Gangliosides GD1b Research Articles

Lactonization of GD1b ganglioside under acidic conditions

Gangliosides that contain the disialosyl residue α-Neu5Ac-(2→8)-α-Neu5Ac-(2→3)- can lactonize in the presence of traces of acid and this reaction has been studied in detail on GD1b {β-Gal-(1→3)-β-GalNAc-(1→4)-[α-Neu5Ac-(2→8)-α-Neu5Ac-(2→3)]-β-Gal-(1→4)-β-Glc-(1→1)-Cer}. Lactonization occurs rapidly at a proton-ganglioside molar ratio of ⪢1. At equilibrium, the ratio of GD1b to its lactone is 3:7. The data suggest the possibility that a proton-driven lactonization of gangliosides may occur in vivo.

Monoclonal Antibody AA4, Which Inhibits Binding of IgE to High Affinity Receptors on Rat Basophilic Leukemia Cells, Binds to Novel α-Galactosyl Derivatives of Ganglioside GD1b

Abstract Mouse monoclonal antibody AA4 inhibits the binding of IgE to high affinity IgE receptors on the rat basophilic leukemia cell line RBL-2H3. As shown by immunostaining of thin layer chromatograms, antibody AA4 binds avidly to two disialogangliosides (antigen I and antigen II) that occur in this cell line. The two antigens were purified by anion exchange chromatography followed by short-bed continuous thin-layer chromatography. About 230 micrograms of antigen I and 60 micrograms of antigen II were obtained from 20 g (wet weight) of leukemia cells. The structures of both purified antigens were determined to be alpha-galactosyl derivatives of the ganglioside GD1b by fast atom bombardment-mass spectrometry, by chemical ionization-mass spectrometry of permethylated samples, by gas chromatography-mass spectrometry of partially methylated alditol acetates, and by treatment with exoglycosidases and mild acid hydrolysis. The structure of antigen I is: (formula; see text) Antigen II has an additional alpha-galactosyl residue as follows: (formula; see text) The ceramide of antigen I contains approximately equal amounts of C24:0, C22:0, C20:0, C18:0, and C16:0 N-acyl fatty acids. The ceramide base is predominantly sphingosine along with a small amount of dihydrosphingosine. In contrast, the ceramide of antigen II contains mainly C24:0 N-acyl fatty acid with much lower amounts of C22:0, C20:0, and C18:0 fatty acids. Moreover, the ceramide base is approximately 55% sphingosine and 45% dihydrosphingosine. No unsaturated N-acyl fatty acids were detected in either antigen.

Ganglioside content and composition of cells from normal and atherosclerotic human aorta

The ganglioside content and composition of cells obtained by enzyme digestion of 2 layers of human aortic intima were investigated. Five gangliosides were identified in cells isolated from the external musculo-elastic intimal layer adjacent to the media: GM 3, GM 1, GD 3, GD 1a, and GT 1b. The same gangliosides plus ganglioside G x, the chromatographic mobility of which corresponded to the mobility of ganglioside GD 1b from human brain, were found in cells from the internal elastic-hyperplastic intimal layer adjacent to the vessel lumen. In both layers, the major cellular ganglioside was GM 3 which represented 60% of the total cellular ganglioside content. The ganglioside content was lower in cells obtained from fatty streaks compared to cells isolated from unaffected intima. The amount of di- and trisialogangliosides in atherosclerotic plaque cells was lower, and that of monosialogangliosides higher than in cells isolated from unaffected intima. The amount of GM 3 was mainly responsible for the difference in the total ganglioside content of cells obtained from different lesion types. On the whole, cells from fatty streaks contained smaller amounts of total gangliosides, whereas cells from plaques had greater total ganglioside content, than cells from unaffected intima.

Identity of GA1, GM1a and GD1b synthase in Golgi vesicles from rat liver

Synthesis of ganglioside GDD1b from ganglioside GD2 was demonstrated using Golgi membranes isolated from rat liver. Competition experiments using gangliosides GA2, GM2 and GD2 as substrates, and as mutual inhibitors for ganglioside galactosyltransferase activity in preparations of Golgi vesicles derived from rat liver, suggested that galactosyl transfer to these three compounds, leading to gangliosides GA1, GM1a and GD1b respectively, is catalyzed by one enzyme. These results strengthen the hypothesis that the main site for the regulation of ganglioside biosynthesis occurs within the reaction sequence LacCer→GA3→GD3→GT3.

