Fluorescent derivatives of ganglioside GM1 function as receptors for cholera toxin

A fluorescent derivative of ganglioside GM1 was prepared by oxidation of the sialic acid residue with sodium periodate and reaction of the resulting aldehyde with Lucifer yellow CH. The biological activity of the fluorescent derivative was compared with that of native GM1 using GM1-deficient rat glioma C6 cells. When the cells were exposed to either native or fluorescent GM1, their ability to bind 125I-labeled cholera toxin was increased to a similar extent. This increase in binding was directly proportional to the amount of ganglioside added to the medium. The affinity of the toxin for cells treated with either native or fluorescent GM1 also was similar. More importantly, the fluorescent GM1 was as effective as native GM1 in enhancing the responsiveness of the cells to cholera toxin. Thus, the ganglioside-treated cells exhibited a 9-fold increase in toxin-stimulated cyclic AMP production over cells not exposed to GM1. There was a similar increase in iodotoxin binding and toxin-stimulated cyclic AMP accumulation in cells treated with other GM1 derivatives containing rhodaminyl or dinitrophenyl groups. On the basis of these results, it is clear that these modified gangliosides retain the ability to function as receptors for cholera toxin. Consequently, fluorescent gangliosides are likely to be useful as probes for investigating the dynamics and function of these membrane components.     

Binding of cholera toxin B-subunits to derivatives of the natural ganglioside receptor, GM1.

In a previous paper we showed that the B-pentamer of cholera toxin (CT-B) binds with reduced binding strength to different C(1) derivatives of N-acetylneuraminic acid (NeuAc) of the natural receptor ganglioside, GM1. We have now extended these results to encompass two large amide derivatives, butylamide and cyclohexylmethylamide, using an assay in which the glycosphingolipids are adsorbed on hydrophobic PVDF membranes. The latter derivative showed an affinity approximately equal to that earlier found for benzylamide ( approximately 0.01 relative to native GM1) whereas the former revealed a approximately tenfold further reduction in affinity. Another derivative with a charged C(1)-amide group, aminopropylamide, was not bound by the toxin. Toxin binding to C(7) derivatives was reduced by about 50% compared with the native ganglioside. Molecular modeling of C(1) and C(7) derivatives in complex with CT-B gave a structural rationale for the observed differences in the relative affinities of the various derivatives. Loss of or altered hydrogen bond interactions involving the water molecules bridging the sialic acid to the protein was found to be the major cause for the observed drop in CT-B affinity in the smaller derivatives, while in the bulkier derivatives, hydrophobic interactions with the protein were found to partly compensate for these losses.     

Second generation mimics of ganglioside GM1 as artificial receptors for cholera toxin: replacement of the sialic acid moiety

In a program directed towards the design and synthesis of mimics of ganglioside GM1, the NeuAc recognition domain was replaced by simple hydroxy acids, and the affinity of the new ligands to the cholera toxin was determined by fluorescence spectroscopy. The (R)-lactic acid derivative 4 was found to display the highest affinity of the series (KD = 190 microM).     

Ganglioside GM1 mimics: lipophilic substituents improve affinity for cholera toxin

Ganglioside GM1 mimics including (R)-2-hydroxy-3-cyclohexylpropionic acid or (R)-2-hydroxy-3-phenylpropionic acid as replacements for NeuAc are stronger cholera toxin binders than the parent ligand 2, which includes (R)-2-hydroxy-propionic acid.     

Neoglycolipid analogues of ganglioside GM1 as functional receptors of cholera toxin.

