Molecular parameters and physical state of neutral glycosphingolipids and gangliosides in monolayers at different temperatures.

The effect of temperature on the behaviour of four different gangliosides (GM3, GM1, GD1a and GT1b), sulphatide, ceramide (Cer) and three neutral glycosphingolipids (GalCer, Gg3Cer, Gg4Cer) was investigated in monolayers at the air-NaCl (145 mM) interface. GM1, GD1a and GT1b are liquid-expanded in the range of temperatures studied (5-65 degrees C). GM3, sulphatide, Cer and neutral glycosphingolipids show isothermal liquid-expanded----liquid-condensed transitions. The collapse pressure of ganglioside monolayers decreases with temperature, whereas neutral glycosphingolipids may show some maximum values at particular temperatures. The reduction of the molecular area of liquid-expanded glycosphingolipids under compression occurs with a favorable positive entropy change and an unfavorable negative enthalpy. By contrast, the compression of interfaces with a two-dimensional phase transition occurs with an unfavorable entropy but a favorable enthalpy change. From the temperature dependence of the surface pressure at which the two-dimensional phase transition takes place, a minimal temperature above which the isotherm becomes totally liquid-expanded can be obtained. For the different glycosphingolipids this temperature decreases in the order Cer greater than GalCer greater than sulphatide greater than Gg3Cer greater than Gg4Cer greater than GM3 greater than GM1 greater than GD1a greater than GT1b. This sequence is similar to that found for the calorimetrically determined transition temperatures (cf. Maggio, B., Ariga, T., Sturtevant, J.M. and Yu, R.K. (1985) Biochemistry 24, 1084-1092).

Neurotransmitter movements in nerve endings. Influence of substances that modify the interfacial potential.

Polysialogangliosides, sulphatides, glycerylmonooleate, unsaturated fatty acids, myelin basic protein and sucrose inhibit the Na+-coupled uptake and induce a Ca2+-dependent release of dopamine from nerve endings. Substances chemically related to those referred to above, such as monosialogangliosides, neutral glycosphingolipids, glycerylmonostearate, saturated fatty acids and albumin, do not show these effects. Mixtures of polysialogangliosides or sulphatides with myelin basic protein or albumin inhibit, to different degrees, the effects of the individual components. The decreased uptake induced by sucrose reverted to control levels upon reduction of the concentration of the perturbing agent. The restoration of the uptake was probably mediated by the Na+-pump reconstituting the transmembrane Na+-gradient necessary for the Na+-coupled cotransport of dopamine. It is suggested that the effects of uptake inhibitor or release inducer agents derive from their ability to decrease the surface potential and modify the molecular organization of phospholipid interfaces which can result in changes of the membrane ionic permeability.

Membrane instability induced by purified myelin components. Its possible relevance to experimental allergic encephalomyelitis.

The fusogenic properties of purified myelin components in a system employing chicken erythrocytes were studied. Sulphatides, myelin basic protein and the apoprotein of Folch-Lees proteolipid were capable of individually inducing membrane fusion in the presence of Ca2+. By contrast, cerebrosides or a mixture of sulphatides and myelin basic protein (molar ratio 19 : 1) did not show such effect. The fusogenic ability of sulphatide was correlated to its behaviour in mixed monolayers with phospholipids at the air-water interface. Mixed films of sulphatides with phosphatidylcholine or sphingomyelin but not with phosphatidylethanolamine showed reductions of molecular packing and surface potential similar to those found for other fusogenic compounds. The effects of myelin components described could be of importance in the membrane instability and vesicular disruption of myelin occurring in demyelinative disorders.

Myelin basic protein domains involved in the interaction with actin.

A fluorescence assay was used to measure the interaction of myelin basic protein (MBP) with monomeric actin labeled with a fluorescent compound (IAEDANS). The complex actin-IAEDANS increase the fluorescence in presence of MBP. The enhancement of the fluorescence has a sigmoidal dependence on the concentration of MBP and the fluorescence maximum is reached at a MBP:actin molar ratio of 1:20. The fluorescence maximum in absence of Ca2+ and ATP is 4 times lower than that in their presence although it is reached at the same MBP:actin molar ratio. Similar behavior is observed when synapsin replaces MBP, while acetylated MBP and bovine serum albumin fail to induce any fluorescence change. To define possible interacting domains on MBP involved in the actin-MBP interaction, experiments were performed using MBP-derived peptides obtained under controlled proteolysis of the whole molecule. The fluorescence changes induced by the different peptides depend on their location in the native protein and can not be explained simply by a difference in the net charge of the peptides. The results suggest that two sites are involved in the interaction. A Ca2+/ATP-dependent site located in the amino-terminal region (peptide 1-44) and a Ca2+/ATP-independent one near the carboxyl terminus of the MBP molecule. The actin-MBP interaction was also observed using immunoblot and ELISA techniques.

Central nervous system lipid alterations in rats with experimental allergic encephalomyelitis and its suppression by immunosuppressive drugs.

