Glycolipid crypticity in membranes — not a simple shielding effect of macromolecules

The potential of membrane-bound macromolecules for shielding glycolipids from involvement in specific binding events was considered in model membranes. Serum albumin and several Dextrans were covalently derivatized with oleic acid so that they adsorbed irreversibly to lipid bilayers. This provided a means of generating bilayer membranes with a considerable layer of attached material. Gangliosides dispersed in such membranes were subjected to attack by the enzyme, neuraminidase, in order to assess their ‘accessibility’. We were surprised to find that we could not demonstrate any significant reduction in ganglioside hydrolysis in phosphatidylcholine bilayers bearing extensive surface coats of protein or polysaccharide. We conclude that non-specific, physical shielding by macromolecules is an unlikely source of the often-observed ‘crypticity’ of glycolipids at the cell surface. Consistent with this interpretation was a relative lack of headgroup motional restriction seen for spin-labelled ganglioside headgroups in the same bilayers and in cell membranes.     

Glycolipid crypticity in membranes--not a simple shielding effect of macromolecules

The potential of membrane-bound macromolecules for shielding glycolipids from involvement in specific binding events was considered in model membranes. Serum albumin and several Dextrans were covalently derivatized with oleic acid so that they adsorbed irreversibly to lipid bilayers. This provided a means of generating bilayer membranes with a considerable layer of attached material. Gangliosides dispersed in such membranes were subjected to attack by the enzyme, neuraminidase, in order to assess their "accessibility'. We were surprised to find that we could not demonstrate any significant reduction in ganglioside hydrolysis in phosphatidylcholine bilayers bearing extensive surface coats of protein or polysaccharide. We conclude that non-specific, physical shielding by macromolecules is an unlikely source of the often-observed "crypticity' of glycolipids at the cell surface. Consistent with this interpretation was a relative lack of headgroup motional restriction seen for spin-labelled ganglioside headgroups in the same bilayers and in cell membranes.     

Gangliosides from human melanoma immunomodulate response of T cells to interleukin-2

The gangliosides expressed by normal melanocytes are predominantly GM3 (greater than 90%) and GD3 (less than 5%). Malignant melanoma can express several other types of gangliosides in significant quantities, including GM2 and GD2. Melanoma patients can develop an immune response against some of these ganglioside antigens on autologous melanoma cells. The four major gangliosides expressed by human melanoma cells (GM3, GD3, GM2, and GD2) were examined for their immunomodulatory effect on lymph node lymphocytes from melanoma patients. Gangliosides were added exogenously to lymphocytes grown in the presence of IL-2. Preferential interactions of specific melanoma gangliosides on IL-2 stimulation were found. While GM2 and GD2 enhanced the lymphocyte response to IL-2, GM3 and GD3 significantly inhibited this response. GM2 and GD2 differ from GM3 and GD3 by the presence of a terminal N-acetylgalactosamine. Since different gangliosides can up-regulate and down-regulate lymphocyte responses to IL-2, the ganglioside phenotype of melanoma cells may play a major role in determining whether an individual tumor causes immune stimulation or suppression.     

Gangliosides induce selective modulation of CD4 from helper T lymphocytes

The cluster designation (CD)4 molecule is one of several nonpolymorphic T lymphocyte surface proteins that have been implicated in T cell-target cell interactions, and is thought to play an important role in regulating T helper cell function. Previously, we found that gangliosides inhibited the function of rat T helper cell lines, and simultaneously inhibited the expression of the rat CD4 molecule identified by the W3/25 antibody. We have now evaluated the generality and mechanism(s) of ganglioside-induced modulation of CD4 expressed by mouse, rat, and human T helper lymphocytes. Ganglioside pretreatment induced rapid and selective disappearance of the CD4 molecule from T helper cells of all three species. The ganglioside effect was temperature- and dose-dependent, reversible within 24 hr of ganglioside removal, azide-insensitive, and was neutralized completely by 10% serum. CD4 modulation appeared to be a general property of gangliosides since the effect could be induced similarly by highly purified individual gangliosides with varying amounts of sialic acid, or by mixed gangliosides. The activity of gangliosides appeared to require both the lipid and sialated oligosaccharide moieties. Gangliosides did not inactivate antibody function, but prevented binding at the cell surface by 12 different monoclonal antibodies specific for a variety of different CD4 epitopes. Preclearance of CD4 by antibody-mediated capping reduced binding of 3H-GM1 to T helper cells. Labeled GM1 bound to several detergent-extracted and transblotted lymphocyte-associated proteins, but apparently did not bind directly to the CD4 molecule under these conditions. These results indicate that gangliosides induce a profound change in the molecular orientation of CD4 within the T helper cell membrane which renders epitopes on the CD4 molecule inaccessible to antibody. This ganglioside effect represents a novel pathway which may contribute to the understanding of the role of CD4 as a regulatory molecule and as a specific receptor for the acquired immune deficiency syndrome virus.     

