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Shiga toxin 1 (Stx1) represents an AB5 toxin produced by enterohemorrhagic Escherichia coli, which cause gastrointestinal diseases in humans that are often followed by potentially fatal systemic complications, such as acute encephalopathy and hemolytic uremic syndrome. The expression of the preferential Stx1 receptor, Gb3Cer/CD77 (Gal alpha1-4Gal beta1-4Glc beta1-1Cer), is one of the primary determinants of susceptibility to tissue injury. Due to the clinical importance of this life-threatening toxin, a combined strategy of preparative high-performance thin-layer chromatography (HPTLC) overlay assay and mass spectrometry was developed for the detection and structural characterization of Stx1-binding glycosphingolipids (GSLs). A preparation of neutral GSLs from human erythrocytes, comprising 21.4% and 59.1% of the high- and low-affinity Stx1-binding ligands Gb3Cer/CD77 and Gb4Cer, respectively, was separated on silica gel precoated HPTLC plates and probed for the presence of Stx1 receptors. Stx1 positive on the one hand and anti-Gb3Cer/CD77 and anti-Gb4Cer antibody positive bands from parallel reference runs on the other hand were extracted with chloroform/methanol/water (30/60/8, v/v/v). These crude extracts were used without any further purification for a detailed structural analysis by nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF-MS) in the negative ion mode. In all extracts investigated, neutral GSLs were detected as singly charged deprotonated molecular ions, [M-H]-, and neither buffer-derived salt adducts nor coextracted contaminants from the overlay assay procedure or the silica gel layer were observed. For the structural characterization of Stx1- and antibody-binding GSLs low-energy collision-induced dissociation (CID) was applied to high and low abundant receptor species of the crude extracts. All MS/MS spectra obtained contained full series of Y-type ions, B-type ions and additional ions generated by ring cleavages of the sugar moiety. Only analytical quantities in the microgram scale of a single GSL species within the complex GSL mixture were required for the structural MS characterization of Stx1 ligands as Gb3Cer/CD77 and Gb4Cer. This effective combined HPTLC/MS procedure offers a broad range of applications, not only for toxins of bacterial origin, but also for any GSL-binding agents such as plant-derived lectins or human proteins with yet unknown binding specificities., (2005 John Wiley & Sons, Ltd.)

The anticancer drug rViscumin, currently under clinical development, has been shown in previous studies to be a sialic acid specific ribosome inactivating protein (RIP). Comparative binding assays with the CD75s-specific monoclonal antibodies HB6 and J3-89 revealed rViscumin to be a CD75s-specific RIP due to identical binding characteristics toward CD75s gangliosides. The receptor gangliosides are IV6nLc4Cer, VI6nLc6Cer, and the newly characterized ganglioside VIII6nLc8Cer, all three carrying the Neu5Acalpha2-6Galbeta1-4GlcNAc motif. To elucidate the clinical potential of the rViscumin targets, CD75s gangliosides were determined in several randomly collected gastrointestinal tumors. The majority of the tumors showed an enhanced expression of CD75s gangliosides compared with the unaffected tissues. The rViscumin binding specificity was further investigated with reference glycoproteins carrying sialylated and desialylated type II N-glycans. Comparative Western blots of rViscumin and ricin, an rViscumin homologous but galactoside-specific RIP, revealed specific recognition of type II N-glycans with CD75s determinants by rViscumin, whereas ricin failed to react with terminally sialylated oligosaccharides such as CD75s motifs and others. This strict binding specificity of rViscumin and the increased expression of CD75s gangliosides in various tumors suggest this anticancer drug as a promising candidate for an individualised adjuvant therapy of human tumors.

A combined strategy of preparative high-performance thin-layer chromatography overlay assay and mass spectrometry was established for the structural characterization of immunostained glycosphingolipids (GSLs) in silica gel extracts. Crude chloroform/methanol/water (30/60/8, v/v/v) extracts of immunostained TLC bands were analyzed by nanoelectrospray low-energy CID mass spectrometry without further purification. The GSL species investigated were isomeric monosialogangliosides of the neolacto series from a ganglioside preparation of human granulocytes, the disialoganglioside GD3 from a human melanoma lipid extract, and ganglio series Gg3Cer of a neutral GSL preparation from murine lymphoreticular MDAY-D2 cells. For the specific detection of lipid-bound oligosaccharides, polyclonal chicken IgY, murine monoclonal IgG3, and IgM antibodies were used. The resulting mass spectra show that only analytical quantities of approximately 1 microg of a single GSL within a complex mixture are required for the structure determination of immunostained GSLs by MS and MS/MS. All species investigated were detected as singly charged deprotonated molecular ions, and neither buffer-derived salt adducts nor coextracted contaminants from the immunostaining procedure or the silica gel layer were observed. This effective HPTLC-MS-joined procedure offers a wide range of applications for any carbohydrate binding agents such as bacterial toxins, plant lectins, and others.

Mistletoe lectin I (ML-I) is a type II ribosome-inactivating protein, which inhibits the protein biosynthesis at the ribosomal level. ML-I is composed of a catalytically active A-chain with rRNA N-glycosidase activity and a B-chain with carbohydrate binding specificities. Using comparative solid-phase binding assays along with electrospray ionization tandem mass spectrometry, ML-I was shown to preferentially bind to terminally alpha2-6-sialylated neolacto series gangliosides from human granulocytes. IV(6)Neu5Ac-nLc4Cer, VI(6)Neu5Ac-nLc6Cer, and VIII(6)Neu5Ac-nLc8Cer were identified as ML-I receptors, whereas the isomeric alpha2-3-sialylated neolacto series gangliosides were not recognized. Only marginal binding of ML-I to terminal galactose residues of neutral glycosphingolipids with a Galbeta1-4Glc or Galbeta1-4GlcNAc sequence was determined, whereas a distal Galalpha1-4Gal, GalNAcbeta1-3Gal, or GalNAcbeta1-4Gal disaccharide did not bind at all. Among the glycoproteins investigated in Western blot and microwell adsorption assays, only those carrying Neu5Acalpha2-6Galbeta1-4GlcNAc residues, exclusively, predominantly, or even as less abundant constituents in an assembly with Neu5Acalpha2-3Galbeta1-4GlcNAc-terminated glycans, displayed high ML-I binding capacity. From our data we conclude that (i) ML-I has to be considered as a sialic acid- and not a galactose-specific lectin and (ii) neolacto series gangliosides and sialoglycoproteins with type II glycans, which share the Neu5Acalpha2-6Galbeta1-4GlcNAc terminus, are true ML-I receptors. This strict preference might help to explain the immunostimulatory potential of ML-I toward certain leukocyte subpopulations and its therapeutic success as a cytotoxic anticancer drug.

The expression of gangliosides and neutral glycosphingolipids (GSLs) in the lymph nodes of mice lacking the gene for the tumour necrosis factor-alpha receptor p55 (TNFR1) has been investigated. GSL expression in the tissues of mice homozygous (TNFR1-/-) or heterozygous (TNFR1+/-) for the gene deletion was analysed by flow cytometry and high-performance thin-layer chromatography (HPTLC) followed by immunostaining with specific antibodies. HPTLC immunostaining revealed that lymph nodes from TNFR1-/- mice had reduced expression of ganglioside GM1b and GalNAc-GM1b, neolacto-series gangliosides, as well as the globo- (Gb3, Gb4 and Gb5) and ganglio-series (Gg3 and Gg4) neutral GSLs. Flow cytometry of freshly isolated lymph node cells showed no significant differences in GSL expression, except for the GalNAc-GM1b ganglioside, which was less abundant on T lymphocytes from TNFR1-/- lymph nodes. In TNFR1-/- mice, GalNAc-GM1b+/CD4+ T cells were twofold less abundant (3.8% vs 7.6% in the control mice), whereas GalNAc-GM1b+/CD8+ T cells were fourfold less abundant (5.0% vs 20.2% in the control mice). This study provides in vivo evidence that TNF signalling via the TNFR1 is important for the activation of GM1b-type ganglioside biosynthetic pathway in CD8 T lymphocytes, suggesting its possible role in the effector T lymphocyte function.

