Saccharide Binding Research Articles

Histidine determination in wheat germ agglutinin isolectin by X-ray diffraction analysis

Electron density maps based on 2·4 Å and 2·2 Å X-ray diffraction data for crystals of two isolectins of wheat germ agglutinin (designated isolectins 1 and 2) were compared in terms of side-chain identities. While the primary structure of wheat germ agglutinin is not available, a partial amino acid sequence for isolectin 2 has been deduced by inspection of the electron density map and through model building. The positions of the two histidines predicted from amino acid composition studies to be present in isolectin 2 but not in isolectin 1, were located by difference Fourier techniques and analysis of the heavy-atom binding properties of these two isolectins. Both histidines were found to reside in the B-domain of the multi-domain wheat germ agglutinin protomer (A, B, C, D). Histidine 57 lies in the contact region between the two subunits near the molecular dimer axis. The side-chain of histidine 64 forms part of the primary saccharide binding site at the interface where B and C-domains of opposite protomers make contact. In addition, this histidine serves as a major target for heavy-atom binding by platinum and mercury compounds.

Lectin immuno tests: Quantitation and titration of antigens and antibodies using lectin-antibody conjugates

We have investigated the possibility of using lectin-antibody conjugates as general reagents in immunological procedures requiring a labeled antigen or antibody. Using these conjugates, labeling is achieved through saccharide binding sites of lectins which operate as acceptors for glycoconjugate marker substances added secondarily. Marker substances used in this work were enzymes, radioactively labeled glycoconjugates and erythrocytes, but other markers can also be used. Using the first two markers, antigens and antibodies were determined with accuracy and sensitivity equal to those of conventional enzyme or radioimmunoassays. Using erythrocytes as a marker, a simple erythro-adsorption procedure, possibly followed by hemolysis, has been developed which allowed the titration of antigens and antibodies to be carried out with a sensitivity at least equal to enzyme or radioimmunoassays.

Location of Mn 2+ in concanavalin A containing only a Mn 2+ ion

The metal-sugar distances in two metallized forms of concanavalin A have been compared by 19F magnetic resonance techniques. Using relaxation times measured at two different frequencies we have shown that the distance between the Mn 2+ ion and the bound sugar in concanavalin A containing only Mn 2+ is essentially identical to that found in concanavalin A containing both Mn 2+ and Ca 2+. Our results rule out the possibility that Mn 2+ activates concanavalin A by binding at the Ca 2+ site (S2) and would suggest that Mn 2+ alone can induce an active saccharide binding conformation by binding at the transition metal site (S1).

Crystallographic elucidation of the saccharide binding mode in wheat germ agglutinin and its biological significance

