Plant Lectin Wheat Germ Agglutinin Research Articles

Cover Feature: Photoswitching Affinity and Mechanism of Multivalent Lectin Ligands (Chem. Eur. J. 27/2022)

Photochemical cis–trans isomerization was used to switch a divalent lectin ligand between its two forms, which show contrasting binding properties to the plant lectin wheat germ agglutinin (WGA), as visualized by the yin-yang symbol. Whereas the E isomer (obtained under irradiation with green light) is able to bridge adjacent binding sites of WGA leading to a high-affinity chelating binding mode, the Z isomer (obtained under UV light irradiation) crosslinks two lectin molecules associated with a much lower binding affinity. More information can be found in the Research Article by V. Wittmann and co-authors (DOI: 10.1002/chem.202200267).

Enhanced cell adhesion on silk fibroin via lectin surface modification

Various tissue engineering (TE) approaches are based on silk fibroin (SF) as scaffold material because of its superior mechanical and biological properties compared to other materials. The translation of one-step TE approaches to clinical application has generally failed so far due to the requirement of a prolonged cell seeding step before implantation. Here, we propose that the plant lectin WGA (wheat germ agglutinin), covalently bound to SF, will mediate cell adhesion in a time frame acceptable to be part of a one-step surgical intervention. After the establishment of a modification protocol utilizing carbodiimide chemistry, we examined the attachment of cells, with a special focus on adipose-derived stromal cells (ASC), on WGA-SF compared to pure native SF. After a limited time frame of 20min the attachment of ASCs to WGA-SF showed an increase of about 17-fold, as compared to pure native SF. The lectin-mediated cell adhesion further showed an enhanced resistance to trypsin (as a protease model) and to applied fluid shear stress (mechanical stability). Moreover, we could demonstrate that the adhesion of ASCs on the WGA-SF does not negatively influence proliferation or differentiation potential into the osteogenic lineage. To test for in vitro immune response, the proliferation of peripheral blood mononuclear cells in contact with the WGA-SF was determined, showing no alterations compared to plain SF. All these findings suggest that the WGA modification of SF offers important benefits for translation of SF scaffolds into clinical applications.

A high‐throughput screening assay to identify bacterial antagonists against Fusarium verticillioides

A high-throughput antagonistic assay was developed to screen for bacterial isolates capable of controlling the maize fungal phytopathogen Fusarium verticillioides. This assay combines a straightforward methodology, in which the fungus is challenged with bacterial isolates in liquid medium, with a novel approach that uses the plant lectin wheat germ agglutinin (WGA) coupled to a fluorophore (Alexa-Fluor® 488) under the commercial name of WGA, Alexa Fluor® 488 conjugate. The assay is performed in a 96-well plate format, which reduces the required laboratory space and streamlines quantitation and automation of the process, making it fast and accurate. The basis of our assay is that fungal biomass can be assessed by WGA, Alexa Fluor® 488 conjugate staining, which recognizes the chitin in the fungal cell wall and thus permits the identification of potential antagonistic bacteria that inhibit fungal growth. This principle was validated by chitin-competition binding assays against WGA, Alexa Fluor® 488 conjugate; confocal laser microscopy confirmed that the fluorescent WGA, Alexa Fluor® 488 conjugate binds to the chitin of the fungal cell wall. The majority of bacterial isolates did not bind to the WGA, Alexa Fluor® 488 conjugate. Furthermore, including washing steps significantly reduced any bacterial staining to background levels, even in the rare cases where bacterial isolates were capable of binding to WGA. Confirmatory conventional agar plate antagonistic assays were also conducted to validate our technique. We are now successfully employing this large-scale antagonistic assay as a pre-screening step for potential fungal antagonists in extensive bacteria collections (on the order of thousands of isolates).

