GANGLIOSIDES IN NERVOUS TISSUE CULTURES AND BINDING OF 125I-LABELLED TETANUS TOXIN, A NEURONAL MARKER

Abstract— —Continuous cell lines, primary cell cultures derived from embryonic CNS, and homogenates made from adult and embryonic CNS were compared with respect to their lipid pattern and their ability to bind ¹²⁵I-labelled tetanus toxin. In parallel experiments de novo synthesis of gangliosides in the cell lines was studied, using [¹⁴C]glucosamine as precursor. Of the total lipid only gangliosides were specifically labelled by [¹⁴C]glucosamine. The patterns of the de novo synthesized gangliosides corresponded to those present in the respective cells.
Pronounced binding of ¹²⁵I-labelled toxin was only detectable in tissues containing long-chain gangliosides (ganglioside C which represents GDIb and GTI).
Accordingly, hybrid (neuroblastoma x glioma) cells, due to their lack of long-chain gangliosides, bound just-discernible amounts of labelled toxin. When previously exposed to gangliosides, their binding of tetanus toxin tremendously increased.
It was concluded that only the long-chain gangliosides in the neuronal cells are functionally involved in the binding of the tetanus toxin and that these acceptors of tetanus toxin can be transplanted.     

Neuronal sensitivity to tetanus toxin requires gangliosides.

Tetanus toxin produces spastic paralysis in situ by blocking inhibitory neurotransmitter release in the spinal cord. Although di- and trisialogangliosides bind tetanus toxin, their role as productive toxin receptors remains unclear. We examined toxin binding and action in spinal cord cell cultures grown in the presence of fumonisin B(1), an inhibitor of ganglioside synthesis. Mouse spinal cord neurons grown for 3 weeks in culture in 20 microM fumonisin B(1) develop dendrites, axons, and synaptic terminals similar to untreated neurons, even though thin layer chromatography shows a greater than 90% inhibition of ganglioside synthesis. Absence of tetanus and cholera toxin binding by toxin-horseradish peroxidase conjugates or immunofluorescence further indicates loss of mono- and polysialogangliosides. In contrast to control cultures, tetanus toxin added to fumonisin B(1)-treated cultures does not block potassium-stimulated glycine release, inhibit activity-dependent uptake of FM1-43, or abolish immunoreactivity for vesicle-associated membrane protein, the toxin substrate. Supplementing fumonisin B(1)-treated cultures with mixed brain gangliosides completely restores the ability of tetanus toxin to bind to the neuronal surface and to block neurotransmitter release. These data demonstrate that fumonisin B(1) protects against toxin-induced synaptic blockade and that gangliosides are a necessary component of the receptor mechanism for tetanus toxin.     

Characterization of nuclear gangliosides in rat brain: concentration, composition, and developmental changes

Nuclear gangliosides were characterized using two distinct fractions of large (N1) and small (N2) nuclear populations from rat brain. The ganglioside concentration of N1 nuclei from adult rat brain was 0.92 microg sialic acid/mg protein, which was about 3.8 times higher than that of N2 nuclei. N1 and N2 nuclear gangliosides showed similar compositional profiles; they contained major gangliosides of GM1, GD1a, GD1b, and GT1b, with GM3 in lesser amounts. c-Series gangliosides such as GT3, GQ1c, and GP1c were also detected in both nuclear preparations. Nuclear localization of gangliosides was confirmed by immunofluorescence with anti-GM1 antibody, cholera toxin B subunit, and c-series ganglioside-specific monoclonal antibody A2B5. Developmental changes of nuclear gangliosides were examined using rats of different ages ranging from embryonic day 14 (E14) to postnatal 7 weeks. The concentration of N1 nuclear gangliosides changed only slightly during development and did not correlate with that of whole-brain gangliosides. The developmental pattern of ganglioside composition of N1 nuclei was also distinguished from that of microsomal membranes; the ganglioside changes in N1 nuclei included reduced expression of di- and polysialogangliosides at E16 and higher proportions of GM3 at early and late stages of the period. These findings suggest that gangliosides in nuclear membranes are developmentally regulated in a distinct manner in brain cells.     

