Tetrameric Concanavalin Research Articles

Binding Affinity of Concanavalin A to Native and Acid-Hydrolyzed Phytoglycogen Nanoparticles.

Phytoglycogen (PG) is a polysaccharide produced in the kernels of sweet corn as soft, highly branched, compact nanoparticles. Its tree-like or dendritic architecture, combined with a high-safety profile, makes PG nanoparticles attractive for use in biological applications, many of which rely on the association or binding of small biomolecules. We have developed a methodology to functionalize surface plasmon resonance (SPR) sensor surfaces with PG nanoparticles, and we demonstrate the utility of the PG-functionalized SPR sensor by measuring the binding affinity of the tetrameric concanavalin A (ConA) protein to both native PG nanoparticles and smaller, softer acid-hydrolyzed PG nanoparticles. We measure comparable values of the equilibrium association constant K for native and acid-hydrolyzed PG, with a slightly smaller value for the acid-hydrolyzed particles that we attribute to unfavorable lateral interactions between the tetrameric subunits of ConA due to the increase in surface curvature of the smaller acid-hydrolyzed PG particles. We also use infrared reflection-absorption spectroscopy (IRRAS) to show that ConA maintains a large fraction of its native conformation, and thus its bioactivity, upon binding to PG, representing an important step toward the realization of PG as a novel bioactive delivery vehicle.

Highly Sensitive Determination of Concanavalin A Lectin Based on Silver-Enhanced Electrogenerated Chemiluminescence of Luminol

ABSTRACTA highly sensitive bioassay based on silver-enhanced luminol electrogenerated chemiluminescence (ECL) is reported for the determination of concanavalin A lectin. A gold electrode modified with the mixed self-assembled monolayer of thiolated mannoside and mercaptohexanol was used to selectively capture a target lectin, concanavalin A, through the specific interaction between mannoside and concanavalin A. Mannoside-functionalized gold nanoparticles were further introduced to the opposite binding sites of the tetrameric concanavalin A to form a sandwich-type complex. Silver enhancement step was performed to coat the surface of mannose-stabilized gold nanoparticles with silver. The deposited silver was dissolved in an acidic solution and further neutralized. The resulting silver ions were finally detected with luminol electrogenerated chemiluminescence, in which the silver ions greatly enhanced the chemiluminescence intensity. The present electrogenerated chemiluminescence bioassay detected concanavalin A from 0.190 to 10.0 µg/mL (r2 = 0.999) with a detection limit of 0.146 µg/mL (signal to noise ratio = 3), which is much lower compared to previously reported methods such as microgravimetry, surface plasmon resonance, and colorimetry. Furthermore, the present bioassay showed good selectivity over possible interfering lectin proteins.

Symmetry of Charge Partitioning in Collisional and UV Photon-Induced Dissociation of Protein Assemblies.

Tandem mass spectrometry can provide structural information on intact protein assemblies, generating mass fingerprints indicative of the stoichiometry and quaternary arrangement of the subunits. However, in such experiments, collision-induced dissociation yields restricted information due to simultaneous subunit unfolding, charge rearrangement, and subsequent ejection of a highly charged unfolded single subunit. Alternative fragmentation strategies can potentially overcome this and supply a deeper level of structural detail. Here, we implemented ultraviolet photodissociation (UVPD) on an Orbitrap mass spectrometer optimized for native MS and benchmark its performance to HCD fragmentation using various protein oligomers. We investigated dimeric β-lactoglobulin, dimeric superoxide dismutase, dimeric and tetrameric concanavalin A, and heptameric GroES and Gp31; ranging in molecular weight from 32 to 102 kDa. We find that, for the investigated systems, UVPD produces more symmetric charge partitioning than HCD. While HCD spectra show sporadic fragmentation over the full protein backbone sequence of the subunits with a bias toward fragmenting labile bonds, UVPD spectra provided higher sequence coverage. Taken together, we conclude that UVPD is a strong addition to the toolbox of fragmentation methods for top-down proteomics experiments, especially for native protein assemblies.