UPEC biomimickry at the urothelial barrier: Lectin-functionalized PLGA microparticles for improved intravesical chemotherapy

The urgent demand for more potent treatment schedules in bladder cancer (BCa) therapy calls for a refinement of the intravesical administration modalities. However, progress on drug delivery systems tailored to the penetration-hostile urothelial barrier lags behind the advancements in comparable fields. This study reports on a multimodal, carrier-based delivery concept that combines biorecognitive targeting with modified release strategies for improved intravesical chemotherapy. The plant lectin wheat germ agglutinin (WGA) was immobilized on poly(lactide-co-glycolide) (PLGA) microparticles (MP) to induce stable cytoadhesion via cellular carbohydrate chains, similar to the specific attachment mechanism utilized by uropathogenic bacteria. A panel of DNA-selective chemotherapeutics with established track record in uro-oncology was screened for physicochemical compatibility with the polymeric carrier formulation. Critical limitations in encapsulation efficiency were found for mitomycin C (MMC), doxorubicin (DOX), and gemcitabine hydrochloride (GEM), despite multiparametric optimization of the preparation conditions. In contrast, the amphiphilic 4-(N)-stearoyl prodrug of gemcitabine (GEM-C18) exhibited excellent processability with PLGA. In vitro bioassays on 5637 human BCa cells showed that the enhanced cytoadhesion of WGA-GEM-C18-PGLA-MP traces back to the specific lectin/carbohydrate interaction, and is not easily disrupted by adverse environmental factors. Owing to several synergistic effects, the combined prodrug/targeting approach resulted in strong cytostatic response even when adjusting the exposure scheme to the confined temporal conditions of instillative treatment. Our results highlight the importance of fine-tuning both pharmacokinetic and pharmacologic parameters to gain adequate impact on urothelial cancer cells, and assign promising potential to glycan-targeted delivery concepts for the intravesical route.

Binding of Antitumor Compounds to Wheat Protein

ABSTRACTBinding of porphyrins to biological macromolecules is important to be studied as an approach to keeping them in stable, effective and safe dosage forms. In this work we show the interaction of three porphyrin compounds: Hematoporphyrin IX, Mn- and Fe- porphyrins with plant lectin Wheat germ agglutinin (WGA). Porphyrin binding to WGA was monitored by the changes in the intrinsic protein fluorescence. Conformational changes due to the formation of WGA-porphyrin complexes, were shown by the hyperbolic binding curves. The calculated dissociation constants KD (0.16 μM to 0.24 μM) indicate high affinity of WGA for the three porphyrins. Most probably they occupy the same high affinity sites. In conclusion, since WGA binds cancer cells and interacts with antitumor compounds, it could be useful as a drug delivery molecule.

Énucléation d’une lésion pancréatique par laparotomie

The urgent demand for more potent treatment schedules in bladder cancer (BCa) therapy calls for a refinement of the intravesical administration modalities. However, progress on drug delivery systems tailored to the penetration-hostile urothelial barrier lags behind the advancements in comparable fields. This study reports on a multimodal, carrier-based delivery concept that combines biorecognitive targeting with modified release strategies for improved intravesical chemotherapy.The plant lectin wheat germ agglutinin (WGA) was immobilized on poly(lactide-co-glycolide) (PLGA) microparticles (MP) to induce stable cytoadhesion via cellular carbohydrate chains, similar to the specific attachment mechanism utilized by uropathogenic bacteria. A panel of DNA-selective chemotherapeutics with established track record in uro-oncology was screened for physicochemical compatibility with the polymeric carrier formulation. Critical limitations in encapsulation efficiency were found for mitomycin C (MMC), doxorubicin (DOX), and gemcitabine hydrochloride (GEM), despite multiparametric optimization of the preparation conditions. In contrast, the amphiphilic 4-(N)-stearoyl prodrug of gemcitabine (GEM-C18) exhibited excellent processability with PLGA. In vitro bioassays on 5637 human BCa cells showed that the enhanced cytoadhesion of WGA-GEM-C18-PGLA-MP traces back to the specific lectin/carbohydrate interaction, and is not easily disrupted by adverse environmental factors. Owing to several synergistic effects, the combined prodrug/targeting approach resulted in strong cytostatic response even when adjusting the exposure scheme to the confined temporal conditions of instillative treatment. Our results highlight the importance of fine-tuning both pharmacokinetic and pharmacologic parameters to gain adequate impact on urothelial cancer cells, and assign promising potential to glycan-targeted delivery concepts for the intravesical route.

