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Gangliosides (sialic acid-containing glycosphingolipids) help regulate many important biological processes, including cell proliferation, signal transduction, and differentiation, via formation of functional microdomains in plasma membranes. The structural diversity of gangliosides arises from both the ceramide moiety and glycan portion. Recently, differing molecular species of a given ganglioside are suggested to have distinct biological properties and regulate specific and distinct biological events. Elucidation of the function of each molecular species is important and will provide new insights into ganglioside biology. Gangliosides are also suggested to be involved in skeletal muscle differentiation; however, the differential roles of ganglioside molecular species remain unclear. Here we describe striking changes in quantity and quality of gangliosides (particularly GM3) during differentiation of mouse C2C12 myoblast cells and key roles played by distinct GM3 molecular species at each step of the process., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The genetic (stable overexpression of sialyltransferase I, GM3 synthase) or pharmacological (selective pressure by N-(4-hydroxyphenyl)retinamide)) manipulation of A2780 human ovarian cancer cells allowed us to obtain clones characterized by higher GM3 synthase activity compared with wild-type cells. Clones with high GM3 synthase expression had elevated ganglioside levels, reduced in vitro cell motility, and enhanced expression of the membrane adaptor protein caveolin-1 with respect to wild-type cells. In high GM3 synthase-expressing clones, both depletion of gangliosides by treatment with the glucosylceramide synthase inhibitor D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol and silencing of caveolin-1 by siRNA were able to strongly increase in vitro cell motility. The motility of wild-type, low GM3 synthase-expressing cells was reduced in the presence of a Src inhibitor, and treatment of these cells with exogenous gangliosides, able to reduce their in vitro motility, inactivated c-Src kinase. Conversely, ganglioside depletion by D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol treatment or caveolin-1 silencing in high GM3 synthase-expressing cells led to c-Src kinase activation. In high GM3 synthase-expressing cells, caveolin-1 was associated with sphingolipids, integrin receptor subunits, p130(CAS), and c-Src forming a Triton X-100-insoluble noncaveolar signaling complex. These data suggest a role for gangliosides in regulating tumor cell motility by affecting the function of a signaling complex organized by caveolin-1, responsible for Src inactivation downstream to integrin receptors, and imply that GM3 synthase is a key target for the regulation of cell motility in human ovarian carcinoma.

A2780 human ovarian carcinoma cells respond to treatment with the synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) with the production of dihydroceramide and with a concomitant reduction of cell proliferation and induction of apoptosis. The derived HPR-resistant clonal cell line, A2780/HPR, is less responsive to HPR in terms of dihydroceramide generation. In this report, we show that the production of sphingosine 1-phosphate (S1P) is significantly higher in A2780/HPR versus A2780 cells due to an increased sphingosine kinase (SK) activity and SK-1 mRNA and protein levels. Treatment of A2780 and A2780/HPR cells with a potent and highly selective pharmacological SK inhibitor effectively reduced S1P production and resulted in a marked reduction of cell proliferation. Moreover, A2780/HPR cells treated with a SK inhibitor were sensitized to the cytotoxic effect of HPR, due to an increased dihydroceramide production. On the other hand, the ectopic expression of SK-1 in A2780 cells was sufficient to induce HPR resistance in these cells. Challenge of A2780 and A2780/HPR cells with agonists and antagonists of S1P receptors had no effects on their sensitivity to the drug, suggesting that the role of SK in HPR resistance in these cells is not mediated by the S1P receptors. These data clearly demonstrate a role for SK in determining resistance to HPR in ovarian carcinoma cells, due to its effect in the regulation of intracellular ceramide/S1P ratio, which is critical in the control of cell death and proliferation.

Human fibroblasts, rat neurons, and murine neuroblastoma cells, cultured in the presence of fetal calf serum, were fed with [1-(3)H]sphingosine to radiolabel sphingolipids. The fate of cell sphingolipids, the release of sphingolipids in the culture medium, the interaction of sphingolipids with the proteins and lipoproteins of fetal calf serum, and the fate of sphingolipids taken up by the cells were investigated. For this latter purpose, the culture medium containing radioactive sphingolipids was delivered to nonlabeled cells. The presence of tritium at position 1 of sphingosine allowed us to follow the extent of sphingolipid catabolism by measuring the production of radioactive phosphatidylethanolamine and proteins by recycling the radioactive ethanolamine formed during sphingosine catabolism and the production of tritiated water. We confirmed that in cells the recycling of sphingosine occurred to a high extent and that only a minor portion of cell sphingolipids was catabolized to the small fragments of ethanolamine and water. Cell sphingolipids were released in the culture medium, where they formed large lipoproteic aggregates at a rate of about 12% per day. Released sphingolipids were taken up by the cells and catabolized to the sphingosine and then to ethanolamine, and recycling of sphingosine was not observed. This suggests that in the presence of fetal calf serum in the culture medium, exogenous sphingolipids directly reach the lysosomes, were they are entirely catabolized. Thus, the trafficking of sphingolipids from cells to the extracellular environment and from this to other cells does not allow the modification of the plasma membrane composition.

