The detection of picomolar quantities of GD1b,GT1b and GQ1b on thin-layer chromatograms by the direct binding of125I-labeled tetanus toxin,fragment C

The¹²⁵I-labeled fragment C of tetanus toxin was found to bind specifically to the gangliosides G_D1b, G_T1b, and G_Q1b when applied to thin-layer chromatograms on which a mixture of gangliosides had been resolved. As little as 2.5 pmoles of these gangliosides could be detected by this method. In addition to factors determined by the sample, namely the amount and species of gangliosides present, optimal binding of the¹²⁵I-labeled fragment C also depended upon the iodination procedure used to generate the probe, the toxin concentration, and the concentration, buffer type, pH, and ionic strength of the binding solution. This new technique was shown to be a sensitive method for the detection and identification of specific gangliosides originating from extraneural or neural cells.Nomenclature: The gangliosides follow the nomenclature system of Svennerholm [Eur J Biochem (1977) 79:11–21] G_M3 II³NeuAc-LacCer - G_D3 II³(NeuAc)₂-LacCer - G_M1 II³NeuAc-GgOse₄Cer - G_D1a IV³NeuAc, II³NeuAc-GgOse₄Cer - G_D1b II³(NeuAc)₂-GgOse₄Cer - G_T1b IV³NeuAc, II³(NeuAc)²-GgOse₄Cer - G_Q1b IV³(Neu-Ac)₂, II³(NeuAc)₂-GgOse₄Cer - G_P1b IV³(NeuAc)₃, II³(NeuAc)₂-GgOse₄Cer     

Susceptibility of infant mice to F5 (K99)E. coli infection: Differences in glycosyltransferase activities in intestinal mucosa of inbred CBA and DBA/2 strains

EnterotoxigenicEscherichia coli (ETEC) strains expressing F5 (K99) fimbriae cause diarrhoea in the young animal through adhesion to specific sialoglycolipids of the small intestine surface. We studied here an infant mouse diarrhoea model, as CBA infant mice are susceptible to F5-positive ETEC infection, whereas DBA/2 ones are resistant. In an attempt to determine an enzymatic basis for susceptibility and resistance, we investigated the intestine ganglioside pattern in relation to the activity of glycosyltransferases responsible for the globo- and ganglio-series. We observed that the intestine of susceptible CBA infant mice displayed a characteristic sialoglycolipid pattern containing mainly the F5 receptors. The two murine strains differed in the relative activities of galactosyltransferases (GbOse₃Cer and GM1 synthases),N-acetylgalactosylaminyltransferases (GA2 and GM2 synthases) and sialyltransferases (GM3 and GD3 synthases). An elevated GM3-synthase activity was observed in the intestine of susceptible CBA infant mice, at the age of high susceptibility. Hence, we conclude that the marked specificity of mouse type correlated with susceptibility and resistance to F5-positive ETEC infection which could be controlled through the regulation of glycosyltransferase activities.Abbreviations NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - Glc glucose - GalNAc N-acetylgalactosamine - Gal galactose - Car ceramide - LacCer lactosylceramide (Galß-4Glcß1-1Cer) - GA2 asialo-GM2 (GgOse₃Cer) - GA1 asialo-GM1 (GgOse₄Cer) - NeuAc/NeuGc-GMla II³ NeuAc/NeuGc-GgOse₄Cer - NeuAc/NeuGc-GM1a IV³ NeuAc/NeuGc-GgOse₄Cer - NeuAc/NeuGc-GM2 II³ NeuAc/neuGc-GgOse₃Cer - NeuAc/NeuGc-GM3, II³ NeuAc/NeuGc-LacCer; NeuAc/NeuGc-GD1a, IV³ NeuAc/NeuGc, II³ NeuAc/NeuGc-GgOse₄Cer; NeuAc/NeuGc-GD1b II³ (NeuAc/NeuGc)₂-GgOse₄Cer - NeuAc/NeuGc-GD1c IV³ (NeuAc/NeuGc)₂-GgOse₄Cer - NeuAc/NeuGc-GD2, II³ (NeuAc/NeuGc)₂-GgOse₃Cer; NeuAc/NeuGc-GD3, II³ (NeuAc/NeuGc)₂-Lac Cer; NeuAc/NeuGcGT1a IV³ (NeuAc/NeuGc)₂, II³ NeuAc/NeuGc-GgOse₄Cer - NeuAc/neuGc-GT1b IV³ NeuAc/NeuGc, II³ (NeuAc/NeuGc)₂-GgOse₄Cer - NeuAc/NeuGc-GT1c II³ (NeuAc/NeuGc)₃-GgOse₄Cer; NeuAc/NeuGc-GT2, II³ (NeuAc/NeuGc)₃-GgOse₃Cer - NeuAc/NeuGc-GT3 II³ (NeuAc/NeuGc)₃-Lac Cer - NeuAc/NeuGc-GQ1b IV³ (NeuAc/NeuGc)₂, II³ (NeuAc/NeuGc)₂-GgOse₄Cer - NeuAc/NeuGc-GQ1c IV³ NeuAc/NeuGc, II³ (NeuAc/NeuGc)₃-GgOse₄Cer - NeuAc/NeuGc-GP1c IV³ (NeuAc/NeuGc)₂, II³ (NeuAc/NeuGc)₃-GgOse₄Cer - GD, GT and GQ di-, tri- and tetra-sialoglangliosides. NeuGc-SPG, IV³ NeuGc-nLcOse₄Cer. Glycosyltransferases assayed in this work areN-acetylgalactosaminyltransferases - UDP-GalNAc lactosylceramide 1-4N-acetylgalactosaminyltransferase or GA2 synthase (EC 2.4.1-) and UDP-GalNAc:(N-acetylneuraminyl)-lactosylceramide 1-4N-acetylgalactosaminyltransferase or GM2 synthase (EC 2.4.1.92) - sialyltransferases CMP-N-acetylneuraminate: lactosylceramide 2–3 sialyltransferase (sialyltransferases I and IV) or GM3 synthase (EC 2.4.99.-) and CMP-N-acetylneuraminate:(N-acetylneuraminyl) lactosylceramide 2-8 sialyltransferase (sialyltransferase II) or GD3 synthase (EC 24.99.8) - galactosyltransferases UDP-galactose:N-acetylgalactosaminyl-(N-acetylneuraminyl) lactosylceramide 1-3 galactosyltransferase (galactosyltransferase II) or GM1a synthase (EC 2.4.1.62) and UDP-galactose:lactosylceramide 1-4 galactosyltransferase or GbOse₃Cer synthase (EC 2.4.1-)     