Formation of Ganglioside GD 1b‐Lactone in Rat Brain from Intracisternally Administered GD 1b

The presence of ganglioside GD1b, in lactone form GD1b-L, was ascertained in rat brain. The possible formation of GD1b-L from GD1b in brain was explored by the intracisternal injection of GD1b, 3H-labelled at the level of the terminal galactose. This was followed by recognition of the radioactive gangliosides formed at different times (1, 3, and 7 days) after injection. Whereas at 0 time after injection the only radioactive ganglioside was GD1b, after 1, 3, and 7 days other radioactive gangliosides were also found, thus indicating GD1b penetration into the brain tissue, followed by metabolic processing. Besides GD1b, the following radioactive gangliosides were recognized: GM1 and GM2, derived from GD1b degradation; GT1b, formed by the direct sialylation of GD1b; and GD1b-L, produced by metabolic lactonization. The radioactivity carried by GD1b-L was maximal 3 days after injection; its time course was different from that of the other gangliosides, suggesting that the process of lactonization is separate from that of both degradation and glycosylation. Under the same experimental conditions, some radioactive gangliosides also appeared in the liver, although in much smaller amounts than in brain. Radioactive GD1b-L could not be detected in liver, thus indicating that metabolic lactonization is a tissue- or organ-specific process.

Brain gangliosides in dementia of the Alzheimer type.

Gangliosides GM1, GD1a, GD1b, and GT1b were measured in nine brain regions of five patients, clinically and neuropathologically diagnosed as having dementia of the Alzheimer type (DAT), and of three control patients. Analysis of variance revealed that mean concentrations of all gangliosides analyzed were significantly lower in DAT than in control brains. The areas affected in DAT included the nucleus basalis, and entorhinal, posterior cingulate, visual, and prefrontal cortices. A significant interaction between ganglioside type and brain area indicated unequal ganglioside concentrations. Individual gangliosides had significantly different concentrations in the hippocampal, entorhinal, posterior cingulate, visual, and prefrontal cortices. Analysis of ratios of "a"-ganglioside (GM1 and GD1a) and "b"-ganglioside (GD1b and GT1b) subtypes indicated that DAT preferentially affected "b"-gangliosides. Ganglioside concentrations in nucleus basalis did not correlate with age at disease onset, age at death, or postmortem interval. Changes in gangliosides, observed in this study, were not correlated with classic DAT neuropathology.

Binding of cholera toxin to pig intestinal mucosa glycosphingolipids: relationship with the ABO blood group system.

A search for compounds from intestinal mucosa of pigs carrying and not carrying blood group A-active substances (A+ and A- pigs, respectively) capable of binding cholera toxin (CT) was performed. Glycolipid extracts from a pool of pig intestinal mucosa resolved in thin-layer chromatography (TLC) revealed the presence of six to eight compounds capable of binding 125I-CT, two of them running as the ganglioside standards GM1 and GD1b. When intestinal mucosa glycolipids from single pigs were assayed by TLC for CT-binding capacity, two different patterns of labeling were observed. The main difference was at the level of compounds running below GD1b. The A+ pigs but not the A- pigs showed CT binding at this level. The major CT-binding compound detected only in A+ pigs was purified and some properties were determined. After TLC developed with different solvent systems, the purified compound bound CT and also immunoreacted with anti-A and anti-AB antisera but not with anti-B antiserum. The compound was also able to inhibit the hemagglutination of human A erythrocytes caused by anti-A antiserum, but inhibition was not observed with the B-anti-B or O (H)-Ulex europaeus lectin systems. A partial chemical characterization indicated that the active compound is a neutral glycosphingolipid containing glucose, fucose, galactose, and hexosamine. The existence of a blood group-active substance(s) able to interact with CT may help to explain the relationship between ABO blood groups and the diarrheal disease caused by infection with Vibrio cholerae.

Modulation of ganglioside biosynthesis in primary cultured neurons.