We describe herein the enzyme behavior of MmNEU3, the plasma membrane-associated sialidase from mouse (Mus musculus). MmNEU3 is localized at the plasma membrane as demonstrated directly by confocal microscopy analysis. In addition, administration of the radiolabeled ganglioside GD1a to MmNEU3-transfected cells, under conditions that prevent lysosomal activity, led to its hydrolysis into ganglioside GM1, further indicating the plasma membrane topology of MmNEU3. Metabolic labeling with [1-(3)H]sphingosine allowed the characterization of the ganglioside patterns of COS-7 cells. MmNEU3 expression in COS-7 cells led to an extensive modification of the cell ganglioside pattern, i.e. GM3 and GD1a content was decreased to about one-third compared with mock-transfected cells. At the same time, a 35% increase in ganglioside GM1 content was observed. Mixed culture of MmNEU3-transfected cells with [1-(3)H]sphingosine-labeled cells demonstrates that the enzyme present at the cell surface is able to recognize gangliosides exposed on the membrane of nearby cells. Under these experimental conditions, the extent of ganglioside pattern changes was a function of MmNEU3 transient expression. Overall, the variations in GM3, GD1a, and GM1 content were very similar to those observed in the case of [1-(3)H]sphingosine-labeled MmNEU3-transfected cells, indicating that the enzyme mainly exerted its activity toward ganglioside substrates present at the surface of neighboring cells. These results indicate that the plasma membrane-associated sialidase MmNEU3 is able to hydrolyze ganglioside substrates in intact living cells at a neutral pH, mainly through cell-to-cell interactions.

In the present work, we studied the effects of fenretinide (N-(4-hydroxyphenyl)retinamide (HPR)), a hydroxyphenyl derivative of all-trans-retinoic acid, on sphingolipid metabolism and expression in human ovarian carcinoma A2780 cells. A2780 cells, which are sensitive to a pharmacologically achievable HPR concentration, become 10-fold more resistant after exposure to increasing HPR concentrations. Our results showed that HPR was able to induce a dose- and time-dependent increase in cellular ceramide levels in sensitive but not in resistant cells. This form of resistance in A2780 cells was not accompanied by the overexpression of multidrug resistance-specific proteins MDR1 P-glycoprotein and multidrug resistance-associated protein, whose mRNA levels did not differ in sensitive and resistant A2780 cells. HPR-resistant cells were characterized by an overall altered sphingolipid metabolism. The overall content in glycosphingolipids was similar in both cell types, but the expression of specific glycosphingolipids was different. Specifically, our findings indicated that glucosylceramide levels were similar in sensitive and resistant cells, but resistant cells were characterized by a 6-fold lower expression of lactosylceramide levels and by a 6-fold higher expression of ganglioside levels than sensitive cells. The main gangliosides from resistant A2780 cells were identified as GM3 and GM2. The possible metabolic mechanisms leading to this difference were investigated. Interestingly, the mRNA levels of glucosylceramide and lactosylceramide synthases were similar in sensitive and resistant cells, whereas GM3 synthase mRNA level and GM3 synthase activity were remarkably higher in resistant cells.

We have investigated the ability of a receptor-mediated gene transfer strategy (cross-correction) to restore ganglioside metabolism in fibroblasts from Tay-Sachs (TS) patients in vitro. TS disease is a GM2 gangliosidosis attributed to the deficiency of the lysosomal enzyme beta-hexosaminidase A (HexA) (beta-N-acetylhexosaminidase, EC ). The hypothesis is that transduced cells overexpressing and secreting large amounts of the enzyme would lead to a measurable activity in defective cells via a secretion-recapture mechanism. We transduced NIH3T3 murine fibroblasts with the LalphaHexTN retroviral vector carrying the cDNA encoding for the human Hex alpha-subunit. The Hex activity in the medium from transduced cells was approximately 10-fold higher (up to 75 milliunits) than observed in non-transduced cells. TS cells were cultured for 72 h in the presence of the cell medium derived from the transduced NIH3T3 cells, and they were analyzed for the presence and catalytic activity of the enzyme. Although TS cells were able to efficiently uptake a large amount of the soluble enzyme, the enzyme failed to reach the lysosomes in a sufficient quantity to hydrolyze the GM2 ganglioside to GM3 ganglioside. Thus, our results showed that delivery of the therapeutic HexA was not sufficient to correct the phenotype of TS cells.

