Location of the N-acetyl-d-neuraminic acid binding site in wheat germ agglutinin: A crystallographic study at 2.8 Å resolution

The crystal structure of the non-covalent complex between wheat germ agglutinin (isolectin no. 2) and N-acetyl-d-neuraminic acid, a saccharide widely found at the termini of carbohydrate chains in membrane glycoproteins and known to interact with wheat germ agglutinin, has been determined from an electron density difference map at 2.8 Å resolution. This map exhibits two strong binding sites on the wheat germ agglutinin dimer molecule which are located in corresponding crevices at the protomer/protomer interface. Amino acid sidechains from B and C-type domains of opposite protomers contribute to the binding site. The N-acetylneuraminic acid molecule is oriented such that its acetyl group becomes essentially buried upon binding, whereas the charged carboxylate and the glycerol groups point away from the protein surface, but are also able to make contact with surface side-chains. Model building shows that substituents of the pyranoside ring which had been predicted as essential for binding from solution studies, are situated favorably to allow interactions to be made with main and side-chain atoms of the protein molecule.     

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.     

Subunit structure of wheat germ agglutinin

Cells isolated by enzymic digestion of embryonic tendon were incubated under N₂ so that they synthesized and accumulated the unhydroxylated form of procollagen which is known as protocollagen and which is largely comprised of pro-α chains linked by interchain disulfide bonds. The cells were then exposed to O₂ so that the intracellular protocollagen was hydroxylated and secreted as procollagen. When the hydroxylation was allowed to proceed at 31° or 34°, the procollagen secreted into the medium was triple-helical but its hydroxyproline content was less than two-thirds and its hydroxylysine content was less than half the control. Even when the hydroxylation was allowed to occur at 37°, the procollagen secreted by the cells was under-hydroxylated by about 15% in terms of its hydroxyproline content and about 45% in terms of its hydroxylysine content. The results may have consequences for collagen synthesis by tendons and similar tissues $\text{in vivo}$, since temporary anoxia in such tissues may well lead to the synthesis of a less stable procollagen or to fibers of decreased tensile strength.     

Distribution of wheat germ agglutinin in young wheat plants

A liquid phase, competition-binding radioimmunoassay for wheat germ agglutinin, with a detection limit of 10 nanograms, was developed in order to determine the distribution of this lectin in young wheat plants. Affinity columns for wheat germ agglutinin removed all antigenically detectable activity from crude extracts of wheat tissue; thus, the antigenic cross-reactivity detected by the assay possesses sugar-binding specificity similar to the wheat germ-derived lectin. The amount of lectin per dry grain is approximately 1 microgram, all associated with the embryo. At 34 days of growth, the level of lectin per plant was reduced by about 50%, with approximately one-third in the roots and two-thirds in the shoot. The data also indicate that actively growing regions of the plant (the bases of the leaves and rapidly growing adventitious roots) contain the highest levels of lectin. Half of the lectin associated with the roots could be solubilized by washing intact roots in buffer containing oligomers of N-acetylglucosamine, whereas the remainder is liberated only upon homogenization of the tissue.     

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.     

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.     

Incorporation of acylated wheat germ agglutinin into liposomes

Purified wheat germ agglutinin (WGA) was derivatized with palmitic acid at an average stoichiometry of one fatty acid per dinner. Palmitoyl WGA was readily incorporated into liposomes with a cholate-dialysis method. Liposome-bound WGA caused agglutination of red blood cells at a concentration eight-fold lower than that of the native lectin. Furthermore, enhanced binding of liposome-bound WGA to mouse spleen cells was also observed. Potential applications of the liposome-bound lectin are discussed.     

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.     

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.     

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.     

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.     

Microcalorimetric study of wheat germ agglutinin binding to N-acetylglucosamine and its oligomers.

The energetics of association of wheat germ agglutinin (WGA) with N-acetylglucosamine (GlcNAc) and its beta(1,4) oligomers have been measured using isothermal titration calorimetry. Association constants of 0.4, 5.3, 11.1, 12.3, and 19.1 mM-1 and enthalpies of binding of -6.1, -15.6, -19.4, -19.3, and -18.2 kcal mol-1 were obtained at 26 degrees C for the titration of WGA with GlcNAc, (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, and (GlcNAc)5, respectively. The term T delta S was always of negative value, indicating that the binding process is enthalpically driven. Titrations of WGA performed at pH 4.5 did not differ significantly from those performed at pH 7.0, suggesting that no groups with a pKa in this range are directly involved in the binding event. Also, performing the titration in a buffer system with a higher enthalpy of protonation did not change the enthalpy of binding confirming that there is no net protonation or deprotonation when WGA binds GlcNAc residues at pH 7. A model of four independent binding sites was found to adequately describe the binding curves, except in the case of (GlcNAc)4 which exhibited positive cooperativity. The energetic values are discussed within the context of the structure of the WGA-(GlcNAc)2 complex.     

