Composition and Metabolism of Gangliosides in Rat Peripheral Nervous System During Development

Abstract: Ganglioside composition of rat trigeminal nerve was studied during development in order to understand the changes that occur as a result of cellular differentiation in the nerve. The ganglioside composition of the trigeminal nerve was entirely different from that of brain. The major gangliosides in adult trigeminal nerve were GM₃, GD₃, and LM₁ (sialosyl-lactoneotetraosylceramide or sialosylparagloboside). The structure of LM₁ and other gangliosides was established by enzymatic degradation and by analysis of the products of acid hydrolysis. At 2 days after birth, when the Schwann cells were immature, GM₃ and GD₃ were the major gangliosides in the nerve, 50 and 18 mol %, respectively. As the nerve developed and Schwann cells proliferated and myelinated the axons, the mol % of GM₃ and GD₃ reduced and that of LM₁ steadily increased. Polysialogangliosides did not change drastically with nerve development. The rate of deposition of LM₁ in the nerve with age was very similar to that of myelin marker lipids, cerebrosides, and sulfatides; thus, deposition appears to be localized mainly in the rat nerve myelin. LM₁ also had long-chain fatty acids 22:0 and 24:0, which are not usually found in CNS gangliosides. The ganglioside pattern of the rat trigeminal nerve was very similar to that of rat sciatic nerve, but was different from that of rabbit and chicken sciatic nerve. The activity of the two key enzymes involved in the metabolism of GM₃, viz., CMP-N-acetylneuraminic acid:lactosylceramide sialyltransferase and UDP-N-acetylgalactosamine:GM₃-N-acetylgalactosaminyltransferase, was also studied during development of the nerve and brain. The developmental profiles of both enzymes were consistent with the amounts of GM₃ present in the nerve.     

Gangliosides Are Important for the Preservation of the Structure and Organization of RBL-2H3 Mast Cells

Gangliosides are known to be important in many biological processes. However, details concerning the exact function of these glycosphingolipids in cell physiology are poorly understood. In this study, the role of gangliosides present on the surface of rodent mast cells in maintaining cell structure was examined using RBL-2H3 mast cells and two mutant cell lines (E5 and D1) deficient in the gangliosides, GM₁ and the α-galactosyl derivatives of the ganglioside GD_1b. The two deficient cell lines were morphologically different from each other as well as from the parental RBL-2H3 cells. Actin filaments in RBL-2H3 and E5 cells were under the plasma membrane following the spindle shape of the cells, whereas in D1 cells, they were concentrated in large membrane ruffles. Microtubules in RBL-2H3 and E5 cells radiated from the centrosome and were organized into long, straight bundles. The bundles in D1 cells were thicker and organized circumferentially under the plasma membrane. The endoplasmic reticulum, the Golgi complex, and the secretory granule matrix were also altered in the mutant cell lines. These results suggest that the mast cell–specific α-galactosyl derivatives of ganglioside GD_1b and GM₁ are important in maintaining normal cell morphology. (J Histochem Cytochem 58:83–93, 2010)     

Effects of Cell Density on Lipids of Human Glioma and Fetal Neural Cells

Abstract: Gangliosides, phospholipids, and cholesterol of human glioma (12-18) and fetal neural cells (CH) were analyzed at specified cell densities, from sparse to confluent. Total ganglioside sialic acid, phospholipid phosphorus, and cholesterol increased in the glioma cells on a per cell, mg protein, or mg total lipid basis two- to threefold as cell density increased 25-fold. These same three constituents in the fetal cells increased with cell density on a per cell and mg protein basis but not on a per mg total lipid basis. In glioma cells, the di- and trisialogangliosides (GD₂+ GD_lb+ GT₁) increased from 1–2% of total ganglioside sialic acid at sparse densities to 7–8% at intermediate (logarithmic phase) densities to 10–13% at confluent densities. The set of simpler gangliosides (GM₄+ GM₃+ GM₂) decreased from 50% of total ganglioside sialic acid at sparse glioma cell densities, to 36% at intermediate and 30% at confluent densities. In the fetal neural cells, the set of gangliosides (GM₄+ GM₃+ GM₂) had about 48% of total ganglioside sialic acid in both sparse and confluent preparations. The fetal cells were twofold higher in GM₃ (32.4 ± 2.1%) than the glioma cells (16.8 ± 1.6%), but lower in GM_t (9.1 ± 0.9% versus 18.2 ± 1.8%), cell densities notwithstanding. Confluent cell preparations of both cell lines were consistently higher in ethanolamine plasmalogen than sparse cells. We conclude that in these two neural cell lines quantitative changes in ganglioside and phospholipid species occurred correlatively as cell densities increased. Higher glioma cell densities were associated with greater proportions of complex ganglioside species. These changes in cell membrane constituents during growth may result from cell contact and may indicate a role for them in cell growth regulation and/or differentiation.     