A combined strategy of thin-layer chromatography immunostaining and negative ion nanoelectrospray low-energy CID mass spectrometry was established for the differentiation of isomeric alpha2-3 and alpha2-6 sialylated neolacto-series monosialogangliosides from human granulocytes. The gangliosides investigated differed in the ceramide moiety by substitution with C16:0 or C24:1 fatty acid and in their oligosaccharide chains due to nLc4 and nLc6 core structures. With respect to the type of sialylation, the homogeneity of the HPLC-purified ganglioside fractions was verified by use of specific anti-Neu5Acalpha2-3Galbeta1-4GlcNAc-R and anti-Neu5Acalpha2-6Galbeta1-4GlcNAc-R antibodies. A clear-cut series of fragment ions for both types of isomeric gangliosides, carrying alpha2-3- and alpha2-6-linked neuraminic acid, respectively, was obtained by low-energy CID. Additionally, a characteristic ring cleavage was detected exclusively in all species with Neu5Acalpha2-6Galbeta1-4GlcNAc terminus, regardless of ceramide fatty acid and oligosaccharide chain lengths. The diagnostic (0,2)X(4/6) ions, generated by ring cleavage of an alpha2-6-linked neuraminic acid are accompanied by a simultaneous decrease of the corresponding Y(4)/Y(6) ions. These results suggest the unequivocal discrimination of individual alpha2-3- and alpha2-6-sialylated neolacto-series monosialogangliosides by distinct fragmentation patterns in low-energy CID tandem MS.

R24, a mouse IgG3 monoclonal antibody (MAb) against ganglioside GD3 (Neu5Acalpha8Neu5Acalpha3Gal beta4Glcbeta1Cer), can block tumor growth as reported in a series of clinical trials in patients with metastatic melanoma. The IgG molecule basically contains an asparagine-linked biantennary complex type oligosaccharide on the C(H)2 domain of each heavy chain, which is necessary for its in vivo effector function. The purpose of this study was to investigate the biotechnological production and particularly the glycosylation of this clinically important MAb in CO(2)/HCO(3) (-) (pH 7.4, 7.2, and 6.9) and HEPES buffered serum-free medium. Growth, metabolism, and IgG production of hybridoma cells (ATCC HB-8445) were analyzed on a 2-L bioreactor scale using fed-batch mode. Specific growth rates (mu) and MAb production rates (q(IgG)) varied significantly with maximum product yields at pH 6.9 (q(IgG) = 42.9 microg 10(-6) cells d(-1), mu = 0.30 d(-1)) and lowest yields in pH 7.4 adjusted batches (q(IgG) = 10.8 microg 10(-6) cells d(-1), mu = 0.40 d(-1)). N-glycans were structurally characterized by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), and electrospray-ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry (MS). The highest relative amounts of agalacto and monogalacto biantennary complex type oligosaccharides were detected in the pH 7.2 (46% and 38%, respectively) and pH 6.9 (44% and 40%, respectively) cultivations and the uppermost quantities of digalacto (fully galactosylated) structures in the pH 7.4 (32%) and the HEPES (26%) buffered fermentation. In the experiments with HEPES buffering, antibodies with a molar Neu5Ac/Neu5Gc ratio of 3.067 were obtained. The fermentations at pH 7.2 and 6.9 resulted in almost equal molar Neu5Ac/Neu5Gc ratios of 1.008 and 0.985, respectively, while the alkaline shift caused a moderate overexpression of Neu5Ac deduced from the Neu5Ac/Neu5Gc quotient of 1.411. Different culture buffering gave rise to altered glycosylation pattern of the MAb R24. Consequently, a detailed molecular characterization of MAb glycosylation is generally recommended as a part of the development of MAbs for targeted in vivo immunotherapy to assure biochemical consistency of product lots and oligosaccharide-dependent biological activity., (Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 321-334, 2003.)

Production of biochemically defined recombinant mistletoe lectin was achieved by cloning and separate expression of the single catalytically active A-chain and the B-chain with carbohydrate binding properties in Escherichia coli, yielding an active heterodimeric protein named rViscumin (Eck et al. [1999] Eur. J. Biochem., 265, 788-797). Employing solid phase binding assays, rViscumin was shown to preferentially bind to terminally alpha2-6-sialylated neolacto-series gangliosides IV(6)Neu5Ac-nLc4Cer, VI(6)Neu5Ac-nLc6Cer, and VIII(6)Neu5Ac-nLc8Cer isolated from human granulocytes. Only marginal binding of rViscumin to galactose-terminated neutral GSLs was determined, whereas reinvestigation of ricin specificity demonstrated this lectin as a galactose-binding protein. Human promyelotic HL-60 cells exhibited an IC(50) value (half maximum cytotoxicity) of 1.16 pM and human bladder carcinoma 5637 cells of 12.1 pM rViscumin; CHO-K1 cells were resistant to rViscumin treatment up to a concentration of 5.26 nM tested. Quantification of the predominant receptor ganglioside IV(6)Neu5Ac-nLc4Cer by means of a specific anti-Neu5Acalpha2-6Galbeta1-4GlcNAc-R antibody revealed 3.68 x 10(6) and 1.54 x 10(6) receptor molecules per HL-60 and 5637 cell, respectively; CHO-K1 cells were negative, lacking alpha2-6-sialylated gangliosides. The data imply a direct correlation of rViscumin cytotoxicity and the expression of receptor ganglioside. Moreover, CHO-K1 cells were rendered susceptible toward rViscumin cytotoxicity after exogenous application of human granulocyte gangliosides. Thus, (1) rViscumin has to be considered as a sialic acid-specific rather than a galactose-specific type II ribosome-inactivating protein, and (2) neolacto-series gangliosides with Neu5Acalpha2-6Galbeta1-4GlcNAc-terminus are true functional and physiologically relevant rViscumin receptors.

This study presents a comparative analysis of gangliosides from lymphoid (spleen and thymus) and other (brain, liver, lungs and muscle) tissues of C57BL/6 mice lacking the gene for beta2-microglobulin (beta2M), a constitutive component of the MHC class I molecule. Ganglioside fractions in the tissues of mice homozygous (beta2M-/-) and heterozygous (beta2M-/+) for the gene deletion were determined by high performance thin-layer chromatography (HPTLC), followed by immunostaining with specific polyclonal antibodies. Ubiquitous gangliosides GM3(Neu5Ac) and GM3(Neu5Gc) were the dominant gangliosides in the lungs of the control beta2M-/+ mice, whereas the homozygous knockout mice had substantially decreased expression of these structures. The lungs of the beta2M-/- mice also had reduced expression of T-lymphocyte-specific GM1b-type gangliosides (GM1b and GalNAc-GM1b). beta2M-deficient mice also had more GM1a and GD1a gangliosides in the liver, and several neolacto-series gangliosides were increased in the brain and lungs. This study provides in vivo evidence that the beta2M molecule can influence the acquisition of a distinct ganglioside assembly in different mouse organs, implicating its non-immunological functions.

