Large scale biosynthesis of ganglioside analogues by RERF-LC-AI cells cultured in HYPERFlask

The efficient production of ganglioside analogues was accomplished using RERF-LC-AI cells cultured in HYPERFlask (High Yield PERformance Flask). Eight kinds of ganglioside analogues (GM3, GM2, sialylparagloboside, GD3, di-sialylated lacto-N-tetraose, and another three kinds of analogues with intricate structures) were synthesized by the saccharide primer method using lung squamous-cell carcinoma line RERF-LC-AI and 12-azidododecyl β-lactoside primer. The yield for each analogue obtained using HYPERFlask was higher than yields obtained from 100-mm dishes.     

Easy Production of a Glycolipid Analogue Using Animal Cells in Culture

A glycolipid analogue, GM4‐type ganglioside, was obtained by a combination of chemical synthesis and biosynthetic processes in animal cells with dodecyl β‐D ‐galactoside (Gal C12) as primer. The primer was conveniently prepared in two steps: glycosylation, followed by deacetylation. The primer was introduced to mouse melanoma B16 cells to serve as substrate for cellular, enzyme‐catalyzed glycosylation. Incubation of the cells in the presence of the primer resulted in sialylation of the galactose residue to afford a GM4 analogue that was released from the cells to the culture medium. The strategy of preparation of the GM4 analogue described in this study is a viable alternative to the existing methods. The saccharide‐primer method is fast, convenient, not requiring expensive enzymes and glycosyl donors, and highly stereoselective.     

Incorporation of ganglioside analogues into fibroblast cell membranes. A spin-label study

The uptake of ganglioside analogues by a permanent mouse fibroblast cell line has been studied by radio-tracer techniques and ESR spectroscopy with 3H- and nitroxide-labeled compounds. Analogues of GM1, GM2, and GM3 monosialogangliosides and of GD1a and GD3 disialogangliosides were synthesized. The spin-label group was situated on the 5-, 9-, or 13-carbon atom of the C18 fatty acid chain, and the 3H label was in the carbohydrate moiety. Part of the ganglioside associated with the cells could be removed by trypsin treatment and was shown to consist of ganglioside micelles attached to the cell surface. The trypsin-resistant component displayed characteristic anisotropic ESR spectra which closely resembled those of the same spin-labeled analogues at low dilution in liposomes prepared from the extracted cell lipids. The flexibility gradient, polarity profile, and temperature dependence displayed by the spectra were similar to those found for fluid phospholipid bilayer model membranes, and the high effective order parameters suggested a location in the cell plasma membrane. Similar results were obtained for all the different ganglioside analogues, indicating a common anchoring region in the hydrophobic interior of the membrane. Under the incubation conditions used the amount of trypsin-resistant ganglioside analogue taken up by the cells was about 15 nmol/mg of cellular protein, irrespective of the nature of the oligosaccharide moiety. By use of the natural ganglioside [3H]GM3, the trypsin-resistant uptake was about 19 nmol/mg of cellular protein. Although these amounts are quite similar, the uptake kinetics differed between the true ganglioside GM3 and the ganglioside analogues.     

Molecular dynamics study of the conformations of glycosidic linkages in sialic acid modified ganglioside GM3 analogues

The objective of the present study is to model the analogues of monosialoganglioside (GM3) by making modifications in its sialic acid residue with different substitutions in aqueous environment and to determine their structural stability based upon computational molecular dynamics. Molecular mechanics and molecular dynamics investigation was carried out to study the conformational preferences of the analogues of GM3. Dynamic simulations were carried out on the analogues of GM3 varying in the substituents at C-1, C-4, C-5, C-8 and C-9 positions of their sialic acid or Neuraminic acid (NeuAc) residue. The analogues are soaked in a periodic box of TIP3P water as solvent and subjected to a 10 ns molecular dynamics (MD) simulation using AMBER ff03 and gaff force fields with 30 ps equilibration. The analogue of GM3 with 9-N-succNeuAc (analogue5, C9 substitution) was observed to have the lowest energy of ?6112.5 kcal/mol. Graphical analysis made on the MD trajectory reveals the direct and water mediated hydrogen bonds existing in these sialic acid analogues. The preferable conformations for glycosidic linkages of GM3 analogues found in different minimum energy regions in the conformational maps were identified. This study sheds light on the conformational preferences of GM3 analogues which may be essential for the design of GM3 analogues as inhibitors for different ganglioside specific pathogenic proteins such as bacterial toxins, influenza toxins and neuraminidases.     

