Ganglioside Headgroups Research Articles

The impact of the glycan headgroup on the nanoscopic segregation of gangliosides

Gangliosides form an important class of receptor lipids containing a large oligosaccharide headgroup whose ability to self-organize within lipid membranes results in the formation of nanoscopic platforms. Despite their biological importance, the molecular basis for the nanoscopic segregation of gangliosides is not clear. In this work, we investigated the role of the ganglioside headgroup on the nanoscale organization of gangliosides. We studied the effect of the reduction in the number of sugar units of the ganglioside oligosaccharide chain on the ability of gangliosides GM1, GM2, and GM3 to spontaneously self-organize into lipid nanodomains. To reach nanoscopic resolution and to identify molecular forces that drive ganglioside segregation, we combined an experimental technique, Förster resonance energy transfer analyzed by Monte-Carlo simulations offering high lateral and trans-bilayer resolution with molecular dynamics simulations. We show that the ganglioside headgroup plays a key role in ganglioside self-assembly despite the negative charge of the sialic acid group. The nanodomains range from 7 to 120 nm in radius and are mostly composed of the surrounding bulk lipids, with gangliosides being a minor component of the nanodomains. The interactions between gangliosides are dominated by the hydrogen bonding network between the headgroups, which facilitates ganglioside clustering. The N-acetylgalactosamine sugar moiety of GM2, however, seems to impair the stability of these clusters by disrupting hydrogen bonding of neighboring sugars, which is in agreement with a broad size distribution of GM2 nanodomains. The simulations suggest that the formation of nanodomains is likely accompanied by several conformational changes in the gangliosides, which, however, have little impact on the solvent exposure of these receptor groups. Overall, this work identifies the key physicochemical factors that drive nanoscopic segregation of gangliosides.

NMR Experiments Shed New Light on Glycan Recognition by Human and Murine Norovirus Capsid Proteins

Glycan–protein interactions are highly specific yet transient, rendering glycans ideal recognition signals in a variety of biological processes. In human norovirus (HuNoV) infection, histo-blood group antigens (HBGAs) play an essential but poorly understood role. For murine norovirus infection (MNV), sialylated glycolipids or glycoproteins appear to be important. It has also been suggested that HuNoV capsid proteins bind to sialylated ganglioside head groups. Here, we study the binding of HBGAs and sialoglycans to HuNoV and MNV capsid proteins using NMR experiments. Surprisingly, the experiments show that none of the norovirus P-domains bind to sialoglycans. Notably, MNV P-domains do not bind to any of the glycans studied, and MNV-1 infection of cells deficient in surface sialoglycans shows no significant difference compared to cells expressing respective glycans. These findings redefine glycan recognition by noroviruses, challenging present models of infection.

Revealing a Dual Role of Ganglioside Lipids in the Aggregation of Membrane-Associated Islet Amyloid Polypeptide.

Amyloid formation of the human islet amyloid polypeptide (hIAPP) correlates with a loss of insulin-producing beta cells in patients with type II diabetes mellitus. In this study, we investigated the binding of hIAPP to bilayers consisting of ganglioside lipids and dioleoylphosphatidylcholine (DOPC), which is a physiologically relevant lipid species for pancreatic beta cell-associated aggregation. The membrane interactions are studied computationally using a combination of coarse-grained, umbrella sampling, and atomistic molecular dynamics simulations. Herein, we demonstrate how the hIAPP peptides accumulate in the areas with a high content of ganglioside lipids. We have characterized two distinct binding modes of hIAPP on ganglioside-rich membranes, with both binding modes formed due to electrostatic interaction between the cationic peptides and the anionic ganglioside headgroup. We observed that binding in the ganglioside headgroup region induced conformational changes of the peptide towards an aggregation prone conformation, rich in β-strands. In contrast, the binding of hIAPP near the ganglioside-enriched areas mobilizes the peptide, preventing it from conformational changes and potentially shields it from interactions with other peptides. This suggests a dual role of ganglioside lipids, affecting the aggregation of hIAPP by either accelerating or inhibiting amyloid formation depending on the membrane binding and the ganglioside concentration.