On inflamed endothelium the cell surface protein E-selectin isexpressed which supports the initial process of attachment -capturing and rolling of leukocytes. A recombinant CHO cell linesecreting a soluble E-selectin-IgG chimera was cultivated competitively under serum free conditions in three different bioreactor systems: a 1 l Super-Spinner, a 2 l stirred tank bioreactor equipped with a spinfilter, and a 100 l stirred tankbioreactor. In the smallest system 25.4 mg E-selectin-IgG wereproduced in 62 days using a repeated batch process whileachieving a maximal viable cell density of 3.7 x 10(6) cells ml(-1). Using continuous perfusion mode a total amount of35.2 mg were produced with a maximal viable cell density of1.65 x 10(7) cells ml(-1) in the 2 l bioreactor within 29 days. Large scale cultivation in a 100 l stirred tankbioreactor yielded 105.6 mg in three batches with a maximal viable cell density of 9.7 x 10(5) cells ml(-1) within 15 days. After removal of the cells by continuous centrifugation and a depth filter clearance step, the supernatants were concentrated via ultra filtration. Purificationwas performed by affinity chromatography with rProtein A. Integrity of the E-selectin-IgG protein was checked with SDS PAGE. Its activity was verified in a cellular adhesion assay performed with HL-60 cells and a recombinant CHO cell line expressing membrane-anchored E-selectin constitutively, and E-selectin expressing HUVECs, respectively. Soluble E-selectin-IgG was used to block adhesion to these cell layerscompetitively. A concentation of 18.8 and 37.5 mug ml(-1)was sufficient to reduce the amount of adhering HL-60 cells to 50% on CHO and HUVEC layers, respectively.

R24 is a mouse IgG3 monoclonal antibody (mab) that reacts with the ganglioside GD3 expressed by cells of neuroectodermal origin. The anti-tumor activity of R24 has been demonstrated in initial phase I and pilot trials in patients suffering from metastatic melanoma. The purpose of this study was to investigate the biotechnological production and particularly the glycosylation of this clinically important antibody. Growth, metabolism, and IgG production of R24 secreting hybridoma cells were analyzed on 1 L bioreactor bench scale using repeated-batch mode. The amount of 57 mg of pure mab was obtained from 1.6 L crude supernatant by protein A chromatography. Western blot binding assays with sugar-specific lectins revealed glycosylation of the heavy chains, whereas no carbohydrates were detectable on the light chains. Because glycosylation is essential for antibody effector functions in vivo (such as complement fixation or binding to macrophage Fc receptors), mab R24 was subjected to both enzymatic deglycosylation using PNGase F and chemical deglycosylation by hydrazinolysis. Released glycans were structurally characterized by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), matrix assisted laser desorption ionization time-of-flight (MALDI-TOF), and electrospray ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry. Six major biantennary chains of the complex glycosylation phenotype were found with variations in galactosylation and core fucosylation. The predominant N-linked structure, indicating the high degree of agalactosyl glycoforms, was the agalacto biantennary chain with a relative percentage of 57% (51% core-fucosylated, 6% nonfucosylated). The second most abundant oligosaccharide was the monogalacto biantennary chain amounting to 30% (26% core- and 4% nonfucosylated). The antibody contained 0.46 microg sialic acid per mg protein, which splits into 0.243 microg Neu5Gc and 0.217 microg Neu5Ac, corresponding to a Neu5Ac:Neu5Gc ratio of 1:1.06. Furthermore, the antigen specificity of R24 was determined by immunodetection of GD3 on thin-layer chromatograms, and real time GD3-antibody binding interactions were measured with an optical biosensor (BIAcore). From the structural data obtained in this study it is concluded that glycosylation of the antibody may be important in the clinical outcome of targeted anti-cancer immunotherapy.

Nano-ESI QTOF MS was used for sensitive mapping and sequencing of single molecular species in complex ganglioside mixtures obtained from human granulocytes, where the fucosylated carbohydrate chains of granulocyte gangliosides carry sLex and VIM-2 epitopes postulated to interact with E-selectin of the blood vessel wall in the early phase of the inflammation process. Functionally relevant components are expressed only at a low level, but using the negative ion detection it is possible to trace and identify such species, which were not detectable even by TLC. Advantage of the low-energy CID fragmentation for low abundance components of the complex ganglioside mixtures in the negative ion mode is to produce clear-cut series of fragment ions for sequencing. Fucosylation analysis carried out for each molecular species by MS/MS permits the clear distinction between sLex and VIM-2 epitope. VIM-2 epitope was expressed in all species with a longer sugar core, while in the short oligosaccharide chain with a sLex only, using biological material at a mid-femtomol level detection.

Cytosolic Chinese hamster ovary (CHO) cell sialidase has been cloned as a soluble glutathione S-transferase (GST)-sialidase fusion protein with an apparent molecular weight of 69 kD in Escherichia coli. The enzyme has then been produced in mg quantities at 25-L bioreactor scale and purified by one-step affinity chromatography on glutathione sepharose (Burg, M.; Müthing, J. Carbohydr. Res. 2001, 330, 335-346). The cloned sialidase was probed for desialylation of a wide spectrum of different types of gangliosides using a thin-layer chromatography (TLC) overlay kinetic assay. Different gangliosides were separated on silica gel precoated TLC plates, incubated with increasing concentrations of sialidase (50 degreesU/mL up to 1.6 mU/mL) without detergents, and desialylated gangliosides were detected with specific anti-asialoganglioside antibodies. The enzyme exhibited almost identical hydrolysis activity in degradation of GM3(Neu5Ac) and GM3(Neu5Gc). A slightly enhanced activity, compared with reference Vibrio cholerae sialidase, was detected towards terminally alpha(2-3)-sialylated neolacto-series gangliosides IV3-alpha-Neu5Ac-nLc4Cer and VI3-alpha-Neu5Ac-nLc6Cer. The ganglio-series gangliosides G(D1a), G(D1b), and G(T1b), the preferential substrates of V. cholerae sialidase for generating cleavage-resistant G(M1), were less suitable targets for the CHO cell sialidase. The increasing evidence on colocalization of gangliosides and sialidase in the cytosol strongly suggests the involvement of the cytosolic sialidase in ganglioside metabolism on intracellular level by yet unknown mechanisms.

The cDNA of Chinese hamster ovary (CHO) cell cytosolic sialidase was amplified by RT-PCR and cloned into the pGEX-2T plasmid vector encoding for glutathione S-transferase (GST). Screening revealed transformed Escherichia coli clones with the constructed plasmid encoding the CHO cell sialidase sequence. After isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, SDS-PAGE of the total protein extracts revealed a new protein of about 70 kDa, correlating with the molecular weight of a fusion protein composed of the GST (26 kDa) and the cloned cytosolic CHO cell sialidase (43 kDa). A soluble fusion protein was purified from sonified E. coli homogenates by one-step affinity chromatography on Glutathione Sepharose 4B, which showed sialidase activity towards 4-methyl-umbelliferyl-alpha-D-N-acetylneuraminic acid (MUF-Neu5Ac) substrate. Induction of cells with 0.1, 0.5, and 1.0 mM IPTG revealed highest total protein amounts after induction with 1.0 mM IPTG, but highest specific activity for affinity chromatography purified eluates from cultures induced with 0.1 mM IPTG. Therefore, large scale production was performed by inducing cells during exponential growth in a 25 L bioreactor for 3 h with 0.1 mM IPTG after chilling the cell suspension to 25 degrees C. The amount of 26.46 mg of 40-fold purified GST-sialidase with a specific activity of 0.999 U/mg protein was obtained from crude protein extracts by one-step affinity chromatography. 2-Deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en) and Neu5Ac were competitive inhibitors for the sialidase, the former being the more effective one using MUF-Neu5Ac as the substrate. The cytosolic sialidase is capable of desialylating a wide spectrum of different types of gangliosides using a thin-layer chromatography overlay kinetic assay without detergents. This is the subject of the accompanying paper (Müthing, J.; Burg, M. Carbohydr. Res. 2001, 330, 347-356).

