Evidence of a distribution difference between two gangliosides in bilayer membranes

Freeze-etch electron microscopy, a platinum shadowing technique, has been used to compare the lateral distribution of several gangliosides in bilayer model membranes by directly visualizing bound lectin molecules. In particular, GM₁ and GD_1a, major components of brain ganglioside, were studied in phase-separated mixtures of dipalmitoyl- and dielaidoylphosphatidylcholines exposed to Ricinus communis agglutinin and wheat germ agglutinin. The distribution of glycolipid showed evidence of microheterogeneity in that bound lectin tended to occur in clusters of several or more molecules. With GD_1a as receptor such clusters were small and very uniformly distributed over the membrane surface. Somewhat larger, irregularly spaced clusters of up to a dozen lectin particles were more typical of membranes bearing GM₁ and, in addition, there were occasional extensive patches of bound lectin coexisting with areas apparently devoid of glycolipid receptor in phase-separated mixtures of dipalmitoyl- and dielaidoylphosphatidylcholine. Gangliosides in the latter mixtures were not obviously influenced in their lateral distribution by the presence of coexisting fluid and rigid domains. These basic observations seem to extend to bilayer membranes containing mixtures of two gangliosides. The patterns of lectin binding were not grossly affected by incubation time or history of warming and cooling. This study was extended to bilayers of pure dipalmitoylphosphatidylcholine in expectation that the distinctive features characteristic of the P_β′ phase of this lipid might accentuate any behavioural differences between GM₁ and GD_1a.GM₁ was found to exist preferentially in the ‘trough’ regions between P_β′ ripples, while GD_1a showed no apparent preferential arrangement. Given that bound lectins adequately reflect glycolipid distribution in membranes, it would appear that structurally different glycolipids from the same host membrane can assume different distributions on the basis of interactions with defined lipid host matrices.     

Asymmetric distribution of gangliosides in rat renal brush-border and basolateral membranes

Highly enriched brush-border and basolateral membranes isolated from rat renal cortex were used to study the distribution of endogenous gangliosides in the two distinct plasma membrane domains of epithelial cells. These two membrane domains differed in their glycolipid composition. The basolateral membranes contained more of both neutral and acidic glycolipids, expressed on a protein basis. In both membranes, the neutral glycolipids corresponding to mono-, di-, tri- and tetraglycosylceramides were present. The basolateral membranes contained more diglycosylceramide than the brush-border membranes. The major gangliosides found were GM4, GM3, and GD3 with minor amounts of GM1 and GD1a. The latter were identified and quantified by sensitive iodinated cholera toxin binding assays. When the distribution of individual gangliosides was calculated as a percent of total gangliosides, the brush-border membranes were enriched with GM3, GM1 and GD1a compared to the basolateral membranes, which were enriched with GD3 and GM4. The observation of a distinct distribution of glycolipids between brush-border and basolateral membranes of the same epithelial cell suggests that there may be a specific sorting and insertion process for epithelial plasma membrane glycolipids. In turn, asymmetric glycolipid biogenesis may reflect differences in glycolipid function between the two domains of the epithelial plasma membrane.     

Visualization of domains in rigid ganglioside/phosphatidylcholine bilayers: Ca2+ effects

