Ganglioside Preparations Research Articles

A synthetic approach to polysialogangliosides containing α-sialyl-(2 → 8)-sialic acid: total synthesis of ganglioside GD 3

A stereocontrolled, facile total synthesis of ganglioside GD 3 is described as an example of a proposed systematic approach to the preparation of gangliosides containing an α-sialyl-(2 → 8)-sialic acid unit α-glycosidically linked to O-3 of a d-galactose reesidue in their oligosaccharide chains. Glycosylation of 2-(trimethylsilyl)ethyl 6- O-benzoyl-, 3- O-benzoyl-, or 3- O-benzyl-β- d-galactopyranosides, or 2-(trimethylsilyl)ethyl 2,3,6,2′,6′-penta- O-benzyl-β-lactoside ( 7), with methyl [phenyl 5-acetamido-8- O-(5-acetamido-4,7,8,9- tetra- O-acetyl-3,5-dideoxy- d- glycero-α- d- galacto-2-nonulopyranosyl-ono-1′,9-lactone)-4,7-di- O-acetyl-3,5-dideoxy-2-thio- d- glycero- d- galacto-2-nonulopyranosid]onate ( 3), using N-iodosuccinimide-trifluoromethanesulfonic acid as a promoter, gave the corresponding α glycosides 8 (32%), 13 (33%), 14 (48%), and 17 (31%), respectively. The glycyl donor 3 was prepared from O-(5-acetamido-3,5-dideoxy- d- glycero-α- d-galacto-2-nonulopyranosylonic acid)-(2 → 8)-5-acetamido-3,5-dideoxy- d- glycero- d- galacto-2-nonulopyranosonic acid by treatment with Amberlite IR-120 (H +) in methanol, O-acetylation, and subsequent replacement of the anomeric acetoxy group with phenylthio. Compound 8 was converted into the methyl β-thioglycoside via O-benzoylation, replacement of the 2-(trimethylsilyl)ethyl group by acetyl, and introduction of the methylthio group by reaction with methylthiotrimethylsilane. Compound 17 was converted, via O-acetylation, selective removal of the 2-(trimethylsilyl)ethyl group, and reaction with trichloroacetonitrile, into the α-trichloroacetimidate, which was coupled with (2S,3R,4E)-2-azido-3 O-benzoyl-4-octadecene-1,3-diol to give the β-glycoside. This glycoside was easily transformed, via selective reduction of the azido group, condensation with octadecanoic acid, O-deacylation, and hydrolysis of the methyl ester and lactone functions, into ganglioside GD 3.

Pancreatic gangliosides delay the onset of insulitis and hyperglycaemia in the low-dose streptozotocin mouse model.

Gangliosides have been shown to modulate autoimmune phenomena in experimental diabetes. The effects of a pancreatic ganglioside preparation or of a commercial brain ganglioside mixture on the insulitis and blood glucose levels in the low-dose streptozotocin mouse model of diabetes have been investigated. Fifty-five C57BL/6J male mice were grouped as follows: Group 1 (n = 20) was injected intraperitoneally with repeated low doses of streptozotocin; Group 2 (n = 10) received streptozotocin as above but was also injected with a pancreatic ganglioside preparation equivalent to 2 micrograms sialic acid 2 h before each streptozotocin dose; Group 3 (n = 15) received streptozotocin and brain-derived gangliosides in the same dose as that of pancreatic gangliosides; Group 4 (n = 10) consisted of normal animals. Half of the mice were killed on day 12 and the others on day 24 from the beginning of treatment. On day 12, among the streptozotocin-injected animals only those treated with pancreatic gangliosides remained normoglycaemic, whereas on day 24 all streptozotocin mice were hyperglycaemic. Such a result paralleled the data pertaining to insulitis scores. In conclusion, pancreatic gangliosides have a short-term protective role on the development of diabetes in the low-dose streptozotocin model, an effect therefore linked to tissue-related differences in the glycosphingolipid composition.

Purification of non-infectious ganglioside preparations from scrapie-infected brain tissue.

