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Keywords:

  • A2B5;
  • c-series gangliosides;
  • gangliosides;
  • phylogeny

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

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.

Abbreviations used
CID

collision-induced dissociation

CTH

Galα1-4Galβ1-4Glcβ1-1′Cer

ESI-MS

electrospray ionization-mass spectrometry

ECL

enhanced chemiluminescence

GalCer

Galβ1-1′Cer

GgOse3Cer

GalNAcβ1-4Galβ1-4Glcβ1-1′Cer

GgOse4Cer or asialo GM1

Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1′Cer

GlcCer

Glcβ1-1′Cer

globoside

GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1′Cer

LacCer

Galβ1-4Glcβ1-1′Cer.

c-Series gangliosides are characterized by a trisialosyl residue at the inner galactose of the hemato- or ganglio-type oligosaccharide structure. They are enriched in adult fish brain of certain species. Cod fish brain has been shown to contain c-series gangliosides such as GT3, GT2, GT1c, GQ1c and GP1c (Ando and Yu 1979; Yu and Ando 1980). c-Series gangliosides are also found in brain tissue of cartilaginous fish including dogfish (Nakamura et al. 1997) and skate fish (Nakamura et al. 2000). The presence of 9-O-acetyl derivatives of GT2 and GT3 in cod fish brain were also reported (Waki et al. 1993a,b). c-Series gangliosides have also been detected in avian and mammalian tissues of neural and extraneural origin. GP1c was identified in embryonic chicken brain (Rosner et al. 1985b) and human brain (Miller-Podraza et al. 1991). The expression of GT3 in hog kidney (Murakami-Murofushi et al. 1981), cat erythrocytes (Ando and Yamakawa 1982) and human lung (Mansson et al. 1986) was also reported.

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.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Materials

All brain tissues were obtained from adult animals. Fresh bonito fish brain (Katsuwonus pelamis) were gifted by a fish processing factory (Yanagiya Honten Co., Ltd, Yaizu, Japan). Other fish brain were obtained from local fish markets; the fish included pacific cod (Gadus macrocephallus); red sea bream (Pagrus major), bluefin tuna (Thunnus thynnus), and pacific mackerel (Scomber japonicus). Sprague–Dawley rats, New Zealand white rabbits, Japanese terrapins (Clemmys japonica) and bullfrogs (Rana catesbeiana) were purchased from Japan SLC (Shizuoka, Japan). After the animals were killed, brain tissue was taken and stored at − 80°C until ganglioside analysis. Bovine and porcine brain were obtained from a local slaughter house.

Neutral glycosphingolipids, i.e. GlcCer, GalCer, LacCer, GgOse3Cer and GgOse4Cer (asialo GM1) were prepared by mild acid hydrolysis of rat brain gangliosides (Svennerholm et al. 1973). Sialylparagloboside (LM1) and fucosyl GM1 were purified from human and rat erythrocytes, respectively (Ando et al. 1973; Iwamori et al. 1984). Purified gangliosides from rat brain or bovine brain were used as standard ganglioside mixtures for TLC. GM3, CTH (globotriosyl ceramide) and globoside were purchased from Matreya (Pleasant Gap, PA, USA). A2B5-Producing hybridomas CRL 1520 were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The culture medium containing the monoclonal antibody A2B5 (IgM-type) was used as antibody preparation (Saito et al. 1999). An anti-GM1 antibody or anti-asialo GM1 antibody (both IgG-type) was prepared by immunizing rabbits with purified ganglioside (Jacobson et al. 1982). These antiglycolipid antibodies possessed strict specificity to GM1 or asialo GM1, and have successfully been employed for specific detection of the glycolipids (Saito and Sugiyama 1999; 2000a,b). Other reagents were purchased from the following companies: Clostridium perfringens sialidase, Arthrobacter ureafaciens sialidase, goat peroxidase-conjugated antibody against mouse IgM or rabbit IgG, and peroxidase-conjugated cholera toxin B subunit (Sigma, St Louis, MO, USA), Salmonella typhimurium sialidase (α2,3 specific, cloned from S. typhimurium LT2 and expressed in Escherichia coli, Takara Shuzo Co. Ltd, Japan), high-performance TLC plates (Merck KGaA, Darmstadt, Germany), and enhanced chemiluminescence (ECL) western blotting detection kits (Amersham Pharmacia Biotech, Buckinghamshire, UK).

