Tissue-specific expression of GM3(NeuGc) and GD3(NeuGc) in epithelial cells of the small intestine of strains of inbred rats. Absence of NeuGc in intestine and presence in kidney gangliosides of brown Norway and spontaneously hypertensive rats

The ganglioside composition of the epithelial cells of the small intestine was investigated in 15 strains of inbred rats. Most of these strains had GM3 as the only detectable ganglioside. In addition to GM3, small amounts of GD3 were found in four strains, AVN, BN, DA, and LE. The fatty acid content of the ceramide portion was composed of a large, although variable, percentage of 2-hydroxy fatty acids. The sphingoid base was always C18-4D-hydroxysphinganine. The highly prominent sialic acid was N-glycolylneuraminic acid (NeuGc) in most strains. However in two strains, Brown Norway (BN) and spontaneously hypertensive rats (SHR), NeuAc was the only sialic acid of the gangliosides of the intestinal epithelium. The analysis of the ganglioside composition of the epithelium of the small intestine of the first generation hybrids of SHR with DA and BN, respectively, demonstrated that the expressions of GM3 (NeuGc) and GD3 were genetically transmitted as dominant traits and that BN and SHR were likely to carry the same deficient gene that led to the expression of GM3(NeuAc) instead of GM3(NeuGc) in the small intestine. For comparison, the sialic acid composition of kidney gangliosides was analyzed in some strains. 21-23% of the kidney gangliosides was GM3(NeuGc) in all tested strains, including BN and SHR. Therefore, the ganglioside composition of the intestinal epithelium could vary in the rat species, and the defect of N-glycolylneuraminic acid was not only strain-specific but also occurred in a tissue-specific way among strains of inbred rats.     

Sialylation of glycoprotein oligosaccharides with N-acetyl-, N-glycolyl-, and N-O-diacetylneuraminic acids

Four common sialic acids (Sia), NeuAc, N-glycolyl-neuraminic acid (NeuGc), 4-O-acetyl-N-acetylneuraminic acid (4-O-Ac-NeuAc), and 9-O-Ac-NeuAc were examined for activation to their corresponding CMP-sialic acid conjugates and subsequently for their transfer to glycoprotein oligosaccharides by purified mammalian sialyltransferases. CMP-sialic acid synthetases from calf brain and from bovine and equine submaxillary glands were found to convert NeuAc, NeuGc, and 9-O-Ac-NeuAc to their corresponding CMP-sailic acids. In contrast, no conversion of 4-O-Ac-NeuAc to CMP-4-O-Ac-NeuAc was observed for any of the three synthetases examined. A new procedure for the preparation of CMP-9-O-Ac-NeuAc, CMP-NeuGc, and CMP-NeuAc in high yield and purity was developed, using the calf brain CMP-sialic acid synthetase. Each of these derivatives was tested as donor substrates for six mammalian sialyltransferases purified from porcine, rat, and bovine tissues, including a bovine GalNAc alpha 2,6 sialyltransferase whose purification is described in this report. The sialyltransferases examined represent those which form the Sia alpha 2,6Gal beta 1,4-GlcNAc-, Sia alpha 2,3Gal beta 1,3(4)GlcNAc-, Sia alpha 2,3Gal beta 1,3-GalNAc- and Sia alpha 2,6GalNAc- sequences found on N-linked and O-linked oligosaccharides of glycoproteins. CMP-NeuAc and CMP-NeuGc were equally good donor substrates for all six sialyltransferases. However, transfer of 9-O-Ac-NeuAc from CMP-9-O-Ac-NeuAc varied from only 10% to nearly 70% that of the transfer of NeuAc from CMP-NeuAc. Results are viewed to define the relative roles of direct transfer of these sialic acids and modification of glycosidically bound NeuAc in glycoproteins.     