Comparison of the glycolipid-binding specificities of cholera toxin and porcineEscherichia coli heat-labile enterotoxin: identification of a receptor-active non-ganglioside glycolipid for the heat-labile toxin in infant rabbit small intestine

The binding specificities of cholera toxin andEscherichia coli heat-labile enterotoxin were investigated by binding of¹²⁵I-labelled toxins to reference glycosphingolipids separated on thin-layer chromatograms and coated in microtitre wells. The binding of cholera toxin was restricted to the GM1 ganglioside. The heat-labile toxin showed the highest affinity for GM1 but also bound, though less strongly, to the GM2, GD2 and GD1b gangliosides and to the non-acid glycosphingolipids gangliotetraosylceramide and lactoneotetraosylceramide. The infant rabbit small intestine, a model system for diarrhoea induced by the toxins, was shown to contain two receptor-active glycosphingolipids for the heat-labile toxin, GM1 ganglioside and lactoneotetraosylceramide, whereas only the GM1 ganglioside was receptor-active for cholera toxin. Preliminary evidence was obtained, indicating that epithelial cells of human small intestine also contain lactoneotetraosylceramide and similar sequences. By computer-based molecular modelling, lactoneotetraosylceramide was docked into the active site of the heat-labile toxin, using the known crystal structure of the toxin in complex with lactose. Interactions which may explain the relatively high toxin affinity for this receptor were found.Abbreviations CT cholera toxin - CT-B B-subunits of cholera toxin - LT Escherichia coli heat-labile enterotoxin - hLT humanEscherichia coli heat-labile enterotoxin - pLT porcineEscherichia coli heat-labile enterotoxin - EI electron ionization     

Changes of the ganglioside pattern and content in human fibroblasts by high density cell population subculture progression

In this study we show that the ganglioside content and pattern of human skin fibroblasts change along the process of cell subculture progression by varying the cell density.GM3, GD3 and GD1a were components of the total cell ganglioside mixtures extracted from cells, but GD1a was in all the extracts a minor component or very scant. Other gangliosides present in traces were not characterised. The fibroblast ganglioside content of 52 pools of cells obtained from 5 different cell lines cultured at variable cell density ranged from 2.0 to 13.1 nmoles per mg of cell protein. The molar ratio between GM3 and GD3 varied from 418 to 0.6 in the ganglioside mixtures, as determined by densitometric quantitative analysis after thin layer chromatographic separation.Both the ganglioside content and the GM3/GD3 molar ratio were constant along several passages of subculture progression performed by plating cells collected at confluence. Instead, when the subculture progression was performed by plating cells collected at a few days after reaching confluence, a progressive increase of the ganglioside content was observed. GD3 increased proportionally more than GM3 so that a progressive decrease of the ratio between GM3 and GD3 was observed. In some experiments, GD3 was very scant at the beginning of the progression, while it was near 30% after 5 passages under these conditions. The progressive increase of GD3 along the high density cell population subculture progression was associated to a moderate increase of the mRNA GD3 synthase. Published in 2003.     

Absence of ganglioside GM1 in astroglial cells from newborn rat brain

An immunohistochemical method utilizing anti-ganglioside GM1 antiserum combined with the peroxidase-antiperoxidase technique was applied to a mixed cell population in primary cultures of newborn rat brain. Ganglioside GM1 was demonstrated to be present in neurons and oligodendroglia, but was absent in astroglia. This demonstration was confirmed using a newly developed biotinylated choleragen-avidin-peroxidase procedure. Primary cultures from newborn rat brain cells that had been subjected to a single treatment with trypsin (first passage) and then cultured for 14 days were predominately (95%) composed of astrocytes that stained positively for glial fibrillary acidic protein but were negative for GM1 ganglioside. This preparation contained only 0.34 nmol ganglioside NeuNAc per mg protein compared to 23.9 nmol gangliosidic NeuNAc/mg protein for a five day culture of newborn rat brain mixed cell culture that had not been subjected to passage. Prolongation of culture time from 5 to 21 days in the latter preparation reduced the ganglioside NeuNAc content to 4.9 nmol gangliosidic NeuNAc/mg protein as the proportion of astrocytes in the culture increased. Ganglioside GM1 could not be detected by TLC analysis of the lipid extract obtained from the “pure” astrocyte culture, although small amounts of GM3 and some polysialogangliosides were detected. About half of the label incorporated upon 24 h incubation of astrocytes in the presence of $\text{N-[}{}^3\text{H]acetylmannosammine}$ appeared in ganglioside GM3. It is concluded that astrocytes in mixed cell primary cultures from newborn rat brain, as well as astrocytes in astroglial preparations derived from such cultures, do not contain ganglioside GM1.     