We synthesized several lipid analogues of ganglioside GM1 by attaching its oligosaccharide moiety (GM1OS) to aminophospholipids, aliphatic amines, and cholesteryl hemisuccinate. We incubated GM1-deficient rat glioma C6 cells with each of the derivatives as well as native GM1 and assayed the cells for their ability to bind and respond to cholera toxin. On the basis of the observed increase in binding of 125I-labeled cholera toxin, it was apparent that the cells took up and initially incorporated most of the derivatives into the plasma membrane. In the case of the aliphatic amine derivatives, the ability to generate new toxin binding sites was dependent on chain length; whereas the C10 derivative was ineffective, C12 and higher analogues were effective. Increased binding was dependent on both the concentration of the neoglycolipid in the medium and the time of exposure. Cells pretreated with the various derivatives accumulated cyclic AMP in response to cholera toxin, but there were differences in their effectiveness. The cholesterol and long-chain aliphatic amine derivatives were more effective than native GM1, whereas the phospholipid derivatives were less effective. The distance between GM1OS and the phospholipid also appeared to influence its functional activity. The neoglycolipid formed by cross-linking the amine of GM1OS to phosphatidylethanolamine (PE) with disuccinimidyl suberate was less effective than the neoglycolipid formed by directly attaching GM1OS to PE by reductive amination. Furthermore, insertion of a C8 spacer in the former neoglycolipid rendered it even less effective.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endocytosis of exogenous GM1 ganglioside and cholera toxin by neuroblastoma cells.

Cholera toxin (CT) covalently linked to horseradish peroxidase (HRP) is a specific cytochemical marker for its receptor, the monosialoganglioside GM1. The binding and endocytosis of exogenous [3H]GM1 by cultured murine neuroblastoma cells (line 2A [CCl-131] ), which contain predominantly GM3, was examined by quantitative electron microscope autoradiography. The relationship between exogenous receptor, [3H]GM1, and CT HRP was studied in double labeling experiments consisting of autoradiographic demonstration of [3H]GM1 and cytochemical visualization of HRP. Exogenous [3H]GM1 was not degraded after its endocytosis by cells for 2 h at 37 degrees C. Quantitative studies showed similar grain density distributions in cells treated with [3H]GM1 alone and in cells treated with [3H]GM1 followed by CT-HRP. Qualitative studies conducted in double labeling experiments showed autoradiographic grains over the peroxidase-stained plasma membrane, lysosomes, and vesicles at the trans aspect of the Golgi apparatus. The findings indicate that exogenous glycolipid is associated with the plasmid membrane of deficient cells and undergoes endocytosis. The quantitative ultra-structural autoradiographic studies are consistent with the hypothesis that the spontaneous endocytosis of exogenous [3H]GM1 controls the subsequent uptake of CT-HRP.     

Effects of ganglioside GM1 on the thermotropic behavior of cholera toxin B subunit

Summary The B, or binding, subunit of cholera enterotoxin forms a pentameric ring structure in the intact toxin, and also when the subunit is isolated from the A subunit. The thermal denaturation of the B subunit ring was examined by differential scanning calorimetry in the presence and absence of ganglioside G_M1, its natural receptor. In the absence of ganglioside an irreversible endotherm was observed with maximal excess apparent heat capacity, C_max, at 74.6° C. When the ganglioside was added in increasing amounts, multiple transitions were observed at higher temperatures, the most prominent having a C_max at 90.8° C. At high ganglioside concentrations, the 74.6° C transition was not observed. In addition to the thermodynamic results a model is proposed for the interaction of G_M1 and B subunit pentamer. This model is derived independently of the calorimetric results (but is consistent with such data) and is based upon considerations of the geometry of the G_M1 micelle-B subunit pentamer.Abbreviations M_r molecular weight in daltons - G_M1 H³Neu-AcGgOse₄Cer^* = Gall 3Ga1NAc1 4Gal-[3 - 2NeuAc]1 4Glc1 1Cer (asterisked form follows the recommendations of the IUPACIUB Commission on Biochemical Nomenclature, Ref. 3) - R molar ratio of G_M1 to B monomer - DSC differential scanning calorimetry - C_max excess apparent heat capacity - C_max maximal value of C_ex - t_m temperature (° C) at C_ex = C_max - t_1/2 peak width in °C at C_ex = C_max/2 - H_cal calorimetric enthalpy - C _p ^d van't Hoff enthalpy - C _p ^d change in specific heat accompanying denaturation     

