Gangliosides and their cell density-dependent changes in control and chemically transformed C3H/10T1/2 cells

The cloned C3H/10T1/2 mouse embryo cells contained a complex pattern of gangliosides. Two cloned chemical transformants obtained from the C3H/10T1/2 cell line by treatment with 7,12-dimethylbenz(a) anthracene (DMBA-TCL1) and 3-methylcholanthrene (MCA-TCL15) also had complex ganglioside patterns; but the transformants had increased levels of the simplest ganglioside, N-acetylneuraminylgalactosylglucosylceramide (GM₃), and reduced levels of more complex gangliosides. Incorporation of [¹⁴C]glucosamine into gangliosides, as cell-to-cell contact increased in C3H/10T1/2 cells, showed that GM₃ synthesis was decreased and that the synthesis of the more complex ganglioside N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide (GD_1a) was increased. In the two transformants the percentage each individual ganglioside was of total labeled gangliosides was only slightly altered with changing cell density. Turnover of [¹⁴C]glucosamine-labeled gangliosides, as cell density increased, was approximately equal in C3H/10T1/2 cells and MCA-TCL15 cells, but more rapid in the DMBA-TCL1 cells. Most individual gangliosides turned over at about the same rate in the respective cell lines. However, GD_1a increased slightly as a percentage of total labeled gangliosides with increasing cell density in both C3H/10T1/2 cells and transformed cells. The labeling data indicated that the majority of GD_1a synthesis was de novo and only a small part occurred by transfer of sialyl or glycosyl residues to simpler gangliosides or catabolism of more complex gangliosides already present in the outer membrane. Exogenous complex gangliosides added to the medium were more effective inhibitors of DMBA-TCL1 cell growth than of C3H/10T1/2 cell growth. Furthermore, gangliosides added to exponentially growing C3H/10T1/2 and DMBA-TCL1 cells caused both cell lines to incorporate a greater percentage of [¹⁴C]glucosamine into gangliosides more complex than GM₃.     

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.     

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.     

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)     

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.     

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.     

Expression of Mouse Sialic Acid on Gangliosides of a Human Glioma Grown as a Xenograft in SCID Mice

Ganglioside sialic acid content was examined in the U87-MG human glioma grown as cultured cells and as a xenograft in severe combined immunodeficiency (SCID) mice. The cultured cells and the xenograft possessed N-glycolylneuraminic acid (NeuGc)-containing gangliosides, despite the inability of human cells to synthesize NeuGc. Human cells express only N-acetylneuraminic acid (NeuAc)-containing gangliosides, whereas mouse cells express both NeuAc- and NeuGc-containing gangliosides. Small amounts of NeuGc ganglioside sialic acid (2-3% of total ganglioside sialic acid) were detected in the cultured cells, whereas large amounts (66% of total ganglioside sialic acid) were detected in the xenograft. The NeuGc in gangliosides of the cultured cells was derived from gangliosides in the fetal bovine serum of the culture medium, whereas that in the U87-MG xenograft was derived from gangliosides of the SCID host. The chromatographic distribution of U87-MG gangliosides differed markedly between the in vitro and in vivo growth environments. The neutral glycosphingolipids in the U87-MG cells consisted largely of glucosylceramide, galactosylceramide, and lactosylceramide, and their distribution also differed in the two growth environments. Asialo-GM1 (Gg4Cer) was not present in the cultured tumor cells but was expressed in the xenograft, suggesting an origin from infiltrating cells (macrophages) from the SCID host. The infiltration of mouse host cells and the expression of mouse sialic acid on human tumor cell glycoconjugates may alter the biochemical and immunogenic properties of xenografts.     

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.     

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.     

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).     

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.     

Cell density-dependent changes of glycosphingolipid biosynthesis in cultured human skin fibroblasts

In this study, the glycosphingolipid biosynthesis was investigated in the sparse and the confluent cell populations of cultured human skin fibroblasts.The human skin fibroblast cell populations were metabolically pulse labeled with ¹⁴C-galactose (48 h). The amounts of ¹⁴C-radioactivity (cpm) incorporated into extracted and purified total cellular glycosphingolipid fractions were counted by -scintillation and the individual glycosphingolipid species were separated by high performance thin layer chromatography and visualized by autoradiography. The relative labeling (%) of individual newly synthesized glycosphingolipid species was detected by densitometric scanning of autoradiographic glycosphingolipid patterns.The incorporation of ¹⁴C-label into total glycosphingolipids per cell increased significantly as the cell-density increased, referring to five fold higher rate of glycosphingolipid biosynthesis de novo in cells at confluency vs. sparse populations. The total newly synthesized glycosphingolipid pattern (100%) of sparse cell populations showed a significant predominance of the gangliosides (70%) over the neutral glycosphingolipids (30%), with ganglioside GM2 as the major species followed by monohexosyl-ceramide. Oppositely, the newly synthesized neutral glycosphingolipids (67%) predominated over the gangliosides (33%) in cells at confluency (contact inhibition). Cells reaching confluency were characterized by: (a) a dramatic increase of absolute amount of all newly synthesized neutral glycosphingolipid species, particularly the most abundant monohexosyl-ceramide and trihexosyl-ceramide, but also of the ganglioside GM3; (b) a drastic decrease of absolute amount of newly synthesized ganglioside GM2. The specific shift in newly synthesized glycosphingolipid pattern in cells reaching confluency suggests a down-regulation of biosynthetic pathway primarily at the level of N-acetylgalactosaminyl-transferase. A possible involvement of glycosphingolipids in cell density-dependent regulation of cell growth through establishment of the direct intermolecular intermembrane interactions is discussed.     

