Use of a photoactivable GM1 ganglioside analogue to assess lipid distribution in caveolae bilayer

A new photoactivable, radioactive derivative of ganglioside GM1 has been utilized to assess lipid distribution in the caveolae bilayer, taking advantage of the ability of the glycolipid, endogenous or exogenously added, to concentrate within this membrane compartment and to crosslink neighboring molecules upon illumination. After insertion into A431 plasma membrane and photoactivation, a membrane-enriched and a detergent-resistant fraction, enriched in gangliosides, sphingomyelin and cholesterol, were isolated. While a few radioactive proteins were detected in the membrane-enriched fraction, only radioactive caveolin was detected in the detergent-resistant fraction, indicating at the same time the enrichment of this fraction in caveolae and the presence of ganglioside within this compartment. Among lipids, crosslinked phosphatidylcholine, sphingomyelin and cholesterol were detected in the membrane-enriched fraction, while only crosslinked sphingomyelin was detected in the detergent-resistant fraction. These results suggest the enrichment in sphingomyelin—along with ganglioside—within the outer leaflet, and the preferential localization of cholesterol within the endoplasmic leaflet, of the caveolae bilayer.     

Use of a photoactivable GM1 ganglioside analogue to assess lipid distribution in caveolae bilayer

A new photoactivable, radioactive derivative of ganglioside GM1 has been utilized to assess lipid distribution in the caveolae bilayer, taking advantage of the ability of the glycolipid, endogenous or exogenously added, to concentrate within this membrane compartment and to crosslink neighboring molecules upon illumination. After insertion into A431 plasma membrane and photoactivation, a membrane-enriched and a detergent-resistant fraction, enriched in gangliosides, sphingomyelin and cholesterol, were isolated. While a few radioactive proteins were detected in the membrane-enriched fraction, only radioactive caveolin was detected in the detergent-resistant fraction, indicating at the same time the enrichment of this fraction in caveolae and the presence of ganglioside within this compartment. Among lipids, crosslinked phosphatidylcholine, sphingomyelin and cholesterol were detected in the membrane-enriched fraction, while only crosslinked sphingomyelin was detected in the detergent-resistant fraction. These results suggest the enrichment in sphingomyelin - along with ganglioside - within the outer leaflet, and the preferential localization of cholesterol within the endoplasmic leaflet, of the caveolae bilayer.     

Properties of ganglioside GM1 in phosphatidylcholine bilayer membranes.

Gangliosides have been shown to function as cell surface receptors, as well as participating in cell growth, differentiation, and transformation. In spite of their multiple biological functions, relatively little is known about their structure and physical properties in membrane systems. The thermotropic and structural properties of ganglioside GM1 alone and in a binary system with 1,2-dipalmitoyl phosphatidylcholine (DPPC) have been investigated by differential scanning calorimetry (DSC) and x-ray diffraction. By DSC hydrated GM1 undergoes a broad endothermic transition TM = 26 degrees C (delta H = 1.7 kcal/mol GM1). X-ray diffraction below (-2 degrees C) and above (51 degrees C) this transition indicates a micellar structure with changes occurring only in the wide angle region of the diffraction pattern (relatively sharp reflection at 1/4.12 A-1 at -2 degrees C; more diffuse reflection at 1/4.41 A-1 at 51 degrees C). In hydrated binary mixtures with DPPC, incorporation of GM1 (0-30 mol%; zone 1) decreases the enthalpy of the DPPC pretransition at low molar compositions while increasing the TM of both the pre- and main transitions (limiting values, 39 and 44 degrees C, respectively). X-ray diffraction studies indicate the presence of a single bilayer gel phase in zone 1 that can undergo chain melting to an L alpha bilayer phase. A detailed hydration study of GM1 (5.7 mol %)/DPPC indicated a conversion of the DPPC bilayer gel phase to an infinite swelling system in zone 1 due to the presence of the negatively charged sialic acid moiety of GM1. At 30-61 mol % GM1 (zone 2), two calorimetric transitions are observed at 44 and 47 degrees C, suggesting the presence of two phases. The lower transition reflects the bilayer gel --> L alpha transition (zone 1), whereas the upper transition appears to be a consequence of the formation of a nonbilayer, micellar or hexagonal phase, although the structure of this phase has not been defined by x-ray diffraction. At > 61 mol % GM1 (zone 3) the calorimetric and phase behavior is dominated by the micelle-forming properties of GM1; the presence of mixed GM1/DPPC micellar phases is predicted.     

