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.     

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.     

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.     

Dopamine release from nerve endings induced by polysialogangliosides

Polysialogangliosides but not monosialoganlioside or a neutral glycosphingolipid induce release of [³H] -dopamine from synaptosomes in presence of Ca⁺⁺, presumably by exocytosis. This effect is discussed in relation to the ability of polysialogangliosides to induce membrane fusion in chicken erythrocytes and to their behaviour in lipid monolayers. It is suggested that characteristic interactions with phosphatidylcholine involving decreases of surface potential are participating in the polysialoganglioside-induced neurotransmitter release.     

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.     

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.     

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.     

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.     

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.     

Endo-beta-galactosidase of Escherichia freundii. Hydrolysis of pig colonic mucin and milk oligosaccharides by endoglycosidic action.

The purified keratansulfate degrading enzyme from $\text{Eschericia freundii}$ could hydrolyze desialyzed pig colonic mucin and milk oligosaccharides. Desialyzed pig colonic mucin was digested to produce GlcNAcβ(1→3)Gal, GlcNAc-6Sβ(1→3)Gal and resistant polymer. Lacto-N-tetraose and lacto-N-tetraitol were hydrolyzed endoglycosidically to release glucose and sorbitol, respectively. Therefore, this enzyme was found to be an endo-β-galactosidase of rather wide specificity.     

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.     

Isolation and identification of a fucose-containing ganglioside from bovine thyroid gland

Bovine thyroid glands are known to contain a complex array of gangliosides. One of the predominant gangliosides was isolated and analyzed by gas-liquid chromatography and mass spectrometry. The carbohydrate composition was fucose, N-acetylneuraminic acid, galactose, N-acetylgalactosamine, and glucose in molar ratios of 1:1:2:1:1. The structure of the ganglioside was identified as:

     

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)     

Biosynthesis of yeast mannan : Diversity of mannosyltransferases in the mannan-synthesizing enzyme system from yeast

1. A microsomal enzyme preparation from the yeast Saccharomyces cerevisiae catalyzes the transfer of mannosyl units from GDPmannose to mannose and a number of mannose-containing oligosaccharides and glycosides whereby different glycosidic bonds are formed.2. Of the compounds tested besides mannose, only those containing an α-linked mannosyl unit at the nonreducing position of their moleculae were effective as receptors. Monodeoxyanalogues of mannose as well as α-mannose phosphates did not serve as receptors in the above reaction.3. The structure of the product formed with mannose as receptor was determined to be O-α-D-mannosyl-(1→2)-mannose; with αMan(1→Man(1→6)mannose as the acceptor, the product was αMan(1→6)αMan(1→6)mannose and with αMan-(1→2)mannose the product was tentatively characterized as a mixture of αMan-(1→3)αMan(1→2)mannose and αMan(1→2)αMan(1→2)mannose.4. The enzymes catalyzing the formation of different types of glycosidic bonds differed in their acceptor specificity, pH-activity curves and rates of heat denaturation.5. Radioactive disaccharids were unable to enter the mannan protein molecule in the cell-free system while free radioactive mannose did incorporate into polysacchride to a minor extent under the same conditions.     

Accumulation of short DNA fragments in hydroxyurea treated mouse L-cells

Treatment of L-cells with hydroxyurea markedly inhibits the incorporation of [³H]thymidine into DNA. The ³H incorporation that persists during hydroxyurea inhibition is largely into 7S DNA chains. The labelled fragments can be chased into higher MW DNA, suggesting that they are intermediates in the replication process. This interpretation concurs with that of earlier reports which describe a similar effect of hydroxyurea on the replication of viral DNA.     

Synthesis and stability assay of 4-methylumbelliferyl (1→3)-β-D-pentaglucoside

The synthesis and stability of 4-methylumbelliferyl (1 → 3)-β-D-pentaglucoside 3 are described. The (1 → 3)-β-D-glucan isolated from the cell walls of Saccharomyces cerevisiae was recovered from the aqueous medium as water-insoluble particles by the spray drying (GS) method. The acid-solubilized (1 → 3)-β-D-oligoglucosides were prepared by partial acid hydrolysis of glucan. The peracetylated (1 → 3)-β-D-pentaglucoside 1 was obtained by isolation of peracetylated (1 → 3)-β-D-oligoglucoside mixture. The peracetylated 4-methylumbelliferyl (1 → 3)-β-D-pentaglucoside 2 was synthesized by treating compound 1 with the 4-methylumbelliferone and a Lewis acid (SnCl₄) catalyst. NaOMe in dry methanol was used for the deacetylation of the blocked derivative, to give the target compound 3 in an overall yield of 35%. Activity assays with β-glucosidase indicated that compound 3 was much more stable than the corresponding pentasaccharide.     

Monosialoganglioside GM1 increases survival of thymocytes

Gangliosides are normal constituents of the plasma membrane. Exogenous gangliosides can be incorporated into the membrane and extensive research in nervous tissue has demonstrated a beneficial effect of gangliosides on the functional recovery of lesioned neurons and protection against neurotoxins. This paper shows that the effect of gangliosides is not restricted to neurons. The monosialoganglioside GM₁ efficiently increases the survival of thymocytes and protects them against both the lytic effect of the glucocorticoid prednisolone and the effect of a thymocytotoxic serum. The protective effect of GM₁ was achieved bothin vitro andin vivo.     

Purification and Partial Characterization of an Endo-(1→3, 1→4)-β-glucanase from Rice,Oryza sativa L.

(1→3, 1→4)-β-Glucanase (EC 3.2.1.73), with a molecular weight of 34, 000 and an isoelectric point of 4.9, was purified to homogeneity from extracts of fresh rice bran. The enzyme specifically hydrolyzed (1→3, 1→4)-β-glucans such as barley β-glucan and lichenans, but laminarins and CM-cellulose were not substrates. Endproduct analysis using barley β-glucan as the substrate suggested that the enzyme is an endo-type (1→3, 1→4)-β-glucanase.