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BackgroundGangliosides are glycosphingolipids highly enriched in the brain, with important roles in cell signaling, cell-to-cell communication, and immunomodulation. Genetic defects in the ganglioside biosynthetic pathway result in severe neurodegenerative diseases, while a partial decrease in the levels of specific gangliosides was reported in Parkinson’s disease and Huntington’s disease. In models of both diseases and other conditions, administration of GM1—one of the most abundant gangliosides in the brain—provides neuroprotection. Most studies have focused on the direct neuroprotective effects of gangliosides on neurons, but their role in other brain cells, in particular microglia, is not known. In this study we investigated the effects of exogenous ganglioside administration and modulation of endogenous ganglioside levels on the response of microglia to inflammatory stimuli, which often contributes to initiation or exacerbation of neurodegeneration.MethodsIn vitro studies were performed using BV2 cells, mouse, rat, and human primary microglia cultures. Modulation of microglial ganglioside levels was achieved by administration of exogenous gangliosides, or by treatment with GENZ-123346 and L–t-PDMP, an inhibitor and an activator of glycolipid biosynthesis, respectively. Response of microglia to inflammatory stimuli (LPS, IL-1β, phagocytosis of latex beads) was measured by analysis of gene expression and/or secretion of pro-inflammatory cytokines. The effects of GM1 administration on microglia activation were also assessed in vivo in C57Bl/6 mice, following intraperitoneal injection of LPS.ResultsGM1 decreased inflammatory microglia responses in vitro and in vivo, even when administered after microglia activation. These anti-inflammatory effects depended on the presence of the sialic acid residue in the GM1 glycan headgroup and the presence of a lipid tail. Other gangliosides shared similar anti-inflammatory effects in in vitro models, including GD3, GD1a, GD1b, and GT1b. Conversely, GM3 and GQ1b displayed pro-inflammatory activity. The anti-inflammatory effects of GM1 and other gangliosides were partially reproduced by increasing endogenous ganglioside levels with L–t-PDMP, whereas inhibition of glycolipid biosynthesis exacerbated microglial activation in response to LPS stimulation.ConclusionsOur data suggest that gangliosides are important modulators of microglia inflammatory responses and reveal that administration of GM1 and other complex gangliosides exerts anti-inflammatory effects on microglia that could be exploited therapeutically.

Simple Summary: The early impairments in Alzheimer's disease are related to neuronal membrane damage. Different lipids are present in biological membranes, playing relevant physiological roles. Some of them, such as sphingomyelin, cholesterol, and ganglioside GM1, interact with each other and, importantly, with the A beta peptide. Here, these interactions are studied using small angle X-ray scattering in model membrane systems, such as large unilamellar liposomes. This technique gives information on the width of the bilayer and reveals structural differences due to the different lipid compositions, as well as some small differences due to the presence of the A beta peptide. The analysis highlights the concentration-dependent effect of GM1 on membrane thickness and the interaction with the A beta-peptide, together with the inhibiting effect that the presence of sphingomyelin has on the GM1-A beta interaction.& nbsp;The early impairments appearing in Alzheimer's disease are related to neuronal membrane damage. Both aberrant A beta species and specific membrane components play a role in promoting aggregation, deposition, and signaling dysfunction. Ganglioside GM1, present with cholesterol and sphingomyelin in lipid rafts, preferentially interacts with the A beta peptide. GM1 at physiological conditions clusters in the membrane, the assembly also involves phospholipids, sphingomyelin, and cholesterol. The structure of large unilamellar vesicles (LUV), made of a basic POPC:POPS matrix in a proportion of 9:1, and containing different amounts of GM1 (1%, 3%, and 4% mol/mol) in the presence of 5% mol/mol sphingomyelin and 15% mol/mol cholesterol, was studied using small angle X-ray scattering (SAXS). The effect of the membrane composition on the LUVs-A beta-peptide interaction, both for A beta(1-40) and A beta(1-42) variants, was, thus, monitored. The presence of GM1 leads to a significant shift of the main peak, towards lower scattering angles, up to 6% of the initial value with SM and 8% without, accompanied by an opposite shift of the first minimum, up to 21% and 24% of the initial value, respectively. The analysis of the SAXS spectra, using a multi-Gaussian model for the electronic density profile, indicated differences in the bilayer of the various compositions. An increase in the membrane thickness, by 16% and 12% when 2% and 3% mol/mol GM1 was present, without and with SM, respectively, was obtained. Furthermore, in these cases, in the presence of A beta(40), a very small decrease of the bilayer thickness, less than 4% and 1%, respectively, was derived, suggesting the inhibiting effect that the presence of sphingomyelin has on the GM1-A beta interaction.

For five decades it has been known that the pentamer of B subunits (choleragenoid) of the cholera toxin (CT) of Vibrio cholerae binds with high preference to the ganglioside GM1 (II3Neu5Ac-Gg4Cer). However, the exact structures of CT-binding GM1 lipoforms of primary human colon epithelial cells (pHCoEpiCs) have not yet been described in detail. The same holds true for generating further GM1 receptor molecules from higher sialylated gangliosides with a GM1 core through the neuraminidase of V. cholerae. To avoid the artificial incorporation of exogenous gangliosides from animal serum harboring GM1 and higher sialylated ganglio-series gangliosides, pHCoEpiCs were cultured in serum-free medium. Thin-layer chromatography overlay binding assays using a choleragenoid combined with electrospray ionization mass spectrometry revealed GM1 lipoforms with sphingosine (d18:1) as the sole sphingoid base linked to C14:0, C16:0, C18:0 or C20:0 fatty acyl chains forming the ceramide (Cer) moieties of the main choleragenoid-binding GM1 species. Desialylation of GD1a (IV3Neu5Ac,II3Neu5Ac-Gg4Cer) and GT1b (IV3Neu5Ac,II3(Neu5Ac)2-Gg4Cer) of pHCoEpiCs by V. cholerae neuraminidase was observed. GD1a-derived GM1 species with stable sphingosine (d18:1) and saturated fatty acyl chains varying in chain length from C16:0 up to C22:0 could be identified, indicating the ingenious interplay between CT and the neuraminidase of V. cholerae recruiting additional GM1 receptors of pHCoEpiCs.

