Binding of Vibrio cholera toxin and the heat-labile enterotoxin of Escherichia coli to GM1, derivatives of GM1, and nonlipid oligosaccharide polyvalent ligands

Vibrio cholera toxin and the heat-labile enterotoxin of Escherichia coli have been shown to differ somewhat in their ligand specificity and in the antigenicity of their binding sites. Therefore, the components of the oligosaccharide portion of GM1 bound by cholera toxin and the heat-labile enterotoxin of E. coli were identified by determining the concentration of GM1, derivatives of GM1, oligosaccharide isolated from GM1, or clustered oligosaccharide needed to inhibit toxin binding to GM1-coated plastic wells. The KIs for GM1, the C(7) sialosyl alcohol [corrected] of GM1, and ethanolamine-sialosyl-GM1 were similar (approximately 30-50 nM) for both toxins. N-Deacetylation of GM1 resulted in a small increase in KI; formation of the sialosyl methyl ester increased the KI 2-5 fold; loss of the terminal galactosyl residue (GM2) increased the KI by 10-15-fold; and removal of the sialosyl moiety (asialo-GM1) resulted in loss of inhibition of both toxins. Oligosaccharide isolated from GM1 had a KI for both toxins that was approximately 100-fold greater than that obtained for GM1 and approximately 1000-fold greater than that for a clustered oligosaccharide derivative having an average of 8 oligosaccharide residues (isolated from GM1) per molecule of poly-L-lysine. These results indicate that both toxins are functionally quite similar in their recognition of GM1 as a ligand in that each requires the free carboxyl group of sialic acid for optimum binding, does not need carbons 8 and 9 of the sialosyl moiety nor the acetyl groups associated with the sialic acid and galactosamine residues, and can have its binding to GM1 blocked by a nonlipid compound, i.e. oligo-GM1-poly-L-lysine.     

Ecto-ganglioside-sialidase activity of herpes simplex virus-transformed hamster embryo fibroblasts

Cellular location of ganglioside-sialidase activity was determined in confluent hamster embryo fibroblasts transformed with herpes simplex virus type 2. Approximately equal specific activities of ganglioside-sialidase activity were found to be associated with the crude lysosomal and crude plasma membrane fractions isolated from whole cell homogenates. Whole transformed cells hydrolyzed exogenous ganglioside substrate, suggesting a partial location of the cellular sialidase on the outer surface of the plasma membrane of these cells. Intact cells were treated with the diazonium salt of sulfanilic acid, a nonpenetrating reagent inhibitory to ecto-enzymes (DePierre, J.W., and M. L. Karnovsky. 1974. J. Biol. Chem. 249:7111-7120). Cytoplasmic lactate dehydrogenase activity was not inhibited by this treatment, and mitochondrial succinate dehydrogenase activity was inhibited only 10%, indicating that intracellular enzymes were not affected. 5'-Nucleotidase activity was diminished 90%, and sialidase very rapidly lost 40% of its exogenously directed activity. These results show that, in herpes simplex virus-transformed fibroblasts, ganglioside-sialidase is both a lysosomal and a plasma membrane enzyme. The plasma membrane sialidase is capable of acting on endogenous plasma membrane sialolipids and also functions in the cultured transformed cell as an ecto-enzyme which can attack exogenous substrates.     

Sialidase activity in mouse neuroblastoma cell lines

Sialidase activity was determined for three different neuroblastoma clonal lines derived from the A/J strain mouse C1300 neuroblastoma line. For each cell line, the endogenous and exogenous activities were less than 1 nmol sialic acid released/mg protein/90 min and 50 min reaction time, respectively. The C1300 tumor had similarly low levels of sialidase activity. The sialidase activity associated with the neuroblastomas is less than that associated with synaptosomes. Each cell line had a distinctly different ganglioside pattern varying in complexity from G_M3 to G_D1a. Treatment of the cells withVibrio cholerae sialidase under isosmotic conditions showed that cell-surface sialyl residues were susceptible to sialidase activity, with some of the susceptible residues coming from the ganglioside constituents. Of the total number ofV. cholerae sialidase-releasable sialyl residues, 50–60% were released by the neuroblastoma sialidase acting on endogenous substrate.     

