Biological roles of anti-GM1 antibodies in patients with Guillain–Barré syndrome for nerve growth factor signaling

To reveal the biological and pathological roles of anti-GM1 antibody in Guillain–Barré syndrome (GBS), we examined its effects on nerve growth factor (NGF) induced TrkA autophosphorylation (NGF-TrkA signaling) in PC12 cells, a sympathetic nerve cell line. The NGF-TrkA signaling is enhanced by exogenous GM1 ganglioside and this phenomenon is regarded as one of the functional aspects of GM1. The IgGs purified from patients' sera inhibited the NGF-TrkA signaling in GM1 pre-incubated PC12 cells. The degrees of inhibition by IgGs from patients paralleled their immunological reactivity to GM1. In addition, the IgGs also inhibited the neurite outgrowth of NGF-treated PC12 cells. Immunoglobulins in the rabbit sera, which were immunized by GM1, also caused a similar suppressive phenomenon. These results suggested that the anti-GM1 antibody could play roles in pathophysiology in anti-GM1 antibody positive GBS through interfering with the neurotrophic action of NGF and GM1 mediated signal modulation including NGF-TrkA signaling. It is suggested that the modulation of GM1 function is one important action of antibodies and could be one of the important mechanisms in GBS.     

Biological roles of anti-GM1 antibodies in patients with Guillain-Barré syndrome for nerve growth factor signaling

To reveal the biological and pathological roles of anti-GM1 antibody in Guillain-Barré syndrome (GBS), we examined its effects on nerve growth factor (NGF) induced TrkA autophosphorylation (NGF-TrkA signaling) in PC12 cells, a sympathetic nerve cell line. The NGF-TrkA signaling is enhanced by exogenous GM1 ganglioside and this phenomenon is regarded as one of the functional aspects of GM1. The IgGs purified from patients' sera inhibited the NGF-TrkA signaling in GM1 pre-incubated PC12 cells. The degrees of inhibition by IgGs from patients paralleled their immunological reactivity to GM1. In addition, the IgGs also inhibited the neurite outgrowth of NGF-treated PC12 cells. Immunoglobulins in the rabbit sera, which were immunized by GM1, also caused a similar suppressive phenomenon. These results suggested that the anti-GM1 antibody could play roles in pathophysiology in anti-GM1 antibody positive GBS through interfering with the neurotrophic action of NGF and GM1 mediated signal modulation including NGF-TrkA signaling. It is suggested that the modulation of GM1 function is one important action of antibodies and could be one of the important mechanisms in GBS.     

Evidence that ganglioside enriched domains are distinct from caveolae in MDCK II and human fibroblast cells in culture.

Cultures of MDCK II and human fibroblast cells were fed radioactive sphingosine and a radioactive GM3 ganglioside derivative containing a photoactivable group. The derived cell homogenates were treated with Triton X-100 and fractionated by sucrose-gradient centrifugation to prepare a detergent-insoluble membrane fraction known to be enriched in sphingolipid and caveolin-1, i.e. of caveolae. The detergent-insoluble membrane fraction prepared after feeding [1-3H]sphingosine to cells, was found to be highly enriched, with respect to protein content, in metabolically radiolabeled sphingomyelin and glycosphingolipids (about 18-fold). By feeding cells photoactivable radioactive GM3, after 2 h-chase, caveolin-1, CAV1, and proteins of high molecular mass became cross-linked to GM3, the cross-linking complexes being highly concentrated in the detergent-insoluble membrane fraction. The interaction between the ganglioside derivative and CAV1 was a time-dependent, transient process so that CAV1 cross-linking to GM3 was hardly detectable after a 24-h chase followed the pulse time. After a 24-h chase, only the high molecular mass proteins cross-linked to GM3 could be clearly observed. These results suggest that a portion of the GM3 administered to cells enters caveolae and moves to the glycosphingolipid domains, or enters caveolae that are then rapidly catabolized. Electron microscopy of cells in a culture immunostained with a monoclonal antibody to GM3 and a secondary gold-conjugated antibody detected several clusters of gangliosides on the plasma membranes separate from caveolae; gangliosides located inside the caveolae could not be detected. Scanning confocal microscopy of cells immunostained with anti-GM3 and anti-CAV1 Ig showed only a very small overlap with the CAV1 and GM3 signals. Thus, the biochemical and microscopic studies suggest that caveolae contain at most a low level of gangliosides and are separate from the GM3 ganglioside enriched domains.     