Sialyloligosaccharides inhibit cholera toxin binding to the GM1 receptor

It is recognised that cholera toxin (Ctx) is a significant cause of gastrointestinal disease globally, particularly in developing countries where access to uncontaminated drinking water is at a premium. Ctx vaccines are prohibitively expensive and only give short-term protection. Consequently, there is scope for the development of alternative control strategies or prophylactics. This may include the use of oligosaccharides as functional mimics for the cell-surface toxin receptor (GM1). Furthermore, the sialic acid component of epithelial receptors has already been shown to contribute significantly to the adhesion and pathogenesis of Ctx. Here, we demonstrate the total inhibition of Ctx using GM1-competitive ELISA with 25mgmL(-1) of a commercial preparation of sialyloligosaccharides (SOS). The IC(50) value was calculated as 5.21mgmL(-1). One-hundred percent inhibition was also observed at all concentrations of Ctx-HRP tested with 500ngmL(-1) GM1-OS. Whilst SOS has much lower affinity for Ctx than GM1-OS, the commercial preparation is impure containing only 33.6% carbohydrate; however, the biantennary nature of SOS appears to give a significant increase in potency over constituent monosaccahride residues. It is proposed that SOS could be used as a conventional food additive, such as in emulsifiers, stabilisers or sweeteners, and are classified as nondigestible oligosaccharides that pass into the small intestine, which is the site of Ctx pathogenesis.     

Interaction of cholera toxin with ganglioside GM1 receptors in supported lipid monolayers

Lipid monolayers formed at the air-water interface containing the ganglioside GM1 in egg yolk phosphatidylcholine have been transferred according to the Langmuir-Blodgett technique to glass cover slips coated with octadecyl- or hexadecyltrichlorosilane and carbon-coated electron microscope grids. Monolayer transfer has been demonstrated with fluorescence microscopy, by the transfer of a fluorescent phospholipid analogue, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine or Lucifer yellow labeled GM1 (LY-GM1), incorporated into the lipid monolayer. Incubation of supported monolayers with solutions of fluorescein-labeled cholera toxin (FITC cholera toxin) resulted in specific binding of the toxin to monolayers containing GM1, as revealed by fluorescence microscopy. Lateral diffusion coefficients were measured for both the receptor (LY-GM1) [(3.9 +/- 2.1) X 10(-8) cm2/s] and the receptor-ligand complex (GM1-FITC cholera toxin) [(8.9 +/- 3.2) X 10(-9) cm2/s] according to the technique of fluorescence recovery after photobleaching. In separate studies, GM1-containing monolayers transferred to electron microscope grids were incubated with solutions containing unlabeled cholera toxin, followed by negative staining with uranyl acetate. Electron microscopy revealed patches of stained cholera toxin molecules (diameter approximately 70 A) in crystalline, two-dimensional hexagonal arrays. Optical diffraction and image reconstruction showed the arrangement of the cholera toxin molecules in a planar hexagonal cell, a = 81 A. These initial reconstructions give structural information to a resolution of approximately 30 A and indicate a doughnut-shaped molecule with a central aqueous channel.     

Liposome fluidity alters interactions between the ganglioside GM1 and cholera toxin B subunit

Cholera toxin is a complex protein with a biologically active protein (A subunit) and a cell targeting portion (B subunit). The B subunit is responsible for specific cell binding and entry of the A subunit. One way to limit potential toxicity of the toxin after exposure is to introduce cellular decoys to bind the toxin before it can enter cells. In this study the ganglioside GM1, a natural ligand for cholera toxin, was incorporated into liposomes and the interaction between fluorescent B subunit and the liposome determined. Liposome membrane fluidity was determined to play a major role in the binding between liposomes and the cholera toxin B subunit. Liposomes with lower fluidity demonstrated greater binding with the B subunit. The findings from this study could have important implications on formulation strategies for liposome decoys of toxins.     

Lipolytic action of cholera toxin on fat cells. Re-examination of the concept implicating GM1 ganglioside as the native membrane receptor.