Interaction of Fibroblast Growth Factor-2 (FGF-2) with Free Gangliosides: Biochemical Characterization and Biological Consequences in Endothelial Cell Cultures

Exogenous gangliosides affect the angiogenic activity of fibroblast growth factor-2 (FGF-2), but their mechanism of action has not been elucidated. Here, a possible direct interaction of sialo-glycolipids with FGF-2 has been investigated. Size exclusion chromatography demonstrates that native, but not heat-denatured, ¹²⁵I-FGF-2 binds to micelles formed by gangliosides GT_1b, GD_1b, or GM₁. Also, gangliosides protect native FGF-2 from trypsin digestion at micromolar concentrations, the order of relative potency being GT_1b > GD_1b > GM₁ = GM₂ = sulfatide > GM₃ = galactosyl-ceramide, whereas asialo-GM₁, neuraminic acid, and N-acetylneuramin-lactose were ineffective. Scatchard plot analysis of the binding data of fluorochrome-labeled GM₁ to immobilized FGF-2 indicates that FGF–2/GM₁ interaction occurs with a K_d equal to 6 μM. This interaction is inhibited by the sialic acid-binding peptide mastoparan and by the synthetic fragments FGF-2(112–129) and, to a lesser extent, FGF-2(130–155), whereas peptides FGF-2(10–33), FGF-2(39–59), FGF-2(86–96), and the basic peptide HIV-1 Tat(41–60) were ineffective. These data identify the COOH terminus of FGF-2 as a putative ganglioside-binding region. Exogenous gangliosides inhibit the binding of ¹²⁵I-FGF-2 to high-affinity tyrosine-kinase FGF-receptors (FGFRs) of endothelial GM 7373 cells at micromolar concentrations. The order of relative potency was GT_1b > GD_1b > GM₁ > sulfatide a = sialo-GM₁. Accordingly, GT_1b,GD_1b, GM₁, and GM₂, but not GM₃ and asialo-GM₁, prevent the binding of ¹²⁵I-FGF-2 to a soluble, recombinant form of extracellular FGFR-1. Conversely, the soluble receptor and free heparin inhibit the interaction of fluorochrome-labeled GM₁ to immobilized FGF-2. In agreement with their FGFR antagonist activity, free gangliosides inhibit the mitogenic activity exerted by FGF-2 on endothelial cells in the same range of concentrations. Also in this case, GT_1b was the most effective among the gangliosides tested while asialo-GM₁, neuraminic acid, N-acetylneuramin-lactose, galactosyl-ceramide, and sulfatide were ineffective. In conclusion, the data demonstrate the capacity of exogenous gangliosides to interact with FGF-2. This interaction involves the COOH terminus of the FGF-2 molecule and depends on the structure of the oligosaccharide chain and on the presence of sialic acid residue(s) in the ganglioside molecule. Exogenous gangliosides act as FGF-2 antagonists when added to endothelial cell cultures. Since gangliosides are extensively shed by tumor cells and reach elevated levels in the serum of tumor-bearing patients, our data suggest that exogenous gangliosides may affect endothelial cell function by a direct interaction with FGF-2, thus modulating tumor neovascularization.     

