Cholera toxin and membrane gangliosides: Binding and adenylate cyclase activation in normal and transformed cells

Summary A virally transformed, ganglioside GM₁-deficient cell line binds 2% of the cholera toxin (choleragen) bound by the parent, line and is less responsive to choleragen with respect to adenylate cyclase stimulation. This biological response is maximal when 10% of choleragen-binding sites in the transformed line, or 0.5% in the parent line, are occupied. In contrast, in isolated fat cells saturation of binding and adenylate cyclase stimulation are seen at very similar concentrations.Incubation of ganglioside GM₁ with intact cells increases choleragen binding (defined here as ganglioside incorporation) in the transformed cell line but does not enhance the biological response to choleragen. Stimulation of adenylate cyclase is enhanced in isolated fat cells, however, by exogenous ganglioside GM₁. The binding and cyclase response in fat cells can be reduced by the addition of the inactive analog and competitive antagonist, choleragenoid, and there is recovery of the enzyme response and binding upon subsequent addition of exogenous GM₁. Failure of enhancement in the transformed cell line is explained by the presence of a five- to tenfold excess of binding sites over the number required for the full biological effect of choleragen. Cells with a large excess of toxin receptors are relatively refractory to the blocking effects of choleragenoid on biological responses. Notably, untransformed cells, which contain large quantities of toxin receptor, cannot incorporate exogenously added ganglioside GM₁. These findings suggest the possible existence in the cytoplasmic membrane of specific molecular structures, present in finite and limited number, for recognizing and accepting ganglioside molecules exposed to the external medium.     

Pasteurella multocida toxin stimulates mitogenesis and cytoskeleton reorganization in Swiss 3T3 fibroblasts

Pasteurella multocida toxin (PMT) causes cytoplasmic retraction in epithelial cells, activates osteoclast neoformation, and is a potent mitogen for Swiss 3T3 fibroblasts. In the present study designed to further investigate the effects of PMT on cell shape and proliferation, we report that the mitogenic effect of affinity-purified PMT on quiescent 3T3 cells was even superior at 5 ng/ml to that of fetal bovine serum or bombesin. This positive effect was inhibited by heat denaturation and methylamine treatment (this agent blocks internalization). Preincubation of PMT with gangliosides GM₁, GM₂, or GM₃ counteracted its effect on DNA synthesis, suggesting that the toxin binds to GM-type ceramides on target cells. The distribution of F-actin was analyzed in control/treated cells using FITC-conjugated phalloidin. In comparison with FBS and bombesin, PMT triggered a more rapid and profound reorganization of cortical actin into prominent stress fibers after only 5–10 min. This event lead to the retraction of cells after only 30 min and ultimately to the induction of mitotic figures. Interestingly, methylamine blocked the effects of PMT on stress fiber formation and cell retraction but not the ruffling response, suggesting that some early events may not require toxin internalization. In summary, these findings indicate that PMT concomitantly exerts a strong mitogenic activity and a rapid stimulation of cytoskeletal rearrangements, possibly after binding to membrane gangliosides and subsequent internalization. We propose that this toxin could be used in the future as a defined inducer of transduction signals involved in cellular proliferation and control of cell shape. © 1996 Wiley-Liss, Inc.     

Monomer of the B Subunit of Heat-Labile Enterotoxin from Enterotoxigenic Escherichia coli Has Little Ability to Bind to GM1 Ganglioside Compared to Its Coligenoid

Coligenoid, composed of the B subunit of heat-labile enterotoxin from enterotoxigenic Escherichia coli, was separated into monomers in the presence of 2% propionic acid containing 6 M urea (pH 3.8). Monomers equilibrated against 0.75% or 0.5% propionic acid containing 3 M urea (pH 3.8) did not reassemble into coligenoid. Complexes of GM₁ ganglioside and coligenoid in these buffers were detected by SDS-polyacrylamide gel electrophoresis, but those of the GM₁ ganglioside and monomers were not. The binding ability of monomer to GM₁ ganglioside in these buffers was about 1% of that of normal coligenoid by GM₁-enzyme-linked immunosorbent assay. Moreover, monomers in these buffers reassembled into coligenoid by buffering against original TEAN buffer, and the binding ability of the resulting coligenoid to GM₁ ganglioside was identical to that of native coligenoid. These data suggest that although coligenoid formation is important for the receptor binding of the B subunit, little binding ability to GM₁ ganglioside remains in monomer of the B subunit.     

