Studies on lectins. XLIV. The pH dependence of lectin interactions with sugars as determined by affinity electrophoresis.

The pH dependence of association constants of the lectin-sugar complexes was determined by means of affinity electrophoresis. All the lectins studied (from the seeds of Dolichos biflorus, Glycine soja, Lens esculenta and Vicia cracca and of the fruiting body of Marasmius oreades) were characterized by a similar course of pH dependence of the association constants, with the maximum values at pH 7--9. For concanavalin A and the L-fucose binding Ulex europaeus lectin only the association constants at three selected pH values were determined. Concanavalin A does not interact with immobilized alpha-D-mannosyl residues at pH 2.3. The association constants vs. pH curves measured for lectins isolated from two different lentil varieties slightly differ in accordance with the differences observed in the interaction of these lectins with the Sephadex gel.     

Studies on lectins XLIV. The pH dependence of lectin interactions with sugars as determined by affinity electrophoresis

The pH dependence of association constants of the lectin-sugar complexes was determined by means of affinity electrophoresis. All the lectins studied (from the seeds of Dolichos biflorus, Glycine soja, Lens esculenta and Vicia cracca and of the fruiting body of Marasmius oreades) were characterized by a similar course of pH dependence of the association constants, with the maximum values at pH 7–9. For concanavalin A and the l-fucose binding Ulex europaeus lectin only the association constants at three selected pH values were determined. Concanavalin A does not interact with immobilized α-d-mannosyl residues at pH 2.3. The association constants vs. pH curves measured for lectins isolated from two different varieties slightly differ in accordance with the differences observed in the interaction of these lectins with the Sephadex gel.     

Frontal affinity chromatography of ovalbumin glycoasparagines on a concanavalin A-sepharose column. A quantitative study of the binding specificity of the lectin

The interactions of Sepharose 4B-immobilized concanavalin A (ConA) with 10 glycoasparagines derived from ovalbumin were investigated quantitatively by frontal affinity chromatography. In this method, a carbohydrate solution is applied continuously to a ConA-Sepharose column and the retardation of the elution front is measured as a parameter of the strength of the interaction. The dissociation constant (Kd) for each saccharide with ConA can be determined. An analysis of the binding of p-nitrophenyl-alpha,D-mannoside has shown that the binding properties of ConA do not change essentially after immobilization on Sepharose 4B. Each of the ovalbumin glycoasparagines was labeled with tritium by the reductive methylation method for analysis. A comparison of the Kd values obtained showed that the binding of ConA varies considerably with very slight structural differences of the glycosyl chain. The results suggest that ConA recognizes a specific glycosyl chain structure, Man alpha 1-6(Man alpha 1-3)Man, in which at least one hydroxyl group at the C-3 position of C-6-linked mannose should be free. The glycoasparagines containing this structure bound strongly to ConA-Sepharose with dissociation constants below 3.4 X 10(-7) M.     

Man alpha1-2 Man alpha-OMe-concanavalin A complex reveals a balance of forces involved in carbohydrate recognition.

We have determined the crystal structure of the methyl glycoside of Man alpha1-2 Man in complex with the carbohydrate binding legume lectin concanavalin A (Con A). Man alpha1-2 Man alpha-OMe binds more tightly to concanavalin A than do its alpha1-3 and alpha1-6 linked counterparts. There has been much speculation as to why this is so, including a suggestion of the presence of multiple binding sites for the alpha1-2 linked disaccharide. Crystals of the Man alpha1-2 Man alpha-OMe-Con A complex form in the space group P2(1)2(1)2(1) with cell dimensions a = 119.7 A, b = 119.7 A, c = 68.9 A and diffract to 2. 75A. The final model has good geometry and an R factor of 19.6% (Rfree= 22.8%). One tetramer is present in the asymmetric unit. In three of the four subunits, electron density for the disaccharide is visible. In the fourth only a monosaccharide is seen. In one subunit the reducing terminal sugar is recognized by the monosaccharide site; the nonreducing terminal sugar occupies a new site and the major solution conformation of the inter-sugar glycosidic linkage conformation is adopted. In contrast, in another subunit the non reducing terminal sugar sits in the so called monosaccharide binding site; the reducing terminal sugar adopts a different conformation about its inter-sugar glycosidic linkage in order for the methyl group to access a hydrophobic pocket. In the third subunit, electron density for both binding modes is observed. We demonstrate that an extended carbohydrate binding site is capable of binding the disaccharide in two distinct ways. These results provide an insight in to the balance of forces controlling protein carbohydrate interactions.     