Anti-idiotypic monoclonal antibodies to GM1 identify ganglioside binding proteins

Ganglioside stimulated neurite outgrowth may be due to gangliosidebinding to membrane proteins or to intercalation into the membrane.To test that ganglioside binding proteins could be found onneuronal surfaces, antiidiotypic ganglioside monoclonal antibodies(AIG mAbs) were generated to mimic the biological propertiesof the GM1 ganglioside. The AIG mAbs were identified by theirability to bind to a known GM1 binding protein, the ß-subunit of cholera toxin. For the two AIG mAbs studied, AIG5 andAIG20, binding to ß-CT was blocked most strongly byGM1. This data also suggests that AIG5 and AIG20 mimic differentbut overlapping epitopes of the ganglioside GM1. Western blottingand immunoprecipitation of mammalian tissues reveals four potentialganglioside binding proteins of molecular weight 93, 66, 57,and 45 kDa. Immunocytochemistry demonstrates neuronal surfacelabel with the AIG mAbs, which suggests that gangliosides, enrichedon the neuronal surface membrane, are co-localized with putativeganglioside binding proteins. In bioassays, the AIG mAbs promoteneuronal sprouting. This shows that these antibodies can beused to study the biological effects of ganglioside bindingto neuronal surface proteins, and the role of gangliosides inthe activation of neurite outgrowth. agonist antibody anti-idiotypic antibody gangliosides ganglioside binding proteins     

Participation of the GM1 ganglioside in the gastrulation of anuran amphibian Bufo arenarum

In the present paper we established the ganglioside composition of the blastula and gastrula stages of the anuran amphibian Bufo arenarum, two relevant stages characterized by dynamic changes in morphology and cellular rearrangements. Densitometric studies evidenced that GD1a and GT1b were the more abundant gangliosides of the blastula embryos whereas GM1 and GM2 were the predominant species in gastrula embryos. Analysis of ganglioside abundance indicates that the "a" and "b" synthesis pathways perform similar biosynthetic activities in the blastula stage, in contrast to the gastrula stage in which a marked predominance of the "a" pathway occurred. The spatio-temporal expression of GM1 and of polygangliotetraosyl ceramides (pGTC) was investigated by wholemount immunocytochemistry using cholera toxin B subunit (CTB) and an affinity purified human anti-GM1 antibody. The pGTC were detected as GM1 after treatment with neuraminidase. Blastomeres from the inner surface of the blastocoelic roof (BCR) of blastula embryos were GM1 and pGTC positive. At midgastrula stage, embryos showed an increased labeling on the inner surface of BCR. To establish whether the GM1 ganglioside was involved in the gastrulation processes, CTB, anti-GM1 antibodies and anti-GM1 Fab' fragments were microinjected into the blastocoel cavity of blastula embryos. Treatment with the probes blocked gastrulation. Scanning electron microscopy analysis of blocked embryos revealed that mesodermal cell migration, radial interdigitation, and convergent extension movements were affected. The blocking of gastrulation was correlated with the absence of fibronectin and EP3/EP4 on the inner surface of blastocoelic roof of CTB- or anti-GM1 treated embryos. Results show that the GM1 ganglioside is differentially expressed by embryonic cells and participates in the morphogenetic processes of amphibian gastrulation. J. Exp. Zool. 286:457-472, 2000.     

