Gangliosides and sulphatide in human cerebrospinal fluid: quantitation with immunoaffinity techniques

A sensitive micromethod involving extraction, purification and thin-layer chromatography (TLC)-enzyme immunostaining was developed for the quantation of gangliosides and sulphatide, as markers for neuronal disorders and myelin disturbances, in individual samples of less than 5 ml of cerebrospinal fluid. The gangliosides of the gangliotetraose series were individually determined with cholera toxin subunit B by TLC-enzyme-linked immunosorbent assay (ELISA) after chromatography and subsequent sialidase hydrolysis to II3NeuAc-GgOse4Cer (GM1). Other gangliosides and sulphatide were determined with specific monoclonal antibodies by TLC-ELISA. The total ganglioside content varied between 100 and 230 nmol/l in ten normal cerebrospinal fluid samples from adults. The major gangliosides were of the gangliotetraose series, represented by GM1, IV3NeuAc,II3NeuAc-GgOse4Cer, (GD1a), II3(NeuAc)2-GgOse4Cer (GD1b) and IV3NeuAc,II3 (NeuAc)2-GgOse4Cer (GT1b) of which the b-series gangliosides dominated, i.e., GD1b and GT1b.     

Detecting Protein–Glycolipid Interactions Using Glycomicelles and CaR-ESI-MS

This study reports on the use of the catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay, combined with glycomicelles, as a method for detecting specific interactions between water-soluble proteins and glycolipids (GLs) in aqueous solution. The B subunit homopentamers of cholera toxin (CTB₅) and Shiga toxin type 1 B (Stx1B₅) and the gangliosides GM1, GM2, GM3, GD1a, GD1b, GT1b, and GD2 served as model systems for this study. The CTB₅ exhibits broad specificity for gangliosides and binds to GM1, GM2, GM3, GD1a, GD1b, and GT1b; Stx1B₅ does not recognize gangliosides. The CaR-ESI-MS assay was used to analyze solutions of CTB₅ or Stx1B₅ and individual gangliosides (GM1, GM2, GM3, GD1a, GD1b, GT1b, and GD2) or mixtures thereof. The high affinity interaction of CTB₅ with GM1 was successfully detected. However, the apparent affinity, as determined from the mass spectra, is significantly lower than that of the corresponding pentasaccharide or when GM1 is presented in model membranes such as nanodiscs. Interactions between CTB₅ and the low affinity gangliosides GD1a, GD1b, and GT1b, as well as GD2, which served as a negative control, were detected; no binding of CTB₅ to GM2 or GM3 was observed. The CaR-ESI-MS results obtained for Stx1B₅ reveal that nonspecific protein-ganglioside binding can occur during the ESI process, although the extent of binding varies between gangliosides. Consequently, interactions detected for CTB₅ with GD1a, GD1b, and GT1b are likely nonspecific in origin. Taken together, these results reveal that the CaR-ESI-MS/glycomicelle approach for detecting protein–GL interactions is prone to false positives and false negatives and must be used with caution.

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High-performance capillary electrophoresis of gangliosides

Gangliosides are sialic acid-containing glycosphingolipids. In aqueous media, these glycolipids have been shown to exist as stable micelles. Ganglioside micelles could be analyzed by high-performance zonal capillary electrophoresis in uncoated fused-silica capillaries within 10 min. The mass sensitivity determined by monitoring the absorption of ultraviolet light at 195 nm was in the order of 10(-11) mol. Increasing the pH of the running buffer from 3.0 to 7.4 or the voltage from 10 to 30 kV increased the relative mobilities of gangliosides. By contrast, increasing the ionic strength of the buffer decreased the migration and broadened the elution peak widths of gangliosides. Ganglioside* micelles including GM1, GD1b, and GT1b were resolved into separate peaks by capillary electrophoresis at physiological pH shortly after mixing. Upon prolonged incubation, the ganglioside peaks merged to form a single species. The fusion process was temperature-dependent. At 50 degrees C, formation of mixed micelles between polysialogangliosides GD1b and GT1b was complete within 30 min. In contrast, no fusion of the ganglioside peaks was observed at 0 degrees C even after 75 h. Formation of mixed micelles between GD1b and other polysialogangliosides including GD1a, GT1b, and GQ1b at 37 degrees C required 1.5, 3.0, and 2.0 h, respectively. Formation of mixed micelles between monosialoganglioside GM1 and polysialogangliosides were 6- to 36-fold slower. No fusion was observed between monosialogangliosides GM1 and GM2 after 2 days of incubation. These findings indicate that polysialogangliosides may have higher propensities than monosialoganglioside to form mixed micelles.     