The specific saccharide binding sites in wheat germ agglutinin have been characterized by difference Fourier analysis of a number of WGA † † Abbreviations used: WGA, wheat germ agglutinin; WGA2, isolectin 2 of wheat germ agglutinin; GLA, glutaraldehyde; ConA, concanavalin A; GlcNAc, N-acetyl- d-glucosamine; NeuNAc, N-acetyl- d-neuraminic acid; INAG, 6-iodo-1,4-dimethyl- N-acety1- d-glucosaminc; MUC, O-4-methylumbelliferyl-di- N-acetyl-β-chitobioside; NeuLac, N-acetyl- d-neuraminic acid lactose (neuramin lactose); NeuLacNAc, N-acetyl- d-neuraminic acid lactosamine; n.m.r. nuclear magnetic resonance; cd., circular dichroism; CHO, Chinese hamster ovary; Bkg, background; F CMP, structure factors for WGA: sugar complexes; F PAR, structure factors for either native or glutaraldehyde-treated WGA crystals; WGM, wheat germ mitogen; MIR, multiple isomorphous replacement. -sugar complexes. All four domains (A,B,C,D) of each WGA protomer (I and II) were found to contribute to sugar binding at four locations in the protomer/protomer contact region: B I C II , B II C I , I D II , A II D I . The two equivalent binding sites involving the B and C domains have been shown to bind N-acetylneuraminic acid, a common cell surface sialic acid (Wright, 1980 a). These two sites, termed the primary binding location, are readily accessible in the crystal to all three saccharides investigated: di- N-acetylglucosamine (2.8 Å data), 6-iodo-1, 4-dimethyl- N-acetylglucosamine (3 Å data) and N-acetylneuraminic acid lactose (4 Å data). The other two sites involving A and D domains are designated as the secondary binding locations, since they are only poorly occupied by di- N-acetylglucosamine in glutaraldehyde-crosslinked crystals and not at all by the sialic acid sugar. The early observation that native WGA crystals disintegrate in the presence of N-acetylglucosamine, is attributed to interference of sugar binding at this secondary site with molecular lattice interactions. Despite differences in their accessibility in the crystal and specificity toward the two types of acetylated sugars, the two unique binding locations exhibit very similar saccharide binding modes. The main contribution to binding of oligosaccharides comes from interactions of the acetamido and one hydroxyl group of the non-reducing terminal sugars with the protein at subsite 1, whereas little or no contribution is provided by subsites 2 and 3. Moreover, oligosaccharides binding at the primary binding location which possess terminal sialic acid, assume a distinctly different orientation from that of N-acetylglucosamine oligomers, although they share subsite 1. This is due to a difference in position of their glycosidic hydroxyl groups. Space limitations in crosslinked crystals prevent oligomers of N-acetylglucosamine larger than the dimer from binding either at the primary or secondary binding locations. These results suggest that differing accessibility and specificity determinants for the two unique binding sites in WGA, as well as protein self-association as seen in the crystal lattice, could explain (1) the co-operative binding behavior of WGA binding to various cells, and (2) the rapid release of cell-bound WGA when N-acetylglucosamine is introduced.

Conformational changes following Mn(II) binding to demetalized concanavalin A

A temperature-dependent conformational change occurs following the binding of only one Mn(II) to a concanavalin A monomer. This change is independent of Ca(II) near pH 7 and is characterized by an activation energy of 22.3 kcal mol −1, a value similar to that attributed to a cis-trans peptide isomerization. Two conformations have been detected in magnetic resonance experiments on solvent water protons where spin lattice relaxation times are influenced by bound Mn(II). Both conformations possess saccharide binding activity and Ca(II) stoichiometrically enhances the rate of conversion to the final, more stable conformation.

Internalization and intracellular fate of Wistaria floribunda agglutinin in BALB/c 3T3 fibroblasts

The accumulation of large lysosomes in BALB/c 3T3 fibroblasts following exposure of the cells to Wistaria floribunda agglutinin (WFA) was accompanied by adsorptive endocytosis of the lectin. The internalization of WFA was examined by a double label immunofluorescence technique that could distinguish internal WFA from cell surface WFA in the same cell. The lectin bound to the plasmalemma of the cell via its saccharide binding site resulting in the aggregation into a surface cap of some lectin-receptor complexes. Lectin was internalized by a temperature-sensitive process. The lectin accumulated within the cell over a 24 h period as shown by an increase with time of the [ 125I]WFA that could not be washed from the cells by the competitive saccharide inhibitor, lactose. The vesicles containing WFA combined with lysosomes as evidenced by (1) the localization of ferritin-WFA conjugates in vesicles having the size and morphology of lysosomes; and (2) the degradation of radiolabeled lectin to an ethanol-soluble form. Lectin treatment had no significant effect on the overall degradation of intracellular proteins in growing or serum-starved cells. It is suggested that WFA either enhances the rate of fusion of lysosomes with other lysosomes or lectin-containing endocytic vesicles or inhibits the process by which lysosomes turn over.