Effect of micellar and bilayer gangliosides on proliferation of interleukin-2-dependent lymphocytes

Micellar gangliosides are potent inhibitors of the proliferation of the murine interleukin-2-dependent cell lines HT-2 and CTLL-2 in vitro. The glycolipids abolished both DNA and protein synthesis, and depressed cellular expansion, without affecting viability. These effects were reversible for at least 12 hr following ganglioside treatment. Highly sialylated gangliosides were more inhibitory, while structurally related molecules, including ganglioside oligosaccharides, simple and complex neutral glycosphingolipids, sulfatides, sphingomyelin, ceramides, and sphingosine had only small suppressive effects. Gangliosides were most effective as inhibitors when added during the first 4 hr of culture with the growth factor. Inhibition of DNA synthesis by gangliosides could be partially reversed by high concentrations of exogenous interleukin-2. Gangliosides incorporated into lipid bilayers, both multilamellar liposomes and unilamellar vesicles, were also effective inhibitors of interleukin-2-induced proliferation. Competition studies showed that both ganglioside micelles and lipid vesicles containing gangliosides prevented binding of 125I-interleukin-2 to high-affinity receptors on the lymphocyte surface. We have recently shown that gangliosides, in both micelles and lipid bilayer vesicles, are able to bind interleukin-2 (J. W. K. Chu and F. J. Sharom, Biochim, Biophys. Acta 1028, 205, 1990). Taken together, these results strongly suggest that inhibition of lymphocyte proliferation by gangliosides in micelles and vesicles arises as a direct result of competition between the glycolipids and high-affinity receptors for available interleukin-2.     

Relationship between the regulation of membrane enzyme activities by gangliosides and a possible ganglioside segregation in membrane microdomains

Laser and neutron scattering experiments showed that in mixed micelles of ganglioside GM2 and GT1b, a membrane mimicking system, the segregation of gangliosides may occur spontaneously. Photolabeling experiments using nitrophenylazide containing ganglioside GM1 proved that gangliosides added to cells in culture enter the cell and bind to its membrane as components of microdomains, which specifically interact with a protein of about 30 kDa. This suggests that ganglioside segregation may be a natural phenomenon. Gangliosides when added to granule cells in culture led to increase in protein phosphorylation, the effect exerted being related to the amount of ganglioside molecules inserted stably into the cell lipid layer and an increase of 0.7% of the cell original ganglioside content promoted an increase of 57% in the incorporation of 32P into cell membrane proteins. From the above results a possible relationship between ganglioside segregation and involvement of ganglioside in enzyme activity control is suggested.     

p56lck, LFA-1 and PI3K but not SHP-2 interact with GM1- or GM3-enriched microdomains in a CD4-p56lck association-dependent manner

We previously showed that the association of CD4 and G(M3) ganglioside induced by CD4 ligand binding was required for the down-regulation of adhesion and that aggregation of ganglioside-enriched domains was accompanied by transient co-localization of LFA-1 (lymphocyte function-associated antigen-1), PI3K (phosphoinositide 3-kinase) and CD4. We also showed that these proteins co-localized with the G(M1) ganglioside that partially co-localized with G(M3) in these domains. In the present study, we show that CD4-p56(lck) association in CD4 signalling is required for the redistribution of p56(lck), PI3K and LFA-1 in ganglioside-enriched domains, since ganglioside aggregation and recruitment of these proteins were not observed in a T-cell line (A201) expressing the mutant form of CD4 that does not bind p56(lck). In addition, we show that although these proteins associated in different ways with G(M1) and G(M3), all of the associations were dependent on CD4-p56(lck) association. Gangliosides could associate with these proteins that differ in affinity binding and could be modified following CD4 signalling. Our results suggest that through these associations, gangliosides transiently sequestrate these proteins and consequently inhibit LFA-1-dependent adhesion. Furthermore, while structural diversity of gangliosides may allow association with distinct proteins, we show that the tyrosine phosphatase SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), also required for the down-regulation of LFA-1-dependent adhesion, transiently and partially co-localized with PI3K and p56(lck) in detergent-insoluble membranes without association with G(M1) or G(M3). We propose that CD4 ligation and binding with p56(lck) and their interaction with G(M3) and/or G(M1) gangliosides induce recruitment of distinct proteins important for CD4 signalling to form a multimolecular signalling complex.     