Gangliosides from histopathologically-defined human cerebrum-resembling remnant and cerebellum from 37 and 30 gestational week-old anencephaluses were identified using mass spectrometry and high performance thin layer chromatography combined with immunochemical analysis in comparison to respective normal newborn/fetal and adult brain regions. A novel strategy of nano-electrospray ionization quadrupole time-of-flight tandem MS has been developed for identification of ganglioside components in complex mixtures. By morphoanatomical and histological investigation the anencephalic cerebral remnant was found to be aberrant, while the anencephalic cerebellum was defined as normal. Total ganglioside concentrations in the anencephalic cerebral remnant and the cerebellum were 34% and 13% lower in relation to the age-matched controls. In the cerebral remnant, GD3, GM2 and GT1b were elevated, while GD1a was decreased in the anencephalic cerebral remnant, but enriched in anencephalic cerebellum. GQ1b was reduced in both anencephalic regions. Gg4Cer, GM1b and GD1alpha, members of the alpha-series biosynthetic pathway, and neolacto-series gangliosides were found to be present in anencephalic, as well as in normal, fetal and adult brain tissues, indicating the occurrence of these biosynthetic pathways in human brain. In both cerebral and cerebellar anencephalic tissues, GM1b, GD1alpha, nLM1 and nLD1 were expressed at a higher rate in relation to normal tissue. It can be demonstrated that the anencephalic cerebral remnant, as a primitive brain structure, represents a naturally-occurring model to study the ganglioside involvement in induction of aberrant brain development.

Expression of neutral glycosphingolipids (GSLs) and gangliosides in normal lymphoid tissues and cells has been studied mostly by biochemical and immunochemical analysis of lipid extracts separated by thin-layer chromatography. GSLs and gangliosides involved in the GM1b biosynthetic pathway were assigned to T-lymphocytes, whereas B-cell gangliosides and GSLs have been poorly characterized in former publications. We used specific polyclonal antibodies in immunohistochemistry and flow cytometry to analyze the distribution of globotriaosylceramide (Gb(3)Cer), globoside (Gb(4)Cer), gangliotriaosylceramide (Gg(3)Cer), gangliotetraosylceramide (Gg(4)Cer), and gangliosides GM3 and GalNAc-GM1b in the mouse thymus, spleen, and lymph node. Immature thymocytes expressed epitopes recognized by all antibodies, except for anti-Gb(4)Cer. Mature thymocytes bound only antibodies to GalNAc-GM1b, Gg(4)Cer, and Gb(4)Cer. In secondary lymphoid organs, antibodies to globo-series GSLs bound to vascular spaces of secondary lymphoid organs, whereas the ganglio-series GSL antibodies recognized lymphocyte-containing regions. In a Western blotting analysis, only GalNAc-GM1b antibody recognized a specific protein band in all three organs. Flow cytometric analysis of spleen and lymph node cells revealed that B-cells carried epitopes recognized by all antibodies, whereas the T-cell GSL repertoire was mostly oriented to ganglio-series-neutral GSLs and GM1b-type gangliosides. The results of immunohistochemistry and flow cytometry were not always identical, possibly because of crossreactivity to glycoprotein-linked oligosaccharides and/or differences between cell surface carbohydrate profiles of isolated cells and cells in a tissue environment.

Cultivation of the new immortalized hepatocyte cell line HepZ was performed with a 1:1 mixture of DMEM and Ham's F12 media containing 5% FCS. The cells were grown in their 40th passage in 100 mL and 1 L volumes in spinner flasks and in a bioreactor, respectively. For the production of adherently growing HepZ cells macroporous CultiSpher G gelatin microcarriers were used in various concentrations from 1 to 3 g/L. The cells were seeded in a density of 2 x 10(5) cells/mL when using a microcarrier concentration of 1 g/L and 5 x 10(5) cells/mL at a microcarrier concentration of 3 g/L. After 7 days of cultivation a maximum cell concentration of 4.5 x 10(6) cells/mL was obtained in the spinner culture using a microcarrier concentration of 1 g/L. With bubble-free aeration and daily medium exchange from day 7, 7.1 x 10(6) cells/mL were achieved in the bioreactor using a microcarrier concentration of 3 g/L. The cells exhibited a maximum specific growth rate of 0.84 per day in the spinner system and 1.0 per day in the bioreactor, respectively. During the growth phase the lactate dehydrogenase (LDH) activity rose slightly up to values of 200 U/L. At the end of cultivation the macroporous carriers were completely filled with cells exhibiting a spherical morphology whereas the hepatocytes on the outer surface were flat-shaped. Concerning their metabolic activity the cells predominantly consumed glutamine and glucose. During the growth phase lactate was produced up to 19.3 mM in the spinner culture and up to 9.1 mM in the bioreactor. Maximal oxygen consumption was 1950 nmol/(10(6) cells. day). HepZ cells resisted a 4-day long chilling period at 9.5 degrees C. The cytochrome P450 system was challenged with a pulse of 7 microgram/mL lidocaine at a cell density of 4.5 x 10(6) cells/mL. Five ng/mL monoethylglycinexylidide (MEGX) was generated within 1 day without phenobarbital induction compared to 26 ng/mL after a preceded three day induction period with 50 microgram/mL of phenobarbital indicating hepatic potency. Thus, the new immortalized HepZ cell line, exhibiting primary metabolic functions and appropriate for a mass cell cultivation, suggests its application for a bioartificial liver support system., (Copyright 2000 John Wiley & Sons, Inc.)

This study presents a comparative analysis of gangliosides from lymphoid (spleen and thymus) and other tissues (brain, liver, lung, muscle) of C57BL/6 mice homozygous (-/-) and heterozygous (+/-) for the tumor necrosis factor receptor 1 (TNFRp55). Quantitative and qualitative differences in the expression of the lipid-bound N-acetylneuraminic (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) and of various ganglioside biosynthesis pathways were detected between the tissues of the TNFRp55 -/- and the control TNFRp55 +/- mice. Sialic acid profiles showed a strong decrease in the absolute amount of sialic acids (Neu5Ac + Neu5Gc) in the lungs and thymus of homozygous (1.41 and 0.3 ng/mg wet weight, respectively) compared with control heterozygous animals (7.18 and 2.05 ng/mg wet weight, respectively). Considerable differences of Neu5Ac/Neu5Gc ratios in the lungs, muscle, spleen, and thymus were also detected. The gangliosides GM3(Neu5Ac) and GM3(Neu5Gc) were the dominant gangliosides in the lungs of the control animals, whereas the knockout mice almost completely lacked these structures in this organ. Reduced expression of GM1b-type gangliosides (GM1b and GalNAc-GM1b) was also found in the lungs, spleen, and thymus of the TNFRp55 knockout mice. On the other hand, neolacto-series gangliosides were more abundant in the lungs, brain, and muscle of the knockout mice, whereas their expression in the liver, spleen, and thymus was similar in both groups of animals. This study provides in vivo evidence that TNF signaling via the TNFRp55 is involved in the acquisition of a distinct ganglioside assembly in different mouse organs. TNFRp55 signaling seems to be especially important for the activation of the GM1b-type ganglioside biosynthetic pathway that is a unique characteristic of the mouse lymphoid tissues.

A preparative anion-exchange high-performance liquid chromatographic method for the separation of the closely allied monosialogangliosides GM3(Neu5Ac) and GM3(Neu5Gc) has been developed. Hybridoma cells, readily available material derived from industrial monoclonal antibody production, were used as ganglioside source and led to fractions with pure GM3(Neu5Ac) and GM3(Neu5Gc) in high milligram quantities. The crude ganglioside extract was loaded onto columns filled with the strong anion-exchanger trimethylaminoethyl (TMAE)-Fractogel. Gangliosides were eluted from the stationary phase with a gradient system of ammonium acetate in methanol. The scaled-up approach ranged over more than one order of magnitude from 20 to 500 mg batches of GM3 gangliosides. Thus, the high-resolution power of the strong anion-exchanger TMAE-Fractogel allowed the preparative isolation by one-step column chromatography of two GM3 specimens which only differ in one hydroxyl group at position 5 of the neuraminic acid (N-acetyl-versus N-glycolylneuraminic acid).

