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)     

The mechanism of wheat germ agglutinin mediated cytolysis of murine mastocytoma cells

The present study was undertaken to test whether cytolysis by wheat germ agglutinin requires lateral mobility of membranal lectin receptor sites into caps.Preincubation of interphase murine mastocytoma cells with 100 μg/ml trypsin promoted cap formation by the agglutinin in about 30% of the cells, followed by cytolysis of these cells. Pretreatment of the cells with NaN₃, low temperature or glutaraldehyde decreased degree of capping and to some extent decreased the degree of cytolusis, while the addition of antibodies to the agglutinin increased the degree of capping and lysis. A linear relationship with a high correlation coefficient exists between the degree of capping and the degree of cytolysis, suggesting that lateral mobility of membrane wheat germ agglutinin receptors is required for cytolysis by the lectin. Further studies have shown the restricted small hole damage followed by osmotic lysis is responsible for the damage induced by the agglutinin of trypsin-treated mastocytoma cells. This was demonstrated (a) by markers of low molecular weight (⁸⁶Rb) which were released from the cells before those of high molecular weight (⁵¹Cr-protein) and (b) by protecting the cells from lysis through incubating them in non-penetrating solutes, such as Dextrans of high molecular weight. It has been calculated the the initial size of the lytic lesion induced by wheat germ agglutinin is $\text{≈ 32}\text{A}\text{?}$.     

Effects of wheat germ agglutinin on membrane transport

(1) Low concentrations of wheat germ agglutinin are cytotoxic toward several tissue culture lines, including Chinese hamster ovary cells, Swiss 3T3 cells, mouse L cells and baby hamster kidney cells. The LD50 ranged from 1 to 5 microgram wheat germ agglutinin per ml. Similar concentrations of the lectin inhibited the transport of the non-utilizable amino acids alpha-aminoisobutyric acid and cycloleucine and inhibited the uptake of thymidine. In contrast, 2-deoxy-D-glucose uptake was not altered and colchicine uptake was enhanced. (2) The inhibition of alpha-aminoisobutyric acid uptake occurred within minutes after lectin addition and was maximal by 1 h. Maximal inhibition ranged from 50 to 70% of control values. Studies of the kinetics of the uptake demonstrated that wheat germ agglutinin decreased the V of the uptake by 70% without affecting the apparent Km. Ovomucoid, a haptene inhibitor of wheat germ agglutinin-binding to cell surface receptors, prevented the wheat germ agglutinin-induced inhibition of alpha-aminoisobutyric acid transport. Three other lectins (Concanavalin A, Phaseolus vulgaris E-phytohemagglutinin and L-phytohemagglutinin) inhibited the uptake by 20% or less at doses up to 50 microgram/ml. (3) We propose that the cytotoxicity of wheat germ agglutinin probably results in part, if not totally, from membrane alterations which impair multiple membrane transport systems.     

Effect of wheat germ agglutinin on the viscoelastic properties of erythrocyte membrane

The elasticity and viscosity of the human erythrocyte membrane were measured as a function of the concentration of wheat germ agglutinin (WGA) in a suspending solution containing 1 mg/ml albumin, approximately 5 X 10(5) cells/ml and between 0.0 and 0.2 microgram/ml WGA. Membrane elasticity was characterized by the elastic shear modulus, which provided a measure of the resistance of the membrane to constant-area elastic deformations that occurred in the membrane plane. The elastic shear modulus was determined by aspirating a portion of the membrane into a micropipette and measuring the extension of the membrane into the pipette as a function of the suction pressure. The results indicated no significant change in shear modulus for concentrations of WGA between 0.0 and 0.2 microgram/ml. Membrane viscosity was characterized by the coefficient of surface viscosity, which, in effect, was a measure of the membrane's resistance to rates of deformation. This coefficient was determined from the time required for an erythrocyte to recover its undeformed shape after it had been elongated by the application of an equal and opposite force applied at diametrically opposite points on the erythrocyte rim. The value for the coefficient of surface viscosity was found to increase by a factor of almost three when the WGA concentration was increased from 0.0 to 0.2 microgram/ml. These results indicated that, in the presence of albumin, WGA can increase membrane dissipation (viscosity) without altering the structural rigidity (elasticity) of the membrane.     

Inhibition of fibronectin receptor function by antibodies against baby hamster kidney cell wheat germ agglutinin receptors