Drosophila cell fusion induced by wheat germ agglutinin.

The effects of wheat germ agglutinin on Drosophila embryonic cell lines growing on cover-glasses was examined by scanning electron microscopy. At low concentrations of the lectin (5-10 mug/ml), cells spread against the glass surface and fused to form syncytia. At high concentration, damage to the cell surface was evidenced as extensive membrane shrivelling and loss of surface microfilaments. Fusion also occurred under these conditions. There was some indication that the morphology of cells in division remains undisturbed by wheat germ agglutinin. The coalescence of cells and morphologic disotrtion induced by wheat germ agglutinin were not inhibited by N-acetylglucosamine, the hapten inhibitor of the lectin, under the conditions utilized in this study.     

The interaction between wheat germ agglutinin and membrane incorporated glycophorin A. An optical binding study

A novel integrated optical technique is used to monitor the kinetics of incorporation of glycophorin A (GPA) from solution into a planar dimyristoylphosphatidylcholine-cholesterol bilayer membrane, and the subsequent binding of wheat germ agglutinin (WGA) to the membrane-incorporated GPA. The technique significantly improves the attainable accuracy of kinetic measurements. The number of bound molecules can be determined to a precision of ca ± 80 mol µm^–2. Our results show that GPA incorporates spontaneously into the bilayer. Binding of WGA to GPA is optimal in the presence of human serum albumin, and can be reversed byN-acetyl-d-glucosamine. The kinetics of the binding are consistent with the presence of two classes of kinetically distinguishable binding sites with association rates of 2.0×10⁴ and 9.6×10² M^–1 s^–1, and dissociation rates of 2.7×10^–3 s^–1 and <10^–5 s^–1, respectively. A stoichiometry of 4 WGA monomers per GPA monomer was determined as characteristic of the overall binding interaction.Abbreviations DMPC dimyristoylphosphatidylcholine - GlcNAc N-acetyl-d-glucosamine - GPA glycophorin A - HSA human serum albumin - NeuNAc N-acetyl-d-neuraminic acid - TE transverse electric - TM transverse magnetic - WGA wheat germ agglutinin     

Fluorescence analysis of hormone binding activities of wheat germ agglutinin

Wheat germ agglutinin (WGA) from embryos of the monocotyledonous plant Triticum vulgaris (Graminaceae) is a carbohydrate binding protein characterized by high specificity to N-acetyl-d-glucosamine and N-acetyl-d-neuraminic acid. In this study we show that parallel to its carbohydrate binding activities, WGA binds with several orders of magnitude higher affinity adenine, adenine-related cytokinins: kinetin, zeatin and isopentenyl-adenine as well as abscisic and gibberellic acids (K(d) 0.43-0.65 microM). Its interactions with these ligands cause conformational rearrangements in the protein molecules and significant enhancement of the protein tryptophan fluorescence (up to 60%) allowing characterization of the protein-hormone complexes. Dimeric WGA molecules possess two different classes of binding sites for the fluorescent hydrophobic probe 2-(p-toluidinyl) naphthalene sulfonic acid (TNS) as suggested by the sigmoid shape of the fluorescence titration curve and the value of the Hill coefficient (n(H) 1.6+/-0.3). The plant hormones displace part of the bound TNS probe and share the higher affinity TNS binding sites. These results characterize WGA as a hormone-binding protein.     

Non-crystallographic symmetry in the crystal dimer of wheat germ agglutinin

Three isomorphous heavy atom derivatives of wheat germ agglutinin crystals, KAu(CN)₂, K₂Pt(NH₃)₂(NO)₂ and mersalyl, have been examined at high resolution. Heavy atom sites were located from difference Patterson maps in three dimensions at 2.15 Å resolution for the gold and platinum derivatives and with less certainty in the centrosymmetric [010] projection for the mersalyl derivative. These sites are distributed in the crystallographic asymmetric unit such that one half of them can be related to the other half by a 180 ° rotation about an axis parallel to a, and an additional translation of about 6.35 Å along that axis. It is suggested that the two subunits of the wheat germ agglutinin dimer, which represent the asymmetric unit of the C2 unit cell, are related by the same symmetry axis, causing heterologous subunit contacts due to the 6.35 Å translation of one relative to the other subunit.     

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.     

Purification of wheat germ agglutinin using affinity chromatography on chitin

Chitin, the naturally occurring polymer of N-acetyl-glucosamine, has been used as a ligand matrix for affinity chromatography of wheat germ agglutinin in a new purification method which is well suited for large scale preparations. The agglutinin obtained is homogeneous with respect to polypeptide chain molecular weight, has a blocked amino terminus and is free of proteolytic and β-N-acetyl-glucosaminidase activities.