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     

Ganglioside from eel serum high density lipoprotein (HDL) and its role as a ligand for HDL binding protein

First, we attempted to isolate glycosphingolipids from eel serum HDL. A single ganglioside containing N-acetylneuraminic acid (NeuAc), which is positive with resorcinol and orcinol reactions, was purified. The mobilities of the purified ganglioside and its lyso-form on high performance TLC were similar as those of authentic GM4 and its lyso-form, respectively. The mass of the purified ganglioside was determined by TOF mass spectrometer, and the mass of its oligosaccharide was the same as that of authentic GM4 from human brain consisting of disaccharide of NeuAc and galactose. The ganglioside from eel HDL was not hydrolyzed by recombinant endoglycoceramidase II, which cannot hydrolyze between galactose and ceramide of gangliosides, but hydrolyzes between glucose and ceramide. We concluded from these results that the ganglioside purified from eel serum HDL is GM4. Second, we investigated the effects of the ganglioside on binding of HDL labeled with fluorescein isothiocyanate (FITC-HDL) to cultured eel hepatocytes and on FITC-HDL ligand blotting by using plasma membrane proteins of the hepatocytes. Stimulatory effect of GM4 on FITC-HDL binding to the hepatocytes and FITC-HDL ligand blotting suggests strongly that GM4 is a ligand for HDL binding protein of eel hepatocytes.     

Characterization of high-affinity binding between gangliosides and amyloid beta-protein

The binding specificities of amyloid beta-protein (A beta) such as A beta 1-40, A beta 1-42, A beta 40-1, A beta 1-38, A beta 25-35, and amyloid beta precursor protein (beta-APP) analogues for different glycosphingolipids were determined by surface plasmon resonance (SPR) using a liposome capture method. A beta 1-42, A beta 1-40, A beta 40-1, and A beta 1-38, but not A beta 25-35, bound to GM1 ganglioside in the following rank order: A beta 1-42 > A beta 40-1 > A beta 1-40 > A beta 1-38. The beta-APP analogues bound to GM1 ganglioside with a relatively lower affinity. Aged derivatives of A beta were found to have higher affinity to GM1 ganglioside than fresh or soluble derivatives. A beta 1-40 bound to a number of gangliosides with the following order of binding strength: GQ1b alpha > GT1a alpha > GQ1b > GT1b > GD3 > GD1a = GD1b > LM1 > GM1 > GM2 = GM3 > GM4. Neutral glycosphingolipids had a lower affinity for A beta 1-40 than gangliosides with the following order of binding strength: Gb4 > asialo-GM1 (GA1) > Gb3 > asialo-GM2 (GA2) = LacCer. The results seem to indicate that an alpha2,3NeuAc residue on the neutral oligosaccharide core is required for binding. In addition, the alpha2-6NeuAc residue linked to GalNAc contributes significantly to binding affinity for A beta.     

Efficient conversion from polysialogangliosides to monosialotetrahexosylganglioside using Oerskovia xanthineolytica YZ-2

A new sialidase-producing strain isolated from soil was identified as Oerskovia xanthineolytica YZ-2. Sialidase was produced when Oerskovia xanthineolytica YZ-2 was exposed to polysialogangliosides. The sialidase of Oerskovia xanthineolytica YZ-2 hydrolyzed sialic acid linkages in polysialogangliosides, and released monosialotetrahexosylganglioside (GM1). The sialidase had the capability of product specificity because it did not attack the sialic acid linkage in GM1. Therefore, Oerskovia xanthineolytica YZ-2 was used for GM1 production from polysialogangliosides. In flasks cultivation phase, it was proved that Oerskovia xanthineolytica YZ-2 could convert polysialogangliosides to GM1 efficiently. Scaling-up the bioprocess with 8% crude ganglioside, polysialogangliosides was converted to GM1 by Oerskovia xanthineolytica YZ-2 in 30 L bioreactor after 18 h. The relative content of GM1 increased from 16.3% in crude ganglioside to 83.7% after Oerskovia xanthineolytica YZ-2 conversion. Therefore, a simple, large-scale conversion process for GM1 production from polysialogangliosides was achieved using Oerskovia xanthineolytica YZ-2 as a biocatalyst.     

