Binding Site Of Cholera Toxin Research Articles

Molecular dynamics of sialic acid analogues complex with cholera toxin and DFT optimization of ethylene glycol-mediated zinc nanocluster conjugation

Cholera is an infectious disease caused by cholera toxin (CT) protein of bacterium Vibrio cholerae. A sequence of sialic acid (N-acetylneuraminic acid, NeuNAc or Neu5Ac) analogues modified in its C-5 position is modelled using molecular modelling techniques and docked against the CT followed by molecular dynamics simulations. Docking results suggest better binding affinity of NeuNAc analogue towards the binding site of CT. The NeuNAc analogues interact with the active site residues GLU:11, TYR:12, HIS:13, GLY:33, LYS:34, GLU:51, GLN:56, HIE:57, ILE:58, GLN:61, TRP:88, ASN:90 and LYS:91 through intermolecular hydrogen bonding. Analogues N-glycolyl-NeuNAc, N-Pentanoyl-NeuNAc and N-Propanoyl-NeuNAc show the least XPGscore (docking score) of −9.90, −9.16, and −8.91, respectively, and glide energy of −45.99, −42.14 and −41.66 kcal/mol, respectively. Stable nature of CT-N-glycolyl-NeuNAc, CT-N-Pentanoyl-NeuNAc and CT-N-Propanoyl-NeuNAc complexes was verified through molecular dynamics simulations, each for 40 ns using the software Desmond. All the nine NeuNAc analogues show better score for drug-like properties, so could be considered as suitable candidates for drug development for cholera infection. To improve the enhanced binding mode of NeuNAc analogues towards CT, the nine NeuNAc analogues are conjugated with Zn nanoclusters through ethylene glycol (EG) as carriers. The NeuNAc analogues conjugated with EG-Zn nanoclusters show better binding energy towards CT than the unconjugated nine NeuNAc analogues. The electronic structural optimization of EG-Zn nanoclusters was carried out for optimizing their performance as better delivery vehicles for NeuNAc analogues through density functional theory calculations. These sialic acid analogues may be considered as novel leads for the design of drug against cholera and the EG-Zn nanocluster may be a suitable carrier for sialic acid analogues.

Molecular modeling of methyl-α-Neu5Ac analogues docked against cholera toxin--a molecular dynamics study.

Molecular modeling of synthetic methyl-α-Neu5Ac analogues modified in C-9 position was investigated by molecular docking and molecular dynamics (MD) simulation methods. Methyl-α-Neu5Ac analogues were docked against cholera toxin (CT) B subunit protein and MD simulations were carried out for three Methyl-α-Neu5Ac analogue-CT complexes (30, 10 and 10ns) to estimate the binding activity of cholera toxin-Methyl-α-Neu5Ac analogues using OPLS_2005 force field. In this study, direct and water mediated hydrogen bonds play a vital role that exist between the methyl-α-9-N-benzoyl-amino-9-deoxy-Neu5Ac (BENZ)-cholera toxin active site residues. The Energy plot, RMSD and RMSF explain that the simulation was stable throughout the simulation run. Transition of phi, psi and omega angle for the complex was calculated. Molecular docking studies could be able to identify the binding mode of methyl-α-Neu5Ac analogues in the binding site of cholera toxin B subunit protein. MD simulation for Methyl-α-9-N-benzoyl-amino-9-deoxy-Neu5Ac (BENZ), Methyl-α-9-N-acetyl-9-deoxy-9-amino-Neu5Ac and Methyl-α-9-N-biphenyl-4-acetyl-deoxy-amino-Neu5Ac complex with CT B subunit protein was carried out, which explains the stable nature of interaction. These methyl-α-Neu5Ac analogues that have computationally acceptable pharmacological properties may be used as novel candidates for drug design for cholera disease.