Fluorescence polarization studies of saccharide binding to Wheat germ agglutinin and lysozyme

N-acetyl-D-glucosamine causes only slight increases in the polarization of Wheat germ agglutinin fluorescence at saturating levels whereas the disaccharide and trisaccharide produce increases in the polarization value from 0.116 to 0.151 and 0.154 respectively. These increases suggest that rotational motions of the tryptophan residue at the binding sites are being restricted by an interaction between these tryptophans and the bound sugars. A model of the nature and location of these interactions is discussed. Comparable results are obtained with lysozyme, which shows a larger effect upon binding of N-acetyl-D-glucosamine, but a maximal increase in polarization upon binding the corresponding disaccharide or trisaccharide.

Activation of saccharide binding in demetalized concanavalin A by transition metal ions.

Activation of saccharide binding in demetalized concanavalin A by transition metal ions.

Cell surface receptors for lymphokines: II. Studies on the carbohydrate composition of the MIF receptor on macrophages using synthetic saccharides and plant lectins

The carbohydrate composition of the surface receptor for macrophage migration inhibitory factor (MIF) on guinea pig macrophages has been studied by examining the interaction of MIF with different saccharides and by testing the ability of plant lectins with known saccharide binding affinities to bind to macrophages and block their response to MIF. Comparison of the effectiveness of a variety of natural and synthetic mono- and disaccharides in inhibiting MIF activity in lymphocyte supernatants revealed that inhibitory activity was confined to natural 5-methylpentose sugars ( l-fucose > l-rhamnose = 6-deoxy- d-glucose) and synthetic saccharides containing α-fucosyl residues. Observations on the MIF inhibitory activity of synthetic fucosyl glycosides containing fucosyl residues of defined configuration at terminal and subterminal positions indicate that MIF interacts preferentially with terminal α- l-fucopyranosyl residues and does not recognize subterminal saccharides. Studies with disaccharides containing α-(1 → 2)-, α-(1 → 3), and α-(1 → 6)-linked l-fucosyl residues failed to reveal preferential interaction of MIF with any one linkage configuration. Incubation of macrophages before exposure to MIF with lectins that bind to terminal fucosyl residues ( Lotus tetragonolobus and Ulex europaeus I, agglutinins) rendered them unresponsive to MIF but lectins which bind to nonterminal fucosyl residues and to other saccharides had no effect. The role of fucosyl residues in the binding of MIF by macrophages is discussed with reference to the possible composition of the MIF receptor and the role of fucose-containing glycolipids as receptors for this lymphokine.

Metal ion induced conformational changes in concanavalin A: Evidence for saccharide binding to one metal free structure

The addition of Mn 2+, Zn 2+, Co 2+, Ca 2+ or Pb 2+ to apo-concanavalin A results in a slow conformational conversion of the protein to the active saccharide binding form. The rates of conversion are dependent upon the sample pH and identity of the ions which occupy the native transition metal and calcium ion sites yet the affinity of each metalloform for the fluorescent sugar, 4-methylumbelliferyl-α-D-mannopyranoside, is independent of these same parameters (above pH 5.6). EDTA quickly removes all metal ions from the active Mn 2+ or Co 2+-concanavalin A samples leaving a metastable metal free structure which retains its high saccharide affinity for several hours at room temperature. This form of apo-concanavalin A and the metallized derivatives have equally high saccharide binding affinities in 1M NaCL but the former dramatically loses its sugar affinity as the ionic strength is lowered.

Evidence for the glycoprotein nature of radish β-fructosidase

Concanavalin A (Con A) was utilized free, bound to Sepharose 4 B or cross-linked to glutaraldehyde to investigate the possibility of binding this lectin to radish beta-fructosidase (E.C.3.2.1.26). The choice of cross-linked Con A as affinoadsorbent is discussed and standard conditions for binding are defined. Specificity of precipitation of this enzyme by the lectin was especially investigated. Thus, the possibility of binding was tested in the presence of high ionic strength, ethylene glycol, alpha-methyl mannoside, alpha-methyl glucoside and during periodate oxidation of the enzyme. Based on the interactions observed between beta-fructosidase and Con A under these conditions it is concluded that the saccharide binding site of the lectin is primarily involved with a secondary contribution from the hydrophobic site. The specificity of binding and the complete precipitation of beta-fructosidase activity by the insolubilized lectin imply that all beta-fructosidase activity measured in Raphanus sativus seedling extracts is linked to (a) glycoprotein form(s) of this enzyme.

