Glycolipids and glycosyltransferases in permanent cell lines established from human medulloblastomas.

Medulloblastoma biopsies are heterogenous and might contain normal brain tissue, which limits the usefulness of such tumor material for biochemical analyses. We have, therefore, examined the gangliosides and their metabolism using the medulloblastoma cell lines. Daoy and D341 Med, cultured both in vitro and as xenografts in nude mice. The ganglioside patterns in the Daoy showed a switch from a high GM2, 70% (mol% of total ganglioside sialic acid) and low lactoseries gangliosides (2%) content in monolayer cultures, to a high proportion of lactoseries gangliosides (50%) and virtually no GM2 (1%) in xenografts, but an increased proportion of other a-series gangliosides. The D341 Med showed a similar change regarding the lacto-series gangliosides from 1% in suspension culture to 10% in xenografts. The activity of five glycosyltransferases, GM3, GD3, GM2, GM1 and LA2 synthases, did not parallel the ganglioside patterns and could not account for the noted variations therein. In the Daoy cell line the LA2 synthase as well as the GM2 synthase activity was relatively high in both culture systems, despite the marked difference in the expression of GM2 and the lactoseries gangliosides. These results suggest that environmental factors play a crucial role for the in vivo activity of the glycosyltransferases.     

Receptor ganglioside content of three hosts for Sendai virus. MDBK, HeLa, and MDCK cells

Specific gangliosides GD1a, GT1b and GQ1b isolated from brain have been shown to function as receptors for Sendai virus by conferring susceptibility to infection when they are incorporated into receptor-deficient cells (Markwell, M.A.K., Svennerholm, L. and Paulson, J.C. (1981) Proc. Natl. Acad. Sci. USA 78, 5406-5410). The endogenous gangliosides of three commonly used hosts for Sendai virus: MDBK, HeLa, and MDCK cells were analyzed to determine the amount and type of receptor gangliosides present. In all three cell lines, GM3 was the major ganglioside component. The presence of GM1, GD1a and the more complex homologs of the gangliotetraose series was also established. In cell lines derived from normal tissue, MDBK and MDCK cells, gangliosides contributed 47-65% of the total sialic acid. In HeLa cells, gangliosides contributed substantially less (17% of the total sialic acid). The ganglioside content of each cell line was shown not to be immutable but instead to depend on the state of differentiation, passage number, and surface the cells were grown on. Thus, the ganglioside concentration of undifferentiated MDCK cells was found to be substantially greater than that of MDBK or HeLa cells, but decreased as the MDCK cells underwent differentiation. Changes in culture conditions that were shown to decrease the receptor ganglioside content of the cells resulted in a corresponding decrease in susceptibility to infection. The endogenous oligosialogangliosides present in susceptible host cells were shown to function as receptors for Sendai virus.     

Ganglioside Composition in Human Meningiomas

The ganglioside composition in meningioma specimens from 20 patients was analyzed to find potential meningioma-associated structures. The characterization was performed by immunological staining with specific monoclonal antibodies to ganglioside antigens and fast atom bombardment-mass spectrometry. The major gangliosides were GM3 and GD3, and most of the meningioma specimens could be divided into a "GM3-rich" or a "GD3-rich" group. Gangliosides of the gangliotetraose series were represented by GM1, GD1a, GD1b, and GT1b, which were found in minor amounts in all the specimens. The ratios of GM1/GD1a and GD1a/GD1b differed from that in normal brain, and therefore existence of this series could not be explained by contamination with brain material. Ganglioside 3'-isoLM1, found in human malignant glioma, could not be detected in any meningioma specimen.     

Gangliosides in serum immune complexes from tumor-bearing patients

Immune complexes in the serum of tumor-bearing patients were adsorbed from whole blood or plasma on a protein A-Sepharose column. The adsorbed material was eluted, precipitated and analyzed for gangliosides. All precipitates obtained from eight patients at different treatment occasions contained gangliosides at concentrations varying from 0.1 to 12.2 nmol sialic acid/mg protein. The compositions of gangliosides were similar among the patients, regardless of the type of cancer, and quite different from that of normal serum. Most (75-85% of total sialic acid) belonged to the gangliotetraose series, of which 26-33% was GM1, 26-34% GD1a, 8-17% GD1b, and 5-13% GT1b. However, the dominant ganglioside in normal serum, GM3, was present in only trace amounts, which ruled out a nonspecific adsorption of serum ganglioside by protein A-Sepharose. Similar results were obtained for whole blood and plasma treatments, and these results suggest a specific interaction between gangliosides of the gangliotetraose series and serum immunoglobulins, either by the gangliosides acting as antigens and forming immune complexes or by their binding to already formed complexes.     