Botulinum Neurotoxin Serotype C Associates with Dual Ganglioside Receptors to Facilitate Cell Entry

Botulinum neurotoxins (BoNTs) cleave SNARE proteins in motor neurons that inhibits synaptic vesicle (SV) exocytosis, resulting in flaccid paralysis. There are seven BoNT serotypes (A–G). In current models, BoNTs initially bind gangliosides on resting neurons and upon SV exocytosis associate with the luminal domains of SV-associated proteins as a second receptor. The entry of BoNT/C is less clear. Characterizing the heavy chain receptor binding domain (HCR), BoNT/C was shown to utilize gangliosides as dual host receptors. Crystallographic and biochemical studies showed that the two ganglioside binding sites, termed GBP2 and Sia-1, were independent and utilized unique mechanisms to bind complex gangliosides. The GBP2 binding site recognized gangliosides that contained a sia5 sialic acid, whereas the Sia-1 binding site recognized gangliosides that contained a sia7 sialic acid and sugars within the backbone of the ganglioside. Utilizing gangliosides that uniquely recognized the GBP2 and Sia-1 binding sites, HCR/C entry into Neuro-2A cells required both functional ganglioside binding sites. HCR/C entered cells differently than the HCR of tetanus toxin, which also utilizes dual gangliosides as host receptors. A point-mutated HCR/C that lacked GBP2 binding potential retained the ability to bind and enter Neuro-2A cells. This showed that ganglioside binding at the Sia-1 site was accessible on the plasma membrane, suggesting that SV exocytosis may not be required to expose BoNT/C receptors. These studies highlight the utility of BoNT HCRs as probes to study the role of gangliosides in neurotransmission.     

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)     

Periodate-Modified Gangliosides Enhance Surface Binding of Tetanus Toxin to PC 12 Pheochromocytoma Cells

The interaction of 125I-labeled tetanus toxin with PC12 pheochromocytoma cells in monolayer cultures has been examined. Under regular growth conditions, the PC12 cells bind 125I-tetanus toxin to a limited degree compared with dissociated cerebral neuron cultures. After exposure to nerve growth factor for 2 days in low serum-containing media with growth factor supplements, binding of toxin increases over twofold compared with untreated PC12 cells. Binding can also be enhanced (greater than 2.5-fold) after treatment of cells with 2 mM sodium metaperiodate for 20 min. Dissociated cerebral neurons but not fibroblasts in cell culture bind more toxin after periodate treatment. The effect of periodate can be abolished by 5 mM sodium borohydride. A ganglioside isolated from periodate-treated PC12 cells and tentatively identified as GT1b [(N-acetylneuraminyl)galactosyl-N-acetylgalactosaminyl(N- acetylneuraminyl-N-acetylneuraminyl)-galactosyl-glucosylceramide] binds 125I-tetanus toxin on silica gel chromatoplates and on nitrocellulose paper. There are no indications to suggest binding to a polypeptide from treated cells after polyacrylamide gel electrophoresis. Cells artificially supplemented with GT1b and subsequently treated with periodate effectively bind the toxin. The data suggest that modified sialyl groups linked to gangliosides, and not to proteins, are preferential targets for tetanus toxin.     

The role of gangliosides in the interaction of human chorionic gonadotropin and cholera toxin with murine Leydig tumor cells

A clonal line of murine Leydig tumor cells (MLTC-1) bound both human chorionic gonadotropin (hCG) and cholera toxin (CT) with high affinity and accumulated cyclic AMP in response to either effector. The major cellular ganglioside was GM3 with small amounts of GM2, GM1, and GD1a. The gangliosides became labeled when the cells were grown in medium containing [3H] galactose or were exposed to galactose oxidase or NaIO4 followed by NaB3H4. CT specifically protected GM1 from surface labeling whereas hCG did not protect any gangliosides from being labeled. When the cells were exposed to sialidase, surface GD1a was eliminated, and GM1 increased with a corresponding increase in CT binding. When sialidase-treated cells were first incubated with the B component of CT, binding and action of CT was blocked. The cells, however, retained their ability to bind and respond to hCG. Addition of purified gangliosides to the medium effectively inhibited the binding and action of CT but not hCG. The cells incorporated the exogenous gangliosides and exhibited increased binding of and responsiveness to CT but not hCG. Both hCG- and CT-receptor complexes were extracted from the cells with nonionic detergent and analyzed by sucrose gradient centrifugation. The hCG-receptor complex had an apparent molecular weight of 190,000 whereas the CT-receptor complex sedimented only slightly faster than CT itself. MLTC-1 gangliosides were separated on thin layer chromatograms which were overlayed with either iodinated CT or hCG. The toxin bound to a ganglioside corresponding to GM1 whereas the hormone did not bind to any of the gangliosides. When the cells were incubated overnight with hCG, they lost their hCG receptors but exhibited an increase in CT binding and gangliosides. Our results indicate that GM1 is the specific receptor for CT whereas gangliosides are not involved in the binding and action of hCG.     