Specific interaction between follitropin and GM1 ganglioside incorporated into lipid membranes.

A specific interaction was demonstrated between a monosialo-ganglioside (galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide) incorporated in glycerol monoleate planar bilayer membranes and follitropin. Conductance of glycerol monoleate planar bilayer membranes containing gangliosides were measured before and after addition of follitropin in the aqueous phase. A 5 fold increase of membrane conductance was observed in presence of a specific monosialo-ganglioside. No significant effect was obtained with di- and trisialogangliosides. Membrane conductance changes were suppressed by the presence of an equimolar mixture of the saccharide residues (galactose, N-acetylgalactosamine and N-acetylneuraminic acid) present in the hydrophilic moeity of the ganglioside. The results favour the hypothesis that gangliosides may contribute to the formation of functional glycoproteins receptors.     

Lateral diffusion of ganglioside GM1 in phospholipid bilayer membranes.

The lateral diffusion coefficient of ganglioside GM1 incorporated into preformed dimyristoylphosphatidylcholine (DMPC) vesicles has been investigated under a variety of conditions using the technique of fluorescence photobleaching recovery. For these studies the fluorescent probe 5-(((2-Carbohydrazino)methyl)thio)acetyl) amino eosin was covalently attached to the periodate-oxidized sialic acid residue of ganglioside GM1. This labeled ganglioside exhibited a behavior similar to that of the intact ganglioside, and was able to bind cholera toxin. The lateral diffusion coefficient of the ganglioside was dependent upon the gel-liquid crystalline transition of DMPC. Above Tm the lateral diffusion coefficient of the ganglioside was 4.7 X 10(-9) cm2 s-1 (with greater than 80% fluorescence recovery). This diffusion coefficient is significantly slower than the one previously observed for phospholipids in DMPC bilayers. The addition of increasing amounts of ganglioside, up to a maximum of 10 mol %, did not have a significant effect on the lateral diffusion coefficient or in the percent recovery. At 30 degrees C, the lateral mobility of ganglioside GM1 was not affected by the presence of 5 mM Ca2+, suggesting that, at least above Tm, Ca2+ does not induce a major perturbation in the lateral organization of the ganglioside molecules. The addition of stoichiometric amounts of cholera toxin to samples containing either 1 or 10 mol % ganglioside GM1 produced only a small decrease in the measured diffusion coefficient. The fluorescence recovery after photobleaching experiments were complemented with excimer formation experiments using pyrene-phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholera toxin assault on lipid monolayers containing ganglioside GM1

Many bacterial toxins bind to and gain entrance to target cells through specific interactions with membrane components. Using neutron reflectivity, we have characterized the structure of mixed DPPE:GM(1) lipid monolayers before and during the binding of cholera toxin (CTAB(5)) or its B-subunit (CTB(5)). Structural parameters such as the density and thickness of the lipid layer, extension of the GM(1) oligosaccharide headgroup, and orientation and position of the protein upon binding are reported. The density of the lipid layer was found to decrease slightly upon protein binding. However, the A-subunit of the whole toxin is clearly located below the B-pentameric ring, away from the monolayer, and does not penetrate into the lipid layer before enzymatic cleavage. Using Monte Carlo simulations, the observed monolayer expansion was found to be consistent with geometrical constraints imposed on DPPE by multivalent binding of GM(1) by the toxin. Our findings suggest that the mechanism of membrane translocation by the protein may be aided by alterations in lipid packing.     