Ganglioside changes in the chicken optic lobes and cerebrum during embryonic development

Summary The developmental profiles of 15 different gangliosides of the optic lobes and cerebrum of the chicken were followed from the 6 th day of incubation to hatching and correlated to morphological development. Five of these gangliosides appearing in both structures between the sixth and tenth day, have not been reported previously in higher vertebrates. Three chromatographed on TLC-plates similarly to G_T3, G_T2, and G_T1c gangliosides, which have been demonstrated in fish brain. One fraction moved just below G_Q1b and is suggested, to contain G_Q1c. These novel gangliosides, which are possibly related to a recently proposed separate and probably phylogenetically older biosynthetic pathway, contained up to 20% of total ganglioside sialic acid. The fifth novel fraction, containing up to 16% of total ganglioside-sialic acid, moved below the penta-sialoganglioside G_P1 and is suggested to contain hexa-sialogangliosides.There were two main changes in ganglioside synthesis, which were identical in both structures.The first occurred from the sixth to the eleventh day, parallel to decreased proliferation, maximal cell migration and neuroblast differentiation, G_D3 and G_D2 decreased rapidly in favour of G_Q1b, G_P1, and to the novel fractions, described above.The second occurred from the eleventh to the eighteenth day, parallel to increased growth and arborization of dendrites and axons as well as functional establishment of synaptic contacts, there was a sharp rise in the amount of G_D1b, G_T1b, and G_D1a. Concomitantly the novel gangliosides decreased. At hatching G_D1a was the predominant ganglioside. G_M3, G_M2, and G_M1 were always minor fractions, each accounting for less than 4% of total ganglioside-sialic acid. G_M4 was never detected, indicating neglegible myelinisation until hatching.Abbreviations NeuAc N-Acetylneuraminic acid - G_M4 I³NeuAc-GalCer - G_M3 II³NeuAc-LacCer - G_M2 II³NeuAc-G_gOse₃Cer - G_M1 II³NeuAc-G_gOse₄Cer - G_D3 II³(NeuAc)₂LacCer - G_D1a IV³NeuAc, II³NeuAc G_gOse₄Cer - G_T3 II³ (NeuAc)₃LacCer - G_D2 II³(NeuAc)₂G_gOse₃Cer - G_D1b II³(NeuAc)₂G_gOse₃Cer - G_D1b II³(NeuAc)₂G_gOse₄Cer - G_T2 II³(NeuAc)₃G_gOse₃Cer - G_T1b IV³NeuAc, II³(NeuAc)₂G_gOse₄Cer - G_T1c II³(NeuAc)₃G_gOse₄Cer - G_Q1b IV³(NeuAc)₂ II³(NeuAc)₂G_gOse₄Cer - G_Q1c IV³NeuAc, II³(NeuAc)₃G_gOse₄Cer - G_P1 IV³(NeuAc)₂, II³(NeuAc)₃G_gOse₄Cer - G_H(?) IV₃(NeuAc)₃, II³(NeuAc)₃G_gOse₄Cer     