GM3 ganglioside induces hamster fibroblast growth inhibition in chemically-defined medium: ganglioside may regulate growth factor receptor function

GM₃ or GM₁ ganglioside exogenously added in chemically-defined medium incorporate equally into cells. However, only GM₃ showed a significant growth inhibition to hamster fibroblasts (BHK). The GM₃-fed cells became refractory to growth stimulation by fibroblast growth factor (FGF) in chemically-defined media. Radiolabeled FGF accumulated on GM₃-fed cells, but not on GM₁-fed cells. Both GM₃ and GM₁ did not directly interact with FGF. These data suggest that GM₃ may regulate the function of the receptors for growth hormones.     

Binding Cooperativity Matters: A GM1-Like Ganglioside-Cholera Toxin B Subunit Binding Study Using a Nanocube-Based Lipid Bilayer Array

Protein-glycan recognition is often mediated by multivalent binding. These multivalent bindings can be further complicated by cooperative interactions between glycans and individual glycan binding subunits. Here we have demonstrated a nanocube-based lipid bilayer array capable of quantitatively elucidating binding dissociation constants, maximum binding capacity, and binding cooperativity in a high-throughput format. Taking cholera toxin B subunit (CTB) as a model cooperativity system, we studied both GM₁ and GM₁-like gangliosides binding to CTB. We confirmed the previously observed CTB-GM₁ positive cooperativity. Surprisingly, we demonstrated fucosyl-GM₁ has approximately 7 times higher CTB binding capacity than GM₁. In order to explain this phenomenon, we hypothesized that the reduced binding cooperativity of fucosyl-GM₁ caused the increased binding capacity. This was unintuitive, as GM₁ exhibited higher binding avidity (16 times lower dissociation constant). We confirmed the hypothesis using a theoretical stepwise binding model of CTB. Moreover, by taking a mixture of fucosyl-GM₁ and GM₂, we observed the mild binding avidity fucosyl-GM₁ activated GM₂ receptors enhancing the binding capacity of the lipid bilayer surface. This was unexpected as GM₂ receptors have negligible binding avidity in pure GM₂ bilayers. These unexpected discoveries demonstrate the importance of binding cooperativity in multivalent binding mechanisms. Thus, quantitative analysis of multivalent protein-glycan interactions in heterogeneous glycan systems is of critical importance. Our user-friendly, robust, and high-throughput nanocube-based lipid bilayer array offers an attractive method for dissecting these complex mechanisms.     

Part I: an x-ray scattering study of cholera toxin penetration and induced phase transformations in lipid membranes

Cholera toxin is a highly efficient biotoxin, which is frequently used as a tool to investigate protein-membrane interactions and as a reporter for membrane rafts. Cholera toxin binds selectively to gangliosides with highest affinity to GM₁. However, the mechanism by which cholera toxin crosses the membrane remains unresolved. Using x-ray reflectivity and grazing incidence diffraction, we have been able to monitor the binding and penetration of cholera toxin into a model lipid monolayer containing the receptor GM₁ at the air-water interface. Very high toxin coverage was obtained allowing precise measurements of how toxin binding alters lipid packing. Grazing incidence x-ray diffraction revealed the coexistence of two monolayer phases after toxin binding. The first was identical to the monolayer before toxin binding. In regions where toxin was bound, a second membrane phase exhibited a decrease in order as evidenced by a larger area per molecule and tilt angle with concomitant thinning of the monolayer. These results demonstrate that cholera toxin binding induces the formation of structurally distinct, less ordered domains in gel phases. Furthermore, the largest decrease in lateral order to the monolayer occurred at low pH, supporting a low endosomal pH in the infection pathway. Surprisingly, at pH = 8 toxin penetration by the binding portion of the toxin, the B₅ pentamer, was also observed.     