Genetic regulation of GM4(NeuAc) expression in mouse erythrocytes

The polymorphic expression of GM4(NeuAc), GM3(NeuGc), GM2(NeuGc), and GM1(NeuGc) was found in erythrocytes of inbred strains of mice [Nakamura, K. et al. (1988) J. Biochem. 103, 201-208]. In this paper, we report the results of genetic analysis of the expression of GM4(NeuAc) and GM2(NeuGc). Ganglioside analysis of the progeny obtained on mating between BALB/c mice [GM4 (+)] and WHT/Ht or C57BL/6 mice [both GM4 (-)] indicated that the expression of GM4(NeuAc) is an autosomal dominant trait, and that WHT/Ht and C57BL/6 mice carry a defect on a single autosomal gene. We named this gene Gsl-4. On quantitative determination of galactosylceramide (GalCer), which is the biosynthetic precursor of GM4(NeuAc), the content of GalCer was found to be quite low in WHT/Ht erythrocytes, compared with in BALB/c erythrocytes. On analysis of GM4(NeuAc) and GalCer in 92 backcross mice produced on mating between BALB/c and WHT/Ht mice, it was found that 45 GM4(+) mice apparently expressed a detectable amount of GalCer and that 47 GM4(-) mice expressed an almost undetectable amount of GalCer. These results suggest that Gsl-4 controls the expression of GM4(NeuAc) by regulating the content of GalCer. Linkage analysis of Gsl-4 and the gene controlling GM2(NeuGc) in erythrocytes indicated that the two genes are not genetically linked. Comparison of the ganglioside expression in liver and erythrocytes of the same backcross mice suggested that the gene controlling GM2(NeuGc) expression in the liver (Ggm-2) is also responsible for the expression of GM2(NeuGc) in erythrocytes.     

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-)     

Participation of cytochrome b5 in CMP-N-acetylneuraminic acid hydroxylation in mouse liver cytosol

The activity of CMP-N-acetylneuraminic acid hydroxylase, that converts CMP-N-acetylneuraminic acid (CMP-NeuAc) to CPM-N-glycolylneuraminic acid (CMP-NeuGc), in mouse liver was determined by a newly developed HPLC method using non-radioactive CMP-NeuAc as a substrate. The activity was detected in the cytosol fraction but not in the microsomal fraction. Either NADH or NADPH was used as an electron donor by the cytosol enzyme, but NADH was much more efficiently used than NADPH. An antibody against cytochrome b5 markedly reduced the CMP-NeuAc hydroxylase activity when added to incubation mixture containing either NADH or NADPH as an electron donor. These data led us to postulate the following electron transport system, which is involved in the CMP-NeuAc hydroxylation in mouse liver cytosol: (formula; see text) where X, Y, and Z are components supposedly involved.     

Liver gangliosides of various animals ranging from fish to mammalian species

Liver gangliosides of different animal species were analyzed. Bony fish liver contained a major ganglioside that migrated faster than GM3 on thin-layer chromatography (TLC). This ganglioside was identified to be GM4 (NeuAc) by methods including product analysis after sialidase treatment and negative-ion electrospray ionization (ESI)-mass spectrometry (MS). The presence of GM4 (NeuGc) in fish liver was also demonstrated. The main ganglioside band of bovine liver consisted of two different molecular species, i.e. GD1a (NeuAc/NeuAc) and GD1a (NeuAc/NeuGc). Major gangliosides of liver tissue exhibited a distinct phylogenetic profile; GM4 was expressed mainly in lower animals such as bony fish and frog liver, whereas mammalian liver showed ganglioside patterns with smaller proportions of monosialo ganglioside species. While c-series gangliosides were consistently expressed in lower animals, they were found only in mammalian liver of particular species. No apparent trend was observed between the concentration of liver gangliosides and the phylogenetic stage of animals. The present study demonstrates the species-specific expression of liver gangliosides.     