High-affinity multivalent wheat germ agglutinin ligands by one-pot click reaction

A series of six mono-, di-, and trivalent N,N’-diacetylchitobiose derivatives was conveniently prepared by employing a one-pot procedure for Cu(II)-catalyzed diazo transfer and Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) starting from commercially available amines. These glycoclusters were probed for their binding potencies to the plant lectin wheat germ agglutinin (WGA) from Triticum vulgaris by an enzyme-linked lectin assay (ELLA) employing covalently immobilized N-acetylglucosamine (GlcNAc) as a reference ligand. IC50 values were in the low micromolar/high nanomolar range, depending on the linker between the two disaccharides. Binding enhancements β up to 1000 for the divalent ligands and 2800 for a trivalent WGA ligand, compared to N,N’-diacetylchitobiose as the corresponding monovalent ligand, were observed. Molecular modeling studies, in which the chitobiose moieties were fitted into crystallographically determined binding sites of WGA, correlate the binding enhancements of the multivalent ligands with their ability to bind to the protein in a chelating mode. The best WGA ligand is a trivalent cluster with an IC50 value of 220 nM. Calculated per mol of contained chitobiose, this is the best WGA ligand known so far.

Structural Basis of Multivalent Binding to Wheat Germ Agglutinin

The inhibition of carbohydrate-protein interactions by tailored multivalent ligands is a powerful strategy for the treatment of many human diseases. Crucial for the success of this approach is an understanding of the molecular mechanisms as to how a binding enhancement of a multivalent ligand is achieved. We have synthesized a series of multivalent N-acetylglucosamine (GlcNAc) derivatives and studied their interaction with the plant lectin wheat germ agglutinin (WGA) by an enzyme-linked lectin assay (ELLA) and X-ray crystallography. The solution conformation of one ligand was determined by NMR spectroscopy. Employing a GlcNAc carbamate motif with alpha-configuration and by systematic variation of the spacer length, we were able to identify divalent ligands with unprecedented high WGA binding potency. The best divalent ligand has an IC(50) value of 9.8 microM (ELLA) corresponding to a relative potency of 2350 (1170 on a valency-corrected basis, i.e., per mol sugar contained) compared to free GlcNAc. X-ray crystallography of the complex of WGA and the second best, closely related divalent ligand explains this activity. Four divalent molecules simultaneously bind to WGA with each ligand bridging adjacent binding sites. This shows for the first time that all eight sugar binding sites of the WGA dimer are simultaneously functional. We also report a tetravalent neoglycopeptide with an IC(50) value of 0.9 microM being 25,500 times higher than that of GlcNAc (6400 times per contained sugar) and the X-ray structure analysis of its complex with glutaraldehyde-cross-linked WGA. Comparison of the crystal structure and the solution NMR structure of the neoglycopeptide as well as results from the ELLA suggest that the conformation of the glycopeptide in solution is already preorganized in a way supporting multivalent binding to the protein. Our findings show that bridging adjacent protein binding sites by multivalent ligands is a valid strategy to find high-affinity protein ligands and that even subtle changes of the linker structure can have a significant impact on the binding affinity.

Changes in the N -Glycome, Glycoproteins with Asn-Linked Glycans, of Giardia lamblia with Differentiation from Trophozoites to Cysts