Synthesis of novel ganglioside GM4 analogues containing N-deacetylated and lactamized sialic acid: probes for searching new ligand structures for human L-selectin

Novel ganglioside GM4 analogues, which contain N-deacetylated or lactamized sialic acid instead of usual N-acetylneuraminic acid, were synthesized in a highly efficient manner. (Methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trifluoroacetamido-D-glycero-alpha-D-galacto-2-nonulopyranosylonate)-(2-->3)-4,6-di-O-acetyl-2-O-benzoyl-D-galactopyranosyl trichloroacetimidate was coupled with 2-(tetradecyl)hexadecanol to give the desired beta-glycoside in high yield. Successive O- and N-deacylation, and saponification of the methyl ester group afforded the N-deacetylated sialyl derivative that was converted by treatment with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in Me2SO into the lactamized sialic acid-containing ganglioside GM4 analogue.     

GANGLIOSIDE1−COMPOSITION OF BRAIN IN TAY-SACHS DISEASE: INCREASED AMOUNTS OF GD2 AND N-ACETYL-β-D-GALACTOSAMINYL GD1a GANGLIOSIDE

Abstract— The ganglioside composition of the brain of a patient with Tay-Sachs disease (TS-brain) was determined by a newly developed ganglioside-mapping procedure and compared with that of an age-matched control brain. GM2 ganglioside was the predominant component in TS-brain and the following gangliosides were also found, GM1, GD1a, GD1b and GT1 (major gangliosides in normal brain), and GM3, GD3, GD2 and GD1a-GAN (minor or undetectable components of normal brain). Individual gangliosides were isolated by column chromatography using a combination of DEAE-Sepharose, Iatrobeads and Silica Gel 60 and their structures were confirmed by comparing them with authentic standards using TLC, analysing their carbohydrate compositions by gas-liquid chromatography and cleaving them sequentially with glycosidases. The amounts of individual components were measured by quantitative densitometric scanning of the thin-layer plates. As a reflection of myelin breakdown, no sialosylgalactosyl ceramide was detectable in TS-brain. Although the total amounts of all gangliosides except GM2 in TS-brain were low, there were normal molar ratios of the main gangliosides in normal brain, that is, GM1, GD1a, GD1b and GT1. In comparison with the amount of GDla ganglioside, the amounts of GM2, GD2 and GD1a-GAN, which contain N-acetylgalactosamine as a terminal carbohydrate residue, were all elevated in TS-brain. The long chain bases of individual gangliosides contained both C-18 and C-20 sphingosine in different ratios and the ratio of C-20 to C-18 increased in the gangliosides in the order: GM2 < GM1 < GD1a < GD1a-GAN < GD1b < GT1 in both normal brain and TS-brain. In contrast, GD2 and GD3 gangliosides consisted mainly of C-18 sphingosine. The C-20 to C-18 ratios of individual gangliosides in the TS-brain were lower than those of age-matched control brain. Hexosaminidase from Turbo cornutus showed the same specific activity and K_m value in catalysing the cleavage of terminal N-acetylgalactosaminyl residues from GM2, GD2 and GD1a-GAN, suggesting that the brain gangliosides that increase in Tay-Sachs disease may be cleaved by the same enzyme.     

Binding specificities of heat-labile enterotoxins isolated from porcine and human enterotoxigenic Escherichia coli for different gangliosides

The binding specificities of heat-labile enterotoxins (LTp and LTh) isolated from porcine and human enterotoxigenic Escherichia coli on human erythrocytes were studied by competitive binding assays using different gangliosides as inhibitors. The binding of 125I-labeled LTp to neuraminidase-treated human type A erythrocytes was most effectively inhibited by ganglioside GM1. Ganglioside GM1 was 11 and 105 times more potent than gangliosides GD1b and GM2, respectively. Gangliosides GD1a, GT1b, and GM3 were much less potent. Similar results were also obtained in competitive binding assays with the 125I-labeled B subunit of LTh and neuraminidase-treated human type B erythrocytes, and in those with 3H-labeled ganglioside GM1 and LTp-coupled Sepharose 4B. The binding of 3H-labeled ganglioside GM1 to LTp was not effectively inhibited by galactose-beta(1----3)N-acetyl-D-galactosamine at the highest concentration used. These findings suggest that the combining sites of LTp and LTh may be specific for at least the galactose-N-acetyl-D-galactosamine-galactose (N-acetyl-neuraminic acid) portion of ganglioside GM1.     

