Line Activity of Ganglioside GM1 Regulates the Raft Size Distribution in a Cholesterol-Dependent Manner.

Liquid-ordered lipid domains, also called rafts, are assumed to be important players in different cellular processes, mainly signal transduction and membrane trafficking. They are thicker than the disordered part of the membrane and are thought to form to compensate for the hydrophobic mismatch between transmembrane proteins and the lipid environment. Despite the existence of such structures in vivo still being an open question, they are observed in model systems of multicomponent lipid bilayers. Moreover, the predictions obtained from model experiments allow the explanation of different physiological processes possibly involving rafts. Here we present the results of the study of the regulation of raft size distribution by ganglioside GM1. Combining atomic force microscopy with theoretical considerations based on the theory of membrane elasticity, we predict that this glycolipid should change the line tension of raft boundaries in two different ways, mainly depending on the cholesterol content. These results explain the shedding of gangliosides from the surface of tumor cells and the following ganglioside-induced apoptosis of T-lymphocytes in a raft-dependent manner. Moreover, the generality of the model allows the prediction of the line activity of different membrane components based on their molecular geometry.

Designed fluorescent probes reveal interactions between amyloid-beta(1-40) peptides and GM1 gangliosides in micelles and lipid vesicles.

A hallmark of the common Alzheimer's disease (AD) is the pathological conversion of its amphiphatic amyloid-beta (Abeta) peptide into neurotoxic aggregates. In AD patients, these aggregates are often found to be tightly associated with neuronal G(M1) ganglioside lipids, suggesting an involvement of G(M1) not only in aggregate formation but also in neurotoxic events. Significant interactions were found between micelles made of newly synthesized fluorescent G(M1) gangliosides labeled in the polar headgroup or the hydrophobic chain and Abeta(1-40) peptide labeled with a BODIPY-FL-C1 fluorophore at positions 12 and 26, respectively. From an analysis of energy transfer between the different fluorescence labels and their location in the molecules, we were able to place the Abeta peptide inside G(M1) micelles, close to the hydrophobic-hydrophilic interface. Large unilamellar vesicles composed of a raftlike G(M1)/bSM/cholesterol lipid composition doped with labeled G(M1) at various positions also interact with labeled Abeta peptide tagged to amino acids 2 or 26. A faster energy transfer was observed from the Abeta peptide to bilayers doped with 581/591-BODIPY-C(11)-G(M1) in the nonpolar part of the lipid compared with 581/591-BODIPY-C(5)-G(M1) residing in the polar headgroup. These data are compatible with a clustering process of G(M1) molecules, an effect that not only increases the Abeta peptide affinity, but also causes a pronounced Abeta peptide penetration deeper into the lipid membrane; all these factors are potentially involved in Abeta peptide aggregate formation due to an altered ganglioside metabolism found in AD patients.

[BODIPY-labeled ganglioside probes for membrane and biological studies].

A series of new fluorescent ganglioside G(M1) derivatives bearing the residue of 4,4-difluoro-4-bora-3a,4a-diaza-s-indecene (BODIPY) either in the polar or nonpolar part of the molecule have been synthesized. Gangliosides G(M1) labeled with the residues of (4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indecenyl)-5-pentanoic (564/570-BODIPY-pentanoic) acid and (4,4-difluoro-5-butadienylphenyl-4-bora-3a,4a-diaza-s-indecenyl)-11-undecanoic (581/591-BODIPY-undecanoic) acid at the polar part of the molecule or with the residue of (4,4-difluoro-5-butadienylphenyl-4-bora-3a,4a-diaza-s-indecenyl)-5-pentanoic (581/591-BODIPY-pentaoic) acid at the nonpolar part of the molecule have been synthesized. The spectral characteristics of the resulting probes and their behavior in ganglioside G(M1) micelles and in sphingomyelin-cholesterol enriched bilayers containing BODIPY-FL-labeled gangliosides G(M1) have been studied. The localization of the probe in the ganglioside molecule has been demonstrated to affect the efficiency of energy transfer in the case of the corresponding donor-acceptor pairs.

Donor-donor energy migration (DDEM) as a tool for studying aggregation in lipid phases.

