Soluble gangliosides in cultured neurotumor cells

Abstract: The biosynthesis and degradation of glycosphingolipids were studied in cytosolic and membrane fractions obtained from rat glioma C6 cells. Both pools had a similar composition of neutral glycosphingolipids but the soluble pool contained only a few percent of the total. The major ganglioside in C6 cells was GM3, of which only 2% was soluble. Whereas the bulk of the membrane GM3 was accessible to surface labeling procedures, the soluble GM3 was not. Mouse neuroblastoma N18 cells also contained small amounts of cytoplasmic gangliosides corresponding to GM3, GM2, GM1, and GDla. When C6 cells were incubated with medium containing [³H]galactose at 37°C, the specific activity of soluble GM3 initially increased more rapidly than that of membrane GM3; by 4 h, the specific activities in both pools became equal. Total incorporation into the membrane pool, however, was always several-fold greater even at the shortest incubation times examined. The labeling pattern of neutral glycosphingolipids in both soluble and membrane fractions indicated the existence of a precursor-product relationship between glucosylceramide and other glycosphingolipids. When labeled cells were transferred to nonradioactive medium, glucosylceramide disappeared the most rapidly, with a 50% loss within <6 h. The turnover rates of other glycosphingolipids were much slower. Although cytosolic GM3 was degraded more rapidly (t_1/2= 26 h) than membrane-bound GM3 (t_1/2= 44 h), its turnover rate was much slower than the time required for transport of GM3 to the cell surface (20–30 min). Our results are consistent with the existence of a small intracellular pool of soluble gangliosides and neutral glycosphingolipids that is stable and independent of the main membrane-bound pool. Although the role of these cytosolic glycolipids is unknown, they do not appear to represent a transport pool between the site of synthesis and the plasma membrane.     

Hormonal effects on the development changes of mouse small intestinal glycolipids

The composition of intestinal glycosphingolipids during normal and hormone-perturbed development was investigated. The concentrations of glycosphingolipids of mouse small intestine were affected by the injection of thyroxine or cortisone during suckling and weaning periods. GDla was reduced by the hormonal treatment among major gangliosides, GM3, GM1 and GD1a, of mouse small intestine during the suckling period. In contrast, asialo GM1 was precociously produced by the treatment, which scarcely found in control suckling mouse small intestine. The results showed that these hormones were related to developmental alteration of small-intestinal glycolipids.     

Binding specificities of the lectins from Helix pomatia, soybean and peanut against different glycosphingolipids in liposome membranes

The binding specificities of the lectins from Helix pomatia, soybean and peanut against glycosphingolipids containing β-linked terminal D-galactose and N-acetyl-D-galactosamine and the role of sialic acid as a modulator of the binding specificity were investigated. The test system used consisted of liposomes containing the glycosphingolipids and lectins coupled to gel columns. Of the investigated glycosphingolipids only ganglioside GM2 bound to soybean agglutinin while Helix pomatia agglutinin was found to bind only GA2. Peanut agglutinin showed good affinity both for ganglioside GM1 and its asialoderivative GA1.     

The glycosphingolipid receptor for Vibrio trachuri in the red sea bream intestine is a GM4 ganglioside which contains 2-hydroxy fatty acids

Three major glycosphingolipids (tentatively designated IGL-1, 2, and 3) were isolated from the intestine of red sea bream (Pagrus major) and were subjected to a TLC-overlay assay with (35)S-labeled Vibrio trachuri which causes vibriosis of fish. The bacteria adhered to IGL-2, which was determined to be a GM4 ganglioside (NeuAcalpha2-3Galbeta1-ceramide). The fatty acid portion of IGL-2 was composed of 2-hydroxy C22:0, C24:0, and C24:1, in addition to the non-hydroxy C16:0 and C18:0, while the sphingoid base was composed exclusively of sphingenine (d18:1). Among glycosphingolipids tested, V. trachuri adhered to GM4 the most strongly followed by adherence to GM3 and GalCer, but the bacteria did not adhere to GM1a, GM2, LacCer, or GlcCer. V. trachuri was found to aggregate with the erythrocytes coated with GM4, but not with those coated with GM1a or GM2, thus indicating that specific adhesion occurs on intact cells. Interestingly, the dynamics for adhesion of V. trachuri to glycosphingolipids was defined by the structure of not only the sugar moiety but also the ceramide moiety, since the bacteria adhered to GM4 which contained 2-hydroxy fatty acids much more strongly than to that which contained non-hydroxy fatty acids.     