Biomimetic particles for isolation and reconstitution of receptor function

Biomimetic particles supporting lipid bilayers are becoming increasingly important to isolate and reconstitute protein function. Cholera toxin (CT) from Vibrio cholerae, an 87-kDa AB₅ hexameric protein, and its receptor, the monosialoganglioside GM₁, a cell membrane glycolipid, self-assembled on phosphatidylcholine (PC) bilayer-covered silica particles at 1 CT/5 GM₁ molar ratio in perfect agreement with literature. This receptor-lig-and recognition represented a proof-of-concept that receptors in general can be isolated and their function reconstituted using biomimetic particles, i.e., bilayer-covered silica. After incubation of colloidal silica with small unilamellar PC vesicles in saline solution, pH 7.4, PC adsorption isotherms on silica from inorganic phosphorus analysis showed a high PC affinity for silica with maximal PC adsorption at bilayer deposition. At 0.3 mM PC, fluorescence of pyrene-labeled GM₁ showed that GM₁ incorporation in biomimetic particles increased as a function of particles concentration. At 1 mg/mL silica, receptor incorporation increased to a maximum of 40% at 0.2–0.3 mM PC and then decreased as a function of PC concentration. At 5 μM GM₁, 0.3 mM PC, and 1 mg/mL silica, CT binding increased as a function of CT concentration with a plateau at 2 mg bound CT/m² silica, which corresponded to the 5 GM₁/1 CT molar proportion and showed successful reconstitution of receptor-ligand interaction.     

Effects of Cell Density on Lipids of Human Glioma and Fetal Neural Cells

Abstract: Gangliosides, phospholipids, and cholesterol of human glioma (12-18) and fetal neural cells (CH) were analyzed at specified cell densities, from sparse to confluent. Total ganglioside sialic acid, phospholipid phosphorus, and cholesterol increased in the glioma cells on a per cell, mg protein, or mg total lipid basis two- to threefold as cell density increased 25-fold. These same three constituents in the fetal cells increased with cell density on a per cell and mg protein basis but not on a per mg total lipid basis. In glioma cells, the di- and trisialogangliosides (GD₂+ GD_lb+ GT₁) increased from 1–2% of total ganglioside sialic acid at sparse densities to 7–8% at intermediate (logarithmic phase) densities to 10–13% at confluent densities. The set of simpler gangliosides (GM₄+ GM₃+ GM₂) decreased from 50% of total ganglioside sialic acid at sparse glioma cell densities, to 36% at intermediate and 30% at confluent densities. In the fetal neural cells, the set of gangliosides (GM₄+ GM₃+ GM₂) had about 48% of total ganglioside sialic acid in both sparse and confluent preparations. The fetal cells were twofold higher in GM₃ (32.4 ± 2.1%) than the glioma cells (16.8 ± 1.6%), but lower in GM_t (9.1 ± 0.9% versus 18.2 ± 1.8%), cell densities notwithstanding. Confluent cell preparations of both cell lines were consistently higher in ethanolamine plasmalogen than sparse cells. We conclude that in these two neural cell lines quantitative changes in ganglioside and phospholipid species occurred correlatively as cell densities increased. Higher glioma cell densities were associated with greater proportions of complex ganglioside species. These changes in cell membrane constituents during growth may result from cell contact and may indicate a role for them in cell growth regulation and/or differentiation.     

Monosialoganglioside GM1 increases survival of thymocytes

Gangliosides are normal constituents of the plasma membrane. Exogenous gangliosides can be incorporated into the membrane and extensive research in nervous tissue has demonstrated a beneficial effect of gangliosides on the functional recovery of lesioned neurons and protection against neurotoxins. This paper shows that the effect of gangliosides is not restricted to neurons. The monosialoganglioside GM₁ efficiently increases the survival of thymocytes and protects them against both the lytic effect of the glucocorticoid prednisolone and the effect of a thymocytotoxic serum. The protective effect of GM₁ was achieved bothin vitro andin vivo.     