GM1-ganglioside-induced Abeta assembly on synaptic membranes of cultured neurons

The cell-surface expression of GM1 ganglioside was studied using various cultured cells, including brain-derived endothelial cells, astrocytes, neuroblastoma cells (SH-SY5Y), and pheochromocytoma cells (PC12). GM1 ganglioside was detected only on the surface of native and nerve-growth-factor (NGF)-treated PC12 cells. We investigated whether GM1 ganglioside on the surface of these cells is sufficiently potent to induce the assembly of an exogenous soluble amyloid beta-protein (Abeta). A marked Abeta assembly was observed in the culture of NGF-treated PC12 cells. Notably, immunocytochemical study revealed that, despite the ubiquitous surface expression of GM1 ganglioside throughout cell bodies and neurites, Abeta assembly initially occurred at the terminals of SNAP25-immunopositive neurites. Abeta assembly in the culture was completely suppressed by the coincubation of Abeta with the subunit B of cholera toxin, a natural ligand for GM1 ganglioside, or 4396C, a monoclonal antibody specific to GM1-ganglioside-bound Abeta (GAbeta). In primary neuronal cultures, Abeta assembly initially occurred at synaptophysin-positive sites. These results suggest that the cell-surface expression of GM1 ganglioside is strictly cell-type-specific, and that expression of GM1 ganglioside on synaptic membranes is unique in terms of its high potency to induce Abeta assembly through the generation of GAbeta, which is an endogenous seed for Abeta assembly in Alzheimer brain.     

GM1-ganglioside-induced Aβ assembly on synaptic membranes of cultured neurons

The cell-surface expression of GM1 ganglioside was studied using various cultured cells, including brain-derived endothelial cells, astrocytes, neuroblastoma cells (SH-SY5Y), and pheochromocytoma cells (PC12). GM1 ganglioside was detected only on the surface of native and nerve-growth-factor (NGF)-treated PC12 cells. We investigated whether GM1 ganglioside on the surface of these cells is sufficiently potent to induce the assembly of an exogenous soluble amyloid β-protein (Aβ). A marked Aβ assembly was observed in the culture of NGF-treated PC12 cells. Notably, immunocytochemical study revealed that, despite the ubiquitous surface expression of GM1 ganglioside throughout cell bodies and neurites, Aβ assembly initially occurred at the terminals of SNAP25-immunopositive neurites. Aβ assembly in the culture was completely suppressed by the coincubation of Aβ with the subunit B of cholera toxin, a natural ligand for GM1 ganglioside, or 4396C, a monoclonal antibody specific to GM1-ganglioside-bound Aβ (GAβ). In primary neuronal cultures, Aβ assembly initially occurred at synaptophysin-positive sites. These results suggest that the cell-surface expression of GM1 ganglioside is strictly cell-type-specific, and that expression of GM1 ganglioside on synaptic membranes is unique in terms of its high potency to induce Aβ assembly through the generation of GAβ, which is an endogenous seed for Aβ assembly in Alzheimer brain.     

Activation of ganglioside GM3 biosynthesis in human monocyte/macrophages during culturing <Emphasis Type="Italic">in vitro</Emphasis>

We found that GM3 levels in human peripheral blood monocytes and cultured monocyte-derived macrophages were 0.37 and 2.7 μg per million cells, respectively. GM3 synthase of monocytes and to a greater extent of monocyte-derived macrophages was shown to be able to sialylate endogenous substrate, lactosylceramide (LacCer), to form GM3. With exogenously added LacCer, GM3 synthase activity was 57.1 and 563 pmol/h per mg protein in monocytes and monocyte-derived macrophages, respectively. The revealed changes in ganglioside GM3 biosynthesis are specific as the activity of some other sialyltransferases under these conditions was not altered. Human anti-GM3 synthase antibody detected in monocytes a main protein with molecular weight of 60 kD and minor proteins with molecular masses of 52 and 64 kD. In monocyte-derived macrophages the amounts of 60 kD protein and especially 64 kD protein sharply rose. Thus, the increase in ganglioside GM3 levels, GM3 synthase activity, and the enzyme amounts during culturing of monocyte/macrophages may be one of the mechanisms of in vivo increased ganglioside GM3 levels in arterial atherosclerotic lesions. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 7, pp. 948–954.     