Poster Session 3

Lipid analysis of gestational day E14.5 mouse brain revealed elevation of ceramide to a tissue concentration that induced apoptosis when added to the medium of neuroprogenitor cells grown in cell culture. Elevation of ceramide was coincident with the first appearance of β-series complex gangliosides (BCGs). Expression of BCGs by stable transfection of murine neuroblastoma (F-11) cells with sialyltransferase-II (ST2) resulted in a 70% reduction of apoptosis that was induced with the novel ceramide analog (NCA) N-oleoyl serinol (S18). This was most likely due to an 80% reduced expression of prostate apoptotis response-4 (PAR-4). PAR-4 expression and apoptosis were restored by preincubation of ST2-transfected cells with N-butyl deoxinojirimycin (NB-DNJ) or PD98059, two inhibitors of ganglioside biosynthesis or p42/44 MAPK-kinase, respectively. In sections of day E14.5 mouse brain, the intermediate zone showed intensive staining for complex gangliosides, but only low staining for apoptosis (TUNEL) and PAR-4. Apoptosis and PAR-4 expression, however, were elevated in the ventricular zone, which only weakly stained for complex gangliosides. Complex gangliosides may thus activate a cell survival mechanism that selectively protects developing neurons against ceramide-induced apoptosis by up-regulation of MAPK and reduction of PAR-4 expression. NCAs may be useful to analyse the molecular mechanisms that underlie or counteract ceramide-induced apoptosis during neuronal development.
Acknowledgements:   Supported by grants MH61934-04 (to E.B.) and NS11853 (to R.K.Y.).     

Oxidation and base-catalyzed elimination of the saccharide portion of GSLs having very different polarities

Glycosphingolipids (GSLs), present in cell membranes, participate in a variety of biological functions. Although their exact role(s) may not be understood, it has been shown that 1) embryos lacking glucosylceramide synthase activity do not develop normally, 2) GSLs can affect neuritogenesis, and 3) they can function as receptors for some pathogens. To study the role of the saccharide portion of a GSL in any of these functions, it is necessary to either isolate it from the intact GSL or synthesize it. Because syntheses are more complex, modifications were made to the oxidation/elimination procedure previously described for the isolation of the saccharide portion of GM1 and GD1a to enable it to be used with GSLs of varying polarity. The key is to use a mixture of GSLs that differ in polarity. This appears to eliminate problems encountered when purified GSLs such as sulfatide or GT1b are used.     

UDP-6-deoxy-6-fluoro-alpha-D-galactose binds to two different galactosyltransferases, but neither can effectively catalyze transfer of the modified galactose to the appropriate acceptor.

The effect of substitution of the HO-6 of D-galactose with fluorine on the ability of alpha-(1-->3)-galactosyltransferase (EC 2.4.1.151) and beta-(1-->4)-galactosyltransferase (EC 2.4.1.22) to catalyze its transfer from UDP to an appropriate acceptor was determined. HPLC analyses indicated that each transferase properly catalyzed formation of the expected product [beta-D-Gal-(1-->4)-D-GlcNAc] for the beta-(1-->4)-galactosyltransferase and alpha-D-Gal-(1-->3)-beta-D-Gal-(1-->4)-D-GlcNAc for the alpha-(1-->3)-D-galactosyltransferase] when UDP-alpha-D-Gal was the substrate. When UDP-6-deoxy-6-fluoro-alpha-D-galactose (6) was used in conjunction with each transferase, no product indicative of transfer of 6-deoxy-6-fluoro-D-galactose to its respective acceptor sugar was identified. 6-Deoxy-6-fluoro-D-galactose (3) was obtained by hydrolysis of methyl 6-deoxy-6-fluoro-alpha-D-galactopyranoside, synthesized by the selective fluorination of methyl alpha-D-galactopyranoside with diethylaminosulfur trifluoride (DAST), with aqueous trifluoroacetic acid. Acetylation of 3 gave crystalline 1,2,3,4-tetra-O-acetyl-6-deoxy-6-fluoro-beta-D-galactopyranose, which was converted to the corresponding 1-alpha-phosphate and used for the synthesis of 6.     