Superparamagnetic adsorbents for high-gradient magnetic fishing of lectins out of legume extracts

This work presents the development, testing, and application in high-gradient magnetic fishing of superparamagnetic supports for adsorption of lectins. Various approaches were examined to produce affinity, mixed mode, and hydrophobic charge induction type adsorbents. In clean monocomponent systems affinity supports created by direct attachment of glucose or maltose to amine-terminated iron oxide particles could bind concanavalin A at levels of up to approximately 280 mg g(-1) support with high affinity ( approximately 1 microM dissociation constants). However, the best performance was delivered by adsorbents featuring coupled tentacular dextran chains displaying a maximum binding capacity of 238 mg g(-1) and a dissociation constant of 0.13 microM. Adsorbents derivatized with mixed mode or hydrophobic charge induction ligands likewise demonstrated very high capacities for both concanavalin A and Lens culinaris agglutinin (> or = 250 mg g(-1)) with dissociation constants in the micromolar range, though neither of these systems showed any selectivity for lectins in leguminous extracts. When the affinity supports were applied to carbohydrate containing legume extracts only the dextran-linked adsorbents supplied sufficient competition to dissolved sugars to selectively bind concanavalin A in an extract of jack beans. The dextran-linked supports were employed in a high-gradient magnetic fishing experiment, in which concanavalin A was purified to near homogeneity from a crude, unclarified extract of jack beans.     

Triplet-state detection of labeled proteins using fluorescence recovery spectroscopy

The experimental procedures for detecting the triplet states of chromophores in solutions (cuvettes) by fluorescence recovery spectroscopy (FRS) are described in detail, together with applications in studies of protein structure and protein-cell interactions in the microsecond to millisecond time domain. The experimental configuration has been characterized by measuring the emission intensities and anisotropies of eosin and erythrosin immobilized in poly(methyl methacrylate). The fluorescence data are compared with those from phosphorescence emission measurements and with theoretical predictions. Triplet-state lifetimes were obtained in 5 mM phosphate buffer, pH 7.0, of concanavalin A labeled with eosin, tetramethylrhodamine, and fluorescein and of alpha 2-macroglobulin labeled with the first two probes. In the case of labeled concanavalin A, iodide quenching measurements gave bimolecular rate constants of approximately 10(9) M-1 s-1. The usefulness of FRS for studying protein-cell interactions is exemplified with eosin-labeled concanavalin A bound to living A-431 human epidermoid carcinoma cells. Finally, the advantages and disadvantages of the technique are compared to those of the alternative phosphorescence emission method.     

Fluorescence-polarization studies on binding of 4-methylumbelliferyl beta-D-galactopyranoside to Ricinus communis (castor-bean) agglutinin.

The binding of Ricinus communis (castor-bean) agglutinin 1 to saccharides was studied by equilibrium dialysis and fluorescence polarization by using the fluorescently labelled sugar 4-methylumbelliferyl beta-D-galactopyranoside. No appreciable change in ligand fluorescence of 4-methylumbelliferyl beta-D-galactopyranoside was considerably polarized on its binding to the lectin. The association constants obtained by Scatchard analysis of equilibrium-dialysis and fluorescence-polarization data do not differ much from each other, and at 25 degrees C, Ka = 2.4 (+/- 0.2) X 10(4)M-1. These values agree reasonably well with that reported in the literature for Ricinus agglutinin 1. The number of binding sites obtained by the different experimental procedures is 1.94 +/- 0.1 per molecule of 120 000 daltons and is equal to the reported value of 2. The consistency in the values of Ka and number of binding sites indicate the absence of additional subsites on Ricinus agglutinin 1 for its specific sugars. In addition, the excellent agreement between the binding parameters obtained by equilibrium dialysis and fluorescence polarization indicate the potential of ligand-fluorescence-polarization measurements in the investigation of lectin-sugar interactions.     