Analysis of gangliosides from carp intestinal mucosa

The gangliosides of carp intestinal mucosa were isolated and analysed by thin-layer chromatography (TLC), TLC immunostaining test, and TLC/secondary ion mass spectrometry (TLC/SIMS). Four species of gangliosides, designated as G-1, G-2, G-3 and G-4, were separated on TLC. The TLC/SIMS analysis of the G-1 ganglioside of carp intestinal mucosa revealed a series of [M-H](-)ions from m/z 1061 to m/z 1131 representing the molecular mass range of GM4-like ganglioside with NeuAc. G-2, G-3 and G-4 gangliosides were analysed by the TLC immunostaining test. G-2 ganglioside was recognised by the monoclonal antibody specific for ganglioside GM1 (AGM-1 monoclonal antibody). However, G-3 ganglioside migrating on TLC between GM3 and GM1 ganglioside was not recognised by anti-GM3 monoclonal antibody and by AGM-1 monoclonal antibody. Furthermore, G-4 ganglioside with a similar TLC mobility as GD1a ganglioside did not show the reactivity to the anti-GD1a monoclonal antibody. In addition using the AGM-1 monoclonal antibody, the expression of GM1 ganglioside in the carp intestinal tissue was studied. GM1 ganglioside was detected on the epithelial cell surface of carp intestinal mucosa.     

Ganglioside GM1 Does Not Initiate, but Enhances Neurite Regeneration of Nerve Growth Factor-Dependent Sensory Neurones

An enzyme-linked immunoadsorbent assay (ELISA) for neurofilament protein was utilised to quantify the effect of exogenous ganglioside on neurite regeneration in cultures of dorsal root ganglion neurones. In contrast to nerve growth factor (NGF), ganglioside GM1 (100 micrograms/ml) failed to support neuronal survival and neurite regeneration as quantified by the ELISA assay and confirmed by morphological criteria. However, the simultaneous presence of GM1 (100 micrograms/ml) and NGF (0.5-5 ng/ml) throughout a 5-day period of culture resulted in an enhancement of previously reported NGF-induced increases in the expression of neurofilament protein. Further, the addition of GM1 (0-200 micrograms/ml) at 48 h in vitro to cultures initially established in the presence of 5 ng/ml NGF substantially increased the subsequent expression of neurofilament protein, this response being both independent of and not potentiated by NGF. The results in the present system suggest that GM1 cannot initiate a programme of neurite regeneration; however, GM1 can enhance this process with the response being secondary to the effect of NGF.     

Sialylpentaosylceramide detected with anti-GM2 monoclonal antibody. Structural characterization and complementary expression with GM2 in gastric cancer and normal gastric mucosa

The ganglioside fraction of human gastric mucosa was analyzed with a newly established anti-GM2 monoclonal antibody KM531. Using this antibody, accumulation of GM2 was observed in all of four cases of gastric carcinoma. In all ganglioside fractions extracted from normal gastric mucosa obtained from eight cases of peptic ulcer GM2 itself was not detected, but three kinds of glycolipid showing slower mobility than GM2 on thin-layer plates were detected by immunostaining with KM531. These glycolipids were assigned as NGM-1, -2, and -3. They were completely lost in all carcinoma tissues and in non-cancerous gastric mucosa from two cases of gastric cancer, and they were also not detected in the ganglioside fraction of small or large intestine. Of these glycolipids, the major one, NGM-1, was isolated from the pooled ganglioside fraction of normal gastric mucosa obtained from cases of peptic ulcer. The structure was determined by proton nuclear magnetic resonance, negative ion fast atom bombardment-mass spectrometry, gas chromatography-mass spectrometry, and treatment with exoglycosidases and mild acid hydrolysis. The structure was GalNAc beta 1----4(NeuAc alpha 2----3) Gal beta 1----4GlcNAc beta 1----3 Gal beta 1----4Glc beta 1----1Cer, which has the same terminal sequence as GM2 but has internal neolacto series structure. This epitope was previously identified as Cad blood group antigen. The decrease of this glycolipid and the increase of GM2 was considered to be a cancer-associated change in gastric mucosa.     