In the present paper, we report on the properties of sphingolipid-enriched domains of rat cerebellar granule cells in culture at different stages of neuronal development. The major lipid components of these domains were glycerophospholipids and cholesterol. Glycerophospholipids were 45-75% and cholesterol 15-45% of total lipids of the domains. This corresponded to 5-17% of total cell glycerophospholipids and 15-45% of total cell cholesterol. Phosphatidylcholine, mainly dipalmitoylphosphatidylcholine, was 66-85% of all the glycerophospholipids associated with these domains. Consequently, the palmitoyl residue was significantly enriched in the domains. The surface occupied by these structures increased during development. 40-70% of cell sphingolipids segregated in sphingolipid-enriched membrane domains, with the maximum ganglioside density in fully differentiated neurons. A high content of ceramide was found in the domains of aging neurons. Then, the sphingolipid/glycerophospholipid molar ratio was more than doubled during the initial stage of development, whereas the cholesterol/glycerophospholipid molar ratio gradually decreased during in vitro differentiation. Phosphorylated phosphoinositides, which were scant in the domains of undifferentiated cells, dramatically increased during differentiation and aging in culture. Proteins were minor components of the domains (0.1-2.8% of all domain components). Phosphotyrosine-containing proteins were selectively recovered in the sphingolipid-enriched domain. Among these, Src family protein-tyrosine kinases, known to participate to the process of neuronal differentiation, were associated with the sphingolipid-enriched domains in a way specific for the type of kinase and for the developmental stage of the cell. Proteins belonging to other signaling pathways, such as phosphoinositide 3-kinase and its downstream target, Akt, were not associated with the domains.

Sphingolipid-enriched membrane domains, characterized by a particular protein and lipid composition, have been detected in a variety of cells. However, limited data are available concerning these domains in neuronal cells. We analyzed the lipid and protein composition of a sphingolipid-enriched membrane fraction prepared from primary rat cerebellar granule cells differentiated in culture. Although the protein content of this fraction was only 1.4% of total cellular protein, 60% of the gangliosides, 67% of the sphingomyelin, 50% of the ceramide, and 40% of the cholesterol were located in this fraction. The protein pattern of the sphingolipid-enriched domain fraction was dramatically different from that associated with the cell homogenate. This fraction contained 25% of the tyrosine-phosphorylated proteins and was enriched in two proteins with apparent molecular masses of 135 and 15 kDa. 12% of cellular glycerophospholipids were located in the fraction, with phosphatidylcholine having the highest enrichment. The molar ratio between proteins, glycerophospholipids, cholesterol, sphingomyelin, ceramide and gangliosides in cerebellar granule cells was 1.6:41.6:6. 1:1.3:0.3:1 in the cell homogenate and 0.04:8.3:4.0:1.4:0.2:1 in the sphingolipid-enriched membrane fraction. These data indicate that selected proteins segregate with sphingolipids in specialized domains in the membrane of cultured neurons.

To understand the reason why, in the absence of GM2 activator protein, the GalNAc and the NeuAc in GM2 (GalNAcbeta1-->4(NeuAcalpha2-->3)Galbeta1-->4Glcbet a1-1'Cer) are refractory to beta-hexosaminidase A and sialidase, respectively, we have recently synthesized a linkage analogue of GM2 named 6'GM2 (GalNAcbeta1-->6(NeuAcalpha2-->3)Galbeta1-->4Glcbet a1-1'Cer). While GM2 has GalNAcbeta1-->4Gal linkage, 6'-GM2 has GalNAcbeta1-->6Gal linkage (Ishida, H., Ito, Y., Tanahashi, E., Li, Y.-T., Kiso, M., and Hasegawa, A. (1997) Carbohydr. Res. 302, 223-227). We have studied the enzymatic susceptibilities of GM2 and 6'GM2, as well as that of the oligosaccharides derived from GM2, asialo-GM2 (GalNAcbeta1-->4Galbeta1--> 4Glcbeta1-1'Cer) and 6'GM2. In addition, the conformational properties of both GM2 and 6'GM2 were analyzed using NMR spectroscopy and molecular mechanics computation. In sharp contrast to GM2, the GalNAc and the Neu5Ac of 6'GM2 were readily hydrolyzed by beta-hexosaminidase A and sialidase, respectively, without GM2 activator. Among the oligosaccharides derived from GM2, asialo-GM2, and 6'GM2, only the oligosaccharide from GM2 was resistant to beta-hexosaminidase A. Conformational analyses revealed that while GM2 has a compact and rigid oligosaccharide head group, 6'GM2 has an open spatial arrangement of the sugar units, with the GalNAc and the Neu5Ac freely accessible to external interactions. These results strongly indicate that the resistance of GM2 to enzymatic hydrolysis is because of the specific rigid conformation of the GM2 oligosaccharide.