The extraction and purification of gangliosides from brains of animals infected with the scrapie agent was evaluated by scaling-down a large-scale procedure currently used with bovine brains. Inactivation experiments employed hamster brains infected with the 263 K strain of scrapie. Residual infectivity was determined at different points of the procedure and in the final preparation by an in vivo animal bioassay. The efficacy of single steps, which included chemicals or physical techniques known to affect the viability of the scrapie agent, was verified by spiking experiments where known amounts of infectivity were added just before each single step. Infectivity was significantly diminished at early stages of the purification, and no infectivity was detected in the final preparation containing purified gangliosides. The significant reduction of infectivity at intermediate steps and the total absence of detectable infectivity in the final product confirmed that a combination of heat and exposure to sodium hydroxide, even in the presence of organic solvents, completely inactivated the scrapie agent.

Production and characterization of a monoclonal antibody (BBH5) directed to ganglioside lactone.

The occurrence of lactones in various ganglioside preparations has been clearly demonstrated, yet the natural occurrence of ganglioside lactones in cells and tissues has been the subject of long debate, since lactones can be formed readily during preparation of gangliosides. We now report the generation of monoclonal antibody (BBH5) that reacts specifically with lactones of disialogangliosides having the NeuAc2-8NeuAc2-3Gal sequence, but does not crossreact with the parent ganglioside. The specificity of the antibody resides on the first lactone ring between two sialic acid residues but not on the second lactone ring between sialic acid and galactose, as evidenced by reactivity with lactonized GD1b having the first lactone ring (L1), and by reactivity with lactonized polysialic acid homo-oligomers ([NeuAc alpha 2-8]nNeuAc). The sialic acid carboxyl involved in the lactone ring was unequivocally determined after ammonolysis followed by methylation and fast atom bombardment mass spectrometry. The antibody BBH5 thus provides a novel tool for studies of the natural occurrence of lactones in cells and tissues.

A simple method to detect contaminating peptides and amino acids in large-scale ganglioside preparations

A reverse-phase liquid chromatography method was developed to analyse the presence of contaminating peptides and amino acids in large-scale monosialoganglioside preparations. Samples were hydrolysed under controlled conditions and derivatized with phenylisothiocyanate. PTC-amino acids were then separated and identified by HPLC. The sensitivity of the method allowed detection of a least 50 pmoles of amino acid per mg of ganglioside with excellent reproducibility and little or no interference of by-products derived from hydrolysis of the glycosphingolipid. The response was consistently linear for each amino acid within the examined analytical range. The ease and speed of performance make this method a useful tool for rapidly monitoring large-scale extraction and purification processes of biologicals obtained from natural sources.

Involvement of gangliosides in the interaction between BK virus and Vero cells

BK virus infectivity was inhibited by gangliosides extracted from Vero cells and by standard preparations of different gangliosides. Gangliosides were also able to restore the susceptibility of glycosidase-treated Vero cells to BK virus infection.

Ganglioside variations in human liver cirrhosis and hepatocellular carcinoma as shown by two-dimensional thin-layer chromatography

Gangliosides isolated from 5 cases of normal liver tissues, 11 cases of liver cirrhosis and 5 cases of hepatocellular carcinoma were compared in their concentrations and compositions. Quantitative analysis revealed no significant change of ganglioside levels between normal and cirrhotic liver tissues or hepatocellular carcinoma. There was also no significant difference (p greater than 0.05) between cirrhotic liver tissues and hepatocellular carcinoma. Two dimensional thin-layer chromatography of the total ganglioside preparations of liver tissues from both liver cirrhosis and hepatocellular carcinoma showed proliferation of GM2, GD3, GD1 and at least two unidentified components, named provisionally spots Nos. 1 and 2 in the present report, and loss of GM3. Sialidase treatment and thin-layer chromatography showed the components of these spots to be sialidase-labile monosialogangliosides and distinctly different from GD3 which was described elsewhere.

Ganglioside interactions with the dopaminergic system of rats.