Lipid patterns of embryonal carcinoma cell lines and their derivatives: Changes with differentiation

The lipid composition of several teratocarcinoma cell lines has been examined by biochemical and immunological methods in order to identify properties that might be correlated with the state of cell differentiation. The data indicate qualitative and quantitative changes in the phospholipid, cholesterol, and glycolipid composition. In particular, the ratios of cholesterol/phospholipid and of sphingomyelin/phosphatidylcholine are higher in differentiated cells. Gangliosides with short glycosidic chains (GM₃ and GD₃) are characteristic of undifferentiated, multipotent, embryonal carcinoma cell lines. More complex gangliosides (GM₁ and GD_1a) appear early during the course of differentiation. Each differentiated cell line presents a unique ganglioside map. Results are tentatively correlated with a stabilization of the membrane bilayer in differentiated cell lines, whereas a more fluid state of the membrane in embryonal carcinoma cell lines would allow maximal flexibility. Subtle differences in ganglioside composition among embryonal carcinoma cell lines are discussed in relation with their potentialities, and their developmental age.     

GANGLIOSIDE COMPOSITION OF CHEMICALLY INDUCED RAT NEURAL TUMORS AND CHARACTERIZATION OF HEMATOSIDE FROM NEURINOMAS

Abstract– Experimental rat neural tumors in offspring were induced transplacentally by a single injection of a chemical carcinogen, ethylnitrosourea, 20mg/kg body wt, in the tail vein of the mother. The ganglioside content and pattern in these tumors and the normal tissues from which the tumors originated are described. The ganglioside content in tumors was reduced, on wet tissue weight basis, compared to normal control. However, there was no significant difference of ganglioside content on dry weight or protein basis. Altered ganglioside composition was found in most of the neural tumors. In central nervous system tumors, there was some increase in GM₃ and GT_1b′ (nomenclature according to Svennerholm , 1963), a marked decrease in GM₁ and some decrease in GD_1a, but no apparent loss in GD_1b. Extreme simplification of ganglioside pattern was seen in tumors originated from peripheral nervous system. Large accumulation of GM₃ with concomitant loss of all the higher gangliosides was seen. GM₃ from neurinomas as well as from normal gray matter was isolated and characterized. GM₃ from neurinomas separated into two bands on thin layer chromatographic plates. Both these GM₃ bands had identical sphingosine and carbohydrate composition but differed in their fatty acid composition. The fast moving band had 77% of the total fatty acids as C_20:0 or longer chain while the slow moving band had only 22% of the long chain fatty acids. Normal gray matter GM₃ had one major band containing 82% of and only 17% of the fatty acids as C_20:0 or higher. It is suggested that in the tumor cells either the specificity of the enzyme cytidine monophosphate-N-acetyl neuraminic acid: ceramide dihexoside sialyltransferase for C_18.0 fatty acid containing glycolipid was altered or that the compartmentation of precursor pools for the simpler glycolipids present in normal tissue did not exist in transformed cells.     

Developmental changes in gangliosides during myogenesis of a rat myoblast cell line and its drug resistant variants.

Cloned cells of a myoblast line show the presence of GM₃, GM₂, GM₁ and GD_1a gangliosides. The amount of GM₃, GM₂ and GM₁ gangliosides does not vary significantly during the differentiation of myoblasts to myotubes. However, the concentration of GD_1a transiently increases almost 3-fold just prior to the fusion of myoblasts and returns to the basal levels in the myotubes. Mutant myoblasts selected for 5-azacytidine resistance and unable to fuse produce only GM₃ and traces of GM₂. We conclude that GD_1a probably participates in the fusion process through yet unknown mechanism.     