Studies on phytohemagglutinins: XXII. Isolation and characterization of a lymphocyte receptor for concanavalin A

A receptor glycopeptide for concanavalin A was isolated from calf thymocytes by a method originally devised for the isolation of a lectin receptor from human erythrocytes (Kubánek, J., Entlicher, G.; and Kocourek, J. [1973] Biochim, Biophys. Acta 304, 93–102). The method consisted of pronase digestion of the lipid-depleted thymocyte membrane material followed by ethanol fractionation, separation on Sephadex and preparative paper electrophoresis. The isolated glycopeptide contains 10.4% of neutral sugar. The molar ratios of the sugar components mannose, galactose, glucosamine, glucose, fucose and sialic acid are 3 : 2 : 2 : 1 : 1 : 1. The minimum molecular weight calculated from the sugar composition is about 12 000.Concanavalin A receptor activity of the glycopeptide was demonstrated in three different ways: (i) Reduction of the ¹²⁵I-labeled concanavalin A binding to thymocytes. (ii) Prevention of concanavalin A induced agglutination of calf thymocytes. (iii) Inhibition of concanavalin A stimulated DNA synthesis in calf and rabbit thymocytes and rabbit lymph node lymphocytes cultivated in vitro.The isolated glycopeptide seems to be involved in the interaction of lymphocytes with concanavalin A and the subsequent stimulation.     

Determination of lectin-sugar binding constants by microequilibrium dialysis coupled with high performance liquid chromatography

We used high performance liquid chromatography to determine the concentrations of free ligands after equilibrium dialysis to examine lectin-sugar interactions. The binding of p-nitrophenyl 1-thio-alpha-mannoside, p-nitrophenyl N-acetyl-1-thio-beta-glucosaminide, and the pyridylamino derivative of Man6GlcNAc2 to concanavalin A and Triticum vulgaris lectin was examined. The binding constant, Ka, and the concentration of total binding sites, [L]t, were calculated from the trace amounts of sugars and lectins using the equation, [S]/[LS] = 1/Ka[L]t + [S]/[L]t, where [S] is the concentration of free ligand and [LS] the concentration of bound ligand.     

Inhibition of cell surface receptor-bound plasmin by alpha 2-antiplasmin and alpha 2-macroglobulin.

The purpose of this investigation was to characterize the reaction of alpha 2-antiplasmin (alpha 2AP) and alpha 2-macroglobulin (alpha 2M) with human plasmin bound to rat C6 glioma cells and human umbilical vein endothelial cells (HUVECs). Binding of plasmin (0.1 microM) to C6 cells at 4 degrees C did not cause cell detachment, decrease viability or change cell morphology. The KD and Bmax for the binding of diisopropyl phosphoryl plasmin (DIP-plasmin) to C6 cells were 0.9 microM and 2.6 x 10(6) sites/cell. The dissociation rate constants (koff) for 125I-plasmin were 9.7 x 10(-4) and 4.0 x 10(-4) s-1 at 4 degrees C in the presence and absence of 0.3 microM DIP-plasmin, respectively. Similar constants were determined for 125I-plasminogen and 125I-DIP-plasmin. Neither alpha 2AP nor alpha 2M affected the dissociation of DIP-plasmin. C6 cell-associated 125I-plasmin reacted slowly with alpha 2AP; however, the inhibition rate constants exceeded the koff. alpha 2AP-plasmin complex formed after the plasmin dissociated into solution (reaction pathway 1) and by direct reaction of alpha 2AP with cell-associated enzyme (reaction pathway 2). High concentrations of alpha 2AP favored pathway 2. C6 cell-associated plasmin was also protected from inhibition by alpha 2M. While the same pathways were probably involved in this reaction, alpha 2M was less effective than alpha 2AP as an inhibitor of nondissociated plasmin (pathway 2). When C6 cell-bound plasmin reacted with alpha 2AP, alpha 2AP-plasmin complex was recovered primarily in the medium, suggesting dissociation of complexes formed on the cell surface. Plasmin-receptor dissociation and inhibition experiments were performed at 22 degrees and 37 degrees C, confirming the conclusions of the 4 degrees C studies. Comparable results were also obtained using HUVEC cultures. These studies demonstrate that cell-associated plasmin is protected from inhibition by alpha 2M as well as alpha 2AP. At least two reaction pathways may be demonstrated for the inhibition of plasmin that is initially receptor-bound; however, neither pathway is highly effective, accounting for the "plasmin-protective" activity of the cell surface.     

