Selective shedding of gangliosides by cells of mouse ascitic sarcoma-37

The profile and content of gangliosides associated with tumour cells of mouse sarcoma-37 and exfoliated from the cell surface were determined. It was shown that 20% of tumour gangliosides shed from the cell surface and only about 7% of all the shed gangliosides were contained in plasma membrane fragments. When incubated in vitro at 37 degrees C for 1 hour, the tumour cells were found to release less than 2% of cellular gangliosides. The main components of cellular gangliosides corresponded in their chromatographic behaviour to GM2 (31-32% of the total ganglioside content), GD3 (17-18%), GD1a (9-11%), GD2 (16-17%) and hematosides (19-21%). The profiles of in vivo and in vitro shed gangliosides were similar but strongly differed from those of cellular gangliosides: the relative content of GM2 was 70-75% and the other gangliosides were found in negligible amounts. The data show that GM2 accumulation in extracellular spaces is rather the result of its selective shedding than the shedding of products of "incomplete" cellular ganglioside synthesis.     

The role of gangliosides in the interaction of a growth inhibitor with mouse LM cells

We have isolated and characterized glycopeptides, derived from mouse and bovine cerebral cortex cells, that inhibit protein synthesis and cell growth of normal but not transformed cells. The inhibitor binds to target cell surfaces, and gangliosides have previously been shown to influence cell sensitivity to the glycopeptides. Preincubation with 3.0 micrograms/ml ganglioside GM1 at 0 degrees C for 3 hr sensitized the mouse L-cell line to the inhibitor, as determined by protein synthesis assays. Preincubation of LM cells with ganglioside GM1 alone did not affect protein synthesis rates. In addition, the gangliosides GD1a and GM3 also sensitized the LM cells to the protein synthesis inhibitory effect of the glycopeptide inhibitor. Binding experiments were performed with 3T3 (sensitive) and LM (insensitive) cells to determine if sensitivity to the glycopeptide inhibitor was reflected in binding of the inhibitor to these cells. Binding of 125I-labeled inhibitor to 3T3 cells was maximal after 60 min at 0 degrees C and saturable at approximately 1 X 10(4) molecules/cell. Furthermore, binding of the inhibitor was dose-dependent, with half-maximal binding at 1.5-2.0 nM and saturation at 8.0-10.0 nM. Scatchard plot analysis indicated that the Kd was about 1 X 10(-9) M and that there are 1 X 10(4) receptors/cell. Binding of the inhibitor to LM cells was maximal after 30 min at 0 degrees C and saturation occurred at 5 X 10(3) molecules/cell. We then examined the possibility that gangliosides are the cellular receptor or co-receptor for the glycopeptide inhibitor. Binding of the inhibitor to ganglioside GM1 was first examined after the ganglioside had been preadsorbed to polystyrene tubes. These experiments indicated that the ganglioside did not bind the inhibitor. Ganglioside-containing liposomes from phosphatidylcholine or LM cell membrane components were also prepared; these artificial membranes did not bind appreciable amounts of the iodinated inhibitor. Competition experiments showed that the gangliosides GM1 and GD1a did not neutralize the protein synthesis inhibitory activity of the glycopeptides, indicating that gangliosides do not directly interact with the glycopeptide inhibitor. In addition, binding of the inhibitor to LM cells preincubated with ganglioside GM1 was studied. Although the binding of the inhibitor to LM cells was one-half that observed for 3T3 cells, incorporation of exogenous gangliosides into LM cells did not result in increased binding of the inhibitor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of Exogenous Gangliosides on Amino Acid Uptake and Na+,K+-ATPase Activity in Superior Cervical and Nodose Ganglia of Rats