Nano-electrospray ionization time-of-flight mass spectrometry of gangliosides from human brain tissue

A general approach for the detection and structural elucidation of brain ganglioside species GM1, GD1 and GT1 by nano-electrospray ionization quadrupole time-of-flight (nanoESI-QTOF) mass spectrometry (MS), using combined data from MS and MS/MS analysis of isolated native ganglioside fractions in negative ion mode and their permethylated counterparts in the positive ion mode is presented. This approach was designed to detect and sequence gangliosides present in preparatively isolated ganglioside fractions from pathological brain samples available in only very limited amounts. In these fractions mixtures of homologue and isobaric structures are present, depending on the ceramide composition and the position of the sialic acid attachment site. The interpretation of data for the entire sequence, derived from A, B, C and Y ions by nanoESI-QTOFMS/MS in the negative ion mode of native fractions, can be compromised by ions arising from double and triple internal cleavages. To distinguish between isobaric carbohydrate structures in gangliosides, such as monosialogangliosides GM1a and GM1b, disialogangliosides GD1a, GD1b and GD1c or trisialogangliosides GT1b, GT1c and GT1d, the samples were analysed after permethylation in the positive ion nanoESI-QTOFMS/MS mode, providing set of data, which allows a clear distinction for assignment of outer and inner fragment ions according to their m/z values. The fragmentation patterns from native gangliosides obtained by low-energy collision induced dissociation (CID) by nanoESI-QTOF show common behaviour and follow inherent rules. The combined set of data from the negative and positive ion mode low-energy CID can serve for the detection of structural isomers in mixtures, and to trace new, not previously detected, components.     

Inhibition effects of gangliosides G(M1), G(D1a) and G(T1b) on base-catalyzed isomerization of prostaglandin A(2)

Micellar inhibition effect of gangliosides on a degradation of drug was investigated, where ganglioside G(M1) (GM1), G(D1a) (GD1a) and G(T1b) (GTlb) whose sialic acid residue is one, two and three, respectively, were used. The base-catalyzed isomerization of prostaglandin A(2) (PGA(2)) to prostaglandin B(2) (PGB(2)) was chosen as a model experiment. The rate for the isomerization of PGA(2) was determined by measuring the concentration of PGA(2) (and PGB(2)) with a high-performance liquid chromatography. Gangliosides micelles inhibited the isomerization of PGA(2). The inhibition effect of GT1b micelles was larger than that of GD1a micelles. This result would be due to the larger absolute value of surface potential of GT1b micelles, which brings about a larger electrostatic repulsion between micellar surface and OH(-). The terminal sialic acid residue of ganglioside was effective to inhibit the isomerization of PGA(2). GM1 micelles without terminal sialic acid residue but with large aggregation number exhibited a superior steric shielding effect rather than an electrostatically repulsive effect. The inhibition effect of GM1 micelles was enhanced by the mixed micellization with the other ganglioside with a terminal sialic acid residue. GM1-GD1a or GM1-GT1b mixed micelles remarkably inhibited the isomerization of PGA(2). The physiological activity of PGs in the biological membranes containing gangliosides was also discussed.     

Highly sensitive localization analysis of gangliosides and sulfatides including structural isomers in mouse cerebellum sections by combination of laser microdissection and hydrophilic interaction liquid chromatography/electrospray ionization mass spectrometry with theoretically expanded multiple reaction monitoring