Gangliosides do not affect ABC transporter function in human neuroblastoma cells

Previous studies have indicated a role for glucosylceramide synthase (GCS) in multidrug resistance (MDR), either related to turnover of ceramide (Cer) or generation of gangliosides, which modulate apoptosis and/or the activity of ABC transporters. This study challenges the hypothesis that gangliosides modulate the activity of ABC transporters and was performed in two human neuroblastoma cell lines, expressing either functional P-glycoprotein (Pgp) or multidrug resistance-related protein 1 (MRP1). Two inhibitors of GCS, D,L-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (t-PPPP) and N-butyldeoxynojirimycin (NB-dNJ), very efficiently depleted ganglioside content in two human neuroblastoma cell lines. This was established by three different assays: equilibrium radiolabeling, cholera toxin binding, and mass analysis. Fluorescence-activated cell sorting (FACS) analysis showed that ganglioside depletion only slightly and in the opposite direction affected Pgp- and MRP1-mediated efflux activity. Moreover, both effects were marginal compared with those of well-established inhibitors of either MRP1 (i.e., MK571) or Pgp (i.e., GF120918). t-PPPP slightly enhanced cellular sensitivity to vincristine, as determined by 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide analysis, in both neuroblastoma cell lines, whereas NB-dNJ was without effect. MRP1 expression and its localization in detergent-resistant membranes were not affected by ganglioside depletion. Together, these results show that gangliosides are not relevant to ABC transporter-mediated MDR in neuroblastoma cells.     

Exogenous gangliosides enhance the interaction of fibronectin with ganglioside-deficient cells

The major cell-surface glycoprotein fibronectin mediates a variety of cellular adhesive interactions that have been reported to be competitively inhibited by gangliosides. These effects suggest a possible function of gangliosides as receptors for fibronectin. To test this hypothesis more directly, we examined the interaction of endogenous fibronectin with a ganglioside-deficient cell line, NCTC 2071. These cells, which grow in serum-free medium, synthesized fibronectin. The fibronectin did not bind to these cells, but instead bound diffusely to the culture substratum. When the cells were cultured in medium containing ganglioside, the fibronectin became bound to the cell surface in fibrillar strands. The order of effectiveness of purified gangliosides was GT1b greater than GD1a greater than GM1 greater than GM2 greater than GM3. The effect with mixed gangliosides was accompanied by a restoration of cellular capacity to bind and to respond to cholera toxin. Treatment of the cells with several phospholipids did not alter fibronectin binding. Our results support the hypothesis that gangliosides can help mediate the binding of fibronectin to fibroblasts.     

Cooperativity of ganglioside-dependent with protein-dependent substratum adhesion and neurite extension of human neuroblastoma cells

The potential involvement of gangliosides in the adherence and neurite extension of human neuroblastoma cells (Platt and La-N1) was investigated on tissue culture substrata coated with the ganglioside GM1-binding protein, cholera toxin B (CTB) subunit, for comparison with similar processes on plasma fibronectin (pFN)-coated substrata. Cells attached with reduced efficiency on CTB substrata as compared with pFN substrata and required a much longer time to form neurite processes for a small percentage of cells on CTB. The specificity of these processes for GM1 binding was tested in a variety of ways. Supplementation of the cells with exogenous GM1, but not GD1a, identified a larger population of cells adherent on CTB (comparable to pFN-adherent cells) and dramatically increased the proportion of cells capable of forming neurites without reducing the time requirement. In ultrastructural studies using the scanning electron microscope (SEM) and immunofluorescence (IF) analyses to discriminate microtubule distributions, neurites of GM1-supplemented cells on CTB were virtually identical with pFN-adherent neurites, whereas unsupplemented cells on CTB generated processes with fine-structural differences. Treatment of cells during the GM1 supplementation period with cycloheximide completely abolished the ability of cells to generate neurites on CTB and decreased the adhesive capacity of cells as well; a similar treatment of cells had no adverse effect on adherence or neurite extension on pFN. The importance of one or more proteins in GM1-dependent processes was further confirmed by demonstrating the trypsin sensitivity of a cell surface component(s) required to achieve maximal attachment on CTB; in contrast, adherence and neurite extension on pFN were much more resistant to this treatment process. Therefore, these experiments demonstrate (a) that certain cell surface gangliosides are capable of mediating adherence and neurite outgrowth of human neuroblastoma cells on a suitable ganglioside-binding substratum; (b) this ganglioside dependence is cooperative with one or more cell surface proteins which can now be analysed. These results are discussed in light of the identification in ref. [16] (Exp cell res 169 (1987) 311) of a second ‘cell-binding’ domain on the pFN molecule competent for adherence and neurite extension of these neuroblastoma cells, as well as the potential role of pFN binding to a complex ganglioside on the surface of these neural tumor cells in these processes.     