Gangliosides are major constituents of the plasma membrane and are known to promote a number of physiological actions in the brain, including synaptic plasticity and neuroprotection. In particular, the ganglioside GM1 was found to have a wide range of preclinical and clinical benefits in brain diseases such as spinal cord injury, Huntington’s disease and Parkinson’s disease. However, little is known about the underlying cellular and molecular mechanisms of GM1 in the brain. In the present study, we show that GM1 exerts its actions through the promotion of glycolysis in astrocytes, which leads to glucose uptake and lactate release by these cells. In astrocytes, GM1 stimulates the expression of several genes involved in the regulation of glucose metabolism. GM1 also enhances neuronal mitochondrial activity and triggers the expression of neuroprotection genes when neurons are cultured in the presence of astrocytes. Finally, GM1 leads to a neuroprotective effect in astrocyte-neuron co-culture. Together, these data identify a previously unrecognized mechanism mediated by astrocytes by which GM1 exerts its metabolic and neuroprotective effects.

Ganglioside GM1 is the most common brain ganglioside enriched in plasma membrane regions known as lipid rafts or membrane microdomains. GM1 participates in many modulatory and communication functions associated with the development, differentiation, and protection of neuronal tissue. It has, however, been demonstrated that GM1 plays a negative role in the pathophysiology of Alzheimer’s disease (AD). The two features of AD are the formation of intracellular neurofibrillary bodies and the accumulation of extracellular amyloid β (Aβ). Aβ is a peptide characterized by intrinsic conformational flexibility. Depending on its partners, Aβ can adopt different spatial arrangements. GM1 has been shown to induce specific changes in the spatial organization of Aβ, which lead to enhanced peptide accumulation and deleterious effect especially on neuronal membranes containing clusters of this ganglioside. Changes in GM1 levels and distribution during the development of AD may contribute to the aggravation of the disease.

To resolve the contribution of ceramide-containing lipids to the aggregation of the amyloid-β protein into β-sheet rich toxic oligomers, we employed molecular dynamics simulations to study the effect of cholesterol-containing bilayers comprised of POPC (70% POPC, and 30% cholesterol) and physiologically relevant concentrations of sphingomyelin (SM) (30% SM, 40% POPC, and 30% cholesterol), and the GM1 ganglioside (5% GM1, 70% POPC, and 25% cholesterol). The increased bilayer rigidity provided by SM (and to a lesser degree, GM1) reduced the interactions between the SM-enriched bilayer and the N-terminus of Aβ42 (and also residues Ser26, Asn27, and Lys28), which facilitated the formation of a β-sheet in the normally disordered N-terminal region. Aβ42 remained anchored to the SM-enriched bilayer through hydrogen bonds with the side chain of Arg5. With β-sheets in the at the N and C termini, the structure of Aβ42 in the sphingomyelin-enriched bilayer most resembles β-sheet-rich structures found in higher-ordered Aβ fibrils. Conversely, when bound to a bilayer comprised of 5% GM1, the conformation remained similar to that observed in the absence of GM1, with Aβ42 only making contact with one or two GM1 molecules. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.

Pig-human xenotransplantation can trigger cell-mediated immune responses. We explored the role of gangliosides in inflammation related to immune rejection in xenotransplantation. Co-culture of xenogeneic cells (pig-MSCs and RAW264.7) was used to emulate xenotransplantation conditions. MTT assay results indicated that cell viability was significantly decreased in pADMSCs co-cultured with RAW264.7 cells. GM1 and GM3 were highly expressed in pADMSCs co-cultured with RAW264.7 cells. pADMSCs co-cultured with RAW264.7 cells strongly expressed pro-inflammatory proteins such as COX-2, iNOS, p50, p65, pIκBα, and TNF-α. GM1-knockdown pADMSCs co-cultured with RAW 264.7 cells did not show significantly altered cell viability, but pro-inflammatory proteins were markedly inhibited. Co-culture of pADMSCs with RAW264.7 cells induced significant phosphorylation (p) of JNK1/2 and pERK1/2. However, pERK1/2 and pJNK1/2 were decreased and MEK1/2 and Raf1 were suppressed in GM1-knockdown pADMSCs co-cultured with RAW264.7 cells. Thus, the Raf-1/MEK1/2/ERK1/2 and JNK1/2 pathways were significantly upregulated in response to increases of GM1 in co-cultured xenogeneic cells. However, the inflammatory response was suppressed in co-culture of GM1-knockdown pADMSCs with RAW264.7 cells via down-regulation of the Raf-1/MEK1/2/ERK1/2 and JNK1/2 pathways. Therefore, the ganglioside GM1 appears to play a major role in the inflammatory response in xenotransplantation via the Raf-1/MEK1/2/ERK1/2 and JNK1/2 pathways.

We compared the lateral structure of giant unilamellar vesicles (GUVs) composed of three pseudo binary mixtures of different glycosphingolipid (GSL), i.e. sulfatide, asialo-GM1 or GM1, with POPC. These sphingolipids possess similar hydrophobic residues but differ in the size and charge of their polar head group. Fluorescence microscopy experiments using LAURDAN and DiIC18 show coexistence of micron sized domains in a molar fraction range that depends on the nature of the GSLs. In all cases, experiments with LAURDAN show that the membrane lateral structure resembles the coexistence of solid ordered and liquid disordered phases. Notably, the overall extent of hydration measured by LAURDAN between the solid ordered and liquid disordered membrane regions show marked similarities and are independent of the size of the GSL polar head group. In addition, the maximum amount of GSL incorporated in the POPC bilayer exhibits a strong dependence on the size of the GSL polar head group following the order sulfatide > asialo-GM1 > GM1. This observation is in full harmony with previous experiments and theoretical predictions for mixtures of these GSL with glycerophospholipids. Finally, compared with previous results reported in GUVs composed of mixtures of POPC with the sphingolipids cerebroside and ceramide, we observed distinctive curvature effects at particular molar fraction regimes in the different mixtures. This suggests a pronounced effect of these GSL on the spontaneous curvature of the bilayer. This observation may be relevant in a biological context, particularly in connection with the highly curved structures found in neural cells. Fil: Rodi, Pablo Marcelo. MEMPHYS - Center for Biomembrane Physics; Dinamarca. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Departamento de Bioquímica y Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Maggio, Bruno. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Bagatolli, Luis Alberto. MEMPHYS - Center for Biomembrane Physics; Dinamarca. Universidad Yachay Tech; Ecuador. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Cholera toxin B-subunit (CTxB) has emerged as one of the most widely utilized tools in membrane biology and biophysics. CTxB is a homopentameric stable protein that binds tightly to up to five GM1 glycosphingolipids. This provides a robust and tractable model for exploring membrane structure and its dynamics including vesicular trafficking and nanodomain assembly. Here, we review important advances in these fields enabled by use of CTxB and its lipid receptor GM1.

