Antioxidant properties of ganglioside micelles

Antioxidant activity of gangliosides GM1 and GT1b in the Fenton type of reaction was investigated by EPR spectroscopy using DMPO as a spin trap. Hydroxyl radical spin adduct signal intensity was significantly reduced in the presence of gangliosides at their micellar concentrations. Mean micellar hydrodynamic diameter was not changed, whereas significant changes in negative Zeta potential values were observed as evidenced by Zetasizer Nano ZS. This study showed that the primary mode of ganglioside action was not due to direct scavenging of OH·, but rather to the inhibition of hydroxyl radical formation. This phenomenon is related to the ability of ganglioside micelles to bind oppositely charged ferrous ions, thus reducing their concentration and consequently inhibiting OH· formation.     

鸟嘌呤碱基与羟基自由基反应的密度泛函理论

羟基自由基 (·OH)进攻嘌呤碱基是破坏核酸造成DNA断链损伤的重要原因之一 .采用密度泛函 (DFT)理论中B3LYP方法在 6— 31G基组水平上对鸟嘌呤 (G)受羟基自由基进攻形成的各种可能产物自由基进行几何全优化 .根据总能量、键长和自旋密度的计算结果 ,从理论上确认了C 5和C 8位加成机制 .得产物自由基G5OH·、G8OH· ,且G5OH·易与N 11位H脱水得一个更稳定的产物自由基 ,而G8OH·不易发生开环反应 ,得到与实验一致的结论 .这些稳定自由基的形成造成DNA断链损伤     

Association of gangliosides with the lymphocyte plasma membrane studied using radiolabels and spin labels

Gangliosides are known to act as potent suppressors of lectin-stimulated lymphocyte activation when added to the culture medium. Since this effect may be mediated via ganglioside association with (or insertion into) the plasma membrane, we have used 3H- and spin-labelled derivatives of mixed gangliosides to probe the nature of this interaction. Gangliosides bind rapidly to the lymphocyte membrane and show no preference for association with either inside-out or right-side-out membrane vesicles. Around 20% of the bound gangliosides can be removed by repetitive washing, and a further 22-28% by treatment with pronase for 1 h, suggesting that this fraction is tightly bound to membrane proteins at the cell surface. The ESR spectrum of membrane-bound gangliosides did not resemble the spin-exchanged spectrum of micellar spin-labelled gangliosides in aqueous solution, but was similar to that seen for 5 mol% ganglioside spin label in liposomes of egg phosphatidylcholine. This suggests that the bulk of the membrane-bound gangliosides are inserted and molecular dispersed in the lymphocyte membrane. Binding of wheat-germ agglutinin to lymphocyte-associated gangliosides results in specific immobilization of the carbohydrate headgroup, while concanavalin A and other lectins have little or no effect on oligosaccharide mobility. Membrane-inserted gangliosides show a response to lectin binding which is qualitatively different from that seen for gangliosides in bilayers of phosphatidylcholine.     

Alpha-fucosidase-ganglioside interactions. Action of alpha-L-fucosidase from the hepatopancreas of Octopus vulgaris on a fucose-containing ganglioside (Fuc-GM1).

alpha-L-Fucosidase, prepared in highly purified form (Mr 70 000-74 000) from Octopus hepatopancreas, was able to hydrolyse a fucose-containing ganglioside, namely Fuc-GM1 (II3NeuAc,IV2Fuc-GgOse4-Cer). The enzyme showed an irregular kinetic behaviour (v/[S] and v/[E] relationships following sigmoidal curves) when working on micellar Fuc-GM1 (Mr of the micelle 500 000), but obeyed regular hyperbolic kinetics when acting on low-Mr substances. It was observed that, on incubation with micellar Fuc-GM1 under the conditions used for the enzyme assay, Octopus alpha-L-fucosidase produced a ganglioside-enzyme complex that was catalytically inactive. This complex had an Mr exceeding 500 000 and a ganglioside/protein ratio of 4:1 (w/w), which is consistent with a stoichiometric combination of one ganglioside micelle with two enzyme molecules. Inactivation of alpha-L-fucosidase by formation of the corresponding complexes was also obtained with micellar gangliosides GM1 (II3NeuAc-GgOse4-Cer), GD1a (II3NeuAc,IV3NeuAc-GgOse4-Cer) and GT1b [II3(NeuAc)2,IV3-NeuAc-GgOse4-Cer], which are not substrates for the enzyme, indicating that the ganglioside micelles per se act as enzyme inhibitors. However, alpha-L-fucosidase easily forms a Fuc-GM1-alpha-L-fucosidase complex, displaying regular Michaelis-Menten kinetics. Therefore the anomalous behaviour exhibited by alpha-L-fucosidase on micellar Fuc-GM1 is likely due to formation of the complex, which separates the fucosyl linkage from the active site of the complexed enzyme, but makes it available to the enzyme in the free form.     