Studies on the turnover and subcellular localization of membrane gangliosides in cultured neuroblastoma cells

To compare the subcellular distribution of endogenously synthesized and exogenous gangliosides, cultured murine neuroblastoma cells (N1E-115) were incubated in suspension for 22h in the presence ofd-[1-³H]galactose or [³H]GM1 ganglioside, transferred to culture medium containing no radioisotope for periods of up to 72 hr, and then subjected to subcellular fractionation and analysis of lipidsialic acid and radiolabeled ganglioside levels. The results indicated that GM2 and GM3 were the principal gangliosides in the cells with only traces of GM1 and small amounts of disialogangliosides present. About 50% of the endogenously synthesized radiolabelled ganglioside in the four major subcellular membrane fractions studied was recovered from plasma membrane and only 10–15% from the crude mitochondrial membrane fraction. In contrast, 45% of the exogenous [³H]GM1 taken up into the same subcellular membrane fractions was recovered from the crude mitochondrial fraction; less than 15% was localized in the plasma membrane fraction. The results are similar to those obtained from previously reported studies on membrane phospholipid turnover. They suggest that exogenous GM1 ganglioside, like exogenous phosphatidylcholine, does not intermix freely with any quantitatively major pool of endogenous membrane lipid.     

Direct visualization of redistribution and capping of fluorescent gangliosides on lymphocytes

Fluorescent derivatives of gangliosides were prepared by oxidizing the sialyl residues to aldehydes and reacting them with fluorescent hydrazides. When rhodaminyl gangliosides were incubated with lymphocytes, the cells incorporated them in a time- and temperature- dependent manner. Initially, the gangliosides were evenly distributed on the cell surface but were redistributed into patches and caps by antirhodamine antibodies. When the cells were then stained with a second antibody or protein A labeled with fluorescein, the fluorescein stain revealed the coincident movement of both the gangliosides and the antirhodamine antibodies. When the cells were treated with both rhodamine and Lucifer yellow CH-labeled gangliosides, the antirhodamine antibodies induced patching and capping of both fluorescent gangliosides but had no effect on cells incubated only with Lucifer yellow CH-labeled gangliosides. In addition, capping was observed on cells exposed to cholera toxin, antitoxin antibodies, and rhodamine- labeled protein A, indirectly showing the redistribution of endogenous ganglioside GM1, the cholera toxin receptor. By incorporating Lucifer yellow CH-labeled GM1 into the cells and inducing capping as above, we were able to demonstrate directly the coordinate redistribution of the fluorescent GM1 and the toxin. When the lymphocytes were stained first with Lucifer yellow CH-labeled exogenous ganglioside GM3, which is not a toxin receptor, there was co-capping of endogenous GM1 (rhodamine) and exogenous GM3 (Lucifer yellow CH). These results suggest that gangliosides may self-associate in the plasma membrane which may explain the basis for ganglioside redistribution and capping.     

Characterization of the receptor for cholera toxin and Escherichia coli heat-labile toxin in rabbit intestinal brush borders.