Lipolytic action of cholera toxin on fat cells. Re-examination of the concept implicating GM1 ganglioside as the native membrane receptor.

The possible role of galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GM1) ganglioside in the lipolytic activity of cholera toxin on isolated fat cells has been examined. Analyses of the ganglioside content and composition of intact fat cells, their membranous ghosts, and the total particulate fraction of these cells indicate that N-acetylneuraminylgalactosylglucosylceramide (GM3) represents the major ganglioside, with substantial amounts of N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GM2) and smaller amounts of other higher homologues also present. Native GM1 was not detected in any of these preparations. Examination of the relative capacities of various exogenously added radiolabeled sphingolipids to bind to the cells indicated that GM2 and glucosylsphingosine were accumulated by the cells to extents comparable to GM1. Galactosylsphingosine and sulfatide also exhibited significant, although lesser, binding affinities for the cells. The adipocytes appeared to nonspecifically bind exogenously added GM1; saturation of binding sites for GM1 could not be observed up to the highest concentration tested (2 X 10(-4) M), wherein about 7 X 10(9) molecules were associated with the cells. Essentially all of this exogenously added GM1 was found bound to the plasma membrane "ghost" fraction. Investigation of the biological responses of the cells confirmed their sensitivities to both cholera toxin and epinephrine-stimulated lipolysis, as well as the lag period displayed during the toxin's action. While we could confirm that the toxin's lipolytic activity can be enhanced by prior treatment of the fat cells with GM1, several of the observed characteristics of this phenomenon differ from earlier reported findings. Accordingly, added GM1 was able to enhance only the subsequent rate, but not the extent, of toxin-stimulated glycerol release (lipolysis) from the cells. We also were unable to confirm the ability of GM1 to enhance the toxin's activity at either saturating or at low toxin concentrations. The limited ability of added GM1 to enhance the toxin's activity appeared in a unique bell-shaped dose-response manner. The inability of high levels of GM1 to stimulate a dose of toxin that was ineffective on native cells suggests that the earlier reported ability of crude brain gangliosides to accomplish this was due to some component other than GM1 in the crude extract. While several glycosphingolipids and some other carbohydrate-containing substances that were tested lacked the ability to mimic the enhancing effect of GM1, 4-methylumbelliferyl-beta-D-galactoside exhibited an effect similar to, although less pronounced than, that of GM1. The findings in these studies are unable to lend support to the earlier hypothesis that (a) GM1 is cholera toxin's naturally occurring membrane receptor on native fat cells, and (b) the ability of exogenously added GM1 to enhance the toxin's lipolytic activity represents the specific creation of additional natural receptors on adipocytes...     

Direct immobilization of gangliosides onto gold-carboxymethyldextran sensor surfaces by hydrophobic interaction: applications to antibody characterization

We describe a novel immobilization technique to investigate interactions between immobilized gangliosides (GD3, GM1, and GM2) and their respective antibodies, antibody fragments, or binding partners using an optical biosensor. Immobilization was performed by direct injection onto a carboxymethyldextran sensor chip and did not require derivatization of the sensor surface or the ganglioside. The ganglioside appeared to bind to the sensor surface by hydrophobic interaction, leaving the carbohydrate epitope available for antibody or, in the case of GM1, cholera toxin binding. The carboxyl group of the dextran chains on the sensor surface did not appear to be involved in the immobilization as evidenced by equivalent levels of immobilization following conversion of the carboxyl groups into acyl amino esters, but rather the dextran layer provided a hydrophilic coverage of the sensor chip which was essential to prevent nonspecific binding. This technique gave better reactivity and specificity for anti- ganglioside monoclonal antibodies (anti-GD3: KM871, KM641, R24; and anti-GM2: KM966) than immobilization by hydrophobic interaction onto a gold sensor surface or photoactivated cross-linking onto carboxymethydextran. This rapid immobilization procedure has facilitated detailed kinetic analysis of ganglioside/antibody interactions, with the surface remaining viable for a large number of cycles (>125). Kinetic constants were determined from the biosensor data using linear regression, nonlinear least squares and equilibrium analysis. The values of kd, ka, and KAobtained by nonlinear analysis (KAKM871 = 1.05, KM641 = 1.66, R24 = 0.14, and KM966 = 0.65 x 10(7) M- 1) were essentially independent of concentration and showed good agreement with data obtained by other analytical methods.      