Gangliosides influence EGFR/ErbB2 heterodimer stability but they do not modify EGF-dependent ErbB2 phosphorylation

Gangliosides are well-known regulators of cell differentiation through specific interactions with growth factor receptors. Previously, our group provided the first evidence about stable association of ganglioside GM₃ to EGFR/ErbB2 heterodimers in mammary epithelial cells. Goals of the present study were to better define the role of gangliosides in EGFR/ErbB2 heterodimerization and receptor phosphorylation events and to analyze their involvement in mammary cell differentiation. Experiments have been conducted using the ceramide analogue (+/−)-treo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride ([D]-PDMP), which inhibits ceramide glucosyltransferase resulting in the endogenous ganglioside depletion, and the lactogenic hormone mix DIP (dexamethasone, insulin, prolactin), which induces cell differentiation and β-casein mRNA synthesis. In addition, treatments of ganglioside-depleted cells with exogenous GM₃ have been carried out to ascertain the specific involvement of this ganglioside. Results from co-immunoprecipitation and Western blot experiments have shown that the endogenous ganglioside depletion resulted in the disappearance of SDS-stable EGFR/ErbB2 heterodimers and in the appearance of tyrosine-phosphorylated EGFR also in the absence of EGF stimulation; exogenous GM₃ added in combination with [D]-PDMP reversed both these effects. In contrast, the tyrosine phosphorylation of ErbB2 in ganglioside-depleted cells occurred only after EGF stimulation. Moreover, when ganglioside-depleted cells were treated with DIP in absence of EGF, β-casein gene expression appeared strongly down-regulated, and β-casein mRNA levels were partially restored by exogenous GM₃ treatment. Altogether, although the involvement of other ganglioside species cannot be excluded, these findings sustain the ganglioside GM₃ as an essential molecule for EGFR/ErbB2 heterodimer stability and important regulator of EGFR tyrosine phosphorylation, but it is not crucial for tyrosine phosphorylation of the heterodimerization partner ErbB2. Moreover, modulation of EGFR phosphorylation may explain how gangliosides contribute to regulate the lactogenic hormone-induced mammary cell differentiation.     

Evidence of a distribution difference between two gangliosides in bilayer membranes

Freeze-etch electron microscopy, a platinum shadowing technique, has been used to compare the lateral distribution of several gangliosides in bilayer model membranes by directly visualizing bound lectin molecules. In particular, GM₁ and GD_1a, major components of brain ganglioside, were studied in phase-separated mixtures of dipalmitoyl- and dielaidoylphosphatidylcholines exposed to Ricinus communis agglutinin and wheat germ agglutinin. The distribution of glycolipid showed evidence of microheterogeneity in that bound lectin tended to occur in clusters of several or more molecules. With GD_1a as receptor such clusters were small and very uniformly distributed over the membrane surface. Somewhat larger, irregularly spaced clusters of up to a dozen lectin particles were more typical of membranes bearing GM₁ and, in addition, there were occasional extensive patches of bound lectin coexisting with areas apparently devoid of glycolipid receptor in phase-separated mixtures of dipalmitoyl- and dielaidoylphosphatidylcholine. Gangliosides in the latter mixtures were not obviously influenced in their lateral distribution by the presence of coexisting fluid and rigid domains. These basic observations seem to extend to bilayer membranes containing mixtures of two gangliosides. The patterns of lectin binding were not grossly affected by incubation time or history of warming and cooling. This study was extended to bilayers of pure dipalmitoylphosphatidylcholine in expectation that the distinctive features characteristic of the P_β′ phase of this lipid might accentuate any behavioural differences between GM₁ and GD_1a.GM₁ was found to exist preferentially in the ‘trough’ regions between P_β′ ripples, while GD_1a showed no apparent preferential arrangement. Given that bound lectins adequately reflect glycolipid distribution in membranes, it would appear that structurally different glycolipids from the same host membrane can assume different distributions on the basis of interactions with defined lipid host matrices.     