The effects of ganglioside treatment on changes in dopaminergic function following 6-hydroxydopamine (6-OHDA) or repeated exposure to haloperidol were studied in male Fischer-344 rats. Rats were injected sc with 30 mg/kg of a mixed ganglioside preparation (GM) at the time of the surgery and for 13 days after receiving an intranigral injection of 6-OHDA. GM treatment attenuated 6-OHDA depletion of striatal dopamine (DA) and DOPAC. Another group of rats was implanted with chronic indwelling cannulas in the lateral cerebroventricles at the time of 6-OHDA administration into the substantia nigra. Daily intraventricular injection of 25 or 50 micrograms GM attenuated depletions of striatal DA and DOPAC. A separate experiment sought to determine the effects of GM1 on the development of receptor supersensitivity produced by repeated exposure to a dopamine receptor antagonist. Rats were injected sc with 1 mg/kg haloperidol for 8 or for 16 days; some rats were coadministered 20 mg/kg GM1 sc. Four days after the last dose, the rats were challenged with 1 mg/kg apomorphine, and activity was counted for 60 min. Treatment with GM1 decreased the behavioral supersensitivity to apomorphine induced by repeated exposure to haloperidol. These experiments suggest that treatment with GM can have a protective effect against 6-OHDA-induced depletion of dopamine if treatment with GM begins at the time of lesioning. These studies also support receptor binding data from other laboratories indicating that treatment with GM1 can affect up-regulation of dopamine receptors.

Ganglioside monoclonal antibody (A2B5) labels Alzheimer's neurofibrillary tangles.

Ganglioside monoclonal antibody (A2B5) labels Alzheimer's neurofibrillary tangles both in isolated neurofibrillary tangle-bearing nerve cells and in partially purified preparations of tangle fibers. Antibody staining was preabsorbed by preincubation of antibody with neuronal ganglioside preparations. These results suggest that Alzheimer's neurofibrillary tangles have a ganglioside associated with them.

Inhibition of BK virus haemagglutination by gangliosides.

The effect of gangliosides extracted from human group O Rh+ erythrocytes on haemagglutination by BK virus was investigated. Experiments were performed on both ganglioside mixtures and isolated fractions separated by column chromatography and characterized by thin-layer chromatography. These results were compared with those obtained with standard preparations of gangliosides, and the inhibiting activity was shown to be confined mainly to gangliosides with a RF lower than GM1. It was also observed that the insertion of gangliosides in liposomes increased the haemagglutination-inhibiting activity and that ganglioside coating restored the ability of glycosidase-treated human red blood cells to agglutinate.

New chemical trends in ganglioside research

A report is given of recent progress in the methodology for isolation of gangliosides from natural sources, for the preparation of molecular species of gangliosides homogeneous in both the oligosaccharide and ceramide portions of the molecule, for chemical manipulation and derivatization of gangliosides, and for the preparation of gangliosides radiolabelled in different parts of the molecule. Particular emphasis has been given to: (a) high performance liquid chromatographic procedures capable to separate gangliosides on the basis of their oligosaccharide or ceramide moieties and yielding completely homogeneous compounds, that is gangliosides with a single oligosaccharide, a single long chain base and a single fatty acid; (b) two-dimensional thin-layer chromatographic procedures, provided with a fully computerized quantification system, particularly suitable to identifying gangliosides containing alkali-labile linkages, including ganglioside lactones; (c) chemical procedures of high yield for reducing gangliosides at the double bond of long chain base, for selective removal of the fatty acyl moiety and replacement with a novel fatty acid, and for the synthesis of ganglioside lactones; (d) chemical procedures for inserting fluorescent, paramagnetic or photoreactive probes at the fatty acyl part of the ganglioside molecule; (e) procedures for chemical isotopic radiolabelling of gangliosides at the level of sialic acid acetyl group and at the fatty acid moiety. Examples are provided evidencing the significance and potential use of a variety of ganglioside derivatives in the study of ganglioside metabolism and functional implications.

A new chemical procedure for the preparation of gangliosides carrying fluorescent or paramagnetic probes on the lipid moiety

A new chemical procedure is described for preparing labelled GM1 molecular species, carrying as acyl moiety pyrene-decanoic acid, 5-doxyl-stearic acid and 16-doxyl-stearic. It makes use of a mixed anhydride formed by ethylchloroformate and the labelled acyl chain, as the reagent for N-acylation of a deacetylated, deacylated GM1 ganglioside, which is prepared by alkaline hydrolysis of natural GM1. The reaction performed with a unitary GM1 derivative/mixed anhydride molar ratio, occurs with a yield of above 40%. The labelled deacetylated GM1 molecular species are then N-acetylated by means of acetic anhydride with quantitative yield. The chemical process of insertion of labelled fatty acid and reconstitution of GM1 ganglioside has been confirmed by GLC-MS and NMR analyses. Fluorescence and electron spin resonance experiments indicate that the labelled gangliosides behave similarly to natural GM1, in both the aggregation properties and the capability to be transferred from micelles to vesicular dispersions of phospholipids.