Coordinate Regulation of Ganglioside Glycosyltransferases in Differentiating NG108-15 Neuroblastoma X Glioma Cells

The enzymatic basis for ganglioside regulation during differentiation of NG108-15 mouse neuroblastoma x rat glioma hybrid cells was studied. This cell line contains four gangliosides that lie along the same biosynthetic pathway: GM3, GM2, GM1, and GD1a. Chemically induced neuronal differentiation of NG108-15 cells led to an 80% drop in the steady-state level of their major ganglioside, GM3, a sixfold increase in the level of a minor ganglioside, GM2 (which became the predominant ganglioside of differentiated cells); and relatively little change in the levels of GM1 and GD1a, which lie further along the same biosynthetic pathway. The enzymatic basis for this selective change in ganglioside expression was investigated by measuring the activity of two glycosyltransferases involved in ganglioside biosynthesis. UDP-N-acetylgalactosamine: GM3 N-acetylgalactosaminyltransferase (GM2-synthetase) activity increased fivefold during butyrate-induced differentiation, whereas UDP-galactose: GM2 galactosyltransferase (GM1-synthetase) activity decreased to 10% of its control level. Coordinate regulation of these two glycosyltransferases appears to be primarily responsible for the selective increase of GM2 expression during NG108-15 differentiation.     

Glycosphingolipid Glycosyltransferases in Human Fetal Brain

The developmental pattern of gangliosides in human fetal brain should reflect the activities of the respective glycosyltransferases. LA2-synthase activity, along with that of GM3-, GD3-, GM2-, and GM1-synthases, was determined in human fetal brain at 10-22 weeks of gestation. LA2-synthase is the pivotal enzyme in lacto series ganglioside formation. LA2-synthase activity decreased during the study period, mirroring a similar temporal decline in levels of the lacto series gangliosides, particularly 3'-isoLM1. The developmental profiles of the ganglio series glycosyltransferase activities demonstrate distinct changes that correspond to the ganglioside pattern between fetal weeks 10 and 22. In particular, the marked increase in GM2-synthase activity at 20 and 22 weeks of gestation and the decline in GD3-synthase activity after 15 weeks could explain the prominent expression of the a series gangliosides in this period of rapid neuronal outgrowth. However, a similar decline (two- to 2.5-fold) in GM3-synthase activity suggests a more likely conclusion, namely, that the two sialyltransferase activities are derived mainly from astroglial cells, which show a marked proliferation during the 10-15th fetal weeks. The data do not negate the hypothesis that GM3- and GD3-synthase are the critical enzymes in the regulation of ganglioside biosynthesis but do indicate a need to reevaluate the significance of GM2-synthase in expression of the a series gangliosides.     

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+.     

Sialyllactotetraosylceramide, a Ganglioside Marker for Human Malignant Gliomas

The ganglioside composition of surgically removed human glioma tissue was shown to differ from that of normal adult brain tissue. First, it contained reduced amounts of the major normal brain gangliosides of the gangliotetraose series. Second, it contained increased proportions of gangliosides not detected in normal brain tissue. One of these was isolated and established as being sialyllactotetraosylceramide 3'-isoLM1. Radioimmunoassay for this ganglioside antigen in human glioma tissue revealed that 14/14 specimens of grades III and IV were positive but only 1/4 of grade II. Normal brain tissue was negative. These results suggest that sialyllactotetraosylceramide is a marker for human malignant gliomas.     

Adhesion of Human Glioma Cell Lines to Fibronectin, Laminin, Vitronectin and Collagen I Is Modulated by Gangliosides in vitro

Adhesion of eight cell lines, derived from human gliomas of different histological types, to fibronectin, collagen I, vitronectin, and laminin was investigated in vitro. The glioma cell lines were found to attach to these substrates to different extents. Interestingly, all cell lines strongly attached to laminin. In addition, glioma cell adhesion was found to be dose dependent. Moreover, adhesion of three cell lines to fibronectin and collagen I was partially inhibited and to vitronectin completely prevented by GRGDTP peptide, indicating the involvement of integrin receptors in glioma cell adhesion. We have demonstrated, recently, that gangliosides play an important role in promoting glioma cell invasion of the reconstituted basement membrane, Matrigel, in vitro. In order to study the mechanism of action of gangliosides in this process, the role of six gangliosides (GM1, GM3, GD3, GD1a, GD1b, and GT1b) in cell adhesion to the four proteins was investigated in three cell lines. Although all gangliosides, with the exception of GM3, were found to enhance cell adhesion to these proteins to different extents, GD3 proved to be the most effective adhesion-promoting ganglioside in all three cell lines. GM3 was found to inhibit cell adhesion to the four proteins in one cell line but enhanced cell adhesion in two other cell lines. The three cell lines were found to express both GD3 and gangliosides recognised by the A2B5 antibody. Furthermore, adhesion of the three cell lines to fibronectin, vitronectin, laminin, and collagen I was inhibited by incubation with A2B5, demonstrating the involvement of intrinsic cell membrane gangliosides in adhesion of glioma cells to these proteins. Taken together with the observation that gangliosides modulate integrin receptor function, these data suggest that gangliosides may play a central role in the control of the adhesive and invasive properties of human glioma cells.     