Rats with experimental allergic encephalomyelitis (EAE) induced with myelin or spinal cord show decreases in the content of sulphatides and cerebrosides and increases in the level of esterified cholesterol in the CNS. In this work it is shown that brain sulphatide changes can be obtained by injection of mixtures containing glycosphingolipids. Alterations in the content of cerebrosides occur when the injection mixture contains cerebrosides. The alterations of sulphatides and cholesterol ester induced by injection of spinal cord could be suppressed by treatment with immunosuppressive drugs (dexamethasone, cyclophosphamide and 6-mercaptopurine) able to prevent clinical signs of EAE.

Surface topography of sulfatide and gangliosides in unilamellar vesicles of dipalmitoylphosphatidylcholine.

The property of the dyes, acridine orange and methylene blue, to exhibit metachromatic changes upon binding to negatively charged groups that are within a defined spatial separation was employed to study the lateral and transverse topography of sulfatide and gangliosides GM1 and GD1a mixed with dipalmitoylphosphatidylcholine (DPPC) in unilamellar vesicles. The spectral changes of the dyes in the presence of liposomes containing anionic glycosphingolipids (GSLs) (hypochromism and frequency shift) are typical of polyanionic lattices while minor changes are found for neutral lipids. The metachromatic changes are abolished by the presence of Ca2+ in the external medium. The proportion of anionic GSLs accessible to the dyes on the external surface of the liposomes is greater as the GSLs are more complex (sulfatide less than GM1 less than GD1a) and as its proportion in the mixture decreases. The number of molecules of anionic GSLs that are laterally distributed on the external surface in a position favorable for the formation of dye dimers (at intermolecular distances not exceeding 1 nm) is greater for sulfatide than for ganglioside. This is correlated to the greater intermolecular distances and delocalization in ganglioside-, compared to sulfatide-containing interfaces. The experimental values indicate that the mixture with DPPC of any of the anionic GSLs studied behaves as if it was more enriched in the GSLs compared to the proportions of the whole mixture.

Interaction of myelin basic protein, melittin and bovine serum albumin with gangliosides, sulphatide and neutral glycosphingolipids in mixed monolayers.

Some parameters that may regulate the miscibility and stability of mixed lipid-protein monolayers at the air-145 mM NaCl interface were studied employing six glycosphingolipids (acidic or neutral), three different types of proteins (soluble, extrinsic or highly amphipathic) and some phospholipids. The results obtained show that the percentage of the total area occupied by the protein at the interface is an important parameter leading to lateral phase separations; the amount and area contribution of the protein accepted in the film before the components become immiscible increase with the complexity of the polar head group of the glycosphingolipids. The interactions occur with progressive reductions of the intermolecular packing as the polar head group of the glycosphingolipid becomes more complex and this is accompanied by more negative values of the excess free energy of mixing. The lipid component seems to be the major responsible for the reduction in mean molecular area.

Molecular interactions and thermotropic behavior of glycosphingolipids in model membrane systems.

The oligosaccharide chain of glycosphingolipids (GSLs) has a marked influence on their thermotropic behavior, intermolecular packing and surface electrical potential. The transition temperature and enthalpy of GSLs decrease proportionally to the complexity of the polar head group and show a linear dependence with the intermolecular spacings. Interactions occurring among GSLs and phospholipids induce changes of the molecular area and surface potential that depend on the type of GSLs. Increasing proportions of phospholipids perturb the thermodynamic properties of the GSLs up to a point where phase separated phospholipid domains separate out but no phase separation of pure GSLs occurs. Heterogeneous equilibria among different structures occur for some systems. Large changes of the molecular free energy, eccentricity, asymmetry ratio and phase state of the GSLs-containing structure can be triggered by small changes of the molecular parameters, lipid composition and lateral surface pressure. The thermotropic behavior of GSLs is considerably perturbed by myelin basic protein. Phase separation occurs depending on the amount of protein and type of GSLs. The protein induces a decrease of the lipid molecular area, the more so the more complex the oligosaccharide chain in the GSLs. These membrane systems can not be described only on the basis of the individual properties of the molecules involved in a simple causal manner. Still scarcely explored long range thermodynamic, geometric and field effects that belong simultaneously to the intervening molecules, to the morphological properties of the structure involved and to the aqueous environment, are important determinants of their behavior.

Gangliosides noncovalently bound to DEAE-Sephadex: application to purification of anti-ganglioside antibodies.

A simple, rapid, effective, and inexpensive method for the purification of ligands having high affinity for gangliosides has been developed. DEAE-Sephadex has a high capacity for binding gangliosides (approx 1/1.6, w/w). The gangliosides, bound to the support by electrostatic and hydrophobic interactions, showed a high resistance, in an aqueous environment, to being detached by eluants commonly employed to desorb ligands (i.e., low or high pH or chaotropic agent solutions) or by nonionic detergent solutions as well as by organic solvents. The DEAE-Sephadex-ganglioside complex was assayed as an immunoadsorbent for purifying anti-GM1 ganglioside antibodies from serum of an immunized rabbit. The specific activity of the purified antibodies was 200- to 400-fold higher, and the recovery of the anti-ganglioside activity was above 50%, with respect to the untreated antiserum. The preparation of the complex and the purification of the antibodies can be done in less than 5 h. The glycolipids from the complex can be recovered by elution with organic solvents containing salt or volatile base solutions, and reused. In principle, this method can be adapted for other anionic amphipathic receptor molecules to purify ligands which bind to them.