Liquid chromatography/electrospray ionization mass spectrometry (LC/ESI‐MS) is suitable for analysis of glycosphingolipids such as fragile gangliosides avoiding the use of the sialic acid elimination. However, it was not possible to distinguish the structural isomers such as GD1a and GD1b with reversed‐phase LC/ESI‐MS by hydrophobic interaction. Here we report an effective method for targeted analysis of theoretically expanded ganglioside molecular species including structural isomers by hydrophilic interaction liquid chromatography (HILIC)/ESI‐MS with multiple reaction monitoring (MRM). As a result of MRM analysis of glycosphingolipid mixtures from porcine brain, each of the lipid classes was detected within 25 min in the following order: sulfatides > GM3 > GM2 > GM1 > GD3 > GD1a > GD2 > GD1b > GT1a > GT1b > GQ1b. For the advanced application, localization analysis of postnatal day 15 (P15) mouse cerebellum layered structures was carried out by combination of MRM and laser microdissection (LMD). As a result, GM3, GD1a, GT1b and GQ1b were abundantly detected in the molecular and granular layers, whereas GM1 was widely presented in each layered structure. These gangliosides were mainly composed of d18:1‐18:0 and d18:1‐20:0, but GM3 was d18:1‐16:0 and d18:1‐20:0. Meanwhile, sulfatide molecular species were mostly localized in the myelinated fibers and scarcely found in the molecular layer. These results suggested that our method is suitable to detect a variety of ganglioside classes and sulfatides with high sensitivity at the molecular species level and effective for localization analysis of these glycosphingolipids from mouse brain sections. Copyright © 2010 John Wiley & Sons, Ltd.     

Improved method for large-scale purification of brain gangliosides by Q-sepharose column chromatography. Immunochemical detection of C-series polysialogangliosides in adult bovine brains

A new chromatographic method for separation of bovine brain gangliosides has been developed using Q-Sepharose. Gangliosides were separated based not only on their sialic acid numbers but also on the sialic acid molecular species and chain lengths of the skeletal oligosaccharide portions. The following results indicate that this column chromatography has practical advantages in separating mixtures of gangliosides, especially positional isomers and molecular species with N-acetyl- or N-glycolylneuraminic acid. (1) the loading capacity of Q-Sepharose for gangliosides was very high; (2) most major gangliosides such as GM1, GD1a, GD1b, GT1b and GQ1b were isolated in a single step; (3) these major gangliosides were clearly separated from gangliosides containing, N-glycolylneuraminic acid when examined using Hanganutziu-Deicher antibody; (4) polysialogangliosides that have four or more sialic acid residues were isolated efficiently. It was shown by the combination of Q-Sepharose column chromatography with thin-layer chromatography/enzyme immunostaining that adult bovine brains possess C-series polysialogangliosides as minor components which are known as embryonic molecules in avian and mammalian brains.     

Dynamic changes of gangliosides expression during the differentiation of embryonic and mesenchymal stem cells into neural cells

Stem cells are used for the investigation of developmental processes at both cellular and organism levels and offer tremendous potentials for clinical applications as an unlimited source for transplantation. Gangliosides, sialic acid-conjugated glycosphingolipids, play important regulatory roles in cell proliferation and differentiation. However, their expression patterns in stem cells and during neuronal differentiation are not known. Here, we investigated expression of gangliosides during the growth of mouse embryonic stem cells (mESCs), mesenchymal stem cells (MSCs) and differentiated neuronal cells by using high-performance thin-layer chromatography (HPTLC). Monosialoganglioside 1 (GM1) was expressed in mESCs and MSCs, while GM3 and GD3 were expressed in embryonic bodies. In the 9-day old differentiated neuronal cells from mESCs cells and MSCs, GM1 and GT1b were expressed. Results from immunostaining were consistent with those observed by HPTLC assay. These suggest that gangliosides are specifically expressed according to differentiation of mESCs and MSCs into neuronal cells and expressional difference of gangliosides may be a useful marker to identify differentiation of mESCs and MSCs into neuronal cells.     

Compositional analysis of the three main gangliosides from adult human myometrium by a rapid capillary gas chromatographic method

A new capillary GC method is described for the compositional analysis of the three main gangliosides isolated from adult human myometrium. The sample was subjected to methanolysis, acetylation and trimethylsilylation which allows all the constituents to be analyzed simultaneously. The predominant ganglioside was found to be GD3, with GM3 and GT1b the next most abundant.     

Chip-nanoelectrospray quadrupole time-of-flight tandem mass spectrometry of meningioma gangliosides: a preliminary study

