GM1 Oligosaccharide Research Articles

Second-generation mimics of ganglioside GM1 oligosaccharide: a three-dimensional view of their interactions with bacterial enterotoxins by NMR and computational methods.

As a step to delineate a strategy of ligand design for cholera toxin (CT), NMR studies were performed on several mimics of the GM1 ganglioside oligosaccharide. The conformation of these analogues was investigated first in solution and then upon binding to cholera toxin by transferred nuclear Overhauser effect (TR-NOE) measurements. It was demonstrated that CT selects a conformation similar to the global minima of the free saccharides from the ensemble of presented conformations. No evidence of major conformational distortions was obtained, but one or two of the available conformers of the hydroxyacid side chain appear to be selected in the bound state. The NMR data were interpreted with the aid of computer models, generated and analyzed by using a combination of different approaches (MacroModels' MC/EM and MC/SD, Autodock, and GRID). Analysis of the NMR data supported by computational studies allowed us to interpret the experimental observations and to derive workable models of the ligand:toxin complexes. These models suggest that the higher affinity of the (R)-lactic acid derivative 3 may stem from lipophilic interactions with a hydrophobic area in the toxin binding site located in the vicinity of the sialic acid side chain binding region of the CT:GM1 complex, and formed by the side chain of Ile-58 and Lys-34. Thus, the models obtained have allowed us to make useful design suggestions for the improvement of ligand affinity.

Simulation of carbohydrate-protein interactions: computer-aided design of a second generation GM1 mimic.

The oligosaccharide of ganglioside GM1 [Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3)Galbeta1-4Glcbeta1-1Cer] is the cellular target of two bacterial enterotoxins: the cholera toxin (CT) and the heat-labile toxin of E. coli (LT). We recently reported that the pseudosaccharide 2 [Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3)DCCHD] is a high-affinity ligand for CT. and thus a functional mimic of GM1 (Bernardi, A., Checchia, A., Brocca, P., Sonnino, S. and Zuccotto, F., J. Am. Chem. Soc., 121 (1999) 2032-2036). In this paper we describe the design of a second-generation mimic, formally obtained from 2 by inverting the configuration of a single stereocenter, thus transforming a N-acetyl galactosamine into a N-acetyl glucosamine. The design process involved modeling of the free ligand and its LT complex, followed by qualitative and quantitative comparison with the corresponding structures of 2. The protocol employed relied on both conformational search and molecular dynamics methodologies to account for the flexibility of both the ligand and the protein receptor. The conformational search of the LT:inhibitor complex showed that, compared to 2, the new compound can insert one more hydroxy group within the protein binding site. Molecular dynamics simulations showed that, in turn, this may trigger a series of rearrangements and reorientations of side chains and crystallographic water molecules in the toxin, leading to new H-bond contacts which may result in enhanced affinity of the new inhibitor. FEP calculations were performed by mutating the structure of 2 in solution and in the protein complex, and the prediction was made that the second-generation mimic should be a stronger binder than its parent compound.

Sugar Mimics: An Artificial Receptor for Cholera Toxin

The paper describes the pseudosugar 2 [Galβ1−3GalNAcβ1−4(NeuAcα2−3)DCCHD], a high affinity binder of cholera toxin (CT). The molecule was designed using molecular modeling techniques to mimic the natural CT membrane receptor, ganglioside GM1. The central residue of GM1, a 3,4-disubstituted galactose unit, was recognized as the ganglioside scaffold element and substituted with a conformationally locked cyclohexanediol (DCCHD). DCCHD was synthesized in enantiopure form using enantioselective Diels Alder methodology and regioselectively α-sialylation at the equatorial position. Glycosylation with a Galβ(1−3)GalNAc donor completed the synthesis of 2. The solution structure of 2 and its binding ability to CT were found to be analogous to those of the GM1 oligosaccharide.