Subcellular distribution and biosynthesis of rat liver gangliosides

Gangliosides have generally been assumed to be localized primarily in the plasma membrane. Analysis of gangliosides from isolated subcellular membrane fractions of rat liver indicated that 76% of the total ganglioside sialic acid was present in the plasma membrane. Mitochondria and endoplasmic reticulum fractions, while containing only low levels of gangliosides on a protein basis, each contained approx. 10% of total ganglioside sialic acid. Gangliosides also were present in the Golgi apparatus and nuclear membrane fractions, and soluble gangliosides were in the supernatant. Individual gangliosides were non-homogeneously distributed and each membrane fraction was characterized by a unique ganglioside composition. Plasma membrane contained only 14 and 28% of the total GD1a and GD3, respectively, but 80-90% of the GM1, GD1b, GT1b and GQ1b. Endoplasmic reticulum, when corrected for plasma membrane contamination, contained only trace amounts of GM1, GD1b, GT1b and GQ1b, but 11 and 5% of the total GD1a and GD3, respectively. The ganglioside composition of highly purified endoplasmic reticulum was similar. Ganglioside biosynthetic enzymes were concentrated in the Golgi apparatus. However, low levels of these enzymes were present in the highly purified endoplasmic reticulum fractions. Pulse-chase experiments with [3H]galactose revealed that total gangliosides were labeled first in the Golgi apparatus, mitochondria and supernatant within 10 min. Labeled gangliosides were next observed at 30 min in the endoplasmic reticulum, plasma membrane and nuclear membrane fractions. Analysis of the individual gangliosides also revealed that GM3, GM1, GD1a and GD1b were labeled first in the Golgi apparatus at 10 min. These studies indicate that gangliosides synthesized in the Golgi apparatus may be transported not only to the plasma membrane, but to the endoplasmic reticulum and to other internal endomembranes as well.     

Properties of a protease-sensitive acceptor component in mouse brain synaptosomes for Clostridium botulinum type B neurotoxin

To characterize an acceptor for Clostridium botulinum type B neurotoxin, its binding kinetics were examined with mouse brain synaptosomes treated with various enzymes. The amount of 125I-labelled neurotoxin bound to synaptosomes decreased upon treatment with lysyl endopeptidase, neuraminidase, or phospholipase C. The binding of the neurotoxin was partially recovered by incubation of neuraminidase-treated synaptosomes with ganglioside GT1b or GD1a. Gangliosides incorporated into untreated, lysyl endopeptidase-treated, and phospholipase C-treated synaptosomes had no effect on the binding of the neurotoxin. These results may suggest that type B neurotoxin binds to gangliosides in cooperation with a certain protease-sensitive substance on the neural membranes.     

Regulation of lymphocyte responses by human gangliosides. I. Characteristics of inhibitory effects and the induction of impaired activation

Gangliosides obtained from normal human brain were found to inhibit the in vitro activation of human lymphocytes by nonspecific mitogens and allogeneic cells at concentrations between 3 to 50 microgram/1.5 to 1.7 X 10(5) lymphocytes/0.2 ml culture. Ganglioside inhibition did not represent cytotoxic effects or altered lectin binding and was independent of the mitogen concentration. In addition to concentration, the degree of inhibition was dependent on the mode of presentation to lymphocytes, since gangliosides incorporated within liposomal membranes displayed a synergistic inhibitory effect greater than predicted from the cultures receiving either gangliosides or liposomes alone. In binding experiments, radiolabeled ganglioside GM1 became associated with human lymphocytes within 10 min. However, approximately 72 hr pre-exposure of human lymphocytes to gangliosides was required to induce impaired lymphocyte responses to mitogens and allogeneic cells. Thus, concentrations of human gangliosides equivalent to the levels occurring in the sera of patients with certain malignancies are capable of actively inhibiting lymphocyte stimulation in addition to inducing impaired lymphocyte responses.     

A model for ganglioside behaviour in cell membranes.