A strategy combining high-performance thin layer chromatography (HPTLC), laser densitometry, and fully automated chip-based nanoelectrospray (nanoESIchip) performed on a NanoMate robot coupled to QTOF-MS was developed, optimized, and for the first time applied for mapping and structural identification of gangliosides (GGs) extracted and purified from a human angioblastic meningioma specimen. While HPTLC pattern indicated only seven fractions migrating as GM3, GM2, GM1, GD3, GD1a (nLD1, LD1), GD1b, GT1b, and possibly GD2, due to the high sensitivity, mass accuracy, and ability to ionize minor species in complex mixtures, nanoESIchip-QTOF MS was able to discover significantly more GG species than ever reported in meningioma. Thirty-four distinct glycosphingolipid components of which five asialo, one GM4, nine GM3, two GM2, two GD3, nine GM1, and six GD1 differing in their ceramide compositions were identified. All structures presented long-chain bases with 18 carbon atoms, while the length of the fatty acid was found to vary from C11 to C25. MS screening results indicated also that the diversity of the expressed GM1 structures is higher than expected in view of the low proportions evidenced by densitometric quantification. Simultaneous fragmentation of meningioma-associated GM1 (d18:1/24:1) and GM1 (d18:1/24:0) by MS/MS using CID confirmed the postulated structures of the ceramide moieties and provided data on the glycan core, which document that for each of the GM1 (d18:1/24:1) and GM1 (d18:1/24:0) forms both GM1a and GM1b isomers are expressed in the investigated meningioma tissue.     

Syntheses of new model compounds related to an antigenic epitope from Bupleurum falcatum L. and their distributions in various ganglioside-phospholipid monolayers

6-N-[2-(Tetradecyl)hexadecanamido]hexyl beta-D-glucopyranosyluronic acid-(1-->6)-beta-D-galactopyranosyl-(1-->6)-beta-D-galactopyranoside (1) and its clustering compound (2) carrying a tetravalent sugar unit, which are new model compounds related to a major antigenic epitope from antiulcer pectic polysaccharide of Bupleurum falcatum L., were synthesized and the distributions of 1 and 2 in mixed ganglioside (GM1, GD1a or GT1b)/phospholipid (DPPC) monolayers were observed using atomic force microscopy (AFM). AFM images showed that 1 was distributed in the GM1, GD1a and GT1b region of the mixed monolayers, in which 1 was miscible with GD1a. Specific distribution of 1 was observed in the mixed GM1/DPPC monolayer. Compound 2 was miscible with GM1, while 2 formed associations with GD1a and GT1b in the mixed monolayers. The distribution mode of 1 and 2 was different among the mixed ganglioside/DPPC monolayers.     

Chemoenzymatic synthesis of biotin-appended analogues of gangliosides GM2, GM1, GD1a and GalNAc-GD1a for solid-phase applications and improved ELISA tests

Biotinylated analogues of gangliosides GM2, GM1, GD1a and GalNAc-GD1a were synthesized in high yields using glycosyltransferases from Campylobacter jejuni. The presence of a biotin moiety in the aglycone part of these mimics allows for attachment of these materials onto various streptavidin-coated surfaces. Analysis of the interaction of biotin-appended GM1 with the B subunit of Escherichia coli heat-labile enterotoxin performed in a modified ELISA procedure shows the potential of this compound to replace the natural GM1 in toxin detection.     

Synthesis of ganglioside epitopes for oligosaccharide specific immunoadsorption therapy of Guillian-Barré syndrome

Guillain-Barré syndrome is a postinfectious, autoimmune neuropathy resulting in neuromuscular paralysis. Auto-antibodies, often induced by bacterial infection, bind to human gangliosides possessing monosialoside and diasialoside epitopes and impair the function of nerve junctions, where these ganglioside structures are highly enriched. Truncated gangliosides representive of GD3, GQ1b and GM2 epitopes have been synthesized as methyl glycosides and as a glycosides of an eleven carbon tether. The synthetic oligosaccharide ligands are structural mimics of these highly complex ganglioside epitopes and via their ability to neutralize or remove auto-antibodies have the potential for therapy, either as soluble blocking ligands administered systemically, or as immuno-affinity ligands for use as extracorporeal immunoadsorbents.     

Ganglioside partitioning and aggregation in phase-separated monolayers characterized by bodipy GM1 monomer/dimer emission