Evaluation of the efficiency of an assay procedure for gangliosides in human serum

An efficiency assessment of a ganglioside assay procedure was carried out on human serum gangliosides from healthy subjects of different sex and age. The analysis of the gangliosides, extracted with chloroform/methanol and purified by lipid partitioning, ion exchange column chromatographic separation and desalting procedures as described by Sennet al. (1989)Eur J Biochem 181: 657–62, was performed by HPTLC followed by densitometric quantification. The yield of the procedure, expressed as radioactivity recovery, was determined by adding GM3 ganglioside, tritium labelled at the sialic acid acetyl group and at the C3 position of sphingosine, to the lyophilized serum or by associating it with the serum lipoproteins. In spite of the fact that the extraction and purification procedures were performed exactly as described we found the radioactivity recovery to be variable (25–50%) and much lower than that proposed. Much of the radioactivity was found in the organic phase after lipid partitioning, whilst all the ganglioside purification steps led to some further loss. After the introduction of some modifications to the procedure the recovery improved, reaching 67–79%.The analyses on 33 samples of 5 ml showed a human serum ganglioside content of about 10 nmol ml^–1 (as corrected for the recovery), and confirmed that GM3 ganglioside is the main component of the total serum ganglioside mixture. Abbreviations: Ganglioside nomenclature is in accordance with Svennerholm (1980) [37] and the IUPAC-IUB Recommendations (1977, 1982) [38]. GM3, II³Neu5AcLacCer, -Neu5Ac-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer; Cer, ceramide; Neu5Ac,N-acetyl-neuraminic acid;erythro-GM3, GM3 containingerythro-sphingosine;threo-GM3, GM3 containingthreo-sphingosine;erythro-C18 sphingosine, (2s,3R,4E)-2-amino-1,3-dihydroxy-octadecene;erythro-C20 sphingosine, (2S,3R,4E)-2-amino-1,3-dihydroxy-eicosene;threo-C18 sphingosine, (2S,3S,4E)-2-amino-1,3-dihydroxy-octadecene;threo-C20 sphingosine, (2S,3S,4E)-2-amino-1,3-dihydroxy-eicosene; DDQ, dichlorodicyano-benzoquinone.     

Studies on the turnover and subcellular localization of membrane gangliosides in cultured neuroblastoma cells

To compare the subcellular distribution of endogenously synthesized and exogenous gangliosides, cultured murine neuroblastoma cells (N1E-115) were incubated in suspension for 22h in the presence ofd-[1-³H]galactose or [³H]GM1 ganglioside, transferred to culture medium containing no radioisotope for periods of up to 72 hr, and then subjected to subcellular fractionation and analysis of lipidsialic acid and radiolabeled ganglioside levels. The results indicated that GM2 and GM3 were the principal gangliosides in the cells with only traces of GM1 and small amounts of disialogangliosides present. About 50% of the endogenously synthesized radiolabelled ganglioside in the four major subcellular membrane fractions studied was recovered from plasma membrane and only 10–15% from the crude mitochondrial membrane fraction. In contrast, 45% of the exogenous [³H]GM1 taken up into the same subcellular membrane fractions was recovered from the crude mitochondrial fraction; less than 15% was localized in the plasma membrane fraction. The results are similar to those obtained from previously reported studies on membrane phospholipid turnover. They suggest that exogenous GM1 ganglioside, like exogenous phosphatidylcholine, does not intermix freely with any quantitatively major pool of endogenous membrane lipid.     

Synthesis of radioactive and photoactivable ganglioside derivatives for the study of ganglioside-protein interactions

The procedures for the preparation of radioactive and photoactivable ganglioside derivatives have been continuously developed from 1989, when for the first time the synthesis of photoactivable tritium labeled GM1 ganglioside was presented. We described previously the synthesis of photoactivable derivatives of GM3 and GM1 gangliosides, tritium-labeled at acetyl group of sugar units, and of photoactivable GM1 and GD1b gangliosides, tritium-labeled at position 6 of the external galactose. These procedures are reviewed in detail in the present paper. The use of these ganglioside derivatives to study the ganglioside-protein interactions and to identify proteins that specifically interact with gangliosides (including GPI-anchored proteins of the outer membrane leaflet, proteins anchored to the cytoplasmic side of the plasma membrane through a fatty acyl chain, transmembrane proteins, and soluble cytoplasmic proteins) is discussed. Published in 2004. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterization of Sialyltransferase-IV Activity and Its Involvement in the c-Pathway of Brain Ganglioside Metabolism