Reprint of “Ganglioside lipids accelerate α-synuclein amyloid formation”

The deposition of α-synuclein fibrils is one hallmark of Parkinson's disease. Here, we investigate how ganglioside lipids, present in high amounts in neurons and exosomes, influence the aggregation kinetics of α-synuclein. Gangliosides, as well as, other anionic lipid species with small or large headgroups were found to induce conformational changes of α-synuclein monomers and catalyse their aggregation at mildly acidic conditions. Although the extent of this catalytic effect was slightly higher for gangliosides, the results imply that charge interactions are more important than headgroup chemistry in triggering aggregation. In support of this idea, uncharged lipids with large headgroups were not found to induce any conformational change and only weakly catalyse aggregation. Intriguingly, aggregation was also triggered by free ganglioside headgroups, while these caused no conformational change of α-synuclein monomers. Our data reveal that partially folded α-synuclein helical intermediates are not required species in triggering of α-synuclein aggregation.

Ganglioside lipids accelerate α-synuclein amyloid formation

The deposition of α-synuclein fibrils is one hallmark of Parkinson's disease. Here, we investigate how ganglioside lipids, present in high amounts in neurons and exosomes, influence the aggregation kinetics of α-synuclein. Gangliosides, as well as, other anionic lipid species with small or large headgroups were found to induce conformational changes of α-synuclein monomers and catalyse their aggregation at mildly acidic conditions. Although the extent of this catalytic effect was slightly higher for gangliosides, the results imply that charge interactions are more important than headgroup chemistry in triggering aggregation. In support of this idea, uncharged lipids with large headgroups were not found to induce any conformational change and only weakly catalyse aggregation. Intriguingly, aggregation was also triggered by free ganglioside headgroups, while these caused no conformational change of α-synuclein monomers. Our data reveal that partially folded α-synuclein helical intermediates are not required species in triggering of α-synuclein aggregation.

Water response to ganglioside GM1 surface remodelling

BackgroundGangliosides are biological glycolipids participating in rafts, structural and functional domains of cell membranes. Their headgroups are able to assume different conformations when packed on the surface of an aggregate, more lying or standing. Switching between different conformations is possible, and is a collective event. Switching can be induced, in model systems, by concentration or temperature increase, then possibly involving ganglioside–water interaction. In the present paper, the effect of GM1 ganglioside headgroup conformation on the water structuring and interactions is addressed. MethodsDepolarized Rayleigh Scattering, Raman Scattering, Quasielastic Neutron Scattering and NMR measurements were performed on GM1 ganglioside solutions, focusing on solvent properties. ResultsAll used techniques agree in evidencing differences in the structure and dynamics of solvent water on different time-and-length scales in the presence of either GM1 headgroup conformations. ConclusionsIn general, all results indicate that both the structural properties of solvent water and its interactions with the sugar headgroups of GM1 respond to surface remodelling. The extent of this modification is much higher than expected and, interestingly, ganglioside headgroups seem to turn from cosmotropes to chaotropes upon collective rearrangement from the standing- to the lying-conformation. SignificanceIn a biological perspective, water structure modulation could be one of the physico-chemical elements contributing to the raft strategy, both for rafts formation and persistence and for their functional aspects. In particular, the interaction with approaching bodies could be favoured or inhibited or triggered by complex-sugar-sequence conformational switch. This article is part of a Special Issue entitled “Science for Life” Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo.

Number of Sialic Acid Residues in Ganglioside Headgroup Affects Interactions with Neighboring Lipids

Monolayers of binary mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and asialo-(GA1), disialo-(GD1b) and trisialo-(GT1b) gangliosides were used to determine the effect of ganglioside headgroup charge and geometry on its interactions with the neighboring zwitterionic lipid. Surface pressure versus molecular area isotherm measurements along with concurrent fluorescence microscopy of the monolayers at the air-water interface were complemented with atomic force microscopy imaging of monolayers deposited on solid substrates. Results were used to further develop a proposed geometric packing model that the complementary geometry of DPPC and monosialoganglioside GM1 headgroups affects their close molecular packing, inducing condensation of the layer at small mol % of ganglioside. For GA1, GD1b, and GT1b, a similar condensing effect, followed by a fluidizing effect is seen that varies with glycosphingolipid concentration, but results do not directly follow from geometric arguments because less DPPC is needed to condense ganglioside molecules with larger cross-sectional areas. The variations in critical packing mole ratios can be explained by global effects of headgroup charge and resultant dipole moments within the monolayer. Atomic force microscopy micrographs further support the model of ganglioside-induced DPPC condensation with condensed domains composed of a striped phase of condensed DPPC and DPPC/ganglioside geometrically packed complexes at low concentrations.