Prion protein (PrPC ) localizes stably in lipid rafts microdomains and is able to recruit downstream signal transduction pathways by the interaction with promiscuous partners. Other proteins have the ability to occasionally be recruited to these specialized membrane areas, within multimolecular complexes. Among these, we highlight the presence of the low-density lipoprotein receptor-related protein 1 (LRP1), which was found localized transiently in lipid rafts, suggesting a different function of this receptor that through lipid raft becomes able to activate a signal transduction pathway triggered by specific ligands, including Tissue plasminogen activator (tPA). Since it has been reported that PrPC participates in the tPA-mediated plasminogen activation, in this study, we describe the role of lipid rafts in the recruitment and activation of downstream signal transduction pathways mediated by the interaction among tPA, PrPC and LRP1 in human neuroblastoma SK-N-BE2 cell line. Co-immunoprecipitation analysis reveals a consistent association between PrPC and GM1, as well as between LRP1 and GM1, indicating the existence of a glycosphingolipid-enriched multimolecular complex. In our cell model, knocking-down PrPC by siRNA impairs ERK phosphorylation induced by tPA. Moreover the alteration of the lipidic milieu of lipid rafts, perturbing the physical/functional interaction between PrPC and LRP1, inhibits this response. We show that LRP1 and PrPC , following tPA stimulation, may function as a system associated with lipid rafts, involved in receptor-mediated neuritogenic pathway. We suggest this as a multimolecular signaling complex, whose activity depends strictly on the integrity of lipid raft and is involved in the neuritogenic signaling.

To investigate the interaction between amyloid assemblies and “lipid-rafts”, we performed functional and structural experiments on salmon calcitonin (sCT) solutions rich in prefibrillar oligomers, proto- and mature-fibers interacting with liposomes made of monosialoganglioside-GM1 (4%), DPPC (48%) and cholesterol (48%). To focus on the role played by electrostatic forces and considering that sCT is positive and GM1 is negative at physiologic pH, we compared results with those relative to GM1-free liposomes while, to assess membrane fluidity effects, with those relative to cholesterol-free liposomes. We investigated functional effects by evaluating Ca2+-influx in liposomes and viability of HT22-DIFF neurons. Only neurotoxic solutions rich in unstructured prefibrillar oligomers were able to induce Ca2+-influx in the “lipid-rafts” model, suggesting that the two phenomena were correlated. Thus, we investigated protein conformation and membrane modifications occurring during the interaction: circular dichroism showed that “lipid-rafts” fostered the formation of β-structures and energy filtered-transmission electron microscopy that prefibrillar oligomers formed pores, similar to Aβ did. We speculate that electrostatic forces between the positive prefibrillar oligomers and the negative GM1 drive the initial binding while the hydrophobic profile and flexibility of prefibrillar oligomers, together with the membrane fluidity, are responsible for the subsequent pore formation leading to Ca2+-influx and neurotoxicity.

Biomembranes are built up from lipid bilayers with two leaflets that typically differ in their lipid composition. Each lipid molecule stays within one leaflet of the bilayer before it undergoes a transition, or flip-flop, to the other leaflet. The corresponding flip-flop times are very different for different lipid species and vary over several orders of magnitude. Here, we use molecular dynamics simulations to elucidate the consequences of this separation of time scales for compositionally asymmetric bilayers. We first study bilayers with two lipid components that do not undergo flip-flops on the accessible time scales. In such a situation, one must distinguish a bilayer state in which both leaflets have the same preferred area from another state in which each leaflet is tensionless. However, when we add a third lipid component that undergoes frequent flip-flops, the bilayer relaxes toward the state with tensionless leaflets, not to the state with equal preferred leaflet areas. Furthermore, we show that bilayers with compositional asymmetry acquire a significant spontaneous curvature even if both leaflets are tensionless. Our results can be extended to lipid bilayers with a large number of lipid components provided at least one of these components undergoes frequent flip-flops. For cellular membranes containing lipid pumps, the leaflet tensions also depend on the rates of protein-induced flip-flops.

To investigate the interaction between amyloid assemblies and &ldquo, lipid-rafts&rdquo, we performed functional and structural experiments on salmon calcitonin (sCT) solutions rich in prefibrillar oligomers, proto- and mature-fibers interacting with liposomes made of monosialoganglioside-GM1 (4%), DPPC (48%) and cholesterol (48%). To focus on the role played by electrostatic forces and considering that sCT is positive and GM1 is negative at physiologic pH, we compared results with those relative to GM1-free liposomes while, to assess membrane fluidity effects, with those relative to cholesterol-free liposomes. We investigated functional effects by evaluating Ca2+-influx in liposomes and viability of HT22-DIFF neurons. Only neurotoxic solutions rich in unstructured prefibrillar oligomers were able to induce Ca2+-influx in the &ldquo, model, suggesting that the two phenomena were correlated. Thus, we investigated protein conformation and membrane modifications occurring during the interaction: circular dichroism showed that &ldquo, fostered the formation of &beta, structures and energy filtered-transmission electron microscopy that prefibrillar oligomers formed pores, similar to A&beta, did. We speculate that electrostatic forces between the positive prefibrillar oligomers and the negative GM1 drive the initial binding while the hydrophobic profile and flexibility of prefibrillar oligomers, together with the membrane fluidity, are responsible for the subsequent pore formation leading to Ca2+-influx and neurotoxicity.

Background: The roles of gangliosides in senescent endothelial cells (ECs) were unknown. Results: Ganglioside GM1 increased on senescent and aged ECs. GM1 contributes to the impairment of insulin signaling in ECs. Conclusion: Increased GM1 with senescence and aging contributes to insulin resistance in ECs. Significance: GM1 is the first known contributor to insulin resistance in ECs., Vascular endothelial cells (ECs) play central roles in physiologically important functions of blood vessels and contribute to the maintenance of vascular integrity. Therefore, it is considered that the impairment of EC functions leads to the development of vascular diseases. However, the molecular mechanisms of the EC dysfunctions that accompany senescence and aging have not yet been clarified. The carbohydrate antigens carried by glycoconjugates (e.g. glycoproteins, glycosphingolipids, and proteoglycans) mainly present on the cell surface serve not only as marker molecules but also as functional molecules. In this study, we have investigated the abundance and functional roles of glycosphingolipids in human ECs during senescence and aging. Among glycosphingolipids, ganglioside GM1 was highly expressed in abundance on the surface of replicatively and prematurely senescent ECs and also of ECs derived from an elderly subject. Insulin signaling, which regulates important functions of ECs, is impaired in senescent and aged ECs. Actually, by down-regulating GM1 on senescent ECs and overloading exogenous GM1 onto non-senescent ECs, we showed that an increased abundance of GM1 functionally contributes to the impairment of insulin signaling in ECs. Taken together, these findings provide the first evidence that GM1 increases in abundance on the cell surface of ECs under the conditions of cellular senescence and aging and causes insulin resistance in ECs. GM1 may be an attractive target for the detection, prevention, and therapy of insulin resistance and related vascular diseases, particularly in older people.