Structure and Function of a Ganglioside Receptor for Porcine Rotavirus

A ganglioside fraction isolated from pooled intestines from newborn to 4-week-old piglets, which we previously partially characterized and showed to specifically inhibit the binding of porcine rotavirus (OSU strain) to host cells (M. D. Rolsma, H. B. Gelberg, and M. S. Kuhlenschmidt, J. Virol. 68:258–268, 1994), was further purified and found to contain two major monosialogangliosides. Each ganglioside was purified to apparent homogeneity, and their carbohydrate structure was examined by high-pH anion-exchange chromatography coupled with pulsed amperometric detection and fast atom bombardment mass spectroscopy. Both gangliosides possessed a sialyllactose oligosaccharide moiety characteristic of GM₃ gangliosides. Compositional analyses indicated that each ganglioside was composed of sialic acid, galactose, glucose, and sphingosine in approximately a 1:1:1:1 molar ratio. Each ganglioside differed, however, in the type of sialic acid residue it contained. An N-glycolylneuraminic acid (NeuGc) moiety was found in the more polar porcine GM₃, whereas the less polar GM₃ species contained N-acetylneuraminic acid (NeuAc). Both NeuGcGM₃ and NeuAcGM₃ displayed dose-dependent inhibition of virus binding to host cells. NeuGcGM₃ was approximately two to three times more effective than NeuAcGM₃ in blocking virus binding. Inhibition of binding occurred with as little as 400 pmol of NeuGcGM₃/50 ng of virus (~2 × 10⁷ virions) and 2 × 10⁶ cells/ml. Fifty percent inhibition of binding was achieved with 0.64 and 1.5 μM NeuGcGM₃ and NeuAcGM₃, respectively. The free oligosaccharides 3′- and 6′-sialyllactose inhibited binding 50% at millimolar concentrations, which were nearly 1,000 times the concentration of intact gangliosides required for the same degree of inhibition. Direct binding of infectious, triple-layer rotavirus particles, but not noninfectious, double-layered rotavirus particles, to NeuGcGM₃ and NeuAcGM₃ was demonstrated by using a thin-layer chromatographic overlay assay. NeuGcGM₃ and NeuAcGM₃ inhibited virus infectivity of MA-104 cells by 50% at concentrations of 3.97 and 9.84 μM, respectively. NeuGcGM₃ (700 nmol/g [dry weight] of intestine) was found to be the predominant enterocyte ganglioside (comprising 75% of the total lipid-bound sialic acid) in neonatal piglets, followed by NeuAcGM₃ (200 nmol/g [dry weight] of intestine). NeuGcGM₃ and NeuAcGM₃ together comprised nearly 100% of the lipid-bound sialic acid in the neonatal intestine, but their quantities rapidly diminished during the first 5 weeks of life. These data support the hypothesis that porcine NeuGcGM₃ and NeuAcGM₃ are physiologically relevant receptors for porcine rotavirus (OSU strain). Further support for this hypothesis was obtained from virus binding studies using mutant or neuraminidase-treated cell lines. Lec-2 cells, a mutant clone of CHO cells characterized by a 90% reduction in sialyllation of its glycoconjugates, bound less than 5% of the virus compared to control cell binding. In contrast, Lec-1 cells, a mutant CHO clone characterized by a deficiency in glycosylation of N-linked oligosaccharides, still bound rotavirus. Furthermore, exogenous addition of NeuGcGM₃ to the Lec-2 mutant cells restored their ability to bind rotavirus in amounts equivalent to that of their parent (CHO) cell line. In the virus-permissive MA-104 cell line, NeuGcGM₃ was also able to partially restore rotavirus infectivity in neuraminidase-treated cells. These data suggest that gangliosides play a major role in recognition of host cells by porcine rotavirus (OSU strain).     