Cholesterol accelerates the binding of Alzheimer's β-amyloid peptide to ganglioside GM1 through a universal hydrogen-bond-dependent sterol tuning of glycolipid conformation

Age-related alterations of membrane lipids in brain cell membranes together with high blood cholesterol are considered as major risk factors for Alzheimer's disease. Yet the molecular mechanisms by which these factors increase Alzheimer's risk are mostly unknown. In lipid raft domains of the plasma membrane, neurotoxic Alzheimer's beta-amyloid (Abeta) peptides interact with both cholesterol and ganglioside GM1. Recent data also suggested that cholesterol could stimulate the binding of Abeta to GM1 through conformational modulation of the ganglioside headgroup. Here we used a combination of physicochemical and molecular modeling approaches to decipher the mechanisms of cholesterol-assisted binding of Abeta to GM1. With the aim of decoupling the effect of cholesterol on GM1 from direct Abeta-cholesterol interactions, we designed a minimal peptide (Abeta5-16) containing the GM1-binding domain but lacking the amino acid residues involved in cholesterol recognition. Using the Langmuir technique, we showed that cholesterol (but not phosphatidylcholine or sphingomyelin) significantly accelerates the interaction of Abeta5-16 with GM1. Molecular dynamics simulations suggested that Abeta5-16 interacts with a cholesterol-stabilized dimer of GM1. The main structural effect of cholesterol is to establish a hydrogen-bond between its own OH group and the glycosidic-bond linking ceramide to the glycone part of GM1, thereby inducing a tilt in the glycolipid headgroup. This fine conformational tuning stabilizes the active conformation of the GM1 dimer whose headgroups, oriented in two opposite directions, form a chalice-shaped receptacle for Abeta. These data give new mechanistic insights into the stimulatory effect of cholesterol on Abeta/GM1 interactions. They also support the emerging concept that cholesterol is a universal modulator of protein-glycolipid interactions in the broader context of membrane recognition processes.