Murine cerebellar cells were pulse labeled with [14C]galactose, and the incorporation of radioactivity into gangliosides and neutral glycosphingolipids was examined under different experimental conditions. In the presence of drugs affecting intracellular membrane flow, as well as at 15 degrees C, labeled GlcCer was found to accumulate in the cells, whereas the labeling of higher glycosphingolipids and gangliosides was reduced. Monensin and modulators of the cytoskeleton effectively blocked biosynthesis of the complex gangliosides GM1, GD1a, GD1b, GT1b, and GQ1b, whereas incorporation of radioactivity into neutral glycosphingolipids, such as glucosylceramide and lactosylceramide, as well as GM3, GM2, and GD3 was either increased or unaltered. As monensin has been reported to interfere with the flow of molecules from the cis to the trans stacks of the Golgi apparatus, this result highlights at least one subcompartmentalization of ganglioside biosynthesis within the Golgi system. Inhibitors of energy metabolism affected, predominantly, the biosynthesis of the b-series gangliosides, whereas a reduced temperature (15 degrees C) more effectively blocked incorporation of radiolabel into the a-series gangliosides, a result suggesting the importance of GM3, as the principal branching point, for the regulation of ganglioside biosynthesis.

Gangliosides mediate inhibitory effects of tetanus and botulinum A neurotoxins on exocytosis in chromaffin cells

P. Marxen, U. Fuhrmann and H. Bigalke. Gangliosides mediate inhibitory effects of tetanus and botulinum A neurotoxins on exocytosis in chromaffin cells. Toxicon 27, 849–859, 1989.—Bovine chromaffin cells in monolayer culture were preloaded with 3H-NA (noradrenaline) and subsequently stimulated with carbachol. Botulinum A neurotoxin partially inhibited the evoked release of 3H-NA and the basal efflux of the hormone. The inhibition of evoked release did not exceed 40%, although the cells were exposed to 10 μg/ml of toxin for 6 days. The inhibitory effect of botulinum A neurotoxin was neutralized by its antibodies. In contrast to botulinum A neurotoxin, tetanus toxin at even higher concentrations did not influence evoked release. This difference in sensitivity could be explained by the ganglioside pattern of chromaffin cells. Ganglioside GD1a, a putative receptor for botulinum A neurotoxin, could be identified in lipophilic extracts, whereas the tetanus toxin binding gangliosides GT1b and GD1b could not be detected by means of thinlayer chromatography. Treatment of the cells with neuraminidase abolished both, GD1a and the inhibitory effect of botulinum A neurotoxin. Incubation of chromaffin cells with a mixture of gangliosides (21% GM1, 44% GD1a, 15% GD1b, 20% GT1b) not only increased the efficacy of botulinum A neurotoxin but also made the cells sensitive towards tetanus toxin. The concentration-response curve of botulinum A neurotoxin was shifted to the left about five-fold and the maximum inhibition of evoked release was increased up to 60%, even though the cells were exposed to the toxin for 3 days only. In contrast, the maximum inhibition that could be achieved by tetanus toxin was 40%. The results indicate that polysialogangliosides are important for the intracellular accumulation of these clostridial neurotoxins.

Immunostaining of motor nerve terminals by IgM M protein with activity against gangliosides GM 1 and GD lb from a patient with motor neuron disease

We demonstrated the binding of an IgM monoclonal protein, obtained from a patient with motor neuron disease, with known antibody activity against gangliosides GM1, GD1b, and asialo GM1, to neuromuscular junctions in guinea pig gastrocnemius muscle, using an indirect immunofluorescence technique. Staining disappeared after delipidation of muscle sections. Denervated muscle sections showed no labeling at the neuromuscular junction after incubation with the patient's serum. This indicates presynaptic binding of the IgM M protein and supports the concept that IgM monoclonal antibody to nerve terminal determinants may underlie a motor neuron disorder.

Generation of Antibodies to Gangliosides GM1 and GD1b

Generation of Antibodies to Gangliosides GM1 and GD1b

Both GA2, GM2, and GD2 synthases and GM1b, GD1a, and GT1b synthases are single enzymes in Golgi vesicles from rat liver.

Competition experiments using lactosylceramide, ganglioside GM3 and ganglioside GD3 as substrates, as well as mutual inhibitors for ganglioside N-acetylgalactosaminyltransferase, in Golgi vesicles derived from rat liver suggested that N-acetylgalactosamine transfer to these three respective compounds, leading to gangliosides GA2, GM2, and GD2, respectively, is catalyzed by one enzyme. Analogous studies with gangliosides GA1, GM1, and GD1b as glycolipid acceptors in sialyltransferase assays indicated GM1b, GD1a, and GT1b synthases to be identical. These results are incorporated into a model for ganglioside biosynthesis and its regulation.