Abstract: To characterize the sialyltransferase-IV activity in brain tissues, the activities of GM1b-, GD1a-, GT1b-, and GQ1c-synthases in adult cichlid fish and rat brains were examined using GA1, GM1, GD1b, or a cod brain ganglioside mixture as the substrate. The GD1a-synthase activity in the total membrane fraction from cichlid fish brain required divalent cations such as Mg²⁺ or Mn²⁺ and Triton CF-54 for its full activity. The V_max value was 1,340 pmol/mg of protein/h at an optimal pH of 6.5, whereas the apparent K_m values for CMP-sialic acid and GM1 were 172 and 78 µM, respectively. Cichlid fish and rat brains also contained GM1b-, GT1b-, and GQ1c-synthase activities. The ratio of GM1b-, GD1a-, and GT1b-synthase activities in fish brain was 1.00:0.89:1.13, respectively, and in rat brain 1.00:0.60:0.63. Incubation of fish brain membranes with a cod brain ganglioside mixture, which contains GT1c, and [³H]CMP-sialic acid produced radiolabeled GQ1c. It is interesting that the adult rat brain also contains an appreciable level of GQ1c-synthase activity despite its very low concentrations of c-series gangliosides. The GD1a- or GQ1c-synthase activity in fish and rat brain was inhibited specifically by coincubation with the glycolipids that serve as the substrates for other sialyltransferase-IV reactions. Thus, the GD1a-synthase activity was inhibited by GA1 and GD1b, but not by LacCer, GM3, or GD3. In a similar manner, the synthesis of GQ1c was suppressed by GA1, GM1, and GD1b, but not by LacCer, GM3, or GD3. The GD1a-synthase activity directed toward endogenous GM1 was inhibited by GA1 or GT1b, whereas the endogenous GT1b-synthase activity was suppressed by GA1 or GM1. GA1, GM1, and GD1b did not affect the endogenous GM3- and GD3-synthase activities. These results clearly demonstrate that sialyltransferase-IV in brain tissues catalyzes the reaction for GQ1c synthesis in the c-pathway as well as the corresponding steps in the asialo-, a-, and b-pathway in ganglioside biosynthesis.     

Interactions between gangliosides and proteins in the exoplasmic leaflet of neuronal plasma membranes: A study performed with a tritium-labeled GM1 derivative containing a photoactivable group linked to the oligosaccharide chain

Interactions between gangliosides and proteins at the exoplasmic surface of the sphingolipid-enriched membrane domains can be studied by ganglioside photolabeling combined with cell surface biotin labeling. In the present paper, we report on the results obtained using a novel radioactive photoactivable derivative of GM1 ganglioside, carrying the photoactivable nitrophenylazide group at the external galactose.After cell photolabeling with the radioactive photoactivable derivative of GM1 and cell surface biotin labeling, sphingolipid-enriched domains were prepared from rat cerebellar neurons differentiated in culture and further purified by immunoprecipitation with streptavidin-coupled beads. Among proteins belonging to the sphingolipid-enriched domains that were biotin labeled, thus bearing an exoplasmic domain, a few were also cross-linked by the radioactive photoactivable ganglioside. In particular, two protein bands showing apparent molecular mass of 135 and 35 kDa were intensely photolabeled. The 135 kDa protein was immunologically identified as the GPI-anchored neural cell adhesion molecule TAG-1. These data suggest that hydrophilic interaction between the exoplasmic domains of the protein and the ganglioside sialooligosaccharide chain could exist. Published in 2004.     

Design and efficient synthesis of novel GM2 analogues with respect to the elucidation of the function of GM2 activator

To elucidate the mechanism underlying the hydrolysis of the GalNAcβ1→4Gal linkage in ganglioside GM2 [GalNAcβ1→4(NeuAcα2→3)Galβ1→4Glcβ1→1′ Cer] by β-hexosaminidase A (Hex A) with GM2 activator protein, we designed and synthesized two kinds of GM2 linkage analogues—6′-NeuAc-GM2 and α-GalNAc-GM2. In this paper, the efficient and systematic synthesis of these GM2 analogues was described. The highlight of our synthesis process is that the key intermediates, newly developed sialyllactose derivatives, were efficiently prepared in sufficient quantities; these derivatives directly served as highly reactive glycosyl acceptors and coupled with GalNTroc donors to furnish the assembly of GM2 tetrasaccharides in large quantities.