Rapid Communication: GM3 Regulates Protein Kinase Systems in Cultured Brain Microvascular Endothelial Cells

Abstract: The barrier function of endothelial cells is known to be positively regulated by protein kinase A (PKA) and negatively regulated by protein kinase C (PKC). We found that exogenously administered GM3(NeuAc) promoted PKA activity in cultured brain microvascular endothelial cells (BMECs). Other glycolipids, including GM1, sulfoglucuronyl paragloboside, and GM3(NeuGc), did not have any effect on the PKA activity of BMECs. PC12 cells did not respond to exogenously applied GM3(NeuAc). GM3(NeuAc) also suppressed the PKC activity of BMECs. Thus, GM3(NeuAc) may function as a modulator of blood-brain barrier function via the two different kinase systems.     

Early murine T-lymphocyte activation is accompanied by a switch from N-Glycolyl- to N-acetyl-neuraminic acid and generation of ligands for siglec-E

It is well established that murine T-lymphocyte activation is accompanied by major changes in cell-surface sialylation, potentially influencing interactions with sialic acid-binding immunoglobulin-like lectins (siglecs). In the present study, we analyzed early activation of murine CD4+ and CD8+ T-lymphocytes at 24 h. We observed a striking and selective up-regulation in the binding of a recombinant soluble form of siglec-E, an inhibitory siglec, which is expressed on several myeloid cell types including antigen-presenting dendritic cells. In contrast, much lower levels of T cell binding were observed with other siglecs, including sialoadhesin, CD22, and siglec-F and the plant lectins Maackia amurensis leukoagglutinin and Sambucus nigra agglutinin. By mass spectrometry, the sialic acid content of 24-h-activated CD4+ and CD8+ T-lymphocytes exhibited an increased proportion of N-acetyl-neuraminic acid (NeuAc) to N-glycolyl-neuraminic acid (NeuGc) in N-glycans. Reduced levels of NeuGc on the surface of activated T cells were demonstrated using an antibody specific for NeuGc and the expression levels of the gene encoding NeuAc- to NeuGc-converting enzyme, CMP-NeuAc hydroxylase, were also reduced. Siglec-E bound a wide range of sialylated structures in glycan arrays, had a preference for NeuAc versus NeuGc-terminated sequences and could recognize a set of sialoglycoproteins that included CD45, in lysates from activated T-lymphocytes. Collectively, these results show that early in T cell activation, glycan remodelling involves a switch from NeuGc- to NeuAc-terminating oligosaccharides on cell surface glycoproteins. This is associated with a strong up-regulation of siglec-E ligands, which may be important in promoting cellular interactions between early activated T-lymphocytes and myeloid cells expressing this inhibitory receptor.     

Escherichia coli K99 binds to N-glycolylsialoparagloboside and N-glycolyl-GM3 found in piglet small intestine

Escherichia coli K12, which possess the K99 plasmid and synthesize K99 fimbriae (E. coli K99), cause severe neonatal diarrhea in piglets, calves, and lambs but not in humans. The organism binds specifically and with high affinity to only two glycolipids in piglet intestinal mucosa as demonstrated by overlaying glycolipid chromatograms with 125I-labeled bacteria. These glycolipids, which are N-glycolyl-GM3 (NeuGc alpha 2-3Gal beta 1-4Glc beta 1-1Cer) and N-glycolylsialoparagloboside (NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer), occur at about 13 and 0.3 micrograms per gram wet weight of mucosa, respectively. E. coli K99 grown at 18 degrees C, a temperature at which the K99 fimbriae are not expressed, do not bind to these glycolipids. Of the standard glycolipids tested in solid phase binding assays, E. coli K99 binds with highest affinity to N-glycolylsialoparagloboside, with less affinity to N-glycolyl-GM3, and with very low affinity to N-acetylsialoparagloboside. The bacteria do not bind to GM3 (NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1Cer), GM2 (GalNAc beta 1-4[Neu-Ac alpha 2-3]Gal beta 1-4Glc beta 1-1Cer), GM1 (Gal beta 1-3GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4Glc beta 1-1Cer), or several other N-acetylsialic acid-containing gangliosides and neutral glycolipids at the levels tested. N-Glycolylsialyl residues are found in the glycoproteins and glycolipids of piglets, calves, and lambs but not in the glycoproteins and glycolipids of humans. Possibly this distribution of sialyl derivatives explains the host range of infection by the organism.     

