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Shiga toxin (Stx) 1 binds to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer/CD77) and injures human endothelial cells. In order to gain insight into Stx1-induced cellular impairment, we analysed in detail the molecular heterogeneity of Stx1 receptors in two endothelial cell lines differing in their Stx1-sensitivity. We observed a moderate sensitivity to Stx1 of human brain microvascular endothelial cells (HBMECs, CD(50) > 200 ng/ml), but a considerably higher mortality rate in cultures of EA.hy 926 cells, a cell line derived from human umbilical vein endothelial cells (CD(50) of 0.2 ng/ml). Immunofluorescence microscopy demonstrated the presence of Gb3Cer in both cell lines, but showed an enhanced content of Gb3Cer in EA.hy 926 cells. Solid phase overlay binding assays of isolated GSLs combined with nanoelectrospray ionization quadrupole time-of-flight mass spectrometry demonstrated a balanced proportion of Gb3Cer and globotetraosylceramide (Gb4Cer) in HBMECs, but an increase of Gb3Cer and absence of Gb4Cer in EA.hy 926 cells. Gb3Cer species with C24:1/C24:0 fatty acids were found to dominate over those with C16:0 fatty acids in EA.hy 926 cells, but were similarly distributed in HBMECs. Reverse transcriptase polymerase chain reaction indicated the concomitant presence of Gb3Cer and Gb4Cer synthases in HBMECs, whereas EA.hy 926 cells expressed Gb3Cer synthase, but completely lacked Gb4Cer synthase. This deficiency, resulting in the accumulation of Gb3Cer in EA.hy 926 cells, represents the most prominent molecular reason that underlies the different Stx1 sensitivities of HBMECs and EA.hy 926 endothelial cells.

The gangliosides G(M1b), GalNAc-G(M1b) and G(D1alpha) are typical compounds of concanavalin A stimulated splenic T lymphoblasts of CBA/J inbred mice. Their structural characterization has been described in previous studies. The intention of this work was the comparative TLC immunostaining analysis of the glycosphingolipid composition of lectin stimulated splenic T lymphoblasts obtained from six genetically different inbred mouse strains. The strains examined were AKR, BALB/c, C57BL/6, CBA/J, DBA/2 and WHT/Ht, which are commonly used for biochemical and immunological studies. The neutral glycosphingolipid GgOse4Cer, the precursor for G(M1b)-type gangliosides, was expressed by all six strains investigated. AKR, C57BL/6 and DBA/2 showed high and BALB/c, CBA/J and WHT/Ht diminished expression in T lymphoblasts, based on single cell calculation. The gangliosides G(M1b) and GalNAc-G(M1b), elongation products of GgOse4Cer, displayed strain-specific differences in their intensities, which were found to correlate with the intensities of GgOse4Cer expression of the same strains. Concerning sialic acid substitution of gangliosides, G(M1b) and GalNAc-G(M1b) predominantly carry N-acetylneuraminic acid, whereas choleragenoid receptors G(M1a) and Gal-GalNAc-G(M1b), which are also expressed by all six strains, are characterized by dominance of N-glycolylneuraminic acid. Two highly polar gangliosides, designated with X and Y, which have not been previously recognized in murine lymphoid tissue, were detected by positive anti-GalNAc-G(M1b) antibody and choleragenoid binding, respectively. Both gangliosides were restricted to AKR, DBA/2 and C57BL/6 mice. The other three strains BALB/c, CBA/J and WHT/Ht are lacking these structures. In summary, the G(M1b)-type pathway is quite active in all six strains analysed in this study. Strain-specific genetic variations in T lymphoblast gangliosides were observed with the occurrence of gangliosides X and Y. This study and data from other groups strongly indicate for G(M1b)-type gangliosides a functional association with T cell activation and leukocyte mediated reactions.