An extremely sensitive method for the quantitative determination of phospholipids on silica gel-precoated thin-layer chromatography plates is reported. The procedure is based on the successive application of two lipid spray reagents. Firstly, phospholipid chromatograms are sprayed with the lipophilic fluorochrome 1,6-diphenyl-1,3,5-hexatriene, followed by application of the phosphorus-specific molybdenum blue (Dittmer-Lester) reagent. Compared to the single use of the respective dyes, the consecutive utilization of both spray reagents resulted in an enormous enhancement in sensitivity of at least one order of magnitude. The procedure was proved with eight phospholipids of animal origin, and visual detection limits of down to 10 ng were achieved. The described technique provides a sensitive and specific means for the analysis of phospholipids on the nanogram scale by augmenting the molybdenum blue staining with the fluorochrome 1,6-diphenyl-1,3,5-hexatriene operating as an enhancer.

Membrane vesicles of rabbit skeletal muscle were prepared and separated by sucrose density gradient centrifugation. The fractions obtained (in the order of increasing density) were sarcolemma (SL), T-tubules (TT), sarcoplasmic reticulum (SR1 and SR2) and triads/mitochondria (Tr/M) as characterized by their specific marker enzymes, ligand binding, and ion flux activities. The distribution of neutral glycosphingolipids and gangliosides in these membrane preparations has been documented in the preceding paper (J. Müthing, U. Maurer, U. Neumann, B. Kniep, and S. Weber-Schürholz, Carbohydr, Res., (1988) 135-145). GM3(Neu5Ac) is the dominant ganglioside, neolacto-series gangliosides are moderately expressed and ganglio-series gangliosides were found in minor quantities, however, all showing different qualitative and quantitative membrane-type specific patterns. The voltage dependent Ca(2+)-channels of skeletal muscle reside prevalently in the triad enriched membrane fractions deduced from highest binding capacity of 1,4-dihydropyridines. Calcium channel complexes of triads were reconstituted into unilamellar phospholipid vesicles of 400 nm defined size and the active 45Ca(2+)-uptake into intravesicular space was measured after incorporation of muscle specific gangliosides into the outer vesicle lipid bilayer in parallel to control liposomes without gangliosides. GM3(Neu5Ac) strongly increased the uptake of 45Ca2+ (+285%) whereas GM3(Neu5Gc) severely inhibited the ion flux (-61%). Neolacto-series gangliosides evoked miscellaneous effects upon 45Ca(2+)-flux depending on isomeric sialic acid configuration, oligosaccharide size and fatty acid chain length of the ceramide portion. VI3Neu5Ac-nLcOse6Cer (C24-fatty acid), IV3Neu5Ac-nLcOse4Cer (C16-fatty acid) and IV6Neu5Ac-nLcOse4Cer (C16-fatty acid) strongly enhanced the 45Ca(2+)-flux (+208, +162, and +120%, respectively, whereas IV3Neu5Ac-nLcOse4Cer (C24-fatty acid), VI3Neu5Ac-nLcOse6Cer (C16-fatty acid) and IV6Neu5Ac-nLcOse4Cer (C24-fatty acid) slightly reduced 45Ca(2+)-flux (-3, -6, and -17%, respectively). Out of all gangliosides tested in this study, GM1 showed the strongest stimulatory effect (+327%). GD1a and GT1b gave rise to remarkable flux-stimulation of +283 and +255%, respectively, whereas GD1b exhibited only a slightly positive effect (+38%). This data suggest a functional role of gangliosides in subcellular muscle membranes giving strong evidence that gangliosides are capable of modulating the cytosolic calcium level of muscle, which regulates muscle contraction.

Membrane vesicles were prepared from rabbit skeletal muscle, separated by sucrose density gradient centrifugation and characterized by their specific marker enzymes, ligand binding, and ion flux activities. The fractions obtained (in the order of increasing density) were sarcolemma (SL), T-tubules (TT), sarcoplasmic reticulum (SR1 and SR2) and triads/mitochondria (Tr/M). Their glycosphingolipid compositions were analyzed by biochemical and immunochemical methods with specific antibodies (TLC immunostaining) and characteristic patterns were obtained from respective membrane fractions, expressed on a protein basis. Glucosylceramide, the main neutral glycosphingolipid of rabbit muscle, was found in SL and TT fractions, whereas SR and Tr/M vesicles lack this compound. Lactosylceramide was selectively recovered in the SR1 fraction. GM3(Neu5Ac), the main ganglioside in rabbit muscle, was found to account for 64% in the SL, 13% in the TT, 7% in the SR1, 3% in the SR2 and 13% in the Tr/M fractions. IV3Neu5Ac-nLcOse4Cer was mostly abundant in SL and decreased in the order SL > TT, Tr/M > SR1, SR2. IV6Neu5Ac-nLcOse4Cer was only detected in the SL and Tr/M fractions in noteworthy quantities. Ganglioseries gangliosides GM1, GD1a, GD1b and GT1b displayed homogeneous distribution patterns in each membrane preparation. They were expressed only in small amounts but mainly in SL, TT and Tr/M vesicles and to less extent in SR1 and SR2 fractions. The presence of GM3(Neu5Ac) in the SL as well as on subcellular level was confirmed in transverse muscle cryosections by means of indirect immunofluorescence microscopy. The SL was brightly stained, but considerable intracellular fluorescence was observed as expected from the biochemical analyses. Thus, the neutral GSL and ganglioside expression of the SL and the intracellular membraneous network is different in skeletal muscle both in terms of quantitative and qualitative GSL composition as demonstrated in details by means of biochemical and immunochemical techniques. The modulatory functions of GM3 and gangliosides of the neolacto- and ganglio-series towards the voltage dependent Ca(2+)-channel, largely preponderant in the triads-containing Tr/M fraction, is the subject of the accompanying paper (J. Müthing, U. Maurer, and S. Weber-Schürholz, Carbohydr. Res., 307 (1998) 147-157).

Neutral glycosphingolipids and gangliosides were isolated from 3.7 x 10(9) primary bovine aortic endothelial cells and structurally characterized by immunological and chemical methods. Glucosyl- and lactosylceramide were detected as the main neutral glycosphingolipids (28% and 40% of total orcinol stain, respectively); LcOse3Cer and nLcOse4Cer were expressed to somewhat minor amounts (16% and 10% of total orcinol stain, respectively), and nLcOse6Cer occurred only in trace quantities. No neutral glycosphingolipids of the ganglio-series (GgOse3Cer and GgOse4Cer) and the globo-series (GbOse4Cer and the Forssman antigen) have been detected; only traces of GbOse3Cer were identified by TLC immunostaining. Positive CD15 bands obtained by TLC overlay with anti-Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-R antibody indicated the presence of lipid bound Lewisx antigen, whereas the isomeric Lewis(a) structure (Gal beta1-3(Fuc alpha1-4)GlcNAc beta1-R) was not detectable. G(M3) substituted with Neu5Gc and Neu5Ac in a 2:1 ratio was the major ganglioside comprising about 95% within the whole ganglioside fraction. G(M3)-structures were further characterized by FAB-MS and GC-MS of the native compounds and their permethylated derivatives. C18-sphingosine was the only long chain base, whereas variation occurred due to C(24:0,24:1) and C16 fatty acids. Terminally alpha2-3 sialylated neolacto-series gangliosides with nLcOse4- and nLcOse6Cer (<5% of total resorcinol stain) were found in almost equal quantities, whereas no alpha2-6 sialylated counterparts were detected. Fucosylated gangliosides with poly-N-acetyllactosaminyl chains (sialyl Lewis[x], sialyl Lewisa, and VIM-2 antigen) and sulfoglucuronylneolacto series structures with HNK-1 epitope were not detectable in the acidic glycosphingolipid fraction by TLC immunostaining. Gangliotetraose-type gangliosides G(M1) and G(D1a) (<1 % of total resorcinol stain) as well as traces of G(D1b) and G(T1b) have been distinctly identified by combined choleragenoid-TLC-immunostaining and previous neuraminidase treatment. The expression of dominant glycosphingolipids lactosylceramide and G(M3)(Neu5Gc) was proved by indirect immunofluorescence microscopy of cell layers grown in chamber slides, each showing different plasma membrane and subcellular distribution patterns. The results provide the basis for investigation of the role of glycosphingolipids as cell surface antigens of cellular interaction as well as receptors for blood components and macromolecules of the extracellular matrix.