Gangliosides from beef brain have been spin-labeled using two different attaching groups and employed to investigate the physical nature of ganglioside behaviour in membranes. Results obtained using EPR spectroscopy indicate that, in phosphatidylcholine bilayers at physiological pH, ganglioside oligosaccharide chains are quite mobile and show a measurable tendency towards cooperative interaction amongst themselves. We suggest that the source of this interaction is the formation of H-bonds between sugar residues in adjacent ganglioside molecules. We present evidence that physiological (extracellular fluid) levels of Ca2+ and Mg2+ lead to cross-linking and condensing of ganglioside headgroups by complexing sialic acid carboxyl residues. Ganglioside headgroup interactions are not very sensitive to changes in the buffer ionic strength, suggesting that ionic interactions are of minor importance. We have found no measurable tendency for headgroup carbohydrate to penetrate hydrophobic regions of lipid bilayers. EPR spectroscopy was also used to follow the interaction of spin-labeled gangliosides with the glycoprotein, glycophorin, and with intact BHK cells. We suggest that these carbohydrate-based interactions should contribute significantly to the properties of the eucaryotic cell glycocalyx. We predict that laterally mobile carbohydrate-bearing components of cell surface will show a tendency to cluster about complex glycoprotein arrays, especially if the species involved bear accessible carboxylic acid functions.     

A model for ganglioside behaviour in cell membranes

Gangliosides from beef brain have been spin-labeled using two different attaching groups and employed to investigate the physical nature of ganglioside behaviour in membranes. Results obtained using EPR spectroscopy indicate that, in phosphatidylcholine bilayers at physiological pH, ganglioside oligosaccharide chains are quite mobile and show a measurable tendency towards cooperative interaction amongst themselves. We suggest that the source of this interaction is the formation of H-bonds between sugar residues in adjacent ganglioside molecules. We present evidence that physiological (extracellular fluid) levels of Ca²⁺ and Mg²⁺ lead to cross-linking and condensing of ganglioside headgroups by complexing sialic acid carboxyl residues. Ganglioside headgroup interactions are not very sensitive to changes in the buffer ionic strength, suggesting that ionic interactions are of minor importance. We have found no measurable tendency for headgroup carbohydrate to penetrate hydrophobic regions of lipid bilayers. EPR spectroscopy was also used to follow the interaction of spin-labeled gangliosides with the glycoprotein, glycophorin, and with intact BHK cells.We suggest that these carbohydrate-based interactions should contribute significantly to the properties of the eucaryotic cell glycocalyx. We predict that laterally mobile carbohydrate-bearing components of cell surfaces will show a tendency to cluster about complex glycoprotein arrays, especially if the species involved bear accessible carboxylic acid functions.     

Inhibition of the UDP-N-Acetylgalactosamine: GM3, N-Acetylgalactosaminyl Transferase by Gangliosides

Gangliosides in the range of 0.1-0.4 mM inhibited the UDP-N-acetylgalactosamine:GM3, N-acetylgalactosaminyl transferase (EC 2.4.1.79) of chicken retina. Other lipids such as phosphatidylethanolamine, sphingomyelin, sulfatides, and phosphatidic acid in concentrations similar to those of gangliosides did not affect the enzyme activity significantly. GM3 has an inhibition capability slightly less than that of gangliosides with two or three sialyl groups in their molecules, while asialo-GM1 is clearly less inhibitory. The inhibitory effect of a constant amount of GT1 ganglioside was higher at low concentrations of membrane preparation, but the inhibition was similar at different concentrations of the substrates GM3 or UDP-N-acetylgalactosamine and at all incubation times studied. The added gangliosides were found attached to the membranes. In this attached state they may act either as substrate or inhibitor. The inhibitory effect of gangliosides was not apparent when a mixture of Triton CF 54-Tween 80 was added to the incubation medium at concentrations greater than 0.33%.     