The effect of binding of metal ions on the three-dimensional structure of demetallized concanavalin A

Concanavalin A (con A) is a saccharide-binding protein of the Jack bean [ 11. The protein contains two different metal-binding sites, which must be occupied in order for saccharide binding to be possible [2,3]. When the metal ions are removed by acid treatment, the demetallized protein can bind only divalent transition metal ions (Mn”, Co”, Ni2+, Zn”, or Cd23 at a site designated S 1, but it cannot bind Ca2+ or saccharide. When Sl is occupied by one of the metal ions listed above, the protein can bind Ca” at a second metalbinding site designated S2 [4]. We have made an X-ray crystallographic determination of the three-dimensional structure of demetallized concanavalin A and we report here the important structural differences in the metal-binding region between the demetallized protein and the native metalloprotein [5-71 (fig.1). We postulate a detailed mechanism whereby binding of the transition metal ion at Sl engenders the Ca2+-binding site (S2). The experimental details, refinement procedure and electron density maps upon which our structure is based will be reported fully [8].

Saccharide binding to transition metal ion free concanavalin A.

Saccharide binding has been observed with demetallized concanavalin A in the presence of Ca(2+) only, using the fluorescent sugar 4-methylumbelliferyl alpha-D-mannopyranoside. At pH 7.2 both the nicked and intact forms of concanavalin A bound 4-methylumbelliferyl alpha-D-mannopyranoside with similar affinities. Competitive binding with methyl alpha-D-mannopyranoside was demonstrated. The association constants at 5 degrees C were 9.6 +/- 0.6 X 10(4) M(-1) for 4-methylumbelliferyl alpha-D-mannopyranoside and 1.1 +/- 0.3 X 10(4) M(-1) for methyl alpha-D-mannopyranoside. 4-Methylumbelliferyl alpha-D-mannopyranoside binding was also observed if demetallized concanavalin A was remetallized with less than stoichiometric amounts of Ca(2+). The association constants with low Ca(2+) concentrations were similar to those determined with saturating Ca(2+). With less than stoichiometric levels of Ca(2+), the number of sugar molecules bound per protein subunit was a reflection of the fraction of activated lectin subunits. These results show that saccharide binding activity of concanavalin A does not require a transition metal ion at pH 7.2; only Ca(2+) is required. At pH values near 5, where most previous studies have been carried out, both a transition metal ion and Ca(2+) are necessary.

Conformation as the determinant of saccharide binding in concanavalin A: Ca2+-concanavalin A complexes.

