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- Materials and methods
To examine the specificity of monoclonal antibody A2B5, four A2B5-reactive gangliosides (designated as G-1, G-2, G-3 and G-4) were purified from bonito fish brain. Ganglioside-1, -2, and -3 migrated above GD1b, below GQ1b, and far below GQ1b on thin-layer chromatography. Ganglioside-4 had the slowest chromatographic mobility and migrated below G-3. The structures of these gangliosides were characterized by overlay analysis with glycolipid-specific ligands, product analysis after sialidase or mild acid treatment, and electrospray ionization-mass spectrometry (ESI-MS). Accordingly, G-1, G-2 and G-3 were identified to be GT3, GQ1c and GP1c, respectively. The ganglioside G-4 was shown to have the following structure: NeuAc-NeuAc-NeuAc-Galβ1-3Gal NAcβ1-4(NeuAc-NeuAc-NeuAcα2-3)Galβ1-4Glcβ1-1′Cer. The antibody A2B5 reacted with these c-series gangliosides, but not with GD3 and other gangliosides and neutral glycosphingolipids. The antigenic epitope for A2B5 was assumed to include the trisialosyl residue connected to the inner galactose of the hemato- or ganglio-type oligosaccharide structure of gangliosides. Phylogenetic analysis of brain gangliosides using the A2B5 preparation demonstrated that c-series gangliosides are enriched in lower animals, especially bony fish of different species. The monoclonal antibody A2B5 would be a useful tool for examining the distribution and function of c-series gangliosides.
In higher animals, c-series gangliosides constitute minor ganglioside components of tissues and cells and are often difficult to separate from major ganglioside species on TLC. Accordingly, specific antibodies directed toward c-series gangliosides have been developed; they include monoclonal antibodies Q211 (Henke-Fahle 1983), 18B8 (Grunwald et al. 1985), M6704 and M7103 (Obata and Tanaka 1988). Using these antibodies, it has been demonstrated that c-series gangliosides and their O-acetyl derivatives are temporarily expressed in avian and mammalian brain at certain embryonic stages, but are hardly detected at adult ages (Dubois et al. 1986; Hirabayashi et al. 1988, 1989; Rosner et al. 1988,1993). Stage- and cell-specific expression of c-series gangliosides in brain tissue has also been suggested by immunohistochemical and immunocytochemical techniques (Rosner et al. 1985a, 1988, 1992; Letinic et al. 1998). The exclusive localization of c-series gangliosides in stellate neurons in adult human cerebellum has also been demonstrated (Heffer-Lauc et al. 1998).
A monoclonal antibody A2B5 was originally prepared by immunizing with chicken embryonic retinal cells (Eisenbarth et al. 1979). Although there has been some controversy about its specificity (Kasai et al. 1983; Kundu et al. 1983; Fredman et al. 1984), recent studies have suggested the specific reactivity of A2B5 to c-series gangliosides. A2B5 was shown to react with GT3 and GQ1c, but not with GD3, GM3 and GD1b (Fenderson et al. 1987). The antibody was demonstrated to react with GT3 and its 9-O-acetylated derivative in embryonic chicken brain (Dubois et al. 1990) or in cultured oligodendrocyte type-2 astrocyte progenitor cells (Farrer and Quarles 1999). The specific binding of A2B5 to GT3, GT1c, GQ1c, GP1c and G′H′ has also been suggested using cod fish brain gangliosides (Freischutz et al. 1994). We have recently examined rat liver gangliosides using an A2B5 preparation and suggested that the antibody reacted with GT3, GT1c and GQ1c in hepatocytes (Saito and Sugiyama 1999). However, the specificity of A2B5 and antigenic epitope of gangliosides still remain to be clarified.
In the present study, we characterized four A2B5-reactive gangliosides in bonito fish brain and examined a relationship between the structures of these gangliosides and their reactivity to A2B5. We also examined the phylogenetic profile of c-series gangliosides in brain tissue using the A2B5 preparation.