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.     

Gangliosides as paramyxovirus receptor. Structural requirement of sialo-oligosaccharides in receptors for hemagglutinating virus of Japan (Sendai virus) and Newcastle disease virus

A sensitive assay system for receptor activity of gangliosides to paramyxovirus was developed. This system involves incorporation of gangliosides into neuraminidase-treated chicken erythrocytes (asialoerythrocytes) followed by estimation of virus-mediated agglutination and hemolysis. The asialoerythrocytes coated with I-active ganglioside (Sia alpha 2-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer) were effectively agglutinated by hemagglutinating virus of Japan (HVJ, Sendai virus). The hemolysis of the asialoerythrocytes mediated by HVJ was restored to the highest level by labeling the cells with gangliosides possessing lacto-series oligosaccharide chains, i.e., I-active ganglioside, N-acetylneuraminosylparagloboside (SiaPG(NeuAc)), and i-active ganglioside (Sia alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer). The specific receptor activity of ganglioside GD1a possessing a gangliotetraose chain was lower than those of the gangliosides described above. Gangliosides GM3, GD3, GM1a, GD1b, SiaPG(NeuGc) showed little effect on the restoration of HVJ-mediated hemolysis. On infection with Newcastle disease virus (NDV), the highest specific restoration of lysis was found in chicken asialoerythrocytes coated with SiaPG(NeuAc or NeuGc) and GM3(NeuAc or NeuGc), whereas those coated with I-active ganglioside, GD3, GM1a, and GD1b showed very low NDV-mediated hemolysis. The above results indicate that the determinants of receptor for HVJ contain sialylated branched and/or linear lacto-series oligosaccharides carried by I,i-active gangliosides and SiaPG(NeuAc) and sialosylgangliotetraose chain carried by GD1a. The determinants for NDV are carried by SiaPG(NeuAc or NeuGc) containing linear lacto-series oligosaccharide and GM3(NeuAc or NeuGc). The absence of detectable binding of free oligosaccharides obtained from I-active ganglioside and sialoglycoprotein GP-2 isolated from bovine erythrocyte membranes as HVJ receptor (Suzuki, Y., et al. J. Biochem. (1983) 93, 1621-1633; (1984) 95, 1193-1200) indicates that HVJ recognizes the sialooligosaccharides oriented out of the lipid bilayer in the cell membranes where the hydrophobic ceramide or peptide backbone of the receptor is integrated.     

A new ganglioside of the lactotetraose series, GalNAc-3'-isoLM1, detected in human meconium

A monoclonal antibody produced by immunization with cells of the human glioma cell line D-54 MG reacted with ganglioside GM2. The binding epitope of the antibody was found to be GalNAc beta 1-4(NeuAc alpha 2-3)Gal. Immunological detection of glycolipid antigens on thin-layer plates with this monoclonal antibody, DMAb-1, revealed the presence of a new ganglioside. This ganglioside, co-migrating with NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer(6'-LM1) and GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-3GalNAc beta 1-4Gla beta 1-4Glc beta 1-1Cer (GalNAc-isoGM1) at chromatographic separation was isolated from human meconium. Its structure was determined by permethylation and fast atom bombardment-mass spectometry analyses. The new ganglioside was found to be a combination of the lacto and ganglio series gangliosides, and the structure found to be GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-3GlcNAc alpha 1-3Gal beta 1-4Glc beta 1-1Cer(GalNAc-3'-isoLM1).     