Previous studies suggest that the baby hamster kidney (BHK) cell fibronectin receptor is also a wheat germ agglutinin receptor (WGA-R). To analyze this possibility further, IgG and Fab fragments of antibodies produced against a BHK cell WGA-R preparation were tested to determine their effects on cell adhesion mediated by fibronectin, wheat germ agglutinin, concanavalin A, and polycationic ferritin. The WGA-R preparation was isolated by octylglucoside extraction of BHK cells followed by chromatography of the extract on WGA-agarose. The antibodies against the WGA-R preparation reacted primarily with polypeptides of molecular weights 48, 61, 83, 105, 120, 165, 210, and 230 kilodaltons (kdaltons). It was concluded that the antibodies interfered with BHK cell fibronectin receptors on the basis of the ability of anti-WGA-R IgG or Fab fragments to (a) inhibit cell spreading on fibronectin-coated substrata; (b) cause rounding and detachment of cells previously spread on fibronectin-coated substrata; and (c) inhibit binding of fibronectin-coated latex beads to the cells. Antibody activity was blocked by treatment of anti-WGA-R with the WGA-R preparation or by absorption of anti-WGA-R with intact BHK cells. The antibodies also appeared to prevent coupling of ligand-receptor complexes (involving concanavalin A or polycationic ferritin) with the cytoskeleton. Finally, cell rounding and detachment caused by the antibodies were found to require metabolic energy since it did not occur in the presence of azide or at 4 degrees C.     

Endogenous abscisic acid and wheat germ agglutinin content in wheat grains during development

Abscisic acid (ABA) and wheat germ agglutinin content of immature wheat grains and embryos was determined by immunoassay throughout the development of a field-grown wheat crop ( Triticum aestivum cv. Timmo). Wheat germ agglutinin accumulation in the embryo was not preceded by an increase in endogenous abscisic acid amount or concentration in either embryos or grains. At a later stage in development the endogenous concentration of abscisic acid in both embryos and grains was found to be two orders of magnitude lower than the endogenous levels required to inhibit precocious germination and promote wheat germ agglutinin accumulation in excised embryos cultured in vitro. These findings are discussed in the context of the control of embryo development in vivo by both ABA and the water status of the grain and embryo.     

Folding and homodimerization of wheat germ agglutinin

Wheat germ agglutinin (WGA) is emblematic of proteins that specialize in the recognition of carbohydrates. It was the first lectin reported to have a capacity for discriminating between normal and malignant cells. Since then, it has become a preferred model for basic research and is frequently considered in the development of biomedical and biotechnological applications. However, the molecular basis for the structural stability of this homodimeric lectin remains largely unknown, a situation that limits the rational manipulation and modification of its function. In this work we performed a thermodynamic characterization of WGA folding and self-association processes as a function of pH and temperature by using differential scanning and isothermal dilution calorimetry. WGA is monomeric at pH 2, and one of its four hevein-like domains is unfolded at room temperature. Under such conditions, the agglutinin exhibits a fully reversible thermal unfolding that consists of three two-state transitions. At higher pH values, the protein forms weak, nonobligate dimers. This behavior contrasts with that observed for the other plant lectins studied thus far, which form strong, obligate oligomers, indicating a distinctly different molecular basis for WGA function. For dimer formation, the four domains must be properly folded. Nevertheless, depending on the solution conditions, self-association may be coupled with folding of the labile domain. Therefore, dimerization may proceed as a rigid-body-like association or a folding-by-binding event. This hybrid behavior is not seen in other plant lectins. The emerging molecular picture for the WGA assembly highlights the need for a reexamination of existing ligand-binding data in the literature.     

Evolution of the multidomain protein wheat germ agglutinin

We compared the homologous amino acid sequences of hevein and each of the four domains (A, B, C, and D) of wheat germ agglutinin and used them to construct a pseudophylogenetic tree relating these sequences to a hypothetical common ancestor sequence. In the crystal structure of the wheat germ agglutinin dimer, six pseudo-two-fold rotational symmetry axes have previously been located in addition to the true twofold axis. Four of these relate two nonidentical domains to each other in each of the four possible pairs constituting the sugar-binding sites (A1D2, A2D1, B1C2, and B2C1). The remaining two relate contiguous unique pairs of sugar-binding sites to each other (A1D2 to B1C2, and A2D1 to B2C1). These latter two sets of pairs are related to each other by the true twofold axis. Side chains that mediate sugar binding in the interfaces of each of the four pairs were found to be largely conserved. The sequence homology, taken together with these pseudo-symmetry elements in the dimer structure, suggests a pathway for the evolution of the four-domain molecule from a single-domain dimer that can be correlated with simultaneous development of the saccharide-binding sites.     

Evolution of the multidomain protein wheat germ agglutinin

Summary We compared the homologous amino acid sequences of hevein and each of the four domains (A, B, C, and D) of wheat germ agglutinin and used them to construct a pseudophylogenetic tree relating these sequences to a hypothetical common ancestor sequence. In the crystal structure of the wheat germ agglutinin dimer, six pseudo-twofold rotational symmetry axes have previously been located in addition to the true twofold axis. Four of these relate two nonidentical domains to each other in each of the four possible pairs constituting the sugar-binding sites (A₁D₂, A₂D₁, B₁C₂, and B₂C₁). The remaining two relate contiguous unique pairs of sugar-binding sites to each other (A₁D₂ to B₁C₂, and A₂D₁ to B₂C₁). These latter two sets of pairs are related to each other by the true twofold axis. Side chains that mediate sugar binding in the interfaces of each of the four pairs were found to be largely conserved. The sequence homology, taken together with these pseudo-symmetry elements in the dimer structure, suggests a pathway for the evolution of the four-domain molecule from a single-domain dimer that can be correlated with simultaneous development of the saccharide-binding sites.     