Molecular specificity of ganglioside effects on neurite regeneration of sensory neurons in vitro

Highly purified preparations of individual gangliosides have been tested for their ability to modulate the survival and morphological differentiation of embryonic chick dorsal root ganglion neurons. When added at 48 h to established cultures of nerve growth factor (NGF)-dependent neurons, all ganglioside species tested increased the expression of neurofilament protein. Poly- and trisialogangliosides were more effective than di- or monosialogangliosides. In contrast, neither NGF nor an antiserum against NGF influenced neurofilament protein expression over this period of culture. In addition, ganglioside-induced expression of neurofilament protein was not inhibited by the anti-NGF serum.

Ganglioside modulation of lipopolysaccharide-initiated complement activation

Highly purified preparations of mouse gangliosides have been demonstrated to bind to purified preparations of lipopolysaccharide (LPS). In some instances, the binding has been demonstrated to be dependent upon the presence of sialic acid in the ganglioside preparation. The binding of gangliosides to LPS from the deep rough Salmonella minnesota Re mutant has suggested that the interaction involves the lipid A-2-keto-3-deoxyoctulosanate region of the LPS macromolecule. The interaction between gangliosides and LPS has been demonstrated to result in an abrogation of lipid A dependent activation of the classical pathway of serum complement by Re LPS. Surprisingly, however, the presence of sialic acid containing glycolipids has been shown to enhance significantly the capacity of LPS to initiate activation of the alternative pathway of complement. These data suggest that sialic acid can enhance as well as inhibit the formation of a stable alternative-pathway C3 convertase.

Further characterization of the tracheal receptor for Pseudomonas aeruginosa.

The purpose of this study was to obtain more information on the nature of the macromolecule to which Pseudomonas aeruginosa adheres. Acid-injured tracheal epithelium was treated with trypsin or lipase to determine whether the receptor molecule was a protein or a lipid. Lipase treatment significantly reduced adherence to these cells, whereas trypsin had no effect. Since the receptor appeared to be a lipid containing sialic acid, gangliosides were used to test whether they would inhibit adherence. Crude ganglioside preparations inhibited adherence in a dose-dependent manner when added to the bacteria before exposure to tracheal cells. Lastly, fibronectin, which presumably binds to gangliosides, significantly reduced the adherence of these organisms. According to these findings Pseudomonas aeruginosa appears to adhere to a sialic acid-containing glycolipid on cell surfaces, probably a ganglioside.

The effect of exogenous gangliosides on neurons in culture: A morphometric analysis

Cultures of isolated neurons have been treated with a purified preparation of gangliosides (10−5M and 10−9M) added to the cell growth medium at the 3rd day in culture and a morphometric analysis of the cells was performed with an image analyzer after 1 and 4 days of treatment. The number of cells and the area of the cell bodies were increased following the treatment. The results indicate as well the ‘sprouting’ effect of the glycolipids on the number of secondary neuronal processes and an increase in the length of the primary neuntes. The present data and other biochemical evidence (Dreyfus et al., 1984, J. Neurosci. Res.) suggest that the addition of exogenous gangliosides may have a trophic effect on neurons, greatly enhances the number of cell to cell contacts, and, possibly, stimulates cell proliferation and differentiation.

Glycolipid-dependent interaction between human migration-inhibitory factor and mononuclear phagocytes

It has been previously established in the guinea pig that the response of peritoneal macrophages to migration inhibitory factor (MIF) is enhanced by a macrophage glycolipid and that gangliosides reversibly bind MIF. This suggests that glycolipids function as cell surface receptors for MIF. In this report, it is demonstrated that the response of human peripheral blood monocytes to human MIF is augmented by preincubation of these cells with glycolipidenriched material extracted from the human macrophage-like cell line U937 or human peripheral blood monocytes and with a purified glycolipid from guinea pig peritoneal macrophages. In addition, a mixed ganglioside preparation from bovine brain shows the same effect. In contrast, the pure gangliosides, G M1 and G D1a, and glycolipids from the HL-60 cell line, which is a MIF-unresponsive cell line, were not able to enhance the response to human MIF. The specificity of enhancement by particular glycolipids could not be attributed to an increased uptake of only enhancing glycolipids since there was no significant difference between the association of monocytes with radioactive liposomes containing biologically active or inactive glycolipids. Pronase treatment did not affect the enhancing activity of the U937 glycolipidenriched material. Incubation of cells with glycolipids results in enhancement only if done at 37 °C and not at 4 °C. Therefore, the association of lipid with the monocyte surface appears to be dependent on temperature. Further evidence for the receptor nature of these enhancing glycolipids is provided by experiments involving affinity purification experiments. Coupling of bovine brain mixed gangliosides to agarose resulted in a matrix capable of reversibly binding MIF. G D1a-agarose was inactive in this respect.

