Peter H. Fishman

Role of membrane gangliosides in the binding and action of bacterial toxins

SummaryGangliosides are complex glycosphingolipids that contain from one to several residues of sialic acid. They are present in the plasma membrane of vertebrate cells with their oligosaccharide chains exposed to the external environment. They have been implicated as cell surface receptors and several bacterial toxins have been shown to interact with them. Cholera toxin, which mediates its effects on cells by activating adenylate cyclase, bind with high affinity and specificity to ganglioside GM1. Toxin-resistant cells which lack GM1 can be sensitized to cholera toxin by treating them with GM1. Cholera toxin specifically protects GM1 from cell surface labeling procedures and only GM1 is recovered when toxin-receptor complexes are isolated by immunoadsorption. These results clearly demonstrate that GM1 is the specific and only receptor for cholera toxin. Although cholera toxin binds to GM1 on the external side of the plasma membrane, it activates adenylate cyclase on the cytoplasmic side of the membrane by ADP-ribosylation of the regulatory component of the cyclase. GM1 in addition to functioning as a binding site for the toxin appears to facilitate its transmembrane movement. The heat-labile enterotoxin ofE. coli is very similar to cholera toxin in both form and function and can also use GM1 as a cell surface receptor. The potent neurotoxin, tetanus toxin, has a high affinity for gangliosides GD1b and GT1b and binds to neurons which contain these gangliosides. It is not yet clear whether these gangliosides are the physiological receptors for tetanus toxin. By applying the techniques that established GM1 as the receptor for cholera toxin, the role of gangliosides as receptors for tetanus toxin as well as physiological effectors may be elucidated.

Induction of glycolipid biosynthesis by sodium butyrate in HeLa cells

Summary Butyrate inhibits the growth of HeLa cells and markedly alters their morphology as the cells acquire a more fibroblast-like shape by greatly extending cellular processes. Butyrate simultaneously causes an increase in cellular sialylactosylceramide content by elevating CMP-sialic acid: lactosylceramide sialyltransferase activity (7–24 fold); whereas other glycolipid glycosyltransferase activities do not increase. Induction of this specific sialyltransferase is blocked by cycloheximide or actinomycin D. This first report on the induced synthesis of glycolipid components suggests that these complex carbohydrates have a role in cell growth and morphology.

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.

Sequence similarity between cholera toxin and glycoprotein hormones: Implications for structure activity relationship and mechanism of action

Abstract The B chain of cholera toxin and the β subunits of thyrotropin, luteinizing hormone, human chorionic gonadotropin, and follicle-stimulating hormone are shown to have a region of sequence analogy believed to correlate with their ability to bind to receptors on cell membranes. A possible sequence analogy is also defined in the α subunits of these glycoprotein hormones and a region of the cholera toxin A 1 chain believed to be responsible for adenylate cyclase activation.

Morphological changes in cultured mammalian cells: Prevention by the calcium ionophore A23187

The morphological changes induced by butyrate in HeLa cells and by monobutyryl or dibutyryl cAMP in CHO cells are prevented by micromolar concentrations of the divalent cation ionophore A23187. The ionophore is unable to prevent such changes in medium from which calcium is omitted. At slightly higher (but nontoxic) concentrations, the ionophore inhibits the butyrate-mediated induction of the ganglioside biosynthetic enzyme, sialyltransferase, in HeLa. In CHO, sialyltransferase activity is normally high and not altered by any of the compounds tested.

Ganglioside biosynthesis in mouse cells: Glycosyltransferase activities in normal and virally-transformed lines

Abstract The activities of four glycosyltransferases involved in ganglioside biosynthesis were measured in two different established mouse cell lines and in the SV40 and polyoma transformed variants of these lines. The only consistent change observed was the very reduced activity of UDP-GalNAc: hematoside N-acetyl-galactosaminyltransferase in all of the transformed cell lines (15–20% of normal cells). There was no significant differences in any of the glycosyltransferases with increased cell density and cell-to-cell contact.

Anomalous behavior of CGP 12177A on beta 1-adrenergic receptors.