A BODIPY-labelled sulfatide (N-(BODIPY-FL-pentanoyl)-galactosylcerebroside-sulfate, hereafter abbreviated as BD-Sulfatide) was solubilised at different concentrations in lipid vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Time-correlated single photon counting experiments show that the fluorescence relaxation is mono-exponential (with a lifetime of 6.5 ns) at molar ratios of BD-Sulfatide: DOPC that are less than 1:100. The fluorescence steady-state anisotropy decreases monotonously at molar ratios smaller than 1:1000, which is compatible with donor-donor energy migration (DDEM) among the BODIPY groups. A model that assumes DDEM across the lipid bilayers, as well as in their planes, was used to analyse the time-resolved fluorescence anisotropy. Only two parameters appear in the model namely: the bilayer thickness (d) and the average number density (C2) distribution of BD-Sulfatide in the lipid bilayers. The extracted d-values vary between 35 and 40 A, which is about the reported thickness of a bilayer of DOPC (38 A). Hence, the BODIPY groups are preferentially located in the water-lipid interface. At low concentration the experimental C2-values and those independently calculated are in good agreement, while the experimental values gradually become lower with increasing BD-Sulfatide concentration. These results are compatible with an aggregation of the sulfatides and self-quenching of BODIPY, which is clearly established at higher concentrations of the BD-Sulfatide.

Immunosuppressive activity of glycosphingolipids. Influence of serum factors on ganglioside inhibition of IL-4-dependent cell proliferation.

Gangliosides have been shown to inhibit proliferation of the interleukin-4 (IL-4) responsive cell line CT.4R. Kinetic analysis has revealed that ganglioside GT1b is a competitive inhibitor of proliferation, while GM and GM3 show a mixed pattern of inhibition, i.e., exhibit more than one inhibition type. Contribution of the competitive cell inhibition for GM1 and GM3 depends on serum factors added: the higher is the percentage of FCS, the larger is the contribution of competitive inhibition. The pattern of proliferation inhibition shown for GT1b does not depend on the FCS content. We have also studied the interaction of the recombinant IL-4 with fluorescent (anthrylvinyl-labelled) gangliosides GM1 and GM3 and lactosylceramide incorporated into liposomes. Dissociation constants of the IL-4-ganglioside complexes have been determined; lactosylceramide does not interact with rIL-4. The K(d) values for the lymphokine complexes with gangliosides support the conclusion based on the kinetic analysis that IL-4 has a higher affinity for GM3 (K(d) = 5 nM) than for GM1 (K(d) = 0.28 microM).

New fluorescent lysolipids: preparation and selective labeling of inner liposome leaflet.

Two new fluorescent lysophosphatidylcholine probes have been synthesized for use as a donor-acceptor pair in fluorescence resonance energy transfer (FRET): 9-anthrylvinyl (LAPC) as donor and 3-perylenoyl (LPPC) as acceptor. The partition coefficients between membrane and aqueous phases were 8.3 x 10(5) and 10.5 x 10(5) for LAPC and LPPC, respectively. The inner leaflets of unilamellar lipid vesicles were labeled with these probes to assess conservation of membrane sidedness after membrane fusion. After medium-sized unilamellar vesicles (MUV) were prepared with a probe in both leaflets, probe in the outer leaflet was removed by repeatedly washing with an excess of unlabeled giant unilamellar vesicles (GUV). MUV and GUV were separated by centrifugation. The probes did not flip-flop across bilayers at 25 degrees C for at least 12 h. MUV containing the ganglioside GT1b were labeled with the LAPC/LPPC pair in the inner leaflet and incubated for 30 min at neutral pH with influenza virus. Fusion was triggered by acidification to pH 5.0 and was monitored by an increase in donor fluorescence in a FRET assay. When the inner leaflets of MUV were labeled by LAPC only, its fluorescence did not change after fusion. However, the fluorescence decreased by 60% when the LAPC was removed from the outer leaflets of the fused membranes by repeated washings with GUV. We conclude that the lipids of the inner and outer leaflets of the fused MUV/virus complexes intermixed.

Selective labeling of the inner liposome leaflet by fluorescent lipid probes, and studies of liposome fusion with influenza virus.

The inner leaflet of unilamellar lipid vesicles was labeled with fluorescent lysophosphatidylcholines. The probes make a donor-acceptor pair in resonance energy transfer (RET), being labeled with 9-anthrylvinyl (L-APC, donor) and 3-perylenoyl (L-PPC, acceptor) fluorophores. They migrate rapidly between bilayers through the water phase: tau 1/2 of equilibration is approximately 5 min at 37 degrees C. The probe(s) can be removed from the outer leaflet of uniformly labeled medium-size unilamellar vesicles (MUV) by repeated washings with excess unlabeled large unilamellar vesicles (LUV) (separation by centrifugation). The probes flip-flop across bilayers rather slowly. MUV containing the ganglioside GT1b and labeled with the L-APC/L-PPC pair in the inner leaflet were fused with an equal amount of influenza virus; the process was monitored by an increase of the donor fluorescence in RET assay. If inner MUV leaflet was labeled with the anthrylvinyl probe only, the probe fluorescence decreased by half when the probe was removed from the outer leaflets of the fused membranes. This shows that the lipids of the inner and outer leaflets of the MUV randomize in the process of fusion.