Interaction of gangliosides with plasma low density lipoproteins

The role of gangliosides in the reception of low density lipoproteins (LDL) was studied using as targets mouse ascites hepatoma 22a (MAH) cells which bind LDL through a specific high affinity receptor. Low density lipoprotein binding and uptake by MAH cells decreased after brief treatment of the cells with neuraminidase to partially remove surface sialic acid residues. The LDL uptake capability of the neuraminidasetreated MAH cells was fully restored after incorporation of exogeneous G_M1- and G_D1a-gangliosides into the cell surface. In contrast, free (extracellular) gangliosides inhibited LDL uptake by native MAH cells. This inhibitory effect was seen at ganglioside concentrations corresponding to the ganglioside content of serum and was most pronounced with gangliosides whose sialic acids were linked to a terminal galactose residue (G_M3, G_D1a, G_T1b) but was smaller or absent with gangliosides whose sialic acids were attached to an internal galactose (G_M1, G_M2). The binding of gangliosides to LDL was structure and concentration dependent, saturable and trypsin sensitive. The LDL-ganglioside interaction was further investigated by steady state fluorescence spectroscopy. Changes in the LDL fluorescence polarization were observed with as little as 0.01 M concentrations of the gangliosides. The magnitude and nature of the effect depended on the type of ganglioside. We conclude that the LDL surface possesses sites recognizing specific carbohydrate sequences of glycoconjugates and that changes in the cell surface concentrations of sialic acids significantly modulate the LDL uptake. It is postulated that shedding of gangliosides into the blood stream may be a factor involved in regulation of cholesterol homeostasis.Abbreviations MAH mouse ascites hepatoma 22a - LDL low density lipoprotein - ASM anthrylvinyl-labeled sphingomyelin [N-12-(9-anthryl-trans-dodecanoyl-sphingosine-1-phosphocholine] - RITC rhodamine isothiocyanate. The designation of gangliosides follows the IUPAC-IUB recommendation [1]: G_M3, II³NeuAc-LacCer, II³-N-acetylneuraminosyllactosylceramide - G_M2 II³-NeuAc-GgOse₃Cer, II³-N-acetylneuraminosylgangliotriaosylceramide - G_M1 II³-NeuAc-GgOse₄Cer, II³-N-acetylneuraminosylgangliotetraosylceramide - G_D1a, II³ IV³(NeuAc)₂-GgOse₄Cer, II³, IV³-di(N-acetylneuraminosyl)gangliotetraosylceramide - G_T1b II³(NeuAc)₂, IV³ NeuAc-GgOse₄Cer, II³-di-N-acetylneuraminosyl, IV³-N-acetylneuraminosylgangliotetraosylceramide     

In vivo growth conditions suppress the expression of ganglioside GM2 and favour that of lacto series gangliosides in the human glioma D-54MG cell line

The human glioma D-54MG cell line grownin vitro primarily expresses ganglio series gangliosides, particularly GM2. Subcutaneous injection of these cells into nude mice produced xenografts with an increased content of the human glioma-associated lacto series gangliosides, primarily 3-isoLM1, an alteration that was dose dependent, with the highest dose (1×10⁸) resulting in a phenotype that was most like that of the inoculum. After one passagein vivo, the lacto series dominated and reached a proportional level that was kept throughout the 10 passages. The mRNA levels of the GM2-synthase clearly coincided with GM2 expression and was 20 times higher in cells grownin vitro than in those grownin vivo. These results support the view that ganglioside expression in human gliomas is strongly influenced by environmental factors. Abbreviations: The gangliosides have been designated according to Svennerholm (Eur J Biochem (1977)79: 11–21) GM3, II³NeuAc-LacCer; GM2, II³NeuAc-GgOse₃Cer; GM1, II³NeuAc-GgOse₄Cer; GD3, II³(NeuAc)₂-LacCer; GD2, II³(NeuAc)₂-GgOse₃Cer; GD1a, IV³NeuAc, II³NeuAc-GgOse₄Cer; GD1b, II³(NeuAc)₂-GgOse₄Cer; GT1b, IV³NeuAc, II³(NeuAc)₂-GgOse₄Cer; 3-LM1, IV³NeuAc-nLcOse₄Cer; 3-isoLM1, IV³NeuAc-LcOse₄Cer; 3,6-isoLD1, IV³NeuAc, III⁶NeuAc-LcOse₄Cer; 38-LM1, IV³(NeuAc)₂-nLcOse₄Cer. MAb(s), monoclonal antibody (ies); the designation LM1 is used when both 3-isoLM1 and 3-LM1 and LD1, when both 36-isoLD1 and 38-LD1 are included.     

Gangliosides,sialoglycoproteins and acetylcholinesterase of the developing mouse brain

Summary The developmental accretion of up to nine individual gangliosides in foetal brains, peri- and postnatal cortices, postnatal cerebelli and olfactory lobes and in the liver and the spleen were investigated in mice and compared with that of glycoprotein-bound sialic acid and the activity of the acetylcholinesterase.In foetal brain and in postnatal liver and spleen more sialic acid was found bound to glycoproteins than to gangliosides. In postnatal brain structures, however, ganglioside-NeuAc predominated and increased between the 7th and 21st d about 2-fold in the olfactory lobes and cerebellum and more than 3-fold in the cortex.During foetal development the relative quantities (mol %) as well as the absolute concentrations (compared with the fresh weight) of GM1, GM2 and GM3 in the brain decreased, whereas those of GD1a, GD1b and GQ increased.This pattern change continued perinatally in the cortex up to the end of the first week. Thereafter the pattern changed little, but the concentration of all gangliosides present increased much more rapidly, especially between the 10th and 13th d.The postnatal cerebellum and olfactory lobes contained higher concentrations of GM1 and GM3 than the cortex, both gangliosides decreasing in favour of their di-, tri- and tetrasialo-homologues during the third postnatal week.In all brains structures the accretion of GD1a and GT1 was proportional to the increase in the activity of the acetylcholinesterase.Unlike the brain structures, the ganglioside pattern in the liver and spleen, characterised by a predominance of monosialogangliosides and of GD3, did not change noticeably during the first three weeks after birth.The coincidence of the changes in ganglioside accretion observed in the different brain structures with successive periods of morphological differentiation further support the suggestion that gangliosides may play an important role in control of the growth and differentiation of developing nerve cells.Abbreviations GM3 II³NeuAc-GgOse₂Cer - GM2 II³NeuAc-GgOse₃Cer - GM1 II³NeuAcGgOse₄Cer - GD1a IV³NeuAc-, II³ NeuAc-GgOse₄Cer - GD3 II³ NeuAc₂-GgOse₂Cer - GD2 II³ NeuAc₂-GgOse₃ Cer - GD1b II³ NeuAc₂-GgOse₄ Cer - GT1 IV³ NeuAc-, II³ NeuAc₂-GgOse₄ Cer - GQ IV³ NeuAc-, II³ NeuAc₃-GgOse₄ Cer - NeuAc N-acetylneuraminic acid (sialic acid) - AChE Acetylcholinesterase     

