Characterization of two molecular species GD3 ganglioside from bovine buttermilk.

Two gangliosides, representing 85% of total lipid-bound sialic acid, have been isolated from bovine buttermilk and characterized. Both contained long-chain base, glucose, galactose and sialic acid in the molar ratio 1:1:1:2, and gave, upon sialidase treatment, a neutral glycolipid, characterized as lactosylceramide. Partial acid hydrolysis, permethylation analysis and chromium trioxide oxidation indicated their basic oligosaccharide portion to be NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----4Glc. The difference between the two forms was exclusively in the ceramide moiety of the molecule, one containing mainly long-chain (C22-C25) fatty acids and an equimolar proportion of C16 and C18 long-chain bases, and the other mainly palmitic acid and C18 long-chain base.

Determination of the affinity of monoclonal human IgM for myelin-associated glycoprotein and sulfated glucuronic paragloboside.

We determined the association constant of eight monoclonal IgM with two of their targets, i.e. the myelin-associated glycoprotein (MAG) or the sulfated glucuronic paragloboside (SGPG). All IgM had a 10- to 100-fold higher affinity for MAG than for SGPG. The affinity of the different IgM for MAG ranged from 1.3 x 10(-6) to 7 x 10(-9) mol/liter. The Scatchard plots for MAG were curvilinear, half of the sites being of high or low affinity. In contrast, the plots were linear in the assay using SGPG. No obvious correlations were found between the fine specificity of these IgM for the glucuronyl sulfate epitope and their affinity, although most IgM with a high affinity reacted exclusively with SGPG derivatives retaining a sulfate group. There was no parallelism between the severity of the neuropathy and the affinity of the IgM for MAG or SGPG.

Interaction of Clostridium perfringens delta toxin with erythrocyte and liposome membranes and relation with the specific binding to the ganglioside GM2.

The specific interaction of the cytolytic Clostridium perfringens delta toxin with membrane GM2 was indicated by: (i) characterization of this glycolipid in the membrane of sheep and goat erythrocytes, which are lysed by the toxin, whereas GM2 was undetectable in insensitive rabbit erythrocytes, (ii) demonstration of 125I-toxin binding to GM2, by autoradiography, following incubation with thin-layer chromatograms containing separated neuroblastoma gangliosides, and (iii) toxin fixation by phospholipid-cholesterol unilamellar vesicles containing either sheep gangliosides or GM2. In order to investigate the intramembrane events leading to membrane disruption following toxin binding, the photoreactive probe 12(4-azido-2-nitrophenoxy)stearoyl 1-14C glucosamine, which inserts into the outer layer and labels integral membrane proteins, was used to establish whether delta toxin penetrates into target cell membrane. No toxin labeling was found, suggesting that toxin action takes place at the membrane surface. This contention is supported by the observation that despite toxin binding, GM2 liposomes did not release entrapped 14C-glucose. Treatment of toxin with carboxypeptidases, but not aminopeptidases, abolished both toxin binding capacity onto erythrocytes and its combination with antitoxin neutralizing antibodies, suggesting that the carboxy terminal end of the toxin is critical for binding to cell membrane.

Low pH induces a hydrophobic domain in the tetanus toxin molecule.

Binding of the non-ionic detergent [3H]Triton X-100 by tetanus toxin, by its fragment C and by its alpha chain has been studied. At pH 4.00 or above, tetanus toxin does not bind Triton X-100. At pH lower than 4.00, binding of detergent to the toxin occurs. At pH 3.00, a maximum of 100 mol bound/mol of protein is reached only when the detergent concentration exceeds its critical micelle concentration. No measurable amount of Triton X-100 is bound by the toxin C fragment at pH 3.00. Most of the tetanus toxin alpha chain precipitates out in Triton X-100 at pH 3.00. Leakage of K+ from single-walled asolectin vesicles loaded with potassium was observed with tetanus toxin at pH lower than 4.00. When ganglioside GDlb was present on the asolectin vesicles, release of K+ was obtained with tetanus toxin between pH 4.00 to 5.00. We suggest that, as for diphtheria toxin, entry of tetanus toxin into an acidic compartment of target cells might be required for the expression of its biological activity.

[Chemically controlled depolymerization of beta hydroxybutyric lipids (PHB) in Bacillus megaterium. Isolation and structure of the oligomers of D(-)beta hydroxybutyric acid]. / Dépolymérisation par voie chimique ménagée, du lipide beta-hydroxybutyrique (ou PHB) de Bacillus megaterium. Obtention et structure d'oligomères de l'acide D(-)beta-hydroxybutyrique

Partial depolymerisation of PHB by chemical means to the appearance of homologous polymers from PHB constituted of short carbon chains and oligomers which represent the first elements of this macromolecule. The chemical structure of these oligomers ranging from dimer to heptamer has been essentially deduced from their mass spectrum and then confirmed by studying their physical, chemical and biological properties.

