Related Gangliosides Research Articles

Gangliosides are neuronal ligands for myelin-associated glycoprotein.

Nerve cells depend on specific interactions with glial cells for proper function. Myelinating glial cells are thought to associate with neuronal axons, in part, via the cell-surface adhesion protein, myelin-associated glycoprotein (MAG). MAG is also thought to be a major inhibitor of neurite outgrowth (axon regeneration) in the adult central nervous system. Primary structure and in vitro function place MAG in an immunoglobulin-related family of sialic acid-binding lactins. We report that a limited set of structurally related gangliosides, known to be expressed on myelinated neurons in vivo, are ligands for MAG. When major brain gangliosides were adsorbed as artificial membranes on plastic microwells, only GT1b and GD1a supported cell adhesion of MAG-transfected COS-1 cells. Furthermore, a quantitatively minor ganglioside expressed on cholinergic neurons, GQ1b alpha (also known as Chol-1 alpha-b), was much more potent than GT1b or GD1a in supporting MAG-mediated cell adhesion. Adhesion to either GT1b or GQ1b alpha was abolished by pretreatment of the adsorbed gangliosides with neuraminidase. On the basis of structure-function studies of 19 test glycosphingolipids, an alpha 2,3-N-acetylneuraminic acid residue on the terminal galactose of a gangliotetraose core is necessary for MAG binding, and additional sialic acid residues linked to the other neutral core saccharides [Gal(II) and GalNAc(III)] contribute significantly to binding affinity. MAG-mediated adhesion to gangliosides was blocked by pretreatment of the MAG-transfected COS-1 cells with anti-MAG monoclonal antibody 513, which is known to inhibit oligodendrocyte-neuron binding. These data are consistent with the conclusion that MAG-mediated cell-cell interactions involve MAG-ganglioside recognition and binding.

Neuronal expression of a minor monosialosyl ganglioside GM1b in rat brain: immunochemical characterization using a specific monoclonal antibody

Monoclonal antibodies (mAbs) against GM1b ganglioside were raised by immunizing NZB/n mice with the antigen purified from bovine brains, and the details of binding specificity of the mAbs were characterized. Anti-GM1b mAb, termed NA-6, reacted specifically with GM1b (NeuAc) and GM1b(NeuGc). NA-6 antibody did not react with other structurally related gangliosides, indicating that the antibody recognizes NeuAc or NeuGc α2-3Galβ1-4GalNAcβ1-4Gal structure. Using NA-6 antibody, GM1b ganglioside in developing rat brain was investigated by TLC/enzyme-immunostaining and detected first on gestational day 16. The specific content of brain GM1b increased until postnatal day 10, and then gradually decreased in later stage of development. Immunohistochemically GM1b was found in proximal dendrites and cell bodies of neurons in extensive regions of adult rat brain. The immunoreactivity tended to be confined in patch-like clusters on cell membranes, as typically found in the hippocampus and neocortex. The GM1b synthase activity, when assayed in the cerebellar microsome proteins, was significantly reduced in lurcher mutant mouse that is devoid of both cerebellar granule and Purkinje cells. These findings demonstrate that GM1b ganglioside exists in neurons and is actively synthesized during the development in rat brain.

Modulation by GM1 ganglioside of β1-adrenergic receptor-induced cyclic AMP formation in Sf9 cells

The effect of ganglioside GM1 on isoproterenol-induced cAMP accumulation was studied in insect Sf9 cells expressing the human β 1 t-adrenergic receptor by infection with recombinant baculovirus. When such Sf9 cells were treated with isoproterenol plus IBMX, intracellular cAMP formation increased approximately 10-fold over the basal level. Preincubation of the baculovirus-infected cells with GM1 for 1 h caused a concentration-dependent inhibition of the isoproterenol-induced CAMP accumulation. Phosphatidylserine, GM3, GTIb and a bovine brain ganglioside preparation lacking GM1 did not cause significant inhibition. Forskolin-induced CAMP accumulation was not affected by the GM1 treatment. Inhibition of isoproterenol-induced cAMP formation by GM1 was not observed in Sf9 cells expressing, β 2-adrenergic receptor instead of the β 1-adrenergic receptor. Binding studies with (−)-[ 3H]CGP12177 showed that preincubation with GM1 significantly reduced the affinity of antagonist binding to the β 1-adrenergic receptor. These results suggest that GM1 or related ganglioside structure(s) may function as natural modulator(s) of the /3,-adrenergic receptor.