Interaction of gangliosides with plasma low density lipoproteins

The role of gangliosides in the reception of low density lipoproteins (LDL) was studied using as targets mouse ascites hepatoma 22a (MAH) cells which bind LDL through a specific high affinity receptor. Low density lipoprotein binding and uptake by MAH cells decreased after brief treatment of the cells with neuraminidase to partially remove surface sialic acid residues. The LDL uptake capability of the neuraminidasetreated MAH cells was fully restored after incorporation of exogeneous G_M1- and G_D1a-gangliosides into the cell surface. In contrast, free (extracellular) gangliosides inhibited LDL uptake by native MAH cells. This inhibitory effect was seen at ganglioside concentrations corresponding to the ganglioside content of serum and was most pronounced with gangliosides whose sialic acids were linked to a terminal galactose residue (G_M3, G_D1a, G_T1b) but was smaller or absent with gangliosides whose sialic acids were attached to an internal galactose (G_M1, G_M2). The binding of gangliosides to LDL was structure and concentration dependent, saturable and trypsin sensitive. The LDL-ganglioside interaction was further investigated by steady state fluorescence spectroscopy. Changes in the LDL fluorescence polarization were observed with as little as 0.01 M concentrations of the gangliosides. The magnitude and nature of the effect depended on the type of ganglioside. We conclude that the LDL surface possesses sites recognizing specific carbohydrate sequences of glycoconjugates and that changes in the cell surface concentrations of sialic acids significantly modulate the LDL uptake. It is postulated that shedding of gangliosides into the blood stream may be a factor involved in regulation of cholesterol homeostasis.Abbreviations MAH mouse ascites hepatoma 22a - LDL low density lipoprotein - ASM anthrylvinyl-labeled sphingomyelin [N-12-(9-anthryl-trans-dodecanoyl-sphingosine-1-phosphocholine] - RITC rhodamine isothiocyanate. The designation of gangliosides follows the IUPAC-IUB recommendation [1]: G_M3, II³NeuAc-LacCer, II³-N-acetylneuraminosyllactosylceramide - G_M2 II³-NeuAc-GgOse₃Cer, II³-N-acetylneuraminosylgangliotriaosylceramide - G_M1 II³-NeuAc-GgOse₄Cer, II³-N-acetylneuraminosylgangliotetraosylceramide - G_D1a, II³ IV³(NeuAc)₂-GgOse₄Cer, II³, IV³-di(N-acetylneuraminosyl)gangliotetraosylceramide - G_T1b II³(NeuAc)₂, IV³ NeuAc-GgOse₄Cer, II³-di-N-acetylneuraminosyl, IV³-N-acetylneuraminosylgangliotetraosylceramide     

Cell surface receptors for lymphokines. I. The possible role of glycolipids as receptors for macrophage migration inhibitory factor (MIF) and macrophage activation factor (MAF).

Incubation of culture supernatants from concanavalin A-stimulated guinea pig and rat lymphocytes with protein-free preparations of bovine brain gangliosides abolished their macrophage migration inhibitory factor (MIF) and macrophage activation factor (MAF) activity. The identity of the MIF/MAF-binding component(s) present in these glycolipid mixtures has yet to be established, but adsorption experiments using purified preparations of mono- (GM₁, GM₂, and GM₃), di- (GD_1a), and trisialogangliosides (GT₁) were negative. Since these gangliosides account for over 90% of the glycolipid content in brain ganglioside mixtures it appears that the MIF-binding component(s) is present only in very small amounts. Treatment of guinea pig peritoneal macrophages with liposomes containing similar brain gangliosides or water-soluble glycolipids extracted from guinea pig macrophages enhanced their responsiveness to MIF. The enhanced response to MIF of liposome-treated macrophages was abolished by incubation of the treated macrophages with fucose-binding lectins (Lotus agglutinin and Ulex europaeus agglutinin I) before exposure to MIF, suggesting that the MIF-binding component donated by the liposomes may be a fucose-containing glycolipid. The possible role of glycolipids as surface receptors for MIF and MAF is discussed.     

Abnormal ganglioside accumulation in cultured fibroblasts from patients with mucolipidosis IV.

Extracts of cultured skin fibroblasts derived from patients with mucolipidosis IV showed a marked increase and altered distribution of GM₃ and GD₃ gangliosides. GD₃ is elevated 1.5–2 times that of normal whereas GM₃ is elevated to a lesser extent. No abnormalities were found in the neutral glycolipids. These two gangliosides apparently comprise most of the accumulated lipid-like material observed on ultrastructural analysis in this disease.     