Molecular parameters and physical state of neutral glycosphingolipids and gangliosides in monolayers at different temperatures

The effect of temperature on the behaviour of four different gangliosides (GM3, GM1, GD1a and GT1b), sulphatide, ceramide (Cer) and three neutral glycosphingolipids (GalCer, Gg3Cer, Gg4Cer) was investigated in monolayers at the air-NaCl (145 mM) interface. GM1, GD1a and GT1b are liquid-expanded in the range of temperatures studied (5-65 degrees C). GM3, sulphatide, Cer and neutral glycosphingolipids show isothermal liquid-expanded----liquid-condensed transitions. The collapse pressure of ganglioside monolayers decreases with temperature, whereas neutral glycosphingolipids may show some maximum values at particular temperatures. The reduction of the molecular area of liquid-expanded glycosphingolipids under compression occurs with a favorable positive entropy change and an unfavorable negative enthalpy. By contrast, the compression of interfaces with a two-dimensional phase transition occurs with an unfavorable entropy but a favorable enthalpy change. From the temperature dependence of the surface pressure at which the two-dimensional phase transition takes place, a minimal temperature above which the isotherm becomes totally liquid-expanded can be obtained. For the different glycosphingolipids this temperature decreases in the order Cer greater than GalCer greater than sulphatide greater than Gg3Cer greater than Gg4Cer greater than GM3 greater than GM1 greater than GD1a greater than GT1b. This sequence is similar to that found for the calorimetrically determined transition temperatures (cf. Maggio, B., Ariga, T., Sturtevant, J.M. and Yu, R.K. (1985) Biochemistry 24, 1084-1092).     

Characterization of the glycosphingolipids of pig cortical bone and cartilage

Neutral glycosphingolipids and gangliosides were extracted from pig cortical bone and cartilage. To ensure the completeness of extraction, the cortical bone was demineralized and reextracted. Globotriaosylceramide and globoside were noted to be present at high content in the cortical bone. It contained glucosylceramide, lactosylceramide, globotriaosylceramide and globoside as neutral glycosphingolipids at a ratio of 1:0.7:3.1:2.7. In articular cartilage, the ratio was 1:0.7:0.4:0.8. GM3 and GD3 were the major gangliosides in both these tissues. GM3, GM1, GD3, GD1 and GT1 were present at ratios of 1:0.9:0.9:0.1:0.1 in the cortical bone and 1:0:1.2:0.06:0.02 in the cartilage. Neutral glycosphingolipids could be extracted from the cortical bone without the need for demineralization, while most of the gangliosides were extracted after this treatment, implying the occurrence of interactions between gangliosides and minerals in the bone.     

Localization of neutral and acidic glycosphingolipids in rat lens

Rat lens was found to contain several neutral and acidic glycosphingolipidsin lens epithelia, cortex and nucleus, and showed developmentalchanges in their content and localization. TLC-immunostainingof gangliosides revealed the enrichment of some ganglio-seriesgangliosides (GM3, GM1, GD3 and GD1b) in lens epithelia andthe presence of GM3 and GD3 in the lens nucleus. Immunohistochemicalstudies confirmed the distribution of GM3 and GM1 in anteriorlens epithelial cells and the cortex, with expression decreasingtoward the lens nucleus. Immunoreaction to GD3 was more intensein the lens nucleus than in epithelial cells. In contrast, theexpression of neolacto-series glycosphingolipids was restrictedto the lens nucleus. In order to investigate the pathologicalchanges of glycosphingolipids in cataract, galactose-inducedcataractous lenses were examined. However, no significant changeswere observed in the content and composition of glycosphingolipids.In addition, Lewis^x epitopes found in human cataractous lenseswere not detected in the cataractous lenses of galactosaemicrats and hereditary cataractous Emory mice. cataract gangliosides glcosphingolipids Lewis^x rat lens     

Glycosphingolipids of human aorta

The structures of the main gangliosides of human aorta (intima and media) were elucidated. The main component (67%) was identified as N-acetylneuraminosyl-lactosylceramide (ganglioside GM3). The aorta tissue contained also gangliosides GM1, GD3, GD1a, and GT1. All sialic acid residues in gangliosides were present as N-acetyl-neuraminosyl derivatives. Among neutral glycosphingolipids of human aorta, the main components were identified as glucosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylceramide. The preliminary data suggest that the composition of the investigated glycosphingolipids in tissue might vary upon atherosclerosis lesions of aorta.     