The effects of some gangliosides on active uptake of nonmetabolizable alpha-aminoisobutyric acid (AIB) and Na+, K+-ATPase and Ca2+, Mg2+-ATPase activities in superior cervical ganglia (SCG) and nodose ganglia (NG) excised from adult rats were examined during aerobic incubation at 37 degrees C for 2 h. In NG, amino acid uptake was greatly accelerated with the addition of galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylgluc osyl ceramide (GM1) (85%) and also with N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide (GM2) or [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetyl- neuraminyl]-galactosylglucosyl ceramide (GD1a) (43% each) compared with a nonaddition control at a 5 nM concentration. Under identical conditions, Na+, K+-ATPase activity was strongly stimulated with GM1 (180%) and GD1a (93%), whereas Ca2+, Mg2+-ATPase activity showed no change. In SCG, on the other hand, AIB uptake was apparently inhibited (-27%) by addition of GM1, with a slight decrease in Na+, K+-ATPase but no change in Ca2+, Mg2+-ATPase activity in the tissue. Both asialo-GM1, in which N-acetylneuraminic acid is deficient, and Forssman glycolipid, which is not present in nervous tissue, failed to produce any significant increase in both SCG and NG not only in amino acid uptake, but also in Na+, K+-ATPase activity. A kinetic study of active AIB uptake showed that GM1 ganglioside produced an increase in Km with no change in Vmax in SCG, whereas it caused a decrease in Km with a slight increase in Vmax in NG. Treatment of NG and SCG with neuraminidase from Vibrio cholerae, an enzyme that split off sialic acid from polysialoganglioside, leaving GM1 intact, caused little inhibition of the amino acid uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible Role of Gangliosides in Regulating an Adenylate Cyclase-Linked 5-Hydroxytryptamine (5-HT1) Receptor

Cultured NCB-20 hybrid cells express adenylate cyclase-coupled receptors for 5-hydroxytryptamine (5-HT) that correspond biochemically and pharmacologically to 5-HT1 receptors in rodent brain membrane preparations, apart from a much-reduced affinity for 5-HT (160 nM compared to less than 5 nM in brain). Since NCB-20 cells also differ from rodent brain both qualitatively and quantitatively in their ganglioside composition, the effects of exogenously added gangliosides on the affinity of the 5-HT1 receptor for 5-HT were tested. Both GM1 ganglioside (the cholera toxin receptor) and tetrasialoganglioside GQ1b produced a 10-fold increase in receptor affinity for [3H]5-HT, measured by binding studies. All gangliosides, at submicromolar concentrations, resulted in significantly reduced EC50 values for 5-HT-mediated elevation of intracellular cyclic AMP levels. GQ1b had the capacity to most dramatically enhance the potency of 5-HT in mediating increases in cyclic AMP levels. Gangliosides had no effect on the potency of DADLE or 3,4-dihydroxyphenylethylamine (dopamine)-mediated depression of cyclic AMP levels, suggesting some specificity for 5-HT. Our data are interpreted as implying a specific role for polysialogangliosides in modulating the affinity of the 5-HT1 receptor and the coupling of the 5-HT1 receptor-guanine nucleotide binding protein adenylate cyclase complex.     

Fibronectin binding to gangliosides and rat liver plasma membranes

Binding of fibronectins to gangliosides was tested directly using several different in vitro models. Using an enzyme-linked immunoabsorbent assay (ELISA), gangliosides were immobilized on polystyrene tubes and relative binding of fibronectin was estimated by alkaline phosphatase activity of conjugated second antibody. Above a critical ganglioside concentration, the gangliosides bound the fibronectin (GT1b congruent to GD1b congruent to GD1a greater than GM1 much greater than GM2 congruent to GD3 congruent to GM3) in approximately the same order of efficiency as they competed for the cellular sites of fibronectin binding in cell attachment assays (Kleinman et al., Proc natl acad sci US 76 (1979) 3367). Alternatively, these same gangliosides bound to immobilized fibronectin. Rat erythrocytes coated with gangliosides GM1, GD1a or GT1b bound more fibronectin than erythrocytes not supplemented with gangliosides. Using fibronectin in which lysine residues were radioiodinated, an apparent Kd for binding to mixed rat liver gangliosides of 7.8 X 10(-9) M was determined. This value compared favorably with the apparent Kd for attachment of fibronectin to isolated plasma membranes from rat liver of 3.7 X 10(-9) M for fibronectin modified on the tyrosine residue, or 6.4 X 10(-9) M for fibronectin modified on lysine residues. As shown previously by Grinnell & Minter (Biochem biophys acta 550 (1979) 92), fibronectin modified on tyrosine residues did not promote spreading and attachment of CHO cells. It did, however, bind to cells. In contrast, lysine-modified fibronectin both bound to cells and promoted cell attachment. Plasma membranes isolated from hepatic tumors in which the higher gangliosides that bind fibronectin were depleted bound 43-75% less [125I]fibronectin than did plasma membranes from control livers. The findings were consistent with binding of fibronectins to gangliosides, including the same gangliosides depleted from cell surfaces during tumorigenesis in the rat.     