Conformational Analysis of GM1 Oligosaccharide in Water Solution with a New Set of Parameters for the Neu5Ac Moiety

Conformational Analysis of GM1 Oligosaccharide in Water Solution with a New Set of Parameters for the Neu5Ac Moiety

Molecular modeling of the conformational and sodium ion binding properties of the oligosaccharide component of ganglioside GM1.

The receptor-like recognition behavior of the GM1 ganglioside has been examined theoretically in terms of conformational and binding properties. Modeling was conducted at two limiting conditions of dielectric constant in order to determine sensitivity to scaling of coulombic interactions. A systematic conformational search of the GM1 oligosaccharide in the absence of explicit solvent molecules indicates that there are many inherently low energy conformational states. Up to 39 conformers were found with energies within 5 kcal/mole of the observed lowest energy conformer. Using a dielectric constant of 80, a systematic search of sodium binding sites on GM1 identified 37 sites where a positively charged group might bind, while at least 12 sites were identified using a dielectric constant of 1. Notably important binding sites include pockets formed by the proximity of glycosidic (O1), sugar ring (O5), and exocyclic methylene hydroxyl (OH6) oxygens on the sugars. The oxygens of acetyl groups attached to sugars also contribute to the binding. Direct coordination with the carboxylate of sialic acid is not a prerequisite for cationic binding. The large number of conformational states and binding sites for the GM1 oligosaccharide are paradoxical to the specific recognition behavior of the molecule. This paradox can be explained in terms of bridging ligands, which are found from molecular dynamics to be capable of stabilizing molecular conformation.

Effects of specific gangliosides on the in vitro proliferation of MPTP-susceptible cells.

To determine which portion of a ganglioside molecule might be necessary for the enhancement of recovery from MPTP-induced lesions, the ability of specific gangliosides to stimulate proliferation of MPTP-treated 140-3 cells was investigated. The results indicate that of the gangliosides tested, GM1 was the most effective. Although GD1a and GT1b were able to enhance the proliferation of MPTP-treated cells, twice as much GT1b was needed to induce the same effect seen with GM1. In contrast, asialo-GM1, GM2, and GM3 were ineffective at promoting proliferation of MPTP-treated cells. The isolated oligosaccharide of GM1 had little effect. These results indicate that in addition to the sialosyl residue, at least the Gal(beta 1-3)Gal-NAc portion of the oligosaccharide chain and the ceramide moiety are essential for the induction of proliferation of the MPTP-treated cells. Investigation of the time of addition of GM1 on its ability to counteract the MPTP-induced inhibition of 140-3 cell proliferation indicated that addition of GM1 before or concomitantly with MPTP resulted in a significant reduction in MPTP-induced inhibition of cell proliferation.

Intoxication of cultured cells by cholera toxin: evidence for different pathways when bound to ganglioside GM1 or neoganglioproteins.

We previously reported that when the oligosaccharide of ganglioside GM1 is covalently attached to cell surface proteins of GM1-deficient rat glioma C6 cells, the cells bind large amounts of cholera toxin (CT) but their cAMP response to CT is not enhanced [Pacuszka, T., & Fishman, P. H. (1990) J. Biol. Chem. 265, 7673-7668]. We now report that when such cells were exposed to CT in the presence of chloroquine, an acidotropic agent, they accumulated cAMP. This raised the possibility that CT bound to cell surface "neoganglioproteins" may be entering the cells through a different pathway from that of CT-bound GM1. To further explore this phenomenon, we covalently attached GM1 oligosaccharide to human transferrin (Tf). The modified protein (GM1OS-Tf) bound with high affinity to Tf receptors on HeLa cells and increased the binding of CT to the cells. The bound CT, however, was unable to activate adenylyl cyclase as measured by cyclic AMP accumulation. By contrast, treatment of HeLa cells with GM1 increased both CT binding and stimulation of cyclic AMP accumulation. Control cells and cells treated with either GM1 or GM1OS-Tf were exposed to CT in the presence of chloroquine. Whereas chloroquine had little or no effect on the response of control or GM1-treated cells to CT, it made the cells treated with GM1OS-Tf responsive to the toxin. Our results indicate that CT bound to its natural receptor GM1 enters the cells through a pathway different from that of toxin bound to neoganglioproteins.