Interaction of Fibroblast Growth Factor-2 (FGF-2) with Free Gangliosides: Biochemical Characterization and Biological Consequences in Endothelial Cell Cultures

Exogenous gangliosides affect the angiogenic activity of fibroblast growth factor-2 (FGF-2), but their mechanism of action has not been elucidated. Here, a possible direct interaction of sialo-glycolipids with FGF-2 has been investigated. Size exclusion chromatography demonstrates that native, but not heat-denatured, ¹²⁵I-FGF-2 binds to micelles formed by gangliosides GT_1b, GD_1b, or GM₁. Also, gangliosides protect native FGF-2 from trypsin digestion at micromolar concentrations, the order of relative potency being GT_1b > GD_1b > GM₁ = GM₂ = sulfatide > GM₃ = galactosyl-ceramide, whereas asialo-GM₁, neuraminic acid, and N-acetylneuramin-lactose were ineffective. Scatchard plot analysis of the binding data of fluorochrome-labeled GM₁ to immobilized FGF-2 indicates that FGF–2/GM₁ interaction occurs with a K_d equal to 6 μM. This interaction is inhibited by the sialic acid-binding peptide mastoparan and by the synthetic fragments FGF-2(112–129) and, to a lesser extent, FGF-2(130–155), whereas peptides FGF-2(10–33), FGF-2(39–59), FGF-2(86–96), and the basic peptide HIV-1 Tat(41–60) were ineffective. These data identify the COOH terminus of FGF-2 as a putative ganglioside-binding region. Exogenous gangliosides inhibit the binding of ¹²⁵I-FGF-2 to high-affinity tyrosine-kinase FGF-receptors (FGFRs) of endothelial GM 7373 cells at micromolar concentrations. The order of relative potency was GT_1b > GD_1b > GM₁ > sulfatide a = sialo-GM₁. Accordingly, GT_1b,GD_1b, GM₁, and GM₂, but not GM₃ and asialo-GM₁, prevent the binding of ¹²⁵I-FGF-2 to a soluble, recombinant form of extracellular FGFR-1. Conversely, the soluble receptor and free heparin inhibit the interaction of fluorochrome-labeled GM₁ to immobilized FGF-2. In agreement with their FGFR antagonist activity, free gangliosides inhibit the mitogenic activity exerted by FGF-2 on endothelial cells in the same range of concentrations. Also in this case, GT_1b was the most effective among the gangliosides tested while asialo-GM₁, neuraminic acid, N-acetylneuramin-lactose, galactosyl-ceramide, and sulfatide were ineffective. In conclusion, the data demonstrate the capacity of exogenous gangliosides to interact with FGF-2. This interaction involves the COOH terminus of the FGF-2 molecule and depends on the structure of the oligosaccharide chain and on the presence of sialic acid residue(s) in the ganglioside molecule. Exogenous gangliosides act as FGF-2 antagonists when added to endothelial cell cultures. Since gangliosides are extensively shed by tumor cells and reach elevated levels in the serum of tumor-bearing patients, our data suggest that exogenous gangliosides may affect endothelial cell function by a direct interaction with FGF-2, thus modulating tumor neovascularization.     

Developmental changes in gangliosides during myogenesis of a rat myoblast cell line and its drug resistant variants.

Cloned cells of a myoblast line show the presence of GM₃, GM₂, GM₁ and GD_1a gangliosides. The amount of GM₃, GM₂ and GM₁ gangliosides does not vary significantly during the differentiation of myoblasts to myotubes. However, the concentration of GD_1a transiently increases almost 3-fold just prior to the fusion of myoblasts and returns to the basal levels in the myotubes. Mutant myoblasts selected for 5-azacytidine resistance and unable to fuse produce only GM₃ and traces of GM₂. We conclude that GD_1a probably participates in the fusion process through yet unknown mechanism.     

