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Phase behavior and structural characteristics of hydrated bovine brain gangliosides.

Hydrated bovine brain gangliosides have been studied by differential scanning calorimetry, X-ray diffraction, and polarized light microscopy. Over the hydration range 18-50 wt.% H2O, mixed brain gangliosides exhibit a hexagonal mesophase structure, in which the ganglioside molecules form hexagonally packed rod-like structures. The apolar lipid chains radiate from the center of the rods, with the sugar groups on the cylinder surface in contact with water. At higher water contents, an isotropic micellar solution is formed. Over the hydration range 20-30 wt.% H2O, two small thermal transitions with peak maxima at 30 degrees C and 46 degrees C are observed by differential scanning calorimetry. These transitions broaden and move apart in temperature as the hydration is increased to 50 wt.% H2O. X-ray diffraction data indicate that this double transition is associated with a hydrocarbon chain rearrangement from a disordered state to another, possibly more disordered, state. Thus, the gangliosides, although membrane lipid components, have physical characteristics which are very different from those of the membrane phospholipids.

Animals↗

Micellar gangliosides mediate the lipid insertion of cholera toxin protomer A.

The topology of the interaction of cholera toxin with ganglioside and detergent micelles was studied with the technique of hydrophobic photolabelling. Cholera toxin alpha and gamma polypeptide chains appear to penetrate into the hydrophobic core of ganglioside micelles. Micelles of SDS cause the labelling also of the beta polypeptide chains, while Triton X-100 micelles have little ability to mediate the labelling of the toxin. The specific reduction of the alpha-gamma disulfide bond allows the penetration of the alpha polypeptide chain into Triton X-100 micelles, but does not affect the interaction of cholera toxin with either ganglioside or SDS micelles. Thus, ganglioside micelles appear to cause a conformational change of the native toxin, such as to induce the penetration of the alpha chain into the micelle hydrophobic core.

Cholera Toxin↗

Thermodynamic and phase characterization of phosphatidylethanolamine and ganglioside GD1a mixtures.

By employing diphenylhexatriene steady-state fluorescence anisotropy, pyrenedecanoic acid excimer formation, and high sensitivity scanning calorimetry we have demonstrated that the liposomes containing phosphatidylethanolamine (PE) and various mole fractions of ganglioside GD1a had a gel-liquid crystalline phase transition between 15 and 25 degrees C. Calorimetric measurements indicated that these phase transitions were broad and centered between 17 and 21 degrees C. The enthalpy change of the transition was linearly dependent on the ganglioside concentration up to 10.0 mol% and plateaued between 11.4-16.2 mol%. The high enthalpy change (37 kcal/mol of GD1a added into the PE bilayer) indicates the existence of PE-GD1a complex structure in the liposomal membrane. It is proposed that semi-fluid domains containing six PE and one ganglioside molecule are present in the PE-GD1a membranes at temperatures above gel-liquid crystalline phase transition. The Sendai virus induced leakage of PE-GD1a liposomes has been investigated by using an entrapped, self-quenching fluorescent dye, calcein. The leakage rate was dependent on the mole fraction of ganglioside GD1a and was maximal at 6.3 mol%. Arrhenius plots of the leakage rates showed breaks in the 20-25 degrees C temperature range, which correspond to the gel-liquid crystalline phase transition of the target liposomes. These data suggest that the rate of Sendai virus-induced leakage can be regulated via fluidity modulation by changing the PE to GD1a ratio at constant temperatures.

Calorimetry, Differential Scanning↗

Asymmetric distribution of gangliosides in rat renal brush-border and basolateral membranes.