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- Materials and methods
It is known that c-series gangliosides constitute major ganglioside components in brain tissue of certain fish species. Yu and Ando (1980) characterized the structures of gangliosides in cod fish brain and identified c-series gangliosides including GT3, GQ1c and GP1c. A ganglioside of unknown structure, i.e. G′H′, was also observed below GP1c. Little information is available about the distribution of c-series gangliosides in other bony fish species. The present study demonstrated that bonito fish brain expresses four major A2B5-reactive gangliosides (i.e. G-1, G-2, G-3 and G-4), which, respectively, corresponded with GT3, GQ1c, GP1c and G′H′ in cod fish brain. Accordingly, these A2B5-reactive gangliosides were purified and characterized for their structures. As a result, G-1, G-2 and G-3 were identified to be GT3, GQ1c and GP1c, respectively. The finding that G-2 was susceptible to the action of S. typhimurium sialidase was in accordance with the fact that GQ1c contains an α2-3 linked sialic acid residue at the terminal galactose of the gangliotetraose oligosaccharide. As for G-4, it was suggested that this ganglioside has the gangliotetraose oligosaccharide structure with two trisialosyl residues, each of which separately connects to the inner and terminal galactose. The position of two trisialosyl residues was successfully determined by ESI-MS of the permethylated derivative of the compound. Although the linkage of sialic acids in the trisialosyl residues remains to be determined, it is likely that they are connected to each other through an α2-8 linkage, as usually observed in other polysialo structures (Schauer 1982). The finding that the treatment of this ganglioside by sialidase produced G-3, i.e. GP1c, suggests that the trisialosyl residue is connected to the terminal galactose via an α2-3 linkage. These results further support our conclusion that this ganglioside is GH1c, i.e. (NeuAcα2-8NeuAcα2-8NeuAcα2-3)Galβ1-3GalNAcβ1-4(NeuAcα2-8NeuAcα2-8NeuAcα2-3)Galβ1-4Glcβ1-1′Cer.
The strict specificity of A2B5 to c-series gangliosides was shown using TLC immunostaining with various gangliosides and neutral glycosphingolipids; the monoclonal antibody reacted with GT3, GQ1c, GP1c and GH1c, but not with other glycolipids including GD3. Based upon the reactivity to A2B5 and chromatographic mobility on TLC, the ganglioside G-x in bonito fish brain was assumed to be GT1c. This assumption was further supported by the observation that the treatment of G-2 (i.e. GQ1c) or G-3 (i.e. GP1c) produced a ganglioside corresponding to G-x.
While an antibody directed toward c-series gangliosides recognizes a common epitopic structure, its reactivity may differ among individual ganglioside species. The monoclonal antibody M6704 almost equally reacts with GT3, GT1c, GQ1c, and GP1c, whereas the antibody M7103 is less reactive to GT2 and practically unreactive with GT3 (Hirabayashi et al. 1988). Thin-layer chromatographic immunostaining of cod fish brain gangliosides suggests that the antibody Q211 has lower reactivity to GT3 than A2B5 (Freischutz et al. 1994). The present study showed that the reactivity of A2B5 is highest in GT3 among c-series gangliosides examined; gangliosides having larger numbers of sialic acid at the terminal galactose of the gangliotetraose oligosaccharide showed lower reactivity to A2B5. Thus, it is very unlikely that the trisialosyl residue connected to the terminal galactose in GH1c also serves as an antigenic epitope for A2B5. While A2B5 has been shown to react with O-acetyl GT3 (Dubois et al. 1990; Farrer and Quarles 1999), its reactivity to O-acetyl derivatives of other c-series gangliosides remains to be clarified.
Although the function of c-series gangliosides remains to be elucidated, evidence has been provided suggesting for their possible involvement in growth, differentiation, and migration of neuronal cells (Seybold et al. 1989; Greis and Rosner 1990; Sonnentag et al. 1992). Involvement of c-series gangliosides in cell adhesion and invasion of glioma cells has also been suggested (Merzak et al. 1994, 1995). A2B5 would be a useful tool for investigation of the expression and function of c-series gangliosides.