Enhancement of hydrolytic activity of sphingolipid ceramide N-deacylase in the aqueous-organic biphasic system

Lysoglycosphingolipids were produced from glycosphingolipids by using sphingolipid ceramide N-deacylase, which cleaves the N-acyl linkage between fatty acids and sphingosine bases in various glycosphingolipids. The enzyme reaction was done in a biphasic media prepared with water;-immiscible organic solvent and aqueous buffer solution containing the enzyme. We investigated the effects of organic solvents and detergents on lysoglycosphingolipid production in the biphasic system. Among the organic solvents tested, n-butylbenzene, cumene, cyclodecane, cyclohexane, n-decane, diisopropylether, n-heptadecane, and methylcyclohexane promoted hydrolysis of GM1, whereas benzene, chloroform, ethyl acetate, and toluene inhibited GM1 hydrolysis. Hydrolysis of asialo GM1, GD1a, GalCer, and sulfatide was also enhanced by the addition of n-decane. The hydrolytic activity of the enzyme was enhanced by the addition of 0.8% sodium taurodeoxycholate or sodium cholate to the aqueous phase. The most effective hydrolysis of various glycosphingolipids by the enzyme was thus obtained in the aqueous-n-decane biphasic system containing 0.8% sodium taurodeoxycholate. Under this condition, the fatty acids released from GM1 by the action of the enzyme were trapped and diffused into the organic phase, while lysoGM1 remained in the aqueous phase.Thus the almost complete hydrolysis of GM1 was achieved using the biphasic system, while at most 70% of hydrolysis was obtained using normal aqueous media possibly due to the inhibition of hydrolysis reaction by accumulation of fatty acids in the reaction mixture.     

Comparison of the glycolipid-binding specificities of cholera toxin and porcineEscherichia coli heat-labile enterotoxin: identification of a receptor-active non-ganglioside glycolipid for the heat-labile toxin in infant rabbit small intestine

The binding specificities of cholera toxin andEscherichia coli heat-labile enterotoxin were investigated by binding of¹²⁵I-labelled toxins to reference glycosphingolipids separated on thin-layer chromatograms and coated in microtitre wells. The binding of cholera toxin was restricted to the GM1 ganglioside. The heat-labile toxin showed the highest affinity for GM1 but also bound, though less strongly, to the GM2, GD2 and GD1b gangliosides and to the non-acid glycosphingolipids gangliotetraosylceramide and lactoneotetraosylceramide. The infant rabbit small intestine, a model system for diarrhoea induced by the toxins, was shown to contain two receptor-active glycosphingolipids for the heat-labile toxin, GM1 ganglioside and lactoneotetraosylceramide, whereas only the GM1 ganglioside was receptor-active for cholera toxin. Preliminary evidence was obtained, indicating that epithelial cells of human small intestine also contain lactoneotetraosylceramide and similar sequences. By computer-based molecular modelling, lactoneotetraosylceramide was docked into the active site of the heat-labile toxin, using the known crystal structure of the toxin in complex with lactose. Interactions which may explain the relatively high toxin affinity for this receptor were found.Abbreviations CT cholera toxin - CT-B B-subunits of cholera toxin - LT Escherichia coli heat-labile enterotoxin - hLT humanEscherichia coli heat-labile enterotoxin - pLT porcineEscherichia coli heat-labile enterotoxin - EI electron ionization     

Role of Host Glycosphingolipids on <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> Adhesion