125I-labelled heat-labile toxin (from Escherichia coli) and 125I-labelled cholera toxin bound to immobilized ganglioside GM1 and Balb/c 3T3 cell membranes with identical specificities, i.e. each toxin inhibited binding of the other. Binding of both toxins to Balb/c 3T3 cell membranes was saturable, with 50% of maximal binding occurring at 0.3 nM for cholera toxin and 1.1 nM for heat-labile toxin, and the number of sites for each toxin was similar. The results suggest that both toxins recognize the same receptor, namely ganglioside GM1. In contrast, binding of 125I-heat-labile toxin to rabbit intestinal brush borders at 0 degree C was not inhibited by cholera toxin, although heat-labile toxin inhibited 125I-cholera toxin binding. In addition, there were 3-10-fold more binding sites for heat-labile toxin than for cholera toxin. At 37 degrees C cholera toxin, but more particularly its B-subunit, did significantly inhibit 125I-heat-labile toxin binding. Binding of 125I-cholera toxin was saturable, with 50% maximal of binding occurring at 1-2 nM, and was quantitatively inhibited by 10(-8) M unlabelled toxin or B-subunit. By contrast, binding of 125I-heat-labile toxin was non-saturable (up to 5 nM), and 2 X 10(-7) M unlabelled B-subunit was required to quantitatively inhibit binding. Neuraminidase treatment of brush borders increased 125I-cholera toxin but not heat-labile toxin binding. Extensive digestion of membranes with Streptomyces griseus proteinase or papain did not decrease the binding of either toxin. The additional binding sites for heat-labile toxin are not gangliosides. Thin-layer chromatograms of gangliosides which were overlayed with 125I-labelled toxins showed that binding of both toxins was largely restricted to ganglioside GM1. However, 125I-heat-labile toxin was able to bind to brush-border galactoproteins resolved by SDS/polyacrylamide-gel electrophoresis and transferred to nitrocellulose.     

GANGLIOSIDE COMPOSITION AND BIOSYNTHESIS IN CULTURED CELLS DERIVED FROM CNS

Abstract— Cultured mouse neuroblastoma cells (clone N18) contained a homologous series of gangliosides, G_M3, G_M2, G_M1 and G_D1a; the total lipid bound sialic acid (LBSA) was 3.3 nmol per mg of protein, of which G_D1a comprised two-thirds. In contrast, neonatal hamster astrocytes (clone NN) and human glioblastoma cells (Cox clone) contained mainly G_M3, which represented 95% of the 2 nmol of LBSA per mg protein in these cells. When the cells were grown in the presence of [¹⁴C]galactose, label was incorporated into all of the gangliosides isolated from the cells. The labeling pattern corresponded to the ganglioside composition of the cell lines; G_D1a was more extensively labeled in N18 cells and G_M3 was the major labeled ganglioside extracted from glial cells. In addition to in rivo biosynthesis, in vitro synthesis of gangliosides was also determined. The activities of five glycosyltransferases of the ganglioside biosynthetic pathway were measured in homogenates of the three cell lines. The neuroblastoma cells contained all five enzyme activities whereas the two glial cell lines were deficient in UDP- N -acetylgalactosamine: G_M3 N -acetylgalactosaminyltransferase activity, which catalyzes the synthesis of G_M2 from G_M3. The results indicated that cells of neuronal origin contain the more complex gangliosides associated with CNS and the requisite biosynthetic enzymes and that cells of glial origin are missing these complex gangliosides and the key glycosyltransferase required for their synthesis.     

Induction of GM1a/GD1b synthase triggers complex ganglioside expression and alters neuroblastoma cell behavior; a new tumor cell model of ganglioside function

Neuroblastoma is the most common extracranial solid tumor in children and tumor ganglioside composition has been linked to its biological and clinical behavior. We recently found that high expression of complex gangliosides that are products of the enzyme GM1a/GD1b synthase predicts a more favorable outcome in human neuroblastoma, and others have shown that complex gangliosides such as GD1a inhibit metastasis of murine tumors. To determine how a switch from structurally simple to structurally complex ganglioside expression affects neuroblastoma cell behavior, we engineered IMR32 human neuroblastoma cells, which contain almost exclusively (89%) the simple gangliosides (SG) GM2, GD2, GM3, and GD3, to overexpress the complex gangliosides (CG) GM1, GD1a, GD1b and GT1b, by stable retroviral-mediated transduction of the cDNA encoding GM1a/GD1b synthase. This strikingly altered cellular ganglioside composition without affecting total ganglioside content: There was a 23-fold increase in the ratio of complex to simple gangliosides in GM1a/GD1b synthase-transduced cells (IMR32-CG) vs. wild type (IMR32) or vector-transfected (IMR32-V) cells with essentially no expression of the clinical neuroblastoma marker, GD2, confirming effectiveness of this molecular switch from simple to complex ganglioside synthesis. Probing for consequences of the switch, we found that among functional properties of IMR32-CG cells, cell migration was inhibited and Rho/Rac1 activities were altered, while proliferation kinetics and cell differentiation were unaffected. These findings further implicate cellular ganglioside composition in determining cell migration characteristics of tumor cells. This IMR32 model system should be useful in delineating the impact of ganglioside composition on tumor cell function.     