Binding of 8-Anilinonaphthalene Sulfonate to Dimeric and Tetrameric Concanavalin A: Energetics and Its Implications on Saccharide Binding Studied by Isothermal Titration Calorimetry and Spectroscopy

The binding of 8-anilinonaphthalene sulfonate to concanavalin A has been investigated. Isothermal titration calorimetry (ITC) and circular dichroism studies have been performed under different experimental conditions to understand the binding quantitatively and evaluate contributions of different forces responsible for it. Isothermal titration calorimetric results of concanavalin A with ANS at pH 5.2 and 2.5, where it exists as a dimer, indicated binding heterogeneity and two classes of noninteracting sites. Enhancement of the binding constants from native to pH 2.5 suggests that the ANS binding is strongly influenced by the protein charge and the favorable alteration in the structure of concanavalin A as suggested by near-UV CD results. No binding was observed with the tetrameric form of concanavalin A, indicating shielding of sites due to dimerization of canonical dimers. The results have also demonstrated existence of a hydrophobic binding site that is distinct from the saccharide binding site.

Denaturant-induced equilibrium unfolding of concanavalin A is expressed by a three-state mechanism and provides an estimate of its protein stability

The urea and guanidine hydrochloride (GdnHCl)-induced denaturation of tetrameric concanavalin A (ConA) at pH 7.2 has been studied by using intrinsic fluorescence, 8-anilino-1-naphthalenesulfonate (ANS) binding, far-UV circular dichroism (CD), and size-exclusion chromatography. The equilibrium denaturation pathway of ConA, as monitored by steady state fluorescence, exhibits a three-state mechanism involving an intermediate state, which has been characterized as a structured monomer of the protein by ANS binding, far-UV CD and gel filtration size analysis. The three-state equilibrium is analyzed in terms of two distinct and separate dissociation (native tetramer↔structured monomer) and unfolding (structured monomer↔unfolded monomer) reaction steps, with the apparent transition midpoints ( C m), respectively, at 1.4 and 4.5 M in urea, and at 0.8 and 2.4 M in GdnHCl. The results show that the free energy of stabilization of structured monomer relative to the unfolded state (−Δ G unf, aq), is 4.4–5.5 kcal mol −1, and that of native tetramer relative to structured monomer (−Δ G dis, aq) is 7.2–7.4 kcal mol −1, giving an overall free energy of stabilization (−Δ G dis&unf, aq) of 11.6–12.9 kcal mol −1 (monomer mass) for the native protein. However, the free energy preference at the level of quaternary tetrameric structure is found to be far greater than that at the tertiary monomeric level, which reveals that the structural stability of ConA is maintained mostly by subunit association.

Presynaptic inhibition by concanavalin A: are alpha-latrotoxin receptors involved in action potential-dependent transmitter release?

Effects of concanavalin A on transmitter release were investigated in primary cultures of chick sympathetic neurons. The lectin reduced electrically evoked [3H]noradrenaline release by up to 30% with half-maximal inhibition at 0.16 microM. Concanavalin A also reduced the release triggered by extracellular Ca2+ in neurons depolarized by 25 mM K or rendered Ca2+-permeable by the ionophore A23187. The inhibitory action of concanavalin A on electrically evoked release was additive to that of the alpha2-adrenergic agonist UK 14,304. Inactivation of Gs and Gi/Go type G proteins by either cholera or pertussis toxin did not alter the inhibitory effect of the lectin. Concanavalin A failed to affect the resting membrane potential, action potential waveforms, or voltage-dependent K+ and Ca2+ currents. In contrast, the lectin efficiently blocked both the Ca2+-dependent and -independent alpha-latrotoxin-induced transmitter release, but only when applied before the toxin. The reduction of electrically evoked, as well as alpha-latrotoxin-evoked, release by concanavalin A was attenuated in the presence of glucose and abolished by methyl alpha-D-mannopyranoside. The dimeric derivative, succinyl-concanavalin A, was significantly less active than tetrameric concanavalin A. In bovine adrenal chromaffin cells, which displayed only weak secretory responses to alpha-latrotoxin, concanavalin A failed to alter K+-evoked catecholamine secretion. These results show that concanavalin A causes presynaptic inhibition in sympathetic neurons and indicate that cross-linking of alpha-latrotoxin receptors may reduce action potential-dependent transmitter release.