Anti-GM3-lactam monoclonal antibodies of the IgG type recognize natural GM3-ganglioside lactone but not GM3-ganglioside

Immunization of mice with a synthetic GM₃-lactam-BSA (bovine serum albumin) conjugate (designed to emulate the corresponding natural GM₃-lactone conjugate), followed by fusion of splenocytes with myeloma cells, gave rise to more than 300 monoclonal hybridomas producing antibodies to GM₃-lactam-BSA, which did not react with Glc-BSA and BSA. Eight antibody clones were randomly chosen from the positive 300 hybridomas. The eight clones, all belonging to the IgG class, were unreactive against GM₃-ganglioside, whereas two antibodies (P5-1 and P5-3, both IgG₁, ) reacted with GM₃-ganglioside lactone. Binding of these two antibodies to the GM₃-lactam-BSA conjugate was inhibited by soluble glycosides of GM₂-, GM₃-, and GM₄-lactam and by GM₃- and GM₄-lactam, respectively, but not by Gb₃ or asialo-GM₁ and GM₂-saccharides. A third antibody (P3; IgG_2b, ) was inhibited by GM₂-, GM₃-, and GM₄-lactam, but did not recognize GM₃-ganglioside lactone.     

Morphological and chromosomal characterization of three new continuous cell lines of Drosophila melanogaster

Three continuous cell lines (GM₁, GM₂ and GM₃) were obtained from embryos of Drosophila melanogaster. Karyotypic analysis revealed characteristics distinguishing each line. Except for some minor variations GM₁ cells had an X and a centric heterochromatic fragment (which is a portion of the Y). GM₂ line was characterized by XO cells showing two new telocentric chromosomes while an autosome of the II pair was missing. GM₃ cells were XY; the Y chromosome, however, was shorter than the normal, having a deletion of the terminal section of the short arm. Several problems concerning the origin of these different genomes are discussed.This work was supported by a grant of the Consiglio Nazionale delle Ricerche, Roma.     

Differential interactions of two local anesthetics with phospholipid membrane and nonerythroid spectrin: Localization in presence of cholesterol and ganglioside,GM1

Interactions of two local anesthetics, dibucaine and tetracaine have been studied with phospholipid vesicles containing cholesterol and/or monosialogangliosides (GM₁) using fluorescence spectroscopy. The fluorescence intensity of tetracaine showed a marked increase with the increasing molar ratio of the phospholipid to tetracaine, while that of dibucaine showed opposite effects. Steady state anisotropy and the wavelength of maximum emission (λ_max) decreased with the increasing phospholipids to tetracaine ratio. The extent of such changes in anisotropy and λ_max in the presence and absence of two important components of neuronal membranes, cholesterol and GM₁ indicated differential membrane localization of the two local anesthetics. To understand the intercellular mode of action of local anesthetics, we have also studied the interactions of dibucaine and tetracaine with brain spectrin which indicate differential spectrin interactions with similar binding strength. Thermodynamic parameters associated with such binding reveal that binding is favored by entropy. Tetracaine brings about distinct structural changes in spectrin compared to dibucaine, as reflected in the tryptophan mean lifetime and far-UV CD spectra. Tetracaine also exhibits a detergent-like property inducing concentration dependent decrease in spectrin anisotropy, further indicating structural changes in brain spectrin with probable implications in its anesthetic potential.     

Line tension at lipid phase boundaries regulates formation of membrane vesicles in living cells

Ternary lipid compositions in model membranes segregate into large-scale liquid-ordered (L_o) and liquid-disordered (L_d) phases. Here, we show μm-sized lipid domain separation leading to vesicle formation in unperturbed human HaCaT keratinocytes. Budding vesicles in the apical portion of the plasma membrane were predominantly labelled with L_d markers 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, 1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate and weakly stained by L_o marker fluorescein-labeled cholera toxin B subunit which labels ganglioside GM₁ enriched plasma membrane rafts. Cholesterol depletion with methyl-β-cyclodextrin enhanced DiI vesiculation, GM₁/DiI domain separation and was accompanied by a detachment of the subcortical cytoskeleton from the plasma membrane. Based on these observations we describe the energetic requirements for plasma membrane vesiculation. We propose that the decrease in total ‘L_o/L_d’ boundary line tension arising from the coalescence of smaller L_d-like domains makes it energetically favourable for L_d-like domains to bend from flat μm-sized surfaces to cap-like budding vesicles. Thus living cells may utilize membrane line tension energies as a control mechanism of exocytic events.     