Chinese hamster ovary (CHO) cells producing the recombinant glycoprotein human antithrombin III (rhAT III) were batch cultivated in a 20-L bioreactor for 13 days. Neuraminidase activity in cell-free supernatant was monitored during cultivation and free sialic acid was determined by HPLC. Neu5Acalpha(2-->3)Gal-specific Maackia amurensis and Galbeta(1-->4)GlcNAc-specific Datura stramonium agglutinin were used for determination of sialylated and desialylated rhAT III, respectively. A commercial test kit was used for evaluation of functional rhAT III activity. Supernatant neuraminidase as well as lactate dehydrogenase activity increased significantly during batch growth. The enhanced number of dead cells correlated with increased neuraminidase activity, which seemed to be principally due to cell lysis, resulting in release of cytosolic neuraminidase. Loss of terminally alpha(2-->3) linked sialic acids of the oligosaccharide portions of rhAT III, analyzed in lectin-based Western blot and lectin-adsorbent assays, correlated with a decrease of activity of rhAT III produced throughout long-term batch cultivation. Thus, structural oligosaccharide integrity as well as the functional activity of recombinant glycoprotein depend on the viability and mortality of the bioreactor culture, and batches with a high number of viable cells are required to guarantee production of glycoproteins with maximum biological activity. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 441-448, 1997.

Staphylococcus saprophyticus, an important cause of urinary tract infections, produces two major surface proteins, the S. saprophyticus surface-associated protein (Ssp) and the hemagglutinin, which mediates fibronectin binding and also functions as the major adhesion of the organism. The hemagglutinating and fibronectin binding functions probably reside on different parts of the molecule. To identify a receptor on eukaryotic cells, binding and inhibition studies with acidic and neutral glycosphingolipids, carbohydrates, and proteins of sheep erythrocyte membranes were conducted. S. saprophyticus did not bind to any glycosphingolipid and no inhibition was observed when hemagglutination assays were done in the presence of carbohydrates or fibronectin. Neither treatment of erythrocytes with galactose oxidase or neuraminidase and galactose oxidase nor mild periodate oxidation of erythrocytes reduced hemagglutination. However, proteinase-treated erythrocytes were no longer agglutinated. Similarly, untreated erythrocyte membranes inhibited hemagglutination, whereas proteinase-treated membranes did not. In addition, only hemagglutinating strains bound to 60- and 21-kDa sheep erythrocyte membrane proteins on ligand blots, and these proteins inhibited hemagglutination. Our data indicate that, in contrast to many other hemagglutinins, the receptor on sheep erythrocytes for S. saprophyticus is a protein.

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.

Enhancement in separation of gangliosides on silica gel precoated high-performance TLC plates has been obtained by automated multiple development chromatography. A less polar mixture of the standard solvent chloroform-methanol-20 mM aqueous CaCl2 (120:85:20, v/v) was used. Lowering the water content achieved separation of two complex monosialoganglioside fractions, isolated from murine YAC 1 T lymphoma and MDAY-D2 lymphoreticular cells. Three-fold chromatography in the solvent chloroform-methanol-20 mM aqueous CaCl2 (120:85:14, v/v) resulted in TLC separation of GM1b-type gangliosides, substituted with C24 and C16 fatty acids and with Neu5Ac and Neu5Gc as well, which could not be achieved by unidirectional standard chromatography. Compared to conventional single chromatography, the technique described allows high-resolution separation of extremely heterogenous ganglioside mixtures and offers a convenient tool for both analytical and preparative TLC.

Influenza A and Sendai viruses are known to bind to various extent to neolacto-series gangliosides IV3Neu5Ac-nLcOse4Cer, IV6Neu5Ac-nLcOse4Cer, and VI3Neu5Ac-nLcOse6Cer, which are the dominant gangliosides of human granulocytes. Recently, minor gangliosides of granulocytes were characterized and found to express sialyl Lewis(x) and VIM-2 epitopes. These long chain linear monosialogangliosides with nLcOse8, and nLcOse10, cores, carrying one to three fucoses, are shown in this study to bind with strong avidity to influenza A/PR/8/34 (H1N1), A/X-31 (H3N2), and Sendai virus (Z-strain) using the overlay technique. These and recent data from other groups imply that selectins and virus hemagglutinins are capable of competing with lipid bound sialyl Lewis(x) and VIM-2 epitopes on myeloid cells during inflammatory reactions.

Chloroform, the predominant constituent of solvents used for lipid extraction and chromatography, is believed to give rise to birth defects and serious damage to health, and may also be carcinogenic. Therefore, simple and successful methods have been developed to replace chloroform throughout the isolation of glycosphingolipids (GSLs) by less harmful solvents. Gangliosides of sheep brain (ganglio-series gangliosides GM1, GDla, GD1b and GT1b) and of lymphocyte-derived mouse hybridoma cells (namely GM3) were extracted with six different solvent mixtures. Chloroform:methanol:water (40:80:30, v/v/v) was employed as reference (solvent I). Combinations without chloroform were: n-propanol:water (40:10, v/v) (II), methylisobutylketone:methanol:water (40:80:30, v/v/v) (III), ethylacetate:methanol:water (40:72:28, v/v/v) (IV), methylacetate:methanol:water (40:72:28, v/v/v) (V) and petroleum ether:isopropanol:water (40:112:38, v/v/v) (VI). After extraction and dialysis, the weight of lipid extract as well as the content of sialic acid, gangliosides, sulphatides and phospholipids were determined. Quantitation of GSL yields in crude extracts obtained by the alternative solvent mixtures II to VI showed recoveries of brain gangliosides from nearly 67% up to 104% compared with the reference solvent I. Extraction of hybridoma cells by means of the alternative combinations without chloroform revealed at least the same and mostly better ganglioside yields in the range from 98% to 116% with regard to the reference solvent I. n-Propanol:water (II) and methylisobutylketone:methanol:water (III) were the recommended extractants for both tissues. Therefore, the methods described offer simple, less hazardous and successful strategies for GSL extraction in excellent yield without the need for using chloroform.

The ganglioside GM3 (II3Neu5Ac-lactosylceramide) represents a minor ganglioside in normal human brain compared to major gangliosides with gangliotetraose-backbone. In this study the presence of GM3 in three 23 and 24 weeks of gestation old human cerebella was demonstrated by immunostaining extracted gangliosides on thin-layer chromatography plate as well as by immunohistochemical analysis of cerebellar cryosections. During this stage of brain development GM3 was found to be dominantly expressed on cells corresponding to Purkinje neurons. Delipidation of histological sections with chloroform/methanol caused significant reduction of anti-GM3 immunostaining, thus confirming the prevalent ganglioside nature of this antigen. These results give evidence that (1) GM3 ganglioside is associated with a specific subset of human fetal cerebellar neurons during the critical development stage, and (2) a definite ganglioside in general is distributed to a specific subset of cells in normal human brain.

In this study a simple and effective procedure for the isolation of individual neutral glycosphingolipids (GSLs) by preparative thin-layer chromatography is described. The method is based on nondestructive visualization of neutral GSLs on silica gel precoated thin-layer chromatography plates with anionic lipophilic fluorochromes. After thin-layer chromatography, individual neutral GSLs were detected by spraying the plate with fluorochrome solution followed by exposure to ultraviolet light. GSL containing silica gel was scraped off and extracted with chloroform:methanol:water (30:60:8, by vol). Neutral GSLs were freed from contaminating anionic fluorochrome by DEAE-Sepharose anion-exchange chromatography. Finally, a stepwise chloroform:methanol gradient chromatography on a small silica gel K60 column was employed to remove non-GSL impurities. Of nine different anionic fluorochromes tested, 2-(N-methylanilino)-naphthalene-6-sulfonic acid was found to be the most suitable for preparative purposes. The method was proved with reference GSL mixtures containing glucosyl-, galactosyl-, lactosyl-, globotriaosyl-, and neolactotetraosylceramides, each substituted with C24- and C16-fatty acids, resulting in isolation of individual GSL fractions. The technique was applied for the purification of neutral GSLs from mouse kidney and human granulocytes carrying Lewisx-epitopes. In summary, the method described offers an easy to handle and successful preparative thin-layer chromatography strategy to obtain pure neutral GSLs in microgram and milligram quantities.