Purification of A2B5-reactive gangliosides from bonito fish brain

Total lipids were extracted from 479 g bonito fish brain with 10 volumes of chloroform/methanol (1 : 1) and applied to a DEAE-Sephadex A-25 column (bed volume 240 mL), which was equilibrated with chloroform/methanol/H2O (30 : 60 : 8). While neutral lipids passed through the column, acidic lipids were retained on the column and recovered by elution with chloroform/methanol/0.8 m sodium acetate (30 : 60 : 8). Acidic lipids were treated with methanolic 0.2 m NaOH at 37°C for 1 h, followed by neutralization with acetic acid. A total ganglioside mixture (125 mg as lipid-bound sialic acid) was obtained after dialysis of the neutralized sample against water.

Four major A2B5-reactive gangliosides in the ganglioside mixture were purified as follows. Total gangliosides were applied to a DEAE-Sephadex A-25 column (column size 1.4 × 100 cm) and eluted with a linear gradient system of methanol containing ammonium acetate (0.1–0.6 m) (total elution volume 2500 mL). Fractions containing each A2B5-reactive ganglioside were combined, desalted by Sephadex LH-20 column chromatography, and applied to Iatrobeads column chromatography (column size, 1.4 × 90 cm, Iatron Laboratories Inc., Tokyo, Japan) with a linear gradient system of chloroform/methanol/water (from 60 : 30 : 4.5 to 60 : 33 : 8) (total volume 2400 mL). A2B5-reactive fractions were collected and subjected to preparative TLC, followed by purification HPLC with a size exclusion column (TSKgel α-2500, 0.8 × 30 cm, Tosoh, Japan) with a solvent of methanol. The purified ganglioside showed a single band on TLC.

Overlay analysis of gangliosides

Overlay with glycolipid-specific ligands was carried out as follows (Saito et al. 1985b). Gangliosides were developed on TLC. After coating with a 0.4% polyisobutylmethacrylate solution, the plate was overlaid successively with an antiglycolipid antibody and peroxidase-conjugated second antibody at room temperature (i.e. 20–25°C) for 1.5 h. When peroxidase-conjugated cholera toxin B subunit was used, the step with second antibody was omitted. The reactive bands were detected using the ECL method. The gangliosides on the plate were then visualized with resorcinol-HCl reagent (Svennerholm 1957). Densitometric analysis of chromatograms was carried out using a densitometric image analyzer (Atto Densitograph AE-6920M, Atto Co., Tokyo, Japan).

In some experiments, gangliosides on TLC plates were treated in situ with Cl. perfringens sialidase at room temperature for 1.5 h. The generated GM1 was detected using GM1-specific ligands, i.e. cholera toxin B subunit or anti-GM1 antibody (Saito et al. 1985a; Wu and Ledeen 1988).

Treatment of gangliosides with sialidase or mild acid in test-tube reaction

A ganglioside was incubated at 37°C with Cl. perfringens sialidase or A. ureafaciens sialidase (at pH 4.8 in both cases) or with S. typhimurium sialidase (pH 5.5) for different periods. Alternatively, a ganglioside was treated in 5.6 mm formic acid at 80°C for 30 min. The reaction product(s) were isolated by Sephadex LH-20 column chromatography, developed on TLC, and immunostained with specific antibodies or visualized with resorcinol-HCl reagent or orcinol-H2SO4 reagent (Sewell 1979).

Electrospray ionization-mass spectrometry of gangliosides

Electrospray ionization-mass spectrometry (ESI-MS) of gangliosides was carried out using an LCQ ion-trap mass spectrometer equipped with an ESI source (Finnigan, MA, USA). A ganglioside was permethylated with methyl iodide using a method reported previously (Tadano-Aritomi et al. 1992). The reaction mixture was partitioned with a solvent system of chloroform/methanol/0.88% KCl (8 : 4 : 3). The permethylated derivative, which was recovered in the lower phase, formed a single band on TLC. The permethylated lipid was dissolved in methanol at a concentration of 10 pmol/µL, and introduced into the electrospray needle by mechanical infusion at a flow rate of 3 µL/min. The ESI capillary was kept at a voltage of + 4 V at 200°C. The tube lens offset was set at − 30 V. The collision-induced dissociation (CID)-MS2 spectra were taken using helium as the collision gas. The relative collision energy scale was set at + 2.5 eV. Mass spectra were averaged over 10 scans.