The possible role of galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GM1) ganglioside in the lipolytic activity of cholera toxin on isolated fat cells has been examined. Analyses of the ganglioside content and composition of intact fat cells, their membranous ghosts, and the total particulate fraction of these cells indicate that N-acetylneuraminylgalactosylglucosylceramide (GM3) represents the major ganglioside, with substantial amounts of N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GM2) and smaller amounts of other higher homologues also present. Native GM1 was not detected in any of these preparations. Examination of the relative capacities of various exogenously added radiolabeled sphingolipids to bind to the cells indicated that GM2 and glucosylsphingosine were accumulated by the cells to extents comparable to GM1. Galactosylsphingosine and sulfatide also exhibited significant, although lesser, binding affinities for the cells. The adipocytes appeared to nonspecifically bind exogenously added GM1; saturation of binding sites for GM1 could not be observed up to the highest concentration tested (2 X 10(-4) M), wherein about 7 X 10(9) molecules were associated with the cells. Essentially all of this exogenously added GM1 was found bound to the plasma membrane "ghost" fraction. Investigation of the biological responses of the cells confirmed their sensitivities to both cholera toxin and epinephrine-stimulated lipolysis, as well as the lag period displayed during the toxin's action. While we could confirm that the toxin's lipolytic activity can be enhanced by prior treatment of the fat cells with GM1, several of the observed characteristics of this phenomenon differ from earlier reported findings. Accordingly, added GM1 was able to enhance only the subsequent rate, but not the extent, of toxin-stimulated glycerol release (lipolysis) from the cells. We also were unable to confirm the ability of GM1 to enhance the toxin's activity at either saturating or at low toxin concentrations. The limited ability of added GM1 to enhance the toxin's activity appeared in a unique bell-shaped dose-response manner. The inability of high levels of GM1 to stimulate a dose of toxin that was ineffective on native cells suggests that the earlier reported ability of crude brain gangliosides to accomplish this was due to some component other than GM1 in the crude extract. While several glycosphingolipids and some other carbohydrate-containing substances that were tested lacked the ability to mimic the enhancing effect of GM1, 4-methylumbelliferyl-beta-D-galactoside exhibited an effect similar to, although less pronounced than, that of GM1. The findings in these studies are unable to lend support to the earlier hypothesis that (a) GM1 is cholera toxin's naturally occurring membrane receptor on native fat cells, and (b) the ability of exogenously added GM1 to enhance the toxin's lipolytic activity represents the specific creation of additional natural receptors on adipocytes...     

Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide.

Cholera toxin (CT) is an AB5 hexameric protein responsible for the symptoms produced by Vibrio cholerae infection. In the first step of cell intoxication, the B-pentamer of the toxin binds specifically to the branched pentasaccharide moiety of ganglioside GM1 on the surface of target human intestinal epithelial cells. We present here the crystal structure of the cholera toxin B-pentamer complexed with the GM1 pentasaccharide. Each receptor binding site on the toxin is found to lie primarily within a single B-subunit, with a single solvent-mediated hydrogen bond from residue Gly 33 of an adjacent subunit. The large majority of interactions between the receptor and the toxin involve the 2 terminal sugars of GM1, galactose and sialic acid, with a smaller contribution from the N-acetyl galactosamine residue. The binding of GM1 to cholera toxin thus resembles a 2-fingered grip: the Gal(beta 1-3)GalNAc moiety representing the "forefinger" and the sialic acid representing the "thumb." The residues forming the binding site are conserved between cholera toxin and the homologous heat-labile enterotoxin from Escherichia coli, with the sole exception of His 13. Some reported differences in the binding affinity of the 2 toxins for gangliosides other than GM1 may be rationalized by sequence differences at this residue. The CTB5:GM1 pentasaccharide complex described here provides a detailed view of a protein:ganglioside specific binding interaction, and as such is of interest not only for understanding cholera pathogenesis and for the design of drugs and development of vaccines but also for modeling other protein:ganglioside interactions such as those involved in GM1-mediated signal transduction.     