Sequencing and two-dimensional structure prediction of a phospholipase A(2) inhibitor from the serum of the common tiger snake (Notechis scutatus)

The complex between concanavalin A (Con A) and alpha1-2 mannobiose (mannose alpha1-2 mannose) has been refined to 1.2 A resolution. This is the highest resolution structure reported for any sugar-lectin complex. As the native structure of Con A to 0.94 A resolution is already in the database, this gives us a unique opportunity to examine sugar-protein binding at high resolution. These data have allowed us to model a number of hydrogen atoms involved in the binding of the sugar to Con A, using the difference density map to place the hydrogen atoms. This map reveals the presence of the protonated form of Asp208 involved in binding. Asp208 is not protonated in the 0.94 A native structure. Our results clearly show that this residue is protonated and hydrogen bonds to the sugar. The structure accounts for the higher affinity of the alpha1-2 linked sugar when compared to other disaccharides. This structure identifies different interactions to those predicted by previous modelling studies. We believe that the additional data presented here will enable significant improvements to be made to the sugar-protein modelling algorithms.     

Peptide inhibitors of HIV-1 integrase dissociate the enzyme oligomers.

Integration of HIV-1 genome into host cell chromosome is mediated by viral integrase (IN). The IN catalytic core (CC, IN(50-212)) dimerizes through mutual interactions of its alpha1 and alpha5 helices. Peptides INH1 and INH5 reproducing these helix sequences strongly inhibited IN. For instance, an IC(50) of 80 nM was determined for INH5 in integration assays using wild-type IN (wtIN). In size exclusion chromatography, INH1 and INH5 perturbed the association-dissociation equilibrium of both dmIN (IN(1-288)/F185K/C280S) and CC, leading to monomers as surviving species, while in circular dichroism, binding of peptides to dmIN altered the protein conformation. Thus, enzyme deactivation, subunit dissociation, and protein unfolding are events which parallel one another. The target of INH5 in the enzyme was then identified. In fluorescence spectroscopy, C(0.5) values of 168 and 44 nM were determined for the binding affinity of INH5 to IN and CC, respectively, at 115 nM subunit concentration, while interaction of INH5 with INH1 was found stronger than interaction of INH5 with itself (23 times larger in term of dissociation constants). These results strongly suggested that the alpha1 helix is the privileged target of INH5. The latter could serve as a lead for the development of new chemotherapeutic agents against HIV-1.     

Influence of placental mannose/N-acetyl glucosamine-binding proteins on the interaction of insulin and insulin-like growth factors with their receptors

Placenta is a source of carbohydrate-binding proteins that function as molecular scavengers, but they could also be involved in interactions that assist in metabolic control. Mannose/N-acetyl-glucosamine (Man/GlcNAc)-binding proteins from placenta were isolated and their reactivity towards placental insulin and insulin-like growth factor receptors (IR and IGF-Rs) was analyzed. The lectins reduced the binding of insulin and IGF-I in a dose-dependent manner, while almost no effect was observed on the binding of IGF-II. The shape of the inhibition curves changed, suggesting altered binding specificity. The presence of sugar could not reverse completely the effect of the lectins, implicating both lectin-sugar and protein-protein conformational recognition. Since biological molecules in our experimental system were those that are in close relation in vivo, placental Man/GlcNAc-specific lectins may be regarded as potential allosteric modulators of lig- and-receptor interactions in a system of homologous ligands, selectively affecting only binding to tyrosine kinase type receptors (IR and IGF-1R).     

The solution structure of the circular trinucleotide cr(GpGpGp) determined by NMR and molecular mechanics calculation.