Highly enriched brush-border and basolateral membranes isolated from rat renal cortex were used to study the distribution of endogenous gangliosides in the two distinct plasma membrane domains of epithelial cells. These two membrane domains differed in their glycolipid composition. The basolateral membranes contained more of both neutral and acidic glycolipids, expressed on a protein basis. In both membranes, the neutral glycolipids corresponding to mono-, di-, tri- and tetraglycosylceramides were present. The basolateral membranes contained more diglycosylceramide than the brush-border membranes. The major gangliosides found were GM4, GM3, and GD3 with minor amounts of GM1 and GD1a. The latter were identified and quantified by sensitive iodinated cholera toxin binding assays. When the distribution of individual gangliosides was calculated as a percent of total gangliosides, the brush-border membranes were enriched with GM3, GM1 and GD1a compared to the basolateral membranes, which were enriched with GD3 and GM4. The observation of a distinct distribution of glycolipids between brush-border and basolateral membranes of the same epithelial cell suggests that there may be a specific sorting and insertion process for epithelial plasma membrane glycolipids. In turn, asymmetric glycolipid biogenesis may reflect differences in glycolipid function between the two domains of the epithelial plasma membrane.

Animals↗

The effect of gangliosides on the lamellar phase behaviour of phosphatidylethanolamines.

The thermotropic properties of aqueous phosphatidylethanolamine dispersions vary with hydration. Measured by EPR-spectroscopy freshly hydrated dimyristoylphosphatidylethanolamine dispersions exhibit a gel to liquid-crystalline phase transition at Tml = 48 degrees C. Dehydration could be induced by prolonged incubation of a hydrated sample at 4 degrees C. The phase transition temperature of the dehydrated phase was determined to be Tmh = 54 degrees C. From the measured phase transition curves we followed the dehydration with time and found a cooperative nucleation process. A 50% dehydration was reached after 5 days. This dehydration process could be prevented by gangliosides: 1.5 mol% of GT1b, 4 mol% of GM1 or 7 mol% of GD1a or GM3 but also 7 mol% of phosphatidic acid were able to stabilize the hydrated phase completely. The effect of gangliosides GM1, GM3, GD1a, GT1b and of the negatively charged phosphatidic acid on the phase behaviour of dimyristoylphosphatidylethanolamine (DMPE) dispersions were investigated. The phase transition temperature of freshly hydrated DMPE samples was successively decreased from 48 to 43 degrees C with increasing amounts of GD1a up to 10 mol% whereby the phase transition was significantly broadened. Gangliosides GM1, GM3 and GT1b as well as phosphatidic acid had minor effects. Dispersions of pure DMPE prepared below the transition temperature Tml form the dehydrated phase again with a melting temperature of Tmh = 54 degrees C. In the presence of 10 mol% GD1a or GT1b this value is reduced to Tml, the phase transition temperature of the hydrated phase. The reduction induced by GM3 is less pronounced. With GM1 or phosphatidic acid the samples remain partially dehydrated and the phase transition curves become biphasic up to 7 mol% ganglioside or phosphatidic acid.

Chemical Phenomena↗

Interaction of ganglioside-containing planar bilayers with serotonin and inorganic cations.

The binding of serotonin and inorganic cations K+, Na+, Ca2+, Mg2+ to planar bilayers formed from mixtures of phosphatidylcholine and mono-, di- and trisialogangliosides was studied by the potentiodynamic and nonactin-induced potassium conductivity method. The theoretical analysis of the results obtained was made taking into account (1) protrusion of the ganglioside charges from the membrane surface and (2) simultaneous adsorption of ions on the bilayer surface and on the ganglioside charges protruding into the solution. It was shown that there was no specific binding of K+ and Na+. The binding constants for Ca2+, Mg2+ were determined. These constants for all the gangliosides studied were equal to 500 M-1. The determined binding constants of serotonin to various gangliosides diminish in the following order: GD3 greater than GT1b greater than GD1a greater than GM1.

Anti-Bacterial Agents↗

Curvature and composition-dependent lipid asymmetry in phosphatidylcholine vesicles containing phosphatidylethanolamine and gangliosides.