Enhancement of cytotoxic and proliferative responses of lymphocytes from melanoma patients by incubation with monoclonal antibodies against ganglioside GD3

Summary Previous studies have shown that monoclonal antibodies (M.Ab) to the ganglioside GD₃ may induce partial remissions in tumour growth in patients with melanoma. In vitro studies demonstrated that M.Abs to GD₃ may also enhance lymphocyte responses to phytohemagglutinin and interleukin 2 (IL2). The present study extended these findings by showing that the IL2-dependent proliferative and cytotoxic response of T cell clones derived from a melanoma patient and a patient with the Vogt-Koyanagi-Harada syndrome were enhanced by pre-incubation of T cells with M.Ab to GD₃. The degree of enhancement increased with the duration of pre-incubation from 2 to 24 h and applied to both T4⁺ and T8⁺ clones. The potentiation of these responses was not specific for M.Abs to GD₃ but was also seen with M.Abs to GD₂ and the T10 structure on T cells but not with M.Abs to the transferrin receptor or an isotype control M.Ab. Incubation of lymphocytes from a melanoma patient with M.Ab to GD₃ during culture with autologous melanoma cells enhanced the proliferative response to the tumour. The expression of IL2 receptors (Tac epitope) on the T cells showed variable enhancement after incubation with M.Ab to GD₃ but the significance of these findings in relation to the mechanism of the enhanced responses is not known. These results suggest that certain M.Abs may stimulate cell-mediated immune responses against tumour cells and that this may provide an additional mode of action of M.Abs against tumours in vivo     

Shedding of Gangliosides by Human Medulloblastoma Cells

Shedding of immunosuppressive gangliosides is an important characteristic of both experimental and human tumors. Using a medulloblastoma cell line, Daoy, with a very high ganglioside expression (141 ± 13 nmol/10⁸cells) and a well-characterized ganglioside complement, we have now studied ganglioside shedding by human brain tumor cells. Shedding of gangliosides, quantified by metabolic radiolabeling, was significant (169 pmol/10⁸cells/h) and was generalized with respect to the major ganglioside carbohydrate structures (G_M2, G_M3, and G_D1a). For each ganglioside, however, shedding was selective for ceramide structures containing shorter fatty acyl chains. Rapid and ceramide-selective shedding was confirmed in two additional human medulloblastoma cell lines, D341 Med and D283 Med (112 and 59 pmol/10⁸cells/h). Significant ganglioside shedding is therefore a common characteristic of human medulloblastoma cells and may influence the biological behavior of this tumor, in view of immunosuppressive and other biological properties of shed gangliosides.     

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.     

Number of Sialic Acid Residues in Ganglioside Headgroup Affects Interactions with Neighboring Lipids

Monolayers of binary mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and asialo-(GA₁), disialo-(GD_1b) and trisialo-(GT_1b) gangliosides were used to determine the effect of ganglioside headgroup charge and geometry on its interactions with the neighboring zwitterionic lipid. Surface pressure versus molecular area isotherm measurements along with concurrent fluorescence microscopy of the monolayers at the air-water interface were complemented with atomic force microscopy imaging of monolayers deposited on solid substrates. Results were used to further develop a proposed geometric packing model that the complementary geometry of DPPC and monosialoganglioside GM₁ headgroups affects their close molecular packing, inducing condensation of the layer at small mol % of ganglioside. For GA₁, GD_1b, and GT_1b, a similar condensing effect, followed by a fluidizing effect is seen that varies with glycosphingolipid concentration, but results do not directly follow from geometric arguments because less DPPC is needed to condense ganglioside molecules with larger cross-sectional areas. The variations in critical packing mole ratios can be explained by global effects of headgroup charge and resultant dipole moments within the monolayer. Atomic force microscopy micrographs further support the model of ganglioside-induced DPPC condensation with condensed domains composed of a striped phase of condensed DPPC and DPPC/ganglioside geometrically packed complexes at low concentrations.     