Binding of yeast forms to human lung fibroblast cultures was analyzed, aiming to better understand the initial steps of Paracoccidioides brasiliensis infection in humans. A significant P. brasiliensis adhesion was observed either to fibroblasts or to their Triton X-100 insoluble fraction, which contains extracellular matrix and membrane microdomains enriched in glycosphingolipids. Since human lung fibroblasts express at cell-surface gangliosides, such as GM1, GM2, and GM3, the role of these glycosphingolipids on P. brasiliensis adhesion was analyzed by different procedures. Anti-GM3 monoclonal antibody or cholera toxin subunit B (which binds specifically to GM1) reduced significantly fungal adhesion to fibroblast cells, by 35% and 33%, respectively. Direct binding of GM1 to yeast forms of P. brasiliensis was confirmed using cholera toxin subunit B conjugated to AlexaFluor^®488. It was also demonstrated that P. brasiliensis binds to polystyrene plates coated with galactosylceramide, lactosylceramide, trihexosylceramide, GD3, GM1, GM3, and GD1a, suggesting that glycosphingolipids presenting residues of beta-galactose or neuraminic acid at non-reducing end may act as adhesion molecules for P. brasiliensis. Conversely, no binding was detected when plates were adsorbed with glycosphingolipids that contain terminal residue of beta-N-acetylgalactosamine, such as globoside (Gb4), GM2, and asialo-GM2. In human fibroblast (WI-38 cells), GM3 and GM1 are associated with membrane rafts, which remain insoluble after treatment with Triton X-100 at 4°C. Taken together, these results strongly suggest that lung fibroblast gangliosides, GM3 and GM1, are involved in binding and/or infection by P. brasiliensis.     

Modulation of phospholipase A2 activity by neutral and anionic glycosphingolipids in monolayers.

The effect of neutral (galactocerebroside and asialo-ganglioside GM1) or anionic (sulphatide and gangliosides GM1, GD1a and GT1b) glycosphingolipids on the activity of phospholipase A2 from pig pancreas was studied in mixed monolayers of dilauroyl phosphatidylcholine with the glycosphingolipids in different molar fractions at various constant surface pressures. The activity of the enzyme depends on the proportion and type of glycosphingolipid in the interface. Sulphatide activates the enzyme at all proportions, whereas galactocerebroside shows inhibition or activation depending on its proportion in the film. Asialo-ganglioside GM1 and gangliosides GM1, GD1a and GT1b can strongly inhibit the enzyme at relatively low molar fractions in the film in the following order: asialo-ganglioside GM1 less than ganglioside GM1 less than ganglioside GT1b less than ganglioside GD1a. The changes of activity are not due to a direct action of the lipids on the active centre or interfacial recognition region of the enzyme.     

High-sensitivity analysis of glycosphingolipids by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight imaging mass spectrometry on transfer membranes

Glycosphingolipids are ubiquitous constituents of cells. Yet there is still room for improvement in the techniques for analyzing glycosphingolipids. Here we report our highly sensitive and convenient analytical technology with imaging mass spectrometry for detailed structural analysis of glycosphingolipids. We were able to determine detailed ceramide structures; i.e., both the sphingosine base and fatty acid, by MS/MS/MS analysis on a PVDF membrane with 10 pmol of GM1, with which only faint bands were visible by primuline staining. The limit of detection was approximately 1 pmol of GM1, which is lower than the value in the conventional reports (10 pmol).     

Characterization of acidic glycolipids in porcine plasma

Acidic glycosphingolipids including two sulfatides and five gangliosides were isolated from porcine plasma. They were characterized by NMR spectrometry as galactosylceramide-I³-sulfate and lactosylceramide-II³-sulfate, gangliosides GM3, GD3, GM1, GD1a and fucosyl GM1.     