Gangliosides as paramyxovirus receptor. Structural requirement of sialo-oligosaccharides in receptors for hemagglutinating virus of Japan (Sendai virus) and Newcastle disease virus

A sensitive assay system for receptor activity of gangliosides to paramyxovirus was developed. This system involves incorporation of gangliosides into neuraminidase-treated chicken erythrocytes (asialoerythrocytes) followed by estimation of virus-mediated agglutination and hemolysis. The asialoerythrocytes coated with I-active ganglioside (Sia alpha 2-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer) were effectively agglutinated by hemagglutinating virus of Japan (HVJ, Sendai virus). The hemolysis of the asialoerythrocytes mediated by HVJ was restored to the highest level by labeling the cells with gangliosides possessing lacto-series oligosaccharide chains, i.e., I-active ganglioside, N-acetylneuraminosylparagloboside (SiaPG(NeuAc)), and i-active ganglioside (Sia alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer). The specific receptor activity of ganglioside GD1a possessing a gangliotetraose chain was lower than those of the gangliosides described above. Gangliosides GM3, GD3, GM1a, GD1b, SiaPG(NeuGc) showed little effect on the restoration of HVJ-mediated hemolysis. On infection with Newcastle disease virus (NDV), the highest specific restoration of lysis was found in chicken asialoerythrocytes coated with SiaPG(NeuAc or NeuGc) and GM3(NeuAc or NeuGc), whereas those coated with I-active ganglioside, GD3, GM1a, and GD1b showed very low NDV-mediated hemolysis. The above results indicate that the determinants of receptor for HVJ contain sialylated branched and/or linear lacto-series oligosaccharides carried by I,i-active gangliosides and SiaPG(NeuAc) and sialosylgangliotetraose chain carried by GD1a. The determinants for NDV are carried by SiaPG(NeuAc or NeuGc) containing linear lacto-series oligosaccharide and GM3(NeuAc or NeuGc). The absence of detectable binding of free oligosaccharides obtained from I-active ganglioside and sialoglycoprotein GP-2 isolated from bovine erythrocyte membranes as HVJ receptor (Suzuki, Y., et al. J. Biochem. (1983) 93, 1621-1633; (1984) 95, 1193-1200) indicates that HVJ recognizes the sialooligosaccharides oriented out of the lipid bilayer in the cell membranes where the hydrophobic ceramide or peptide backbone of the receptor is integrated.     

Two pathways for GM2(NeuGc) expression in mice: genetic analysis

We have reported that WHT/Ht mice express neither GM2(NeuGc) nor GM1(NeuGc) in the liver or erythrocytes due to a defect on the Ggm-2 gene, which was demonstrated to control the activity of UDP-GalNAc:GM3(NeuGc) N-acetylgalactosaminyltransferase in mouse liver, and, in addition, WHT/Ht mice do not express a detectable amount of GM2(NeuGc) but do express GM1(NeuGc) in tissues other than the liver and erythrocytes, such as the spleen, thymus, heart, lung, kidney, and testis [Nakamura et al. (1988) J. Biochem. 103, 201-208]. In order to determine whether the phenotype of WHT/Ht mice exhibiting an undetectable amount of GM2(NeuGc) in these tissues is genetically controlled or not, we analyzed the expression of gangliosides in the progeny obtained on backcross mating between (BALB/c X WHT/Ht)F1 and WHT/Ht mice, and in a GM2(NeuGc) congenic mouse, WHT.C. Concerning the expression of GM2(NeuGc) in the liver, lung, and kidney, 102 backcross mice could be segregated into two types. One type expressed a detectable amount of GM2(NeuGc) in the liver, lung, and kidney, and the other type did not. The ratio of the numbers of mice exhibiting these two types was 42: 60, indicating that the two phenotypes were genetically determined by the involvement of a single autosomal gene. Recombination as to GM2(NeuGc) expression in the liver, lung, and kidney was not detected among the 102 backcross mice. Analysis of the GM2(NeuGc) congenic mouse indicated that a detectable amount of GM2(NeuGc) was expressed in the liver, erythrocytes, lung, kidney, heart, spleen, and small intestine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of N-acetylneuraminic acid and of CMP-N-acetylneuraminic acid in the rat liver cell.