Synthesis of a photoreactive, radiolabelled derivative of the oligosaccharide of GM1 ganglioside.

A photoreactive, radioiodinatable derivative of the oligosaccharide (GM1OS) of ganglioside GM1 was synthesized as follows: GM1OS was generated from GM1 by ozonolysis and alkaline fragmentation, and reductively aminated to GM1OSNH2 (1-amino-1-deoxymonosialogangliotetraitol). The latter compound was then reacted with N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA) to form GM1OSNH-ASA [1-(4-azidosalicoylamido)-1-deoxymonosialogangliotetraitol], which was radioiodinated and further purified. To test the [125I]GM1OSNH-IASA [1-(4-iodoazidosalicoylamido)-1-deoxymonosialogangliotetraitol+ ++] as a probe for ganglioside-binding proteins, the derivative was incubated with cholera toxin, which specifically binds GM1, followed by photolysis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The probe only labelled the B or binding subunit of cholera toxin, but not the A or adenylyl cyclase activating subunit. Labelling was inhibited by excess GM1OS, but not by the oligosaccharides from gangliosides GD1a and GD1b. [125I]GM1OSNH-IASA and analogous oligosaccharide derivatives may be valuable probes for detecting ganglioside-binding proteins.

Characterization of a nonspecific activator protein for the enzymatic hydrolysis of glycolipids.

We have studied the substrate specificities of a non-specific activator protein on the enzymatic hydrolyses of the following compounds: GM1 and GM2, as well as several of their derivatives including oligosaccharides, GgOse3Cer-II3-sulfate and LacCer-II3-sulfate, Gb-Ose3Cer and GbOse4Cer, three neolacto-series glycosphingolipids, and two non-ceramide glycolipids. Our results show that this activator protein has a broad spectrum of activity and exhibits the properties of a nonspecific natural detergent. The evidence of non-specificity was the ability of this activator protein to stimulate the hydrolyses of glycolipids, regardless of glycosphingolipids or non-ceramide glycolipids, carried out by glycosidases from animals, plants, and microorganisms. Its activity was, however, limited to substrates that had a lipid moiety. The oligosaccharide of GM1 and deacetyl-fatty acid free GM1 (II3-NeuGg-Ose4-sphingosine) were hydrolyzed by beta-galactosidase in the absence of this activator protein.

Antigenic Determinants of the Cholera/Coli Family of Enterotoxins

Hybridoma-derived monoclonal antibodies were raised to enterotoxins of the cholera family and to chimeric B-subunit proteins in which individual amino acid residues of a heat-labile, cholera-related enterotoxin from an Escherichia coli strain of porcine origin (P-LT) were substituted with corresponding residues from such an enterotoxin from an E. coli strain of human origin (H-LT). Single amino acid substitutions were found to have profound effects on the physicochemical behavior of the proteins and on their immunologic reactivity. With the use of enzyme-linked immunosorption assays (ELISAs) with and without the GM1 ganglioside receptor for these toxins, several distinct epitopes in GM1-binding domains were identified by different monoclonal antibodies. Polyclonal rabbit antisera to synthetic peptides of the cholera enterotoxin B subunit were cross-reactive to various degrees with the proteins in our library, which include two different cholera enterotoxins, two H-LTs, P-LT, and four chimeric proteins. Some of these reactions were blocked by GM1 ganglioside but not the oligosaccharide of GM1, a finding suggesting that the peptides generated antibodies to epitopes near, but not in, a GM1-binding domain. A hypothetical evolutionary tree based on the reported amino acid sequences of the various enterotoxins is constructed. As additional enterotoxins are described, it will be interesting to determine if and where they fit in this scheme.