GM1 Ganglioside Treatment of PC 12 Cells Stimulates Ganglioside, Glycolipid, and Lipid, but Not Glycoprotein Synthesis Independently from the Effects of Nerve Growth Factor

The incorporation of radioactive precursors into gangliosides and other glycolipids, glycoproteins, and total lipids has been studied in rat pheochromocytoma PC12 cells. Starting with the same PC12 cell pool, cultures displaying different degrees of neuritic expression in response to nerve growth factor (NGF) and combinations of serum ganglioside GM1 were produced. Attempts were then made to correlate neuritic regulation with biochemical performances of these cells. NGF stimulates the incorporation of [3H]galactose into gangliosides and other glycolipids and glycoproteins and [14C]acetate into total lipids, regardless of the serum concentration. NGF both increased their initial labeling rates and promoted additional and more extensive labeling from culture day 4 onward. Unexpectedly, exogenous GM1 also elicited an increase in ganglioside labeling as well as that of the other lipid classes, but not of glycoproteins. The GM1-induced increase was evident at higher serum concentrations (1%) regardless of the presence or absence of NGF, but not apparent in low (0.15%) serum. Serum levels themselves did not affect labeling patterns in the absence of NGF and GM1. GM1-induced stimulation of labeling reflects an increase in the synthetic activities of the cells, and not increased precursor uptake or reduced product degradation. For all constituents stimulated by GM1, concurrent treatment with NGF produces cumulative effects, suggesting independent mechanisms of action by the two molecules.     

Cholera Toxin and Dibutyryl Cyclic AMP Inhibit the Expression of Neurofilament Protein Induced by Nerve Growth Factor in Cultures of Naive and Primed PC12 Cells

The effects of nerve growth factor (NGF), dibutyryl cyclic AMP (db cAMP), and cholera toxin on neurofilament protein expression in cultures of PC12 rat pheochromocytoma cells were examined using an enzyme-linked immunoadsorbent assay (ELISA). Morphological differentiation induced by NGF was associated with up to 30-fold increases in the level of neurofilament protein recognised by monoclonal antibody RT97. A more rapid response was apparent from primed as compared to naive PC12 cells. Cholera toxin and db cAMP both induced morphological differentiation of naive PC12 cells, but failed to promote neurite regeneration from primed cells. Neither response was associated with a significant induction of neurofilament protein. Both cholera toxin and db cAMP, but not B-cholera toxin nor antibodies to the toxin receptor, were found to inhibit the neurofilament protein response induced by NGF. Primed cells were more susceptible to this inhibition, and both cholera toxin and db cAMP inhibited neurite regeneration from these cells. These data suggest that increased intracellular cyclic AMP can suppress the expression of neuronal differentiation antigens induced by NGF, and are consistent with a role for neurofilament protein in promoting or facilitating the formation of a stable neuritic network.     

Glycolipid markers of murine lymphocyte subpopulations.

We have shown previously that purified antibodies to ganglioside GM1 react with peripheral T cells and most thymocytes in several strains of mice, independent of Thy-1 phenotype. GM1 and the Thy-1.2 antigen cap independently on C3H thymocytes, which provides additional evidence that GM1 is not the Thy-1.2 antigen. In C3H and nude mice antibodies to GM1 also react with a population of cells, comprising about 25% of lymphocytes from lymph nodes or spleen, that bear surface immunoglobulin. After removal of immunoglobulin from these cells by digestion with proteolytic enzyme, the GM1+ cells regenerate their surface immunoglobulin during 18 hr in culture, which indicates that these double-labeled cells synthesize their surface immunoglobulin. Protease treatment of lymphocytes reveals receptors for antibodies to GM1 on most cells. These data indicate that T and B cells differ in the accessibility of GM1 to antibody, and not necessarily in their content of GM1. Purified antibodies to asialo GM1 react with mature T cells in all strains of mice tested. In contrast to anti-GM1, these antibodies do not react with most thymocytes, with immunoglobulin-bearing lymphocytes of C3H or nude mice, nor with pronase-treated B cells.     