Molecular interactions between micellar polysialogangliosides and affinity-purified tetanotoxins in aqueous solution

Two-affinity purified tetanotoxin forms, TeToA and TeToB, with different affinities for gangliosides were characterized by analytical ultracentrifuge, circular dichroism (CD), and amino acid composition. Both toxin forms share a common sedimentation coefficient of about 6-7 S and similar alpha-helicity values, but they vary in amino acid composition. Incubation of TeToB with micellar polysialogangliosides results in formation of high (21-24 S) and medium (13-15 S) size toxin-micellar ganglioside aggregates as revealed by analytical ultracentrifuge technique. At TeToB/[N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl- [N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosylceramide (GT1b) molar ratios of greater than 26, high molecular weight aggregates (Mr greater than or equal to 700,000) which contain between 3 and 5 toxin monomers are formed, whereas at molar ratios less than 15, about 1-2 monomers are present. TeToA does not form aggregates in the presence of gangliosides. A marked increase in the alpha-helix from about 20 to 39% is apparent in the CD spectrum of TeToB after interaction with ganglioside mixture (G1b). Cerebrosides, sulfatides, sphingomyelin, and phosphatidylserine also increase the alpha-helix, presumably because of an overall effect of lipids on the protein. TeToA and fragment B but not C also undergo similar changes in the presence of G1b, suggesting that the effect of ganglioside is not specific. The polarity of the CD spectra of a number of gangliosides is shifted from a negative to a positive value after interaction with tetanotoxin. The data are consistent with the interpretation of a discrete hydrophobic domain on the toxin heavy chain which interacts with micellar gangliosides to form macromolecular complexes.     

Thermotropic behavior and electronmicroscopic structures of mixtures of gangliosides and dipalmitoylphosphatidylcholine

The calorimetric properties and morphological structures of dispersed mixtures of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and highly purified human brain gangliosides, GM2, GM1, GD1a, GD1b, and GT1b, were studied using a high-sensitivity differential scanning calorimeter and an electron-microscope, as a function of the ganglioside molar fraction. No thermal phase transitions of pure gangliosides in aqueous dispersions could be detected. In the mixtures of DPPC and gangliosides, the gel to liquid crystalline phase transition occurred at a higher temperature than in pure DPPC dispersions and progressed over a wide temperature range. As increasing amounts of the pure ganglioside species were added to DPPC, the temperature for the main transition gradually increased. The phase transition progressed differently among different gangliosides/DPPC mixtures. The enthalpy values were found to decrease linearly as the number of sialic acid residues increased. Electron-microscopically the ganglioside/DPPC mixtures formed multilamellar structures at lower concentrations of the gangliosides, and the structures changed to cylindrical and spherical micelles as the ganglioside concentration was increased. The polysialoganglioside/DPPC mixtures showed the micellar form even at lower ganglioside concentrations, contrary to the case of the monosialoganglioside/DPPC mixtures. The morphological changes of gangliosides/DPPC mixtures corresponded with changes in the calorimetric properties. These results show that individual gangliosides have different physicochemical effects on model membranes, possibly because of the interaction of their negatively charged head groups.     

Uptake and metabolism of exogenous glycosphingolipids by cultured cells

Exogenous glycosphingolipids, especially gangliosides, are used to study transport and metabolism of their endogenous counterparts as well as their role in cell adhesion, cell recognition and signal transduction. Unlike monodispersed solutes, in aqueous media ganglioside molecules aggregate into micelles (or bilayer structures) with a very low critical micellar concentration. Upon addition to cells in culture, exogenous gangliosides bind to the cell surface in three operationally defined modes: loosely associated micelles removable by serum; tightly attached micelles removable by proteases such as trypsin; and ganglioside molecules inserted into the outer leaflet of the plasma membrane. As shown by a biotin-labeled derivative of the ganglioside GM1 these inserted molecules are endocytosed and transported to intralysosomal membranes for catabolism. The benefit from using (partially) nondegradable as well as semi-truncated glycosphingolipids in transport studies is discussed.     