Lipids in HIV's Envelope Help the Virus to Spread

​DendriticDendritic cells (DCs) are a type of immune cell that patrol tissues, on the lookout for microbial invaders. When DCs encounter a pathogen, they chop it up into tiny pieces and then carry samples of it to local lymph nodes. There, they display their finds to another kind of immune cell, the T cell, which then mounts a full-fledged immune response against the invader. Left: Gangliosides in HIV's lipid envelope (red) promote uptake of the virus (or experimental substitutes) by dendritic cells. Unfortunately, HIV, the virus that causes AIDS, can exploit the DC surveillance network to its own advantage: HIV is picked up by DCs that patrol mucosal tissues, but avoids being killed by them, and instead hitches a ride to lymph nodes. There, the virus transfers into its favorite host cell type, the T cell. Until now, it wasn't clear how DCs recognize HIV for uptake—a mystery that's now been solved thanks to a joint effort by Spanish and German research groups. The groups, headed by Nuria Izquierdo-Useros and Javier Martinez-Picado in Spain, and Maier Lorizate and Hans-Georg Krausslich in Germany, uncover the culprit in this month's issue of PLoS Biology. It's no mystery how DCs detect many pathogens: the microbes betray themselves by displaying certain pathogen-specific compounds on their surfaces, which DCs recognize using dedicated receptors. HIV also displays a few viral proteins on its surface (it hides the rest away under a lipid bilayer envelope that it acquires from the plasma membrane of infected cells). However, previous studies indicated that viral proteins displayed on the surface may not be essential for DC capture. Instead, prior work from Martinez-Picado's group led the authors to theorize that host cell glycosphingolipids incorporated into the virus envelope could be critical in triggering DC recognition. There are many types of glycosphingolipids, and one kind had previously been observed in the membranes of several retroviruses, including HIV. This lipid, GM3, is from a subclass of glycosphingolipids called gangliosides. To follow up on these findings, Izquierdo-Useros, Lorizate, and colleagues first examined whether other gangliosides might also be present in HIV's envelope. Indeed, they found several other gangliosides there—an observation that prompted the researchers to examine what role gangliosides might play in HIV recognition and capture by DCs. To determine whether gangliosides are needed for HIV uptake by DCs, the authors examined DC uptake of artificial virus-like particles (VLPs), which carry a lipid envelope and the inner structural proteins, but lack HIV surface glycoproteins and viral genetic material. They found that VLPs containing gangliosides were able to get into DCs, but those lacking gangliosides could not do so. Next, the researchers tested which gangliosides can promote DC uptake by using artificial lipid globules called liposomes, each containing a different ganglioside. Similar to VLPs, DCs easily took up liposomes containing gangliosides, but were unable to take up liposomes devoid of all gangliosides. Both liposomes and VLPs, the authors showed, use the same method to enter DCs—a method that depends not on viral proteins but on the presence of gangliosides in the viral envelope. These data suggest that there is some special feature of gangliosides that promotes their specific recognition by DCs. While much of a ganglioside is buried in the lipid bilayer, a portion of it, called the head group, is exposed at the membrane surface. Ganglioside head groups contain one or more copies of sialic acid, a sugar, with different ganglioside types each sporting distinct arrangements of their sialic acids. The authors' investigation revealed that it's these head groups—particularly sialic acids attached to a lactose group—that promote DC recognition. But, while DC uptake requires sialic acid molecules, gangliosides whose head groups are too complex can't be recognized by DCs. These findings all point to the idea that gangliosides are essential for HIV uptake by DCs. In fact, the authors showed that addition of free GM3 head groups to cell cultures prevented DCs from picking up intact HIV. They also demonstrated that DCs exposed to ganglioside-depleted HIV can't pick up the virus and so can't efficiently transfer it to T cells. Therefore, gangliosides may also affect HIV's ability to establish infection in the body. Of course, further studies are needed to understand how DCs recognize gangliosides in the viral envelope, how HIV avoids destruction after ganglioside-mediated DC uptake, and how the virus later escapes to infect T cells. Nonetheless, gangliosides may represent a useful therapeutic target for preventing or limiting infection by HIV and other ganglioside-containing viruses. Izquierdo-Useros N, Lorizate M, Contreras F-X, Rodriguez-Plata MT, Glass B, et al. (2012) Sialyllactose in Viral Membrane Gangliosides Is a Novel Molecular Recognition Pattern for Mature Dendritic Cell Capture of HIV-1. doi:10.1371/journal.pbio.1001315

Condensing And Fluidizing Effects Of Structurally Related Gangliosides On Phospholipid Films

In model membrane mixtures that mimic lipid raft compositions, the more ordered domains are enriched in the ganglioside, GM1, which contains four neutral sugars and a negatively charged sialic acid. To understand the organization and partitioning of GM1 in cell membranes, the outer leaflet of the cell membrane was modeled using Langmuir monolayers of DPPC and varying concentrations of GM1. At low biologically relevant concentrations, GM1 condenses the DPPC monolayer while at higher concentrations, it acts to fluidize, with a switch-over point between the two behaviors at a ratio of 3:1 DPPC:GM1. To examine phase morphology and organization of the components, the monolayers were transferred onto solid substrates and imaged with atomic force microscopy. At concentrations below the switch-over point, GM1 is located in nanoscale clusters within the condensed DPPC domains. The total surface area of these nanosize domains is larger than that attributable to GM1 molecules alone, suggesting the regions are due to GM1 and DPPC packing preferentially in condensed geometric complexes.To pinpoint the structural region of GM1 giving rise to the condensation effect, parallel experiments were run with ceramide and PEGylated lipids. Our results indicate that the bulky, rigid sugar ganglioside headgroup is necessary for the significant phase behavior effects on the surrounding lipid molecules. Ganglioside headgroup geometry and charge were further explored with binary mixtures of asialo-, disialo- and trisialo-gangliosides (containing zero, two, and three sialic acids) with DPPC. In all cases, a similar condensing and fluidizing effect that varies with ganglioside concentration is seen, suggesting the negatively charged sialic acid residue is not critical for the close-packing phenomenon. Variations in mole ratio of critical packing between different ganglioside molecules can be explained by global effects of headgroup geometry, charge, and resultant molecular dipole moments.