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.     

Calmodulin interaction with mesocaine-modified lipid bilayer

Calmodulin (CaM) interactions with bilayer lipid membranes (BLM) were studied by measuring modulus of elasticity in direction perpendicular to the membrane plane (E perpendicular) and intramembrane potential delta psi. Upon interaction of CaM with egg phosphatidylcholine and asolectin BLM the parameter E perpendicular grew slightly (not more than by 10% as compared to the respective vale for nonmodified BLM), suggesting a weak effect on the ordering of the hydrophobic moiety of the lipid bilayer. In the presence of mesocaine (Mes), a calmodulin inhibitor, CaM affected the incorporation of Mes into the membrane. It can be concluded that CaM affects the ordering of the polar (superficial) membrane region.     

GM1 ganglioside: another nuclear lipid that modulates nuclear calcium. GM1 potentiates the nuclear sodium-calcium exchanger

The nuclear envelope (NE) enclosing the cell nucleus, although morphologically and chemically distinct from the plasma membrane, has certain features in common with the latter including the presence of GM1 as an important modulatory molecule. This ganglioside influences Ca(2+) flux across both membranes, but by quite different mechanisms. GM1 in the NE contributes to regulation of nuclear Ca(2+) through potentiation of a Na(+)/Ca(2+) exchanger in the inner nuclear membrane, whereas in the cell membrane, it regulates cytosolic Ca(2+) through modulation of a nonvoltage-gated Ca(2+) channel. Studies with neuroblastoma cells suggest GM1 concentration becomes elevated in the NE with onset of axonogenesis. However, the nuclear GM1/exchanger complex is not limited to neuronal cells but also occurs in NE of astrocytes, C6 cells, and certain non-neural cells. Immunoprecipitation and immunoblot experiments have shown high affinity association of the nuclear Na(+)/Ca(2+) exchanger with GM1, in contrast to Na(+)/Ca(2+) exchangers of the plasma membrane, which bind GM1 less avidly or not at all. This is believed to be due to different isoforms of the exchanger and a difference in topology of GM1 relative to the large inner loop of the exchanger in the 2 membranes. Cultured neurons from mice genetically engineered to lack GM1 suffered Ca(2+) dysregulation as seen in their high vulnerability to Ca(2+)-induced apoptosis. They were rescued by GM1 and more effectively by LIGA20, a membrane-permeant derivative of GM1. The mutant animals were highly susceptible to kainate-induced seizures, which are also a reflection of Ca(2+) dysregulation. The seizures were effectively attenuated by LIGA20 in parallel with the ability of this agent to enter brain cells, insert into the NE, and potentiate Na(+)/Ca(2+) exchange activity in the nucleus. The Na(+)/Ca(2+) exchanger of the NE, in association with nuclear GM1, is thus seen contributing to independent regulation of Ca(2+) by the nucleus in a manner that provides cytoprotection against Ca(2+)-induced apoptosis.     

On the interaction of hemocyanin with lipid bilayer membranes

Osmotic jump experiments were used to measure the ionic permeability induced in lipid vesicles by Megathura crenulata hemocyanin. It was found that this protein strongly increases the conductance of K⁺ and Cl^- through these membranes but not that of SO ₄ ⁼ . These effects were attributed to the formation of ionic channels in the vesicles. We have found that a simple first-order binding model can explain the dependence of the number of pore-containing vesicles both on the time after exposure to hemocyanin and on the protein concentration. Milder effects were attributed to a non-specific adhesion of the protein to the membrane surface. Consistent with the hypothesis of reversible association, vesicles which retained hemocyanin after step sucrose density gradient centrifugation at low ionic strength, lost most of the protein upon recentrifugation at high ionic strength. Consistent with the hypothesis of channel formation bot the above vesicle preparations transferred voltage-dependent hemocyanin channels into planar bilayers when they were made to fuse with them. It is concluded that hemocyanin can interact both specifically, by forming pores within the hydrophobic core of lipid membranes, and non-specifically, probably by means of electrostatic interaction with the surface of the same membrane.Abbreviations Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PS phosphatidylserine - DOC sodium deoxycholate     