Loss of choleragen receptors and ganglioside upon differentiation of 3T3-L1 preadipocytes

3T3-L1 preadipocytes differentiate in culture into cells having the enzymatic and morphological characteristics of adipocytes. Differentiation is accompanied by a decrease in total cellular ganglioside content; the ganglioside level is 1.8 to 2.5-fold higher in undifferentiated than in differentiated cells. Gangliosides GM3 and GD1a constitute a majority of total cell gangliosides in both cell types, while ganglioside GM1, the putative choleragen receptor, constitutes less than 5%. Differentiation results in a 75 to 85% decrease in ganglioside GM1. An inverse correlation exists between the percentage of adipocytes in the cell population and: 1) total ganglioside and ganglioside GM1 content, and 2) surface ganglioside GM1 as estimated by choleragen binding or fluorescent staining of bound choleragen. Nondifferentiating 3T3-C2 control cells do not exhibit changes in total ganglioside, ganglioside GM1, or choleragen binding that are observed with 3T3-L1 cells.     

Patterns of endogenous gangliosides and metabolic processing of exogenous gangliosides in cerebellar granule cells during differentiation in culture

The qualitative and quantitative pattern of endogenous gangliosides and the routes of metabolic processing of exogenous GM1,³H labeled in the sphingosine moiety (Sph-³H GM1) were studied in cerebellar granule cells during differentiation in vitro. During the first 7–8 days in culture the ganglioside content markedly increased, and the qualitative pattern showed, in percentage terms, a drastic decrease of GD3 and a marked increase of GD2, O-Ac-GT1b, O-Ac-GQ1b and GQ1b. After pulse with (Sph-³H) GM1, at all the investigated days in culture, different radiolabelled lipids were formed indicating that taken up exogenous GM1 was degraded and that its catabolic fragments, and partly GM1 itself, were used for biosynthetic purposes; moreover radioactive water was measured in the culture medium during chase indicating that labelled sphingosine underwent also degradation. The uptake of exogenous GM1 and the extent of its metabolic processing per cell unit increased during differentiation: a) GM2 was the major metabolic product and was relatively more abundant at 2 than 7 days in culture; b) the percentage of metabolites of biosynthetic origin over total metabolites increased during differentiation, especially at the short pulse times; c) among the metabolites of anabolic origin sphingomyelin equalled gangliosides at 2 days, whereas it was largely overcome by gangliosides at 7 days in culture; d) at 4 and 7 days in culture a radioactive substance, not yet identified, was present, whereas no trace of it was found at 2 days. In conclusion, cerebellar granule cells in culture feature a different pattern of endogenous gangliosides and display different ability to metabolically process exogenous GM1 ganglioside in the undifferentiated and fully differentiated stage.Abbreviations used: this article follows the ganglioside nomenclature of Svennerholm [J. Lipid Res., 5, 145–155, (1964)] and the IUPAC-IUP recommendations for lipid nomenclature [Lipids, 12, 455–468, (1977)] NeuAc N-Acetylneuraminic acid; sph, sphingosine - O-Ac O-acetylated - TLC thin layer chromatography     

Altered ganglioside composition in virally transformed rat embryo fibroblasts.

The composition of gangliosides was examined in a normal rat embryo fibroblast cell line (REF52) and in two viral transformants: a polyoma transformant (REF52-PyMLV) and a simian viral 40 transformant (REF52-SV40). The distribution of gangliosides in the cell lines was determined using gas-liquid chromatography and high-performance thin-layer chromatography. N-acetylneuraminic acid was the predominant sialic acid species detected in the three cell lines. The total ganglioside concentration (microgram/100 mg dry weight of cells) in the normal, PyMLV, and SV40 lines was 144.7 +/- 10.4, 153.8 +/- 9.2, and 86.1 +/- 6.8, respectively. Gangliosides GM3, GM2, GM1, and GD1a were the major species in the normal and transformed lines. The distribution of these gangliosides, however, differed markedly between the normal and the transformed lines and also between the transformed lines themselves. The transformed cells also differed from the normal cells in growth rate, morphology, and social behavior. The cell line with highest GM3 content (PyMLV) formed islands, whereas the normal and SV40 cell lines, which had lower GM3 levels, grew as monolayers. The findings suggest that PyMLV and SV40 transformation can have multiple and different effects on cellular ganglioside distribution and growth behavior.     