Structural studies of gangliosides from the YAC-1 mouse lymphoma cell line by immunological detection and fast atom bombardment mass spectrometry

YAC-1 cells were propagated in bioreactors in 11 and 7.51 volumes. The cells were metabolically labelled withd-[1-¹⁴C]galactose andd-[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 G_M1 and G_D1a. 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 G_M1b ganglioside. As well as small amounts of G_M2 and G_M1, the major gangliosides found in the complex mixture were G_M1b and GalNAc-G_M1b. The structural heterogeneity of these gangliosides was cased by (a) substitution of the ceramide moiety by fatty acids of different chain length and degree of unsaturation (C_16:0, C_24:0, C_24:1) 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 G_M1b or GgOse₄Cer was observed. Abbreviations: FAB-MS, fast atom bombardment mass spectrometry; GSL(s), glycosphingolipid(s); HPLC, high performance liquid chromatography, HPTLC, high performance thin layer chromatography; NK, natural killer; SIM, selective ion monitoring; TIC, total ion current. NeuAc,N-acetylneuraminic acid; NeuGc,N-glycolylneuraminic acid. The designation of the following glycosphingolipids follows the IUB-IUPAC recommendations. GgOse₃Cer or gangliotriaosylceramide or asialo G_M2, GalNAc1-4Gal1-4GlcCer; GgOse₄Cer or gangliotetraosylceramide or asialo G_M1, Gal1-3GalNAc1-4Gal1-4GlcCer; GgOse₅Cer organgliopentaosylceramide, GalNAc1-4Gal1-3GalNAc1-4Gal1-4GlcCer; II³NeuAc-GgOse₄Cer or G_M1; IV³NeuAcGgOse₄Cer or G_M1b; IV³NeuAc-GgOse₅Cer or GalNAc-G_M1b; IV³NeuAc, II³NeuAc-GgOse₄Cer or G_D1a; II³(NeuAc)₂-GgOse₄Cer or G_D1b; IV³(NeuAc)₂-GgOse₄Cer or G_D1c; IV³NeuAc,III⁶NeuAc-GgOse₄Cer or G_D1a; IV³NeuAc,II³(NeuAc)₂-GgOse₄Cer or G_T1b;Vibrio cholerae and Arthrobacter ureafaciens neuraminidase (EC 3.2.1.18).     

ON THE STRUCTURE OF A NEW, FUCOSE CONTAINING GANGLIOSIDE FROM PIG CEREBELLUM

A new ganglioside, provisionally named GLIVa, was isolated in pure form from pig cerebellum. Ganglioside GLIVa is a disialoganglioside containing fucose. Its basic neutral glycosphingolipid core is the gangliotetraose ceramide: Gal, β 1 → 3 GalNAc, β 1 → 4 Gal, β 1 → 4 Glc, β 1 → Cer. Fucose is α-glycosidically linked to the 2-position of external galactose and one N-acetylneuraminic acid is linked to the other one by an α, 2 → 8 linkage. Thus the total structure of ganglioside GLIVa is the following: Fuc, α 1 → 2 Gal, β 1 → 3 GalNAc, β 1 → 4 (NeuAc, α 2 48 NeuAc, α 2 → 3) Gal, β 1 → 4 Glc, β 1 → Ceramide. According to the IUPAC-IUB Commission on Biochemical Nomenclature is indicated as II³α(NeuAc)₂ IV²αFuc-GgOse₄Cer.     

Fucosyl-GM1 — A ganglioside associated with small cell lung carcinomas

Characterization of monosialogangliosides of a small cell lung carcinoma showed a unique composition. The tumour contained G_M2 and Fucosyl-G_M1 (Fuc-G_M1) with 2-hydroxy fatty acids as major ganglioside components. Three out of four other small cell carcinomas analysed contained also Fuc-G_M1 as a characteristic ganglioside. Fuc-G_M1 is suggested to be a small-cell lung carcinoma associated ganglioside antigen.Nomenclature: The gangliosides have been designated according to Svennerholm [25] G_M3 II³NeuAc-Lac-Cer - G_D3 II³(NeuAc)₂-LacCer - G_M2 II³NeuAc-GgOse₃Cer - G_M1 II³NeuAc-GgOse₄Cer - Fuc-G_M1 FuclV²Neu-AcII³-GgOse₄Cer - 3-L_M1 IV³NeuAc-nLcOse₄Cer - 6-L_M1 IV⁶NeuAc-nLcOse₄Cer     

Concanavalin A-stimulated expression of gangliosides with GalNAcβ1-4(NeuAcα2-3)Galβ structure in murine thymocytes