Giardia lamblia is present in the intestinal lumen as a binucleate, flagellated trophozoite or a quadranucleate, immotile cyst. Here we used the plant lectin wheat germ agglutinin (WGA), which binds to the disaccharide di-N-acetyl-chitobiose (GlcNAc(2)), which is the truncated Asn-linked glycan (N-glycan) of Giardia, to affinity purify the N-glycomes (glycoproteins with N-glycans) of trophozoites and cysts. Fluorescent WGA bound to the perinuclear membranes, peripheral acidified vesicles, and plasma membranes of trophozoites. In contrast, WGA bound strongly to membranes adjacent to the wall of Giardia cysts and less strongly to the endoplasmic reticulum and acidified vesicles. WGA lectin-affinity chromatography dramatically enriched secreted and membrane proteins of Giardia, including proteases and acid phosphatases that retain their activities. With mass spectroscopy, we identified 91 glycopeptides with N-glycans and 194 trophozoite-secreted and membrane proteins, including 42 unique proteins. The Giardia oligosaccharyltransferase, which contains a single catalytic subunit, preferred N glycosylation sites with Thr to those with Ser in vivo but had no preference for flanking amino acids. The most-abundant glycoproteins in the N-glycome of trophozoites were lysosomal enzymes, folding-associated proteins, and unique transmembrane proteins with Cys-, Leu-, or Gly-rich repeats. We identified 157 secreted and membrane proteins in the Giardia cysts, including 20 unique proteins. Compared to trophozoites, cysts were enriched in Gly-rich repeat transmembrane proteins, cyst wall proteins, and unique membrane proteins but had relatively fewer Leu-rich repeat proteins, folding-associated proteins, and unique secreted proteins. In summary, there are major changes in the Giardia N-glycome with the differentiation from trophozoites to cysts.

Probing multivalent carbohydrate–lectin interactions by an enzyme-linked lectin assay employing covalently immobilized carbohydrates

We report here the synthesis of a series of mono- to trivalent N-acetylglucosamine (GlcNAc) derivatives as ligands for the plant lectin wheat germ agglutinin (WGA). Their WGA binding potencies were determined by an established enzyme-linked lectin assay (ELLA) employing microtiter plates with non-covalently immobilized porcine stomach mucin (PSM) as reference ligand and an ELLA with a new GlcNAc derivative covalently immobilized via a thiourea linkage. Comparison of both assays revealed that the type of presentation of GlcNAc residues on the microtiter plates either as part of a glycoprotein or as a covalently immobilized monosaccharide derivative strongly influences the outcome of the assay. Although the apparent dissociation constants K D ELLA for the interaction of peroxidase-labeled WGA with the microtiter plates are comparable for both surfaces, IC 50 values obtained with the PSM-free ELLA were substantially lower. Even more strikingly, this ELLA displayed a better differentiation between ligands of different valency leading to significantly higher relative inhibitory potencies of multivalent ligands compared to monovalent. Additionally, problems associated with the use of PSM, such as maximum inhibition at considerably less than 100% and poor reproducibility of IC 50 values could be overcome with this type of ELLA.

The plant lectin wheat germ agglutinin inhibits the binding of pemphigus foliaceus autoantibodies to desmoglein 1 in a majority of patients and prevents pathomechanisms of pemphigus foliaceus in vitro and in vivo.

Pemphigus foliaceus (PF) is a life-threatening autoimmune blistering skin disease caused by pathogenic IgG autoantibodies against desmoglein 1 (dg1), a desmosomal cadherin-type adhesion glycoprotein. Using lectins and glycosidases, we have shown that dg1 displays an N-glycosylation pattern of the complex triantennary type. We have found that lectins and glycosidases interfere with N-bound sugar residues on the amino-terminal ectodomain of dg1 and completely abolish, in vitro, the antigenicity of dg1 in most of the patients' sera. Moreover, in an ex vivo model using punch biopsies from normal human skin, we demonstrate that preincubation of the epidermis in wheat germ agglutinin (WGA) prevents PF autoantibody binding, acantholysis, and subcorneal blistering. In addition, we show that topical treatment with WGA inhibits PF autoantibody binding to keratinocytes in both newborn BALB/c mice and in organotypic human epidermis grafted onto the back of SCID mice. The epidermis of these pretreated animals displays a regular morphology, whereas control animals develop the immunopathologic phenotype of PF. These findings suggest that WGA may interfere with autoantibody binding to dg1, preventing experimental PF without affecting the adhesive function of dg1. Our observations may provide a new approach to the therapy of PF.