Identification as synapsin of a synaptosomal protein immunoreacting with anti-myelin basic protein antiserum.

Rat brain proteins able to react with anti-myelin basic protein antiserum, raised under conditions to induce experimental allergic encephalomyelitis in rabbits, were examined by immunoblot methods after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Apart from the four forms of myelin basic protein present in rat brain, the antiserum detected other proteins of higher molecular weight. Subcellular fractionation shows that these high-molecular-weight proteins are relatively concentrated in a synaptosome-enriched fraction compared to a myelin fraction. A major protein fraction immunorelated to myelin basic protein migrated in the gels as a doublet with apparent molecular weights of approximately 80K and 86K; these proteins were tentatively identified as synapsin Ia and Ib. A purified synapsin preparation analyzed by immunoblot after two-dimensional gel electrophoresis also reacted with anti-myelin basic protein antisera. When the serum was purified by affinity chromatography on a myelin basic protein-conjugated Sepharose column the nonadsorbed material lost this activity whereas the eluted antibodies reacted with myelin basic protein and synapsin. In addition, sequence amino acid comparison of decapeptides showed some homology between these two proteins. A possible implication of immunological agents against myelin basic protein cross-reacting with extra-myelin proteins in the process of experimental allergic encephalomyelitis is considered.

Ganglioside-cholera toxin interactions: a binding and lipid monolayer study.

On t.l.c. plates 125I-cholera toxin binds to a disialoganglioside tentatively identified as GD1b with about 10 times less capacity then to ganglioside GM1. Binding of labeled toxin to both gangliosides was abolished in presence of excess amounts of unlabeled B subunit. Ganglioside extracts from human or pig intestinal mucosa showed toxin binding to gangliosides GM1 and GD1b. In ganglioside-containing lipid monolayers the penetration of the toxin was independent of the ganglioside binding capacity.

Surface behaviour of gangliosides and related glycosphingolipids.

1. The surface behaviour of six different gangliosides and eight chemically related glycosphingolipids was investigated in monolayers at the air-water interface. 2. Mono-, di-, tri and tetra-hexosylceramides had force-area isotherms showing similar limiting molecular areas on 145 mM-NaCl, pH 5.6. The increasing number of negatively charged sialosyl residues in mono-, di- and tri-sialogangliosides induced a progressive increase in the liquid-expanded character of the films and in the limiting area occupied per molecule, owing to electrostatic repulsions. When the ganglioside monolayers were spread on subphases at pH 1.2, the limiting area per molecule was similar to that found for neutral glycosphingolipids. 3. The monolayer collapse pressure at pH 5.6 increased with the number of uncharged carbohydrate units up to when the polar head group contained 3-4 residues. For gangliosides the collapse pressures were lower and decreased from mono- to tri-sialogangliosides. Ganglioside monolayers on subphases at pH 1.2 showed increases in their collapse pressure. 4. The glycosphingolipid monolayers studied had various surface in their collapse pressure. 4. The glycosphingolipid monolayers studied had various surface potentials according to the complexity of the polar head group of the lipid. Attempts to calculate the dipolar contributions to the surface potential from each carbohydrate residue suggest that the second and third sialosyl residues in di- and tri-sialogangliosides contributed with a vertical dipole moment opposite to that of the first sialosyl residue.

Interactions of gangliosides with phospholipids and glycosphingolipids in mixed monolayers.

1. The interactions among five different gangliosides and three chemically related glycosphingolipids and their behaviour in mixed monolayers with six different phospholipids were investigated at the air/145 mM-NaCl interface at pH 5.6. 2. The mixed monolayers of any of the different gangliosides showed an immiscible behaviour at high surface pressures, with absence of interactions among them revealed by an ideal behaviour for mean molecular area and surface potential per molecule. 3. This behaviour was probably the consequence of steric hindrance and electrostatic repulsions between their polar head groups. 4. Di- and tri-sialogangliosides could be differentiated from neutral sphingolipids and monosialogangliosides on the basis of their interactions with phospholipids, which were correlated to the perpendicular electric field at the interface contributed by the carbohydrate residues. 5. The presence of the phosphocholine polar head group in phosphatidylcholine was important to establish interactions with di- and tri-sialogangliosides revealed by negative deviations from the ideal behaviour for mean molecular areas and mean surface potential per molecule. 6. The possible significance of these observations is discussed in relation to the participation of gangliosides in the organization of membranes and to their capability of inducing membrane fusion.