We analysed the glycolipids of mouse thymocytes before and after Concanavalin A (Con A) or recombinant interleukin-2 (rIL-2) stimulation by TLC-immunostaining with carbohydrate-specific antiglycolipid antibodies. The thymocytes were cultured in serum-free medium in the presence of 500 ng ml^–1 Con A, 10 U ml^–1 rIL-2 or Con A plus rIL-2 for 6, 12, 24, 48, and 72 h, and were found to start proliferating 24 h after cultivation in the presence of Con A or Con A plus rIL-2, the maximum levels being reached at 72 h and 48 h, respectively, in a thymidine uptake experiment. The concentrations of II³Neu-Gg₄Cer, Gg₄Cer and IV³GalNAc-Gb₄Cer after 48 h Con A stimulation were found to be at almost the original levels. Conversely, II³Neu-Gg₃Cer, which was not detected in the thymocytes at the start, began to appear after 48 h stimulation with Con A and Con A plus rIL-2, and IV³Neu-Gg₅Cer in the cells 48 h after stimulation with Con A and Con A plus rIL-2 has increased to 41 and 44 times higher than in the original cells, respectively, as judged on TLC-immunostaining with monoclonal antibody YHD-06, which detects the GalNAc1-4(NeuAc or NeuGc2-3)Gal-structure. These results indicate that the increased synthesis of both gangliosides, in other words, the activation ofN-acetylgalactosaminyltransferase, is associated with the mitogen-induced proliferation.N-Acetylneuraminic acid was the sole sialic acid in II³Neu-Gg₃Cer which newly appeared in the cells on stimulation, whereas the sialic acid of IV³Neu-Gg₅Cer was a mixture ofN-acetyl- andN-glycoloylneuraminic acids. This result may suggest that the substrates for the two differentN-acetylgalactosaminyltransferases must be different. This GalNAc1-4(NeuAc or NeuGc2-3)Gal-structure was also detected on the surface of the Con A or Con A plus rIL-2 stimulated mouse thymocytes on flow cytometric analysis of cells indirectly stained with monoclonal antibody YHD-06. Abbreviations: carbohydrate and glycolipid nomenclature and abbreviations follow the IUPAC-IUB recommendations or the nomenclature system of Svennerholm L. (1963)J Neurochem 10:613–23.     

Different binding capacities of influenza A and Sendai viruses to gangliosides from human granulocytes

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 G_M3 (II³Neu5Ac-LacCer), neolacto-series gangliosides (IV³Neu5Ac-nLcOse₄Cer, IV⁶Neu5Ac-nLcOse₄Cer and VI³Neu5Ac-nLcOse₆Cer) containing C_24:1, and to some extent C_22:0; and C_16:0 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 Neu5Ac2-3Gal and/or Neu5Ac2-6Gal 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. Abbreviations: FAB-MS, fast atom bombardment mass spectrometry; GC/EIMS, gas chromatography/electron impact mass spectrometry; GSL(s) glycosphingolipids; HPTLC, high performance thin-layer chromatography; Neu5Ac,N-acetylneuraminic acid [26], PFU, plaque forming unit. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations, and the ganglioside nomenclature system of Svennerholm was used. LacCer or lactosylceramide, Gal1-4Glc1-1Cer gangliotetraosylceramide or GgOse₄Cer, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; lacto-N-tetraosylceramide or nLcOse₄Cer, Gal1-4GlcNAc1-3Gal1-4-Glc1-1Cer; lacto-N-norhexaosylceramide or nLcOse₆Cer, Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal 1-4-Glc1-1Cer; G_M3, II³Neu5Ac-LacCer; G_M1, II³Neu5Ac-GgOse₄Cer; G_D1a, IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer; G_D1b, II³(Neu5Ac)₂-GgOse₄Cer; G_T1b, IV³Neu5Ac, II³(Neu5Ac)₂-GgOse₄Cer; G_Q1b, IV³(Neu5Ac)₂, II³(Neu5Ac)₂-GgOse₄Cer; sialyllacto-N-tetraosylceramide, IV³Neu5Ac/IV⁶Neu5Ac-nLcOse₄Cer; sialyllacto-N-norhexaosylceramide or i-active ganglioside, VI³Neu5Ac-nLcOse₆Cer.     

Glycosphingolipid biosynthesis during myogenesis of rat L6 cells in vitro

Summary Glycosphingolipid biosynthesis was examined using [³H]-galactose as a precursor as rat L6 myoblasts fused to form multinucleated myotubes. Incorporation of label into neutral glycolipids decreased steadily as the population of myotubes increased, so that final biosynthesis was one-half that observed with myoblasts (p < 0.02). Conversely, ganglioside biosynthesis doubled during myoblast confluency (p < 0.02) and then decreased as myotubes formed. Qualitatively, L6 cells synthesized large amounts of ganglioside GM3 during all myogenic phases. The major neutral glycosphingolipid products were lactosylceramide and paragloboside (nLcOse₄Cer). Few changes in TLC autoradiographic patterns were noted during differentiation, with the exception of a slight decrease in ganglioside GM1. The results indicate that the biosynthesis of glycosphingolipids is tightly regulated during myogenesis in vitro and suggest a role for membrane gangliosides in muscle cell differentiation.Abbreviations GM1 II³NeuAc-GgOse₄Cer - GM3 II³NeuAc-GgOse²Cer - MG4 IV³NeuAc-nLcOse₄Cer - MG6 VI³NeuAc V⁴Gal-IV³GlcNAc-nLcOse⁴Cer - TLC Thin-Layer Chromatography - DMEM Dulbecco's Modified Eagles' Medium     