Cultured fibroblasts from normal subjects and from subjects affected by Salla disease, characterized by the lack or misfunction of the membrane carrier responsible for the egress of sialic acid from lysosomes, were fed with ganglioside GM3 labeled at the sialic acid acetyl group, [Neu5Ac-3H]GM3, or at C-3 of sphingosine (Sph), [Sph-3H]GM3, or at C-1 of stearoyl chain, [stearoyl-14C]GM3. After a 15-h pulse the total amount of cell-bound GM3 corresponded to about 2% of the endogenous ganglioside content. Cells were then subjected to a 72-h chase, and the radioactive products from both ganglioside catabolism and salvage processes of catabolic fragments were measured. These data indicated that about 50% of the cell-bound ganglioside underwent metabolic processing, suggesting a ganglioside half-life of 2-3 days. [Neu5Ac-3H] formed from [Neu5Ac-3H]GM3 degradation was mostly re-cycled for the biosynthesis of gangliosides and sialoglycoproteins, only a minor part being degraded to [3H]water, which constituted only 1.6% of total metabolite linked radioactivity. [Sph-3H] from the [Sph-3H]GM3 degradation was partly re-cycled for the biosynthesis of gangliosides, neutral glycosphingolipids and sphingomyelin, and partly (about 20% of the total metabolite linked radioactivity) degraded to [3H]water. In Salla fibroblasts metabolic processing of [Neu5Ac-3H]GM3 produced large amounts of free [3H]Neu5Ac, and a reduced incorporation of radioactivity into glycoconjugates (as compared to normal cells). However, the accumulation of free Neu5Ac was not accompanied by an increase of tritiated water. LacCer and Cer from [stearoyl-14C]GM3 catabolism were found to accumulate in Salla fibroblasts, an indication that the enzymes of glycosphingolipid metabolism were affected by the impairment of Neu5Ac egress from lysosomes. Particularly relevant was the accumulation of ceramide which was hardly detectable in control cells.

GM2 activator protein is a protein cofactor which stimulates the enzymatic hydrolysis of both GalNAc and NeuAc from GM2. We have previously isolated two cDNA clones, GM2 activator cDNA and GM2A cDNA, for human GM2 activator protein (Nagarajan, S., Chen, H.-C., Li, S.-C., Li, Y.-T., and Lockyer, J. M. (1992) Biochem. J. 282, 807-813). GM2A mRNA is an RNA alternative splicing product that contains exons 1, 2, 3, and intron 3 of the genomic DNA sequence of GM2 activator protein (Klima, H., Tanaka, A., Schnabel, D., Nakano, T., Schröder, M., Suzuki, K., and Sandhoff, K. (1991) FEBS Lett. 289, 260-264). GM2A cDNA encodes a protein (GM2A protein) containing 1-109 of the 160 amino acids of human GM2 activator protein, plus a tripeptide (VST) encoded by intron 3 at the COOH terminus. Thus, GM2A protein can be regarded as a form (truncated version) of GM2 activator protein. We have expressed GM2A cDNA in Escherichia coli using pT7-7 as the vector. The recombinant GM2A protein was purified to an electrophoretically homogeneous form and was found to stimulate the hydrolysis of NeuAc from GM2 by clostridial sialidase, but not the hydrolysis of GalNAc from GM2 by beta-hexosaminidase A. Like GM2 activator protein, GM2A protein also specifically recognized the terminal GM2 epitope in GalNAc-GD1a and stimulated the hydrolysis of only the external NeuAc from this ganglioside by clostridial sialidase. These results enabled us to discern the enzymatic hydrolyses of GalNAc and NeuAc from the GM2 epitope and established that the NeuAc recognition domain of GM2 activator protein is located within amino acids 1-109. The presence of GM2A mRNA in human tissues and the selective stimulation of NeuAc hydrolysis by GM2A protein indicate that this activator protein may be involved in the catabolism of GM2 through the asialo-GM2 pathway.