Binding of amyloid β-peptide to ganglioside micelles is dependent on histidine-13

Amyloid beta-peptide (Abeta) is a major component of plaques in Alzheimer's disease, and formation of senile plaques has been suggested to originate from regions of neuronal membrane rich in gangliosides. Here we demonstrate using NMR on 15N-labelled Abeta-(1-40) and Abeta-(1-42) that the interaction with ganglioside G(M1) micelles is localized to the N-terminal region of the peptide, particularly residues His13 to Leu17, which become more helical when bound. The key interaction is with His13, which undergoes a G(M1)-specific conformational change. The sialic acid residue of the ganglioside headgroup is important for determining the nature of the conformational change. The isolated pentasaccharide headgroup of G(M1) is not bound, suggesting the need for a polyanionic surface. Binding to heparin confirms this suggestion, since binding is of similar affinity but does not produce the same conformational changes in the peptide. A comparison of Abeta-(1-40) and Abeta-(1-42) indicates that binding to G(M1) micelles is not related to oligomerization, which occurs at the C-terminal end. These results imply that binding to ganglioside micelles causes a transition from random coil to alpha-helix in the N-terminal region, leaving the C-terminal region unstructured.

Comparative analysis of ganglioside conformations by MD simulations: implications for specific recognition by proteins

The accessible conformations of ganglioside headgroups belonging to the GM-(GM3, GM2, GM1, GM1b), GD-(GD3, GD2, GD1a, GD1b) and GT-(GT1a, GT1b) series have been determined using molecular dynamics (MD) simulations. Three to five 1 ns simulations have been run for each oligosaccharide by considering water molecules explicitly. The conformational space accessed by individual glycosidic linkages is the same in all the gangliosides and is similar to that in corresponding disaccharides. Transitions between the different minimum energy regions in the disaccharide map were also observed, especially in the gangliosides of GM- and GD-series. Some of the conformers that correspond to high-energy minima in disaccharide maps are accessed for longer durations probably due to the stabilising effect of the remote-residue interactions possible in these conformations. Comparison of the conformational preferences of specific pairs of gangliosides shows that the addition/deletion of residues (i.e. GM2 vs. GM3, etc.) does not seem to bring about any significant change in the accessible conformations of the rest of the molecule. The α2→3- and the β1→4- branches do not have a significant effect on each other's conformational freedom even though they are on the adjacent hydroxyl groups of galactose. The ganglioside headgroups can thus present a variety of shapes and surfaces to the receptor for binding due to the flexibility associated with glycosidic linkages, especially the branches. The results from the present MD simulations have been able to provide a stereochemical explanation for the observed specificities of some of the ganglioside-binding proteins such as bacterial toxins.

Mimicking gangliosides by design: mimics of GM1 headgroup.

Detailed knowledge of the three-dimensional structure of ganglioside headgroups has allowed the successful design of structural and functional mimics of ganglioside GMI oligosaccharide. Our recent work in this area is reviewed in this paper.

Dynamics of Ganglioside Headgroup in Lipid Environment: Molecular Dynamics Simulations of GM1 Embedded in Dodecylphosphocholine Micelle

Recognition of membrane-anchored glycosphingolipid receptors by various ligands is the key event in several biological phenomena. However, mere presence of these cell-surface receptors does not always ensure their recognition and binding by their respective ligands, a phenomenon termed “crypticity”. Earlier studies have suggested that different glycan headgroup orientations exposing and/or masking different epitopes may explain the crypticity of glycolipids. The effect of lipid environment on the orientation and conformation of GM1 ganglioside has been investigated in the present study by MD simulation technique in an attempt to understand the structural basis of crypticity. In addition, the effect of the length of the ceramide hydrocarbon tails on the headgroup conformation has also been investigated. MD simulations for 1 ns were performed with explicit water molecules for both GM1 headgroup (GM1-Os) and GM1 embedded in dodecylphosphocholine micelle. The simulations show that the conformations of the hyd...