Adult male rats, under starving and normal conditions, were injected intravenously with N-acetyl[3H]mannosamine and after various time intervals the specific radioactivities of free N-acetylneuraminic acid (NeuAc) and CMP-N-acetylneuraminic acid were determined in the liver. The specific radioactivity of free NeuAc was high even within 20s after injection; the maximum was reached between 7 and 10 min. The specific radioactivity of CMP-NeuAc showed a lag phase of approx. 1 min. Thereafter it increased quickly and rose above the specific radioactivity of free NeuAc, reaching a maximum about 20 min after injection. These results point to a channelling of the newly synthesized NeuAc molecules into a special compartment, from which they are preferentially used by the enzyme CMP-sialic acid synthetase. It is suggested that the cytosolic enzyme N-acetylneuraminic acid 9-phosphate phosphatase is working in concert with the nuclear localized enzyme CMP-N-acetylneuraminic acid synthetase. Incorporation of radioactive sialic acid into sialoglycoproteins in liver occurred 2 min after injection, and after 10 min bound radioactivity began to appear in the circulation, indicating a transport time of 8 min of sialoglycoproteins from the point of attachment of sialic acid to the point of excretion.     

Purification and properties of GM2 synthase, UDP-N-acetylgalactosamine: GM3 beta-N-acetylgalactosaminyltransferase from rat liver

A UDP-N-acetylgalactosamine:ganglioside GM3 beta-N-acetylgalactosaminyltransferase which catalyzes the conversion of ganglioside GM3 to GM2 has been purified over 6300-fold from a Triton X-100 extract of rat liver particulate fractions by hydrophobic chromatography and affinity chromatography on GM3-acid-Sepharose. The purified enzyme has two identical subunits of 64,000 daltons. The enzyme has a pH optimum of pH 6.7-6.9 and requires divalent cations such as Mn2+ and Ni2+. In studies on substrate specificity GM3 containing N-acetylneuraminic acid (GM3(NeuAc] and GM3 containing N-glycolylneuraminic acid were both good acceptors for the purified enzyme. The plots of the activity of transferase as a function of GM3(NeuAc) showed sigmoidal relationships. The oligosaccharide of GM3, sialyllactose, was also a good acceptor, which indicates that the preferred acceptor substrate has the possible structure NeuAc alpha 2- or NeuGc alpha 2-3 Gal beta 1-4Glc-OR.     

Glycosyltransferase Activities in Cultured Endothelial Cells of Bovine Brain Microvascular Origin

Bovine brain microvascular endothelial cells (BMECs) express GM3 (NeuAc) and GM3 (NeuGc) as the major gangliosides, and GM1, GD1a, GD1b, GT1b as well as sialosylparagloboside and sialosyllactosaminylparagloboside as the minor species. To investigate the metabolic basis of this ganglioside pattern, the activities of eight glycosyltransferases (GM3-, GD1a-, GD3-, LM1-, GM2 (NeuAc)-, GM2 (NeuGc)-, LacCer-, and GM1-synthases) in cultured BMECs were studied. It was found that BMECs possessed high activities of GM3- and GD1a-synthases, and low activities of GM2-, GM1-, and GD3-synthases. Thus, the present study provides evidence that endothelial cells are capable of synthesizing gangliosides in situ and that the high content of GM3 in BMEC is closely associated with high activities of GM3-synthase and low activities of GM2-, GM1-, and GD3-synthases.     

