Ilya Mikhalyov

Self-aggregation : an intrinsic property of GM1 in lipid bilayers

We demonstrate that the ganglioside GM1 in lipid bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) exhibits a non-uniform lateral distribution, i.e., enriched regions of GM1 molecules are formed, which is an argument in favour of self-aggregation of GM1 being an intrinsic property of GM1 ganglioside. This was concluded from energy transfer/migration studies of BODIPY-labelled gangliosides by means of time-resolved fluorescence lifetime and depolarization experiments. Three fluorophore-labelled gangliosides were synthesized to include either of two spectroscopically different BODIPY groups. These were specifically localized either in the polar headgroup region or in the non-polar region of the lipid bilayer. An eventual ganglioside-ganglioside affinity/aggregation induced by the BODIPY groups was experimentally excluded, which suggests their use in examining the influence of GM1 in more complex systems.

Synthesis and characterization of new fluorescent glycolipid probes. Molecular organisation of glycosphingolipids in mixed-composition lipid bilayers

Abstract The synthesis and properties of new fluorescent analogs of glycosphingolipids (galactosylcerebrosides, gangliosides GM1, GM3, GD1a and GD3 bearing a 9-anthrylvinyl or 3-perylenoyl residue in the acyl chain are described. The synthesis includes steps: (i) hydrolytic removal of the fatty acyl from the corresponding natural compound; (ii) reacylation of the sphingosine amino group with trans-12-(9-anthryl)-11-dodecenoic or 9-(3-perylenoyl)nonanoic acid; (iii) reacetylation of the neuraminic amino group (for gangliosides). Studies of the behavior of the fluorescent glycolipids in model membranes (multilayer liposomes and sonicated vesicles) revealed that the probes are largely lipid-specific, i.e. they behave in many aspects similar to their corresponding natural counterparts. Fluorescence anisotropy measurements and studies of fluorescence energy transfer from anthryvinyl- to perylenoyl-labeled probes suggest that in mixed bilayer systems containing approx. 10 mol% glycolipid, galactosylceramide is partly segregated from phospholipids even above the temperature of gel/liquid crystal transition of the phospholipid matrix. By contrast, no indication of lipid demixing was found for phospholipid/ganglioside systems containing up to 15 mol% glycolipid.

Ganglioside binding pattern of CD33-related siglecs

Our study deals with the interaction of CD33 related-siglecs-5,-7,-8,-9,-10 with gangliosides GT1b, GQ1b, GD3, GM2, GM3 and GD1a. Siglec-5 bound preferentially to GQ1b, but weakly to GT1b, whereas siglec-10 interacted only with GT1b ganglioside. Siglec-7 and siglec-9 displayed binding to gangliosides GD3, GQ1b and GT1b bearing a disialoside motif, though siglec-7 was more potent; besides, siglec-9 interacted also with GM3. Siglec-8 demonstrated low affinity to the gangliosides tested compared with other siglecs. Despite high structural similarity of CD33 related siglecs, they demonstrated different ganglioside selectivity, in particular to the Neu5Acalpha2-8Neu5Ac motif.

On multivalent receptor activity of GM1 in cholesterol containing membranes

Gangliosides located at the outer leaflet of plasma membrane are molecules that either participate in recognizing of exogenous ligand molecules or exhibit their own receptor activity, which are both essential phenomena for cell communication and signaling as well as for virus and toxin entry. Regulatory mechanisms of lipid-mediated recognition are primarily subjected to the physical status of the membrane in close vicinity of the receptor. Concerning the multivalent receptor activity of the ganglioside GM1, several regulatory strategies dealing with GM1 clustering and cholesterol involvement have been proposed. So far however, merely the isolated issues were addressed and no interplay between them investigated. In this work, several advanced fluorescence techniques such as Z-scan fluorescence correlation spectroscopy, Förster resonance energy transfer combined with Monte Carlo simulations, and a newly developed fluorescence antibunching assay were employed to give a more complex portrait of clustering and cholesterol involvement in multivalent ligand recognition of GM1. Our results indicate that membrane properties have an impact on a fraction of GM1 molecules that is not available for the ligand binding. While at low GM1 densities (~1 %) it is the cholesterol that turns GM1 headgroups invisible, at higher GM1 level (~4 %) it is purely the local density of GM1 molecules that inhibits the recognition. At medium GM1 content, cooperation of the two phenomena occurs. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.