Incorporation and metabolism of ganglioside GM2 in skin fibroblasts from normal and GM2 gangliosidosis subjects

Ganglioside GM2, 3H-labeled in the sphingoid base, was added to the culture medium of normal and GM2 gangliosidosis fibroblasts. Ganglioside was found to adsorb rapidly to the cell surface, most of it could however be removed by trypsination. The trypsin-resistant incorporation was about 10 nmol/mg cell protein, after 48 h. The rates of adsorption and incorporation depended strongly on the concentration of fetal calf serum in the medium, higher serum concentrations being inhibitory. After various incubation times, the lipids were extracted, separated by thin-layer chromatography and visualized by fluorography. In normal cells a variety of degradation products as well as sphingomyelin was found whereas in GM2 gangliosidosis cells, only trace amounts of such products (mainly GA2) were found. In contrast, the higher gangliosides GM1 and GD1a were formed in comparable amounts (2.2-3.6% of total radioactivity after 92 h) in normal and pathologic cell lines. Supplementation of cells from GM2 gangliosidosis, variant AB, with purified GM2-activator protein restored ganglioside GM2 degradation to almost normal rates but had no effect on its glycosylation to gangliosides GM1 and GD1a. From these results we conclude that the synthesis of higher gangliosides from incorporated GM2 can occur by direct glycosylation and not only via lysosomal degradation and resynthesis from [3H]sphinganine-containing degradation products. Preliminary studies with subcellular fractionation after various times of [3H]ganglioside incorporation indicated biphasic kinetics for the net transport of membrane-inserted ganglioside to lysosomes, compatible with the notion that a portion of the glycolipids can also escape from secondary lysosomes and migrate to Golgi compartment or cell surface.     

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)     

Analysis of gangliosides from carp intestinal mucosa

The gangliosides of carp intestinal mucosa were isolated and analysed by thin-layer chromatography (TLC), TLC immunostaining test, and TLC/secondary ion mass spectrometry (TLC/SIMS). Four species of gangliosides, designated as G-1, G-2, G-3 and G-4, were separated on TLC. The TLC/SIMS analysis of the G-1 ganglioside of carp intestinal mucosa revealed a series of [M-H](-)ions from m/z 1061 to m/z 1131 representing the molecular mass range of GM4-like ganglioside with NeuAc. G-2, G-3 and G-4 gangliosides were analysed by the TLC immunostaining test. G-2 ganglioside was recognised by the monoclonal antibody specific for ganglioside GM1 (AGM-1 monoclonal antibody). However, G-3 ganglioside migrating on TLC between GM3 and GM1 ganglioside was not recognised by anti-GM3 monoclonal antibody and by AGM-1 monoclonal antibody. Furthermore, G-4 ganglioside with a similar TLC mobility as GD1a ganglioside did not show the reactivity to the anti-GD1a monoclonal antibody. In addition using the AGM-1 monoclonal antibody, the expression of GM1 ganglioside in the carp intestinal tissue was studied. GM1 ganglioside was detected on the epithelial cell surface of carp intestinal mucosa.     

Determination of Gangliosides in Six Human Primary Medulloblastomas

The ganglioside composition of six human medulloblastomas was analyzed. The characterization was performed by thin-layer chromatography, sialidase hydrolysis, and immunological staining with a panel of characterized antiganglioside monoclonal antibodies. The total ganglioside content ranged from 60 to 1,130 nmol of ganglioside sialic acid/g wet weight. Neuronal gangliosides (gangliotetraose series) were found in varying amounts in all medulloblastomas. Gangliosides of the neolactotetraose series (3'-LM1 and LD1) were present in all specimens, and the lactotetraose series ganglioside 3'-isoLM1 was found in all cases showing astrocytic differentiation. This supports our previous findings that 3'-isoLM1 is a marker for proliferating astroglial cells.     

Synthesis and uptake of gangliosides by choleragen-responsive human fibroblasts.

Human fibroblasts, cultured in medium containing 10% fetal calf serum, responded dramatically to choleragen with an increase in cyclic adenosine monophosphate content to greater than 48 times basal levels. Analysis of these cells for gangliosides indicated that the major ganglioside was N-acetylneuraminylgalactosylglucosylceramide (GM3) with trace amounts (less than or equal to 100 pmol/mg of protein) of other gangliosides including GM1, the putative choleragen receptor. Although the cells contained three glycosyltransferases required for ganglioside synthesis, the N-acetylgalactosaminyltransferase activity necessary for the conversion of GM3 to more complex gangliosides was not detected. When the cells were grown in medium containing [14C]galactose or N-acety[3H]mannosamine, however, all of the gangliosides became labeled, indicating that the cells can synthesize complex gangliosides. Although fetal calf serum contains gangliosides including GM1, [3H]GM1 was taken up poorly from the growth medium and uptake at the rate observed could have accounted for less than 2% of the GM1 content of the cells. When the cells were incubated in chemically defined medium containing [3H]GM1 at the concentrations present in fetal calf serum, rapid uptake of the ganglioside occurred and the total GM1 content of the cells increased threefold in less than 3 h. Thus, although the cells are capable of binding exogenous gangliosides, the gangliosides in fetal calf serum are in a form not readily available to the cells.