Light scattering measurements on gangliosides: dependence of micellar properties on molecular structure and temperature

Static and dynamic laser light scattering measurements on micellar aqueous solutions of gangliosides GM2, GM1, GD1a are reported. The aggregation number, the hydrodynamic radius and the micellar shape depend on the type of ganglioside and the unsaturation degree of the hydrocarbon chains. At a temperature of 25 degrees C the molecular weights of GM2, GM1 and GD1a are 740,000, 470,000 and 418,000 DA respectively. A significant decrease of micellar size with temperature has been found for saturated GM1 in the region 25 degrees-40 degrees C. The strong sensitivity of the micellar parameters to the ganglioside structure is explained by making reference to some simple model which takes into account geometrical packing considerations. By measuring the scattered light intensity at low ionic strength of the solution (0.1-30 mE) it was possible to evaluate the ganglioside micellar charge, 100 electronic units for GM2, 48 for GM1 and 60 for GD1a.     

Phase behavior of ganglioside-lecithin mixtures. Relation to dispersion of gangliosides in membranes.

Ganglioside GM1 and mixed brain gangliosides were mixed with 1-stearoyl-2-oleoyl lecithin (SOPC) and examined by differential scanning calorimetry as a function of ganglioside content and temperature. Low mole fractions of ganglioside GM1 and of mixed brain gangliosides are shown to be miscible with SOPC in the gel phase up to X = 0.3, with the possible exception of a small region of immiscibility for the mixed brain gangliosides system centered around X = 0.05. Above X = 0.3, the low-temperature phases demix into a (gel) phase of composition X = 0.3 and a (micellar) phase of composition X = 1.0. Above the endothermic phase transition temperature, no phase boundaries are discerned. It is pointed out that phase structures need to be determined in each domain delineated in the phase diagrams, and that cylindrical phases may exist at higher temperatures and intermediate compositions. The effects of addition of wheat germ agglutinin, which binds to ganglioside GM1, on a ganglioside GM1-SOPC mixture (X = 0.5), are described and interpreted in terms of partial demixing of ganglioside and lecithin. Behavior of the ganglioside-SOPC system is discussed with respect to the kinetics of cholera toxin action in lymphocytes, as well as to other physiological roles of gangliosides in membranes.     

The production of hydroxyl radical from hydrogen peroxide

The formation of hydroxyl radical (OH·) from the oxidation of glutathione, ascorbic acid, NADPH, hydroquinone, catechol, and riboflavin by hydrogen peroxide was studied using a range of enzymes and copper and iron complexes as possible catalysts. Copper-1,10-phenanthroline appears to catalyze the production of OH· from hydrogen peroxide without superoxide radical being formed as an intermediate, and without the involvement of a catalyzed Haber-Weiss (Fenton) reaction. Superoxide radical is involved, however, in the Cu²⁺ -catalyzed decomposition of hydrogen peroxide, and in the oxidation of glutathione by atmospheric oxygen. For this latter oxidation, copper-4,7-dimethyl-1,10-phenanthroline was found to be a much more effective catalyst than the copper complex of 1,10-phenanthroline, which is normally used. Mechanisms for these reactions are proposed, and the toxicological significance of the ability of a variety of biological reductants to provide a prolific source of OH· when oxidized by hydrogen peroxide is discussed.     

Hydrolysis of di- and trisialo gangliosides in micellar and liposomal dispersion by bacterial neuraminidases

The hydrolysis of di- and trisialo gangliosides by bacterial neuraminidases was investigated. Slow rates of hydrolysis were obtained with micellar dispersions of the pure gangliosides; the rates increased considerably with mixtures of ganglioside and phospholipids, such as phosphatidylcholine or sphingomyelin. The greatest rates of hydrolysis were obtained with mixtures containing 5-10 mol% ganglioside and 90-95% phospholipid. With the aid of the nonpenetrating reagent trinitrobenzenesulfonic acid, it was ascertained that this mixture consisted of sealed, unilamellar vesicles in which the ganglioside was distributed symmetrically between the two layers of the liposome. When the relative proportion of the ganglioside was increased, the dispersions contained liposomes admixed with micelles of ganglioside and phospholipid. The rates of hydrolysis of the ganglioside could be correlated with the percentage of sealed vesicles in each mixture. Experiments in which another ganglioside (GM1) or cholesterol was incorporated into the mixed dispersions further supported this conclusion. It is suggested that the rate of hydrolysis is affected predominantly by interactions between the carbohydrate chains of ganglioside molecules. The data emphasize that ganglioside metabolism can be best studied when the latter are part of biological or model membranes.     