The Effect of Apical and Basolateral Lipids on the Function of the Band 3 Anion Exchange Protein

Although many polarized proteins are sorted to the same membrane domain in all epithelial tissues, there are some that exhibit a cell type–specific polarity. We recently found that band 3 (the anion exchanger AE1) was present in the apical membrane of a renal intercalated cell line when these cells were seeded at low density, but its targeting was reversed to the basolateral membrane under the influence of an extracellular matrix protein secreted when the cells were seeded at high density. Because apical and basolateral lipids differ in epithelia, we asked what effect might these lipids have on band 3 function. This question is especially interesting since apical anion exchange in these cells is resistant to disulfonic stilbene inhibitors while basolateral anion exchange is quite sensitive. Furthermore, the apical anion exchanger cannot be stained by antibodies that readily identify the basolateral protein.

We used short chain sphingolipid analogues and found that sphingomyelin was preferentially targeted to the basolateral domain in the intercalated cell line. The ganglioside GM₁ (Gal 1β1, 3GalNAcβ1, 4Gal-NeuAcα2, 3Galβ1, 4Glc ceramide) was confined to the apical membrane as visualized by confocal microscopy after addition of fluorescent cholera toxin to filter grown cells. We reconstituted erythrocyte band 3 into liposomes using apical and basolateral types of lipids and examined the inhibitory potency of 4,4′-dinitorsostilbene-2,2′-disulfonic acid (DNDS; a reversible stilbene) on ³⁵SO₄/SO₄ exchange. Although anion exchange in sphingomyelin liposomes was sensitive to inhibition, the addition of increasing amounts of the ganglioside GM₁ reduced the potency of the inhibitor drastically. Because these polarized lipids are present in the exofacial surface of the bilayer, we propose that the lipid structure might influence the packing of the transmembrane domains of band 3 in that region, altering the binding of the stilbenes to these chains. These results highlight the role of polarized lipids in changing the function of unpolarized proteins or of proteins whose locations differ in different epithelia.

     

Effects of Ganglioside GM3 on Phospholipid Turnover of Human Leukemic J6-2 Cells

Ganglioside GM₃ was reported to induce the differentiation of HL-60 cells to differentiate along the macrophage-monocytic route. We used human monocytoid leukemia J6-2 cells and successfully induced differentiation by GM₃. Because differentiation is accompanied by retarded growth rate and cell cycle is intimately related to phospholipid metabolism, so we explored how GM₃ was related to phospholipid metabolism. By using [³²P]Pi, [³H-CH₃]choline, [³H-CH₃]SAM, and [³H]inositol as radioactive tracers, we studied the turnover changes of phospholipids and their metabolites induced by GM₃. For the morphological changes of differentiation to occur, the cells had to be treated with GM₃ at a concentration of 50 M for 5-6 days, but the phospholipid changes occurred at a very early stage of GM₃ treatment (only 1 h). Our results indicate that GM₃ stimulated PE methylation pathway inhibited both CDP-choline pathway and PI cycle. The phospholipid changes may constitute the early events in differentiation induced by GM₃.     

Gangliosides Are Important for the Preservation of the Structure and Organization of RBL-2H3 Mast Cells

Gangliosides are known to be important in many biological processes. However, details concerning the exact function of these glycosphingolipids in cell physiology are poorly understood. In this study, the role of gangliosides present on the surface of rodent mast cells in maintaining cell structure was examined using RBL-2H3 mast cells and two mutant cell lines (E5 and D1) deficient in the gangliosides, GM₁ and the α-galactosyl derivatives of the ganglioside GD_1b. The two deficient cell lines were morphologically different from each other as well as from the parental RBL-2H3 cells. Actin filaments in RBL-2H3 and E5 cells were under the plasma membrane following the spindle shape of the cells, whereas in D1 cells, they were concentrated in large membrane ruffles. Microtubules in RBL-2H3 and E5 cells radiated from the centrosome and were organized into long, straight bundles. The bundles in D1 cells were thicker and organized circumferentially under the plasma membrane. The endoplasmic reticulum, the Golgi complex, and the secretory granule matrix were also altered in the mutant cell lines. These results suggest that the mast cell–specific α-galactosyl derivatives of ganglioside GD_1b and GM₁ are important in maintaining normal cell morphology. (J Histochem Cytochem 58:83–93, 2010)     

