolecule for the rapid detection of Listeria monocytogenes. A selective biomolecular interaction of wheat germ agglutinin (WGA) lectin with surface carbohydrate components of L. monocytogenes was used as a biorecognition principle. From the previous studies based on agglutination and sugar inhibition principle, we have selected WGA as a highly selective ligand for L. monocytogenes-WGA biomolecular interaction study using a SPR (Biacore 3000) sensor. Under optimized assay conditions, L. monocytogenes strains have generated maximum response of 479 ± 49 resonance unit (RU) at 7.4 log cfu/100 μl with a least cross-reactivity in presence of both G+ve and G−ve Bacterial contaminants. The limit of detection of 3.25 log CFU/100 μl was obtained for the detection of L. monocytogenes by lectin immobilized WGA CM5 chip surface using SPR. Further, the WGA-immobilized chip was evaluated with milk samples enriched in Listeria selective enrichment medium and the response (RU) was again found to be significant up to 3.0 log CFU/100 μl.

Interaction of Wheat Germ Agglutinin with Porphyrin Compounds—Potential Anticancer Agents

ABSTRACTWheat germ agglutinin (WGA) is the first studied plant lectin shown to bind more strongly to tumour cells than to normal ones. WGA interacts with prostate cancer cells, human pancreatic cells and colon-cancer cells.Our results showed the interaction of WGA with two porphyrin compounds—Fe porphyrin and Pdporphyrin. The dissociation constants for the porphyrin binding was kD (0.08–1.02 μM) showing high affinity for the two porphyrins for this protein. The hyperbolic titration curve indicated the presence of a single porphyrin binding site.In conclusion, we show that two photosentisizers with anticancer activity Fe porphyrin and Pd porphyrin, have high affinity for WGA. Since WGA binds to cancer cells, the results suggest that it may have utility in the targeted delivery of drugs for cancer.

Enzymatic Synthesis of Spacer-Linked Divalent Glycosides Carrying N-Acetylglucosamine and N-Acetyllactosamine: Analysis of Cross-Linking Activities with WGA

Divalent glycosides carrying N-acetyl-d-glucosamine (GlcNAc) and N-acetyllactosamine (LacNAc) were designed and prepared as glycomimetics. First, hexan-1,6-diyl bis-(2-acetamido-2-deoxy-beta-d-glucopyranoside) (GlcNAc-Hx-GlcNAc) and 3,6-dioxaoct-1,8-diyl bis-(2-acetamido-2-deoxy-beta-d-glucopyranoside) (GlcNAc-Doo-GlcNAc) were enzymatically synthesized by transglycosylation of an N,N'N'',N'''-tetraacetylchitotetraose [(GlcNAc)(4)] donor with a primary diol acceptor, utilizing a chitinolytic enzyme from Amycolatopsis orientalis. The resulting divalent glycosides were further converted to the respective hexan-1,6-diyl bis-[beta-d-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-d-glucopyranoside] (LacNAc-Hx-LacNAc) and 6-(2-acetamido-2-deoxy-beta-d-glucopyranosyl)-hexyl beta-d-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-d-glucopyranoside (LacNAc-Hx-GlcNAc), and respective 3,6-dioxaoct-1,8-diyl bis-[beta-d-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-d-glucopyranoside] (LacNAc-Doo-LacNAc) and 8-(2-acetamido-2-deoxy-beta-d-glucopyranosyl)-3,6-dioxaoctyl beta-d-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-d-glucopyranoside (LacNAc-Doo-GlcNAc) by galactosyltransferase. The interaction of wheat germ agglutinin (WGA) with a series of divalent glycosides and related compounds were studied using a biosensor based on surface plasmon resonance (SPR) and by precipitation analysis. Our results demonstrated that divalent glycosides carrying GlcNAc on both sides and GlcNAc and LacNAc on each side are capable of precipitating WGA as divalent ligands, but that the corresponding monovalent controls and divalent glycosides carrying LacNAc on both sides are unable to precipitate the lectin and bind as univalent ligands.