GM2 Activator is a low molecular weight protein cofactor that stimulates the enzymatic conversion of GM2 into GM3 by human beta-hexosaminidase A and also the conversion of GM2 into GA2 by clostridial sialidase (Wu, Y.-Y., Lockyer, J.M., Sugiyama, E., Pavlova, N.V., Li, Y.-T., and Li, S.-C. (1994) J. Biol. Chem. 269, 16276-16283). Among the five known activator proteins for the enzymatic hydrolysis of glycosphingolipids, only GM2 activator is effective in stimulating the hydrolysis of GM2. However, the mechanism of action of GM2 activator is still not well understood. Using a unique disialosylganglioside, GalNAc-GD1a, as the substrate, we were able to show that in the presence of GM2 activator, GalNAc-GD1a was specifically converted into GalNAc-GM1a by clostridial sialidase, while in the presence of saposin B, a nonspecific activator protein, GalNAc-GD1a was converted into both GalNAc-GM1a and GalNAc-GM1b. Individual products generated from GalNAc-GD1a by clostridial sialidase were identified by thin layer chromatography, negative secondary ion mass spectrometry, and immunostaining with a monoclonal IgM that recognizes the GM2 epitope. Our results clearly show that GM2 activator recognizes the GM2 epitope in GalNAc-GD1a. Thus, GM2 activator may interact with the trisaccharide structure of the GM2 epitope and render the GalNAc and NeuAc residues accessible to beta-hexosaminidase A and sialidase, respectively.

We have studied the substrate specificities of a non-specific activator protein on the enzymatic hydrolyses of the following compounds: GM1 and GM2, as well as several of their derivatives including oligosaccharides, GgOse3Cer-II3-sulfate and LacCer-II3-sulfate, Gb-Ose3Cer and GbOse4Cer, three neolacto-series glycosphingolipids, and two non-ceramide glycolipids. Our results show that this activator protein has a broad spectrum of activity and exhibits the properties of a nonspecific natural detergent. The evidence of non-specificity was the ability of this activator protein to stimulate the hydrolyses of glycolipids, regardless of glycosphingolipids or non-ceramide glycolipids, carried out by glycosidases from animals, plants, and microorganisms. Its activity was, however, limited to substrates that had a lipid moiety. The oligosaccharide of GM1 and deacetyl-fatty acid free GM1 (II3-NeuGg-Ose4-sphingosine) were hydrolyzed by beta-galactosidase in the absence of this activator protein.

A new ganglioside containing an inner ester linkage was extracted from adult brain specimens, obtained at the time of surgery on 51-70-year-old subjects, purified, and analyzed. It contains glucose, galactose, N-acetylgalactosamine, an N-acetylneuraminic acid in the molar ratio 1:2:1:2, but, on ion-exchange chromatography, behaves as a monosialoganglioside. Structural analyses showed its basic neutral glycosphingolipid core to be ganglio-N-tetraose ceramide, carrying a disialosyl residue on the 3-position of internal galactose. Sialidase degradation and chemical analysis of the products obtained after alkaline treatments suggested one sialic acid residue to be involved in an ester linkage. Fast atom bombardment-mass spectrometry indicated the presence of an inner ester linkage between the carboxyl group of the external sialic acid residue and a hydroxyl group of the internal one. On these bases, the new ganglioside can be assumed to be a GD1b in lactonic form. This ganglioside is present only in trace amounts in the brain of infants, but its content increases with age, reaching a value of 3.5% of total sialic acid in 51-70-year-old subjects.

The antigen defined by a monoclonal antibody, MBr1, was found to be expressed in normal human mammary gland epithelia and human mammary carcinoma cells (Ménard, S., Tagliabue, E., Canevari, S., Fossati, G., and Colnaghi, M. I. (1983) Cancer Res. 43, 1295-1300). The antigen has been isolated from breast cancer cell line MCF-7, which was used as immunogen, and its structure was determined by methylation analysis, NMR spectroscopy, direct probe mass spectrometry, and enzymatic degradation as identified below. Fuc alpha 1----2Gal beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1Cer The antibody cross-reacted weakly with fucosylasialo-GM1 (IV2FucGg4), which shares the same terminal sequence, Fuc alpha 1----2Gal beta 1----3GalNAc, with this antigen. However, various other structures, including lacto-series H structure (Fuc alpha 1----2 Gal beta 1----4/or 3GlcNAc beta 1----3Gal), did not show any reactivity with this antibody. Therefore, this antigen represents a blood group H antigen with a globo-series structure which is abundant in human teratocarcinoma (Kannagi, R., Levery, S. B., Ishigami, F., Hakomori, S., Shevinsky, L. H., Knowles, B. B., and Solter, D. (1983) J. Biol. Chem. 258, 8934-8942), although its presence must be limited in normal adult human tissue.