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.     

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.     

Structural and functional changes associated with cyanogen bromide treatment of wheat germ agglutinin

The lectin, wheat germ agglutinin (WGA), has been shown to have a significant larvicidal effect on the European corn borer, a major insect pest of corn. In order to characterize this toxic effect, we have undertaken structure-function studies on WGA. To this extent, the effect of cyanogen bromide (CNBr) on the conformation, subunit interactions, and biological activity of WGA has been investigated. The CNBr-modified lectin exhibits no toxicity to the ECB, cannot dimerize, does not bind to N-acetylglucosamine or its polymers, has no or vastly reduced hemagglutinating activity against red blood cells of different animals, and shows loss of an antigenic determinant by immunodiffusion. The CD spectrum of CNBr-WGA is not significantly different from that of native WGA, although the intrinsic fluorescence shows about 30% quenching. Our results suggest that the integrity of the N-terminal domain of WGA is essential for dimer formation. Furthermore, toxicity of WGA to ECB may be intrinsically related to its ability to dimerize and bind to sugar residues.     

Differential involvement of cell surface sialic acid residues in wheat germ agglutinin binding to parental and wheat germ agglutinin-resistant Chinese hamster ovary cells

Two Chinese hamster ovary (CHO) cell mutants selected for resistance to wheat germ agglutinin (WGA) have been shown to exhibit defective sialylation of membrane glycoproteins and a membrane glycolipid, GM3. The mutants (termed WgaRII and WgaRIII) have been previously shown to belong to different genetic complementation groups and to exhibit different WGA-binding abilities. These mutants and a WGA-resistant CHO cell mutant termed WgaRI (which also possesses a surface sialylation defect arising from a deficient N-acetylglucosaminyltransferase activity), have enabled us to investigate the role of sialic acid in WGA binding at the cell surface. Scatchard plots of the binding of 125I- WGA (1 ng/ml to 1 mg/ml) to parental and WgaR CHO cells before and after a brief treatment with neuraminidase provide evidence for several different groups of sialic acid residues at the CHO cell surface which may be distinquished by their differential involvement in WGA binding to CHO cells.     

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.     

Isolation and characterization of agglutinin receptor sites: II. Isolation and partial purification of a surface membrane receptor for wheat germ agglutinin

Four different chemical extraction procedures for the isolation of wheat germ agglutinin receptor sites from L1210 cells are described. Fractionation of the biologically active material on Sephadex G-200 columns in pyridine results in two major peaks, the lower molecular weight fraction having a higher inhibitory activity. Electrophoresis in polyacrylamide sodium dodecyl sulfate gels yields four bands. The most active fraction from Sephadex G-200 has an approximate molecular weight between 40000–60000. A preliminary analysis of the active material indicates the presence of sialic acid, neutral sugars and amino sugars, including N-acetylglucosamine.     

Lepidopteran peritrophic membranes and effects of dietary wheat germ agglutinin on their formation and structure

Peritrophic membrane (PM) structure and the effects of dietary wheat germ agglutinin (WGA) on PM formation were studied in larvae of the European corn borer (ECB), Ostrinia nubilalis, and the tobacco hornworm (THW), Manduca sexta. Growth of ECB was strongly inhibited by low amounts of WGA in the diet (0.05%), whereas THW was not affected by amounts of up to 2%. In ECB larvae, chitin microfibrils were secreted to form an orthogonal network within the apical region of the anterior midgut microvilli. The network then moved to the tips of the microvilli where proteinacious matrix was added prior to delamination of a single PM into the lumen to enclose the food bolus. Multiple PMs rapidly appeared as the food moved posteriorly and some of these became greatly thickened in the middle and posterior regions of the midgut. WGA in the diet caused hypersecretion of unorganized PM in the anterior midgut lumen, disintegration of microvilli, and cessation of feeding. It was also shown to bind to both the chitinous network and to several PM proteins, perhaps causing voids in the PM and sparse matrix material. This allowed the passage of food particles through a defective PM into the ectoperitrophic space and penetration into the microvillar brush border. Stimulation of PM secretion and cessation of feeding may have been a response to damage to the brush border. Unlike ECB, the chitinous network of THW is a randomly organized felt-like structure embedded in a proteinaceous matrix. This PM is secreted as a thin multilayered structure in the anterior region of the midgut, but multiple and thickened PMs occur in the middle and posterior lumens of the midgut. THW tolerated high amounts of WGA in its diet with no disruption of PM formation or inhibition of growth. WGA did accumulate as large masses embedded in the PM, but caused no voids that would allow the penetration of food particles and subsequent damage to the brush border. Therefore, differences in PM formation and structure between ECB and THW appeared to affect how WGA interacts with chitinous and proteinaceous components of the PM and subsequent effects on larval feeding and growth.     

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.