Lipid raft detecting in membranes of live erythrocytes

The fluorescent probe N-(BODIPY(®)-FL-propionyl)-neuraminosyl-GM(1) (BODIPY-GM(1)) was used to detect lipid rafts in living red blood cells (RBCs) membranes. The probe was detected with fluorescence video microscopy and was found to be uniformly distributed along plasma membrane at room temperature (23°C). At 4°C some probe clearly phase-separated to yield detectable bright spots that were smaller than spatial resolution. As measured by spectrofluorometry, in addition to a major fluorescence peak caused by emissions from monomers, the probe exhibited a red-shifted peak that is characteristic of a BODIPY fluorophore at high local concentrations, indicating that some probe had clustered. Red-shifted fluorescence was the greatest at 4°C, intermediate at 23°C, and the smallest at 37°C. Treating the RBCs with methyl-β-cyclodextrin to remove cholesterol eliminated the red-shifted peak. This strongly indicates that the presence of cholesterol was essential for phase separation of the probe. Fluorometry experiments indicate that rafts exist at 23°C and at 37°C, even though the membrane appears to be uniform at the resolution of microscope. The distinct GM(1) patches distributed over entire membrane of the erythrocytes were observed at both 23°C and at 37°C in RBCs stained with Alexa FL 647 cholera toxin subunit B conjugate (CTB-A647 ). Based on both fluorometry and fluorescence microscopy, some rafts clearly exist at 37°C.

GM1 Ganglioside Inhibits β-Amyloid Oligomerization Induced by Sphingomyelin

Abstract β‐Amyloid (Aβ) oligomers are neurotoxic and implicated in Alzheimers disease. Neuronal plasma membranes may mediate formation of Aβ oligomers in vivo. Membrane components sphingomyelin and GM1 have been shown to promote aggregation of Aβ; however, these studies were performed under extreme, non‐physiological conditions. We demonstrate that physiological levels of GM1, organized in nanodomains do not seed oligomerization of Aβ40 monomers. We show that sphingomyelin triggers oligomerization of Aβ40 and that GM1 is counteractive thus preventing oligomerization. We propose a molecular explanation that is supported by all‐atom molecular dynamics simulations. The preventive role of GM1 in the oligomerization of Aβ40 suggests that decreasing levels of GM1 in the brain, for example, due to aging, could reduce protection against Aβ oligomerization and contribute to the onset of Alzheimers disease.

Fluorescent BODIPY-labelled GM1 gangliosides designed for exploring lipid membrane properties and specific membrane-target interactions

New fluorophore-labelled G(M1) gangliosides have been synthesised and spectroscopically characterised. Spectroscopically different BODIPY groups were covalently linked, specifically to either the polar or the hydrophobic part of the ganglioside molecule. The absorption and fluorescence spectroscopic properties are reported for 564/571-BODIPY- and 581/591-BODIPY-labelled G(M1). Each of the different BODIPY groups is highly fluorescent and depolarisation experiments provide molecular information about the spatial distribution in lipid bilayers, as well as order and dynamics. From experiments performed on two spectroscopically different BODIPY:s, specific interactions can be revealed by monitoring the rate/efficiency of donor-acceptor electronic energy transfer. Systems of particular interest for applying these probes are e.g. mixtures of lipids, and peptides/proteins interacting with lipid membranes.

Designed Fluorescent Probes Reveal Interactions between Amyloid-β(1–40) Peptides and GM1 Gangliosides in Micelles and Lipid Vesicles

A hallmark of the common Alzheimers 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.

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.

Distribution of BODIPY-labelled phosphatidylethanolamines in lipid bilayers exhibiting different curvatures

In this paper we have investigated the behaviour of newly synthesised mono-palmitoyl- and dipalmitoyl-phosphatidylethanolamine probes (abbreviated as mPE and dPE, respectively) labelled in the polar headgroup region by either the FL-BODIPY or the 564/570-BODIPY fluorophore and solubilised in lipid systems that exhibit different curvatures. Because of the bulky BODIPY-groups, the monoacyl-form derivatives have a conic-like shape, whereas that for the diacyl derivatives is rather cylindrical. A careful analysis of time-resolved resonance energy transfer experiments by means of analytical models as well as Monte Carlo simulations shows that the mPE derivatives have a comparable affinity to highly curved bilayer regions (torroidal pores formed by magainin-2 in lipid bilayers, or the rims of discoid bicelles) and to planar bilayer regions (i.e. the flat region of lipid bilayers and bicelles). Furthermore, the monoacyl-probes are as compared to the diacyl-probes effectively closer to each other in a lipid bilayer, while none of these probes seems to be randomly distributed. Self-aggregation is most efficiently induced by the larger aromatic 564/570-BODIPY chromophore, but it is suppressed when using the diacyl instead of the monoacyl-form, and/or by attaching BODIPY-groups to the acyl-chain.