Stimulation by Gangliosides of Viability of Rat Brain Neurons and of Neuronal PC12 Cell Line under Conditions of Oxidative Stress

We studied effect of gangliosides on viability of brain neurons and neuronal PC12 cell line exposed to toxic concentrations of compounds activating free radical reactions. It is found that preincubation of cerebellar granule cells and PC12 cells with micromolar concentrations of ganglioside GM1 increases statistically significantly viability of these cells submitted to inductors of oxidative stress, such as hydrogen peroxide and the Fe²⁺-ascorbate system However, the effect of ganglioside GM1 in the PC12 cells failed to be revealed 1–2 days after treatment of the cells with trypsin, which indicates an importance of interaction of gangliosides with surface proteins for realization of their protective action. GM1, GD1a, and other gangliosides were shown to produce the neuroprotective effect on cerebellar granule cells in the presence of toxic glutamate concentrations. Not only micro-, but also nanomolar concentrations of these gangliosides increased statistically significantly the neuronal viability, although at micromolar concentrations this effect as a rule was more pronounced. The obtained data allow suggesting that the neuroprotective action of gangliosides is determined to a considerable degree by their ability to inhibit free-radical reactions in nerve cells.     

Dansylated thyrotropin as a probe of hormone-receptor interactions.

A strongly fluorescent 5-dimethylamino-1-naphthalene sulfonate (dansyl) derivative of bovine thyrotropin has been prepared. The dye-conjugated hormone is bioactive and shares, essentially unchanged, the membrane binding and adenylate cyclase stimulatory activities of the native hormone. Binding of 125I-labeled dansyl-thyrotropin to thyroid plasma membranes is sensitive to inhibition by gangliosides and, as is the case for the binding of 125I-thyrotropin, galactosyl-N-acetylgalactosaminyl[N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosylceramide (GDIb) is the most potent binding inhibitor. Gangliosides interact with dansyl-thyrotropin, causing a large increase of the quantum yield and a 5- to 10-nm blue shift of the emission maximum of the hormone-bound naphthalene chromophore; gangliosides cause no change in the fluorescent properties of the free dye. The fluorescence enhancement caused by gangliosides can be specifically reversed by unlabeled thyrotropin. The effect of gangliosides on dansyl-thyrotropin fluorescence is strongly salt-dependent; salts cannot, however, reverse the formation of the dansyl-thyrotropin.ganglioside complex once it has formed. The salt data suggest that the association of the ganglioside with dansyl-thyrotropin is dominated by electrostatic interactions, but that salt-independent, short range interactions, most likely hydrophobic, dominate the dissociation of the dansyl-thyrotropin-ganglioside adduct. Sucrose gradient centrifugation, ultracentrifugation, and fluorescence polarization data indicate that the gangliosides are micellar in nature under the conditions of these experiments. Acid titration of dansyl-thyrotropin causes a marked quenching of dansyl fluorescence which in part reflects dissociation of the hormone into its constituent alpha and beta subunits. In the presence of GDIb, but not N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GDIa), pH-dependent quenching and subunit dissociation are essentially eliminated. Circular dichroism results and fluorescence polarization studies support the interpretation that the ganglioside interaction causes a conformational change in the thyrotropin molecule. The acid titration data together with differences in the ability of gangliosides to influence the tyrosine fluorescence of the thyrotropin molecule indicate that different gangliosides induce different conformational perturbations in the thyrotropin molecule.     

Recycling of glucosylceramide and sphingosine for the biosynthesis of gangliosides and sphingomyelin in rat liver.