The existence of two conformational states of concanavalin A (Con A) with different metal ion binding properties has been recently demonstrated (Brown, R. D., Brewer, C. F., & Koenig, S. H. (1977) Biochemistry 16, 3883). Introduction of Mn2+ to the S1 site and Ca2+ to the S2 site of apo-Con A was shown to induce a conformational change in the protein, ascribed to a cis-trans isomerization of a peptide bond in the secondary structure, which results in extremely tight binding of the metal ions. This induced conformation is referred to as "locked" and the initial conformation as "unlocked". The locked ternary complex is identical with the native protein. In the present paper, we report evidence for the formation of a relatively stable, locked, ternary Ca2+-Con A complex that possesses properties similar to those of native Ca2+-Mn2+Con A. The experimental technique involves measurement of the magnetic field and time dependence of the nuclear magnetic relaxation rate (1/T1) of solvent water protons in solutions of Ca2+-Con A, after the addition of Mn2+ ion which slowly bind to the protein. The kinetic data can be fit by a model for Ca2+ interactions with Con A which indicates that Ca2+, in the absence of Mn2+, can bind at both the S1 and S2 sites of the protein and, furthermore, can induce the protein to undergo the unlocked to locked conformational transition. In terms of this model, the time-dependent binding of the Mn2+ ions is due to replacement of Ca2+ ions at the S1 sites in the locked protein. The off-rate of Ca2+ from the S2 site of the locked ternary Ca2+-Con A complex is much greater than that from the locked Ca2+-Mn2+-Con A complex. From the effects of added alpha-methyl D-mannopyranoside on the rate of replacement of Ca2+ by Mn2+ at the S1 site of the locked ternary Ca2+-Con A complex, it is concluded that the latter complex binds saccharides as strongly as the locked Ca2+-Mn2+-Con A complex. In addition, analysis of the data indicates that apo-Con A in the locked conformation binds alpha -methyl D-mannopyranoside with approximately 7% of the affinity of the fully metallized locked form of the protein. This strong saccharide-binding activity of locked apo-Con A, compared with that of the unlocked apo-Con A, was further demonstrated by equilibration of unlocked apo-Con A with alpha-methyl D-mannopyranoside, which resulted in the formation of the locked apo-Con A-saccharide complex. These results demonstrate that it is the locked conformation of Con A that is primarily responsible for saccharide-binding activity, and that the function of the bound metals is primarily to maintain the protein in the locked conformation.

Binding of galactose and lactose to ricin. Equilibrium studies

The interaction of ricin, one of the two lectins of Ricinus sanguineus, with its specific ligands galactose and lactose ( 4-O-β- d- galactopyranosyl- d- glucopyranose ) has been studied by means of equilibrium dialysis, analytical ultracentrifugation and fluorescence polarization. In the studied concentration range, only one molecule of galactose is bound per molecule of ricin with an association constant, K a = 6900 m −1 at 4°C. Scatchard plots of equilibrium dialysis data show that two molecules of lactose bind to one molecule of ricin, without modification of molecular weight of the lectin. Together with results of microcalorimetric experiments and agglutination of erythrocytes by ricin, equilibrium dialysis data indicate that the lectin contains two distinct saccharide binding sites. Regardless of the existence of extended sites, it is not possible to select between the two models: (a) two independent sites ( K a1 = 35 000 M −1, K a2 = 2800 M −1 at 4°C) or (b) two identical sites with negative cooperativity.

Changes in the three-dimensional structure of concanavalin A upon demetallization

When the Mn(2+) and Ca(2+) ions normally present in concanavalin A are removed, the protein becomes incapable of binding saccharides. To explore the structural differences between the native and demetallized forms and their effects on the saccharide-binding properties of the protein, we have refined and compared the crystal structures of both forms. Refinement, carried out by automated difference Fourier methods, has revealed a number of differences between the two structures as well as minor differences between the two crystallographically independent monomers in the demetallized structure. Significant differences between the holo- and apoproteins are confined to the region where the metals are bound. These differences include a reorganization and disordering of the loop, consisting of residues 7-25, that contains all of the direct metal ligands of the protein. In some molecules, the side chain of arginine-228 appears to move into the metal-binding region, possibly compensating in part for the absence of the metal's positive charge. The cis peptide observed in the native protein at alanine-207 is apparently not present in the demetallized protein. The conformational differences affect many of the residues currently thought to be involved in the specific binding of saccharides.

Transglutaminase activity in human lymphocytes: early activation by phytomitogens.

Transglutaminase activity is present in human peripheral lymphocytes and is enhanced up to 15-fold within 10-30 min after treatment of the cells with concanavalin A. Phytohemagglutinin has a similar effect. The enzyme is not detected when intact cells are assayed; it is detected only in cell lysates. Concanavalin A enhances transglutaminase activity only when it is incubated with intact cells; concanavalin A treatment of cell lysates has no effect, alpha-Methyl-D-mannoside specifically inhibits the enhancement of transglutaminase activity in cells treated with concanavalin A results from the specific interaction of the lectin with its saccharide binding site on the cell surface, rather than by direct interaction with the enzyme itself. The increased activity of transglutaminase in cells treated with concanavalin A, as compared to unstimulated cells, is maintained under assay conditions in which saturating levels of Ca2+ are present. Transglutaminase may be involved in early cellular events leading to lymphocyte blastogenesis.