Identification and Characterization of Gangliosides Reacting with IgM Paraproteins in Three Patients with Neuropathy Associated with Biclonal Gammopathy

IgM monoclonal antibodies from three patients with polyneuropathy associated with biclonal gammopathy reacted with monosialoganglioside GM1 on thin-layer chromatograms. An IgM paraprotein in one of the patients with a predominantly motor neuropathy also reacted strongly with the ganglioside GD1b and asialo-GM1. All three of these antigenic lipids have a Gal(beta 1-3)GalNAc moiety in common which would appear to be the antigenic determinant. However, this IgM also cross-reacted weakly with paragloboside which has an N-acetyllactosaminyl [Gal(beta 1-4)GlcNAc] terminal structure. The specificity of the other paraprotein in this patient is not known. The IgM paraproteins reacting with GM1 in both of the other patients exhibited different specificity because they did not react with GD1b and asialo-GM1, but reacted strongly with GM2 ganglioside. The data suggest that the epitope for both of these IgMs is in the GalNAc(beta 1-4)(NeuAc alpha 2-3)Gal(beta 1-4)Glc region of the gangliosides that is common to both GM2 and GM1. The second IgM paraproteins in both of these latter patients react with the myelin-associated glycoprotein (MAG) and two 3-sulfoglucuronyl glycolipids that share antigenic determinants with MAG.

Many pulmonary pathogenic bacteria bind specifically to the carbohydrate sequence GalNAc beta 1-4Gal found in some glycolipids.

Pneumonia is one of the most common causes of death from infectious disease in the United States. To examine the possible role of carbohydrates as adhesion receptors for infection, several pulmonary pathogenic bacteria were studied for binding to glycosphingolipids. Radiolabeled bacteria were layered on thin-layer chromatograms of separated glycosphingolipids, and bound bacteria were detected by autoradiography. The classic triad of infectious bacteria found in cystic fibrosis, Pseudomonas aeruginosa, Haemophilus influenzae, and Staphylococcus aureus, along with other bacteria commonly implicated in typical pneumonia, such as Streptococcus pneumoniae, Klebsiella pneumoniae, and certain Escherichia coli, bind specifically to fucosylasialo-GM1 (Fuc alpha 1-2Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Cer), asialo-GM1 (Gal beta 1-3GalNAc beta 1-4Gal beta-1-4Galc beta 1-1Cer), and asialo-GM2 (GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer). Bacteria maintained in nutrient medium bind better than the same cells suspended in buffer. They do not bind to galactosylceramide, glucosylceramide, lactosylceramide, trihexosylceramide, globoside, paragloboside, Forssman glycosphingolipid, or several other glycosphingolipids tested, including the gangliosides GM1, GM2, GM3, GD1a, GD1b, GT1b, and Cad. The finding that these pathogens do not bind to lactosylceramide suggests that beta 1-4-linked GalNAc, which is positioned internally in fucosylasialo-GM1 and asialo-GM1 and terminally in asialo-GM2, is required for binding. beta-N-Acetylgalactosamine itself, however, is not sufficient for binding, as the bacteria did not bind to globoside, which contains the terminal sequence GalNAc beta 1-3Gal. These data suggest that these bacteria require at least terminal or internal GalNAc beta 1-4Gal sequences unsubstituted with sialyl residues for binding. Other bacteria, including Mycoplasma pneumoniae, Streptococcus pyogenes, Salmonella species, and some E. coli, do not bind to the GalNAc beta 1-4Gal sequence. The biological relevance of these data is suggested by our finding that substantial amounts of asialo-GM1 occur in human lung tissue.

Antibodies to glycoconjugates in human motor neuron disease.

IgM monoclonal gammopathy has been reported in some patients with motor neuron disease. The monoclonal IgMs in several of the patients bind to the carbohydrate epitope Gal (beta 1-3) GalNAc, which is shared by gangliosides GM1 and GD1b and glycoproteins in the nervous system and crossreacted with Gal (beta 1-3) GlcNAc. They also immunostain spinal cord and gray matter and presynaptic terminals of motor neurons at the neuromuscular junction. The role and mechanisms of action of these antibodies in motor neuron disease is under investigation.