Gangliosides affect membrane-channel activities dependent on ambient temperature.

1. The functional properties of biological membranes depend on their molecular composition. In regard to this, charged glycosphingolipids play an outstanding role in the functional adaptation of membranes to different temperatures. 2. In order to shed some light on the respective functional properties of complex membraneous glycosphingolipids, the effects of altered temperatures (5-40 degrees C) on planar lipid bilayers made from diphytanoylphosphatidylcholine (DPPC) and alamethicin as an ion channel was analyzed in the presence of either a sialoglycosphingolipid (less polar disialoganglioside GD1a or highly polar tetrasialoganglioside GQ1b) or phosphatidylserine (PS; as control). 3. Different to the control bilayers made from DPPC or DPPC + PS, the bilayers containing gangliosides had specific maxima in alamethicin conductance and stabile life times. Changes in pore-state conductances indicate structural effects based on an interaction of the large (negatively charged) ganglioside headgroups with the alamethicin pores. 4. The results concerning open time and closed time of channels seem to be based on the gangliosides changing the viscosity of the bilayer and possibly introducing phase transitions. 5. Thus, the findings suggest that gangliosides (1) directly affect channel molecules via their headgroups and (2) may additionally affect the fluidity of membranes in order to maintain membrane homeoviscosity in areas surrounding ion channels independent from the environmental temperature. 6. The effects of gangliosides may be of special interest in describing the ability of neuronal adaptation of vertebrates to temperature and more general regarding the functional adaptation of neurons.

Modeling ganglioside headgroups by conformational analysis and molecular dynamics.

The conformations and dynamics of gangliosides GM1, GM2, 6'-GM2 and GM4 have been studied by computational means, and the results compared to NMR data. Unconstrained conformational searches were run using the AMBER* force field augmented by MNDO derived parameters for the Neu5Ac anomeric torsion, the GB/SA water solvation model, and the MC/EM alogorithm; extended (10-12 ns) dynamic simulations in GB/SA water were performed with the MC/SD protocol, and the stored structures were minimized. The overall mobility of the Neu5Ac alpha2,3Gal linkage and the position of its minimum energy conformation have been shown to depend mainly on the presence or the absence of a GalNAc residue at the adjacent position. The best quantitative agreement with the available NOE data was achieved after minimization of the structures stored during the MC/SD dynamic runs. The latter protocol appears to reproduce satisfactorily the available experimental data, and can be used with confidence to build three-dimensional models of ganglioside headgroups.

Interaction of the GM2-activator protein with phospholipid-ganglioside bilayer membranes and with monolayers at the air-water interface.

Differential scanning calorimetry (DSC) and film balance measurements were performed to study the interactions of the GalNAcbeta1-->4(NeuAcalpha2-->3)Galbeta1-->4Glc1 -->1'Cer (GM2)-activator protein with phospholipid/ganglioside vesicles and monolayers. The nonglycosylated form of the GM2-activator protein, added to unilamellar lipid vesicles of different composition, causes differential effects on the gel to liquid-crystalline phase transition peaks. The phase transition temperature (Tm) of pure dimyristoylglycerophosphocholine (DMPC) bilayer is slightly decreased. When lipids which specifically bind the GM2-activator protein are incorporated into the vesicles (e.g. a sulfatide or gangliosides) a shoulder in the thermograms at higher temperatures is observed, indicating an increase of the stability of the gel phase in relation to the liquid-crystalline phase. We also studied the surface activity of a glycosylated and a nonglycosylated GM2-activator protein at the air-water interface. The glycosylated form showed a slightly lower surface activity than the GM2-activator protein without oligosaccharide moiety. When the GM2-activator protein is added to the sub-phase of a surface covered with a lipid monolayer, it can only insert into the monolayer and reach the air-water interface below a monolayer pressure of 25 mN.m-1, depending on the lipid composition, and not when the monolayers are at the bilayer equivalence pressure of 30-35 mN.m-1. Particularly for Galbeta1-->3GalNAcbeta1-->4(NeuAcalpha2-->3)Galbeta 1-->4Glc1-->1'Cer (GM1) and GM2 containing films, the critical pressures (picrit) when no additional increase in surface pressure is observed after addition of the protein into the subphase, are much lower. This leads to the conclusion that binding of the GM2 activator protein to the ganglioside headgroups prevents the protein from reaching the air-water interface. The protein is then located preferentially at the lipid-water interface and cannot penetrate into the chain region.