Mammalian sialyltransferase ST3Gal-II: its exchange sialylation catalytic properties allow labeling of sialyl residues in mucin-type sialylated glycoproteins and specific gangliosides

While glycosyltransferases are known to display unidirectional enzymatic activity, recent studies suggest that some can also catalyze readily reversible reactions. Recently, we found that mammalian sialyltransferase ST3Gal-II can catalyze the formation of CMP-NeuAc from 5'-CMP in the presence of a donor containing the NeuAcα2,3Galβ1,3GalNAc unit [Chandrasekaran, E. V., et al. (2008) Biochemistry 47, 320-330]. This study shows by using [9-(3)H]- or [(14)C]sialyl mucin core 2 compounds that ST3Gal-II exchanges sialyl residues between CMP-NeuAc and the NeuAcα2,3Galβ1,3GalNAc unit and also radiolabels sialyl residues in gangliosides GD1a and GT1b, but not GM1. Exchange sialylation proceeds with relative ease, which is evident from the following. (a) Radiolabeleling of fetuin was ~2-fold stronger than that of asialo fetuin when CMP- [9-(3)H]NeuAc was generated in situ from 5'-CMP and [9-(3)H]NeuAcα2,3Galβ1,3GalNAcβ1,3Galα-O-Me by ST3Gal-II. (b) ST3Gal-II exchanged radiolabels between [(14)C]sialyl fetuin and [9-(3)H]NeuAcα2,3Galβ1,3GalNAcβ1,3Galα-O-Me by generating CMP-[(14)C]- and -[9-(3)H]NeuAc through 5'-CMP; only 20.3% (14)C and 28.0% (3)H remained with the parent compounds after the sialyl exchange. The [9-(3)H]sialyl-tagged MN glycophorin A, human chorionic gonadotropin β subunit, GlyCAM-1, CD43, fetuin, porcine Cowper's gland mucin, bovine casein macroglycopeptide, human placental glycoproteins, and haptoglobin were analyzed by using Pronase digestion, mild alkaline borohydride treatment, Biogel P6, lectin agarose, and silica gel thin layer chromatography. Sulfated and sialylated O-glycans were found in GlyCAM-1 and human placental glycoproteins. This technique has the potential to serve as an important tool as it provides a natural tag for the chemical and functional characterization of O-glycan-bearing glycoproteins.     

Generation of murine monoclonal antibodies specific for N-glycolylneuraminic acid-containing gangliosides.

We generated two murine monoclonal antibodies (MAbs) specific for mono- and disialylgangliosides having N-glycolylneuraminic acid (NeuGc) as their sialic acid moiety, respectively, by immunizing C3H/HeN mice with these purified gangliosides adsorbed to Salmonella minnesota followed by fusion with mouse myeloma cells. By use of a wide variety of glycolipids, including NeuGc-containing gangliosides, the precise structures recognized by these two antibodies were elucidated through enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatography. One MAb, GMR8, which was generated by immunizing the mice with purified GM3(NeuGc), reacted specifically with gangliosides having NeuGc alpha 2----3Gal- terminal structures, such as GM3(NeuGc), IV3NeuGc alpha-Gg4Cer, IV3NeuGc alpha-nLc4Cer, V3NeuGc alpha-Gb5Cer, and GD1a(NeuGc, NeuGc). None of the other gangliosides having internal NeuGc alpha2----3Gal- sequences, such as GM2(NeuGc) and GM1(NeuGc), nor corresponding gangliosides having NeuAc alpha 2----3Gal- sequences, nor neutral glycolipids were recognized. Thus, the epitope structures recognized by the MAb were found to be strictly NeuGc alpha 2----3Gal- terminal structures. In contrast, the other MAb, GMR3, which was generated by immunizing the mice with purified GD3(NeuGc-NeuGc-) adsorbed to the bacteria, reacted specifically with gangliosides having NeuGc alpha 2----8NeuGc alpha 2----3Gal- terminal sequences, such as GD3(NeuGc-NeuGc-), IV3NeuGc alpha 2-Gg4Cer, IV3NeuGc alpha 2-nLc4Cer, and V3NeuGc alpha 2-Gb5Cer, but did not react with corresponding gangliosides having NeuAc as their sialic acid moiety or with the neutral glycolipids tested. The epitope structures recognized by the MAb were suggested to be NeuGc alpha 2----8NeuGc alpha 2----3Gal- terminal structures. Using these MAbs, we determined the distribution of such gangliosides in the spleen, kidney, and liver of several mice strains. Novel gangliosides reactive with these MAbs were detected in these tissues.     