At the IVth and Vth Workshop on Human Leukocyte Differentiation Antigens a group of monoclonal antibodies recognizing myeloid cells was found to bind to the ganglioside X3-NeuAcVII3FucnLc10Cer (VIM-2 dodecasaccharide). These antibodies were given the provisional cluster of differentiation designation CDw65. Three antibodies of this cluster (VIM-2, VIM-8, and VIM-11) have now been studied in detail at the molecular and the cellular level. Binding of VIM-2 is abolished after treatment of cells with Vibrio cholerae neuraminidase, whereas VIM-8 and VIM-11 show enhanced binding to neuraminidase-treated cells. We investigated binding of the three mAbs to glycolipid antigens with shorter carbohydrate chains. Distinct differences were observed in the binding of CDw65 antibodies to VIII3-NeuAcV3FucnLc8Cer (VIM-2 decasaccharide). VIM-2 strongly bound to this antigen, whereas no binding was observed with the other two mAbs. Conversely, the asialoganglioside of the VIM-2 decasaccharide, V3FucnLc8Cer, was not recognized by VIM-2, but this antigen bound strongly VIM-8 and VIM-11. Thus, VIM-2 and the other CDw65 antibodies represented two different antigen specificities.

The isolation and structural characterization of fucosylated neolacto-series gangliosides with linear poly-N-acetyllactosaminyl chains from normal human granulocytes is described. Gangliosides were purified by consecutive use of anion exchange HPLC on Fractogel TMAE-650(S), adsorption and reversed phase HPLC on Nucleosil 50-7 and Nucleosil 7C18 columns, respectively. TLC immunostaining with carbohydrate specific monoclonal antibodies, fast atom bombardment-mass spectrometry (FAB-MS) of the permethylated derivatives and gas chromatography-electron impact mass spectrometry (GC-EIMS) of partially methylated alditol acetates were used for structure elucidations. One ganglioside was identified as sialyl Lewis(x) antigen with nLcOse6Cer core, Neu5-Ac alpha 2-3Gal beta 1-4 (Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc NAc beta 1-3 Gal beta 1-4Glc beta 1-1Cer. Furthermore, monosialylated ceramide deca-, undeca-, dodeca- and tridecasaccharides with three (nLcOse8Cer) and four (nLcOse10Cer) linear lactosaminyl repeats were identified, carring one to three fucoses. The ceramide portions were found to contain C18 sphingosine and predominantly C16:0 fatty acids. All monosialogangliosides were homogenous concerning their terminal alpha 2-3 Neu5Ac-sialylation, but different in their fucosylation status. Beside VI3Neu5Ac, V3Fuc-nLcOse6Cer, in two of the fucosylated polylactosaminyl ganglioside fractions the sialyl Lewis(x) epitope was found, whereas five species expressed the terminal VIM-2 motif. The role of protein linked sialy Lewis(x) epitope of human granulocytes as a ligand for endothelial leukocyte adhesion molecule-1 (ELAM-1; E-selectin) and platelet activation-dependent granule external membrane protein (PADGEM; P-selectin) is well documented. However, the involvement of endothelial cells E-and/or P-selectin mediated cell-cell adhesion via lipid bound sialyl Lewis(x) and/or VIM-2 epitopes on human granulocytes has to be proved in further investigations.

In this review an updated overview of current improvements on thin-layer chromatography (TLC) of gangliosides over the past decade is provided. Basic general techniques and special advice is given for successful separation of glycosphingolipids. New approaches concerning continuous and multiple development, and several preparative TLC methods are also included. Emphasis is placed on TLC immunostaining and related techniques, i.e. practical applications of carbohydrate-specific antibodies, toxins and bacteria, viruses, lectins and eukaryotic cells. Thus, this review on ganglioside TLC summarizes its power as an analytical tool for a wide range of purposes.

Five types of dextran-based microcarriers (Dormacell(™), Pfeifer and Langen) with different concentrations of dimeric DEAE anion-exchange groups (nitrogen contents from 1.2 up to 2.9%) were tested as growth substrates for the cultivation of human umbilical vein endothelial cells (HUVECs). All microcarriers were gelatinized before use to improve cell adhesion. The one with the highest DEAE-group density was found to be most suitable for HUVEC propagation reaching final cell densities of 8×10(5) viable cells ml(-1) (95% viability) using microcarrier concentrations of 3 g l(-1). Furthermore, metabolic data of glucose/lactate and amino acid metabolism are presented in this study. The concentrations of 18 amino acids were monitored throughout cultivation. A considerable decrease of glutamine and inverse increase of glutamate was observed. Cultivation with initial glucose concentration of 16.5 mmol l(-1) resulted in high glutamine consumption rates, whereas high glucose-supplemented starting culture medium (30 mmol l(-1)) gave considerably lowered rates, indicating altered glutamine metabolism due to different glucose feeding. The glucose consumption and lactate production rates increased 2.6 fold and 3.5 fold, respectively, due to switch over from low to high glucose supplemented cultures. The rate of glucose metabolism was found not to be directly related to cell growth, because almost identical growth rates and doubling times were obtained. Considering the remaining 16 amino acids measured, serine concentrations considerably declined and glycine as well as alanine concentrations raised strongly. Most amino acid values were found insignificantly altered during 14 days of cultivation. Spinner vessel cultures served as inoculum for up scale propagation of HUVECs in membrane stirred 2 liter bioreactors. About 5×10(9) HUVECs were produced, which were used for the isolation and structural characterization of glycosphingolipids, cell membrane compounds, which are suggested to be involved in e.g. selectin-carbohydrate interaction (cell-cell adhesion), carcinogenesis and atherogenesis.

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.

Primary bovine aortic endothelial cells were cultivated in serum supplemented medium without any additional growth factors. The anchorage dependent cells were propagated on Dormacell(®) microcarriers with covalently bound dimeric DEAE-groups at the surface of the dextrane beads. Cultivations were performed in 200 ml spinner cultures containing 1 g l(-1) to 3 g l(-1) of microcarriers. Out of five types of Dormacell(®) microcarriers with different ion exchange capacities ranging from 0.30 up to 0.65 meq g(-1), corresponding to nitrogen contents from 1.2% to 2.9%, respectively, optimal attachment and growth of endothelial cells were obtained with beads of highest nitrogen content (2.9%). Cells were seeded withca. 5 viable cells per microcarrier being sufficient to achieve fully confluent microcarriers after 4 to 5 days. Glucose concentrations decreased from 21 mM to uppermost half of the original concentrations. 4 mM glutamine was rapidly consumed and virtually exhausted after the cells reached confluency. Lactate concentrations raised to a maximum of 7 mM in spinner cultures, but was found to be reutilized in the stationary phase after glutamine limitation occurred. Serine was found to be the second most prominent amino acid being almost exhausted at confluency whereas alanine was produced in noteworthy amounts. Considerable decrease was determined for threonine, lysine and arginine; low consumption rates were observed for leucine, phenylalanine and methionine. All other amino acids did not alter significantly throughout cultivation. These data support that bovine aortic endothelial cells are capable to utilize glucose and glutamine as well as lactic acid (after glutamine exhaustion) as energy and/or carbon source. Finally, batch cultures in a 2 liter membrane stirred bioreactor with bubble-free aeration were performed to produce large quantities of endothelial cells using microcarrier concentrations of 3 g l(-1).

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.