It was previously shown that sphingomyelin and gangliosides can be biosynthesized starting from sphingosine or sphingosine-containing fragments which originated in the course of GM1 ganglioside catabolism. In the present paper we investigated which fragments were specifically re-used for sphingomyelin and ganglioside biosynthesis in rat liver. At 30 h after intravenous injection of GM1 labelled at the level of the fatty acid ([stearoyl-14C]GM1) or of the sphingosine ([Sph-3H]) moiety, it was observed that radioactive sphingomyelin was formed almost exclusively after the sphingosine-labelled-GM1 administration. This permitted the recognition of sphingosine as the metabolite re-used for sphingomyelin biosynthesis. Conversely, gangliosides more complex than GM1 were similarly radiolabelled after the two treatments, thus ruling out sphingosine re-utilization for ganglioside biosynthesis. For the identification of the lipid fragment re-used for ganglioside biosynthesis, we administered to rats neutral glycosphingolipids (galactosylceramide, glucosylceramide and lactosylceramide) each radiolabelled in the sphingosine moiety or in the terminal sugar residue. Thereafter we compared the formation of radiolabelled gangliosides in the liver with respect to the species administered and the label location. After galactosylceramide was injected, no radiolabelled gangliosides were formed. After the administration of differently labelled glucosylceramide, radiolabelled gangliosides were formed, regardless of the position of the label. After lactosylceramide administration, the ganglioside fraction became more radioactive when the long-chain-base-labelled precursors were used. These results suggest that glucosylceramide, derived from glycosphingolipid and ganglioside catabolism, is recycled for ganglioside biosynthesis.     

Interactions of pig brain cytosolic sialidase with gangliosides. Formation of catalytically inactive enzyme-ganglioside complexes

Cytosolic sialidase A was extracted from pig brain and purified about 2000-fold with respect to the starting homogenate (about 550-fold relative to the cytosolic fraction). The enzyme preparation provided a single peak on Ultrogel AcA-34 column chromatography and had an apparent molecular weight of 4 x 10(4). On incubation with micellar ganglioside GT1b, (molecular weight of the micelle, 3.5 x 10(5)) under the conditions used for the enzyme assay, brain cytosolic sialidase A formed two ganglioside-enzyme complexes, I and II, which were isolated and characterized. Complex II had a molecular weight of 4.2 X 10(5), and a ganglioside/protein ratio (w/w) of 4:1. This is consistent with a stoichiometric combination of one ganglioside micelle and two enzyme molecules. Complex I was probably a dimer of complex II. In both complexes I and II cytosolic sialidase was completely inactive. Inactivation of cytosolic sialidase by formation of the corresponding complexes was also obtained with gangliosides GD1a and GD1b, which, like GT1b, are potential substrates for the enzyme and GM1, which is resistant to the enzyme action. Therefore, the enzyme becomes inactive after interacting with ganglioside micelles. GT1b-sialidase complexes acted as excellent substrates for free cytosolic sialidase, as did the complexes with GD1a and GD1b.     

Nerve growth factor-induced neurite formation in PC12 cells is independent of endogenous cellular gangliosides

The PC12 rat pheochromocytoma cell line is an established model for nerve growth factor (NGF)-induced neurite formation. It has been shown that when gangliosides are added to the culture medium of PC12 cells, NGF-induced neurite formation of PC12 cells is enhanced. To determine the role of endogenous cellular gangliosides themselves in NGF-elicited neurite formation, we depleted cellular gangliosides using the new specific glucosylceramide synthase inhibitor, d, l-threo-1-phenyl-2- hexadecanoylamino-3-pyrrolidino-1-propanol.HCl (PPPP). 0.5-2 microM PPPP rapidly inhibited ganglioside synthesis and depletedcellular gangliosides. Nonetheless, over a concentration range of 5-100 ng/ml NGF, in both low serum and serum-free medium, neurite formation was normal. Even pretreatment of PC12 cells for up to 6 days with 1 microM PPPP followed by cotreatment with PPPP and NGF for 10 days, still did not inhibit neurite formation. The conclusion that ganglioside depletion did not block neurite formation stimulated by NGF was supported by the lack of effect of PPPP, under these same conditions, on cellular acetylcholine esterase activity, a neuronal differentiation marker (73.8 +/- 12.1 versus 67.2 +/- 4.6 nmol/min/mg protein at 50 ng/ml NGF; control versus 1 microM PPPP). These findings, together with previous studies showing enhancement of NGF-induced neurite formation by exogenous gangliosides, underscore the vastly different effects that exogenous gangliosides and endogenous gangliosides may have upon cellular functions.      