The effect of curvature on transbilayer lipid asymmetry in vesicles is investigated using vesicles of different sizes (30-140 nm) prepared by sonication and polycarbonate filter extrusion techniques. The transbilayer distributions of phosphatidylethanolamine and gangliosides are measured using 2,4,6-trinitrobenzenesulphonic acid and Clostridium perfringens neuraminidase as non-penetrating probes, respectively. The distribution of phosphatidylethanolamine in a phosphatidylcholine/phosphatidylethanolamine (4:1, molar ratio) system is more or less symmetric and curvature seems to have little effect. However, the distribution of gangliosides in a phosphatidylcholine/ganglioside (10:1, molar ratio) system is asymmetric in favour of the outer layer in smaller vesicles, the asymmetry disappearing as the degree of curvature decreases. In a phosphatidylcholine/phosphatidylethanolamine/ganglioside (8:2:1, molar ratio) system, both phosphatidylethanolamine and gangliosides distribute asymmetrically, indicating a composition-dependent asymmetric distribution of phosphatidylethanolamine. In this system asymmetry also increases with increasing curvature. The asymmetric distribution of gangliosides in vesicles of low curvature may be due to their long headgroup and larger headgroup surface area in accordance with the theoretical predictions of Israelachvili et al. (Biochim. Biophys. Acta 470 (1977) 185-201).

Chromatography, Gel↗

Effect of sulfatide and gangliosides on phospholipase C and phospholipase A2 activity. A monolayer study.

The effect of sulfatide and gangliosides GM1, GD1a and GT1b on the activity of phospholipase C from Clostridium perfringens on dilauroylphosphatidylcholine and of porcine pancreatic phospholipase A2 on dilauroylphosphatidic acid was studied in lipid monolayers containing different proportions of glycolipids under zero-order kinetics at various constant surface pressures. The presence of sulfatide in the monolayer increases the activity of phospholipase C at high surface pressures. Gangliosides shift the cut-off pressure to lower values and inhibit the action of phospholipase C. In mixed monolayers with dilauroylphosphatidic acid, sulfatide at a molar fraction of 0.5 increases the activity of phospholipase A2 at surface pressures below 18 mN/m and shows an inhibitory effect at higher pressures. Ganglioside GM1 at a molar fraction of 0.25 completely inhibits the enzyme above 20 mN/m and markedly reduces its activity at lower pressures. Gangliosides GD1a and GT1b abolish the enzyme activity at all pressures at molar fractions of 0.25 and 0.15, respectively. The modified velocity of the enzymatic reaction in the presence of glycosphingolipids is not due to an irreversible alteration of the catalytic activity.

Adsorption↗

Purification of G-M-1-ganglioside and ceramide lactoside beta-galactosidase from rabbit brain.

The major beta-galactosidase of rabbit brain has been purified over 400-fold. The enzyme converts G-M-1-ganglioside; Gal beta-1 yields 3 GalNAc beta-1 yields 4 (NANalpha-2 yields 3) Gal beta-1 yields 4 Glc yields ceramide (G-M-1) into Tay Sachs ganglioside GalNAc beta-1 yields 4 (NANalpha-2 yields 3) Gal beta-1 yields 4 Glc yields ceramide (G-M-2-ganglioside) and ceramide lactoside, Gal beta-1 yields 4 Glc yields ceramide (Gal-Glc-Cer) into glucocerebroside, Glc yields ceramide (Glc-Cer). The enzyme also hydrolyzes the synthetic substrates NPh-Gal and MeUmb-Gal. It is eluted as a single peak from Sephadex G-200 columns when natural and synthetic substrates were used and has an isoelectric point of 6.3. We were unable to resolve activity towards G-M-1-ganglioside and Gal-Glc-Cer by polyacrylamide electrophoresis in two buffer systems. With G-M-1 the pH optimum was 4.3 in acetate buffer and the K-m value 78 mu-M while with Gal-Glc-Cer, a pH optimum of 4.5 and a K-m of 17 mu-M were found. Hydrolysis of both natural and synthetic substrates was inhibited by gamma-D-galactonolactone, D-galactose and lactose. The data strongly suggest that a single beta-galactosidase hydrolyzes all the substrates tested.

Animals↗

Different reactivities of monoclonal antibodies to ganglioside lactones.

Six murine monoclonal antibodies were found to react with ganglioside GD2 lactone as well as purified ganglioside GD2. However, the reactivities of these antibodies to various ganglioside lactones were found to differ from each other. Four antibodies only reacted with GD2 lactones, while the other two cross-reacted with lactones of other gangliosides such as GD1b and GT1b.

Animals↗

Human cancer-associated gangliosides defined by a monoclonal antibody (IB9) directed to sialosyl alpha 2 leads to 6 galactosyl residue: a preliminary note.