A new monoclonal antibody directed to sialyl alpha 2-3lactoneotetraosylceramide and its application for detection of human gastrointestinal neoplasms

A new monoclonal antibody (NS24) directed to the N-acetylneuraminyl alpha 2-3Gal beta 1-4GlcNAc residue in type II sugar chain of N-acetylneuraminyllactoneotetraosylceramide [sialylparagloboside, IV3(NeuAc)nLc4Cer] was prepared by hybridoma technique. Liposomes composed of dipalmitoylphosphatidylcholine, cholesterol, IV3(NeuAc)nLc4Cer, and lipopolysaccharides from Salmonella minnesota R595 were used for immunization with IV3(NeuAc)nLc4Cer isolated from human erythrocytes. This method allowed the fusion of spleen cells of immunized mouse with myeloma cells only three days after immunization. NS24 reacted specifically to both naturally occurring and chemically synthesized IV3-(NeuAc)nLc4Cer, whereas it has no reactivity to structurally related gangliosides, such as IV6(NeuAc)nLc4Cer, N-glycolylneuraminyl alpha 2-3lactoneotetraosylceramide [IV3(NeuGc)-nLc4Cer], i-active ganglioside [VI3(NeuAc)nLc6Cer], I-active ganglioside [VIII3(NeuAc)-VI3(NeuAc)IV6kladoLc8Cer], GM4(NeuAc), GM3(NeuAc), GM3(NeuGc), GM1b(NeuAc), GD3-(NeuAc), other ganglio-series gangliosides, sulfatide, and paragloboside (nLc4Cer). Synthetic N-acetylneuraminyl alpha 2-3lactotetraosylceramide [IV3(NeuAc)Lc4Cer] and its asialo-derivative (Lc4Cer) carrying type I sugar chain also showed no reaction with NS24. One to 100 pmol of IV3(NeuAc)nLc4Cer was detected dose-dependently by a thin-layer chromatography/enzyme immunostaining procedure. Human gastric carcinomas showed positive reactions with NS24 immunochemically and histochemically. NS24 reacted preferentially with poorly differentiated adenocarcinomas rather than well differentiated ones.     

A sialidase-susceptible ganglioside, IV3 alpha(NeuGc alpha 2-8NeuGc)-Gg4Cer, is a major disialoganglioside in WHT/Ht mouse thymoma and thymocytes.