Tetanus toxin interaction with human erythrocytes. II. Kinetic properties of toxin association and evidence for a ganglioside-toxin macromolecular complex formation

The properties of tetanus toxin interaction with human erythrocytes supplemented with disialo- and trisialo-gangliosides have been investigated. Binding of toxin is linear with time for 1 h and is 3-4-fold higher at 37 degrees C than at 4 degrees C during incubation of long duration. It exhibits saturation at toxin concentrations between 0.1 and 1 microgram/ml; however, it is nonsaturable between 1 and up to 50 micrograms/ml. It is effectively prevented by free gangliosides and antibodies or by pretreatment with sialidase but is unaffected by a number of closely related ligands including toxoid and toxin fragments. NaCl (1 M) removes a great portion (86%) of cell-associated toxin while Triton X-100 extracts an additional fraction (30%) of the salt-resistant cell-bound toxin. The residual sequestred toxin after detergent extraction is sensitive to proteolytic degradation. The trypsin-stable fraction (1.5%) is biotoxic and may be indicative of internalization of toxin. A macromolecular complex of about 700 kDa containing toxin and gangliosides has been isolated and characterized by Sephacryl S-300 gel permeation chromatography, SDS-gel electrophoresis, immunoprecipitability and biotoxicity. This complex is obtained only in ganglioside-supplemented cells and not when free 3H-labeled GD1b is reacted with 125I-labeled toxin in solution in the absence of cells. The hydrophobicity properties acquired as a result of ganglioside-toxin interaction, presumably at the cell surface, suggest a conformational change of the toxin which may enable its penetration into the bilayer.     

Cerebellar granule cells in culture exhibit a ganglioside-sialidase presumably linked to the plasma membrane

Cerebellar granule cells differentiated in culture were incubated with ganglioside [3H-Sph]GD1a in order to have it inserted into the plasma membrane, internalized by endocytosis, and metabolized. The metabolites formed included GM1, product of GD1a desialosylation. No GM1 or other metabolites were present in the incubation medium, whereas with the lysosomal apparatus blocked by chloroquine, or GD1a endocytosis prevented at 4 degrees C, the only metabolite formed was GM1. These results suggest that GD1a desialosylation did not occur either extracellularly or intracellularly but likely, at the membrane level. Similar results were obtained with [3H-Gal]GD1b, whereas no degradation of [3H-NeuAc]GM1 took place in the presence of chloroquine or at 4 degrees C. In conclusion, cerebellar granule cells express in vivo a sialidase, presumably located on the cell surface, that affects GD1a and GD1b but not GM1.     

Mutant NG108-15 cells (NG-CR72) deficient in GM1 synthase respond aberrantly to axonogenic stimuli and are vulnerable to calcium-induced apoptosis: they are rescued with LIGA-20

The neuroblastoma x glioma NG108-15 hybrid cell line, a widely used model for the study of neuronal differentiation, contains a variety of gangliosides including GM1 and its sialosylated derivative, GD1a. To investigate the role of these a-series gangliotetraose gangliosides in neuritogenesis, we have obtained a mutated subclone of NG108-15 that is deficient in that family of gangliosides. NG108-15 cells were grown in the presence of cholera toxin, which killed the large majority of cells, and from the cholera-resistant survivors we isolated a clone, NG-CR72, that lacks GM1 and GD1a in the plasma and nuclear membranes. GM2 concentration was significantly higher in the plasma membrane. Enzyme assay indicated deficiency of UDP-Gal:GM2 galactosyltransferase (GM1 synthase), which was confirmed by incorporation studies with [3H]sphingosine. These cells resembled wild-type NG108-15 in extending dendritic processes in response to dendritogenic agents (retinoic acid, dibutyryl cAMP) but responded aberrantly to axonogenic stimuli (KCl, ionomycin) by extending unstable neurites that showed the cytoskeletal staining characteristic of dendrites. Moreover, mutant cells treated with the Ca2+ elevating axonogenic agents underwent apoptosis over time, attributed to dysfunction of Ca2+ regulatory mechanisms normally mediated by GM1. Such agents caused dramatic and sustained elevation of intracellular Ca2+ in mutant cells, in contrast to modest and temporary elevation in wild-type cells. Exogenous GM1, inserted into the plasma membrane, had no discernable protective effect on NG-CR72 cells whereas LIGA-20, a membrane-permeant derivative of GM1 that entered both plasma and nuclear membranes, blocked apoptosis, permitted extension of stable neurites, and attenuated the abnormal elevation of intracellular Ca2+.     