Binding of 4-methylumbelliferyl alpha-D-mannopyranoside to tetrameric concanavalin A Fluorescence temperature-jump relaxation study.

The kinetics of saccharide binding to the treatment form of concanavalin A have been studies at pH 7.2 with the temperature-jump method. 4-Methylumbelliferyl alpha-D-mannopyranoside was used as a ligand; its fluorescence is totally quenched upon binding. A single relaxation of ligand fluorescence (tau = 20-400 ms) was observed and was investigated at three different temperatures, using kinetic titration and dilution types of experiments. The concentration dependence of the relaxation time and amplitude was consistent with a single-step bimolecular association and independent binding sites. In the temperature range 13-24 degrees C the association and dissociation rate parameters are in the range (6-10) X 10(4) M-1 s-1 and (1.4-3.2)s-1 respectively, corresponding to activation energies for the forward and reverse reactions equal to approx. 13 and 8 kcal/mol (54 and 33 kJ/mol) respectively. Two additional relaxations of protein fluorescence (3 ms and larger than 1 s at 25 degrees C) were unaffected by carbohydrate binding. Tetrameric concanavalin A shows carbohydrate binding parameters that are almost identical to those of native or derivatized dimeric concanavalin A.

Microcalorimetric measurements of ricin—saccharide binding

Microcalorimetric measurements of ricin—saccharide binding

Mechanism of lysozyme catalysis: role of ground-state strain in subsite D in hen egg-white and human lysozymes.

The association constants for the binding of various saccharides to hen egg-white lysozyme and human lysozyme have been measured by fluorescence titration. Among these are the oligosaccharides GlcNAc-beta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-GlcNAc, GlcNAc-beta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-N-acetyl-D-xylosamine, and GlcNAc-beta(1 leads to 4-GlcNAc-beta(1 leads to 4)-MurNAc, prepared here for the first time. The binding constants for saccharides which must have N-acetylmuramic acid, N-acetyl-D-glucosamine, or N-acetyl-D-xylosamine bound in subsite D indicate that there is no strain involved in the binding of N-acetyl-D-glycosamine in this site, and that the lactyl group of N-acetylmuramic acid (rather than the hydroxymethyl group) is responsible for the apparent strain previously reported for binding at this subsite. For hen egg-white lysozyme, the dependence of saccharide binding on pH or on a saturating concentration of Gd(III) suggests that the conformation of several of the complexes are different from one another and from that proposed for a productive complex. This is supported by fluorescence difference spectra of the various hen egg-white lysozyme-saccharide complexes. Human lysozyme binds most saccharides studied more weakly than the hen egg-white enzyme, but binds GlcNAc-beta(1 leads to 4)-MurNAc-beta(1leads to 4)-GlcNAc-beta(1 leads to 4)-MurNAc more strongly. It is suggested that subsite C of the human enzyme is "looser" than the equivalent site in the hen egg enzyme, so that the rearrangement of a saccharide in this subsite in response to introduction of an N-acetylmuramic acid residue into subsite D destabilizes the saccharide complexes of human lysozyme less than it does the corresponding hen egg-white lysozyme complexes. This difference and the differences in the fluorescence difference spectra of hen egg-white lysozyme and human lysozyme are ascribed mainly to the replacement of Trp-62 in hen egg-white lysozyme by Tyr-63 in the human enzyme. The implications of our findings for the assumption of superposition and additivity of energies of binding in individual subsites, and for the estimation of the role of strain in lysozyme catalysis, are discussed.