Differences in the binding affinities of dimeric concanavalin A (including acetyl and succinyl derivatives) and tetrameric concanavalin A with large oligomannose-type glycopeptides

Dimeric derivatives of concanavalin A (Con A) such as acetyl- and succinyl-Con A have been used for years as probes of cellular membranes. The altered binding and biological activities of these derivatives relative to native tetrameric Con A have generally been attributed to their reduced valence. However, the present study shows that acetyl- and succinyl-Con A possess lower affinities than tetrameric Con A toward certain oligomannose-type glycopeptides which are found on the surface of cells. It has previously been shown that native tetrameric Con A possesses 5-30-fold enhanced affinities toward Man7-Man9 oligomannose-type glycopeptides, respectively, relative to Man5 and Man6 oligomannose-type glycopeptides [Bhattacharyya, L., & Brewer, C. F. (1989) Eur. J. Biochem. 178, 721-726]. Using titration microcalorimetry and hemagglutination inhibition measurements, methyl alpha-D-mannopyranoside, methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside (which binds with about 60-fold higher affinity than methyl alpha-D-mannopyranoside and is the major Con A binding epitope on oligomannose-type carbohydrates), and a Man5 oligomannose-type oligosaccharide are shown to bind to underivatized dimeric Con A at pH 5.2 and acetyl- and succinyl-Con A at pH 7.2 with affinities equal to those of native tetrameric Con A. However, a mixture of Man7 and Man8 glycopeptides and a Man9 oligomannose-type glycopeptide were shown to bind to underivatized dimeric Con A and acetyl- and succinyl-Con A with affinities only about 2-fold higher than the Man5 oligosaccharide, in contrast to the higher affinities of native tetrameric Con A for these carbohydrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of CuCl2 on the dimer-tetramer equilibrium of concanavalin A.

Tetrameric concanavalin A at neutral pH dissociated into the dimer when CuCl2 was added in a concentration range comparable to protomer concentration. This effect of CuCl2 was largely suppressed when NaCl concentration was increased. Neither the conformation of the protein nor its binding activity to 4-methylumbelliferyl alpha-D-mannopyranoside was affected on addition of CuCl2.

Changes in the distribution of platelet membrane proteins revealed by energy transfer

The redistribution of platelet membrane proteins in response to platelet activation was studied. To investigate this process we prepared a variety of platelet ligands, including di- and tetrameric concanavalin A, IgG, thrombin, wheat-germ agglutinin and other lectins. These ligands were conjugated either with acceptor (rhodamine isothiocyanate) or donor (fluoresceine isothiocyanate) fluorophore. Platelets exposed to various combinations of ligand species were stimulated with different aggregating agents, and changes in sensitized fluorescence emission or donor quenching were recorded. Energy transfer was observed with thrombin, dimeric concanavalin A after addition of thrombin and various combinations of dimeric concanavalin A with other membrane ligands. The preincubation of platelets with colchicine prevented energy transfer between appropriate ligand pairs and platelet activator. Our studies show that nonradiative energy transfer can be used to analyze redistribution of membrane receptor sites in platelets.

Release of liposomal markers by [formula omitted] toxin A-III

Marker release from liposomes induced by the cytolytic protein Cerebratulus lacteus toxin A-III was studied. No phospholipid specificity was apparent, but the sensitivity of liposomes to A-III varied with the membrane fluidity. With dioleylphosphatidylcholine liposomes, complete release occurred at 10–20 μg toxin per ml, depending on marker size. Kinetic experiments showed that release was rapid and exhibited no lag phase. The diameter of the A-III produced membrane lesion must exceed 90 Å, as tetrameric Concanavalin A is quantitatively released from A-III treated liposomes.

Insulin-like activity of photochemically obtained monovalent monomeric concanavalin A in the presence of anti-concanavalin A antibodies: dependence on multivalency for stimulation of glucose oxidation of rat fat cells.

Insulin-like action of monovalent monomeric concanavalin A (m-Con A) was examined in rat adipocytes in the presence of anti-m-Con A antiserum. The antisera from rabbits injected with m-Con A reacted with not only monovalent monomeric but also tetravalent tetrameric concanavalin A (alpha-Con A) in Ouchterlony double diffusion analysis. m-Con A alone did not show any appreciable effect on glucose oxidation of adipocytes while it slightly inhibited glycerol release stimulated by epinephrine. In contrast, exposure of adipocytes to m-Con A in the presence of antibodies to m-Con A resulted in stimulation of glucose oxidation and inhibition of epinephrine-stimulated lipolysis. The stimulation and the inhibition with m-Con A in the presence of the antibodies were of the same degree as those with alpha-Con A. Both alpha- and m-Con A were slightly active in inhibiting 125I-labeled insulin binding. These results demonstrate that the ability of anti-m-Con A antiserum to aggregate m-Con A bound to receptors on the isolated-adipocyte plasma membrane allowed m-Con A to mimic the biological activity of insulin and that the aggregation of receptors for ligands other than insulin can induce insulin-like action in rat fat cells.

Monovalent monomer derivative of concanavalin A produced by photochemically induced alkylation.