Background: Multifocal motor neuropathy (MMN) is associated with IgM antibodies to GM1 ganglioside. The importance of the lipid milieu that might facilitate or inhibit antibody binding to GM1 in immunoassays is well recognised. Existing studies, using a range of different approaches, generally concur that anti-GM1 IgM antibody detection rates are improved by the addition of galactocerebroside (GalC) to the GM1 assay.\ud \ud Objective: The current study sought to formally evaluate the clinical utility of the GM1:GalC complex assay in the diagnosis of MMN.\ud \ud Methods: Anti-GM1 and -GM1:GalC antibodies were examined using ELISA and glycoarray (dot blot) in a fully blinded study design, consisting of 100 MMN patients, 100 ALS cases and 100 healthy controls.\ud \ud Results: The detection of anti-GM1 Abs using glycoarray was 67% sensitive and 85% specific. The addition of GalC to GM1, (1:1 weight to weight ratio), increased the sensitivity to 81% , whilst dropping specificity to 80% . Increasing the GalC content to a 1:5 ratio (or higher) further decreased specificity, and in doing so limited the usefulness of the GM1:GalC assay to the level of GM1 alone. The addition of GalC to the ELISA method also significantly increased sensitivity compared with GM1 alone, albeit with a significant decrease in specificity.\ud \ud Conclusions: This study indicates that the GM1:GalC assay is an advantageous assay adaptation for detecting anti-GM1 antibodies in MMN, using either glycoarray or ELISA, and warrants introduction into clinical diagnostic practice.

Gangliosides and the cell adhesion molecule neuroplastin (Np) are abundant constituents of neuronal plasma membranes and important for brain functions as evidenced in studies from knock-out animals. We demonstrated that hippocampal expression of Np is affected in two transgenic mouse strains each of them with specific alterations in ganglioside composition. Additionally, we observed that Np distribution in membrane sub-domains depends on the ganglioside environment. Here, we aim to visualize the molecular relationship of gangliosides and Np in rat hippocampal neurons during different maturation stages in vitro (6-42 DIV). For that purpose, we performed live cell staining using primary antibodies to identify the two Np isoforms (Np65 and Np55) and the four major brain ganglioside species (GM1, GD1a, GD1b and GT1b) in hippocampal neurons. Co-localization of Np with each of the four gangliosides was analyzed by confocal microscopy. Although all four main brain gangliosides were unequivocally detected in the cell surface of mature hippocampal neurons (≥21DIV), both Np isoforms co-localized predominately with GM1 and only to a much lesser extent with GD1a. Neuroplastin and the four ganglioside species are expressed but do not co- localize in immature neurons (≤12DIV). Our results not only corroborate that the Nps distribute preferentially into isolated GM1- containing lipid rafts but also they suggest that neuroplastin is tightly and largely localized in selective GM1 nanodomains in intact plasma membrane of mature neurons. Therefore, we propose that some of the main functions of Np in mature neurons may be defined by its positioning within a GM1-enriched membrane milieu.

Monosialotetrahexosylganglioside (GM1) has good activity on brain diseases and was developed to be a drug applied in clinics for neurological disorders and nerve injury. It is difficult to isolate GM1 in industry scale from the brains directly. In this work, a simple and highly efficient method with high yield was developed for the isolation, conversion, and purification of GM1 from a pig brain. Gangliosides (GLS) were first extracted by supercritical CO2 (SCE). The optimum extraction time of GLS by SCE was 4 h, and the ratio of entrainer to acetone powder from the pig brain was 3:1 (v/w). GM1 was then prepared from GLS by immobilized sialidase and purified by reverse-phase silica gel. Sodium alginate embedding was used for the immobilization of sialidase. Under the optimized method, the yield of high-purity GM1 was around 0.056%. This method has the potential to be applied in the production of GM1 in the industry.

Growing evidence shows that GM1 ganglioside is involved in amyloid deposition and toxicity. By means of real-time single particle tracking, we show that amyloid oligomers and aggregates formed by A beta 1-42 and amylin, two peptides associated, respectively, with the development of Alzheimer's disease and type II diabetes, interact with GM1 and decrease dramatically its lateral diffusion on the plasma membrane of living neuroblastoma cells. The confinement of GM1, a constituent of membrane rafts involved in neuroprotection, at the level of both types of amyloid aggregates can interfere with cell signaling pathways and contribute to the loss of neuroprotection. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Driven by interactions between lipids and proteins, biological membranes display lateral heterogeneity that manifests itself in a mosaic of liquid-ordered (Lo) or raft, and liquid-disordered (Ld) or non-raft domains with a wide range of different properties and compositions. In giant plasma membrane vesicles and giant unilamellar vesicles, specific binding of Cholera Toxin (CTxB) to GM1 glycolipids is a commonly used strategy to label raft domains or Lo membrane environments. However, these studies often use acyl-chain labeled bodipy-GM1 (bdGM1), whose headgroup accessibility and membrane order or phase partitioning may differ from those of GM1, rendering the interpretation of CTxB binding data quite problematic. To unravel the molecular basis of CTxB binding to GM1 and bdGM1, we explored the partitioning and the headgroup presentation of these gangliosides in the Lo and Ld phases using atomistic molecular dynamics simulations complemented by CTxB binding experiments. The conformation of both GM1 and bdGM1 was shown to be largely similar in the Lo and Ld phases. However, bdGM1 showed reduction in receptor availability when reconstituted into synthetic bilayer mixtures, highlighting that membrane phase partitioning of the gangliosides plays a considerable role in CTxB binding. Our results suggest that the CTxB binding is predominately modulated by the partitioning of the receptor to an appropriate membrane phase. Further, given that the Lo and Ld partitioning of bdGM1 differs from those of GM1, usage of bdGM1 for studying GM1 behavior in cells can lead to invalid interpretation of experimental data. publishedVersion