Interaction of wheat germ agglutinin with an N-acetylglucosamine-carrying telomer brush accumulated on a colloidal gold monolayer

A disulfide-carrying telomer with many pendent N-acetylglucosamine (GlcNAc) residues (Cys-PMHGlcNAc) was obtained by photo-polymerization of 1-(6′-methacryloylaminohexyl)-2- N-acetoamido-2-deoxy d-glucopyranoside) (MHGlcNAc) using a benzyl N, N-diethyldithiocarbamoyl (BDC) derivative that shows abilities of initiation, trans fer, and termination ( iniferter). The disulfide-carrying telomer was accumulated on a monolayer of colloidal Au on a glass substrate, and the interaction of wheat germ agglutinin (WGA) with GlcNAc residue at the polymer brush-solution interface was examined by using the localized surface plasmon resonance (LSPR) technique. For comparison, an amphiphile carrying many pendent GlcNAc residues was also prepared with MHGlcNAc and a lipophilic radical initiator and was incorporated in a phospholipid liposome to examine interaction of the GlcNAc residue with WGA on the liposome surface using turbidity measurements. Both the colloidal gold optical device and the liposome showed a concentration-dependent specific binding of WGA, and the GlcNAc-carrying liposome had a detection limit of 100 nM for WGA, whereas that of the colloidal gold device was 10 nM. The sugar-carrying telomer-coated device examined here is not only useful as a simple biosensor chip but is also expected to expand our knowledge of bio-related phenomena at the liquid-telomer brush interfaces on a colloidal Au.

Colorimetric detection of WGA in carbohydrate-functionalized polydiacetylene Langmuir–Schaefer films

In this paper, carbohydrate-modified polydiacetylene (PDA) films prepared by Langmuir–Schaefer (LS) technique were used as biosensors for studying the interaction of wheat germ agglutinin (WGA) with the carbohydrate. Studies showed that the addition of WGA, specific to N-acetylglucosamine, induced the films to change their color from blue to red. UV–vis and Resonance Raman (RR) spectra confirmed the occurrence of molecular recognition. Surface pressure–area isotherms showed that glycolipid and polydiacetylene molecules had good miscibility on pure water subphase, which was significant for further applications as biosensor.

Lectin–sugar interaction

Although a steadily increasing number of protein--ligand docking experiments have been performed successfully, there are only few studies concerning protein--sugar interactions. In this study, we investigate the interaction of wheat germ agglutinin (WGA) with N-acetylglucosamine and a number of its derivatives and predict the binding free energies using flexible docking techniques. To assess the quality of our predictions, we also determined those binding free energies experimentally in cell-binding studies. The predicted binding site, ligand orientation, and details of the binding mode are in perfect agreement with the known crystal structure of WGA with a sialoglycopeptide. Furthermore, we obtained an excellent linear correlation of our predicted binding free energies with both our own data and experimental data from the literature [Monsigny, M., Roche, A.C., Sene, C., Maget Dana, R. & Delmotte, F. (1980) Eur. J. Biochem. 104, 147-153.]. In both cases, predicted energies were within 1.0 kJ x mol(-1) of the experimental value. These results illustrate the usefulness of docking-based methods for the qualitative and quantitative prediction of protein--carbohydrate interactions. The insights gained from such theoretical studies may be used to complement the results from the still scarce crystal structures.

Interaction of GM 1 glycolipid in phospholipid monolayers with wheat germ agglutinin: effect of phospholipidic environment and subphase

Mixed monolayers of GM 1 glycolipid and stearoyl-oleoyl-phosphatidylcholine (SOPC) or dipalmitoyl-phosphatidycholine (DPPC) phospholipids were studied by surface pressure measurements. The effects induced by GM 1 on the mean molecular areas of mixtures and DPPC phase transition were followed for GM 1 concentrations ranging from 1 to 20 mol.%. Under our experimental conditions, one main parameter influencing the behavior of phospholipid–GM 1 monolayers is the ionic strength of the subphase. Mixed monolayers are in a more expanded state on buffer than on pure water. This could be due to a change of GM 1 orientation at the interface. The interaction of wheat germ agglutinin (WGA), a lectin recognizing specifically GM 1, with these monolayers was quantified in terms of the Gibbs equation. Specific WGA–GM 1 interactions are clearly reduced in the presence of DPPC as compared with SOPC, probably because of the higher packing density of these monolayers. Phospholipid–GM 1 monolayers could also undergo some rearrangements induced by WGA binding.