Glycosphingolipids of human plasma

A number of glycosphingolipids, including 10 gangliosides, not previously identified in human plasma have been characterized. The plasma contains 2 micrograms of lipid-bound sialic acid/ml plasma and 54% of the gangliosides are monosialo, 30% disialo, 10% trisialo, and 6% tetrasialo. Individual glycosphingolipids were purified by high-performance liquid chromatography and thin-layer chromatography, and were characterized on the basis of their chromatographic mobility, carbohydrate composition, hydrolysis by glycosidases, methylation analysis, and immunostaining with anti-glycosphingolipid antibodies. The monosialogangliosides were identified as GM3, GM2, sialosyl(2-3)- and sialosyl(2-6)lactoneotetraosylceramides, sialosyllacto-N-nor-hexaosylceramide, and sialosyllacto-N-isooctaosylceramide. The major gangliosides in the polysialo fractions contained a ganglio-N-tetraose backbone and were identified as GD3, GD1a, GD1b, and GQ1b. The most abundant neutral glycosphingolipids were glucosyl, lactosyl, globotriaosyl, globotetraosyl and lactoneotetraosylceramides. The other neutral glycosphingolipids, tentatively identified by immunostaining with monoclonal antibodies, contained H1, Lea, Leb, and lacto-N-fucopentose III (X hapten) structures.     

Hyperacidification of trans-Golgi network and endo/lysosomes in melanocytes by glucosylceramide-dependent V-ATPase activity

Sphingolipids are considered to play a key role in protein sorting and membrane trafficking. In melanocytic cells, sorting of lysosomal and melanosomal proteins requires the sphingolipid glucosylceramide (GlcCer). This sorting information is located in the lumenal domain of melanosomal proteins. We found that two processes dependent on lumenal pH, protein sialylation and lysosomal acid lipase (LAL) activity were aberrant in GM95 melanocyte cells, which do not produce glycosphingolipids. Using fluorescence lifetime imaging microscopy (FLIM), we found that the lumenal pH in the trans-Golgi network and lysosomes of wild-type melanocyte MEB4 cells are >1 pH unit lower than GM95 cells and fibroblasts. In addition to the lower pH found in vivo, the in vitro activity of the proton pump, the vacuolar-type H(+) -translocating ATPase (V-ATPase), was twofold higher in MEB4 compared to GM95 cells. The apparent K(i) for inhibition of the V-ATPase by concanamycin A and archazolid A, which share a common binding site on the c-ring, was lower in glycosphingolipid-deficient GM95 cells. No difference between the MEB4 and GM95 cells was found for the V-ATPase inhibitors apicularen A and salicylihalimide. We conclude that hyperacidification in MEB4 cells requires glycosphingolipids and propose that low pH is necessary for protein sorting and melanosome biogenesis. Furthermore, we suggest that glycosphingolipids are indirectly involved in protein sorting and melanosome biogenesis by stimulating the proton pump, possibly through binding of GlcCer. These experiments establish, for the first time, a link between pH, glycosphingolipids and melanosome biogenesis in melanocytic MEB4 cells, to suggest a role for glycosphingolipids in hyperacidification in melanocytes.     

Glycosphingolipid specificity of the human sulfatide activator protein

The interaction of the sulfatide activator protein with different glycosphingolipids have been studied in detail. The following findings were made. 1. The sulfatide activator protein forms water-soluble complexes with sulfatides [Fischer, G. and Jatzkewitz, H. (1977) Hoppe-Seyler's Z. Physiol. Chem. 356, 6588-6591] and various other glycospingolipids. 2. In the absence of degrading enzymes the activator protein acts in vitro as a glycosphingolipid transfer protein, transporting glycosphingolipids from donor to acceptor liposomes. Lipids having less than three hexoses, e.g. galactosylceramide, sulfatide and ganglioside GM3 were transferred at very slow rates, whereas complex lipids such as gangliosides GM2, GM1 and GD1a were transferred much faster than the former. The transfer rate increased with increasing length of the carbohydrate chain of the lipid molecules. 3. Both the acyl residue in the ceramide moiety and the nature of the carbohydrate chain are significant for recognition of the glycosphingolipids by the sulfatide activator protein. Apparently, both residues serve as an anchor and the longer they are the better they are recognized by the protein. 4. In the absence of activator protein, degradation rates of sulfatide derivatives by arylsulfatase A, and of ganglioside GM1 derivatives by beta-galactosidase, increase with decreasing length of acyl residues in their hydrophobic ceramide moiety. Addition of activator protein stimulates the degradation of only those GM1 and sulfatide derivatives that have long-chain fatty acids in their hydrophobic ceramide anchor.     