Analysis of sialic acids in gangliosides

Lipid-bound sialic acids were analyzed using a fluorometric HPLC method (Hara et al. 1987; Saito and Sugiyama 2000b).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Major and A2B5-reactive gangliosides in bonito fish brain

The compositions of major and A2B5-reactive gangliosides of bonito fish brain were examined and compared with those of cod fish brain. Cod fish brain contained high proportions of polysialo gangliosides including c-series gangliosides such as GT3, GQ1c, GP1c and G′H′(Fig. 1a, lane 1). Bonito fish brain had a similar ganglioside composition and possessed gangliosides corresponding to these c-series gangliosides in cod fish brain (Fig. 1a, lane 2). Moreover, the A2B5-reactive ganglioside pattern of bonito fish brain resembled that of cod fish brain (Fig. 1b). Cod fish brain showed five A2B5-reactive bands, four of which corresponded with GT3, GQ1c, GP1c and G′H′. Bonito fish brain also possessed five A2B5-reactive gangliosides. Four major A2B5-reactive gangliosides, respectively, migrated above GD1b (designated as G-1), below GQ1b (G-2), far below GQ1b (G-3) and between G-3 and origin on TLC (G-4). G-1, G-2, G-3 and G-4 had the same mobility of GT3, GQ1c, GP1c and G′H′, respectively. Bonito fish brain also had one minor A2B5-reactive band (G-x) between GT1b and GQ1b.

image

Figure 1.  The compositions of major and A2B5-reactive gangliosides of bonito and cod fish brain. Gangliosides were isolated from brain tissue, developed on TLC with a solvent system of chloroform/methanol/0.2% CaCl2·2H2O (40 : 40 : 11), and visualized with resorcinol-HCl reagent (a) or immunostained with a monoclonal antibody A2B5 (b). In panel (a), lane 1, cod fish brain gangliosides; lane 2, bonito fish brain gangliosides; lane 3, standard ganglioside mixture. In panel (b), lanes 1 and 2, cod and bonito fish brain gangliosides. The amount of gangliosides per lane is equivalent to 1 µg (a) or 0.1 µg sialic acid (b).

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Overlay and product analyses of A2B5-reactive gangliosides in bonito fish brain

The major A2B5-reactive gangliosides G-1, G-2, G-3 and G-4 of bonito fish brain were purified and examined for their structures. The ganglioside G-1 did not react with either cholera toxin B subunit or anti-GM1 antibody before or after in situ treatment with Cl. perfringens sialidase on TLC (data not shown). The treatment of G-1 with Cl. perfringens sialidase in test-tube reaction generated two ganglioside products that had the same chromatographic mobility of GD3 and GM3, respectively (Fig. 2). These findings suggested that G-1 was a member of the hemato-type ganglioside family and contained the same oligosaccharide structure of GM3.

image

Figure 2.  Product analysis of G-1 after sialidase treatment. The ganglioside G-1 in bonito fish brain was treated with Cl. perfringens sialidase (200 mU/mL, pH 4.8) at 37°C for 45 min. The reaction products were developed on TLC with a solvent system of chloroform/methanol/0.2%, CaCl2·2H2O (50 : 45 : 10) and visualized with resorcinol-HCl reagent. Lane 1, standard ganglioside mixture; lane 2, purified G-1; lane 3, reaction products from G-1 after sialidase treatment; lane 4, authentic GM3.

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The gangliosides G-2, G-3 and G-4 were developed on TLC and examined for its reactivity with GM1-specific ligands. While G-2, G-3 and G-4 did not react with cholera toxin B subunit initially (data not shown), they became reactive with the toxin after treatment with Cl. perfringens sialidase (Fig. 3). These gangliosides also reacted with anti-GM1 antibody after sialidase treatment (data not shown). The test-tube reaction of G-2 with Cl. perfringens sialidase produced a ganglioside corresponding to GM1 and other several gangliosides (Fig. 4a, lane 3). In contrast, the treatment of G-2 with α2,3-specific S. typhimurium sialidase generated a single reaction product that migrated between GT1b and GQ1b on TLC (Fig. 4a, lane 2). Hydrolysis of G-2 by A. ureafaciens sialidase produced two glycolipids, which were identified, respectively, to be GM1 and asialo GM1 by TLC immunostaining with specific antibodies (Fig. 4b). The ganglioside G-3 was hydrolyzed by Cl. perfringens sialidase; the reaction products corresponded with those from G-2 and G-2 itself (Fig. 4c). While G-3 was resistant to the treatment with S. typhimurium sialidase, it was susceptible to the action of A. ureafaciens sialidase, producing GM1 and asialo GM1 (data not shown).

image

Figure 3.  Overlay analysis of G-2, G-3 and G-4 with cholera toxin B subunit after sialidase treatment. The gangliosides G-2, G-3 and G-4 in bonito fish brain were purified, developed on TLC and visualized with resorcinol-HCl reagent (a) or overlaid with cholera toxin B subunit after in situ treatment with Cl. perfringens sialidase (b). In both panels, lane 1, standard ganglioside mixture; lane 2, bonito fish brain gangliosides; lane 3, G-2; lane 4, G-3; lane 5, G-4.