Monoclonal antibody A2B5, which detects cell surface antigens, binds to ganglioside GT3 (II3 (NeuAc)3LacCer) and to its 9-O-acetylated derivative.

The monoclonal antibody A2B5 recognizes antigens at the surface of neuronal and glial cells but also at the surface of thymus epithelia and pancreatic islet cells. Although these antigens have been characterized as polysialogangliosides, A2B5 also reacts with other unidentified gangliosides. In order to characterize further the epitope of A2B5, two new ganglioside antigens isolated from chicken brain are identified in this study. One is the ganglioside NeuAc alpha 2-8NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1ceramide (GT3) and the other is a 9-O-acetylated derivative of GT3). This derivative was purified from 10-day embryonic chicken brain. Acetyl groups substituted on sialic acid were removed either by alkali treatment or by incubation with influenza virus C, which contains receptor-destroying enzyme (a neuraminidate 9-O-acetyl esterase). The product of alkali treatment or viral action was detected by the antibody 18B8 which is specific for GT3. The deacetylated product still reacts with A2B5. These data and the results of mild oxidation of the antigen with sodium periodate suggest that the epitope recognized by antibody A2B5 contains the trisialyl structure found in GT3 but does not include the polyalcohol chain of the terminal sialic acid which can be oxidized by periodate or acetylated without modifying the affinity for the antibody. The epitope recognized by A2B5 is different from the epitope recognized by the antibody 18B8 in that 18B8 requires the three sialic acids with an intact and unsubstituted polyalcohol chain. Antibody 18B8 does not bind to 9-O-acetylated GT3 or GT3 oxidized by sodium periodate.

Inhibition of rabies virus infection by a soluble membrane fraction from the rat central nervous system.

This paper describes the inhibitory effect of a normal rat brain solubilized membrane preparation (RBSM-liposomes) on rabies virus infection. Rabies virus was incubated with RBSM-liposomes or their separated components (proteins, phospholipids, gangliosides) before infection of CER or neuroblastoma cells. In addition, both RBSM-liposomes and target cells were treated with enzymes prior to the infection step. All these experimental procedures showed that the active components were mainly lipids.

Characterization of membrane components of the erythrocyte involved in vesicular stomatitis virus attachment and fusion at acidic pH.

Goose erythrocyte membranes were isolated and tested for their ability to compete with red cell receptors for vesicular stomatitis virus (VSV) attachment and fusion at acidic pH. Crude membranes, solubilized with Triton X-100, Tween 80 and octyl-beta-D-glucopyranoside, showed a dose-dependent inhibitory effect on virus binding and haemolysis. The chemical nature of the active molecules was investigated by enzyme digestion and by separation of purified components. Only the lipid moiety, specifically phospholipid and glycolipid, was found to inhibit VSV attachment; a more detailed analysis of these molecules showed that phosphatidylinositol, phosphatidylserine and GM3 ganglioside were responsible for the inhibitory activity and could therefore represent VSV binding sites on goose erythrocyte membranes. Removal of negatively charged groups from these molecules by enzymic treatment significantly reduced their activity, suggesting that electrostatic interactions play an important role in the binding of VSV to the cell surface. Enzymic digestion of whole erythrocytes confirmed the involvement of membrane lipid molecules in the cell surface receptor for VSV.

Ganglioside spinal cord changes in chronic relapsing experimental allergic encephalomyelitis induced in the Lewis rats.

Chronic relapsing experimental allergic encephalomyelitis (CREAE) was induced in Lewis rats by inoculation with guinea-pig myelin and complete Freund's adjuvant followed by treatment with low-dose cyclosporin A. Rats were sacrified at different phases of the disease (just before the onset of clinical signs, during the first clinical episode of CREAE and during the first recovery). Gangliosides were extracted from the spinal cord, analysed after purification by two-dimensional chromatography and quantified densitometrically. An increase of GM 1, the main rat myelin ganglioside, and a decrease of GT1b, suggested to play a role in mediating the interactions between oligodendroglia and axons, were observed during the development of the CREAE. These findings indicating significant ganglioside changes in CREAE give further support to the concept concerning the involvement of gangliosides in autoimmune demyelination.

[Study of a correlation between glycolipids and tumorigenesis in 6 cell lines derived from human brain tumors]. / Recherche d'une corrélation entre glycolipides et tumorigénicité dans six lignées cellulaires dérivées de tumeurs cérébrales humaines.

By silica gel chromatography we have studied the various glycolipids of six established lines derived from human brain tumors. Among the glycolipids, the GM3 ganglioside appeared as one of the major components in the three lines rejected by athymic nude Mice. On the contrary, GM3 was absent, or present in trace, in the three tumorigenic lines.

Inhibition by gangliosides of the specific binding of lipopolysaccharide (LPS) to human monocytes prevents LPS-induced interleukin-1 production.