Irradiation of tetrameric concanavalin A with a high-pressure mercury lamp in the presence of chloroacetamide and fractionation of the reaction product by affinity chromatography gave a lectin derivative showing a monomeric molecular weight at pH 7.4, with an overall yield of 25%. It retained the intact binding ability against methyl-alpha-D-glucopyranoside. Hemagglutination and glycogen precipitation data suggested the monovalent nature of the derivative. The results of amino acid composition analyses indicated that the photochemically induced alkylation at one or two tryptophan residues per subunit of concanavalin A may be responsible for the dissociation into monovalent monomers which are stable at physiological pH.

Divergent actions of monomeric, dimeric, and tetrameric concanavalin A on lymphocyte traffic

The autoradiographic analysis of the localization of [ 3H]adenosine-labeled cells exposed to concanavalin A (Con A) in vitro has confirmed that the altered migration of Con A-treated lymphocytes is a consequence of their slower rate of migration and delay in normal areas of traffic (5, 6). The mechanisms through which Con A alters cell migration were further investigated by studying the effects of several derivatives of Con A on the distribution of 51Cr-labeled lymph node cells. The results obtained show that the monomeric and dimeric forms of Con A were unable to modify cell traffic, a condition that was partially reversed when succinyl Con A-treated cells were exposed to divalent antibodies to Con A. This suggests that Con A may alter lymphocyte recirculation by actively modifying the membrane fluidity or the surface lateral transport of the lymphocyte. Whatever the exact mechanisms responsible for the altered migration are, they probably involve complex active processes that can be related to the heterogeneity of Con A receptors, the existence of subsets of cells with different reactivities to the lectin, or simply the result of a passive phenomenon dependent on the presence of Con A on the cell surface.

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.

Binding of 4-methylumbelliferyl alpha-D-mannopyranoside to tetrameric and unmodified or derivatized dimeric concanavalin A: equilibrium studies.

The binding of 4-methylumbelliferyl alpha-D-mannopyranoside (MUM) and concanavalin A, composed of intact polypeptide chains, was studied by equilibrium dialysis, difference spectroscopy, and fluorescence titration (Dean, B.R., and Homer, R.B. (1973), Biochim. Biophys. Acta. 322, 141-144), measured either at a fixed wavelength or above 350 nm. Dimeric and tetrameric concanavalin A samples were used under conditions of apparently full metal saturation. The results are consistent with a single carbohydrate-specific site per protomer, without interaction between sites; no indication for additional unspecific binding could be obtained. The values of the association constant are independent of the method or of the saturation range used and 4-methylumbelliferyl alpha-D-mannopyranoside, bound at a fractional saturation of 0.91 can be totally displaced by methyl alpha-D-mannopyranoside. The thermodynamic binding parameters for acetylated or succinylated concanavalin A, composed of intact polypeptide chains, were obtained by titration of total MUM fluorescence in the temperature range 9-39 degrees C. For unmodified dimeric concanavalin A at 25.0 degrees C, the values are K = (3.36 +/- 0.04) 10(4) M-1 with delta H degrees = -8.3 +/- 0.1 kcal mol-1 and delta S degrees = 7.2 +/- 0.3 eu; for tetrameric concanavalin A, the affinity is increased by 25% and within experimental error the values of delta H degrees and delta S degrees are identical to those for the dimeric protein. Derivatized concanavalin A shows binding characteristics that are entirely comparable to those of the native protein.

Precipitin and mitogenic behavior of dimeric and tetrameric concanavalin A.

The results of double diffusion and quantitative precipitin tests show that in contrast to the concanavalin A tetramer, the dimer is incapable of precipitating glycogen. The results support previous work suggesting that dimers composed of two fragmented monomer units possess only one binding site for carbohydrate (McKenzie and Sawyer, J. Biol. Chem. 1973. 248: 549). On the other hand, dimer and tetramer species had similar mitogenic activity as measured by the incorporation of radioactive thymidine by mouse spleen lymphocytes and induced cap formation to similar degrees. Results are discussed in terms of the affinity and valency of the polymeric forms and their stability at the cell surface.

Modulation of lymphocyte mitogenesis.