The distribution of Bodipy GM1 in monolayers of binary and ternary lipid mixtures with coexisting fluid and ordered phases has been examined using a combination of atomic force microscopy and near-field scanning optical microscopy. Monolayers deposited at high (30 mN/m) and low (5 or 10 mN/m) surface pressures were examined and compared to those containing the same concentration of unlabeled ganglioside. Measurements of monomer and dimer Bodipy emission were used to distinguish aggregated from dilute ganglioside levels. For binary DPPC/DOPC monolayers, Bodipy GM1 is distributed throughout both the fluid and ordered phases at low surface pressures, and both labeled and unlabeled gangliosides result in a reduction in the size of ordered DPPC domains at 0.4% and the appearance of small aligned ganglioside-rich domains at 4%. In agreement with earlier studies, GM1 is heterogeneously distributed in small islands in the condensed DPPC domains at high surface pressure. By contrast, Bodipy GM1 causes the disappearance of large DPPC domains at 0.4% and the formation of a new GM1-rich phase at 4%. The addition of both gangliosides leads to a comparable loss of large ordered domains at low surface pressure and the appearance of a new GM1-rich phase at 30 mN/m for ternary lipid mixtures containing cholesterol. The results demonstrate the complexity of GM1 partitioning and illustrate the utility of complementary AFM and high spatial resolution two-color fluorescence experiments for understanding Bodipy GM1 aggregation and distribution.     

Estrogen-induced cholestasis results in a dramatic increase of b-series gangliosides in the rat liver

Hepatic ganglioside composition was investigated in normal and cholestatic Wistar rats. Cholestasis was induced by 17alpha-ethinylestradiol (EE; 5 mg/kg body weight s.c. for 18 days). As compared with controls, the EE administration resulted in severe cholestasis, as indicated by biochemical as well as morphological signs. Gangliosides isolated from the liver tissue were separated by TLC, with resorcinol-HCl detection and densitometric evaluation. As compared with controls, the total hepatic lipid sialic acid content in cholestatic rats was increased almost 2-fold (44.3 +/- 15.2 vs 79.1 +/- 9.0 nmol/g wet weight of liver tissue, p < 0.01). This increase was primarily due to the increase of ganglioside GD1a (3.6 +/- 1.0 vs 11.8 +/- 3.0 nmol/g wet weight of liver tissue, p = 0.001), as well as to the enormous up-regulation of b-series gangliosides GD3 (0.08 +/- 0.03 vs 2.0 +/- 1.2 nmol/g wet weight of liver tissue, p = 0.002), GD1b (0.1 +/- 0.06 vs 5.4 +/- 1.6 nmol/g wet weight of liver tissue, p = 0.002) and GT1b (0.06 +/- 0.03 vs 6.4 +/- 2.6 nmol/g wet weight of liver tissue, p = 0.002). As the majority of gangliosides are concentrated in cell membranes, our findings suggest that dramatic increase of b-series gangliosides might contribute to the protection of hepatocytes against the deleterious effects of cholestasis.     

Determination of ganglioside composition and structure in human brain hemangioma by chip-based nanoelectrospray ionization tandem mass spectrometry

We report here on a preliminary investigation of ganglioside composition and structure in human hemangioma, a benign tumor in the frontal cortex (HFC) in comparison to normal frontal cortex (NFC) tissue using for the first time advanced mass spectrometric methods based on fully automated chip-nanoelectrospray (nanoESI) high-capacity ion trap (HCT) and collision-induced dissociation (CID). The high ionization efficiency, sensitivity and reproducibility provided by the chip-nanoESI approach allowed for a reliable MS-based ganglioside comparative assay. Unlike NFC, ganglioside mixture extracted from HFC was found dominated by species of short glycan chains exhibiting lower overall sialic acid content. In HFC, only GT1 (d18:1/20:0), and GT3 (d18:1/25:1) polysialylated species were detected. Interestingly, none of these trisialylated forms was detected in NFC, suggesting that such components might selectively be associated with HFC. Unlike the case of previously investigated high malignancy gliosarcoma, in HFC one modified O-Ac-GD2 and one modified O-Ac-GM4 gangliosides were observed. This aspect suggests that these O-acetylated structures could be associated with cerebral tumors having reduced malignancy grade. Fragmentation analysis by CID in MS² mode using as precursors the ions corresponding to GT1 (d18:1/20:0) and GD1 (d18:1/20:0) provided data corroborating for the first time the presence of the common GT1a and GT1b isomers and the incidence of unusual GT1c and GT1d glycoforms in brain hemangioma tumor.      