In this study the comparative TLC immunostaining investigation of neutral GSLs and gangliosides from human skeletal and heart muscle is described. A panel of specific polyclonal and monoclonal antibodies as well as the GM1-specific choleragenoid were used for the overlay assays, combined with preceding neuraminidase treatment of gangliosides on TLC plates. This approach proved homologies but also quantitative and qualitative differences in the expression of ganglio-, globo- and neolacto-series neutral GSLs and gangliosides in these two types of striated muscle tissue within the same species. The main neutral GSL in skeletal muscle was LacCer, followed by GbOse3Cer, GbOse4Cer, nLcOse4Cer and monohexosylceramide, whereas in heart muscle GbOse3Cer and GbOse4Cer were the predominant neutral GSLs beside small quantities of LacCer, nLcOse4Cer and monohexosylceramide. No ganglio-series neutral GSLs and no Forssman GSL were found in either muscle tissue. GM3(Neu5Ac) was the major ganglioside, comprising almost 70% in skeletal and about 50% in cardiac muscle total gangliosides. GM2 was found in skeletal muscle only, while GD3 and GM1b-type gangliosides (GM1b and GD1 alpha) were undetectable in both tissues. GM1a-core gangliosides (GM1, GD1a, GD1b and GT1b) showed somewhat quantitative differences in each muscle; lactosamine-containing IV3Neu5Ac-nLcOse4Cer was detected in both specimens. Neutral GSLs were identified in TLC runs corresponding to e.g. 0.1 g muscle wet weight (GbOse3Cer, GbOse4Cer), and gangliosides GM3 and GM2 were elucidated in runs which corresponded to 0.2 g muscle tissue. Only 0.02 g and 0.004 g wet weight aliquots were necessary for unequivocal identification of neolacto-type and GM1-core gangliosides, respectively. Muscle is known for the lowest GSL concentration from all vertebrate tissues studied so far. Using the overlay technique, reliable GSL composition could be revealed, even from small muscle probes on a sub-orcinol and sub-resorcinol detection level.

The expression of neutral glycosphingolipids (GSLs) and gangliosides was investigated in cryosections of normal mouse skeletal muscle and in muscle of mice with neuromuscular diseases using indirect immunofluorescence microscopy. Transversal and longitudinal sections were immunostained with specific polyclonal antibodies against lactosylceramide, lacto-N-neotetraosylceramide, globoside, GM3(Neu5Ac), GM3(Neu5Gc) and Gm1(Neu5Ac) as well as monoclonal anti-Forssman GSL antibody. In normal CBA/J mouse muscle (control) the main immunohistochemically detected ganglioside was GM3(Neu5Ac) followed by moderately expressed GM3(Neu5Gc) and GM1. The neutral GSLs lactosylceramide and globoside were stained with almost identical, high fluorescence intensity. Low amounts of lacto-N-neotetraosylceramide and trace quantities of Forssman GSL were immunostained. All GSLs were detected in the sarcolemma, but also in considerable amounts at the intracellular level. Mice with neuromuscular diseases were the A2G-adr mouse mutant (a model for human recessive myotonia of Becker type), the BL6-wr mutant (a model for motor neuron disease) and the BL10-mdx mouse mutant (a model for human Duchenne muscular dystrophy). No changes in GSL expression were found in the A2G-adr mouse, while muscle of the BL6-wr mouse showed increased intensity of immunofluorescence in stainings with anti-lactosylceramide and anti-GM3(Neu5Ac) antibodies. Muscle of BL10-mdx mice showed the most prominent changes in GSL expression with reduced fluorescence intensity for all antibodies. Major differences were not observed in the intensities of GSLs, but there were significant differences in the patterns of distribution on plasma membrane and at the subcellular level. The exact nature and pathogenesis of these changes should be elucidated since such investigations could furnish advances in understanding the functional role of neutral GSLs and gangliosides in normal as well as in diseased muscle.

Influenza A and Sendai viruses bind to neolacto-series gangliosides isolated from human granulocytes. Differences in receptor specificity of influenza viruses A/PR/8/34 (H1N1), A/X-31 (H3N2), and parainfluenza Sendai virus (HNF1, Z-strain) were determined by two direct solid phase binding assays: the overlay technique, which combines high-resolution in the separation of gangliosides on thin-layer chromatograms with direct binding; and the microwell adsorption assay as a convenient binding assay which is performed in microtitre wells to estimate the avidity of binding to an isolated ganglioside. Both methods were applied for comparative binding studies. Viruses were found to exhibit specificity for oligosaccharides and sialic acids as well as for chain length of the neutral carbohydrate backbone, whereas differing fatty acids (C24:1 and C16:0) in the ceramide portion had no impact on virus adsorption. Terminal sialyloligosaccharides Neu5Ac alpha 2-3Gal beta 1-4Glc-R of GM3, and Neu5Ac alpha 2-3Gal beta 1-4GlcNAc-R as well as Neu5Ac alpha 2-6Gal beta 1-4GlcNAc-R of neolacto-series gangliosides with nLcOse4Cer and nLcOse6Cer backbone, exhibited significant specific receptor activity towards the different viruses. To compare the data revealed from both test systems, values of virus binding were ascertained by a non-parametric statistical approach based on rank correlation. The rank correlation coefficient rs was calculated according to Spearman from each virus binding towards GM3, IV3Neu5Ac-nLcOse4Cer, IV6Neu5Ac-nLcOse4Cer and VI3Neu5Ac-nLcOse6SCer. The rank correlation coefficients 0.74, 0.95 and 0.92, which were determined for A/PR/8/34 (H1N1), A/X-31 (H3N2) and Sendai virus (HNF1, Z-strain), respectively, indicated that both assays generate highly correlated experimental data.(ABSTRACT TRUNCATED AT 250 WORDS)