A new monoclonal antibody (IB9) was prepared by hybridoma technique directed specifically to sialosyl alpha 2 leads to 6 galactosyl residue. With this reagent, accumulation of two major gangliosides in human colonic and liver adenocarcinoma has been detected, and these gangliosides were isolated and characterized as structures A and B (below). Another ganglioside with a ceramide nonasaccharide structure, which reacted to anti-X-hapten antibody after desialylation, was also isolated and partially characterized. (formula; see text) These gangliosides were absent or present in very small quantity in normal tissue and may represent human cancer-associated markers.

Adenocarcinoma↗

Competitive binding of gangliosides and glycophorin to wheat germ agglutinin.

Gangliosides and glycophorin are receptors for wheat germ agglutinin. The competitive binding of these molecules to wheat germ agglutinin is studied by electron spin resonance spectroscopy with spin labels attached to the oligosaccharide chains of gangliosides. Evidence shows that glycophorin is more accessible to wheat germ agglutinin binding than gangliosides. The interactions of gangliosides and glycophorin in liposomes is disrupted on low level binding of WGA.

Animals↗

A chemical study on the development of the human forebrain and cerebellum during the brain 'growth spurt' period. I. Gangliosides and plasmalogens.

Following upon previous studies on the lipid composition of the developing human brain, a further study is presented with the main object of tracing the chemical changes underlying the period of brain 'growth spurt'. Gangliosides and plasmalogens were selected as approximate markers of synaptogenesis and myelination, respectively, and these lipids were compared in cerebrum and cerebellum to establish the time, if any, at which their rate of accretion increases in a significant way. In the forebrain the rate of increase in concentration of these lipids accelerated at about the 32nd week of gestational age. Although there were too few postnatal cases to draw very firm conclusions, it seemed that the ganglioside concentration levelled off at about two months postnatal age and that the plasmalogen concentration reached a plateau between the 4th and the 6th postnatal months. In the cerebellum the concentration of gangliosides was clearly lower than that in the forebrain until about one year of age, the maximum rate of increase occurring between the last weeks of gestation and the second postnatal month. The plasmalogen concentration was somewhat higher in the cerebellum than in the forebrain but the concentration profile was similar to that followed by the gangliosides. In clear contrast with the concentration profiles in the cerebrum, in the cerebellum both lipids apparently continued to increase up to the second postnatal year. A mainly perinatal period of vulnerability is suggested for the forebrain, and a more prolonged one (probably until the second year of life) for the cerebellum.

Cerebellum↗

Cholera toxin-B subunit blocks excitatory effects of opioids on sensory neuron action potentials indicating that GM1 ganglioside may regulate Gs-linked opioid receptor functions.

In a previous study, we demonstrated that cholera toxin-A subunit, as well as the whole toxin, selectively blocks opioid-induced prolongation of the Ca2+ component of the action potential duration (APD) in dorsal root ganglion (DRG) neurons, indicating mediation of this excitatory effect by Gs-linked opioid receptors. The present study shows that pretreatment of DRG neurons with the B subunit of cholera toxin (1-10 ng/ml; greater than 15 min) can also block mu/delta and kappa opioid-induced APD prolongation, but not shortening. Since the B subunit binds selectively to GM1 ganglioside located on the cell surface, these results suggest that this ganglioside may regulate Gs-linked excitatory opioid receptor functions in DRG neurons. Possible contamination of purified B subunit preparations of cholera toxin with traces of the more potent A subunit was eliminated by heating the stock solution to 56 degrees C for 20 min. Exposure of DRG neurons to an affinity-purified anti-GM1 antiserum also blocked opioid-induced APD prolongation, providing further evidence that GM1 ganglioside may play an essential role in excitatory opioid modulation of the action potential of these cells. The blockade by cholera toxin-B subunit and anti-GM1 antibodies of opioid-induced APD prolongation is best accounted for by the following hypothesis: CTX-B interferes with an endogenous GM1 ganglioside component of the excitatory, but not inhibitory, opioid receptor complex on DRG neurons that may allosterically regulate coupling of the receptors via Gs to adenylate cyclase/cyclic adenosine monophosphate-dependent ionic conductances.