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.     

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.     

Glycosphingolipids in detergent-insoluble substrate attachment matrix (DISAM) prepared from substrate attachment material (SAM) : Their possible role in regulating cell adhesion

The glycosphingolipids isolated from the detergent-insoluble material (DIM) of whole cells as well as from a similar detergent-insoluble substrate attachment matrix (DISAM) have been investigated in comparison with the glycosphingolipids of whole cells. The proportion of glycolipids in the total lipid extract was enriched in the DISAM as well as DIM fractions as compared to whole cells. The ratio of ganglioside (GM₃) to neutral glycolipids was also higher in the DISAM fractions than in whole cells. The radioactivity incorporated into DISAM glycolipids of BHK cells, metabolically labeled with radioactive glucosamine, was greater in confluent cells than in sparsely growing cells; however, label incorporation into glycolipids of the DISAM fraction of BHKpy cells was 2–3-fold higher than that of confluent BHK cells, although the chemical quantity of GM₃ in whole cells was much lower in BHKpy cells than in BHK cells. In order to confirm the enhanced label in DISAM glycolipids of BHKpy cells by other procedures, the labeled cells were detached by EGTA, washed, and reattached on plates. The amount of label in DISAM glycolipids of the reattached matrix of BHKpy cells was much higher than that of BHK cells.Cell spreading and cell attachment on plastic plate were inhibited by inclusion of GM₃ in the medium. These data suggest that: (i) glycolipids, particularly GM₃, at the cell attachment site have different metabolic activity from those of whole cells; the label in glycolipids goes preferentially into cell attachment sites, and may have some functional role in regulating cell attachment of BHK cells; (ii) metabolic activity and turnover of GM₃ in cell attachment sites of confluent cells are higher than actively growing cells, yet those of transformed cells are much higher than any state of non-transformed cells.     

Gangliosides in the human glioma cell line U-118 MG grown in culture or as xenografts in nude rats

This study was undertaken to characterize gangliosides in the human glioma cell line U-118 MG. The cell line was grown both in cell culture and as xenografts in nude rats. A common finding in both culture and xenograft cells was the high proportion of the lactoseries ganglioside 3'-LM1, approximately one third of the total ganglioside sialic acid. Otherwise, there were marked differences between the two cell sources. The cells grown in culture had a more simple ganglioside pattern than those grown in xenografts. In the latter instance, more complex gangliosides of the lactoseries, including 3'8'-LD1, sialyllactonorhexaosylceramide and a branched structure with two terminal NeuAc alpha 2-3Gal beta 1- 4GlcNAc chains, and the gangliotetraose series were found. Another marked difference involved GM2, which in the cultured cells was a major fraction, indicating that the synthesis of the gangliotetraose series gangliosides in the former stopped at the level of GM2. These results show that the ganglioside composition of a glioma cell line is strongly influenced by environmental factors.     

Effects of exogenous gangliosides on intracellular Ca2+ mobilization and functional responses in human platelets

Gangliosides, highly expressed in the outer leaflet of plasmamembranes, mediate a variety of biological processes, includingcell-cell and cell-matrix interactions. We examined the effectsof exogenous gangliosides on intracellular Ca²⁺ mobilizationand functional responses in human platelets. Gangliosides (GM₃and GM₁) induced rapid and reversible elevation of intracellularCa²⁺ in fura2-loaded platelets in a concentration-dependentmanner. The Ca²⁺mobilizing effect of gangliosides was not mimickedby de-N-acetyl-GM₃, lactosylceramide, or free sialic acid, suggestingthat structural integrity as ganglioside is essential for thiseffect. GM₃ and GM₁ also induced platelet shape change by themselvesand elicited aggregation in combination with epinephrine. Ourobservations suggest the involvement of ganglioside-activatedplatelets in atherosclerosis, in view of the high observed gangliosidelevels in atherosclerotic lesions of human aorta. de-N-acetyl-GM₃ ganglioside GM₃ intracellular Ca²⁺ mobilization platelet activation