The expression of neutral glycosphingolipids and gangliosides has been studied in human skeletal and heart muscle using indirect immunofluorescence microscopy. Transversal and longitudinal cryosections were immunostained with specific monoclonal and polyclonal antibodies against the neural glycosphingolipids lactosylceramide, globoside, Forssman glycosphingolipid, gangliotetraosylceramide, lacto-N-neotetraosylceramide and against the gangliosides GM3(Neu5Ac) and GM1(Neu5Ac). To confirm the lipid nature of positive staining, control sections were treated with methanol and chloroform:methanol (1:1) before immunostaining. These controls were found to be either negative or strongly reduced in fluorescence intensity, suggesting that lipid bound oligosaccharides were detected. In human skeletal muscle, lactosylceramide was found to be the main neutral glycosphingolipid. Globoside was moderately expressed, lacto-N-neotetraosylceramide and gangliotetraosylceramide were minimally expressed and Forssman glycosphingolipid was not detected in human skeletal muscle. The intensities of the immunohistological stains of GM3 and GM1 correlated to the fact that GM3 is the major ganglioside in skeletal muscle whereas GM1 is expressed only weakly. In human heart muscle globoside was the major neutral glycosphingolipid. Lactosylceramide and lacto-N-neotetraosylceramide were moderately expressed, gangliotetraosylceramide was weakly expressed and the Forssman glycosphingolipid was not expressed at all in cardiac muscle. GM3 and GM1 were detected with almost identical intensity. All glycosphingolipids were present in plasma membranes as well as at the intracellular level.

The aim of the present study was to investigate the ganglioside expression of the highly metastatic murine lymphoreticular tumour cell line MDAY-D2. Cells were propagated under controlled pH conditions and oxygen supply in bioreactors of 1 and 7.5 l volumes by repeated batch fermentation. Gangliosides were isolated from 2.7 x 10(11) cells, purified by silica gel chromatography and separated into mono- and disialoganglioside fractions by preparative DEAE anion exchange high performance liquid chromatography. Individual gangliosides were obtained by preparative thin layer chromatography. Their structural features were established by immunostaining, fast atom bombardment and gas chromatography mass spectrometry. In addition to gangliosides of the GM1a-pathway (GM2, GM1a and GD1a) and GM1b (IV3Neu5Ac-GgOse4Cer) and GalNAc-GM1b of the Gm1b-pathway, the disialoganglioside GD1 alpha (IV3Neu5Ac, III6Neu5Ac-GgOse4Cer) was found in equal amounts compared to GD1a (IV3Neu5Ac, II3Neu5Ac-GgOse4Cer). All gangliosides were substituted with C24:0, 24:1 and C16:0 fatty acids, sphingosine and N-acetylneuraminic acid as the sole sialic acid.

The mammalian placenta is a unique organ for the study of developmental changes. Placentas of laboratory animals such as the mouse allow for the determination of the exact stage of pregnancy, which cannot be achieved with human placenta. In this study, neutral glycosphingolipids were isolated from mouse (inbred strain C57BL/6) placentas, from day 10 to day 18 of gestation, and were separated by high performance thin layer chromatography. Densitometric measurements after orcinol staining showed, at day 10 of gestation, the presence of mono-, tetra-, tri- and dihexosylceramide in decreasing quantities, as well as four unidentified spots. On day 12, the glycosphingolipid composition changed with the disappearance of the unidentified spots and the appearance of an orcinol positive migrating similarly to the Forssman antigen; no further changes occurred between days 12 and 18 of gestation. The identity of the Forssman-like glycosphingolipid with the Forssman antigen was established by binding of 125I labelled Helix pomatia agglutinin (alpha-GalNAc specific) to glycosphingolipids separated on high performance thin layer chromatography plates, and by the reaction of the isolated glycosphingolipid with a monoclonal anti-Forssman antibody. The appearance of the Forssman antigen at day 12 of gestation coincided with the day of final maturation of the mouse placenta and subsequent cessation of growth, suggesting a possible role of the glycosphingolipid during embryonic development.