We have considered the extent to which details of lectin binding directly visualized by freeze-etch electron microscopy are consistent with current concepts of ganglioside arrangement in phosphatidylcholine bilayer membranes. Native lectins in general seem appropriate labels for this type of study. Wheat germ agglutinin, Ricinus communis agglutinin, and peanut agglutinin are adequately resolved on membrane surfaces as spherical particles of diameters 6 nm, 10 nm, and 13 nm, respectively (uncorrected for platinum shadow thickness). The finite areas covered by these markers correspond to some 56, 157, and 265 lipid molecules, respectively, on the surfaces of the shadowed rigid phosphatidylcholine matrices employed here; and this constitutes a basic limitation to the precision with which one can localize a given glycolipid receptor. Ricinus communis agglutinin provides a marker whose size permits adequate quantitation of bound material while minimally obscuring detail. Using it we estimated the size limits of GM1-enriched domains, since this is the ganglioside which has shown the greatest evidence of discontinuous distribution in our hands (Peters, M.W., Mehlhorn, I.E., Barber, K.R. and Grant, C.W.M. (1984) Biochim. Biophys. Acta 778, 419-428). Results of such analyses indicate the probable existence of phase separated domains selectively enriched in GM1 up to 60 nm in extent (5600 lipid molecules) for rigid dipalmitoylphosphatidylcholine membranes bearing up to 14 mol% GM1. Similar observations were true of rigid bilayers of dimyristoylphosphatidylcholine; however, if domains enriched in GM1 exist in fluid dimyristoylphosphatidylcholine, they are on the order of 6 nm or less in diameter (or are dispersed by lectin binding). Employing the small lectin, wheat germ agglutinin, which binds to all gangliosides, we then examined the effect of exposure to Ca2+ ions (while in the fluid state) on the ganglioside 'domain structure' referred to above in rigid dipalmitoylphosphatidylcholine host matrices. GM1, GD1a and GT1b were studied at 0, 2 and 10 mM Ca2+ concentrations. It was demonstrated by spin label measurements that the dipalmitoylphosphatidylcholine matrix retained its basic melting characteristics in the presence of added Ca2+ and ganglioside under these conditions. Within the technique's functional resolution limit of some 6 nm we were unable to identify any effect of Ca2+ in physiological concentration on ganglioside topography as reflected by bound lectin distribution.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of N-acetylneuraminic (sialic) acid in the pH dependence of influenza virion fusion with planar phospholipid membranes

It is known that fusion of influenza virus to host cell membranes is strongly promoted by acidic pH. We have determined conditions required to obtain pH-dependent fusion of influenza virus to planar bilayer membranes. The rate of viral fusion was determined from the flash rate of R18-labeled virions delivered to the surface of the planar membrane by pressure-ejection from a pipette. For a bilayer formed only of phospholipids and cholesterol, the fusion rate was independent of pH and unaffected by the phospholipid composition. When the gangliosides GD1a + GT1b were included in the planar membrane, however, the fusion rate varied steeply with pH. The rate at pH 7.4 in the presence of the gangliosides was about an order of magnitude less than in their absence. At pH less than approximately 5.5, the rate was about an order of magnitude greater in the presence of gangliosides than in their absence. The fusion rate with planar membranes containing globoside, a ceramide-backboned glycolipid, was also independent of pH, indicating that the pH dependence required sialic acid on the carbohydrate moiety of the glycolipid. The gangliosides GM1a and GM3, both of which possess sialic acid, produced the same pH-dependent fusion rate as seen with GD1a + GT1b, indicating that the presence, but not the location, of terminal sialic acids is critical. Incubating virus with soluble sialyllactose blocked fusion to both ganglioside-free and ganglioside-containing planar membranes. These results show that the pH dependence of influenza virion fusion arises from the interaction of the sialic acid receptor with the influenza hemagglutinin. A model for sialic acid-hemagglutinin interactions accounting for pH-dependent fusion is presented.     

Effect of gangliosides on the distribution of a glycosylphosphatidylinositol-anchored protein in plasma membrane from Chinese hamster ovary-K1 cells