Fluorescent gangliosides as probes for the retention and organization of fibronectin by ganglioside-deficient mouse cells

Ganglioside-deficient transformed mouse fibroblasts (NCTC 2071A cells), which grow in serum-free medium, synthesize fibronectin but do not retain it on the cell surface. When fluorescent derivatives of gangliosides, containing either rhodamine or Lucifer yellow CH attached to the sialic acid residues, were added to the culture medium, the cells incorporated the derivatives and their surfaces became highly fluorescent. When the cells were stained with anti-fibronectin antibodies and a fluorescent second antibody, fibrillar strands of fibronectin were observed to be attached to the cell surface, with partial coincidence of the patterns of direct ganglioside fluorescence and indirect fibronectin immunofluorescence at the cell surface. When the cells were exposed to bacterial neuraminidase during the time of ganglioside insertion, similar patterns of fluorescence were observed. Because the fluorescent gangliosides are resistant to the enzyme, these results suggest that neuraminidase-sensitive endogenous glycoconjugates were not involved in the ganglioside-mediated retention and organization of endogenous fibronectin. After cells were exposed to exogenous chicken fibronectin, most of the fibronectin was attached to the substratum and only a few fibrils were attached to the cells. When exogenous gangliosides were included in the incubation, there was a striking increase in cell-associated exogenous fibronectin, which was highly organized into a fibrillar network. Conversely, cells incubated for 18 h with exogenous unmodified gangliosides exhibited a highly organized network of endogenously derived fibronectin. Upon further incubation of the cells for 2 h with fluorescent gangliosides, there was considerable co-distribution of the fluorescent gangliosides with the fibronectin network as revealed by immunofluorescence. Our results support the concept that gangliosides can mediate the attachment of fibronectin to the cell surface and its organization into a fibrillar network.     

Gangliosides modulate sodium transport in cultured toad kidney epithelia

Cultured A6 epithelial cells from toad kidney form confluent monolayers with tight junctions separating the apical and basolateral membranes. These two membrane domains have distinct compositions and functions. Thus, sodium is actively transported across the epithelia from the apical to basolateral surface via amiloride-inhibitable sodium channels located in the apical membrane. Sodium transport is stimulated by vasopressin, cholera toxin, and 8-bromo-cAMP applied to the basolateral surface where the receptors, adenylate cyclase, and Na+/K+-ATPase are located. In a previous study (Spiegel, S., Blumenthal, R., Fishman, P.H., and Handler, J.S. (1985) Biochim. Biophys. Acta 821, 310-318), we demonstrated that exogenous gangliosides inserted into the apical membrane of A6 epithelia do not redistribute to the basolateral membrane. With the ability to vary selectively the ganglioside composition of the apical membrane, we examined the effects of gangliosides on sodium transport in A6 epithelia. When the apical surface of A6 epithelia were exposed to exogenous gangliosides, sodium transport in response to vasopressin, cholera toxin, and 8-bromo-cAMP was enhanced compared to epithelia not exposed to gangliosides. The effect was observed with bovine brain gangliosides, NeuAc alpha 2----3Gal beta 1----3GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4Glc beta 1----Cer (GD1a) and Gal beta-1----3GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4Glc beta 1----Cer (GM1), but not with the less complex ganglioside, Neu-Ac alpha 2----3Gal beta 1----4Glc beta 1----Cer (GM3). We examined A6 cells for endogenous gangliosides and found that, whereas GM3 was a major ganglioside, only trace amounts of GM1 and GD1a were present. Based on cell surface and metabolic labeling studies, these gangliosides were synthesized by the cells and were present on the apical as well as the basolateral surface. Bacterial sialidase, which hydrolyzes more complex gangliosides to GM1, was used to modify the endogenous gangliosides on the apical surface; after sialidase treatment, the epithelia were more responsive to vasopressin, cholera toxin, and 8-bromo-cAMP. Thus, gangliosides may be modulators of sodium channels present in the apical membrane of epithelial cells.     

Characterization of the cholera toxin receptor on Balb/c 3T3 cells as a ganglioside similar to, or identical with, ganglioside GM1. No evidence for galactoproteins with receptor activity.