Production of monoclonal antibodies directed to Hanganutziu-Deicher active gangliosides, N-glycolylneuraminic acid-containing gangliosides.

We have established three kinds of monoclonal antibodies against gangliosides containing N-glycolylneuraminic acid (NeuGc) by immunization of BALB/c mice with the purified gangliosides inserted into liposomes comprising Salmonella minnesota R595 lipopolysaccharides, and fusion of spleen cells with a mouse myeloma cell line. One monoclonal antibody, SHS-1, which was generated by immunizing mice with purified i-active ganglioside(NeuGc), reacted specifically with the i-active ganglioside(NeuGc) used as an immunogen. Structurally related gangliosides, such as GM3(NeuGc), sialosylparagloboside (SPG) (NeuGc), or I-active ganglioside(NeuGc), corresponding gangliosides [GM3 containing N-acetylneuraminic acid (NeuAc), SPG(NeuAc), i-active ganglioside(NeuAc), and I-active ganglioside(NeuAc)], other gangliosides, or neutral glycosphingolipid (GSL) were not recognized by the monoclonal antibody. These findings indicate that the SHS-1 monoclonal antibody may be specific for NeuGc-containing i-active ganglioside. On the other hand, the other two monoclonal antibodies, MSG-1 and SPS-20, which were generated by immunizing mice with purified ganglioside GM3(NeuGc) and SPG(NeuGc), respectively, showed cross-reactivity to structurally related gangliosides. The MSG-1 monoclonal antibody exhibited reactivity to ganglioside GM3(NeuAc). The SPS-20 monoclonal antibody also cross-reacted with SPG(NeuAc), i-active ganglioside(NeuGc), and i-active ganglioside(NeuAc). Neither MSG-1 nor SPS-20 reacted with corresponding gangliosides, other gangliosides, or neutral GSLs tested. Using the SHS-1 antibody specific for i-active ganglioside(NeuGc), we studied the expression of NeuGc-containing antigen in human colon cancer tissue. An NeuGc-containing glycoconjugate was detected in the colon cancer tissue.

GANGLIOSIDE GD(2) EXPRESSION IN THE HUMAN NERVOUS-SYSTEM AND IN NEUROBLASTOMAS - AN IMMUNOHISTOCHEMICAL STUDY

The distribution of the disialoganglioside GD2 in the human nervous system was studied by immunostaining using monoclonal antibodies 3F8 and 3A7. Positive staining was detected throughout the central nervous system, predominantly in neuronal cell bodies and neuropil of the gray matter. The specific localization of reactivity in such structures as cerebellum and hippocampus differs from that of related gangliosides, suggesting that GD2 may have a distinct functional role(s) in the brain, and is not merely a metabolic intermediate. GD2 expression was also analyzed in a series of 39 neuroblastomas. Staining of both primitive neuroblastic and differentiating ganglioneuromatous elements was seen, although some tumor cell heterogeneity was noted. The results have implications for immunotherapy of neuro-blastomas using anti-GD2 antibodies.

A New Monoclonal Antibody Directed to Sialylα2-31acto<italic>neo</italic>tetraosylceramide and Its Application for Detection of Human Gastrointestinal Neoplasmas<xref ref-type="fn" rid="fn1"><sup>1</sup></xref>

A new monoclonal antibody (NS24) directed to the N-acetylneuraminyl alpha 2-3Gal beta 1-4GlcNAc residue in type II sugar chain of N-acetylneuraminyllactoneotetraosylceramide [sialylparagloboside, IV3(NeuAc)nLc4Cer] was prepared by hybridoma technique. Liposomes composed of dipalmitoylphosphatidylcholine, cholesterol, IV3(NeuAc)nLc4Cer, and lipopolysaccharides from Salmonella minnesota R595 were used for immunization with IV3(NeuAc)nLc4Cer isolated from human erythrocytes. This method allowed the fusion of spleen cells of immunized mouse with myeloma cells only three days after immunization. NS24 reacted specifically to both naturally occurring and chemically synthesized IV3-(NeuAc)nLc4Cer, whereas it has no reactivity to structurally related gangliosides, such as IV6(NeuAc)nLc4Cer, N-glycolylneuraminyl alpha 2-3lactoneotetraosylceramide [IV3(NeuGc)-nLc4Cer], i-active ganglioside [VI3(NeuAc)nLc6Cer], I-active ganglioside [VIII3(NeuAc)-VI3(NeuAc)IV6kladoLc8Cer], GM4(NeuAc), GM3(NeuAc), GM3(NeuGc), GM1b(NeuAc), GD3-(NeuAc), other ganglio-series gangliosides, sulfatide, and paragloboside (nLc4Cer). Synthetic N-acetylneuraminyl alpha 2-3lactotetraosylceramide [IV3(NeuAc)Lc4Cer] and its asialo-derivative (Lc4Cer) carrying type I sugar chain also showed no reaction with NS24. One to 100 pmol of IV3(NeuAc)nLc4Cer was detected dose-dependently by a thin-layer chromatography/enzyme immunostaining procedure. Human gastric carcinomas showed positive reactions with NS24 immunochemically and histochemically. NS24 reacted preferentially with poorly differentiated adenocarcinomas rather than well differentiated ones.