A novel inhibitory action of wheat germ agglutinin on phospholipase C in HEL and MEG-01 cell lines

Stimulation of HEL megakaryocytic cells by Fc γRIIA crosslinking is associated with tyrosine phosphorylation of syk and phospholipase C γ2 (PLC γ2) and is accompanied by formation of inositol phosphates and release of intracellular Ca 2+. These responses are inhibited by the kinase inhibitors, staurosporine and ST271. In contrast, the G-protein receptor agonist, thrombin induces formation of inositol phosphates and release of intracellular calcium without an increase in tyrosine phosphorylation. The plant lectin wheat germ agglutinin (WGA) stimulates tyrosine phosphorylation of syk and PLC γ2 but surprisingly does not stimulate formation of inositol phosphates and induce release of intracellular Ca 2+. WGA also inhibited formation of inositol phosphates and release of intracellular Ca 2+ by Fc γRIIA crosslinking and thrombin-stimulation. A similar inhibitory effect of WGA was observed against elevation of Ca 2+ by the same two stimuli in MEG-01 megakaryotic cells. The results demonstrate a novel pathway of inhibition of PLC on crosslinking of cell surface proteins that is not present in platelets.

Polysialosides scaffolded on p- Tert-butylcalix[4]arene

p- Tert-butylcalix[4]arene ( 1) was transformed into known tetraethyl ester 2 to provide cone-shaped calix[4]arene used for the scaffolding of tetravalent α-sialoside. Ester 2 was transformed into acid chloride 4 which after treatment with mono-Boc-1,4-butanediamine, trifluoroacetolysis and N-chloroacetylation afforded tetraamine 7. Conjugation of α-thiosialoside 9 by nucleophilic substitution and deprotection furnished water-soluble tetravalent α-thiosialoside 12 which bound strongly to the plant lectin wheat germ agglutinin.

13C NMR studies of wheat germ agglutinin interactions with N-acetylglucosamine at a magnetically oriented bilayer surface.

The orientation of synthetic 13C-labeled glycolipid receptors and their interaction with the plant lectin wheat germ agglutinin have been studied in an oriented membrane system using NMR spectroscopy. A series of 2-[1,2-13C2]acetamido-2-deoxy-beta-D-glucopyranosides were synthesized with between zero and four hydrophilic ethoxy units between the headgroup and an alkyl chain which anchors the receptors in the bilayers. The chemical shift anisotropy of the 13C carbonyl and a 13C-13C dipolar coupling between the labeled carbons provide information about the orientation and dynamics of the receptor headgroup in oriented membrane systems. It was found that the headgroups of the receptors with two, three, or four ethoxy units appeared isotropic when incorporated in the oriented bilayers, but those of the receptors with zero or one ethoxy units were significantly ordered by the bilayers. The average orientations consistent with measured spectral parameters were determined for the receptors with zero and one ethoxy units and were found to coincide with low-energy conformations from molecular modeling. When the plant lectin wheat germ agglutinin was added to the sample, only the receptors with two, three, or four ethoxy units separating the headgroup from the alkyl chain showed evidence of binding by the lectin. Although the 13C-labeled resonances broadened when the protein bound, no changes in dipolar couplings or chemical shift anisotropies could be detected, suggesting that the motion of the headgroup was slowed by protein binding, but average orientation and overall order changed little. Competition studies demonstrated that none of the lectin/receptor complexes are more stable than the complex of the lectin and N-acetylglucosamine in solution. These results suggest that the membrane does not stabilize the interactions of wheat germ agglutinin with these cell-surface receptors. Furthermore, molecular modeling demonstrates that the zero- and one-spacer receptors may not bind wheat germ agglutinin because the orientations of the N-acetyl groups in these receptors would result in significant steric contacts between the lectin/receptor complex and the membrane.

Novel glycosylation-defective baby hamster kidney cells

The plant lectin wheat germ agglutinin (WGA) has previously been used to select more than ten different glycosylation-defective phenotypes in a variety of mammalian somatic cells. Three WGA-resistant phenotypes have now been obtained spontaneously from baby hamster kidney (BHK) cells. These mutant BHK cells exhibit a pattern of cross resistance and sensitivity to multiple plant lectins, suggesting that the cell surface carbohydrates of these cells are altered. Two WGA-resistant BHK phenotypes appear similar to WGA-resistant CHO cells that lack terminal sialic acid and galactose residues on their cell surface carbohydrates. The third WGA-resistant BHK cell phenotype has not previously been seen in WGA-resistant mammalian cells.