Huntington Disease (HD) is a genetic neurodegenerative disorder characterized by broad types of cellular and molecular dysfunctions that may affect both neuronal and non-neuronal cell populations. Among all the molecular mechanisms underlying the complex pathogenesis of the disease, alteration of sphingolipids has been identified as one of the most important determinants in the last years. In the present study, besides the purpose of further confirming the evidence of perturbed metabolism of gangliosides GM1, GD1a, and GT1b the most abundant cerebral glycosphingolipids, in the striatal and cortical tissues of HD transgenic mice, we aimed to test the hypothesis that abnormal levels of these lipids may be found also in the corpus callosum white matter, a ganglioside-enriched brain region described being dysfunctional early in the disease. Semi-quantitative analysis of GM1, GD1a, and GT1b content indicated that ganglioside metabolism is a common feature in two different HD animal models (YAC128 and R6/2 mice) and importantly, demonstrated that levels of these gangliosides were significantly reduced in the corpus callosum white matter of both models starting from the early stages of the disease. Besides corroborating the evidence of aberrant ganglioside metabolism in HD, here, we found out for the first time, that ganglioside dysfunction is an early event in HD models and it may potentially represent a critical molecular change influencing the pathogenesis of the disease.

The effect of Aβ25–35 peptide, in its fibrillar and non-fibrillar forms, on ganglioside expression in organotypic hippocampal slice cultures was investigated. Gangliosides were endogenously labeled with D-[1-C14] galactose and results showed that Aβ25–35 affected ganglioside expression, depending on the peptide aggregation state, that is, fibrillar Aβ25–35 caused an increase in GM3 labeling and a reduction in GD1b labeling, whereas the non-fibrillar form was able to enhance GM1 expression. Interestingly, GM1 exhibited a neuroprotective effect in this organotypic model, since pre-treatment of the hippocampal slices with GM1 10 μM was able to prevent the toxicity triggered by the fibrillar Aβ25–35, when measured by propidium iodide uptake protocol. With the purpose of further investigating a possible mechanism of action, we analyzed the effect of GM1 treatment (1, 6, 12 and 24 h) upon the Aβ-induced alterations on GSK3β dephosphorylation/activation state. Results demonstrated an important effect after 24-h incubation, with GM1 preventing the Aβ-induced dephosphorylation (activation) of GSK3β, a signaling pathway involved in apoptosis triggering and neuronal death in models of Alzheimer’s disease. Taken together, present results provide a new and important support for ganglioside participation in development of Alzheimer’s disease experimental models and suggest a protective role for GM1 in Aβ-induced toxicity. This may be useful for designing new therapeutic strategies for Alzheimer’s treatment.

Gangliosides are target receptors for bacterial entry, yet those present in human milk exhibit a protective role against bacterial infection. Here, we show that treatment with ganglioside mixture at a concentration of 100 microg/mL resulted in significant inhibition of the vacuole formation activity of Helicobacter pylori vacuolating cytotoxin (VacA) in gastric epithelial cancer AZ-521 cells. All gangliosides (GM1, GM2, GM3, GD1a, GD1b, GD3 and GT1b) examined showed good neutralizing capacity against VacA. A pull-down assay was performed using lyso-GM1 coupled to Sepharose as the tagged polysaccharide polymer to capture VacA from H. pylori culture supernatant. GM1-VacA complexes were successfully precipitated, suggesting that GM1 binds directly to VacA. The hydrodynamic binding of lyso-GM1 and VacA measured by fluorescence correlation spectroscopy had a K(d) value of 190 nM. VacA also bound to lyso-GM1 at pH 2 corresponding to the physiological pH of human stomach. Collectively, these results showed that direct binding of H. pylori VacA to free gangliosides neutralizes the toxin activity of VacA. These findings offer an alternative insight into the role of gangliosides in VacA toxicity and the pathogenesis of H. pylori., Glycobiology, 20(6), pp.668-678; 2010

Cholesterol is a prominent modulator of the integrity and functional activity of physiological membranes and the most abundant sterol in the mammalian brain. DHCR24-knock-out mice lack cholesterol and accumulate desmosterol with age. Here we demonstrate that brain cholesterol deficiency in 3-week-old DHCR24−/− mice was associated with altered membrane composition including disrupted detergent-resistant membrane domain (DRM) structure. Furthermore, membrane-related functions differed extensively in the brains of these mice, resulting in lower plasmin activity, decreased β-secretase activity and diminished Aβ generation. Age-dependent accumulation and integration of desmosterol in brain membranes of 16-week-old DHCR24−/− mice led to the formation of desmosterol-containing DRMs and rescued the observed membrane-related functional deficits. Our data provide evidence that an alternate sterol, desmosterol, can facilitate processes that are normally cholesterol-dependent including formation of DRMs from mouse brain extracts, membrane receptor ligand binding and activation, and regulation of membrane protein proteolytic activity. These data indicate that desmosterol can replace cholesterol in membrane-related functions in the DHCR24−/− mouse. Electronic supplementary material The online version of this article (doi:10.1007/s11064-008-9893-4) contains supplementary material, which is available to authorized users.

GM1 ganglioside is known to be involved in the amyloid-associated diseases and it is a crucial factor for the assembly of amyloid proteins on lipid-rafts, which are lipid structures located on the synaptic plasma membranes. Due to its slow aggregation rate, we employed salmon calcitonin (sCT) as a suitable probe representative of amyloid proteins, to study the interaction between this class of proteins and a membrane model. Here, we prepared a neuronal membrane model by depositing onto mica two Langmuir-Blodgett films in liquid-condensed phase: the outer monolayer was characterized by high content of GM1 (50%) and minority parts of cholesterol and POPC (25–25%), while the inner one by plain POPC. To deeply investigate the interaction of sCT with this model and the role-played by GM1, we prepared the outer leaflet adding sCT at a concentration such that the number of proteins equals that of GM1. Atomic Force Microscopy revealed the occurrence of two distinct kinds of flat surfaces, with globular aggregates localized exclusively on top of the highest one. To unravel the nature of the interaction, we studied by ζ-potential technique liposomes composed as the outer leaflet of the model. Results demonstrated that an electrostatic interaction sCT-GM1 occurred. Finally, to investigate the interaction thermodynamics between sCT and the outer leaflet, Langmuir films as the outer monolayer and containing increasing content of sCT were studied by compression isotherms and Brewster Angle Microscopy experiments. Based on the all body of results we propose an interaction model where GM1 plays a pivotal role., Highlights • Membrane models mimicking the occurrence of lipid-rafts and sCT, were investigated. • Atomic Force Microscopy shows sCT globular aggregates onto very flat surfaces. • ξ-potential data indicate that electrostatic interaction occurs between sCT and GM1. • Thermodynamic data of the outer leaflet reveal that sCT aggregate outside the film. • GM1, one component of the lipid-rafts, result to trigger the interaction.