The Interaction between wheat germ agglutinin and other plant lectins with prostate cancer cells Du-145

The bioadhesive properties of fluorescein-labeled plant lectins with different carbohydrate specificities were investigated by flow cytometry at 4 and 37°C using Du-145 prostate cancer cells. At both temperatures the lectin association rate increased following the order: Dolichos biflorus agglutinin (DBA)<peanut agglutinin< Ulex europaeus isoagglutinin I< Lens culinaris agglutinin< Solanum tuberosum lectin≪wheat germ agglutinin (WGA), reflecting the glycosylation pattern of Du-145 cells. Both, the BSA-binding capacity of the cells referring to nonspecific binding and inhibition studies using the complementary carbohydrate, assured specificity of the lectin–cell interactions except for DBA. The WGA-association rate of Du-145 cells was dependent on temperature indicative for cellular uptake of membrane-bound WGA. Intracellular enrichment of WGA was confirmed by confocal microscopy. As resulted from experiments in presence of ouabain active transport mechanisms were involved in cellular uptake of WGA. Equilibration of the intracellular pH with monensin pointed to accumulation of intracellular located WGA within acidic compartments of Du-145 cells such as the lysosomes or the trans-Golgi complex. Consequently the interaction of WGA with Du-145 cells at 37°C is a one way process due to immediate active transport of membrane-bound lectin into acidic compartments of prostate cancer cells.

Wheat germ agglutinin inhibits the C5a receptor interaction: implications for receptor microheterogeneity and ligand binding site.

Wheat germ agglutinin (WGA) has been shown to inhibit the interaction of C5a with the C5a receptor on both polymorphonuclear neutrophils (PMNs) and the histiocytic cell line U937. The level of inhibition with isolated receptor preparations is 100%, and on intact cells 10 to 20% of the receptor population appear to retain their ability to bind C5a in the presence of WGA. In contrast, this lectin completely inhibits the C5a-mediated degranulation of PMN primary and secondary granules, suggesting that the population of C5a receptors responsible for mediating degranulation is also recognized by WGA. More than 50% of the receptors appear to be blocked before an effect on degranulation occurs. This inhibition by WGA does not appear to be due to down-regulation of C5a receptors from the cell surface, excessive aggregation of receptor sites, or interaction of WGA with the carbohydrate portion of the C5a molecule. The inhibition is reversed by N-acetylglucosamine but not by sialic acid. This effect appears to be specific for WGA because various other lectins do not inhibit the C5a receptor interaction. That the inhibition by WGA is due to direct binding of the lectin to N-acetylglucosamine residues on the C5a receptor is strongly supported by the ability of the cross-linked C5a-receptor complex to bind to and be specifically eluted from a WGA-Affigel affinity matrix. These observations are consistent with hypothesis that the population of C5a receptors on leukocytes exhibits microheterogeneity with respect to structure (carbohydrate content) and/or function.

Wheat germ agglutinin is mitogenic, nonmitogenic and anti-mitogenic for human lymphocytes.

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 oligomers of N-acetyl-D-glucosamine. Significant mitogenic activity was dependent on the use of human (as opposed to fetal calf) serum to supplement the culture medium. Purified T cells responded to WGA very weakly and the incorporation of thymidine into non-T cells in response to WGA was less than the lectin-free control. 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 T cells to respond to WGA; interleukin 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.