New Insights on Non-Enzymatic Oxidation of Ganglioside GM1 Using Mass Spectrometry

Gangliosides are acidic glycosphingolipids that are present in cell membranes and lipid raft domains, being particularly abundant in central nervous systems. They participate in modulating cell membrane properties, cell–cell recognition, cell regulation, and signaling. Disturbance in ganglioside metabolism has been correlated with the development of diseases, such as neurodegenerative diseases, and in inflammation. Both conditions are associated with an increased production of reactive oxidation species (ROS) that can induce changes in the structure of biomolecules, including lipids, leading to the loss or modification of their function. Oxidized phospholipids are usually involved in chronic diseases and inflammation. However, knowledge regarding oxidation of gangliosides is scarce. In order to evaluate the effect of ROS in gangliosides, an in vitro biomimetic model system was used to study the susceptibility of GM1 (Neu5Acα2-3(Galβ1-3GalNAcβ1-4)Galβ1-4Glcβ1Cer) to undergo oxidative modifications. Oxidation of GM1 under Fenton reaction conditions was monitored using high resolution electrospray ionization-mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS/MS). Upon oxidation, GM1 underwent oxidative cleavages in the carbohydrate chain, leading to the formation of other gangliosides GM2 (GalNAcβ1-4Gal(Neu5Acα2-3)1-4Glcβ1Cer), GM3 (Neu5Acα2-3Galβ1-4Glcβ1Cer), asialo-GM1 (Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1Cer), asialo-GM2 (GalNAcβ1-4Galβ1-4Glcβ1Cer), of the small glycolipids lactosylceramide (LacCer), glucosylceramide (GlcCer), and of ceramide (Cer). In addition, oxygenated GM1 and GM2 (as keto and hydroxy derivatives), glycans, oxidized glycans, and oxidized ceramides were also identified. Nonenzymatic oxidation of GM1 under oxidative stress contributes to the generation of other gangliosides that may participate in the imbalance of gangliosides metabolism in vivo, through uncontrolled enzymatic pathways and, consequently, play some role in neurodegenerative processes.

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Chemoenzymatic synthesis of GM3 and GM2 gangliosides containing a truncated ceramide functionalized for glycoconjugate synthesis and solid phase applications

Analogues of GM3 and GM2 gangliosides were chemoenzymatically synthesized on a multifunctional ceramide-type tether designed to facilitate diverse strategies for glycoconjugate synthesis. The truncated ceramide aglycon maintains the stereogenic centres of natural ceramide while avoiding extensive hydrophobicity that can hamper synthesis and purification of the glycolipids. Tetanus toxoid and BSA glycoconjugates of these two gangliosides were prepared for immunization of mice, and for solid phase assays to screen for ganglioside-specific antibodies. Inhibition experiments showed that antibodies generated by tetanus toxoid conjugates of GM3 and GM2 exhibited specificity for the carbohydrate epitope and the stereogenic centres of the ceramide.     

Separation of gangliosides by anion-exchange chromatography on Mono Q

A new type of strong anion-exchange resin, Mono Q, has been used in the separation of brain gangliosides. The resin consists of monodisperse particles (9.8 micron) and was used in prepacked columns with a bed volume of 1 ml. The gangliosides were separated into mono-, di-, tri- and tetrasialoganglioside fractions by a discontinuous gradient of potassium acetate in methanol. The separation was complete in a volume of 50 ml. The major advantages of the new procedure compared to conventional methods are the shorter separation time, higher loading capacity and recovery of separated ganglioside fractions in small solvent volumes. The procedure was applied to the separation of gangliosides from normal human and GM2-gangliosidosis brain.     

Intermicellar interactions may induce anomalous size behavior in micelles carrying out bulky heads with multiple spatial arrangements

We report experimental and theoretical results on the concentration dependence of the micellar size of GM1 and GM1acetyl gangliosides, five-sugar-headed anionic glycolipids. Contrary to one of the mainstays of colloid science, that the aggregation number of amphiphile aggregates grows with concentration, an anomalous region is found at intermediate concentrations, where a sharp decrease of the aggregation number occurs. Experiments were performed by small-angle X-ray and neutron scattering (SAXS and SANS). Two models are discussed, reproducing the observed behavior of either GM1acetyl or GM1. The first one is a conventional picture of interacting micelles where a reduction in the molecular surface area, leading to an increase of the aggregate dimension, is paid to reduce intermicellar interactions: it foresees a monotonous increase of the aggregation number with concentration. The second one accounts for a conformational bistability of the bulky headgroups of GM1, modifying the amphiphilic molecular surface area and protrusion from the aggregate surface, and contributing to the inter- and intramicellar interaction balance. Energy minimization leads to a complex behavior of the aggregation number, which is consistent with the anomalous behavior of GM1.