Mast cell heterogeneity. Differential synthesis and expression of glycosphingolipids by mouse serosal mast cells as compared to IL-3-dependent bone marrow culture-derived mast cells before or after coculture with 3T3 fibroblasts

The synthesis and intracellular expression of glycosphingolipids by mouse serosal mast cells (SMC) have been characterized by radiolabeling and TLC and by immunodetection in situ. Chromatographic analysis of purified glycosphingolipids from SMC intrinsically labeled with [14C]galactose and [14C]glucosamine hydrochloride revealed the predominant synthesis of only the simplest neutral glycosphingolipid and ganglioside, glucosylceramide and ganglioside GM3, respectively. Intracellular indirect immunofluorescence staining of permeabilized SMC demonstrated the absence of the more complex neutral glycosphingolipids lactosylceramide, globotriosylceramide, globotetraosylceramide, and globopentaosylceramide, the absence of ganglioside GM1, and the presence of ganglioside GM3. By contrast, permeabilized mouse IL-3-dependent bone marrow culture-derived mast cells (BMMC) and mast cells recovered after 21 days of coculture of BMMC with mouse 3T3 fibroblasts expressed lactosylceramide, globotriosylceramide, globotetraosylceramide, ganglioside GM1, and ganglioside GM3, but not globopentaosylceramide intracellularly as determined by immunofluorescence. The findings indicate a loss of biosynthetic capacity and epitope maintenance for glycosphingolipids with in vivo differentiation of SMC from IL-3-dependent BMMC progenitors. Thus, although mast cells derived after coculture of these progenitors for 21 days with fibroblasts assume multiple SMC-like properties in terms of their histochemical staining and their secretory granule proteoglycan and neutral protease constituents, they do not lose the ability to express complex glycosphingolipids. The finding that glycosphingolipid composition does not change coordinately with other secretory granule markers defines a new stage of mouse mast cell development between the BMMC and SMC and provides evidence that mast cell development is more complex than previously appreciated.     

A unique glycosphingolipid-splitting enzyme (ceramide-glycanase from leech) cleaves the linkage between the oligosaccharide and the ceramide

A novel type of enzyme which hydrolyzes the linkage between the ceramide and the sugar chain in various glycosphingolipids has been found in the leech, Hirudo medicinalis. This enzyme releases the intact oligosaccharide from LacCer, GbOse3Cer, GbOse4Cer, GbOse5Cer, nLcOse4Cer, GM3, GM2, GM1, GD1a and GT1 with the concurrent release of ceramides. By using tritium-labeled GM1 as substrate we found the optimum pH of this enzyme to be between pH 4 and 5. Since the enzyme cleaves the linkage between the ceramide and the sugar chain in various glycosphingolipids with no apparent preference toward the sugar chain, we propose to call this enzyme ceramide-glycanase.     

The glycosphingolipid composition of the human hepatoma cell line,Hep-G2

The origin of plasma glycosphingolipids in normal individuals and the mechanisms by which tumor-associated glycosphingolipid antigens enter the plasma in patients with cancer are largely unknown. The Hep-G2 human hepatoma cell line retains many of the characteristics of differentiated hepatocytes including the ability to synthesize and secrete lipoproteins. Preliminary results indicated that newly synthesized Hep-G2 cell glycosphingolipids are coupled to the secreted lipoproteins. This suggests that this cell line may offer an interesting model for studying glycosphingolipid secretion, transfer, and shedding. We now report on the chemical and immunological characterization of Hep-G2 cell glycosphingolipids. Five major glycosphingolipids were purified and biochemically characterized: glycosylceramide, lactosyl ceramide, ceramide trihexoside, ganglioside GM3, and lactosyl sulfatide. Four additional minor components (3-fucosyl-lactosamine containing glycolipids, asialo GM2, galactosylgloboside, and ganglioside GM1) were identified using a combination of exoglycosidase digestion and immunostaining of thin-layer chromatography plates with specific carbohydrate binding proteins. This demonstrates that although this cell line synthesizes a limited number of major glycosphingolipids, it retains the ability to produce at least small amounts of structures in the lactoneo, globo, and ganglio series of glycosphingolipids. These studies show that it will be possible to investigate the mechanisms of secretion by Hep-G2 cells of different classes of these molecules such as neutral glycosphingolipids, gangliosides, and sulfatides.