Glycosylphosphatidylinositol (GPI)-anchored proteins are clustered mainly in sphingolipid-cholesterol microdomains of the plasma membrane. The distribution of GPI-anchored fusion yellow fluorescent protein (GPI-YFP) in the plasma membrane of Chinese hamster ovary (CHO)-K1 cells with different glycolipid compositions was investigated. Cells depleted of glycosphingolipids by inhibiting glucosylceramide synthase activity or cell lines expressing different gangliosides caused by stable transfection of appropriate ganglioside glycosyltransferases or exposed to exogenous GM1 were transfected with GPI-YFP cDNA. The distribution of GPI-YFP fusion protein expressed at the plasma membrane was studied using the membrane-impermeable cross-linking agent bis(sulfosuccinimidyl)suberate. Results indicate that GPI-YFP forms clusters at the surface of cells expressing GM3, or cells depleted of glycolipids, or transfected cells expressing mainly GD3 and GT3, or GM1 and GD1a, or mostly GM2, or highly expressing GM1. However, no significant changes in membrane microdomains of GPI-YFP were detected in the different glycolipid environments provided by the membranes of the cell lines under study. On the other hand, wild type CHO-K1 cells exposed to 100 microm GM1 before cross-linking with bis(sulfosuccinimidyl)suberate showed a dramatic reduction in the amount of GPI-YFP clusters. These findings clearly indicate that manipulating the glycolipid content of the cellular membrane, just by changing the ganglioside biosynthetic activity of the cell, did not significantly affect the association of GPI-YFP on the cell surface of CHO-K1 cells. The effect of exogenous GM1 gangliosides on GPI-YFP plasma membrane distribution might be a consequence of the ganglioside level reached in plasma membrane and/or the effect of particular ganglioside species (micelles) that lead to membrane architecture and/or dynamic modifications.     

Inhibitory effects of gangliosides on immune reactions of antibodies to neutral glycolipids in liposomes

Specific immune damage to liposomes containing Forssman or globoside glycolipid was inhibited when the liposomes also contained ganglioside. The activity of a human monoclonal Waldenstr?m macroglobulin antibody to Forssman glycolipid was inhibited by each of three gangliosides tested, GM3, GD1a and GD1b. Inhibition of the monoclonal antibody was dependent on the amount of ganglioside in the liposomes, and was diminished by reducing the relative amount of ganglioside. Inhibition also correlated positively with the number of ganglioside sialic acid groups, with inhibition by GT1b greater than GD1a greater than GM3. Naturally occurring human antibodies to globoside glycolipid were detected in 18% (9 out of 50) of normal human sera tested. Immune damage to liposomes induced by each of the three highest-reacting human anti-globoside sera was blocked by liposomal GM3. We conclude that gangliosides can strongly influence immune damage to membranes induced by antibody interactions with adjacent neutral glycolipids.     

Fibronectin binding to gangliosides and rat liver plasma membranes

Binding of fibronectins to gangliosides was tested directly using several different in vitro models. Using an enzyme-linked immunoabsorbent assay (ELISA), gangliosides were immobilized on polystyrene tubes and relative binding of fibronectin was estimated by alkaline phosphatase activity of conjugated second antibody. Above a critical ganglioside concentration, the gangliosides bound the fibronectin (GT1b congruent to GD1b congruent to GD1a greater than GM1 much greater than GM2 congruent to GD3 congruent to GM3) in approximately the same order of efficiency as they competed for the cellular sites of fibronectin binding in cell attachment assays (Kleinman et al., Proc natl acad sci US 76 (1979) 3367). Alternatively, these same gangliosides bound to immobilized fibronectin. Rat erythrocytes coated with gangliosides GM1, GD1a or GT1b bound more fibronectin than erythrocytes not supplemented with gangliosides. Using fibronectin in which lysine residues were radioiodinated, an apparent Kd for binding to mixed rat liver gangliosides of 7.8 X 10(-9) M was determined. This value compared favorably with the apparent Kd for attachment of fibronectin to isolated plasma membranes from rat liver of 3.7 X 10(-9) M for fibronectin modified on the tyrosine residue, or 6.4 X 10(-9) M for fibronectin modified on lysine residues. As shown previously by Grinnell & Minter (Biochem biophys acta 550 (1979) 92), fibronectin modified on tyrosine residues did not promote spreading and attachment of CHO cells. It did, however, bind to cells. In contrast, lysine-modified fibronectin both bound to cells and promoted cell attachment. Plasma membranes isolated from hepatic tumors in which the higher gangliosides that bind fibronectin were depleted bound 43-75% less [125I]fibronectin than did plasma membranes from control livers. The findings were consistent with binding of fibronectins to gangliosides, including the same gangliosides depleted from cell surfaces during tumorigenesis in the rat.     