Conformations of Higher Gangliosides and Their Binding with Cholera Toxin—Investigation by Molecular Modeling, Molecular Mechanics, and Molecular Dynamics

Molecular mechanics and molecular dynamics studies are performed to investigate the conformational preference of cell surface higher gangliosides (GT1A and GT1B) and their interaction with Cholera Toxin. The water mediated hydrogen bonding network exists between sugar residues in gangliosides. An integrated molecular modeling, molecular mechanics, and molecular dynamics calculation of cholera toxin complexed with GT1A and GT1B reveal that, the active site of cholera toxin can accommodate these higher gangliosides. Direct and water mediated hydrogen bonding interactions stabilize these binding modes and play an essential role in defining the order of specificity for different higher ganglioside towards cholera toxin. This study identifies that the binding site of cholera toxin is shallow and can accommodate a maximum of two NeuNAc residues. The NeuNAc binding site of cholera toxin may be crucial for the design of inhibitors that can prevent the infection of cholera.

Differential Activities of Plant Polyphenols on the Binding and Internalization of Cholera Toxin in Vero Cells

Plant polyphenols, RG-tannin, and applephenon had been reported to inhibit cholera toxin (CT) ADP-ribosyltransferase activity and CT-induced fluid accumulation in mouse ileal loops. A high molecular weight fraction of hop bract extract (HBT) also inhibited CT ADP-ribosyltransferase activity. We report here the effect of those polyphenols on the binding and entry of CT into Vero cells. Binding of CT to Vero cells or to ganglioside GM1, a CT receptor, was inhibited in a concentration-dependent manner by HBT and applephenon but not RG-tannin. These observations were confirmed by fluorescence microscopy using Cy3-labeled CT. Following toxin binding to cells, applephenon, HBT, and RG-tannin suppressed its internalization. HBT or applephenon precipitated CT, CTA, and CTB from solution, creating aggregates larger than 250 kDa. In contrast, RG-tannin precipitated CT poorly; it formed complexes with CT, CTA, or CTB, which were demonstrated with sucrose density gradient centrifugation and molecular weight exclusion filters. In agreement, CTA blocked the inhibition of CT internalization by RG-tannin. These data suggest that some plant polyphenols, similar to applephenon and HBT, bind CT, forming large aggregates in solution or, perhaps, on the cell surface and thereby suppress CT binding and internalization. In contrast, RG-tannin binding to CT did not interfere with its binding to Vero cells or GM1, but it did inhibit internalization.

Disialogangliosides and Their Interaction with Cholera Toxin—Investigation by Molecular Modeling, Molecular Mechanics and Molecular Dynamics

Molecular mechanics and molecular dynamics studies are performed to investigate the conformational preference of cell surface disialogangliosides (GD1A, GD1B and GD3) in aqueous environment. The molecular mechanics calculation reveals that water mediated hydrogen bonding network plays a significant role in the structural stabilization of GD1A, GD1B and GD3. These water mediated hydrogen bonds not only exist between neighboring residues but also exist between residues that are separated by 2 to 3 residues in between. The conformational energy difference between different conformational states of gangliosides correlates very well with the number of water mediated and direct hydrogen bonds. The spatial flexibility of NeuNAc of gangliosides at the binding site of cholera toxin is worked out. The NeuNAc has a limited allowed eulerian space at the binding site of Cholera Toxin (2.4%). The molecular modeling, molecular mechanics and molecular dynamics of disialo- ganglioside-cholera toxin complex reveal that cholera toxin can accommodate the disialo- ganglioside GD1A in three different modes. A single mode of binding is permissible for GD1B and GD3. Direct and water mediated hydrogen bonding interactions stabilizes these binding modes and play an essential role in defining the order of specificity for different disialogangliosides towards cholera toxin. This study not only provides models for the disialoganglioside-cholera toxin complexes but also identifies the NeuNAc binding site as a site for design of inhibitors that can restrict the pathogenic activity of cholera toxin.

Monosialogangliosides and Their Interaction with Cholera Toxin—Investigation by Molecular Modeling and Molecular Mechanics

Molecular mechanics and molecular dynamics studies are performed to investigate the conformational preference of cell surface monosialogangliosides (GM3, GM2 and GM1) in aqueous environment. Water mediated hydrogen bonding network plays a significant role in the structural stabilization of GM3, GM2 and GM1. The spatial flexibility of NeuNAc of gangliosides at the binding site of cholera toxin reveals a limited allowed eulerian space of 2.4% with a much less allowed eulerian space (1.4%) for external galactose of GM1. The molecular mechanics of monosialoganglioside-cholera toxin complex reveals that cholera toxin can accommodate the monosialogangliosides in three different modes. Direct and water mediated hydrogen bonding interactions stabilize these binding modes and play an essential role in defining the order of specificity for different monosialogangliosides towards cholera toxin. This study identifies the NeuNAc binding site as a site for design of inhibitors that can restrict the pathogenic activity of cholera toxin.