Action Potentials↗

Anti-ganglioside antibodies reveal subsets of cultured rat dorsal root ganglion neurons.

Subsets of cultured dorsal root ganglion neurons were identified by using the anti-ganglioside monoclonal antibodies A2B5, D1.1, R24 and JONES. A2B5 and D1.1 labelled a population of cells that was relatively stable between 2 and 20 days in vitro, while the population of cells labeled with both R24 and JONES decreased with time, suggesting that the gangliosides recognized by Jones and R24 are developmentally regulated. Given the observation that the relative proportions of ganglioside species changes with time in culture, it is very important to carefully define the stability of ganglioside antigens before using them as cell markers.

Animals↗

GM1 ganglioside partially rescues cultured dopaminergic neurons from MPP(+)-induced damage: dependence on initial damage and time of treatment.

GM1 ganglioside is believed to be important in promoting the recovery of neurons from injury. The present study assesses the ability of GM1 to repair or prevent the damage of dopamine neurons caused by the neurotoxin 1-methyl-4-phenylpyridinium (MPP+). Treatment of mesencephalic cell cultures with 2.5 microM MPP+ resulted in the loss of 30% of tyrosine hydroxylase (TH) immunoreactive neurons. In contrast, cultures administered 100 microM GM1 ganglioside for 3 days after toxin treatment contained nearly control numbers of TH+ neurons (97%). This reparative effect of GM1 was reflected in parallel increases in TH enzyme activity, dopamine and dopac levels. Cultures sustaining greater insult from higher doses of MPP+ (5.0-10.0 microM) did not benefit from ganglioside treatment, suggesting that rescue by GM1 depended on the degree of initial damage to cells. Moreover, the timing of ganglioside treatment was critical; pretreatment with GM1 alone did not prevent or attenuate the damage caused by subsequent incubation in 2.5 microM MPP+.

3,4-Dihydroxyphenylacetic Acid↗

Interaction of gangliosides with B cells in splenic fragment cultures.

The effect of gangliosides upon murine adult B cells at the single precursor cell level was examined using the splenic focus assay. Adult B cells were stimulated in in vitro organ fragment culture by a hapten-modified carrier protein in the presence of an excess of carrier-primed T cells. The addition of a potential tolerogen in the form of antigen coupled to a carrier not previously presented to the T cells resulted in a temporary unresponsiveness of the onset of antibody production in adult B cells, but not a permanent state of tolerance. This delay could be eliminated by the addition of purified murine gangliosides during the presentation of the hapten coupled to the unrecognized carrier. The ganglioside preparation was fractionated by ion-exchange chromatography and the active fraction was found to be a disialoganglioside. These results suggest that the ganglioside may interfere with membrane receptor-related events occurring during or after antigen binding or cross-linking to responding B cells.

Animals↗

Gangliosides block the inhibition of macrophage Fc-dependent phagocytosis by lipopolysaccharide.

The mechanism whereby bacterial lipopolysaccharide (LPS) exerts its biologic effects on mammalian cells is unknown. Plasma membrane gangliosides bind bacterial toxins and have been implicated in modulating the effects of a variety of immunoregulatory substances. We investigated the possibility that gangliosides can inhibit the effect of lipopolysaccharide on Fc-dependent phagocytosis by murine peritoneal macrophages. Protein-free lipopolysaccharide preparations significantly inhibited Fc-mediated phagocytosis (less than 71% of control) at concentrations of 100 ng/ml or greater after 90 min of incubation. The inhibitory effect of LPS (1 micrograms/ml) was blocked when macrophages were incubated with mono-, di-, or trisialogangliosides (25-50 micrograms/ml). Neither asialoganglioside nor sialic acid alone were capable of blocking the effect of LPS. When chromatographed separately on a Sepharose 4B column, LPS and trisialoganglioside had different elution profiles. LPS and trisialoganglioside coeluted, however, when premixed at 37 degrees C for 60 min and then applied to the column. Therefore, abrogation of the effect of LPS on Fc-dependent phagocytosis may occur as a consequence of direct interaction between LPS and gangliosides. These data suggest that gangliosides may modulate the response of macrophages to bacterial lipopolysaccharide.

Animals↗