The disialogangliosides of WHT/Ht mouse thymomas, which were obtained by subcutaneous transplantation of a thymoma that developed spontaneously in a WHT/Ht mouse, were purified and characterized. From the results of sugar-composition analysis, a permethylation study, enzymatic hydrolysis followed by TLC-immunostaining, negative-ion fast atom bombardment mass spectrometry (FAB/MS), and 1H-NMR spectroscopy, the structure of one of the five purified disialogangliosides was determined to be IV3 alpha(NeuGc alpha 2-8NeuGc)-Gg4Cer. The other 4 disialogangliosides were tentatively characterized on the basis of sialidase treatment followed by TLC-immunostaining with cholera toxin B subunit and anti-Gg4Cer antibody to be IV alpha(NeuAc alpha-NeuGc)-Gg4Cer, IV alpha(NeuGc alpha-NeuAc)-Gg4Cer, IV alpha NeuAc,II3 alpha NeuAc-Gg4Cer, and IV alpha NeuGc,II3 alpha NeuGc-Gg4Cer. In addition, another component exhibiting one spot on TLC was a mixture of IV alpha NeuGc,II3 alpha NeuAc-Gg4Cer and IV alpha NeuAc,II3 alpha NeuGc-Gg4Cer. Then the occurrence of these gangliosides in WHT/Ht mouse thymocytes was examined. As one of two major disialogangliosides, the thymocytes contained IV3 alpha(NeuGc alpha 2-8NeuGc)-Gg4Cer, which was characterized with a mass spectrum and mass chromatograms obtained by micro high-performance liquid chromatography-FAB/MS. The other major disialoganglioside was tentatively characterized to be II3 alpha-(NeuGc alpha-NeuGc)-Gg4Cer by sialidase treatment followed by TLC-immunostaining. A sialidase-susceptible monosialoganglioside, IV3 alpha NeuGc-Gg4Cer [GM1b(NeuGc)], had been reported to be characteristic of mouse immune tissues [Nakamura, K. et al. (1988) J. Biochem, 103, 201-208]. Taken together, the results suggest that the pathway from Gg4Cer to IV3 alpha(NeuGc alpha 2-8NeuGc)-Gg4Cer through GM1b(NeuGc) is quite active in mouse immune tissues.     

Genetic polymorphism of ganglioside expression in mouse organs

In previous studies it was demonstrated that there are three variations as to the expression of liver gangliosides in inbred strains of mice; the first group expresses GM3(NeuGc) as a major component, the second group, GM2(NeuGc), and the third group, GM2(NeuGc), GM1 (NeuGc), and GD1a(NeuGc). In the present study, we attempted to determine which organs, if any, exhibit the same polymorphic variations as those observed in the liver. Thus, the gangliosides in spleen, thymus, heart, lung, kidney, testis, and erythrocytes, as well as those in liver, were examined using a TLC-mapping technique or by one-dimensional TLC. WHT/Ht, BALB/c, and ICR mice, which are typical strains as to the polymorphic expression of liver gangliosides, were used for the analysis. The presence of GM1 was confirmed by not only chemical detection on TLC plates but also with a TLC-immunostaining procedure using choleragenoid. These comparative studies indicated that only erythrocytes exhibited the same polymorphic variations of ganglioside expression as those in the liver, but the other six organs showed specific patterns which were not polymorphic. In addition to this, there were the following two interesting findings. Firstly, WHT/Ht mice, in which GM2(NeuGc) and GM1(NeuGc) are not expressed in the liver and erythrocytes, did not express a detectable amount of GM2(NeuGc) but expressed GM1(NeuGc) in all the other organs. Secondly, marked polymorphic variation was found in the expression of GM4(NeuAc) in the erythrocytes.     

Activation of CMP-N-acetylneuraminic acid:lactosylceramide sialyltransferase during the differentiation of HL-60 cells induced by 12-O-tetradecanoylphorbol-13-acetate

We previously reported that the synthesis of NeuAc(alpha 2-3)Gal(beta 1-4)GlcCer (GM3) ganglioside was preferentially enhanced during the differentiation of HL-60 cells into a monocyte/macrophage lineage induced by 12-O-tetradecanoylphorbol-13-O-acetate (TPA). Since exogenously added GM3 ganglioside was shown to be able to induce the differentiation of HL-60 cells into the monocyte/macrophage lineage in a synthetic medium, the functional role of the GM3 ganglioside increase during the differentiation of HL-60 cells has become the subject of much interest. In the present study, we investigated the activity of CMP-NeuAc:lactosylceramide sialyltransferase, which catalyzes the synthesis of GM3 ganglioside from lactosylceramide, in cells undergoing differentiation induced by two different reagents, TPA and 1 alpha,25-dihydroxy-vitamin D3, which induce the differentiation of HL-60 cells into the monocyte/macrophage lineage through different modes of action. We showed that the activation of CMP-NeuAc:lactosylceramide sialyltransferase and the increase in GM3 ganglioside were not related to the differentiated lineage but to the specific action of TPA, i.e. activation of protein kinase C.