The ganglioside GD1α, IV3Neu5Ac,III6Neu5Ac-GgOse4Cer,is a major disialoganglioside in the highly metastatic murine lymphoreticular tumour cell line MDAY-D2

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 7.5l volumes by repeated batch fermentation. Gangliosides were isolated from 2.7×10¹¹ 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 G_M1a-pathway (G_M2, G_M1a and G_D1a) and G_M1b (IV³Neu5Ac-GgOse₄Cer) and GalNAc-G_M1b of the G_M1b-pathway, the dis8aloganglioside G_D1 (IV³Neu5Ac, III⁶Neu5Ac-GgOse₄Cer) was found in equal amounts compared to G_D1a (IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer). All gangliosides were substituted with C_24:0,24:1 and C_16:0 fatty acids, sphingosine andN-acetylneuraminic acid as the sole sialic acid. Abbreviations: FAB-MS, fast atom bombardment-mass spectrometry; GC-MS, gas chromatography-mass spectrometry; GSL(s), glycosphingolipid(s); HPLC, high performance liquid chromatography; HPTLC, high performance thin layer chromatography; Neu5Ac,N-acetylneuraminic acid; Neu5Gc,N-glycoloylneuraminic acid [57]. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations [58] and the nomenclature of Svennerholm [59]. Gangliotriaosylceramide or GgOse₃Cer, GalNAc1-4Gal1-4Glc1-1Cer; gangliotetraosylceramide or GgOse₄Cer, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer gangliopentaosylceramide or GgOse₅Cer, GalNAc1-4Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; G_M2, II³Neu5Ac-GgOse₃Cer; G_M1a, II³Neu5Ac-GgOse₄Cer; G_M1b, IV³Neu5Ac-GgOse₄Cer; GalNAc-G_M1b, IV³Neu5Ac-GgOse₅Cer; G_D1a, IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer; G_D1b, II³(Neu5Ac)₂-GgOse₄Cer; G_D1 or G_D1e, IV³Neu5Ac, III⁶Neu5AcGgOse₄Cer; G_D1e, IV³(Neu5Ac)₂-GgOse₄Cer; G_T1b, IV³Neu5Ac, II³(Neu5Ac)₂-GgOse₄Cer.     

Novel polysialogangliosides of skate brain structural determination of tetra, penta and hexasialogangliosides with a NeuAc-GalNAc linkage.

The gangliosides in the brain of a cartilaginous fish, skate (Bathyraja smirnovi), have been isolated and characterized by means of methylation analysis, antibody binding, enzymatic hydrolysis and MALDI-TOF MS. In addition to gangliosides with known structures (GM2, fucosyl-GM1, GD3, GD2, GT3 and GT2), five polysialogangliosides were isolated and characterized as having the following structures. (1) IV3NeuAc, III6NeuAc, II3NeuAc-Gg4Cer; (2) IV3NeuAc2, III6NeuAc, II3NeuAc-Gg4Cer; (3) IV3NeuAc, III6NeuAc, II3NeuAc2-Gg4Cer; (4) IV3NeuAc, III6NeuAc, II3NeuAc3-Gg4Cer; and (5) IV3NeuAc2, III6NeuAc, II3NeuAc3-Gg4Cer. These structures are 'hybrid-type' which comprise combinations of alpha-series and either a, b or c-series structures. Three gangliosides (2), (4) and (5), were novel. The main features of the ganglioside composition of skate brain were an abundance of gangliotriaosyl species, a lack of gangliotetraosyl species (except fucosyl-GM1), and an abundance of hybrid-types. These characteristics closely resemble those in shark brain which we reported previously [Nakamura, K., Tamai, Y. & Kasama, T. (1997) Neurochem. Int. 30, 593-604]. Two of the hybrid-type gangliosides (1) and (4), were examined for their neuritogenic activity toward cultured neuronal cells (Neuro-2A), and were found to have more potent activity than nonhybrid-type gangliosides such as GM1.     

Significant inhibition of hybridoma cells by exogenous application of ganglioside G M3, a possible modulator of cell growthin vitro