Ganglioside molecular species containing C18- and C20-sphingosine in mammalian nervous tissues and neuronal cell cultures

Gangliosides exist as a very complex mixture of species differing in both the hydrophilic and hydrophobic moieties. They are particularly abundant in the central nervous system (CNS), where they have been associated with development and maturation of the brain, neuritogenesis, synaptic transmission, memory formation and synaptic aging. Today, many data suggest that some of the effects exerted by gangliosides are due to interactions with proteins that participate in the transduction of signals through the membrane in membrane microdomains. A specific characteristic of CNS gangliosides is the structure of their long-chain base (LCB). In fact, considering all the mammalian cell sphingolipids, gangliosides, sulphatides, neutral glycosphingolipids, sphingomyelin and ceramides, it would seem that while the LCB with 18 carbons is the main component of all sphingolipids, only CNS gangliosides contain significant amounts of LCB with 20 carbons. C18-Sphingosine is always present in cell gangliosides; the individual ganglioside species containing C18-sphingosine increase during cell differentiation then remain constant during cell aging. Gangliosides containing C20-sphingosine are absent, or present only in traces, in undifferentiated cells but with the onset of cell differentiation they appear, their content slowly but continuously increasing throughout the life span. In this review we discuss the chemistry, physico-chemistry and metabolism of ganglioside species differing in LCB length and introduce the hypothesis that the varying ratio between C18- and C20-gangliosides during CNS development and aging can be instrumental in modulating membrane domain organisation and cell properties.     

The plasma membrane ganglioside sialidase cofractionates with markers of lipid rafts

Gangliosides of the plasma membrane are important modulators of cellular functions. Recent reports have shown their enrichment in glycosphingolipid-containing membrane microdomains, called glycosphingolipid-signaling domain or rafts, which can be isolated due to their insolubility in Triton X-100 and flotation through a sucrose gradient. In previous work on neuroblastoma cells we had found that a ganglioside-specific sialidase activity of the plasma membrane controlled proliferation and differentiation through selective ganglioside desialylation. Assuming the ganglioside sialidase to be close to its substrates in the membrane, we investigated its association with detergent-insoluble microdomains in the neuroblastoma cell line SK-N-MC. The results show that the ganglioside sialidase codistributes with the raft markers ganglioside GM1, flotillin, src family kinases, and glycosylphosphatidylinositol-anchored proteins in a fraction containing about 2% of cellular protein. The association of the ganglioside sialidase with glycosphingolipid-enriched membrane fractions therefore is in support of a role of this glycosidase in ganglioside-dependent signaling processes.     

Gangliosides of rat eye lens: a severe reduction in the content of C-series gangliosides following streptozotocin treatment

Gangliosides of eye lenses from normal and experimentally induced diabetic rats were investigated by methods including glycolipid-overlay techniques. Adult rat eye lens showed a complex ganglioside pattern that consisted of six major ganglioside components. These gangliosides were identified as GM3, GD3, GD1a, GD1b, GT1b, and GQ1b based upon their reactivity to anti-GM1 antibody after in situ sialidase treatment and mobility on thin-layer chromatography (TLC). Gangliosides in eye lens were further characterized by TLC-immunostaining with A2B5, a specific monoclonal antibody directed toward c-series gangliosides. Eye lens contained GT3 as the main c-series ganglioside component. Unexpectedly, the relative concentration of GT3 in total gangliosides of eye lens was highest among neural and extra-neural tissues examined. Administration of streptozotocin to rats caused a severe reduction in the GT3 content in eye lenses as early as day 3 without apparent changes in the composition of major gangliosides. Alloxan failed to produce such an effect despite producing similar hyperglycemic conditions. These results suggest that rat eye lens probably contains a streptozotocin-susceptible cell type(s), which is highly enriched with c-series gangliosides.     

Recent advances in identifying the functions of gangliosides

The recent development of several new approaches has proven extremely useful in identifying functions for gangliosides, the sialic-acid containing glycosphingolipids. The first is the incorporation of exogenous gangliosides into the plasma membrane of ganglioside-deficient cells. Using this approach, specific gangliosides have been identified as the receptors for certain bacterial toxins and viruses and as important factors in the organization of fibronectin into an extracellular matrix. The second approach has been a ligand blotting technique which allows detection of ganglioside-binding proteins such as toxins and antibodies. Gangliosides are separated by thin-layer chromatography and overlain with the protein of interest. Specific binding of the ligand to gangliosides can then be detected by either direct or indirect methods. The third approach is the use of the B or binding subunit of cholera toxin as a specific probe for endogenous plasma membrane ganglioside function. The ability of the B subunit to alter the growth of cells directly demonstrates a role for gangliosides as biotransducers of signals for the regulation of cell growth.     