Localization of wheat germ agglutinin and antibody binding sites on the plasma membranes of sea urchin sperm heads as revealed by label‐fracture and fracture‐flip

Freeze-fracture electron microscopy reveals that intramembrane particles are concentrated in a band encircling the posterior portion of the acrosome of Strongylocentrotus purpuratus sperm. Two colloidal gold labeling methods, label-fracture and replica-staining fracture-flip, were employed to show that the plant lectin wheat germ agglutinin, which recognizes a 210 kDa sperm surface glycoprotein, binds to this localized band of intramembrane particles. Monoclonal antibody J18/2, which also recognizes the 210 kDa surface glycoprotein, shows this localized binding in approximately 20% of the sperm observed in this study. The majority of sperm displayed a uniform distribution of receptor sites for monoclonal antibody J18/2. Since wheat germ agglutinin and monoclonal antibody J18/2 are known to agglutinate Strongylocentrotus purpuratus sperm but not sperm of another sea urchin, Lytechinus pictus, similar determinations were made for the latter species. Lytechinus pictus sperm are not labeled with wheat germ agglutinin and are only sparsely labeled with monoclonal antibody J18/2. The acrosomal localizations of wheat germ agglutinin and monoclonal antibody J18/2 receptors in Strongylocentrotus purpuratus sperm are consistent with the involvement of the 210 kDa surface glycoprotein in an egg jelly-induced sperm acrosome reaction. Low-temperature post-embed labeling of thin sections with wheat germ agglutinin and monoclonal antibody J18/2 show concentrations of label within the acrosomal vesicle of Strongylocentrotus purpuratus sperm, suggesting the presence of an intracellular storage site for the 210 kDa glycoprotein.

Fibroblast growth factor-induced decrease in the phosphorylation of Nsp100 mediated through a calcium-dependent mechanism and blocked by lectins.

Separate treatment of PC12h cells with basic fibroblast growth factor (bFGF) and with epidermal growth factor (EGF) induced a selective decrease in the incorporation of radioactive phosphate into a 100,000-dalton soluble protein during phosphorylation with (gamma-32P)ATP of soluble extracts from the cells, as was seen previously with nerve growth factor (NGF). This 100,000-dalton soluble protein was designated in earlier studies as nerve growth factor-sensitive protein 100 (Nsp100). The inhibitory effects of bFGF and EGF on Nsp100 phosphorylation were prevented by pretreatment of PC12h cells with the calcium chelator, EGTA. Treatment of PC12h cells with the plant lectin wheat germ agglutinin (WGA), which binds to N-acetylglucosamine and sialic acid residues on glycoconjugates, blocked the inhibitory effects of bFGF, EGF, and NGF on Nsp100 phosphorylation. The blockage by WGA was reversed by the addition of the lectin-specific sugar N-acetylglucosamine to the PC12h cultures. Although pretreatment of PC12h cells with succinylated WGA, which has the ability to bind to N-acetylglucosamine but not to sialic acid residues, failed to block the inhibitory effect of NGF on Nsp100 phosphorylation as described previously, it did prevent the inhibitory effect of bFGF on this phosphorylation. These data suggest that in PC12h cells bFGF and EGF induce a decrease in the phosphorylation of Nsp100 mediated through a Ca2(+)-dependent mechanism, as in the case of NGF. Furthermore, the blockage of the bFGF-induced inhibition of Nsp100 phosphorylation by WGA and its succinylated form indicates that N-acetylglucosamine residues of bFGF receptor molecules might be involved in the mechanism by which bFGF inhibits the phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Association of nerve growth factor receptors with the triton X-100 cytoskeleton of PC12 cells