Exogenous application of gangliosides changes the state of excitability of retinal tissue as demonstrated by retinal spreading depression experiments.

Gangliosides are amphiphilic, sialic acid-containing glycosphingolipids which are found preferentially in complex composition in the cellular membranes of the nervous system of vertebrates, including the vertebrate retina as well as in other membranes. They are always exposed to the extracellular side of the membranes. By virtue of the negative charges they carry at their headgroup, they contribute to the surface charge of the membrane and may affect ion distribution, mainly that of protons and calcium ions, at the outer side of the membranes. Using retinal spreading depression (RSD) as a tool, we show in this study that the addition of exogenous gangliosides to the extracellular space can change the state of excitability of the retinal tissue. In RSD experiments it reduces the propagation velocity as well as the intrinsic optical signal of RSD waves. These effects are concentration dependent (IC50 about 20 microM) and increase with the increasing negative charge of the ganglioside headgroup. As a possible mechanistic basis of the changes found, the change of the calcium homeostasis of the extracellular space by the exogenously added gangliosides is discussed. Gangliosides have been reported to be useful in the treatment of some neuropathological syndromes, including migraine, although experimental verification has not been possible up to now. Taking into account that the retina is a true part of the CNS, our data may be interpreted as the requested verification.

Gangliosides in phospholipid bilayer membranes: interaction with tetanus toxin

The interaction between tetanus toxin and its fragments with gangliosides and negatively charged phosphatidylglycerols has been studied in phosphatidylcholine host membranes by protein circular dichroism measurement, calorimetry to determine lipid phase transitions, and by fluorescence spectroscopy to follow the toxin-induced pore formation by measuring the release of intravesicular entrapped dye. CD-spectroscopic secondary structure analysis showed conformational change of the toxin only in the presence of GT1b clearly demonstrating the involvement of the ganglioside headgroups for this lipid-protein-interaction. In a dot-blot analysis we showed that fragment C binds to GT1b in reconstituted vesicles and that this fragment is then accessible to a fragment C specific antibody which is only possible if fragment C is exposed at least partially on the surface of the vesicle. Our calorimetric study demonstrates the preferential binding of tetanus toxin to ganglioside GT1b. However, this protein is also able to bind to other gangliosides and also to negatively charged phospholipids causing phase separation due to electrostatic interaction. Since tetanus toxin preferentially binds short chain phosphatidylglycerol, we conclude that the protein adopts lipids with respect to charge, head group structure and chain length from the bulk phase. One consequence of this lipid-protein interaction is the ability of tetanus toxin to permeabilize lipid vesicles. Pore formation is favoured in the presence of GT1b in phosphatidylcholine membranes but only at a sufficiently high enough ganglioside content. Gangliosides others than GT1b are less effective in pore formation. In the presence of negatively charged phosphatidylglycerol tetanus toxin causes a dye release which in contrast to GT1b-containing vesicles is not saturable. We conclude that tetanus toxin acts in combination with a given number of GT1b molecules. Twenty ganglioside molecules are found to be necessary to form the stable pore. Other negatively charged lipids also cause the toxin to intercalate into the membrane but in this case the release velocity is determined by the formation of membrane defects.

The Surface Properties of Gangliosides: II. Direct Measurement of the Interaction between Bilayers Deposited on Mica Surfaces

The interaction between dipalmitylphosphatidylcholine, DPPC, the gangliosides G M1 and G T1b, and ganglioside/DPPC mixtures on mica surfaces have been studied using the surface forces apparatus. The results for DPPC were similar to those reported previously, showing a weak adhesion between the surfaces. For the gangliosides a long range force due to the electrical double layer was noted. No adhesion was observed. The ganglioside/DPPC mixtures, however, exhibit both repulsive electrostatic forces and adhesive contacts, indicating the possible presence of attractive interactions between the ganglioside headgroup and the DPPC matrix.