The 3'-5' circular trinucleotide cr(GpGpGp) was studied by means of 1D and 2D high resolution NMR techniques and molecular mechanics calculations. Analysis of the J-couplings, obtained from the 1H and 13C-NMR spectra, allowed the determination of the conformation of the sugar rings and of the 'circular' phosphate backbone. In the course of the investigations it was found that the Karplus-equation most recently parametrized for the CCOP J-coupling constants could not account for the measured J(C4'P) of 11.1 Hz and a new parametrization for both HCOP and CCOP coupling constants is therefore presented. Subsequent analysis of the coupling constants yielded 'fixed' values for the torsion angles beta and delta (with beta = 178 degrees and delta = 139 degrees). The value of the latter angle corresponds to an S-type sugar conformation. The torsion angles gamma and epsilon are involved in a rapid equilibrium in which they are converted between the gauche(+) and trans and between the trans and gauche(-) domain respectively. We show that the occurrence of epsilon in the gauche(-) domain necessitates S-type sugar conformations. Given the aforementioned values for beta, gamma, delta and epsilon the ring closure constraints for the ring, formed by the phosphate backbone can only be fulfilled if alpha and zeta adopt some special values. After energy minimization with the CHARMm force field only two combinations of alpha and zeta result in energetically favourable structures, i.e. the combination alpha (t)/zeta(g-) in case gamma is in a gauche(+) and epsilon is in a trans conformation, and the combination alpha (t)/zeta (g+) for the combination gamma (t)/epsilon (g-). The results are discussed in relation to earlier findings obtained for cd(ApAp) and cr(GpGp), the latter molecule being a regulator of the synthesis of cellulose in Acetobacter xylinum.     

Characterizing high-affinity antigen/antibody complexes by kinetic- and equilibrium-based methods

Two biophysical methods, Biacore and KinExA, were used to kinetically and thermodynamically characterize high-affinity antigen/antibody complexes. Three to five independent experiments were performed on each platform with three different antigen/antibody complexes possessing nanomolar to picomolar equilibrium dissociation constants. By monitoring the dissociation phase on Biacore for 4 h, we were able to measure dissociation rate constants (kd) on the order of 1 x 10(-5)s(-1). To characterize high-affinity interactions by KinExA, samples needed to be equilibrated for up to 35 h to reach equilibrium. In the end, we show that similar kinetic rate constants and affinities were determined with both solution-phase and solid-phase methodologies. These results help further validate both interaction technologies and illustrate their suitability for characterizing extremely high-affinity interactions.     

Studies of lectin-carbohydrate interactions by quantitative affinity chromatography: systems with galactose and ovalbumin as saccharidic ligand

The potential of affinity chromatography for characterizing lectin-carbohydrate interactions is investigated. First, the effect of galactose on the chromatographic behavior of Ricinus communis phytohemagglutinin on Sepharose 4B is used to establish that quantitative affinity chromatography on polysaccharide matrices affords an unequivocal means of characterizing the interactions of lectins with monosaccharides in solution. Second, a method of characterizing lectin-glycoprotein interactions by affinity chromatography is illustrated in an experimental study with Sephadex G-50 as affinity matrix for examination of the interaction between concanavalin A and ovalbumin. Third, although no general solution to the problem of ligand multivalency in quantitative affinity chromatography has been found, an experimental protocol has been devised for the situation in which the partitioning solute (lectin) is univalent.     

Kinetic studies on the role of elongation factors 1 beta and 1 gamma in protein synthesis

An equilibrium isotope exchange technique was used to measure in an Artemia system the catalytic influence of elongation factor (EF) 1 beta gamma on the dissociation of GDP from the complex of elongation factor 1 alpha.[3H] GDP in the presence of an excess of free GDP. The kinetic data demonstrate that, in analogy to procaryotes, dissociation of GDP occurs via the formation of a transient ternary complex of EF-1 alpha.GDP.EF-1 beta gamma. The rate constants for the dissociation of GDP from EF-1 alpha.GDP and from the ternary complex EF-1 alpha.GDP.EF-1 beta gamma were found to be 0.7 x 10(-3) and greater than or equal to 0.7 s-1, respectively. The equilibrium association constants of GDP to EF-1 alpha.EF-1 beta gamma and of EF-1 beta gamma to EF-1 alpha.GDP were found to be 2.3 x 10(5) and 4.2 x 10(5) M-1, respectively. Judged from the known elongation rate in vivo and kinetic constants of nucleotide exchange, it was estimated that the recycling of EF-1 alpha may be a rate-controlling step in eucaryotic translation. As a model for GTP exchange, the formation of the ternary EF-1 alpha.guanylyl (beta gamma-methylene)diphosphonate.EF-1 beta gamma complex was also studied. It was observed that both an increase of the level of aminoacyl-tRNA and of temperature favored the dissociation of this complex, thereby enabling EF-1 beta gamma to recycle as a catalyst. This behavior would explain the frequent occurrence of a heavy form of elongation factor 1 in extracts of the eucaryotic cell.     