Structure-based exploration of the ganglioside GM1 binding sites of Escherichia coli heat-labile enterotoxin and cholera toxin for the discovery of receptor antagonists

Ganglioside GM1 is the natural receptor for cholera toxin (CT) and heat-labile enterotoxin (LT), which are the causative agents of cholera and traveler's diarrhea, respectively. This observation suggests that small molecules interfering with this recognition process may prevent entry of the toxins into intestinal cells, thereby averting their devastating effects. Here, the terminal sugar of ganglioside GM1, galactose, was chosen as a lead in designing such receptor antagonists. Guided by the experimentally determined binding mode of galactose, we selected a "substructure" for searching the Available Chemicals Database, which led to the purchase of 35 galactose derivatives. Initial screening of these compounds in an LT ELISA revealed that 22 of them have a higher affinity for LT than galactose itself. A structurally diverse subset of these galactose derivatives was selected for determination of IC50 values in the LT ELISA and IC50 values in a CT assay, as well as for the determination of Kd's using the intrinsic fluorescence of LT. The best receptor antagonist found in this study was m-nitrophenyl alpha-galactoside with an IC50 of 0.6 (2) mM in the LT ELISA and 0.72 (4) mM in the CT assay, 100-fold lower than both IC50 values of galactose. Careful analysis of our binding data and comparison with crystal structures led to the derivation of correlations between the structure and affinity of the galactose derivatives. These characteristics will be used in the design of a second round of LT and CT receptor antagonists.     

Conditions of two-dimensional crystallization of cholera toxin B-subunit on lipid films containing ganglioside GM1

A systematic study of the lipid-layer two-dimensional crystallization technique has been carried out on the system composed of cholera toxin B-subunit and monosialoganglioside GM1, by electron microscopy, image analysis, and lipid film surface pressure measurements. Concentrations of protein and lipid components required for two-dimensional crystallization of toxin-GM1 complexes have been determined. Crystals were only obtained in the presence of mixed lipid films, composed of GM1 and of unsaturated lipids, such as dioleoylphosphatidylcholine or dioleoylphosphatidylethanolamine, in agreement with a previous report [D. S. Ludwig et al., (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8585–8588]. Crystals were obtained with cholera toxin B-subunit concentration as low as 5 μg/ml, as well as in the presence of protein contaminants. They were obtained over a wide range of concentrations of both GM1 and unsaturated lipids. The minimal lipid amount needed for crystallization corresponded to a lipid monolayer at, or near, the maximal spreading pressure (50 mN/m). The use of an excess of lipid resulted in a stabilization of lipid monolayers and in a higher reproducibility or crystallization experiments.     

Thermal stability and intersubunit interactions of cholera toxin in solution and in association with its cell-surface receptor ganglioside GM1

The thermal stability of cholera toxin free in solution and in association with its cell-surface receptor ganglioside GM1 has been studied by using high-sensitivity differential scanning calorimetry and differential solubility thermal gel analysis. In the absence of ganglioside GM1, cholera toxin undergoes two distinct thermally induced transitions centered at 51 and 74 degrees C, respectively. The low-temperature transition has been assigned to the irreversible thermal denaturation of the active A subunit. The second transition has been assigned to the reversible unfolding of the B subunit pentamer. The isolated B subunit pentamer exhibits a single transition also centered at 74 degrees C, suggesting that the attachment of the A subunit does not contribute to the stability of the pentamer. In the intact toxin, the A subunit dissociates from the B subunit pentamer at a temperature that coincides with the onset of the B subunit thermal unfolding. In aqueous solution, the denatured A subunit precipitates after dissociation from the B subunit pentamer. This phenomenon can be detected calorimetrically by the appearance of an exothermic heat effect. In the presence of ganglioside GM1, the B subunit is greatly stabilized as indicated by an increase of 20 degrees C in the transition temperature. In addition, ganglioside GM1 greatly enhances the cooperative interactions between B subunits. In the absence of ganglioside, each monomer within the B pentamer unfolds in an independent fashion whereas the fully ganglioside-bound pentamer behaves as a single cooperative unit. On the contrary, the thermotropic behavior of the A subunit is only slightly affected by the presence of increasing concentrations of ganglioside GM1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid phase separations induced by the association of cholera toxin to phospholipid membranes containing ganglioside GM1