Isolation and characterization of extremely minor gangliosides, GM1b and GD1 alpha, in adult bovine brains as developmentally regulated antigens.

In addition to ganglioside GM1b, an unusual and extremely minor ganglioside, GD1 alpha, was efficiently isolated from bovine brain by combination of Q-Sepharose and Iatrobeads column chromatographies. In the course of purification steps, the presence of the sialidase-labile ganglioside was proved by a highly sensitive TLC/enzyme-immunostaining method. The structure was characterized by gas-liquid chromatography, permethylation study, sialidase degradation, immunostaining with specific antibodies, fast atom bombardment-mass spectrometry, and proton magnetic resonance spectrometry. The content of the ganglioside was very small (0.016%) in the total gangliosides. This finding suggests that a synthetic pathway of asialo GM1----GM1b----GD1 alpha may exist in mammalian brains. A monoclonal antibody NA-6 that was obtained by immunizing mice with purified GM1b reacted specifically with GM1b but showed no cross-reactivity with other structurally related gangliosides such as GM1a, GD1a, and so on. Using the method of TLC/immunostaining with NA-6, GM1b was found to be strongly expressed during embryonic days 14-17 in chick brains. Thus, it is assumed that extremely minor gangliosides like GM1b and GD1 alpha found in adult brains are characterized as embryonic molecules.

Human IgM monoclonal proteins that bind 3-fucosyllactosamine, asialo-GM1, and GM1.

Analysis of monoclonal human Ig that occur in association with lymphoproliferative diseases has provided valuable information about antibody structure and idiotypes. We analyzed 940 human sera that contained monoclonal IgM proteins for their ability to bind to four carbohydrate epitopes. Ten sera bound asialo-GM1, five of these sera also bound GM1, 10 bound to 3-fucosyllactosamine (3-FL), and one each bound to levan and galactan. Although the antibody activity in each serum was associated with a single L chain isotype, both kappa and lambda isotypes were represented among the proteins that bound to asialo-GM1 and to 3-FL. Some antibodies against asialo-GM1 were highly specific for this compound, whereas others cross-reacted with the structurally related gangliosides GM1 and GD1b. The antibodies to asialo-GM1 also varied considerably in their ability to lyse liposomes that contain asialo GM1. An association of IgM mAb against gangliosides with peripheral neuropathies has been reported recently, but only one of five patients whose antibodies reacted with GM1 ganglioside had a neuropathy. The antibodies that bound 3-FL exhibited narrower specificity, and less than 10% cross reactivity was noted with structurally related carbohydrates. The frequency of monoclonal proteins that bound 3-FL and asialo-GM1, approximately 1:100 sera for each specificity, was surprisingly high in view of the fact that both of these epitopes are expressed in human tissues. We suggest that these antibodies may be poly-specific and/or that the subset of B lymphocytes that synthesizes these anti-carbohydrate antibodies undergoes malignant transformation more frequently than other B lymphocytes.