Interaction of gangliosides with B cells in splenic fragment cultures

The effect of gangliosides upon murine adult B cells at the single precursor cell level was examined using the splenic focus assay. Adult B cells were stimulated in in vitro organ fragment culture by a hapten-modified carrier protein in the presence of an excess of carrier-primed T cells. The addition of a potential tolerogen in the form of antigen coupled to a carrier not previously presented to the T cells resulted in a temporary unresponsiveness of the onset of antibody production in adult B cells, but not a permanent state of tolerance. This delay could be eliminated by the addition of purified murine gangliosides during the presentation of the hapten coupled to the unrecognized carrier. The ganglioside preparation was fractionated by ion-exchange chromatography and the active fraction was found to be a disialoganglioside. These results suggest that the ganglioside may interfere with membrane receptor-related events occurring during or after antigen binding or cross-linking to responding B cells.

Evidence for a cryptic lectin site in the cell-binding domain of plasma fibronectin.

Various proteolytic fragments from the central region of the fibronectin subunit chains containing the main cell-affinity site were applied in cell binding studies using peritoneal macrophages of guinea pigs. A 125I-labelled 23-kDa peptide was relatively well bound by the cells. Attachment to cells was partially inhibited by wheat germ lectin, suggesting a lectin-like site in the cell-binding domain which recognizes oligosaccharide groups with terminal N-acetylglucosamine or N-acetylneuraminic acid. Binding was inhibited by N-acetylneuraminic acid with half-maximal effect at 2 X 10(-3) M. Other inhibitors were a sialic acid rich ganglioside preparation and fetuin, a sialic acid-containing glycoprotein. In contrast to the 23-kDa peptide a 125I-labelled 125-kDa fragment was only weakly bound, although it included the sequence of the 23-kDa peptide on its C-terminus. The residual binding was weakly inhibited by low concentrations of wheat germ lectin and was remarkably improved by higher concentrations. The behavior of the peptide was explained by the presence of a sialic acid-containing oligosaccharide side chain localized outside of the 23-kDa region and interacting with the lectin-like site in the cell-binding sequence. In accord with this suggestion a 95-kDa fragment representing the oligosaccharide-containing part of the 125-kDa peptide was capable of inhibiting at least partially the cell attachment of the 23-kDa piece. The results indicate a lectin-like affinity site in the cell-binding region of fibronectin which is accessible in the 23-kDa peptide, but is masked in the 125-kDa fragment and in fibronectin by a sialic acid-containing oligosaccharide moiety.

Ganglioside‐Induced Neuritogenesis: Verification That Gangliosides Are the Active Agents, and Comparison of Molecular Species

Gangliosides were previously reported to induce neuritogenesis in primary neuronal cultures and in some neurally derived cell lines. Because isolated gangliosides usually contain variable quantities of peptides, we investigated the possibility that neurite-stimulating activity could be caused by these contaminants. Ganglioside preparations from bovine brain and other sources were subjected to a three-step purification procedure that eliminated at least 95% of the contaminating peptides. These purified preparations retained their capacity to induce extensive neurite growth in neuro-2A murine neuroblastoma. Proteolytic digestion and a number of additional procedures were used to reduce residual contamination further without loss of activity. Several crude ganglioside samples had negative effects on neurite development until freed of their inhibitory factors, which were derived from the tissue and/or introduced during laboratory operations. This was particularly evident for bovine white matter gangliosides whose activity increased in proportion to peptide removal. When carefully purified, virtually all of 11 different gangliosides tested were highly active, with the possible exception of GM4, which demonstrated only moderate activity in a limited number of tests. All of the neutral glycolipids tested, as well as sulfatides and free sialic acid, were inactive.