Interaction of wheat germ agglutinin with human platelets: A model for studying platelet response

Binding of wheat germ agglutinin (WGA) to human platelets and their activation were studied. The binding of fluoresceinyl-substituted WGA (F-WGA) was saturable, specific, reversible and cooperative. The apparent association constant (K a) was 2.4 × 10 6 M −1. Activation of platelets was measured by change in light transmission (LT) (aggregation) and by assays of 14C-serotonin and beta-thromboglobulin (secretion). The maximum platelet activation was obtained with 15 μg/ml WGA. At this concentration only 17% of all available binding sites were occupied. Increasing the WGA concentration diminished the change in LT and the secretion from alpha granules but not from dense bodies. The addition of EDTA partially reduced the platelet aggregation without any effect on the secretion. Remaining LT change was insensitive to all metabolic inhibitors tested (CP/CPK, arginine, indomethacin, PGE 1, chlorpromazine, colchicine and cytochalasin B). The secretion measured in the presence of EDTA was only reduced by preincubation with PGE 1 and CPZ. Succinyl-WGA which only binds glucoconjugates containing G1cNAc did not induce any platelet activation, whereas limulin, which binds to glucoconjugates containing NeuAc did induce platelet agglutination and secretion. These results indicate that: (1) the platelet aggregation and secretion induced by WGA occur with only partial occupancy of the membrane binding sites by the lectin; (2) the platelet LT change is due in part to the agglutinating properties of the lectin bound to the platelet surface, and in another part to the aggregation reaction mediated by a platelet constituent secreted from the storage sites in response to the binding of the lectin.

Horseradish peroxidase (HRP) conjugates of cholera toxin and lectins are more sensitive retrogradely transported markers than free HRP

Horseradish peroxidase (HRP) conjugates of 8 different lectins (wheat germ agglutinin, ricinus communis I and II, peanut agglutinin, lens culinaris, soybean agglutinin, limulus polyhemus, ulex europaeus I) and cholera toxin (CT) or free HRP (FHRP) were individually injected into the submandibular gland (SMG) or anterior chamber (AC) of the eye and the retrogradely labeled neurons in the superior cervical ganglion (SCG) were quantitated. The effect of using 3 different cross-linking reagents (glutaraldehyde, p-benzoquinone and periodic acid) on the results obtained with HRP conjugates of wheat germ agglutinin (WG) was also examined. The results in 100 rats demonstrated the superior sensitivity of ligand-HRP conjugates over FHRP as retrogradely transported markers. After SMG injections, HRP conjugates of CT, WG and soybean agglutinin were 20–50 times more sensitive than FHRP. After AC injections, HRP conjugates of CT and WG consistently yielded labeled SCG neurons while FHRP failed to do so even when the amount of FHRP injected was increased 10-fold. The sensitivity of HRP conjugates of WG was similar after SMG injections using each of the 3 cross-linking reagents, but AC injections of conjugates produced with p-benzoquinone yielded twice as many labeled SCG neurons as the other WG conjugates. Mixtures of conjugates with and without FHRP were no more sensitive than the most sensitive individual ligand-HRP conjugates, except after SMG injections of a conjugate mixture with FHRP. Additional experiments demonstrated the specificity of the ligand-‘receptor’ interaction of WG and CT and that the superior sensitivity of these ligand-HRP conjugates does not depend on the tissue destruction produced by the injection procedure.

Lectin receptors as markers for Trypanosoma cruzi. Developmental stages and a study of the interaction of wheat germ agglutinin with sialic acid residues on epimastigote cells.