The interactions of cholera toxin and their isolated binding and active subunits with phospholipid bilayers containing the toxin receptor ganglioside GM1 have been studied by using high-sensitivity differential scanning calorimetry and steady-state and time-resolved fluorescence and phosphorescence spectroscopy. The results of this investigation indicate that cholera toxin associates with phospholipid bilayers containing ganglioside GM1, independent of the physical state of the membrane. In the absence of Ca2+, calorimetric scans of intact cholera toxin bound to dipalmitoylphosphatidylcholine (DPPC) large unilamellar vesicles containing ganglioside GM1 result in a broadening of the lipid phase transition peak and a slight decrease (less than 5%) in the transition enthalpy. In the presence of Ca2+ concentrations sufficient to cause ganglioside phase separation, the association of the intact toxin to the membrane results in a significant decrease of enthalpy change for the lipid transition, indicating that under these conditions the toxin molecule perturbs the hydrophobic core of the bilayer. Calorimetric scans using isolated binding subunits lacking the hydrophobic toxic subunit did not exhibit a decrease in the phospholipid transition enthalpy even in the presence of Ca2+, indicating that the binding subunits per se do not perturb the hydrophobic core of the bilayer. On the other hand, the hydrophobic A1 subunit by itself was able to reduce the phospholipid transition enthalpy when reconstituted into DPPC vesicles. These calorimetric observations were confirmed by fluorescence experiments using pyrene phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of intersubunit interactions in cholera toxin upon binding to the oligosaccharide portion of its cell surface receptor, ganglioside GM1

The binding and the energetics of the interaction of cholera toxin with the oligosaccharide portion of ganglioside GM1 (oligo-GM1), the toxin cell surface receptor, have been studied by high-sensitivity isothermal titration calorimetry and differential scanning calorimetry. Previously, we have shown that the association of cholera toxin to ganglioside GM1 enhances the cooperative interactions between subunits in the B-subunit pentamer [Goins, B., & Freire, E. (1988) Biochemistry 27, 2046-2052]. New experiments presented in this paper reveal that the oligosaccharide portion of the receptor is by itself able to enhance the intersubunit cooperative interactions within the B pentamer. This effect is seen in the protein unfolding transition as a shift from independent unfolding of the B promoters toward a cooperative unfolding. To identify the origin of this effect, the binding of cholera toxin to oligo-GM1 has been measured calorimetrically under isothermal conditions. The binding curve at 37 degrees C is sigmoidal, indicating cooperative binding. The binding data can be described in terms of a nearest-neighbor cooperative interaction binding model. In terms of this model, the association of a oligo-GM1 molecule to a B protomer affects the association to adjacent B promoters within the pentameric ring. The measured intrinsic binding enthalpy per protomer is -22 kcal/mol and the cooperative interaction enthalpy -11 kcal/mol. The intrinsic binding constant determined calorimetrically is 1.05 x 10(6) M-1 at 37 degrees C and the cooperative Gibbs free energy equal to -850 cal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic AMP accumulation in HeLa cells induced by cholera toxin. Involvement of the ceramide moiety of GM1 ganglioside.

The influence of ceramide composition on the rate of GM1 association to HeLa cells has been investigated by incubating the cells in the presence of either native ganglioside or molecular species carrying highly homogeneous long chain base moieties, fractionated from native GM1. The GM1 ganglioside species carrying the unsaturated C18 long chain base moiety proved to have the fastest rate of association, whereas the saturated species carrying 20 carbon atoms had the slowest rate. After having increased the GM1 cell content (65-fold) by incubation with the various ganglioside species, the cells were incubated with cholera toxin and the time course of cyclic AMP accumulation was monitored. Remarkable differences among cells enriched with the various molecular species were found in the duration of the lag time preceding the accumulation of cyclic AMP, the shortest being displayed by the unsaturated C18 species. Moreover, the amount of cyclic AMP accumulated after a given time of incubation with cholera toxin was significantly higher when the C18:1-GM1 species was present than with native GM1. Fluorescence anisotropy experiments, carried out using the probe 1,3-diphenylhexatriene, show that the GM1 ganglioside ceramide moiety was also modifying the cell membrane fluidity of the host.     

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].     

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.