Comparative analysis of the action of thymic and cerebral GD3 gangliosides on the sensitivity of tumor cells to natural splenic effectors

Thymic gangliosides GM3 and GD3 and LacCer incorporated into the membrane of the tumor target cell leukemia (YAC) increase its sensitivity to the membrane toxic action of spleen effectors. Unlike thymic gangliosides GD3, ganglioside GD3 of the brain origin substantially reduces tumor cell sensitivity to spleen effectors. Some other brain glycosphingolipids differing essentially in the structure of the carbohydrate part of the molecule exert the same action. It has been shown in model experiments with incorporation into the tumor cell membrane of brain ganglioside GD3 combined with thymic LacCer or with egg phosphatidylcholine that the increase in the sensitivity of the tumor cell membrane to spleen effectors is linked with a change in the properties of the lipid membrane matrix under the effect of unsaturated fatty acids (e.g. in experiments with phosphatidylcholine). It follows from the data presented that the capability of influencing the sensitivity of tumor cells to natural spleen effectors largely depends on the differences in the structure of the cearamide part of brain and thymic GD3.     

Dansylated thyrotropin as a probe of hormone-receptor interactions.

A strongly fluorescent 5-dimethylamino-1-naphthalene sulfonate (dansyl) derivative of bovine thyrotropin has been prepared. The dye-conjugated hormone is bioactive and shares, essentially unchanged, the membrane binding and adenylate cyclase stimulatory activities of the native hormone. Binding of 125I-labeled dansyl-thyrotropin to thyroid plasma membranes is sensitive to inhibition by gangliosides and, as is the case for the binding of 125I-thyrotropin, galactosyl-N-acetylgalactosaminyl[N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosylceramide (GDIb) is the most potent binding inhibitor. Gangliosides interact with dansyl-thyrotropin, causing a large increase of the quantum yield and a 5- to 10-nm blue shift of the emission maximum of the hormone-bound naphthalene chromophore; gangliosides cause no change in the fluorescent properties of the free dye. The fluorescence enhancement caused by gangliosides can be specifically reversed by unlabeled thyrotropin. The effect of gangliosides on dansyl-thyrotropin fluorescence is strongly salt-dependent; salts cannot, however, reverse the formation of the dansyl-thyrotropin.ganglioside complex once it has formed. The salt data suggest that the association of the ganglioside with dansyl-thyrotropin is dominated by electrostatic interactions, but that salt-independent, short range interactions, most likely hydrophobic, dominate the dissociation of the dansyl-thyrotropin-ganglioside adduct. Sucrose gradient centrifugation, ultracentrifugation, and fluorescence polarization data indicate that the gangliosides are micellar in nature under the conditions of these experiments. Acid titration of dansyl-thyrotropin causes a marked quenching of dansyl fluorescence which in part reflects dissociation of the hormone into its constituent alpha and beta subunits. In the presence of GDIb, but not N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GDIa), pH-dependent quenching and subunit dissociation are essentially eliminated. Circular dichroism results and fluorescence polarization studies support the interpretation that the ganglioside interaction causes a conformational change in the thyrotropin molecule. The acid titration data together with differences in the ability of gangliosides to influence the tyrosine fluorescence of the thyrotropin molecule indicate that different gangliosides induce different conformational perturbations in the thyrotropin molecule.     

Exogenous glycosphingolipids suppress growth rate of transformed and untransformed 3T3 mouse cells

Gangliosides added to culture media reduced both the growth rate and saturation density of SV40-virus transformed and untransformed 3T3 cells. Monosialogangliosides were much more effective than disialogangliosides in inhibiting growth rate. These gangliosides caused little or no cell damage or significant morphological alteration of the individual cells. Trisialoganglioside markedly reduced growth rate but in some experiments also caused cell damage and lysis. The isolated carbohydrate moiety of the ganglioside G_Gtet1, the sialo-oligosaccharide galactopyranosyl-N-acetyl-galactosaminyl-(N-acetylneuraminyl)-galactosyl-glucose, did not inhibit growth of SV40 3T3 cells in culture. Ceramide alone was also ineffective as a growth inhibitor. However, the tetrahexosyl ceramide derived from the above ganglioside was equally as effective as the parent compound in retarding growth of SV40 3T3 cells. Similarly, mono-, di- and trihexosyl ceramides were also effective in inhibiting growth of these cells. Gangliosides added to the culture media were rapidly accumulated by cells, apparently at the plasma membrane. The accumulated ganglioside was not degraded by the cells. However, the accumulated ganglioside could be distinguished from gangliosides synthesized in vivo by the lability of the former to neuraminidase.