In a previous work we have reported that gangliosides inhibit interleukin 1 (IL-1) release by human monocytes stimulated with lipopolysaccharides (LPS). In the present study we extend this work to IL-1 production and we correlate these observations with the capacity of gangliosides to inhibit the binding of radiolabeled LPS to its specific receptor on human monocytes. Preincubation of 3H-LPS with crude bovine brain gangliosides, as well as purified human brain mono, di, and trisialogangliosides (GM1, GD1a, and GT1b, respectively), led to an inhibition of the specific binding of LPS to the cell surface. Neither ceramide nor N-acetyl neuraminic acid, two constituents of gangliosides, was able by itself to inhibit the specific binding. A strict parallelism was observed with respect to inhibition on LPS-induced IL-1 production and release. Asialoganglioside (asialo-GM1) was inactive in both assays, suggesting that the N-acetyl neuraminic acid plays a role within the ganglioside molecule, with respect to inhibitory activity. We conclude that LPS-induced production and release by human monocytes is not due to a signal triggered by nonspecific absorption and/or intercalation of LPS into cell membrane which occur through hydrophobic interaction mediated by the lipid A region. Addition of exogenous sialogangliosides which blocked LPS-induced IL-1 production and release, did not modify significantly the nonspecific binding of 3H-LPS, whereas it did inhibit the specific binding which is mediated by the polysaccharide moiety of the LPS molecule. These results establish a relationship between the specific endotoxin receptor on monocytes and a LPS-induced cellular function.

Involvement of gangliosides in rabies virus infection.

The role of gangliosides in rabies virus infection of chick embryo-related (CER) cells was investigated. Cultured cells were pretreated with neuraminidase to render the cells transiently non-susceptible to viral infection. Incubation of these desialylated cells with gangliosides allowed them to incorporate exogenous gangliosides and they recovered their susceptibility to rabies virus infection. Infection of CER cells was monitored by specific fluorescence 24 h after virus inoculation. The use of individual purified gangliosides or mixtures of two gangliosides to restore cellular susceptibility to viral infection showed that GT1b and GQ1b were the most effective. The disialogangliosides were also active, principally GD1b, whereas GM1, GM3 were poorly active and GD3 inactive. Incubation of rabies virus with gangliosides prior to virus infection reduced the percentage of infected cells. The results indicate that highly sialylated gangliosides are part of the cellular membrane receptor structure for the attachment of infective rabies virus. However, it is possible that other glycoconjugates such as glycoproteins or glycolipids also participate as components of a receptor structure for rabies virus.

Reactivity of human monoclonal IgM with nerve glycosphingolipids.

We examined the reactivity of monoclonal IgM of sera from patients with neuropathy and monoclonal IgM, with or without antibody activity to myelin-associated glycoprotein (MAG), as well as sera from non-neurologic patients with Waldenström's macroglobulinaemia, with various nerve glycolipids extracts or with purified gangliosides. As expected from previous studies, all (five cases) anti-MAG IgM stained two glycolipids, the chemical characteristics of which corresponded to sulphated glucuronyl-paragloboside (SGPG) and sulphated glucuronyl-lactosaminyl-paragloboside (SGLPG). Five of 12 sera from patients with neuropathy whose IgM was devoid of anti-MAG reactivity stained nerve extracts greatly enriched (98%) with SGPG and SGLPG. Three of these five sera reacted with additional glycolipids and/or gangliosides. Two of 16 sera from patients with macroglobulinaemia without neuropathy reacted strongly with both SGPG and SGLPG. The latter finding as well as the detection of low titre of anti-sphingolipid antibodies in normal sera may cast a doubt on the pathogenetic significance of this antibody activity.

Changes in ganglioside metabolism during in vitro differentiation of quail embryo myoblasts.

The metabolism of gangliosides was studied during the in vitro differentiation of both normal quail myoblasts and myoblasts which have been transformed by a temperature-sensitive mutant of Rous sarcoma virus (RSV). These transformed cells can be maintained undifferentiated if incubated at 35 degrees C, but they will differentiate when shifted to 41 degrees C. (D. Montarras and M. Y. Fiszman (1983) J. Biol. Chem. 258, 3882-3888). The analysis of [14C]Glucosamine-labeled gangliosides by two-dimensional thin-layer chromatography reveals variations in the metabolism of the gangliosides during the process of differentiation. During the formation of myotubes, it was observed that the accumulation of GD1a is reduced, while the accumulation of GD3 is increased. Therefore, this results in the variation of the ratio GD3/GD1a which increases from 1.8 to 25 in the case of clones of transformed myoblasts, and from 0.5 to 1.7 in the case of uninfected myoblasts. These variations which have been observed seem to be specific of the myogenic differentiation since they cannot be reproduced when differentiation is inhibited by BUdR treatment or when fibroblasts reach confluency and are blocked in the G1 phase of cell cycle. Furthermore, the transformed myoblasts in vitro are shown to be a good model system since their gangliosides composition is very similar to that of muscle cells in vivo.