Triton X-100 solubilizes membranes of PC12 cells and leaves behind a nucleus and an array of cytoskeletal filaments. Nerve growth factor (NGF) receptors (10% of those found in intact cells) are associated with this Triton X-100-insoluble residue. Two classes of NGF receptors are found on PC12 cells which display rapid and slow dissociating kinetics. Although rapidly dissociating binding is predominant (greater than 75%) in intact cells, the majority of binding to the Triton X-100 cytoskeleton is slowly dissociating (greater than 75%). Rapidly dissociating NGF binding on intact cells can be converted to a slowly dissociating form by the plant lectin wheat germ agglutinin (WGA). This lectin also increases the number of receptors which associate with the Triton X-100 cytoskeleton by more than 10-fold. 125I-NGF bound to receptors can be visualized by light microscopy autoradiography in Triton X-100-insoluble residues of cell bodies, as well as growth cones and neurites. The WGA-induced association with the cytoskeleton, however, is not specific for the NGF receptor, since greater than 90% of cell surface glycoprotein receptors for WGA become associated with Triton X-100-insoluble material at lectin concentrations greater than 33 micrograms/ml. Concentrations of WGA which change the Triton X-100 solubility of membrane glycoproteins are similar to those required to alter the kinetic state of the NGF receptor. Both events may be related to the crossbridging of cell surface proteins induced by this multivalent lectin.

Induction of human monocyte-mediated tumor cell killing by a plant lectin, wheat germ agglutinin.

Human peripheral blood monocytes from healthy donors were separated by discontinuous gradient centrifugation and adherence to yield highly purified adherent cell populations (greater than 99% monocytes). Five different plant lectins were tested for ability to induce lectin-dependent monocyte-mediated cytotoxicity (LDMC). Only one lectin, wheat germ agglutinin (WGA), induced significant and reproducible LDMC activity. All the tumor target cells tested were sensitive to variable extents to cytotoxicity mediated by WGA-treated monocytes. Pretreatment of monocytes with WGA did not result in development of LDMC. N-Acetylglucosamine, which specifically binds WGA, inhibited WGA-dependent monocyte-mediated cytotoxicity. Treatment of adherent monocyte-rich monolayers with monoclonal anti-natural killer cell antibody (anti-Leu-11b) and complement did not affect the LDMC activity induced by WGA. These results indicate that the plant lectin WGA, which binds specifically to both human monocytes and tumor cells, renders human blood monocytes cytotoxic to human tumor cells.

Adhesivity and rigidity of erythrocyte membrane in relation to wheat germ agglutinin binding.

Binding of the plant lectin wheat germ agglutinin (WGA) to erythrocyte membranes causes membrane rigidification. One of our objectives has been to directly measure the effects of WGA binding on membrane rigidity and to relate rigidification to the kinetics and levels of WGA binding. Our other objective has been to measure the strength of adhesion and mechanics of cell separation for erythrocytes bound together by WGA. The erythrocyte membrane rigidity was measured on single cells by micropipette aspiration. The slope of the suction pressure-length data for entry into the pipette provided the measure of the membrane extensional modulus. Data were collected for cells equilibrated with WGA solutions in the range of concentrations of 0.01-10 micrograms/ml. Erythrocyte-erythrocyte adherence properties were studied by micropipette separation of two-cell aggregates. First, a "test" cell was selected from a WGA solution by aspiration into a small micropipette, then transferred to a separate chamber that contained erythrocytes in WGA-free buffer. Here, a second cell was aspirated with another pipette and maneuvered into close proximity of the test cell surface, and adhesive contact was produced. The flaccid cell was separated from the test cell surface in steps at which the force of attachment was derived from the pipette suction pressure and cell geometry. In addition, we measured the time-dependent binding and release of fluorescently labeled WGA to single erythrocytes with a laser microfluorometry system. The results showed that the stiffening of the erythrocyte membrane and binding of fluorescently labeled WGA to the membrane surface followed the same concentration and time dependencies. The threshold concentration for membrane stiffening was at approximately 0.1 microgram/ml where the time course to reach equilibrium was close to 1 h. The maximal stiffening (almost 30-fold over the normal membrane elastic modulus) occurred in concentrations greater than 2 micrograms/ml where the time to reach equilibrium took less than 1 min. The WGA binding also altered the normal elastic membrane behavior into an inelastic, plastic-like response which indicated that mechanical extension of the membrane caused an increase in cross-linking within the surface plane. Similar to the stiffening effect, we observed that the membrane adhesivity of cells equilibrated with WGA solutions greatly increased with concentration greater than 0.1 microgram/ml.(ABSTRACT TRUNCATED AT 400 WORDS)