A new anion-exchange HPLC method for separation of gangliosides has been developed using TMAE (trimethylaminoethyl)-Fractogel as the stationary phase and a gradient system of ammonium acetate in methanol for elution. Chromatography of mouse brain gangliosides resulted in separation according not only to their degree of sialylation into mono-, di-, tri- and tetrasialogangliosides, but also in separation of positional isomers within the four elution domains, e.g. GD1a (IV3Neu5Ac,II3Neu5Ac-GgOse4Cer) and GD1b (II3(Neu5Ac)2-GgOse4Cer) of the disialoganglioside fraction. Furthermore, base-line separation of alpha 2-3 and alpha 2-6 sialylated neolacto-series gangliosides e.g. IV3Neu5Ac-nLcOse4Cer and IV6Neu5Ac-nLcOse4Cer, isolated from human granulocytes, was achieved and pure fractions of each ganglioside were obtained. Thus, the high-resolution power of the strong anion-exchanger TMAE-Fractogel allowed the isolation of pure gangliosides on a preparative scale by one-step column chromatography.

Automated multiple development chromatography has been utilized to enhance separation of gangliosides on silica-gel precoated high-performance TLC plates. Three-fold chromatography of a complex mixture of neolacto-series monosialogangliosides in the solvent chloroform-methanol-water (120:85:14, v/v, 2 mM CaCl2) resulted in a ca. three-fold increase in separation distance of e.g. alpha 2-3 and alpha 2-6 sialylated ganglioside isomers compared to conventional single chromatography in the standard solvent chloroform-methanol-water (120:85:20, v/v, 2 mM CaCl2). An extremely heterogenous murine disialoganglioside mixture was developed three times in chloroform-methanol-water (120:85:16, v/v, 2 mM CaCl2) leading to a more than two-fold increase in separation distance. Chloroform-methanol-water (120:85:22, v/v, 2 mM CaCl2) was the solvent of choice for multiple chromatography of ganglio-series polysialogangliosides from embryonic chicken brain.

In this study gangliosides from various myelomas and hybridomas of mouse, rat, and human origin were characterized by thin-layer and high-performance liquid chromatography, immunological methods (overlay technique) and fast atom bombardment mass spectrometry. Exclusively GM3 substituted with C24:1- and C16:0-fatty acid, was found in all B cell-derived cell lines. C18 sphingosine was the single long chain base in each GM3 ceramide portion. The mouse myeloma (NS-1) and all hybridomas, obtained by fusion of mouse, rat, or human B lymphocytes with murine myelomas, showed high GM3 (NeuGc) content (> 75%) and low GM3 (NeuAc) expression. Absolute amounts of GM3 ranged from 0.2 up to 0.8 mg x 10(-9) cells. Normally, human cells do not express NeuGc, and an Epstein-Barr virus-transformed human B lymphocyte line analyzed in this study retained this sialylation status, expressing exclusively GM3 (NeuAc) (100%). The fusion of human B lymphocytes with mouse myelomas led to high GM3 (NeuGc) expression (average about 85%) in all mouse/human heterohybridomas examined. Our results indicate the chromosomal gene "transfer" and/or the activation of enzymes involved in NeuGc-biosynthesis due to the somatic cell fusion process, which might explain the mouse dominance in the manifestation of the NeuGc-phenotype in hybridomas of human origin.

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 expression of neutral glycosphingolipids and gangliosides was investigated in mouse and rabbit skeletal muscle by means of biochemical and immunochemical techniques. Neutral glycosphingolipids from muscle of the inbred rabbit strain used in this study showed a simple TLC pattern, comprising mainly monohexosylceramide. In addition to this compound, lactosylceramide, lacto-N-neotetraosylceramide, globoside and Forssman GSL were detected in mouse muscle. The major ganglioside in both species was GM3; GM3 (Neu5Ac) and GM3(Neu5Gc) were found in a 3:1 ratio in mouse muscle, whereas the absence of GM3(Neu5Gc) is characteristic of rabbit muscle. As a general structural feature of all muscle GM3 gangliosides investigated, a C18 fatty acid and C18 sphingosine were the major components besides minor C22 and C24:1 fatty acids of the respective ceramide portions, as revealed by positive and negative ion FAB-MS. alpha 2-3 sialylated lacto-N-neotetraosyl-ceramide (sialylparagloboside) was expressed in both species, whereas the alpha 2-6 sialylated isomeric compound was found only in mouse muscle. Minute quantities of ganglio-series GM1, GD1a, GD1b, and GT1b were detected in muscles from both species. Glycosphingolipid expression could be confirmed immunohistochemically by examining transverse and longitudinal cryosections of skeletal muscle samples. The results provide the basis for the investigation of muscle specific glycosphingolipids that might modulate membrane protein functions in muscle.

Gangliosides of the mouse-rat hybridoma cell line 187.1, which secretes an antibody against kappa-light chain of mouse IgG, were isolated and structurally characterized by biochemical and immunological methods (overlay technique), and fast atom bombardment-mass spectrometry. Exclusively GM3, substituted with C24:1 and C16:0 fatty acid and C18:1 sphingosine, was found in this B cell derived cell line. A GM3(NeuGc) to GM3(NeuAc) ratio (80 to 20), was characteristic for 187.1 cells, and absolute GM3 amounts of about 0.3 mg 10(-9) viable cells were determined. Exogenous application of GM3, which has been isolated from large cell preparations, to 187.1 cells showed growth inhibition in a concentration dependent manner. Using the MTT-assay and the [3H]thymidine incorporation assay, the cells exhibited a strong reduction in metabolic and proliferative activity, respectively, after exposure of cells to GM3. GM3 was applied in concentrations between 3 microM and 30 microM, giving evidence for strong inhibitory effects at 30 microM GM3 and less but significant suppression after application of GM3 concentrations lower than 20 microM. No cellular response was observed at the lowest concentration (3 microM) used in this study. Hybridoma cells as well as other cell types like fibroblasts, muscle cells and endothelial cells, are in general characterized by high expression of the GM3 ganglioside, which is known to act as a modulator of cellular growth in monolayer cultures of adherent cells. Since gangliosides are released to the culture medium by cell lysis, i.e. cell death, and/or by active membrane shedding, the results obtained in this study suggest a growth regulatory role of GM3 in high density hybridoma cell cultures.

B lymphocytes from CBA/J mice were stimulated in splenocyte cultures for 72 h with various endotoxins. Bisphosphoryl lipid A from Escherichia coli had the highest stimulatory effect followed by LPS of Citrobacter freundii and Salmonella minnesota as measured by [3H]thymidine uptake. Gangliosides of stimulated B cells (metabolically labeled with D-[1-14C]galactose and D-[1-14C]glucosamine) and unlabeled gangliosides from resting B cells (prepared from spleens without stimulus) were analyzed by high-performance TLC, DEAE anion-exchange HPLC, and immunostaining procedures. Contents of ganglioside-derived sialic acids, quantified by HPLC as their fluorescent derivatives, decreased from stimulated to resident B lymphocytes in the following order: LPS S. minnesota > LPS C. freundii > bisphosphoryl lipid A E. coli > resting B cells. Gangliosides of resting B cells contained more N-glycolyl- than N-acetylneuraminic acid, whereas inverse ratios were found in activated cells, indicating a shift from N-glycolyl- to N-acetylneuraminic acid due to stimulation. Furthermore, a higher disialoganglioside content was characteristic for activated B cells. Fast atom bombardment mass spectrometry was performed with permethylated mono- and disialoganglioside fractions of LPS S. minnesota and LPS C. freundii stimulated B cells. Major gangliosides were GM1a and GD1a beside minute amounts of GD1b. The structural heterogeneity in the gangliosides was caused by (a) N-substitution of the sialic acids with either acetyl or glycolyl groups, (b) variation in the long-chain base (sphingosine, sphinganine), and (c) substitution of the ceramide moiety by fatty acids of different chain length and degree of unsaturation (C16:0, C24:0,24:1).2+ p6