Neurons within different brain regions have varying levels of vulnerability to external stress and therefore respond differently to injury. A potential reason to explain this may lie within a key lipid class of the cell’s plasma membrane called gangliosides. These glycosphingolipid species have been shown to play various roles in the maintenance of neuronal viability. The purpose of this study is to use electrospray ionization mass spectrometry (ESI-MS) technique and immunohistochemistry to evaluate the temporal changes in the expression profiles of various ganglioside species during the course of neurodegeneration in rat primary cortical neurons exposed to glutamate toxicity. Primary embryonic (E18) rat cortical neurons were cultured to DIV14. Glutamate toxicity was induced for 1, 3, 6 and 24 h. Immunofluorescence was used to stain for GM1 and GM3 species and ESI-MS was used to quantify the ganglioside species expressed within these injured neurons, which were compared to expression profiles of healthy neurons. Neurons were also pretreated with GM1 24 h before glutamate exposure to assess the level of neuroprotection conferred by GM1. Microglia were also activated using amyloid-beta oligomer and stained for GM1 and GM3 expression. ESI-MS data revealed that d16:1 and d18:1 GM1 species were upregulated in neurons exposed to glutamate while no significant changes were observed for GM2 and GM3 expression. Furthermore, neurons that were pretreated with GM1 showed increased viability compared to untreated neurons when exposed to glutamate. Immunofluorescence revealed an elevated expression of GM3 in activated microglia compared to controls. These data suggests that different gangliosides and cells within the CNS play diverse roles in the process of neurodegeneration.

Gangliosides and the urokinase plasminogen activator receptor (uPAR) tipically partition in specialized membrane microdomains called lipid-rafts. uPAR becomes functionally important in fostering angiogenesis in endothelial progenitor cells (EPCs) upon recruitment in caveolar-lipid rafts. Moreover, cell membrane enrichment with exogenous GM1 ganglioside is pro-angiogenic and opposite to the activity of GM3 ganglioside. On these basis, we first checked the interaction of uPAR with membrane models enriched with GM1 or GM3, relying on the adoption of solid-supported mobile bilayer lipid membranes with raft-like composition formed onto solid hydrophilic surfaces, and evaluated by surface plasmon resonance (SPR) the extent of uPAR recruitment. We estimated the apparent dissociation constants of uPAR-GM1/GM3 complexes. These preliminary observations, indicating that uPAR binds preferentially to GM1-enriched biomimetic membranes, were validated by identifying a pro-angiogenic activity of GM1-enriched EPCs, based on GM1-dependent uPAR recruitment in caveolar rafts. We have observed that addition of GM1 to EPCs culture medium promotes matrigel invasion and capillary morphogenesis, as opposed to the anti-angiogenesis activity of GM3. Moreover, GM1 also stimulates MAPKinases signalling pathways, typically associated with an angiogenesis program. Caveolar-raft isolation and Western blotting of uPAR showed that GM1 promotes caveolar-raft partitioning of uPAR, as opposed to control and GM3-challenged EPCs. By confocal microscopy, we have shown that in EPCs uPAR is present on the surface in at least three compartments, respectively, associated to GM1, GM3 and caveolar rafts. Following GM1 exogenous addition, the GM3 compartment is depleted of uPAR which is recruited within caveolar rafts thereby triggering angiogenesis.

Lipid rafts are assumed to undergo biologically important size-modulations from nanorafts to microrafts. Due to the complexity of cellular membranes, model systems become important tools, especially for the investigation of the factors affecting “raft-like” Lo domain size and the search for Lo nanodomains as precursors in Lo microdomain formation. Because lipid compositional change is the primary mechanism by which a cell can alter membrane phase behavior, we studied the effect of the ganglioside GM1 concentration on the Lo/Ld lateral phase separation in PC/SM/Chol/GM1 bilayers. GM1 above 1mol % abolishes the formation of the micrometer-scale Lo domains observed in GUVs. However, the apparently homogeneous phase observed in optical microscopy corresponds in fact, within a certain temperature range, to a Lo/Ld lateral phase separation taking place below the optical resolution. This nanoscale phase separation is revealed by fluorescence spectroscopy, including C12NBD-PC self-quenching and Laurdan GP measurements, and is supported by Gaussian spectral decomposition analysis. The temperature of formation of nanoscale Lo phase domains over an Ld phase is determined, and is shifted to higher values when the GM1 content increases. A “morphological” phase diagram could be made, and it displays three regions corresponding respectively to Lo/Ld micrometric phase separation, Lo/Ld nanometric phase separation, and a homogeneous Ld phase. We therefore show that a lipid only-based mechanism is able to control the existence and the sizes of phase-separated membrane domains. GM1 could act on the line tension, “arresting” domain growth and thereby stabilizing Lo nanodomains.

The lipid A component of Campylobacter jejuni lipopolysaccharides (LPSs) contains the same architectural principle as that found in other bacterial species; however, unlike the case with other bacterial species, the lipid A backbone of C. jejuni strains is composed of a phosphorylated beta(1'-6)-linked disaccharide containing 2,3-diamino-2,3-dideoxy-D-glucose and D-glucosamine as the major molecular species. Despite a backbone that differs structurally from that of classic enterobacterial lipid A, C. jejuni lipid A is antigenically similar to enterobacterial lipid A, and the respective LPSs have comparable endotoxic activities. Structural variability is greater in the core oligosaccharide than in lipid A of C. jejuni LPS. Nevertheless, the inner core oligosaccharides of C. jejuni strains share a common unique tetrasaccharide and also possess a trisaccharide that occurs in the inner core of other bacterial species. Ganglioside mimicry in the outer core is a common feature shared by a number of C. jejuni serotypes, but this mimicry is not conserved in all serotypes.