The interaction of detergents with bilayer lipid membranes

Detergents are widely used for extracting and purifying membrane proteins. Four such detergents have been studied to find the extent to which they alone can alter black lipid film conductances. The slope of the plot of conductivity versus concentration for Triton X-100 is 4.54 in the range 0.025–0.15 mM; dodecyl sulphate 0.82 in the range 0.01–1 mM; sodium deoxycholate 1.03 in the range 0.01–1 mM and sodium cholate 1.37 in the range 0.1–10 mM. These ranges are below the respective critical micelle concentrations; above these concentrations the membranes break. Bilayer lipid membrane conductivity measured at constant detergent concentration increases with the conductivity of the bathing salt solution with a slope greater than 1, indicating an effect on the putative pore structures induced by detergents.     

Association of GM4 ganglioside with the membrane surrounding lipid droplets in shark liver

By TLC, GM4 was found to be the major ganglioside in the liver of six shark species examined: Odontaspis taurus, Negaprion brevirostris, Sphyrna lewini, Mustelus griseus, Mustelus manazo, and Prionace glauca. A detailed analysis of the glycosphingolipids (GSLs) in the liver of O. taurus (sand tiger shark) showed that it contained approximately 110 nmol of lipid-bound sialic acid per gram of wet tissue, of which 80% was GM4. By extracting the liver of O. taurus with chloroform/methanol, followed by chromatographic separation of GSLs using DEAE-Sephadex A-25 and Iatrobeads columns, we have isolated GM4 in pure form with a yield of approximately 5 mg per 100 g of wet tissue. The structures of both the sugar chain and the ceramide moiety of this GM4 were analyzed by chemical analysis, mass spectrometry, and NMR spectroscopy. Similar to GM4 isolated from other sources, 92% of fatty acids in the ceramide of this GM4 were 2-hydroxylated. However, unlike the long-chain bases found in other GSLs, the total long-chain bases in this GM4 were found to contain 43% octadecasphingenine and 50% nonadecasphingenine. Immunohistochemical analysis using a monoclonal antibody against GM4 revealed that the hepatocytes of both M. griseus (spotless smooth hound) and M. manazo (smooth hound) were filled with lipid droplets and GM4 was primarily associated with the membrane structure surrounding lipid droplets.     

The interaction of anionic detergents with lipid bilayer membranes

Summary The time course of the reaction of anionic surfactants with lipid bilayers is followed and analyzed. The distribution of detergents in the membrane phase gives rise to an asymmetry potential followed by a diffusion potential. Detergent-doped membranes are cation-permselective. It is postulated that a variable profile of mobile charges in the membrane account for the cation-permselectivity, the intercation selectivity, and the voltage-dependent gating phenomena observed in excitable membranes.Supported by a grant from the Medical Research Council of Canada.I thank Mr. G. Beauchesme for his technical assistance in part of this work.     