Density-Dependent Changes in Gangliosides and Sialidase Activity of Murine Neuroblastoma Cells

Abstract: Density-dependent changes in ganglioside composition, Vibrio cholerae neuraminidase (VCN)-susceptible sialyl residues, and membrane- associated sialidase activity were determined for the cholinergic murine neuroblastoma cell line S20Y. A decrease in total ganglioside sialic acid and VCN-releasable sialic acid was observed with increasing cell density. G^M3 was the major ganglioside component of preconfluent S20Y cells, whereas G_DIA was predominant in postconfluent cells. Sialidase activity increased in confluent and postconfluent cells and may account for the reduction in total ganglioside sialic acid observed with increasing cell density. In contrast, while adrenergic N115 cells showed a decrease in VCN-susceptible sialic acid residues with increasing cell density, there was no significant change in ganglioside composition or ganglioside sialic acid levels.     

Glycosphingolipids—Sweets for botulinum neurotoxin

A number of viruses, bacteria, and bacterial toxins can only act on cells that express the appropriate glycosphingolipids (GSLs) on the outer surface of their plasma membranes. An example of this dependency is provided by botulinum neurotoxin (BoNT) which is synthesized by Clostridium botulinum and inhibits neurotransmission at the neuromuscular junction by catalyzing hydrolysis of a SNARE protein, thereby inducing a flaccid paralysis. Haemagglutinin components of progenitor forms of BoNT mediate its adherence to glycosphingolipids (GSLs) on intestinal epithelial cells while the cellular activity of most isolated serotypes requires the presence of certain gangliosides, especially those of the Gg1b family. This review discusses available information about the identity and the roles of GSLs in the activity of BoNT. Observations that serotypes A-F of BoNT require gangliosides for optimum activity (serotype G apparently does not), permits the hypothesis that it should be possible to develop an antagonist of this interaction thereby inhibiting/reducing its effect.     

Murine Neuroblastoma Cells Express Ganglioside Binding Sites on Their Cell Surface

The ability of S20Y cholinergic, and N115 adrenergic, murine neuroblastoma cells to adhere to immobilized gangliosides was studied. Viable S20Y cells adhered more strongly to GM1-coated plastic wells than to those coated with GM2, GD1a, or GT1b. The oligosaccharide portion of GM1 inhibited adherence of S20Y cells to GM1-coated wells, indicating that the carbohydrate moiety of GM1 bore the recognition site. Analysis of S20Y cell adherence to wells coated with derivatives of GM1 indicated that the cells did not adhere to asialo-GM1 and adherence to the methyl ester or de-N-acetyl derivatives was significantly reduced. Expression of the GM1 binding sites by S20Y cells appears to be density dependent; cells harvested at the confluent stage of growth were more adherent than those harvested at the preconfluent stage. Trypsin treatment of the S20Y and N115 cells resulted in a loss of binding to GM1-coated wells, suggesting that the cell surface GM1 binding site is a protein. In contrast, N115 cells showed no significant difference in their adherence to wells coated with GM1, GD1a, GT1b, Gal-Cer, asialo-GM1, or the methyl ester of GM1 when assayed under the same conditions as those imposed on the S20Y cells. The N115 cells did show a reduction in adherence to GM2-coated wells, suggesting that they recognized the terminal galactosyl moiety.