Lysosomal sialidase deficiency: increased ganglioside content in autopsy tissues of a sialidosis patient

Organs obtained at autopsy from a patient with sialidosis were analyzed for 'bound' sialic acid and their ganglioside and neutral glycolipid patterns determined. The water-soluble bound sialic acid was increased between 10- and 17-fold in visceral organs, but only about 2-fold in the brain, when compared to normal controls. Lipid-bound sialic acid was increased up to 8-fold in visceral organs due to elevated amounts of gangliosides GM3, GD3 and probably GM4 and LM1, whereas the brain showed no deviation from controls. An alteration of the neutral glycolipid pattern was also observed. The results indicate an impaired catabolism of gangliosides in sialidosis in addition to that of sialyloligosaccharides and sialoglycoproteins.     

Detection of gangliosides of the GM1b type on high-performance thin-layer chromatography plates by immunostaining after neuraminidase treatment

A method for the detection of GM1b-type gangliosides in complex mixtures of gangliosides was developed. The procedure involves separation of gangliosides on high-performance thin-layer chromatography plates, fixation of the silica gel, treatment with neuraminidase from Vibrio cholerae in the absence of detergent, and incubation of the plates with GgOse4Cer-specific antibodies. Alkaline phosphatase-conjugated second antibodies are used to visualize bound first antibodies by generating a blue dye from 5-bromo-4-chloro-3-indolylphosphate. The procedure is capable of detecting as little as 30 ng of gangliosides. Gangliosides from murine T lymphocytes and from human brain served as examples. Besides GM1b, GD1 alpha is also detectable by this method, whereas the human brain gangliosides GM1a, GD1a, GD1b, and GT1b are not, because they are neuraminidase resistant. Since terminally sialylated gangliosides such as GM1b were described as virus receptors, and certain other terminally sialylated gangliosides are discussed as tumor markers, this method should be useful to screen gangliosides from different tissues or cell lines for the presence of such components, especially if only small amounts of material are available.     

Specific staining on thin-layer chromatograms of glycosphingolipids of neolacto series and gangliosides with a terminal N-acetylneuraminyl residue by different procedures with wheat germ agglutinin

Sensitive staining methods with wheat germ agglutinin were developed for the detection of glycosphingolipids of neolacto series (A) and gangliosides with a terminal N-acetylneuraminyl residue (B) on thin-layer chromatograms. (A) Neolacto series glycosphingolipids were treated by beta-galactosidase on the chromatograms in the presence of taurodeoxycholate. Then the chromatograms were incubated with biotinated wheat germ agglutinin followed by incubation with a complex of avidin and biotinated horseradish peroxidase, and the reaction was detected by 4-chloro-1-naphthol. In the case of gangliosides, sialidase treatment on the chromatograms was performed before the beta-galactosidase treatment. The sensitivity of the method for Lc3Cer, nLc4Cer, sialyl-nLc4Cer, and sialyl-nLc6Cer was 4 pmol, 7.6 pmol, 2.9 pmol and 1.4 pmol, respectively. (B) The gangliosides on the chromatograms were oxidized by periodic acid and reduced by NaBH4. Then the chromatograms were stained with wheat germ agglutinin as mentioned above. As little as 0.5 pmol of GM3, NeuAc-nLc4Cer, and NeuAc-nLc6Cer was detected by this method, whereas the detected limits for these gangliosides were 10 pmol, 10 pmol and 2 pmol, respectively, when periodate oxidation was omitted. GM4, GD3 and GD1a were an order less reactive than GM3, GM2, GM1 or GD1b were not stained under the same condition. In contrast to NeuAc-containing gangliosides, any gangliosides with N-glycolylneuraminic acid were not stained by the method in (B).     