Monoclonal antibody Leo Mel 3, which inhibits killing of human melanoma cells by anomalous killer cells, binds to a sugar sequence in GD2 (II3(NeuAc)2-GgOse3Cer) and several other gangliosides

Human anomalous killer (AK) cells lyse freshly isolated human melanoma cells which are insensitive to human natural killer cell-mediated lysis. Monoclonal antibody Leo Mel 3, an IgM (k), produced by a hybridoma obtained from a mouse immunized with human melanoma cells, binds to melanoma cells and inhibits their conjugate formation with AK cells as well as their AK cell-mediated lysis. Other IgM antibodies from the same fusion that bind melanoma cells do not inhibit (Werkmeister, J. A., Triglia, T., Andrews, P., and Burns, G. F. (1985) J. Immunol. 135, 689-695). Leo Mel 3 binds several different gangliosides from melanoma cells, as determined by immunostaining thin layer chromatograms. Binding is abolished by treatment of the gangliosides with neuraminidase. In solid-phase radioimmunoassay, Leo Mel 3 binds strongly to ganglioside GD2 and less strongly to gangliosides GT3, GD3, and GQ1b. It does not bind to other gangliosides including GM1, GM2, GM3, GD1a, GD1b, and GT1b. Thus, the epitope recognized by antibody Leo Mel 3 is found in the sugar sequence of ganglioside GD2, GalNAc beta 1-4[NeuAc alpha 2-8NeuAc alpha 2-3]Gal beta 1-4Glc beta 1 .... This sequence may contain a target in melanoma cells recognized by AK cells.     

Gangliosides Have a Functional Role during Rotavirus Cell Entry

Cell entry of rotaviruses is a complex process, which involves sequential interactions with several cell surface molecules. Among the molecules implicated are gangliosides, glycosphingolipids with one or more sialic acid (SA) residues. The role of gangliosides in rotavirus cell entry was studied by silencing the expression of two key enzymes involved in their biosynthesis—the UDP-glucose:ceramide glucosyltransferase (UGCG), which transfers a glucose molecule to ceramide to produce glucosylceramide GlcCer, and the lactosyl ceramide-α-2,3–sialyl transferase 5 (GM3-s), which adds the first SA to lactoceramide-producing ganglioside GM3. Silencing the expression of both enzymes resulted in decreased ganglioside levels (as judged by GM1a detection). Four rotavirus strains tested (human Wa, simian RRV, porcine TFR-41, and bovine UK) showed a decreased infectivity in cells with impaired ganglioside synthesis; however, their replication after bypassing the entry step was not affected, confirming the importance of gangliosides for cell entry of the viruses. Interestingly, viral binding to the cell surface was not affected in cells with inhibited ganglioside synthesis, but the infectivity of all strains tested was inhibited by preincubation of gangliosides with virus prior to infection. These data suggest that rotaviruses can attach to cell surface in the absence of gangliosides but require them for productive cell entry, confirming their functional role during rotavirus cell entry.     

Analysis of gangliosides from carp intestinal mucosa

The gangliosides of carp intestinal mucosa were isolated and analysed by thin-layer chromatography (TLC), TLC immunostaining test, and TLC/secondary ion mass spectrometry (TLC/SIMS). Four species of gangliosides, designated as G-1, G-2, G-3 and G-4, were separated on TLC. The TLC/SIMS analysis of the G-1 ganglioside of carp intestinal mucosa revealed a series of [M-H](-)ions from m/z 1061 to m/z 1131 representing the molecular mass range of GM4-like ganglioside with NeuAc. G-2, G-3 and G-4 gangliosides were analysed by the TLC immunostaining test. G-2 ganglioside was recognised by the monoclonal antibody specific for ganglioside GM1 (AGM-1 monoclonal antibody). However, G-3 ganglioside migrating on TLC between GM3 and GM1 ganglioside was not recognised by anti-GM3 monoclonal antibody and by AGM-1 monoclonal antibody. Furthermore, G-4 ganglioside with a similar TLC mobility as GD1a ganglioside did not show the reactivity to the anti-GD1a monoclonal antibody. In addition using the AGM-1 monoclonal antibody, the expression of GM1 ganglioside in the carp intestinal tissue was studied. GM1 ganglioside was detected on the epithelial cell surface of carp intestinal mucosa.     

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

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