Bioactive gangliosides: analysis of functional structures of the tetrasialoganglioside G Q1b which promotes neurite outgrowth

Previous reports from our laboratory showed that tetrasialoganglioside G Q1b, when exogenously added, can promote cell proliferation and neurite outgrowth in two human neuroblastoma cell lines, GOTO and NB-1 (Tsuji, S., Arita, M. and Nagai, Y. (1983) J. Biochem. 94, 303–306). To clarify the activity-associated structure of G Q1b, we analyzed the structure-activity relationships using the G Q1b, molecule, derivatives of it, and related gangliosides. When the G Q1b molecule was divided into two parts, the ceramide and oligosaccharide moieties, no activity was found with the former, while with the latter the activity could be seen, although the level of activity obtained never exceeded half that of G Q1b itself and an optimal concentration of 100–200 ng/ml of 20–40-times that of native G Q1b (5 ng/ml) was required. Furthermore, the activity of G Q1b was completely abolished by neuraminidase treatment, which converted G Q1b to G M1, so we examined other molecular species of gangliosides having a common gangliotetraose backbone but linked differently with two to four sialic acids (i.e., G D1a, G D1b, G T1a G Q1c G Q1b and G Q1c). Among them, only G Q1b was found to be active. The results disclosed the interesting fact that deletion of any sialic acid residue from either of the two disialosyi residues of G Q1b results in a complete loss of activity and that the mere existence of the tetrasialosyl structure does not lead to activity; this indicates the absolute necessity of the G Q1b oligosaccharide structure for the expression of activity. For full expression of the activity, both the ceramide and oligosaccharide moieties were necessary. It was also found that the G Q1b oligosaccharide inhibited the activity of G Q1b at a concentration a few times greater than that of G Q1b, suggesting the involvement of a receptor-like mechanism in the action of G Q1b at the cell membrane.

A monoclonal antibody reacting specifically with ganglioside GD1b in human brain

A mouse monoclonal antibody directed against one of the major human brain gangliosides, GD1b, has been produced. The antibody binds specifically to the carbohydrate structure of GD1b as it does not react with structurally related gangliosides like GM1, GD2, GT1b or Fuc-GM1, or any other ganglioside of human brain. The results further indicate that terminal galactose as well as the disialosyl group linked to the inner galactose moiety are involved in binding to the antibody. Monoclonal antibody Brain ganglioside Ganglioside GD1b-specificity

Interaction of cholera toxin and toxin derivatives with lymphocytes. I. Binding properties and interference with lectin-induced cellular stimulation.

The interaction of cholera toxin and a number of toxin derivatives, containing different proportions of light and heavy toxin-composing subunits (L and H), with mouse lymphocytes was studied. Experiments with [(125)I]toxin showed that a single cell can rapidly, within minutes, bind up to 40,000 molecules of toxin, the association constant was estimated to 7 +/- 4 x 10(8) liters/mol, and binding was found to be very similar at 37 degrees C and 5 degrees C. Immunofluorescence studies revealed that the toxin attachment is located on the cell surface, and that purified L subunit but not H subunit binds to the cells. A natural cholera toxoid, built up by aggregated L subunits, showed almost identical binding properties as toxin to the cells. Pure G(M1) ganglioside, the proposed membrane receptor structure for toxin, prevented entirely the cellular binding of both toxin and toxoid. Cholera toxin in concentrations down to approximately 5 x 10(-11) mol/liter (corresponding to 10 bound molecules/cell) inhibited thymus cells from being stimulated to DNA synthesis by concanavalin A (con A), and spleen cells from such stimulation by phytohemagglutinin. The G(M1) ganglioside but not a series of other pure structurally related gangliosides and neutral glycosphingolipids neutralized this toxin activity. Toxin derivatives which, in similarity with toxin, possessed H as well as L subunits but in other proportions, were potent inhibitors of con A-induced thymocyte stimulation, whereas the natural toxoid (aggregated L subunits), purified toxin L subunit and purified toxin H subunit were up to 300-fold less active on a weight basis. The capacity of cholera proteins to inhibit con A-induced thymocyte stimulation correlated well with their activity in the rabbit intradermal toxicity assay. The inhibitory action of cholera toxin on con A-induced thymocyte stimulation did not depend on decreased cell viability from the toxin treatment, nor was it caused by a reaction between toxin and con A. [(125)I]con A bound equally well to the cells when toxin was present as when it was absent, which proves that the toxin did not compete for cellular con A receptors. Nor did the toxin seem to disturb the general mobility of membrane receptors or their ability to accumulate in caps. It is concluded that the L type of subunit confers rapid and firm binding of cholera toxin to lymphocyte membranes, probably to G(M1) ganglioside receptors. For biologic activity the additional presence of H subunit is important. One manifestation of toxin action on lymphocytes is inhibition of lectin-induced DNA synthesis; probably this effect relates to the ability of cholera toxin to raise the levels of intracellular cyclic 3'5'-adenosine monophosphate.