Monoclonal antibody Leo Mel 3, which inhibits killing of human melanoma cells by anomalous killer cells, binds to a sugar sequence in GD2 (II3(NeuAc)2-GgOse3Cer) and several other gangliosides

Human anomalous killer (AK) cells lyse freshly isolated human melanoma cells which are insensitive to human natural killer cell-mediated lysis. Monoclonal antibody Leo Mel 3, an IgM (k), produced by a hybridoma obtained from a mouse immunized with human melanoma cells, binds to melanoma cells and inhibits their conjugate formation with AK cells as well as their AK cell-mediated lysis. Other IgM antibodies from the same fusion that bind melanoma cells do not inhibit (Werkmeister, J. A., Triglia, T., Andrews, P., and Burns, G. F. (1985) J. Immunol. 135, 689-695). Leo Mel 3 binds several different gangliosides from melanoma cells, as determined by immunostaining thin layer chromatograms. Binding is abolished by treatment of the gangliosides with neuraminidase. In solid-phase radioimmunoassay, Leo Mel 3 binds strongly to ganglioside GD2 and less strongly to gangliosides GT3, GD3, and GQ1b. It does not bind to other gangliosides including GM1, GM2, GM3, GD1a, GD1b, and GT1b. Thus, the epitope recognized by antibody Leo Mel 3 is found in the sugar sequence of ganglioside GD2, GalNAc beta 1-4[NeuAc alpha 2-8NeuAc alpha 2-3]Gal beta 1-4Glc beta 1 .... This sequence may contain a target in melanoma cells recognized by AK cells.     

Binding of Clostridium botulinum neurotoxin to gangliosides

The binding characteristics of Clostridium botulinum neurotoxins of types B, C1, and F to gangliosides was studied by thin layer chromatography plate and microtiter plate methods at low (10 mM NaCl in 10 mM Tris-HCl buffer, pH 7.2) or high (150 mM NaCl in 10 mM Tris-HCl buffer, pH 7.2) ionic strengths and at 0 or 37 degrees C. The three types of toxins bound exclusively to three kinds of gangliosides, GD1a, GD1b, and GT1b, in both the thin layer chromatography plate and the microtiter plate methods. Type C1 toxin bound to the three gangliosides under all the conditions, while type B and F toxins bound only at low ionic strength and 37 degrees C. At low ionic strength, the binding kinetics for the three toxins was monophasic in Scatchard plots, and the association constants obtained in the microtiter plate system were 2-4 X 10(8) M-1. In contrast, the binding kinetics of type C1 toxin in high ionic strength was biphasic in the Scatchard plot, and two association constants were obtained in the microtiter plate system. The heavy chain facilitated the binding of the toxin to the gangliosides. These results indicate that different types of botulinum toxins bind to the gangliosides under different optimal conditions and that gangliosides may not be the common receptor for all types of botulinum toxins. The gangliosides may bind to type C1 toxin together with other potential receptor(s) on synaptosomal membranes.     

Molecular interactions between micellar polysialogangliosides and affinity-purified tetanotoxins in aqueous solution

Two-affinity purified tetanotoxin forms, TeToA and TeToB, with different affinities for gangliosides were characterized by analytical ultracentrifuge, circular dichroism (CD), and amino acid composition. Both toxin forms share a common sedimentation coefficient of about 6-7 S and similar alpha-helicity values, but they vary in amino acid composition. Incubation of TeToB with micellar polysialogangliosides results in formation of high (21-24 S) and medium (13-15 S) size toxin-micellar ganglioside aggregates as revealed by analytical ultracentrifuge technique. At TeToB/[N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl- [N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosylceramide (GT1b) molar ratios of greater than 26, high molecular weight aggregates (Mr greater than or equal to 700,000) which contain between 3 and 5 toxin monomers are formed, whereas at molar ratios less than 15, about 1-2 monomers are present. TeToA does not form aggregates in the presence of gangliosides. A marked increase in the alpha-helix from about 20 to 39% is apparent in the CD spectrum of TeToB after interaction with ganglioside mixture (G1b). Cerebrosides, sulfatides, sphingomyelin, and phosphatidylserine also increase the alpha-helix, presumably because of an overall effect of lipids on the protein. TeToA and fragment B but not C also undergo similar changes in the presence of G1b, suggesting that the effect of ganglioside is not specific. The polarity of the CD spectra of a number of gangliosides is shifted from a negative to a positive value after interaction with tetanotoxin. The data are consistent with the interpretation of a discrete hydrophobic domain on the toxin heavy chain which interacts with micellar gangliosides to form macromolecular complexes.     