Association of Engrailed homeoproteins with vesicles presenting caveolae-like properties.

We report here that the homeoproteins Engrailed-1 and Engrailed-2 are present in specific non-nuclear subcellular compartments. Using electron microscopy, we observed that chick-Engrailed-2 expressed in COS-7 cells associates with membrane fractions that are characterized as caveolae. This characterization is based on morphological, biochemical and immunological criteria such as, in particular, the absence of clathrin coat and the presence of caveolin and cholera toxin-binding sites. These data are fully confirmed by subcellular fractionation experiments, which demonstrate that transfected chick-Engrailed-2 is present in low density membrane fractions that are resistant to Triton X-100, enriched in caveolin and solubilized by the addition of a cholesterol-binding detergent, a set of properties highly characteristic of caveolae. The association of Engrailed-2 with specific membrane fractions observed after transfection in COS-7 cells is also observed for endogenous Engrailed-1 and Engrailed-2 expressed at late embryonic stages in the cerebellum and posterior mesencephalon of the rodent. Indeed, the two proteins are present in membrane fractions that bear all the characteristics of microdomains or caveolae-like domains, i.e. Triton X-100 resistance, saponin solubilization, low density on sucrose gradients, enrichment in glycosphingolipid GM1, absence of transmembrane Neural Cell Adhesion Molecule, presence of the glypiated (GPI-anchored) glycoprotein F3/F11 and of the acylated growth-associated protein GAP-43. Finally we demonstrate that part of the membrane-associated Engrailed, either expressed in COS-7 cells or endogenously present in neural tissues, is not accessible to proteolytic enzymes unless the membranes have been permeabilized with detergent. This study suggests that, in addition to their well-known presence in the nucleus, Engrailed proteins are also associated with caveolae-like vesicles that are primarily transported anterogradely into the axon, and that they can get access to a compartment compatible with secretion.

Codistribution of choleratoxin binding sites and tyrosine hydroxylase/FGF-2 immunoreactive nigral nerve cells.

By means of light and confocal laser microscopical analysis of choleratoxin (CT) binding sites indicating the localization of the ganglioside GM1, evidence has been obtained for the presence of ganglioside GM1 in discrete nerve terminals, some of them identified by synapsin-1 immunoreactivity (IR), with a focal distribution in the nerve terminal membrane. Double immunolabelling studies demonstrate that GM1 positive nerve terminals are associated with tyrosine hydroxylase/fibroblast growth factor-2 (TH/FGF-2) immunoreactive dopamine (DA) perikarya in the zona compacta of the rat substantia nigra. It is suggested that GM1 may be released from these terminals to become incorporated into the nerve cell membrane of the FGF-2-containing DA nigral nerve cells, where they may enhance the activity of neurotrophic factor receptors such as those for FGF-2.

On the cellular localization and distribution of the ganglioside GM1 in the rat brain as revealed by immunofluorescence histochemistry of cholera toxin binding sites.

La localisation et la distribution du ganglioside GM1 est etudiee en utilisant des anticorps monoclonaux contre la toxine du cholera chez le rat. Les protomeres B des recepteurs de la toxine du cholera sont detectes par immunofluorescence

Identification of cholera toxin-binding sites in the nucleus of intestinal epithelial cells

Post-embedding immunogold electron microscopy shows several binding sites for cholera toxin in mouse intestinal epithelial cells, particularly in the heterochromatin of the nucleus as well as in the plasma membrane. Anti-ganglioside GM1 antibodies also bound to the nucleus, but did not interfere with the binding of toxin. 125I-labelled toxin bound specifically to a nuclear preparation from rabbit intestinal cells.

Selective expression of cholera toxin-receptor in rat medullary thymocytes.