Interaction of influenza virus with gangliosides and liposomes containing gangliosides

It has already been shown that influenza virus binds unspecifically to liposomes containing ganglioside GM1 wheras with gangliosides GD1b and GT1b binding occurs in a specific and saturable manner [Slepushkin et al. (1986) Biol. Membr. 3, 229-235]. In the present study the mode of interaction between influenza virus and various gangliosides or phospholipid liposomes containing cholesterol and gangliosides has been investigated. The influence of exogenous gangliosides on the structure of the viral envelope was studied using fluorescent and photoactivatable phospholipids incorporated into the viral membrane. With both types of probes maximal effects of gangliosides were caused by GT1b. Addition of that ganglioside resulted in a marked decrease in the fluorescence polarization (P) of fluorescent labeled virus as well as in substantial changes in the binding of photoactivatable analogues of sphingomyelin and phosphatidylcholine to virus proteins, mainly hemagglutinin. The effects of GT1b and GD1b on P value were comparable, whereas gangliosides with other oligosaccharide chains caused much smaller changes in P. Furthermore GT1b but not GM1 influenced phospholipid-hemagglutinin cross-linking. Interaction of the virus with large unilamellar liposomes was monitored by two fluorescence assays based on resonance-energy transfer from the tryptophans and tyrosines of viral proteins to vesicles labeled with a triacylglycerol (anthrylvinyldioleoylglycerol) or from these labeled vesicles to virions labeled with a perylenoyl derivative of galactosylcerebroside (PGalSph). A third fluorescence assay was based on relief of self-quenching in PGalSph-labeled virions, upon low-pH-induced virus-liposome fusion. With all three fusion assays the changes of fluorescence caused by GT1b were more pronounced than those induced by GM1. On the other hand, virus-induced release of [14C]glucose from multilamellar liposomes was enhanced by GM1 but not by GT1b or GD1b. It is concluded that the interaction of GT1b or GD1b with virus hemagglutinin induces a rearrangement of the viral lipids rendering lipid bilayer areas of the viral envelope significantly fluid, which in turn promotes fusion of the virus with target membranes. Probably virus-liposome fusion and virus-induced liposome leakage are brought about by different mechanisms depending on specific or unspecific binding of the virions to the target.

Monoclonal IgM with unique specificity to gangliosides GM 1 and GD 1b and to lacto‐ N ‐tetraose associated with human motor neuron disease

IgM lambda monoclonal antibodies in two patients with motor neuron disease showed the same unique antigenic specificity. They bound to gangliosides GM1 and GD1b and to lacto-N-tetraose-BSA. By immunofluorescence microscopy they bound to central and peripheral nerve tissue and to motor end-plates at the neuromuscular junction. Sera from control subjects did not contain antibodies of similar specificity. Monoclonal IgMs with the same unique specificity could be responsible for motor neuron disease in some patients with monoclonal gammopathies.

Generation of antibodies to gangliosides GM1 and GD1b; Genetic control of fine antigenic specificity

Generation of antibodies to gangliosides GM1 and GD1b; Genetic control of fine antigenic specificity

Myelin Basic Protein as an Encephalitogen in Encephalomyelitis and Polyneuritis Following Rabies Vaccination

Encephalitis and polyneuritis occurring after rabies vaccination are believed to be immunologically mediated. We studied antibody responses to neural antigens in 36 patients with major neurologic complications, 25 with minor complications, and 39 with no complications after immunization with a brain-derived, Semple rabies vaccine. Patients with major complications had significantly elevated levels of antibody to brain white matter as compared with the other groups (P less than 0.001). Assays for antibody to selected central nervous system antigens showed that high levels of serum and cerebrospinal fluid antibody to myelin basic protein correlated with the presence of major neurologic complications (both central and peripheral nervous systems). The level of antibody to cerebroside correlated best with the number of injections of vaccine, but like antibody to myelin basic protein, the antibody to cerebroside was present in the cerebrospinal fluid of patients with major complications. Some patients with major complications also had antibodies directed to the gangliosides GD1b and GT1b. No antibodies to myelin-associated glycoprotein were detected in any of the samples. These data implicate myelin basic protein as an encephalitogen in these autoimmune diseases of the human nervous system, but suggest that immune responses to cerebroside and certain gangliosides may have an augmentative role in severe disease.

Ganglioside lactones:1H-NMR determination of the inner ester position of GD1b-ganglioside lactone naturally occurring in human brain or produced by chemical synthesis

The complete definition of the chemical structure of GD1b-ganglioside (GD1b) lactone isolated from human brain has been given by means of spectrometric and spectroscopic analyses. GD1h lactone contains a single ester linkage involving the external sialic acid carboxyl group and the C-9 hydroxyl group of the internal sialic acid unit. A synthetic lactone of GD1b prepared treating GD1b with glacial acetic acid characterized in the same way showed an identical chemical structure.