Comparisons of the stimulation of normal lymphocytes and lymphoma cells by tetrameric concanavalin a (Con ta) and dimeric succinyl-Con A suggest that both stimulatory and inhibitory signals operate to modulate mitogenesis. Synergistic effects can be obtained for the stimulatory event using lectins, phorbol esters, and calcium ionophores, all of which are independently mitogenic for lymphocytes. The inhibitory effects of high doses of Con A could be mimicked by the simultaneous addition of the phorbol ester and Con A under conditions in which both reagents are optimally mitogenic when used alone. No inhibition of stimulation was found, however, when succinyl-Con A was used with phorbol ester under the same conditions. Moreover, when lymphocytes were cultured with Con A in the presence of succinyl-Con A, the inhibitory effect of the native lectin was seen at lower doses than in the absence of the derivative. These observations suggest that the stimulatory and inhibitory portions of the dose-response curve can be manipulated independently and may be mediated by two distinct signals. It is likely the signal for the inhibition of cell proliferation is regulated by the same cell surface modulating assembly that controls the mobility of cell surface receptors.

On the specificity of carbohydrate-lectin recognition. The interaction of a lectin from Ricinus communis beans with simple saccharides and concanavalin A.

A galactose‐specific protein (RC1) isolated from Ricinus communis beans was found to give a precipitin reaction with concanavalin A. Its carbohydrate content amounted to 8–9% of the total protein and was found to be rich in mannose. The interaction of RC1 with galactose and lactose was measured in 0.05 M phosphate buffer containing 0.2 M NaCl (pH 6.8) by the method of conventional equilibrium dialysis. From the analysis of the binding data according to Scatchard method the association constant (Ka) at 5°C was calculated as 3.8 mM−1 and 1.2 mM−1 for lactose and galactose, respectively. In both cases the number of binding sites per molecule of RC1 with molecular weight of 120000 was found to be 2. From the temperature‐dependent Ka values for the binding of lactose, the values of –5.7 kcal/mol and –4.3 cal × mol−1× K−1 were calculated for ΔH and ΔS, respectively.The addition of concanavalin A to RC1 or vice versa led to the formation of the insoluble complex RC1· ConA4 containing one molecule of RC1 and one molecule of tetrameric concanavalin A (ConA4) which could be dissociated upon addition of concanavalin A‐specific sugars. The complex formation results in a time‐dependent appearance of turbidity in the time range from 10s to 10 min. From the measurement of the time‐dependent appearance and disappearance of the turbidity the formation (kf) and dissociation (kd) rate constants were calculated as 3 mM−1× s−1 and 0.07 ks−1 respectively. The ratio kf/kd (43μM −1), that corresponds to the association constant of complex RC1· ConA4, is higher than that of mannoside · ConA4 and thereby suggests that protein‐protein interaction contributes significantly in stabilising glycoprotein · lectin complexes. The relevance of this finding to the understanding of the chemical specificities that are involved in a model cell‐lectin interaction is discussed.

Concanavalin A derivatives with altered biological activities.

Chemical derivatization of tetrameric concanavalin A (Con A) with succinic anhydride or acetic anhydride converts the protein to a dimeric molecule without altering its carbohydrate-binding specificity. At low concentrations, the dose-response curves for the mitogenic stimulation of mouse spleen cells by native Con A and succinyl-Con A are similar. Above lectin concentrations of 10 mug/ml, however, the response to Con A is diminished, while that for succinyl-Con A does not decrease until much higher doses are reached. We have attributed this difference mainly to the higher rate of cell death induced by the native Con A molecule. Con A also shows a greater capacity than succinyl-Con A to agglutinate sheep erythrocytes and to inhibit cap formation by immunoglobulin receptors on spleen cells. Moreover, at low concentrations, Con A induced its glycoprotein receptors to form caps, but succinyl-Con A did not induce cap formation. Addition of antibodies directed against Con A to succinyl-Con A bound on cells restored the properties of agglutination, inhibition of immunoglobulin receptor cap formation, and induction of cap formation by Con A receptors. Similar results have been obtained for acetyl-Con A. These data suggest that the altered biological activities of succinyl-Con A and acetyl-Con A are attributable to their reduced valence.

Location of the Saccharide Binding Site of Concanavalin A

Abstract Crystals of the lectin concanavalin A containing the saccharide β-(o-iodophenyl)-d-glucopyranoside have been prepared. Difference electron density projections at 2.8-A resolution have revealed the positions of the iodine atoms, directly within prominent cavities on the surface of the protein. One saccharide is bound to each 27,000 molecular weight subunit, and thus 4 sugar molecules are bound to symmetry equivalent positions on the tetrameric concanavalin A molecule. The iodine atoms of the four bound saccharides are separated by distances of 24.6, 25.1, and 35.1 A.