Targeted analysis of ganglioside and sulfatide molecular species by LC/ESI-MS/MS with theoretically expanded multiple reaction monitoring

Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) has been applied primarily to the analysis of glycosphingolipids separated from other complex mixtures by TLC, but it is difficult to obtain quantitative profiling of each glycosphingolipid among the different spots on TLC by MALDI-MS. Thus, the development of a convenient approach that utilizes liquid chromatography/electrospray ionization (LC/ESI)-MS has received interest. However, previously reported methods have been insufficient to separate and distinguish each ganglioside class. Here we report an effective method for the targeted analysis of theoretically expected ganglioside molecular species by LC/ESI tandem mass spectrometry (LC/ESI-MS/MS) in combination with multiple reaction monitoring (MRM). MRM detection specific for sialic acid enabled us to analyze ganglioside standards such as GM1, GM2, GM3, GD1, and GT1 at picomolar to femtomolar levels. Furthermore, other gangliosides, such as GD2, GD3, GT2, GT3, and GQ1, were also detected in glycosphingolipid standard mixtures from porcine brain and acidic glycolipid extract from mouse brain by theoretically expanded MRM. We found that this approach was also applicable to sulfatides contained in the glycosphingolipid mixtures. In addition, we established a method to separate and distinguish regioisomeric gangliosides, such as GM1a and -1b, GD1a, -1b, and -1c, and GT1a, -1b, and -1c with diagnostic sugar chains in the MRM.     

The effects of development on activity, specificity and endogenous substrates of synaptic membrane sialidase

Synaptic plasma membranes were prepared from cortices of rats varying in post-natal age between 4 and 30 days. Sialic acid associated with synaptic plasma membrane glycoproteins and gangliosides increased 75% and 50%, respectively, between 4 and 30 days. The amount of sialic acid released from these membrane constituents by intrinsic synaptic sialidase increased 2-4-fold over the same period. Incubation of synaptic plasma membranes with exogenous gangliosides or glycopeptides demonstrated a 2-3-fold increase in sialidase activity during development. The major gangliosides present in synaptic plasma membranes at all ages were GT1, GD1a, GD1b and GM1. Intrinsic sialidase hydrolyzed 50-70% of endogenous GT1 and GD1a gangliosides at all ages. Endogenous GD1b ganglioside was poorly hydrolyzed in young rats and its susceptibility to enzymic hydrolysis increased during development. When exogenous GD1a and GD1b were used as substrates a preferential increase in activity against GD1b occurred during development, the ratio of activity (GD1a/GD1b) decreasing from 3.6 to 1.6 between 7 and 30 days. 10- and 30-day-old synaptic plasma membranes contained complex mixtures of sialoglycoproteins, an increase in the relative concentrations of lower molecular weight sialoglycoproteins occurring during development. Intrinsic sialidase present in 10- and 30-day-old synaptic plasma membranes acted upon all molecular weight classes of sialoglycoproteins.     

Thermotropic behavior and electronmicroscopic structures of mixtures of gangliosides and dipalmitoylphosphatidylcholine

The calorimetric properties and morphological structures of dispersed mixtures of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and highly purified human brain gangliosides, GM2, GM1, GD1a, GD1b, and GT1b, were studied using a high-sensitivity differential scanning calorimeter and an electron-microscope, as a function of the ganglioside molar fraction. No thermal phase transitions of pure gangliosides in aqueous dispersions could be detected. In the mixtures of DPPC and gangliosides, the gel to liquid crystalline phase transition occurred at a higher temperature than in pure DPPC dispersions and progressed over a wide temperature range. As increasing amounts of the pure ganglioside species were added to DPPC, the temperature for the main transition gradually increased. The phase transition progressed differently among different gangliosides/DPPC mixtures. The enthalpy values were found to decrease linearly as the number of sialic acid residues increased. Electron-microscopically the ganglioside/DPPC mixtures formed multilamellar structures at lower concentrations of the gangliosides, and the structures changed to cylindrical and spherical micelles as the ganglioside concentration was increased. The polysialoganglioside/DPPC mixtures showed the micellar form even at lower ganglioside concentrations, contrary to the case of the monosialoganglioside/DPPC mixtures. The morphological changes of gangliosides/DPPC mixtures corresponded with changes in the calorimetric properties. These results show that individual gangliosides have different physicochemical effects on model membranes, possibly because of the interaction of their negatively charged head groups.     