Hydration and thermal reversibility of glycolipids depending on sugar chains

To elucidate a relationship between the structural properties and hydration characteristic of gangliosides, time-resolved small-angle X-ray scattering measurements using synchrotron radiation have been performed on aqueous dispersions of various types of gangliosides (GM1, GD1a, GD1b and GM3) under a constant heating (5-65 degrees C) and cooling (65-5 degrees C) rate. In the case of GM3, they formed a vesicular aggregate with a high structural reversibility in the heating-and-cooling process. For the micelles of GM1, GD1a and GDlb, we found an evident thermal hysteresis in the structural changes of their headgroups and evaluated quantitatively the amounts of water molecules occluded in the micellar hydrophilic regions by using the shell modeling method reported previously. For all cases of GM1, GD1a and GD1b, the thickness of the hydrophilic region of the micelle shrunk after the heating process, and stayed mostly constant over the entire cooling range. On the other hand, the amounts of water molecules and the behavior of the GM1, GD1a and GD1b micelles in the heating-and-cooling process greatly depended on the number of sialic acid residues in the sugar chain, that is, the penetration of water molecules was much more reversible for the GM1 micelle compared with those for the GD1a and GD1b micelles. The observed clear hysteresis and the hydration characteristics of GD1 gangliosides would relate to their role in neuronal membranes, where GD1 gangliosides show the greatest concentrations.     

Gangliosides released by the perfused rat liver are associated to high density lipoprotein.

We examine here the delivery of gangliosides from the perfused rat liver into the perfusate. One hour after the administration of [3H]GM1 to recirculating perfused livers, almost 80% of the perfusate radioactive gangliosides were recovered associated to the HDL fraction. This fraction was relatively enriched in radioactive GD1a. The pattern of endogenous gangliosides from perfused livers, rat serum and perfusates were very different: GM3 was the main liver ganglioside, GM1 and GD1a were the most abundant in perfusates being GM3 almost absent; GM3, GM1 and GD1a were present in rat serum in similar proportions. Using a non-recirculating perfusion protocol, radioactive gangliosides were found in the HDL fraction since 15 minutes after the administration of [3H]GM1. These results suggest that rat liver supplies the perfusates with some gangliosides and that they are associated to HDL. These facts arise the possibility that the liver is one of the source of serum gangliosides.     

Reevaluation of the Role of Gangliosides as Receptors for Tetanus Toxin

Binding of tetanus toxin to rat brain membranes was of lower affinity and capacity when binding was determined in 150 mM NaCl, 50 mM Tris-HCl (pH 7.4) than in 25 mM Tris-acetate (pH 6.0). Binding under both conditions was reduced by treating the membranes with neuraminidase. Pronase treatment, however, reduced toxin binding only in the Tris-saline buffer (pH 7.4). In addition, the concentration of gangliosides required to inhibit toxin binding was 100-fold higher in Tris-saline compared to Tris-acetate buffer. The toxin receptors in the membranes were analyzed by ligand blotting techniques. Membrane components were dissolved in sodium dodecyl sulfate, separated by polyacrylamide gel electrophoresis, and transferred to nitrocellulose sheets, which were overlaid with 125I-labeled toxin. Tetanus toxin bound only to material that migrated in the region of the dye front and was extracted with lipid solvents. Gangliosides isolated from the lipid extracts or other sources were separated by TLC on silica gel and the chromatograms were overlaid with labeled tetanus toxin. The toxin bound to areas where the major rat brain gangliosides migrated. When equimolar amounts of different purified gangliosides were applied to the chromatogram, binding of the toxin was in the order GD1b approximately equal to GT1b approximately equal to GQ1b greater than GD2 greater than GD3 much greater than GD1a approximately equal to GM1. Thus, the toxin appears to have the highest affinity for gangliosides with a disialyl group linked to the inner galactosyl residue. When binding of tetanus toxin to transfers and chromatograms was determined in the Tris-saline buffer (pH 7.4), the toxin bound to the same components but the extent of binding was markedly reduced compared with the low-salt and -pH conditions. Our results indicate that the interaction of tetanus toxin with rat brain membranes and gangliosides is greatly reduced under more physiological conditions of salt and pH and raise the possibility that other membrane components such as sialoglycoproteins may be receptors for the toxin under these conditions.     