Cholera toxin binding sites in frozen sections of rat thymus were studied by indirect immunofluorescence and were found to be localized selectively in the medulla of thymic lobules. By flow cytometric analysis, two populations of thymocytes differing in size and reactivity towards cholera toxin were detected; the small thymocytes comprising 15% of the total thymocytes were found to express the receptor. The two kinds of cells isolated were not however, distinctly different in morphology. But ganglioside GM1, the cholera toxin binding site in the small thymocytes, when quantified by TLC-immunostaining, was contained at a higher concentration than in the large thymocytes. These results indicate that GM1 should be a useful marker for analysis of thymocyte differentiation, and that cholera toxin-mediated thymocyte proliferation might only occur in the small medullary thymocytes.

Structural features of the binding site of cholera toxin inferred from fluorescence measurements.

The dependence on pH of the fluorescence of cholera toxin and its A and B subunits has been studied at 25 degrees C. The fluorescence intensity of cholera toxin is highly pH-dependent. In the pH range 7-9.5 it reaches a maximum corresponding to a quantum yield of 0.076. In the pH range 4-7 a strong increase in fluorescence intensity is observed (delta Q/Qmax = 0.64). Evaluation of the pH sensitivity of the fluorescence intensity of the A and B subunits reveals that the B subunit is mainly responsible for the observed pH effect (delta Q/Qmax for B subunit = 0.64). The intensity changes are paralleled by similar although less pronounced changes in the average fluorescence excited state life-time tau (delta tau/tau max = 0.33 for cholera toxin). Fluorimetric titration of the B subunit, which is related to the indole fluorescence of the lone Trp-88, reveals that the fluorescence intensity changes in the pH range 4-7 are due to reaction of two types of ionizable quencher displaying apparent pKa values of 4.4 and 6.2, respectively. It is suggested that the increase in fluorescence intensity with a midpoint at pH 6.2 is the result of deionization of the imidazolium side-chain of one or two out of the four histidine residues present in each beta-polypeptide chain, whereas a deionized carboxyl group is responsible for the quenching with midpoint at pH 4.4. Complex formation of cholera toxin or B subunit with the monosialoganglioside GM1 or the oligosaccharide moiety of GM1 (oligo-GM1) completely prevents the quenching by both quenchers. Addition of 6 M urea also eliminates the pH effect. The quenching is not the result of the dissociation of the B subunit into its constituent monomers. Upon fluorimetric titration of cholera toxin or B subunit above pH 9, a progressive drop in both fluorescence intensity and tau occurs. This decrease could be due to energy transfer from the indole moiety of Trp-88 to ionized tyrosines or by quenching through an unprotonated epsilon-amino group of lysine. Fluorimetric titration of the A subunit indicates that the tryptophan fluorescence is only moderately altered by ionizable groups displaying a pKa in the range 4 to 9. Activation of A subunit does not affect this lack of pH sensitivity. Above pH 9, however, a much more significant drop in the fluorescence intensity of activated A subunit occurs. The structural implications of the results are discussed.

Comparison of receptors for cholera and Escherichia coli enterotoxins in human intestine

Extraction of lipids from human small intestinal epithelial cells or brush borders removed specific binding sites for cholera toxin completely, but only about 50% of the receptor sites for Escherichia coli heat-labile enterotoxin. Both cholera toxin and E. coli heat-labile enterotoxin bound strongly to ganglioside GM1 in the lipid extract and, to a lesser extent, to another monosialoganglioside and to GD1b. The results suggest that E. coli heat-labile enterotoxin binds to both ganglioside and glycoprotein receptor sites of the human small intestinal epithelium, whereas cholera toxin binding was restricted to the ganglioside receptors.