CGP 12177A originally was developed as a hydrophilic antagonist to detect cell surface beta 1- and beta 2-adrenergic receptors, and subsequently was found to be a partial agonist for the atypical or beta 3-adrenergic receptor. Using hamster cells stably expressing either the human beta 1-, human beta 2- or rat beta 1-adrenergic receptor, we found that CGP 12177A behaved as an agonist of beta 1-adrenergic receptors. Whereas at low concentrations, CGP 12177a behaved as an antagonist and inhibited isoproterenol stimulation of adenylyl cyclase activity, at higher concentrations, it stimulated a response even in the absence of isoproterenol. The agonistic properties of CGP 12177A were positively correlated with the level of beta 1-adrenergic receptor expression. Thus, at low receptor of densities, CGP 12177A behaved as a weak, partial agonist whereas as high receptor densities, the drug was a full agonist. At similar high densities of the beta 2-adrenergic receptor, CGP 12177A acted only as a partial agonist. Competition binding studies to membranes from cells expressing beta 1-adrenergic receptors indicated that approximately 90% of the receptors were in a high affinity, guanine nucleotide-insensitive state for CGP 12177A whereas approximately 10% of the receptors were in a lower affinity, guanine nucleotide-sensitive state for CGP 12177A. We propose that the latter receptors are precoupled to stimulatory G proteins and recognize CGP 12177A as an agonist whereas the high affinity, uncoupled receptors recognize CGP 12177A as an antagonist.

Mechanism of action of cholera toxin: Studies on the lag period

SummaryThe lag period for activation of adenylate cyclase by choleragen was shorter in mouse neuroblastoma N18 cells than in rat glial C6 cells. N18 cells have 500-fold more toxin receptors than C6 cells. Treatment of C6 cells with ganglioside GM1 increased the number of toxin receptors and decreased the lag phase. Choleragen concentration also effected the lag phase, which increased as the toxin concentration and the amount of toxin bound decreased. The concentration, however, required for half-maximal activation of adenylate cyclase depended on the exposure time; at 1.5, 24, and 48 hr, the values were 200, 1.1., and 0.35pm, respectively. Under the latter conditions, each cell was exposed to 84 molecules of toxin.The length of the lag period was temperature-dependent. When exposed to choleragen at 37, 24, and 20 °C, C6 cells began to accumulate cyclic AMP after 50, 90, and 180 min, respectively. In GM1-treated cells, the corresponding times were 35, 60, and 120 min. Cells treated with toxin at 15 °C for up to 22 hr did not accumulate cAMP, whereas above this temperature they did. Antiserum to choleragen, when added prior to choleragen, completely blocked the activation of adenylate cyclase. When added after the toxin, the antitoxin lost its inhibitory capability in a time and temperature-dependent manner. Cells, however, could be preincubated with toxin at 15 °C, and the antitoxin was completely effective when added before the cells were warmed up. Finally, cells exposed to choleragen for >10 min at 37 °C accumulated cyclic AMP when shifted to 15 °C. Under optimum conditions at 37°C, the minimum lag period for adenylate cyclase activation in these cells was 10 min. These findings suggest that the lag period for cholerage action represents a temperature-dependent transmembrane event, during which the toxin (or its active component) gains access to adenylate cyclase.

Muscarinic receptor-mediated increase in cAMP levels in SK-N-SH human neuroblastoma cells

Stimulation of muscarinic cholinergic receptors in SK-N-SH human neuroblastoma cells resulted in a 1.5-4 fold increase in intracellular cAMP levels. This unusual response was sensitive to atropine and pirenzepine but insensitive to pertussis toxin. It was observable regardless of whether basal, PGE1- or forskolin-stimulated cAMP levels were measured. The half-maximal concentration for carbachol-stimulation of cAMP levels (6 microM) was similar to that for the previously determined carbachol-induced stimulation of phosphoinositide turnover in these cells, suggesting that the former is mediated by the latter. These data indicate that cross-talk between the phosphoinositide turnover system and the adenylate cyclase system results in increased cAMP levels in SK-N-SH cells in response to muscarinic receptor stimulation.

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.