Evaluation of alpha-D-mannopyranoside glycolipid micelles-lectin interactions by surface plasmon resonance method

It is established that achieving higher binding affinities in carbohydrate-protein interactions requires multivalent presentations of the sugar ligands at the receptor binding site. Several inhibition, calorimetric, mass balance, and other studies have reiterated the beneficial effects of molecular level clustering of the sugar ligands for tight binding to the receptors. We have undertaken an effort to study the multivalent effects involving larger assemblies, represented by micelles, and their lectin interactions. The micelles were constituted with monomer bearing one- or two-sugar moieties at the monomolecular level and with varying the distances between the sugar moieties. Micellar aggregation studies and dynamic light scattering (DLS) studies afforded details of the aggregation numbers and the hydrodynamic diameters of various glycolipid (GL) micelles. The GL micelles were used as analytes of surface plasmon resonance (SPR) experiments on a lectin concanavalin A (Con A)-immobilized surface. SPR studies of the micelle-lectin interactions demonstrate that the ligand-receptor binding can be fit into the bivalent analyte model of interaction. Furthermore, micelles formed from two-sugar containing GLs are able to elicit favorable kinetic association rate constants in comparison to the micelles constituted with one-sugar containing GLs. The kinetic rate constants across the micelles and the effect of the sugar valencies in the GLs are discussed.     

Mechanism of lectin-cell interactions: thermodynamic and kinetic analysis of the binding of winged bean acidic lectin (WBA II) to human erythrocytes.

In order to identify the forces involved in the binding and to understand the mechanism involved, equilibrium and kinetic studies were performed on the binding of the winged bean acidic lectin to human erythrocytes. The magnitudes of delta S and delta H were positive and negative respectively, an observation differing markedly from the lectin-simple sugar interactions where delta S and delta H are generally negative. Analysis of the sign and magnitudes of these values indicate that ionic and hydrogen bonded interactions prevail over hydrophobic interactions resulting in net -ve delta H (-37.12 kJ.mol-1) and +ve delta S (14.4 J.mole-1 K-1 at 20 degrees C), thereby suggesting that this entropy driven reaction also reflects conformational changes in the lectin and/or the receptor. Presence of two kinds of receptors for WBA II on erythrocytes, as observed by equilibrium studies, is consistent with the biexponential dissociation rate constants (at 20 degrees C K1 = 1.67 x 10(-3) M-1 sec-1 and K2 = 11.1 x 10(-3) M-1 sec-1). These two rate constants differed by an order of magnitude accounting for the difference in the association constants of the two receptors of WBA II. However, the association process remains monoexponential suggesting no observable difference in the association rates of the lectin molecule with both the receptors, under the experimental conditions studied. The thermodynamic parameters calculated from kinetic data correlate well with those observed by equilibrium. A two-step binding mechanism is proposed based on the kinetic parameters for WBA II-receptor interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lysine-sensitive aspartokinase of Escherichia coli K12. Synergy and autosynergy in an allosteric V system.

The interactions of the lysine-sensitive aspartokinase of E. coli K12 with lysine and leucine, as evidenced in the inhibition and binding curves, are well explained by the equations of an allosteric V model. Mathematical treatments of such a model lead to new linearized plots. These representations are applied to our experimental results and allow the direct determination of some parameters of the model (equilibrium constant L' and leucine dissociation constants). The other parameters are obtained by an optimization method. The theoretical curves drawn according to this model account well for the synergistic inhibition between lysine and leucine and for the role of the two nonequivalent lysine binding sites ("autosynergy").