Exogenous glycosphingolipids suppress growth rate of transformed and untransformed 3T3 mouse cells

Gangliosides added to culture media reduced both the growth rate and saturation density of SV40-virus transformed and untransformed 3T3 cells. Monosialogangliosides were much more effective than disialogangliosides in inhibiting growth rate. These gangliosides caused little or no cell damage or significant morphological alteration of the individual cells. Trisialoganglioside markedly reduced growth rate but in some experiments also caused cell damage and lysis. The isolated carbohydrate moiety of the ganglioside G_Gtet1, the sialo-oligosaccharide galactopyranosyl-N-acetyl-galactosaminyl-(N-acetylneuraminyl)-galactosyl-glucose, did not inhibit growth of SV40 3T3 cells in culture. Ceramide alone was also ineffective as a growth inhibitor. However, the tetrahexosyl ceramide derived from the above ganglioside was equally as effective as the parent compound in retarding growth of SV40 3T3 cells. Similarly, mono-, di- and trihexosyl ceramides were also effective in inhibiting growth of these cells. Gangliosides added to the culture media were rapidly accumulated by cells, apparently at the plasma membrane. The accumulated ganglioside was not degraded by the cells. However, the accumulated ganglioside could be distinguished from gangliosides synthesized in vivo by the lability of the former to neuraminidase.     

Number of Sialic Acid Residues in Ganglioside Headgroup Affects Interactions with Neighboring Lipids

Monolayers of binary mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and asialo-(GA₁), disialo-(GD_1b) and trisialo-(GT_1b) gangliosides were used to determine the effect of ganglioside headgroup charge and geometry on its interactions with the neighboring zwitterionic lipid. Surface pressure versus molecular area isotherm measurements along with concurrent fluorescence microscopy of the monolayers at the air-water interface were complemented with atomic force microscopy imaging of monolayers deposited on solid substrates. Results were used to further develop a proposed geometric packing model that the complementary geometry of DPPC and monosialoganglioside GM₁ headgroups affects their close molecular packing, inducing condensation of the layer at small mol % of ganglioside. For GA₁, GD_1b, and GT_1b, a similar condensing effect, followed by a fluidizing effect is seen that varies with glycosphingolipid concentration, but results do not directly follow from geometric arguments because less DPPC is needed to condense ganglioside molecules with larger cross-sectional areas. The variations in critical packing mole ratios can be explained by global effects of headgroup charge and resultant dipole moments within the monolayer. Atomic force microscopy micrographs further support the model of ganglioside-induced DPPC condensation with condensed domains composed of a striped phase of condensed DPPC and DPPC/ganglioside geometrically packed complexes at low concentrations.     

The alpha-galactosyl derivatives of ganglioside GD(1b) are essential for the organization of lipid rafts in RBL-2H3 mast cells

Gangliosides are complex glycosphingolipids that are important in many biological processes. The present study investigated the role of gangliosides in the organization of lipid rafts in RBL-2H3 mast cells and in the modulation of mast cell degranulation via FcRI. The role of gangliosides was examined using two ganglioside deficient cell lines (B6A4A2III-E5 and B6A4C1III-D1) as well as the parent cell line (RBL-2H3). All three cell lines examined express FcRI, Lyn, Syk and LAT. However, only in RBL-2H3 cells were FcRI, LAT and α-galactosyl derivatives of ganglioside GD_1b mobilized to lipid raft domains following FcRI stimulation. The inhibition of glycosphingolipid synthesis in RBL-2H3 cells also resulted in a decrease in the release of β-hexosaminidase activity after FcRI activation. The two mutant cell lines have a reduced release of β-hexosaminidase activity after FcRI stimulation, but not after exposure to calcium ionophore. These results indicate that the α-galactosyl derivatives of ganglioside GD_1b are important in the initial events of FcRI signaling upstream of Ca²⁺ influx. Since the initial signaling events occur in lipid rafts and in the mutant cell lines the rafts are disorganized, these results also suggest that these gangliosides contribute to the correct assembly of lipid rafts and are essential for mast cell activation via FcRI.