GM1 and GM2 gangliosides are important components of the cell membrane and play an integral role in cell signaling and metabolism. In this conceptual overview, we discuss recent developments in our understanding of the basic biological functions of GM1 and GM2 and their involvement in several diseases. In addition to a well-established spectrum of disorders known as gangliosidoses, such as Tay-Sachs disease, more and more evidence points at an involvement of GM1 in Alzheimer’s and Parkinson’s diseases. New emerging methodologies spanning from single-molecule imaging in vivo to simulations in silico have complemented standard studies based on ganglioside extraction.

International audience; : Previously, we demonstrated that eicosapentaenoic acid enhanced ethanol-induced oxidative stress and cell death in primary rat hepatocytes via an increase in membrane fluidity and lipid raft clustering. In this context, another n-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA), was tested with a special emphasis on physical and chemical alteration of lipid rafts. Pretreatment of hepatocytes with DHA reduced significantly ethanol-induced oxidative stress and cell death. DHA protection could be related to an alteration of lipid rafts. Indeed, rafts exhibited a marked increase in membrane fluidity and packing defects leading to the exclusion of a raft protein marker, flotillin. Furthermore, DHA strongly inhibited disulfide bridge formation, even in control cells, thus suggesting a disruption of protein-protein interactions inside lipid rafts. This particular spatial organization of lipid rafts due to DHA subsequently prevented the ethanol-induced lipid raft clustering. Such a prevention was then responsible for the inhibition of phospholipase C-γ translocation into rafts, and consequently of both lysosome accumulation and elevation in cellular low-molecular-weight iron content, a prooxidant factor. In total, the present study suggests that DHA supplementation could represent a new preventive approach for patients with alcoholic liver disease based upon modulation of the membrane structures.

International audience; 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.

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 modelling 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.

In two-component phosphatidylcholine bilayers with coexisting liquid and P beta' gel phases, the distribution between phases of low concentrations of glycosphingolipids can be determined by freeze-etch electron microscopy after labeling the glycolipid with a suitable protein. We have found that the distribution depends upon the glycosphingolipid species (Rock, P. et al., (1991) Biochemistry 30, 19-25). Using this technique with cholera toxin as the protein label and bilayers formed from dipalmitoyl- and dielaidoylphosphatidylcholine (1:1) containing < 1 mol% GM1, we have studied the distribution of a family of GM1 homologues differing in the acyl chain and sphingoid base moieties. The GM1 preference for the P beta' ripple phase decreases with decreasing acyl chain length and increasing unsaturation. GM1 with either a C18:1 or C20:1 sphingoid base shows similar distributions in liquid and gel phases. When the molecules are preferentially found in the P beta' phase, they are positioned along unique loci in both A and A/2 forms of the ripple structure. This localization and acyl chain dependence reflect the volume, shape and localization of molecular packing defects in the P beta' phase. The ganglioside inclusions stabilize the P beta' phase and form compositional domains of unique topography.

Liposomes containing negatively-charged phospholipid, N-palmitoylphosphatidylethanolamine (NPPE) were examined for stability in the presence of human serum, using the release of the entrapped 5,6-carboxyfluorescein as an aqueous marker. Either small unilamellar vesicles (SUV) or large unilamellar vesicles (LUV) were used. Incorporation of NPPE into PC SUV decreases leakage in the presence of serum or phosphate-buffered saline, no strictly related to size increase observed and to the surface negative charge present. The stabilizing effect of NPPE and Chol were synergistic. Inhibition of destabilization induced by serum of PC/Chol liposomes was observed when NPPE concentrations were above 12 mol%. Change in the membrane fluidity or incorporation of a monosialoganglioside into liposomes do not significantly change the half-life of liposomes in the presence of a high NPPE concentration. Incorporation of NPPE into PC/Chol liposomes increases membrane rigidity which does not change after serum incubation. The presence of NPPE in liposomes decreases lipid transfer/exchange between liposomes and lipoproteins although the same amount of serum proteins were incorporated as in PC/Chol liposomes. As expected, these proteins are accessible to trypsin digestion. In accordance with these results, the liposome agglutination assay shows no steric barrier activity. As a whole, the results obtained in this paper suggest a complex mechanism for stabilization of NPPE containing liposomes in human serum.

Mammalian sperm undergo several maturation steps after leaving the testis to become competent for fertilization. Important changes occur in sperm within the female reproductive tract, although the molecular mechanisms underlying these processes remain unclear. To investigate sperm membrane remodeling upon sperm maturation, we developed transgenic mouse lines carrying glycosylphosphatidylinositol (GPI)-anchored enhanced green fluorescent protein (EGFP–GPI) and traced the fate of this fluorescent protein during the fertility-acquiring process in sperm in vitro and in vivo. When the GFP-labeled sperm were treated with compounds for promoting the acrosome reaction, EGFP–GPI was released from the sperm surface crosslinked with characteristic relocation of a lipid raft marker ganglioside GM1. Sperm ejaculated into the uterus strongly expressed EGFP–GPI in the head region, whereas a part of the oviductal sperm lost fluorescence in a manner that was dependent on the presence of angiotensin-converting enzyme (ACE). Moreover, sperm on the zona pellucida of eggs in the oviduct were all found to have low levels of GFP. These results suggest that sperm undergoing GPI-anchored protein release associated with reorganization of lipid rafts and the acrosome reaction acquire fertilization potential.

Parkinson's disease-associated alpha-synuclein is an amyloidogenic protein not only expressed in the cytoplasm of neurons, but also secreted in the extracellular space and internalized into glial cells through a lipid raft-dependent process. We previously showed that alpha-synuclein interacts with raft glycosphingolipids through a structural motif common to various viral and amyloidogenic proteins. Here we report that alpha-synuclein also interacts with cholesterol, as assessed by surface pressure measurements of cholesterol-containing monolayers. Using a panel of recombinant fragments and synthetic peptides, we identified two distinct cholesterol-binding domains in alpha-synuclein. One of these domains, which corresponds to the tilted peptide of alpha-synuclein (67-78), bound cholesterol with high affinity and was toxic for cultured astrocytes. Molecular modeling suggested that cholesterol binds to this peptide with a tilt angle of 46 degrees. alpha-synuclein also contains a cholesterol recognition consensus motif, which had a lower affinity for cholesterol and was devoid of toxicity. This motif is encased in the glycosphingolipid-binding domain (34-45) of alpha-synuclein. In raft-like model membranes containing both cholesterol and glycosphingolipids, the head groups of glycosphingolipids prevented the accessibility of cholesterol to exogenous ligands. Nevertheless, cholesterol appeared to 'signal' its presence by tuning glycosphingolipid conformation, thereby facilitating alpha-synuclein binding to raft-like membranes. We propose that the association of alpha-synuclein with lipid rafts involves both the binding of alpha-synuclein (34-45) to glycosphingolipids, and the interaction of the fusogenic tilted peptide (67-78) with cholesterol. Coincidentally, a similar mechanism is used by viruses (HIV-1, HTLV-I, Ebola) which display a tilted peptide and fuse with host cell membranes through a sphingolipid/cholesterol-dependent process. (C) 2011 Elsevier B.V. All rights reserved.