GANGLIOSIDE COMPOSITION OF CHEMICALLY INDUCED RAT NEURAL TUMORS AND CHARACTERIZATION OF HEMATOSIDE FROM NEURINOMAS

Abstract– Experimental rat neural tumors in offspring were induced transplacentally by a single injection of a chemical carcinogen, ethylnitrosourea, 20mg/kg body wt, in the tail vein of the mother. The ganglioside content and pattern in these tumors and the normal tissues from which the tumors originated are described. The ganglioside content in tumors was reduced, on wet tissue weight basis, compared to normal control. However, there was no significant difference of ganglioside content on dry weight or protein basis. Altered ganglioside composition was found in most of the neural tumors. In central nervous system tumors, there was some increase in GM₃ and GT_1b′ (nomenclature according to Svennerholm , 1963), a marked decrease in GM₁ and some decrease in GD_1a, but no apparent loss in GD_1b. Extreme simplification of ganglioside pattern was seen in tumors originated from peripheral nervous system. Large accumulation of GM₃ with concomitant loss of all the higher gangliosides was seen. GM₃ from neurinomas as well as from normal gray matter was isolated and characterized. GM₃ from neurinomas separated into two bands on thin layer chromatographic plates. Both these GM₃ bands had identical sphingosine and carbohydrate composition but differed in their fatty acid composition. The fast moving band had 77% of the total fatty acids as C_20:0 or longer chain while the slow moving band had only 22% of the long chain fatty acids. Normal gray matter GM₃ had one major band containing 82% of and only 17% of the fatty acids as C_20:0 or higher. It is suggested that in the tumor cells either the specificity of the enzyme cytidine monophosphate-N-acetyl neuraminic acid: ceramide dihexoside sialyltransferase for C_18.0 fatty acid containing glycolipid was altered or that the compartmentation of precursor pools for the simpler glycolipids present in normal tissue did not exist in transformed cells.     

Evidence of a distribution difference between two gangliosides in bilayer membranes

Freeze-etch electron microscopy, a platinum shadowing technique, has been used to compare the lateral distribution of several gangliosides in bilayer model membranes by directly visualizing bound lectin molecules. In particular, GM₁ and GD_1a, major components of brain ganglioside, were studied in phase-separated mixtures of dipalmitoyl- and dielaidoylphosphatidylcholines exposed to Ricinus communis agglutinin and wheat germ agglutinin. The distribution of glycolipid showed evidence of microheterogeneity in that bound lectin tended to occur in clusters of several or more molecules. With GD_1a as receptor such clusters were small and very uniformly distributed over the membrane surface. Somewhat larger, irregularly spaced clusters of up to a dozen lectin particles were more typical of membranes bearing GM₁ and, in addition, there were occasional extensive patches of bound lectin coexisting with areas apparently devoid of glycolipid receptor in phase-separated mixtures of dipalmitoyl- and dielaidoylphosphatidylcholine. Gangliosides in the latter mixtures were not obviously influenced in their lateral distribution by the presence of coexisting fluid and rigid domains. These basic observations seem to extend to bilayer membranes containing mixtures of two gangliosides. The patterns of lectin binding were not grossly affected by incubation time or history of warming and cooling. This study was extended to bilayers of pure dipalmitoylphosphatidylcholine in expectation that the distinctive features characteristic of the P_β′ phase of this lipid might accentuate any behavioural differences between GM₁ and GD_1a.GM₁ was found to exist preferentially in the ‘trough’ regions between P_β′ ripples, while GD_1a showed no apparent preferential arrangement. Given that bound lectins adequately reflect glycolipid distribution in membranes, it would appear that structurally different glycolipids from the same host membrane can assume different distributions on the basis of interactions with defined lipid host matrices.