Balb/c 3T3 cells contain a large number [(0.8-1.6) x 10(6)] of high-affinity (half-maximal binding at 0.2 nM) binding sites for cholera toxin that are resistant to proteolysis, but are quantitatively extracted with chloroform/methanol. The following evidence rigorously establishes that the receptor is a ganglioside similar to, or identical with, ganglioside GM1 by the galactose oxidase/NaB3H4 technique on intact cells was inhibited by cholera toxin. (2) Ganglioside GM1 was specifically adsorbed from Nonidet P40 extracts of both surface- (galactose oxidase/NaB3H4 technique) and metabolically ([1-14C]palmitate) labelled cells in the presence of cholera toxin, anti-toxin and Staphylococcus aureus. (3) Ganglioside GM1 was the only ganglioside labelled when total cellular gangliosides separated on silica-gel sheets were overlayed with 125I-labelled cholera toxin, although GM3 and GD1a were the major gangliosides present. In contrast no evidence for a galactoprotein with receptor activity was obtained. Cholera toxin did not protect the terminal galactose residues of cell-surface glycoproteins from labelling by the galactose oxidase/NaB3H4 technique. No toxin-binding proteins could be identified in Nonidet P40 extracts of [35S]-methionine-labelled cells by immunochemical means. After sodium dodecyl sulphate/polyacrylamide-gel electrophoresis none of the major cellular galactoproteins identified by overlaying gels with 125I-labelled ricin were able to bind 125I-labelled cholera toxin. It is concluded that the cholera toxin receptor on Balb/c 3T3 cells is exclusively ganglioside GM1 (or a related species), and that cholera toxin can therefore be used to probe the function and organisation of gangliosides in these cells as previously outlined [Critchley, Ansell, Perkins, Dilks & Ingram (1979) J. Supramol. Struct. 12, 273-291].     

The interaction of tetanus toxin with intact bovine adrenal chromaffin cells: binding of toxin and subsequent inhibition of catecholamine release.

Tetanus toxin (about 1 nM) inhibits 70% of the nicotine-evoked release of catecholamines from intact adrenal medullary chromaffin cells after 20 h of incubation and 30% of the K(+)-evoked release. Inhibition of Ca(2+)-evoked release from detergent-permeabilized cells requires higher concentrations of toxin (about 1 microM) toxin, but is maximal after 12 min. Preincubation of the intact cells with ganglioside GT1 in the absence of toxin also inhibits evoked secretion. 125I-labelled toxin bound specifically to these cells; the binding capacity was greater at pH 6 (about 1 pmol toxin/mg cell protein) than at pH 7.4 (about 0.25 pmol). In both cases there were at least two binding components: one of high affinity (Kd about 1 nM) accounting for about 20% of total binding and one of lower affinity (Kd 10-20 nM). Preincubation of the cells with ganglioside increased the binding capacity, but did not affect the Kd of the lower affinity component. Similar observations could be made when binding was measured immunocytochemically. Extraction of gangliosides from chromaffin cells and overlay experiments with radiolabelled toxin showed that, as well as GM3, the major ganglioside component of chromaffin cell membranes, a ganglioside having the chromatographic mobility of GT1 was a major ligand for toxin.     

Gangliosides of sea urchin embryos. Their localization and participation in early development

The influence of antibodies to gangliosides of sea urchin Strongylocentrotus intermedius eggs on early embryos of this species was studied. gamma-Globulins were isolated from rabbit anti-ganglioside serum by micropreparative electrophoresis. These gamma-globulins produced anomalies in the development of embryos permeabilized in Triton X-100. The anomalies were not observed when anti-ganglioside gamma-globulins were added to the incubation medium together with gangliosides or when the permeabilized embryos were incubated with gamma-globulins of normal rabbit serum. Pretreatment of S. intermedius embryos with serotonin, tryptamine or some other indole derivatives led to the disappearance of ganglioside determinants from the cell surface and sharply increased immunofluorescence within the cell. Such pretreatment of embryos increased the amount of cell-associated gangliosides more than threefold as compared to untreated embryos. Serotonin was shown to bind specifically to sea urchin gangliosides immobilized on octyl-Sepharose. These observations suggest that cell-surface gangliosides, after binding drugs, are internalized and that serotonin and its antagonists inhibit the transport of newly synthesized gangliosides to the cell-surface membrane.