Cell surface glycosphingolipids (GSLs) including gangliosides play a key role in the regulation of the conformation, oligomerization, and fibrillation of amyloidogenic proteins. Correspondingly, most amyloidogenic proteins possess a functional GSL-binding motif (GBM). Sequence alignments of GSL-binding proteins against the GBM of alpha-synuclein allowed the establishment of a consensus GBM sequence defined as K/H/R/-X(1-4)-Y/F-X(4-5)-K/H/R, where at least one of the X(1-4) residues is glycine. The GBMs of alpha-synuclein (34-KEGVLYVGSKTK-45) and Alzheimer's disease beta-amyloid peptide (A beta) (5-RHDSGYEVHHQK-16) consist of a structurally related loop centered on tyrosine (Y39 for alpha-synuclein, Y10 for A beta). Surface pressure measurements of GSL monolayers at the air water interface allowed us to determine the following order for alpha-synuclein GSL interactions: GM3 > Gb3 > GalCer-NFA > GM1 > sulfatide > GalCer-HFA > LacCer > GM4 > GM2 > asialo-GM1 > GD3, indicating a marked preference for GSLs with one, three, or five sugar units. The insertion of alpha-synuclein into sphingomyelin-containing monolayers was strongly stimulated by the presence of GM3. This effect was not observed with phosphatidylcholine monolayers, suggesting that the ganglioside facilitated the insertion of alpha-synuclein into raft-like membrane domains. Molecular dynamics simulations suggested that the side chain of Y39 was deeply inserted between GM3 head groups. Monolayer experiments with mutant GBM peptides showed that Y39, K34, and K45 were important for GM3 binding, whereas only Y39 appeared critical for GM1 recognition. The interaction of A beta 5-16 with GM1 involved R5, H13, H14, and K16, but not Y10. These data indicate that subtle amino acid variations in the consensus GBM of alpha-synuclein and A beta conferred distinct GSL-binding properties. (C) 2011 Elsevier Ltd. All rights reserved.

Gangliosides are characteristically enriched in various membrane domains that can be isolated as low density membrane fraction insoluble in detergents (detergent-resistant membranes, DRMs) or obtained after homogenization and sonication in 0.5 M sodium carbonate (low-density membranes, LDMs). We assessed the effect of the ceramide structure of four [3H]-labeled GM1 ganglioside molecular species (GM1s) taken up by HL-60 cells on their occurrence in LDMs, and compared it with our previous observations for DRMs. All GM1s contained C18 sphingosine, which was acetylated in GM1(18:1/2) or acylated with C14, C18 or C18:1 fatty acids (Fas)

Galactose-containing dendrimers with long spacer arms inhibit cholera toxin binding as strongly as the natural ganglioside GM1 oligosaccharide does.

The dependence of CMP-N-acetylneuraminate:GM1 sialyltransferase (SAT IV) activity of rat liver Golgi apparatus on GM1 ganglioside ceramide composition was evaluated. SAT IV activity was assayed on GM1 molecular species carrying homogeneous ceramide moieties containing long chain bases of different length (18 or 20 C atoms) unsaturated or not, linked to 14:0, 16:0, 18:0 or 22:0 fatty acids. The results obtained in the presence of the detergent Triton CF-54, when enzyme and substrate are presumably part of the same supramolecular structure, show that either the long chain base or the fatty acid composition can affect enzyme activity. This feature was not displayed when GM1 was embedded in dipalmitoylphosphatidylcholine vesicles in the absence of detergent. Under the latter conditions, the enzyme was not sensitive to the lipid composition of GM1 but to the ganglioside/phospholipid ratio in the vesicles. These results indicate for the first time that SAT IV is affected by the lipid composition of the substrate and strengthen the hypothesis that glycosyltransferases may contribute to control the cellular glycosphingolipid ceramide pattern.

A CMP-NeuAc:GM1 alpha 2-3 sialyltransferase (GD1a synthase, 2.4.99.2) has been purified from the Triton extract of rat brain. The enzyme was purified and resolved by affinity chromatography on CDP-Sepharose column by a linear NaCl gradient elution. Final purification was achieved by elution from a 'GM1-acid'-Sepharose column. SDS-PAGE of the enzyme revealed a single protein band with an apparent Mr 44 kDa. It catalyzed specifically the sialylation of GD1b, GM1 and asialo-GM1. Enzyme products were identified by TLC in three different solvent systems. The Km value for GM1 was 7.5 x 10(-2) M, and for CMP-NeuAc it was 6.5 x 10(-5) M.

Cholera and the related AB5-subunit toxins co-opt plasma membrane (PM) glycolipids to move retrograde into the endoplasmic reticulum (ER) of the host cell where a portion of the toxin is retro-translocated to the cytosol to induce disease. Only glycolipids that associate strongly with detergent insoluble membrane microdomains can sort the toxins backwards from PM to ER. The way certain lipids and proteins are clustered in the plane of the membrane to form lipid rafts likely explains how the glycolipids can function as sorting motifs for the toxins.

G-protein-coupled receptors (GPCRs) are commonly pharmacologically modulated due to their ability to translate extracellular events to intracellular changes. Previously, studies have mostly focused on protein–protein interactions, but the focus has now expanded also to protein–lipid connections. GM1, a brain-expressed ganglioside known for neuroprotective effects, and GPR37, an orphan GPCR often reported as a potential drug target for diseases in the central nervous system, have been shown to form a complex. In this study, we looked into the functional effects. Endogenous GM1 was downregulated when stably overexpressing GPR37 in N2a cells (N2aGPR37-eGFP). However, exogenous GM1 specifically rescued N2aGPR37-eGFP from toxicity induced by the neurotoxin MPP+. The treatment did not alter transcription levels of GPR37 or the enzyme responsible for GM1 production, both potential mechanisms for the effect. However, GM1 treatment inhibited cAMP-dependent signaling from GPR37, here reported as potentially consecutively active, possibly contributing to the protective effects. We propose an interplay between GPR37 and GM1 as one of the many cytoprotective effects reported for GM1.