Epitopes of the Cholera Family of Enterotoxins

Hybridoma-derived monoclonal antibodies were raised to enterotoxins of the cholera family and to chimeric B-subunit proteins in which individual amino acid residues of a heat-labile, cholera-related enterotoxin from an Escherichia coli strain of porcine origin (P-LT) were substituted with corresponding residues from such an enterotoxin from an E. coli strain of human origin (H-LT). Single amino acid substitutions were found to have profound effects on the physicochemical behavior of the proteins and on their immunologic reactivity. With the use of enzyme-linked immunosorption assays (ELISAs) with and without the GM1 ganglioside receptor for these toxins, several distinct epitopes in GM1-binding domains were identified by different monoclonal antibodies. Polyclonal rabbit antisera to synthetic peptides of the cholera enterotoxin B subunit were cross-reactive to various degrees with the proteins in our library, which include two different cholera enterotoxins, two H-LTs, P-LT, and four chimeric proteins. Some of these reactions were blocked by GM1 ganglioside but not by the oligosaccharide of GM1, a finding suggesting that the peptides generated antibodies to epitopes near, but not in, a GM1-binding domain. A hypothetical evolutionary tree based on the reported amino acid sequences of the various enterotoxins is constructed. As additional enterotoxins are described, it will be interesting to determine if and where they fit in this scheme.

Interaction of cholera toxin with gangliosides: differential effects of the oligosaccharide of ganglioside GM1 and of micellar gangliosides

Ultraviolet difference absorption spectra of cholera toxin and its B protomer produced by the oligosaccharide moiety of the monosialoganglioside GM1 were measured as a function of the oligosaccharide concentration. In the presence of oligosaccharide, the spectrum is characterized by three peaks at 282, 288, and 292 nm. A linear increase in difference absorption was observed at these wavelengths vs. oligosaccharide concentration; a saturation effect occurred when the molar ratio of oligosaccharide to cholera toxin was higher than 5. The features of the spectra indicated that the binding with the oligosaccharide affected the environment of tryptophan and tyrosine residues of protomer B. In good agreement with the above results, circular dichroic spectra indicated also a local effect of the binding, mostly restricted to protomer B, while the residues of protomer A remained largely unperturbed. Difference absorption spectra were also measured for cholera toxin in the presence of ganglioside and detergent micelles. The employed gangliosides GD1a and GT1b, unable to bind cholera toxin, interact with the protein by way of contaminating traces of GM1. The preparations of GD1a and GT1b contained 0.8-1.0% (w/w) and 0.4-0.5% (w/w) of GM1, respectively. The results obtained with ganglioside GD1a and GT1b in contrast with the observations made with the oligosaccharide of GM1 indicated a major conformational change of the toxin structure. Upon comparison of the conformational change induced by ganglioside micelles with that induced by sodium dodecyl sulfate it may be suggested that the ganglioside micelle, behaving as a detergent, alters the structure of the toxin such as to induce the penetration of protomer A into the lipid milieu.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative description of the binding of GM1 oligosaccharide by cholera enterotoxin.

We present a quantitative molecular interpretation of binding between the five B subunits of cholera enterotoxin and the oligosaccharide of ganglioside GM1, based on the currently accepted quaternary structure of the toxin and principles of multiple equilibria. A sequential binding equation is derived and fitted to published binding data obtained by equilibrium dialysis. In one study of binding to reduced toxin (I), intact toxin (II), and isolated B subunits (III) at low concentrations, analysis by the Hill equation suggested that binding was positively cooperative and that there were only four binding sites per toxin molecule; individual affinity constants could not be estimated because of the empirical nature of the Hill equation. Our analysis suggests that the evidence for positive cooperativity is stronger for I and III than for II. Affinity constants for the first binding step are about 2.0-2.1 microM-1 for I and 2.5-2.7 microM-1 for II and III; those for the second binding step are about 3.5-5.0 microM-1 and for I and III, but only 2.5 microM-1 for II. Constants for later binding steps are apparently within the range of 2-7 microM-1. Predictions of the sequential model at higher ligand concentrations diverge substantially from those of the Hill equation, and are supported by data obtained at higher protein and ligand concentrations. Thus all available equilibrium dialysis data are consistent with a single set of affinity constants and with the hypothesis of five equivalent binding sites.