Ganglioside localization on myelinated nerve fibres by cholera toxin binding

GM1 ganglioside has been localized on the surfaces of myelinated, peripheral nerve fibres by using immunofluorescence to detect cholera toxin receptors. Unfixed, mouse sciatic nerves were teased into individual, intact fibres in order to expose their extracellular surfaces. Cholera toxin binding sites were abundant at all nodes of Ranvier; they were scarce on the internodal fibre surfaces. The nodal receptors were resistant to various degradative enzymes, including trypsin and proteinase K. Proteases did not unmask receptors on the internodal surfaces. Exogenous GM1 successfully competed for the toxin binding sites on the fibres. From this evidence and the specificity of cholera toxin binding, we conclude that GM1 ganglioside is abundantly present on the membrane surfaces of peripheral nodes of Ranvier and is not present on the internodal Schwann cell surfaces in an appreciable amount. The patterns of fluorescence within the node suggest that the axon and Schwann cell structures are sites where GM1 is localized. Treatment of the teased fibres with Vibrio cholerae neuraminidase, which is known to reduce polysialogangliosides to the monosialoganglioside GM1, induced cholera toxin binding on the internodal Schwann cell surfaces. The induced receptors, as well as their precursors, were resistant to trypsin and proteinase K. We conclude that the internodal Schwann cell surface is rich in an unidentified polysialoganglioside(s) that can be converted to GM1 by neuraminidase.

Theoretical studies on the conformation of monosialogangliosides and disialogangliosides

The preferred conformation of gangliosides GM3, GM2, GM1, GD1a and GD1b have been studied by computing their potential energies. The conformation of NeuNAc in GM3 differs from that expected for the same residue in GM2 and GM1. The NeuNAc residues in GM2 and GM1 exhibit identical conformations. Theory predicts that the terminal NeuNAc of GD1a is conformationally similar to that of GM3 and that the internal one is similar in conformation to those present in GM2 and GM1 in agreement with NMR studies. The differences in chemical shifts of the C2 and C3 carbons of the internal and terminal NeuNAc of GD1a have been attributed to differences in orientation. The present studies suggest that the binding site of cholera toxin is much smaller than that of tetanus toxin. The preferred shape of these gangliosides correlate well with their biological properties.

Rabbit intestinal glycoprotein receptor for Escherichia coli heat-labile enterotoxin lacking affinity for cholera toxin.

The receptors for cholera toxin and Escherichia coli heat-labile toxin (LT) in rabbit small intestinal epithelium were characterized and compared. (i) In vivo studies in ligated intestinal loops showed that whereas LT B subunits could block the fluid secretogenic action of purified LT as well as cholera toxin, cholera toxin B subunits did not inhibit the LT response even when tested in a concentration 100-fold higher than one which gave complete blocking of cholera toxin action. (ii) In vitro studies indicated that isolated intestinal epithelial cells or brush-border membranes could bind about 10-fold more of E. coli LT than of cholera toxin. (iii) All binding sites for cholera toxin in duodenal, jejunal, or ileal mucosal cells or brush-border membranes were extracted by chloroform-methanol-water (4:8:3), which removed lipids quantitatively but did not extract glycoproteins. The extracted cholera toxin binding sites were to greater than 95% recovered in a monosialoganglioside fraction; quantitatively these sites closely corresponded to the concentration of chromatographically identified mucosal GM1 ganglioside (1 nmol of cholera toxin was bound per 1 to 2 nmol of GM1). In contrast, a substantial fraction of mucosal binding sites for E. coli LT remained in the delipidized tissue residue, and these sites had properties consistent with a glycoprotein nature. Thus, whereas cholera toxin appeared to bind highly selectively to GM1 ganglioside receptor sites of rabbit small intestine, E. coli LT bound both to GM1 ganglioside and to a main glycoprotein receptor for which cholera toxin lacks affinity.

Immunocytochemical study on cholera toxin binding sites by monoclonal anti-cholera toxin antibody in neuronal tissue culture

An indirect method of immunocytochemistry showed that cholera toxin and its B-subunit served as specific neuronal surface markers in conjunction with monoclonal anti-cholera toxin and FITC labeled anti-mouse Fab. The cell types which get specifically stained in culture were peripheral neurons from dorsal root ganglion cells, superior cervical ganglion cells, and cerebral neurons, all of which were rat tissue, and NGF-treated PC12 cells. Non-neuronal cells, i.e. Schwann cells, fibroblasts and glia cells, were not stained. This method can, therefore, be used to distinguish neurons from non-neuronal cells in neuronal tissue cultures, as in the case of tetanus toxin described in the literature 5,10,23,25,28,29.