Campylobacter pylori colonization factor shows specificity for lactosylceramide sulfate and GM3 ganglioside

The specificity of Campylobacter pylori cell surface lectin, a presumptive colonization factor, was investigated using various sulfated and sialic acid containing glycolipids. C. pylori cells, cultured from human antral mucosal biopsies, were incubated with intact and modified glycolipid preparations and examined for agglutination inhibition of human erythrocytes. Titration data revealed that the inhibitory activity was highest with lactosylceramide sulfate and GM3 ganglioside, while galactosylceramide sulfate GM1, GD1a and GD1b gangliosides were less effective. A strong inhibitory activity towards C. pylori hemagglutin was also observed with an antiulcer agent, sucralfate. The inhibitory effect of both types of glycolipids was abolished by the removal of sialic acid and sulfate ester groups, thus indicating that sulfated and sialic acid containing glycolipids with terminal lactosyl moieties serve as mucosal receptors for colonization of gastric epithelium by C. pylori.     

Visualization of domains in rigid ganglioside / phosphatidylcholine bilayers: Ca2+ effects

We have considered the extent to which details of lectin binding directly visualized by freeze-etch electron microscopy are consistent with current concepts of ganglioside arrangement in phosphatidylcholine bilayer membranes. Native lectins in general seem appropriate labels for this type of study. Wheat germ agglutinin, Ricinus communis agglutinin, and peanut agglutinin are adequately resolved on membrane surfaces as spherical particles of diameters 6 nm, 10 nm, and 13 nm, respectively (uncorrected for platinum shadow thickness). The finite areas covered by these markers correspond to some 56, 157, and 265 lipid molecules, respectively, on the surfaces of the shadowed rigid phosphatidylcholine matrices employed here; and this constitutes a basic limitation to the precision with which one can localize a given glycolipid receptor. Ricinus communis agglutinin provides a marker whose size permits adequate quantitation of bound material while minimally obscuring detail. Using it we estimated the size limits of G_M1-enriched domains, since this is the ganglioside which has shown the greatest evidence of discontinuous distribution in our hands (Peters, M.W., Mehlhorn, I.E., Barber, K.R. and Grant, C.W.M. (1984) Biochim. Biophys. Acta 778, 419–428). Results of such analyses indicate the probable existence of phase separated domains selectively enriched in G_M1 up to 60 nm in extent (5600 lipid molecules) for rigid dipalmitoylphosphatidylcholine membranes bearing up to 14 mol% G_M1. Similar observations were true of rigid bilayers of dimyristoylphosphatidylcholine; however, if domains enriched in G_M1 exist in fluid dimyristoylphosphatidylcholine, they are on the order of 6 nm or less in diameter (or are dispersed by lectin binding). Employing the small lectin, wheat germ agglutinin, which binds to all gangliosides, we then examined the effect of exposure to Ca²⁺ ions (while in the fluid state) on the ganglioside ‘domain structure’ referred to above in rigid dipalmitoylphosphatidylcholine host matrices. G_M1, G_D1a and G_T1b were studied at 0, 2 and 10 mM Ca²⁺ concentrations. It was demonstrated by spin label measurements that the dipalmitoylphosphatidylcholine matrix retained its basic melting characteristics in the presence of added Ca²⁺ and ganglioside under these conditions. Within the technique's functional resolution limit of some 6 nm we were unable to identify any effect of Ca²⁺ in physiological concentration on ganglioside topography as reflected by bound lectin distribution. The rigid dipalmitoylphosphatidylcholine host matrix had been selected to minimize receptor redistribution (ganglioside aggregation or disaggregation) caused by lectin probe binding or sample preparation for electron microscopy. However the above Ca²⁺-related observations were basically unaltered in a matrix of intermediate fluidity and zero cooperativity obtained by the addition of 30 mol% cholesterol. In none of our samples did we see bilayer disruption that might indicate significant patches of very high local glycosphingolipid concentration.     