The structures of gangliosides from human granulocytes were elucidated by fast atom bombardment mass spectrometry and by gas chromatography/mass spectrometry as their partially methylated alditol acetates. In human granulocytes besides GM3 (II3Neu5Ac-LacCer), neolacto-series gangliosides (IV3Neu5Ac-nLcOse4Cer, IV6Neu5Ac-nLcOse4Cer and VI3Neu5Ac-nLcOse6Cer) containing C24:1, and to some extent C22:0; and C16:0 fatty acid in their respective ceramide portions, were identified as major components. In this study we demonstrate that gangliosides from human granulocytes, the second most abundant cells in peripheral blood, can serve as receptors for influenza viruses A/PR/8/34 (H1N1), A/X-31 (H3N2), and a parainfluenza virus Sendai virus (HNF1, Z-strain). Viruses were found to exhibit specific adhesion to terminal Neu5Ac alpha 2-3Gal and/or Neu5Ac alpha 2-6Gal sequences as well as depending on the chain length of ganglioside carbohydrate backbones from human granulocytes, these important effector cells which represent the first line of defence in immunologically mediated reactions.

Gangliosides of the 'GM1b-pathway' (GM1b and GalNAc-GM1b) have been found to be highly expressed by the mouse T lymphoma YAC-1 grown in serum-supplemented medium, whereas GM2 and GM1 ('GM1a-pathway') occurred only in low amounts [Müthing, J., Peter-Katalinić, J., Hanisch, F.-G., Neumann, U. (1991) Glycoconjugate J 8:414-23]. Considerable differences in the ganglioside composition of YAC-1 cells grown in serum-supplemented and in well defined serum-free medium were observed. After transfer of the cells from serum-supplemented medium (RPMI 1640 with 10% fetal calf serum) to serum-free medium (RPMI 1640 with well defined supplements), GM1b and GalNAc-GM1b decreased and only low amounts of these gangliosides could be detected in serum-free growing cells. The expression of GM1a was also diminished but not as strongly as that of GM1b and GalNAc-GM1b. These growth medium mediated ganglioside alterations were reversible, and the original ganglioside expression was achieved by readaptation of serum-free growing cells to the initial serum-supplemented medium. On the other hand, a 'new' ganglioside, supposed to represent GalNAc-GD1a and not expressed by serum-supplemented growing cells, was induced during serum-free cultivation, and increased strongly after readaptation. These observations reveal that the ganglioside composition of in vitro cultivated cells can be modified by the extracellular environment due to different supplementation of the basal growth medium.

YAC-1 cells were propagated in bioreactors in 1 l and 7.5 l volumes. The cells were metabolically labelled with D-[1-14C]galactose and D-[1-14C]glucosamine. The ganglioside fraction, purified by DEAE-Sepharose and silica gel column chromatography, showed on thin layer chromatography four major bands with mobilities between GM1 and GD1a. Gangliosides, obtained by further purification steps including high performance liquid chromatography on silica gel 60 columns with a gradient system of isopropanol:hexane:water, and preparative high performance TLC were characterized by (1) immunostaining of corresponding asialogangliosides obtained by mild acid hydrolysis and neuraminidase treatment and (2) fast atom bombardment mass spectrometry of native and permethylated samples and methylation analysis of GM1b ganglioside. As well as small amounts of GM2 and GM1, the major gangliosides found in the complex mixture were GM1b and GalNAc-GM1b. The structural heterogeneity of these gangliosides was caused by (a) substitution of the ceramide moiety by fatty acids of different chain length and degree of unsaturation (C16:0, C24:0, C24:1) and (b) N-substitution of the sialic acid moieties with either acetyl or glycolyl groups. Disialogangliosides were detected only in low amounts and will be the subject of further investigation. A polyclonal chicken antiserum was raised against IVNeuAc-GgOse5Cer. The antiserum was highly specific for gangliosides (IVNeuAc and IVNeuGc) and asialogangliosides with a GgOse5Cer backbone. No cross-reaction with GM1b or GgOse4Cer was observed.