Gangliosides of the mouse-rat hybridoma cell line 187.1, which secretes an antibody against -light chain of mouse IgG, were isolated and structurally characterized by biochemical and immunological methods (overlay technique), and fast atom bombardment-mass spectrometry. Exclusively G _M3, substituted with C₂₄₁ and C₁₆₀ fatty acid and C₁₈₁ sphingosine, was found in this B cell derived cell line. A G _M3 (NeuGc) to G _M3(NeuAc) ratio (80 to 20), was characteristic for 187.1 cells, and absolute G _M3 amounts of about 0.3 mg 10^–9 viable cells were determined. Exogenous application of G _M3, 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 G _M3. G _M3 was applied in concentrations between 3M and 30M, giving evidence for strong inhibitory effects at 30M G _M3 and less but significant suppression after application of G _M3 concentrations lower than 20M. No cellular response was observed at the lowest concentration (3M) 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 G _M3 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 G _M3 in high density hybridoma cell cultures.Abbreviations DMB 1,2-diamino-4,5-methylenedioxybenzene - FAB-MS fast atom bombardment-mass spectrometry - GSL(s) glycosphingolipid(s) - HPLC high performance liquid chromatography - HPTLC high performance thin layer chromatography - MTT 3,(4,5 dimethylthiazol-2-yl)2,5 diphenyl tetrazolium bromide - NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - PBS phosphate buffered saline The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations (1977) and the nomenclature of Svennerholm (1963). Lactosylceramide or LacCer, Galß1–4Glcß1-1Cer; gangliotriaosylceramide or GgOse₃Cer; GalNAcß1–4Galß1–4Glcß1-1 Cer; gangliotetraosylceramide or GgOse₄Cer, Galß1–3GalNAcß1–4Galß1–4Glcß1-1Cer; G _M3(NeuAc), II³NeuAc-LacCer; G _M3(NeuGc), II³NeuGc-LacCer; G _M2(NeuGc), II³NeuGc-GgOse₃Cer; G _M1 or G _M1a, II³NeuAc-GgOse₄Cer; G _M1b, IV³NeuAc-GgOse₄Cer.     

Ganglioside alterations in YAC-1 cells cultivated in serum-supplemented and serum-free growth medium

Gangliosides of the G_M1b-pathway (G_M1b and GalNAc-G_M1b) have been found to be highly expressed by the mouse T lymphoma YAC-1 grown in serum-supplemented medium, whereas G_M2 and G_M1 (G_M1a-pathway) occurred only in low amounts [Müthing, J., Peter-Katalini, 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), G_M1b and GalNAc-G_M1b decreased and only low amounts of these gangliosides could be detected in serum-free growing cells. The expression of G_M1a was also diminished but not as strongly as that of G_M1b and GalNAc-G_M1b. 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-G_D1a 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 ofin vitro cultivated cells can be modified by the extracellular environment due to different supplementation of the basal growth medium. Abbreviations: BSA, bovine serum albumin GSL(s), glycosphingolipid(s); HPTLC, high-performance thin-layer chromatography; LDL, low density lipoprotein; NeuAc,N-acetylneuraminic acid; NeuGc,N-glycoloylneuraminic acid. The designation of the following glycosphingolipids follows IUPAC-IUB recommendations. GgOse₃Cer or gangliotriaosylceramide, GalNAc1-4Gal1-4GlcCer; GgOse₄Cer or gangliotetraosylceramide, Gal1-3GalNAc1-4Gla1-4GlcCer; GgOse₅Cer or gangliopentaosylceramide, GalNAc1-4Gal1-3GalNAc1-4Gal1-4GlcCer; GgOse₆Cer or gangliohexaosylceramide, Gal1-3GalNAc1-4Gal1-3GalNAc1-4Gal1-4GlcCer or GgOse₆Cer; II³NeuAc-GgOse₃Cer or G_M2; II³NeuAc-GgOse₄Cer or G_M1 or G_M1a; IV³NeuAc-GgOse₄Cer or G_M1b; IV³NeuAc-GgOse₅Cer or GalNAc-G_M1b; IV³NeuAc-GgOse₆Cer or Gal-GalNAc-G_M1b; IV³NeuAc, II³NeuAc-GgOse₄Cer or G_D1a; II³(NeuAc)₂-GgOse₄Cer or G_D1b; IV³NeuAc, III⁶NeuAc-GgOse₄Cer or G_D1a; IV³NeuAc, II³NeuAc-GgOse₅Cer or GalNAc-G_D1a. Enzymes: Vibrio cholerae andArthrobacter ureafaciens neuraminidase (EC 3.2.1.18).     

Glycosphingolipids in feces of germ-free rats as a source for studies of developmental changes of intestinal epithelial cell surface carbohydrates

Non-acid and acid glycosphingolipids were isolated from feces of one litter of germ-free rats from day 17 to day 51. Quantitative and qualitative changes described for small intestine of conventional rats [Bouhours D, Bouhours J-F (1981) Biochem Biophys Res Commun 99:1384–89] were also found in the feces of these germ-free rats. A decrease in lactosylceramide and sialyllactosylceramide excretion and a change fromN-acetylneuraminic acid toN-glycoloylneuraminic acid, as well as an appearance of type 1 chain blood group H-active penta- and decaglycosylceramides were observed during the weaning period. Thus the dramatic changes seen in rat intestinal glycosphingolipids postnatally seem to be primarily regulated by non-microbial factors.Abbreviations G_M3 G_M3-ganglioside, II³NeuAc-LacCer or II³NeuGc-LacCer - SPG IV³NeuAc-nLcOse₄Cer - G_M1 G_M1-ganglioside, II³NeuAc-GgOse₄Cer     

Neutral glycosphingolipids and gangliosides from spleen T lymphoblasts of genetically different inbred mouse strains