The interaction of hydrophobic ions with lipid bilayer membranes

Summary Electrical relaxation studies have been made on lecithin bilayer membranes of varying chain length and degree of unsaturation, in the presence of dipicrylamine. Results obtained are generally consistent with a model for the transport of hydrophobic ions previously proposed by Ketterer, Neumcke, and Läuger (J. Membrane Biol. 5:225, 1971). This model visualizes as three distinct steps the interfacial adsorption, translocation, and desorption of ions. Measurements at high electric field yield directly the density of ions adsorbed to the membrane-solution interface. Variation of temperature has permitted determination of activation enthalpies for the translocation step which are consistent with the assumption of an electrostatic barrier in the hydrocarbon core of the membrane. The change of enthalpy upon adsorption of ions is, however, found to be negligible, the process being driven instead by an increase of entropy. It is suggested that this increase may be due to the destruction, upon adsorption, of a highly ordered water structure which surrounds the hydrophobic ion in the aqueous phase. Finally, it is shown that a decrease of transient membrane conductance observed at high concentration of hydrophobic ions, previously interpreted in terms of interfacial saturation, must instead be attributed to a more complex effect equivalent to a reduction of membrane fluidity.Research performed while on sabbatical leave April-September, 1974.     

The interaction of bee melittin with lipid bilayer membranes

The influence of melittin and the related 8-26 peptide on the stability and electrical properties of bilayer lipid membranes is reported. Melittin, unlike the 8-26 peptide, has a dramatic influence on lipid membranes, causing rupture at dilute concentrations. The circular dichroism of melittin demonstrated that under physiological conditions, in water, melittin is in extended conformation, which is enhanced in aqueous ethanol. However in 'membrane-like' conditions it is essentially alpha-helical. Secondary structure predictions were used to locate possible alpha-helical nucleation centres and a model of melittin was built according to these predictions. It is postulated that melittin causes a wedge effect in membranes.     

Characterization of the conformational and orientational dynamics of ganglioside GM1 in a dipalmitoylphosphatidylcholine bilayer by molecular dynamics simulations

The structure and dynamics of a single GM1 (Gal5-beta1,3-GalNAc4-beta1,4-(NeuAc3-alpha2,3)-Gal2-beta1,4-Glc1-beta1,1-Cer) embedded in a DPPC bilayer have been studied by MD simulations. Eleven simulations, each of 10 ns productive run, were performed with different initial conformations of GM1. Simulations of GM1-Os in water and of a DPPC bilayer were also performed to delineate the effects of the bilayer and GM1 on the conformational and orientational dynamics of each other. The conformation of the GM1 headgroup observed in the simulations is in agreement with those reported in literature; but the headgroup is restricted when embedded in the bilayer. NeuAc3 is the outermost saccharide towards the water phase. Glc1 and Gal2 prefer a parallel, and NeuAc3, GalNac4 and Gal5 prefer a perpendicular, orientation with respect to the bilayer normal. The overall characteristics of the bilayer are not affected by the presence of GM1; however, GM1 does influence the DPPC molecules in its immediate vicinity. The implications of these observations on the specific recognition and binding of GM1 embedded in a lipid bilayer by exogenous proteins as well as proteins embedded in lipids have been discussed.     

Characterization of the conformational and orientational dynamics of ganglioside GM1 in a dipalmitoylphosphatidylcholine bilayer by molecular dynamics simulations

The structure and dynamics of a single GM1 (Gal5-β1,3-GalNAc4-β1,4-(NeuAc3-α2,3)-Gal2-β1,4-Glc1-β1,1-Cer) embedded in a DPPC bilayer have been studied by MD simulations. Eleven simulations, each of 10 ns productive run, were performed with different initial conformations of GM1. Simulations of GM1-Os in water and of a DPPC bilayer were also performed to delineate the effects of the bilayer and GM1 on the conformational and orientational dynamics of each other. The conformation of the GM1 headgroup observed in the simulations is in agreement with those reported in literature; but the headgroup is restricted when embedded in the bilayer. NeuAc3 is the outermost saccharide towards the water phase. Glc1 and Gal2 prefer a parallel, and NeuAc3, GalNac4 and Gal5 prefer a perpendicular, orientation with respect to the bilayer normal. The overall characteristics of the bilayer are not affected by the presence of GM1; however, GM1 does influence the DPPC molecules in its immediate vicinity. The implications of these observations on the specific recognition and binding of GM1 embedded in a lipid bilayer by exogenous proteins as well as proteins embedded in lipids have been discussed.