Observation by 13C NMR of interactions between cholera toxin and the oligosaccharide of ganglioside GM1.

The initial event in the action of cholera toxin on intact cells is its recognition of cell-surface receptors, molecules of ganglioside GM1. We have studied details of this interaction by 13C NMR, which enables us to examine simultaneously both the protein and the ganglioside or, as in the present instance, its oligosaccharide portion. 13C NMR spectra of the toxin are consistent with the long correlation times expected for this 84,000-dalton protein. They show, however, some resolved resonances (including one tentatively assigned to the epsilon 2 carbon of tryptophan, 138.3 ppm downfield from tetramethylsilane). When oligosaccharide is added to the toxin this resonance broadens or moves further upfield to reside under phenylalanine resonances at 136.7 ppm. Of the seven tryptophan residues in cholera toxin, five are in the B subunits which bind GM1, so that the data are consistent with a resonance shift for the epsilon 2 carbons of these residues on binding the oligosaccharide. Resonances arising from the anomeric and methylene carbons of the sialic acid moiety of the oligosaccharide are also shifted. Comparison with corresponding chemical shifts in a series of model compounds suggest that the latter effects may originate in a toxin-induced conformational change in the oligosaccharide. At high resolution, anomeric carbon resonances of terminal galactose residues in free and bound oligosaccharide are also resolved.

Mechanism of action of cholera toxin: effect of receptor density and multivalent binding on activation of adenylate cyclase.

Choleragen (cholera toxin) activates adenylate cyclase in HeLa cells, which contain less than 15,000 toxin receptors per cell, in a time- and concentration-dependent manner. Activation is blocked by the addition of the oligosaccharide chain of the ganglioside GM1, the receptor for the toxin. When the cells are preincubated with choleragen at 4 degrees C and then incubated with oligosaccharide at 37 degrees C, adenylate cyclase is activated less than 10%. When the preincubation phase is above 18 degrees C, adenylate cyclase becomes activated and the amount of activation depends on the time of preincubation. This inhibitory effect of the oligosaccharide is also observed with human lymphocytes and rat glial C6 cells but not with Friend erythroleukemic and mouse neuroblastoma N18 cells. The latter two cell lines have large numbers ot toxin receptors, whereas the former two cell lines have few receptors. When the number of toxin receptors in HeLa and C6 cells is increased by treating the cells with GM1, activation of adenylate cyclase by choleragen is no longer blocked by the oligosaccharide. The oligosaccharide has a corresponding effect on the displacement of bound 125I-choleragen. When bound to cells at 4 degrees C, most of the radiotoxin is displaced from HeLa, C6, and lymphocytes but not from Friend, N18, or HeLa cells pretreated with GM1. In untreated HeLa cells, dissociation of toxin-receptor complexes by the oligosaccharide depends on the time and temperature of complex formation; above 18 degrees C, the toxin rapidly becomes stably bound to the cells. The inhibitory effect of GM1 oligosaccharide us reversible, as, once it is removed, the small amount of toxin that remains bound can activate adenylate cyclase. These results are consistent with a model in which choleragen, which is multivalent, must bind to several GM1 molecules on the cell surface in order to subsequently activate adenylate cyclase. Lateral mobility of toxin-receptor complexes may be required only to achieve multivalent binding in cells with few receptors.

Interaction of choleragen with the oligosaccharide of ganglioside GM1: evidence for multiple oligosaccharide binding sites.

Interaction of choleragen with the oligosaccharide of ganglioside GM1: evidence for multiple oligosaccharide binding sites.