Monoclonal antibody directed to a Hanganutziu-Deicher active ganglioside, GM2 (NeuGc)

Spleen cells from NZB mouse immunized with a membrane fraction of rabbit thymus tissue were fused with BALB/c 6-thioguanine-resistant myeloma cells, P3-X63-Ag8.653. One hybridoma clone (Y-2-HD-1) produced IgM immunoglobulin that bound to an N-glycolylneuraminic acid-containing GM2 ganglioside, GM2(NeuGc), which is known to be a Hanganutziu-Deicher antigen. The specificity of the Y-2-HD-1 monoclonal antibody was examined, using authentic glycosphingolipids structurally related to GM2(NeuGc), by means of an enzyme-linked immunosorbent assay and thin-layer chromatography/enzyme immunostaining, respectively. The monoclonal antibody was found to be highly specific to GM2(NeuGc) and the epitope was a non-reducing terminal GalNAc beta 1-4[NeuGc alpha 2-3]Gal structure. This monoclonal antibody (Y-2-HD-1) bound to native mouse erythrocytes, in which GM2(NeuGc) is a major ganglioside. These results indicate that GM2(NeuGc) is located on the surface of mouse erythrocytes.     

Detection of antibodies in neuropathy patients by synthetic GM1 mimics

Antibodies to the ganglioside GM1 are associated with various forms of acute and chronic immune-mediated neuropathy, including Guillain-Barré syndrome (GBS) and multifocal motor neuropathy. In diagnostics and research, these antibodies are usually detected by GM1 preparations derived from bovine brain tissue, which are non-covalently attached to solid carriers such as enzyme-linked immunosorbent assay (ELISA) plates. Such brain-derived GM1 preparations are potentially contaminated with other glycolipids. In the current study, uncontaminated mono- and divalent synthetic analogs of the ganglioside GM1 were successfully attached via covalent bonds onto the surface of ELISA plates. The resulting modified diagnostic tool showed strong affinities and good specificities for binding of monoclonal mouse and human anti-GM1 antibodies and cholera toxin, as well as for the anti-GM1 antibodies in serum samples from neuropathy patients. While these proof-of-principle experiments reveal the potential of synthetic ganglioside mimics in diagnostics, they show the necessity of further studies to overcome certain limitations, specifically the non-specific interactions in the negative control assays with synthetic GM1.     

Isolated Bovine Spinal Motoneurons Have Specific Ganglioside Antigens Recognized by Sera from Patients with Motor Neuron Disease and Motor Neuropathy

The gangliosides GM1 and GD1b have recently been reported to be potential target antigens in human motor neuron disease (MND) or motor neuropathy. The mechanism for selective motoneuron and motor nerve impairment by the antibodies directed against these gangliosides, however, is not fully understood. We recently investigated the ganglioside composition of isolated bovine spinal motoneurons and found that the ganglioside pattern of the isolated motoneurons was extremely complex. GM1, GD1a, GD1b, and GT1b, which are major ganglioside components of CNS tissues, were only minor species in motoneurons. Among the various ganglioside species in motoneurons, several were immunoreactive to sera from patients with MND and motor neuropathy. One of these gangliosides was purified from bovine spinal cord and characterized as N-glycolylneuraminic acid-containing GM1 [GM1(NeuGc)] by compositional analysis, fast atom bombardment mass spectra, and the use of specific antibodies. Among seven sera with anti-GM1 antibody activities, five sera reacted with GM1(NeuGc) and two did not. Two other gangliosides, which were recognized by another patient's serum, appeared to be specific for motoneurons. We conclude that motoneurons contained, in addition to the known ganglioside antigens GM1 and GD1b, other specific ganglioside antigens that could be recognized by sera from patients with MND and motor neuropathy.     

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.