The gangliosides GM1b, GalNAc-GM1b and GD1α 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 GM1b-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 GM1b and GalNAc-GM1b, 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, GM1b and GalNAc-GM1b predominantly carry N-acetylneuraminic acid, whereas choleragenoid receptors GM1a and Gal-GalNAc-GM1b, 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-GM1b 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 GM1b-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 GM1b-type gangliosides a functional association with T cell activation and leukocyte mediated reactions. Abbreviations: ConA, concanavalin A; GSL(s), glycosphingolipid(s); HPTLC, high-performance thin-layer chromatography; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic acid. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations (1977) [48] and the ganglioside nomenclature system of Svennerholm [49] for GM1a-type gangliosides. Glucosylceramide or GlcCer, Glcβ1-1Cer; lactosylceramide or LacCer, Galβ1-4Glcβ1-1Cer; gangliotriaosylceramide or GgOse3Cer or Gg3, GalNAcβ1-4Galβ1-4Glcβ1-1Cer; gangliotetraosylceramide or GgOse4Cer or Gg4, Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1Cer; gangliopentaosylceramide or GgOse5Cer, GalNAcβ1-4Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1Cer; gangliohexaosylceramide or GgOse6Cer, Galβ1-3GalNAcβ1-4Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1Cer. GM3, II3NeuAc-LacCer; GM1 or GM1a, II3NeuAc-GgOse4Cer; GM1b, IV3NeuAc-GgOse4Cer; GalNAc-GM1b, IV3NeuAc-GgOse5Cer; GD1a, IV3NeuAc, II3NeuAc-GgOse4Cer; GD1b, II3(NeuAc)2-GgOse4Cer; GD1c, IV3(NeuAc)2-GgOse4Cer; GD1α, IV3NeuAc, III6NeuAc-GgOse4Cer. Only NeuAc-substituted gangliosides are presented in this list of abbreviations This revised version was published online in November 2006 with corrections to the Cover Date.     

Expression of neutral glycosphingolipids and gangliosides in human skeletal and heart muscle determined by indirect immunofluorescence staining

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 neutral glycosphingolipids lactosylceramide, globoside, Forssman glycosphingolipid, gangliotetraosylceramide, lacto-N-neotetraosylceramide and against the gangliosides G_M3(Neu5Ac) and G_M1(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 glycosphinogolipid. 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 G_M3 and G_M1 correlated to the fact that G_M3 is the major ganglioside in skeletal muscle whereas G_M1 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. G_M3 and G_M1 were detected with almost identical intensity. All glycosphingolipids were present in plasma membranes as well as at the intracellular level. Abbreviations used: BSA, bovine serum albumin; DAPI, 4,6-diamidine-2-phenylindole-dihydrochloride; DTAF, fluorescein isothiocyanate derivative; GSL(s), glycosphingolipid(s); Neu5Ac,N-acetylneuraminic acid [50]; PBS, phosphate buffered saline. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations [51] and the nomenclature of Svennerholm [52]. Lactosylceramide or LacCer, Gal1-4Glc1-1Cer; gangliotriaosylceramide or GgOse₃Cer, GalNAc1-4Gal1-4Glc1-1Cer; globotriaosylceramide or GbOse₃Cer, Gall-4Gall-4Glcl-1Cer; gangliotetraosylceramide or GgOse₄Cer, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; globotetraosylceramide or GbOse₄Cer, GalNAc1-3Gal1-4Gal1-4Glc1-1Cer; lacto-N-neotetraosylceramide or nLcOse₄Cer, Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer; Forssman GSL or GbOse₃Cer, GalNAc1-3GalNAc1-3Gal1-4Gal1-4Gle1-1Cer; G_M3, II³Neu5Ac-LacCer; G_M2, II³Neu5Ac-GgOse₃Cer; G_M1, II³Neu5Ac-GgOse₄Cer; G_D3 II³(Neu5Ac)₂-LacCer; G_D2, II³(Neu5Ac)₂-GgOse₃Cer; G_D1a, IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer; G_D1b, II³(Neu5Ac)₂-GgOse₄Cer.     

THE SITE OF SYNTHESIS OF GANGLIOSIDES IN THE CHICK OPTIC SYSTEM

Abstract– In the retinas of 1-day-old chickens that received an intraocular injection of N-[³H]acetylmannosamine the labelling of N-acetylneuraminic acid and CMP-N-acetylneuraminic acid increased for at least 8 h and that of gangliosides for at least 24 h after injection. In the optic tectum contralateral to the injected eye at 8 h after the intraocular injection, the labelling of gangliosides exceeded the labelling of gangliosides in the ipsilateral tectum by approx 20-fold. In the contralateral tectum the highest concentration of labelled gangliosides was in subfractions enriched in synaptosomes and synaptic plasma membranes. No significant contralateral ipsilateral differences were found in the acid soluble substances of the tectum. In the optic tectum, labelled gangliosides appeared earlier in the neuronal perikarya than in synaptosomes when the injection was intracranial. Conversely, when the injection was intraocular the labelling appeared earlier in the synaptosomes than in the neuronal perikarya. The radioactivity pattern of the optic tectum gangliosides resembled the pattern of retina gangliosides when N-[³H]acetylmannosamine was injected intraocularly, but when N-[³H]acetylmannosamine was given intracerebrally the radioactivity pattern resembled that of optic tectum gangliosides. Intraocular injection of colchicine or vinblastine did not affect the labelling of retinal gangliosides from N-[³H]acetylmannosamine injected into the same eye but prevented the appearance of labelled gangliosides in the optic tectum. In vitro the ganglioside glycosylating activity of optic tectum synaptosomes and synaptic plasma membranes was between 6 and 10-fold lower than that found in the optic tectum neuronal perikarya. These findings support the notion that the main subcellular site of synthesis of neuronal gangliosides is in the neuronal perikarya, from which they are translocated to the nerve endings.