Characterization of the cholera toxin receptor on Balb/c 3T3 cells as a ganglioside similar to, or identical with, ganglioside GM1. No evidence for galactoproteins with receptor activity.

Balb/c 3T3 cells contain a large number [(0.8-1.6) x 10(6)] of high-affinity (half-maximal binding at 0.2 nM) binding sites for cholera toxin that are resistant to proteolysis, but are quantitatively extracted with chloroform/methanol. The following evidence rigorously establishes that the receptor is a ganglioside similar to, or identical with, ganglioside GM1 by the galactose oxidase/NaB3H4 technique on intact cells was inhibited by cholera toxin. (2) Ganglioside GM1 was specifically adsorbed from Nonidet P40 extracts of both surface- (galactose oxidase/NaB3H4 technique) and metabolically ([1-14C]palmitate) labelled cells in the presence of cholera toxin, anti-toxin and Staphylococcus aureus. (3) Ganglioside GM1 was the only ganglioside labelled when total cellular gangliosides separated on silica-gel sheets were overlayed with 125I-labelled cholera toxin, although GM3 and GD1a were the major gangliosides present. In contrast no evidence for a galactoprotein with receptor activity was obtained. Cholera toxin did not protect the terminal galactose residues of cell-surface glycoproteins from labelling by the galactose oxidase/NaB3H4 technique. No toxin-binding proteins could be identified in Nonidet P40 extracts of [35S]-methionine-labelled cells by immunochemical means. After sodium dodecyl sulphate/polyacrylamide-gel electrophoresis none of the major cellular galactoproteins identified by overlaying gels with 125I-labelled ricin were able to bind 125I-labelled cholera toxin. It is concluded that the cholera toxin receptor on Balb/c 3T3 cells is exclusively ganglioside GM1 (or a related species), and that cholera toxin can therefore be used to probe the function and organisation of gangliosides in these cells as previously outlined [Critchley, Ansell, Perkins, Dilks & Ingram (1979) J. Supramol. Struct. 12, 273-291].

Ultracytochemistry of cholera-toxin binding sites in ciliary processes.

Cholera toxin reduces the rate of aqueous humor in concentrations (10-11M) that do not disturb the morphology of the aqueous-humor forming epithelial cells of the ciliary processes of the rabbit eye. The search for an endogenous mediator of aqueous-humor formation comparable to cholera toxin in its mode of operation prompted us to map the distribution of cell surface receptors for cholera toxin in the ciliary processes of the eyes of rabbits. Cytochemical studies were carried out with the use of conjugates of cholera toxin to fluorescein isothiocyanate (CT-FITC) and to horseradish peroxidase (CT-HRP), and of the B subunit of cholera toxin to horseradish peroxidase (B-HRP). Multiple fluorescent CT-FITC binding sites were observed on the outer nonpigmented epithelial layer near the crests of the processes. Processes incubated with CT-HRP in vitro showed surface staining of 30-40% of the nonpigmented epithelial cells. A prominent reaction product was observed along the basal and lateral plasma membranes of these cells. In vivo studies carried out after arterial infusion of B-HRP showed a reproducible dense reaction product between the apical surfaces of the pigmented epithelium (PE) and of the nonpigmented epithelium (NPE) facing each other. Aggregations of reaction product were observed with the electron microscope in the extracellular space between the apices of PE and NPE. The apical plasma membrane of the endothelium of the blood vessels near the crests of the ciliary processes was stained after either in vivo or in vitro exposure to peroxidase conjugates. These findings indicate that the cell-surface receptors which mediate the action of cholera toxin on aqueous humor formation are very likely localized in the apical plasma membranes of the epithelium of the ciliary processes.

Differential expression of cell surface binding sites for cholera toxin in acute and chronic leukaemias.

Binding of purified cholera toxin to cell surface receptors has been visualized by an indirect immunofluorescence procedure. Normal nucleated cells from blood, bone marrow and lymphoid tissues, express these receptors with the possible exception of erythroid precursors. Cells from patients with chronic lymphoid or myeloid leukaemias have a normal receptor expression but acute leukaemic cells showed a marked deficiency in cholera toxin binding. Insertion of purified Gm ganglioside into membranes of acute leukaemic cells provided cellular binding sites for the toxin.