Trypanosoma cruzi at various stages of maturation and differentiation have been isolated by conventional cellular fractionation procedures and characterized by cell surface markers using 30 highly purified lectins encompassing all known sugar specificities. Cell surface carbohydrates of the various T. cruzi stages were analyzed by agglutination and lectin-binding assays. Specific receptors for wheat germ agglutinin (WGA), Helix pomatia, Sophora japonica, and Bandeiraea simplicifolia lectin II were found only in culture epimastigotes, whereas peanut agglutinin (PNA) sites were present exclusively in amastigotes, those for Phaseolus vulgaris in bloodstream trypomastigotes and amastigotes, and for Wistaria floribunda hemagglutinin predominantly in culture forms of T. cruzi. The N-acetylgalactosamine (DGalNAc)-binding lectin from Bauhinia purpurea agglutinated and inhibited the movement of epimastigotes and bloodstream trypomastigotes, but it only inhibited--without agglutinating--culture trypomastigotes. Because both the agglutination and inhibition of movement were reversed by specific sugar haptens, Bauhinia purpurea sites were present in all the flagellated parasites. On the other hand, PNA sites were detectable on epimastigotes after the cells were treated with sialidase, whereas, at the same time, WGA receptors were completely removed and those for the other sialic acid-binding proteins, Aaptos papillata lectin II and Limulus polyphemus, were partially eliminated; moreover, the activity of Wistaria floribunda hemagglutinin, a DGalNAc-binding lectin, increased 4,000 times. Trypsinization and lyzozyme treatment of epimastigote cells did not significantly affect lectin agglutination or lectin binding. WGA reacted solely with sialic acid residues on epimastigote cell surface with an apparent association constant of 2 x 10(6) M-1, each epimastigote having an estimated average of 3 x 10(6) WGA sites, as determined by binding experiments and a minimum of 7.7 x 10(6) sialic acid residues, as calculated by colorimetric method after sialidase digestion. Evidences are presented that the sialyl residues are rapidly regenerated (in approximately 4 h) and that they, at least for the most part, are not adsorbed from the culture medium. The receptor for the D-mannose-binding lectins (concanavalin A [Con A] and Lens culinaris) must either be on the same carbohydrate moiety having the WGA site, or, if in a distinct molecule, both carrier molecules of Con A and WGA sites must be located close to each other in the plasma membrane of the parasite.

Interaction of wheat germ agglutinin with human peripheral blood mononuclear cells. Binding kinetics and flow microfluorometric analysis

We have used binding experiments with radiolabeled lectin and flow microfluorometry to characterize the interaction of wheat germ agglutinin (WGA) with human peripheral blood mononuclear cell preparations. Results indicated that WGA bound to these cells in a complex fashion described by a curvilinear (upwardly concave) Scatchard plot and a nonlinear dissociation curve. Binding was rapid and reversible and was inhibited 80–90% by 0.1 M N-acetylglucosamine, a specific haptenic inhibitor of WGA binding. Approximately 1 5 of the total binding was attributable to monocytes, (but phagocytic uptake was excluded), and the remainder was due to lymphoid cell variants. Kinetics of binding were only modestly altered by formalin fixation of the cells mitigating against a role for site-site interactions in the generation of the non-linear Scatchard plot. Flow microfluorometric analysis revealed that the capability to bind WGA was a characteristic of only 35% of blood mononuclear cells and further allowed identification of a small cell subclass constituting less than 10% of the total that nonetheless accounted for fully one half of the entire WGA binding potential of these cells.

Interaction of wheat germ agglutinin with sialic acid.

Interaction of wheat germ agglutinin with sialic acid.

Role of surface sialic acid in the interaction of wheat germ agglutinin with human platelets

Wheat germ agglutinin binds to the surface of human platelets and leads to their agglutination. Unlike other aggregating agents, the clumping ability of platelets by the lectin was markedly reduced upon removal of the cell surface sialic acid. This reduction in agglutination of neuraminidase-treated platelets was due to a lower capacity of the cells to bind the lectin. Since the terminal sialic acid has been implicated in the clearance of platelets from the circulation, wheat germ agglutinin may prove to be a useful tool to explore those clinical conditions in which platelet survival is shortened.

Interaction of Wheat-germ Agglutinin with Streptococci and Streptococcal Cell Wall Polymers

Wheat-germ agglutinin agglutinates all strains of group A and group C streptococci, a part of the strains tested of groups Band E and some strains of other streptococcal groups. It also agglutinates peptidoglycan of several streptococcal groups and precipitates some group specific carbohydrates of streptococcal cell walls as well as some teichoic acid extracts. The basis for agglutination and precipitation of streptococci and streptococcal cell wall polymers appears to be the interaction of the combining sites of the agglutinin with N-acetyl D-glucosamine residues present in the cell wall polymers, either in terminal or in internal positions respectively.

The interaction of wheat germ agglutinin with sialoglycoproteins. The role of sialic acid.

The interaction of wheat germ agglutinin with sialoglycoproteins. The role of sialic acid.

Interaction of wheat germ agglutinin with rat platelets: Observations on stimulus-response coupling

Interaction of wheat germ agglutinin with rat platelets: Observations on stimulus-response coupling