Ganglioside patterns and cholera toxin--peroxidase labeling of aggregating cells from the chick optic tectum.

2The ganglioside compositions of the chick optic tectum and aggregating tectal cell cultures were examined. Both showed similar trends in changes in ganglioside patterns during development. GD3 and GD1b were the predominant gangliosides early in development, while GD1a and several other multisialogangliosides increased in relative amounts with increasing age in vivo and in vitro. Four gangliosides were present early in development which have not previously been reported. These gangliosides are not present at later developmental times suggesting a possible role for them during the critical early stages of nervous tissue differentiation. Some differences were noted when comparing in vivo versus in vitro ganglioside patterns; these differences may possibly be due to the lack of normal retinotectal connections in the cultures. Cytochemical studies on the localization of the presumed cholera toxin--peroxidase binding site GM1 showed conjugate binding correlates with increasing levels of GM1 in the cultures. In older cultures, the conjugate was uniformly localized on all cells and processes in the aggregates. The conjugate also bound to synaptic membranes and intensely stained the synaptic cleft. This latter observation suggests an enrichment of GM1 in the synaptic cleft region.     

Congregation of gangliosides at the junction between two model membranes.

The diversity and distribution of gangliosides in vertebrate tissue suggests an important role in cellular recognition. Two types of experiments are reported to test the hypothesis that gangliosides can congregate to form an adhesive junction between two membranes. First, to monitor ganglioside distribution and mobility in different regions of two large spherical bilayer membranes, fluorescent derivatives of natural gangliosides were synthesized. Second, the cation carrier nonactin was used as a conductance probe to measure the membrane surface potential, which would be altered if there were a redistribution of the charged gangliosides. These studies were conducted in large spherical artificial membranes made from egg phosphatidylcholine or oleoylpalmitoylphosphatidylcholine with 0-12 mol % bovine brain gangliosides dissolved in n-decane. The fluorescent gangliosides utilized were lucifer yellow adducts to the sialic acids (LY-gangliosides) or a cis-paranaric acid substitution of the N-acyl moiety in the ceramide portion of gangliosides GM1 and GD1a (paranaryl-GM1 and paranaryl-GD1a). The polarized fluorescence from the adhesive junction between two membranes containing LY-gangliosides or either paranarylganglioside was compared to that in nonadhesive regions. For LY-gangliosides, total fluorescence in the junction decreased with time, possibly due to electrostatic repulsion of this highly charged derivative. For paranarylgangliosides, fluorescence in the junction increased 7-fold with time, suggesting congregation of this ganglioside. In both cases, a measure of rotational mobility, fluorescence anisotropy, increased dramatically, about 2-fold, as expected for restricted mobility of adhesive compounds. Independent evidence for congregation of charge-bearing gangliosides was found with the conductance probe nonactin.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-affinity anti-ganglioside IgG antibodies raised in complex ganglioside knockout mice: reexamination of GD1a immunolocalization