Quantification of gangliotetraose gangliosides with cholera toxin

A procedure is described for assay of GM1 and other gangliotetraose-type gangliosides at the picomole level. The gangliosides are absorbed onto polystyrene microwells and treated with neuraminidase and then with cholera toxin B subunits conjugated to horseradish peroxidase. Color is developed and quantified spectrophotometrically. Omission of neuraminidase gives a measure of GM1 alone. Linearity was obtained between 0.5 and 3 pmol. This procedure was applied to ganglioside mixtures isolated fron neuro-2A neuroblastoma and PC12 pheochromocytoma cells. For the latter, an additional step involving reaction with fucosidase increased the yield of GM1 due to the presence of fucosylated gangliosides. Application of the same reagents as a TLC overlay procedure to the gangliosides from neuro-2A cells revealed the presence of GD1a, GD1b, and GT1b in addition to GM1, thus confirming the presence of a family of gangliotetraose gangliosides.     

Occurrence of glycosylation and deglycosylation of exogenously administered ganglioside GM1 in mouse liver.

Ganglioside GM1, 3H-labelled at the level of terminal galactose or of sphingosine, was intravenously injected into Swiss albino mice and some steps in its metabolic fate in the liver were investigated. After administration of [3H]sphingosine-labelled GM1 all major liver gangliosides [GM3, GM2, GM1, GD1a-(NeuAc,NeuGl)] became radioactive, the radioactivity residing in all cases on the sphingosine moiety. The specific radioactivity was highest in GM1, which carried about 53% of the radioactivity incorporated into gangliosides, followed by GM2, with 34.5% of incorporated radioactivity, GM3 and GD1a-(NeuAc,NeuGl), both with about 5% of incorporated radioactivity. After administration of [3H]galactose-labelled GM1 the only radioactive gangliosides present in the liver were GM1 and GD1a-(NeuAc,NeuGl), the former carrying about 95% of the total ganglioside-incorporated radioactivity, the latter about 3%. Both gangliosides were radioactive exclusively in the terminal galactose residue. According to these results exogenously administered GM1, after being taken up by the liver, is mainly degraded to GM2 and GM3, a part being, however, sialylated to GD1a-(NeuAc,NeuGl). All this suggests that exogenous GM1 may be involved in the metabolic routes of endogenous liver gangliosides.     

Effects of Brefeldin A on Synthesis and Intracellular Transport of Ganglioside GT3 by Chick Embryo Retina Cells

Abstract: Ganglioside GT3 is the precursor of c-series gangliosides. It is synthesized by sialylation of GD3 and is expressed in nervous tissue of birds and mammals at early stages of development. In this study we examined the sub-Golgi location of GT3 synthesis and the mechanism of its transport from the site of synthesis to the plasma membrane in chicken embryo retina cells in culture. Neural retina cells from 10-day-old chick embryo were cultured with [³H]galactose in the absence (control cells) or in the presence of 1 µg/ml brefeldin A (BFA). At the end of the labeling period, the fraction of labeled gangliosides transported to the plasma membrane was determined. For this, cells were treated with C . perfringens neuraminidase in conditions to desialylate only those gangliosides that were transported to the plasma membrane and consequently accessible to the enzyme. After neuraminidase treatment of cells, gangliosides were isolated, purified, and the pattern of radioactivity analyzed by HPTLC-fluorography. It was found that BFA blocked the synthesis of complex gangliosides without affecting the synthesis of GM3, GD3, and GT3. Furthermore, in BFA-treated cells, GM3, GD3, and GT3 were protected from the action of added neuraminidase, indicating an intracellular localization and, hence, an inhibition of their transport to the plasma membrane. The results indicate that synthesis of the first intermediates of a-, b-, and c- series gangliosides occurs in a proximal Golgi compartment and that the proximal Golgi-synthesized gangliosides (GM3, GD3, and GT3) use a transport mechanism that is dependent on ADP ribosylation factor and coatomer proteins.     

A new approach to the modification of cell membrane glycosphingolipids: Ganglioside composition of JTC-12 P3 cells altered by feeding with galactose as a sole carbohydrate source in protein- and lipid-free synthetic medium

A significant difference in the glycosphingolipid composition of JTC-12 P3 cells established from monkey kidney tissue was observed when cells cultured in a protein- and lipid-free synthetic medium containing glucose (DM-160) as a sole carbohydrate source were transferred and cultured in the same medium containing galactose and pyruvic acid (DM-170) in place of glucose. In particular, the amounts of gangliosides GM3, GM2, and GD3 in the cells cultured in DM-170 were 5.3-, 17.8-, and more than 8-fold those in the cells cultured in DM-160, respectively, indicating that anabolism of gangliosides is greatly enhanced in cells cultured in the presence of galactose and pyruvic acid, as compared with cells cultured in the presence of glucose. In fact, after cultivation of cells in the medium with N-acetyl-D-[14C]mannosamine for 96 h, the radioactivity incorporated into the gangliosides of the cells in DM-170 was 10-fold that of the cells in DM-160. Among the gangliosides of the cells in DM-170, highly sialylated molecules such as GD3, GD1a, GD1b, and GT1b were preferentially labeled, indicating that the sialyltransferases responsible for the synthesis of gangliosides are significantly more activated in cells cultured in DM-170 than in DM-160. These observations reveal that the glycosphingolipid composition of the plasma membrane can be modified epigenetically under well-defined conditions and provide important clues for clarifying the roles of glycosphingolipids associated with particular cell functions.     

Asymmetric distribution of gangliosides in rat renal brush-border and basolateral membranes

Highly enriched brush-border and basolateral membranes isolated from rat renal cortex were used to study the distribution of endogenous gangliosides in the two distinct plasma membrane domains of epithelial cells. These two membrane domains differed in their glycolipid composition. The basolateral membranes contained more of both neutral and acidic glycolipids, expressed on a protein basis. In both membranes, the neutral glycolipids corresponding to mono-, di-, tri- and tetraglycosylceramides were present. The basolateral membranes contained more diglycosylceramide than the brush-border membranes. The major gangliosides found were GM4, GM3, and GD3 with minor amounts of GM1 and GD1a. The latter were identified and quantified by sensitive iodinated cholera toxin binding assays. When the distribution of individual gangliosides was calculated as a percent of total gangliosides, the brush-border membranes were enriched with GM3, GM1 and GD1a compared to the basolateral membranes, which were enriched with GD3 and GM4. The observation of a distinct distribution of glycolipids between brush-border and basolateral membranes of the same epithelial cell suggests that there may be a specific sorting and insertion process for epithelial plasma membrane glycolipids. In turn, asymmetric glycolipid biogenesis may reflect differences in glycolipid function between the two domains of the epithelial plasma membrane.     

Modulation of Ganglioside Biosynthesis in Primary Cultured Neurons

Murine cerebellar cells were pulse labeled with [14C]galactose, and the incorporation of radioactivity into gangliosides and neutral glycosphingolipids was examined under different experimental conditions. In the presence of drugs affecting intracellular membrane flow, as well as at 15 degrees C, labeled GlcCer was found to accumulate in the cells, whereas the labeling of higher glycosphingolipids and gangliosides was reduced. Monensin and modulators of the cytoskeleton effectively blocked biosynthesis of the complex gangliosides GM1, GD1a, GD1b, GT1b, and GQ1b, whereas incorporation of radioactivity into neutral glycosphingolipids, such as glucosylceramide and lactosylceramide, as well as GM3, GM2, and GD3 was either increased or unaltered. As monensin has been reported to interfere with the flow of molecules from the cis to the trans stacks of the Golgi apparatus, this result highlights at least one subcompartmentalization of ganglioside biosynthesis within the Golgi system. Inhibitors of energy metabolism affected, predominantly, the biosynthesis of the b-series gangliosides, whereas a reduced temperature (15 degrees C) more effectively blocked incorporation of radiolabel into the a-series gangliosides, a result suggesting the importance of GM3, as the principal branching point, for the regulation of ganglioside biosynthesis.