Gangliosides, sialic acid-bearing glycosphingolipids, are highly enriched in the vertebrate nervous system. Anti-ganglioside antibodies are associated with various human neuropathies, although the pathogenicity of these antibodies remains unproven. Testing the pathogenic role of anti-ganglioside antibodies will be facilitated by developing high-affinity IgG-class complement-fixing monoclonal anti-bodies against major brain gangliosides, a goal that has been difficult to achieve. In this study, mice lacking complex gangliosides were used as immune-naive hosts to raise anti-ganglioside antibodies. Wild-type mice and knockout mice with a disrupted gene for GM2/GD2 synthase (UDP-N-acetyl-D-galactosamine : GM3/GD3 N-acetyl-D-glactosaminyltransferase) were immunized with GD1a conjugated to keyhole limpet hemocyanin. The knockout mice produced a vigorous anti-GD1a IgG response, whereas wildtype littermates failed to do so. Fusion of spleen cells from an immunized knockout mouse with myeloma cells yielded numerous IgG anti-GD1a antibody-producing colonies. Ganglioside binding studies revealed two specificity classes; one colony representing each class was cloned and characterized. High-affinity monoclonal antibody was produced by each hybridoma : an IgG1 that bound nearly exclusively to GD1a and an IgG2b that bound GD1a, GT1b, and GT1aalpha. Both antibodies readily readily detected gangliosides via ELISA, TLC immune overlay, immunohistochemistry, and immunocytochemistry. In contrast to prior reports using anti-GD1a and anti-GT1b IgM class monoclonal antibodies, the new antibodies bound avidly to granule neurons in brain tissue sections and cell cultures. Mice lacking complex gangliosides are improved hosts for raising high-affinity, high-titer anti-ganglioside IgG antibodies for probing for the distribution and physiology of gangliosides and the pathophysiology of anti-ganglioside antibodies.     

Analysis of gangliosides using fast atom bombardment mass spectrometry

The native gangliosides GM3, GM1, Fuc-GM1, GD1a, GD1b, Fuc-GD1b, GT1b and GQ1b were analysed by fast atom bombardment mass spectrometry (FAB-MS) in the negative ion mode in a matrix of thioglycerol. After permethylation the same gangliosides were analysed by electron impact (EI) and FAB-MS in the positive ion mode. The negative ion mass spectra furnished information on the molecular weight, the ceramide moiety and the sequence of carbohydrate residues. The sites of attachment and the number of sialic acids present could be deduced directly from the pattern of sequence ions. After addition of sodium acetate positive ion FAB-spectra of the permethylated samples show intense pseudomolecular ions M + Na, that provide evidence on the homogeneity of the samples. In addition, the ceramide part, the oligosaccharide moiety obtained after cleavage of the glycosidic bond of the hexosamine residue, the whole carbohydrate chain and the sialic acids are represented by specific fragment ions. With EI-MS further information can be obtained on the sphingosine and fatty acid components of the ceramide residue. The data show, that the combination of soft ionization mass spectrometry with classical EI-MS gives valuable information on the structure and homogeneity of gangliosides. The method is also applicable to the structural elucidation or quantitation of more complex gangliosides or glycolipid mixtures using only micrograms of material.     

Gangliosides interact directly with plasminogen and urokinase and may mediate binding of these fibrinolytic components to cells

Receptors for the fibrinolytic molecules plasminogen and urokinase are expressed at high capacity on a wide variety of peripheral blood cells and transformed cell lines. We have considered whether gangliosides, components of the outer leaflets of cell membranes, may modulate the interactions of these fibrinolytic ligands with cells. Radiolabeled plasminogen and urokinase bound directly to insolubilized gangliosides. The interactions were saturable and were 50% inhibited by 2.2 microM unlabeled plasminogen or 12 nM unlabeled urokinase, respectively. A panel of gangliosides inhibited binding of both ligands to U937 monocytoid cells, and the order of decreasing inhibitory effectiveness was GD1a greater than GM1 greater than GT1b greater than GM2, while GM3 was minimally effective. The individual components of gangliosides, hexoses, hexosamines, sialic acid, GM1 pentasaccharide, ceramides, and glucocerebrosides were ineffective in in inhibiting the binding of plasminogen and urokinase either to cells or to insolubilized gangliosides. Binding of both ligands to endothelial cells and granulocytes and binding of plasminogen to platelets were also inhibited by gangliosides. U937 cells were cultured with gangliosides to allow incorporation of these glycolipids into the cell membranes. After 3 days of culture, both urokinase binding and plasminogen binding to the cells became enhanced. These results suggest that gangliosides can directly bind to these fibrinolytic components and may mediate or modulate the interactions of plasminogen and urokinase with a variety of cell types.