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image

Figure 4.  Product analysis of G-2 and G-3 after treatment with different sialidases. (a) G-2 was treated with S. typhimurium sialidase (250 mU/mL) at 37°C for 2 h (lane 2) or Cl. perfringens sialidase (125 mU/mL) for 15 min (lane 3). The reaction products were developed on TLC and visualized with resorcinol-HCl reagent. Lane 1, standard ganglioside mixture. (b) G-2 was treated with A. ureafaciens sialidase (500 mU/mL) at 37°C for 1 h. The reaction products were developed on TLC and visualized with orcinol-H2SO4 reagent (left-hand panel) or immunostained using glycolipid-specific ligands (right-hand panel). In left-hand panel, lane 1, standard ganglioside mixture; lane 2, the reaction products from G-2 after sialidase treatment; and lane 3, authentic asialo GM1 (i.e. GA1). In right-hand panel: the reaction products from G-2 were immunostained using anti-GM1 antibody (lane 1) or antiasialo GM1 antibody (lane 2). (c) G-2 and G-3 were treated with Cl. perfringens sialidase in the following conditions: G-2, 125 mU/mL for 15 min; and G-3, 250 mU/mL for 30 min. The reaction products were developed on TLC and visualized with resorcinol-HCl reagent. Lane 1, standard ganglioside mixture; lane 2, reaction products from G-2; lane 3, reaction products from G-3.

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The ganglioside G-4 was less susceptible to the action of Cl. perfringens sialidase than G-2 and G-3. Accordingly, G-4 was subjected to mild acid hydrolysis. The reaction products from G-4 corresponded with those that were generated from G-3 after treatment with Cl. perfringens sialidase (Fig. 5). Like G-3, G-4 was totally resistant to the action of S. typhimurium sialidase, but was hydrolyzed by A. ureafaciens sialidase, producing GM1 and asialo GM1 (data not shown). These findings suggested that G-2, G-3 and G-4 were the members of gangliotetraose gangliosides and contained the same oligosaccharide structure of GM1.

image

Figure 5.  Product analysis of G-4 after mild acid hydrolysis. G-4 was treated with 5.6 mm formic acid at 80°C for 30 min. The reaction products were developed on TLC and visualized with resorcinol-HCl reagent. Lane 1, purified G-4; lane 2, reaction products from G-4 after mild acid hydrolysis; lane 3, standard ganglioside mixture.

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The gangliosides G-2, G-3 and G-4 were treated with Cl. perfringens sialidase, and the reaction products were immunostained with A2B5. As shown in Fig. 6, G-2 produced an A2B5-reactive ganglioside corresponding to G-x, whereas G-3 generated two A2B5-reactive products corresponding to G-x and G-2. The treatment of G-4 with sialidase produced two A2B5-reactive gangliosides corresponding to G-2 and G-3. Thus, it was suggested that G-x, G-2, G-3 and G-4 shared a common structure with different numbers of sialic acid.

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Figure 6.  Thin-layer chromatographic immunostaining with A2B5 of reaction products from G-2, G-3, and G-4 after sialidase treatment. G-2, G-3 and G-4 were treated with Cl. perfringens sialidase (200 mU/mL) at 37°C for 15 min. The reaction products were developed on TLC and visualized with resorcinol-HCl reagent (a) or immunostained with A2B5 (b). In panel (a): lane 1, standard ganglioside mixture; and lanes 2–4, reaction products from G-2, G-3 and G-4, respectively. In panel (b): lanes 1–3, immunochromatograms of reaction products from G-2, G-3 and G-4, respectively.

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Analysis of sialic acids in G-1, G-2, G-3 and G-4 demonstrated that N-acetylneuraminic acid was the sole molecular species of sialic acid in all cases (data not shown).

Electrospray ionization-mass spectrometry of G-1, G-2 and G-3

Based upon the results described above, it was reasonably assumed that the gangliosides G-1, G-2 and G-3 might be GT3, GQ1c and GP1c, respectively. To examine this hypothesis, G-1, G-2 and G-3 were further characterized by ESI-MS of permethylated derivatives of the compounds (Table 1). The positive-ion ESI-MS spectrum of G-1 showed a double-charged ion of [M + Na + K]2+ at m/z 1081, which corresponded with a hemato-type oligosaccharide structure having three sialic acids and a ceramide with the molecular mass of 566 (as a non-methylated structure). The collision-induced dissociation (CID)-MS2 spectrum of the double-charged ion produced characteristic fragment ions of GT3, including the one that corresponded with a trisialosyl residue (Fig. 7a).

Table 1.   Electrospray ionization-mass spectrometry (ESI-MS) of A2B5-reactive gangliosides G-1, G-2 and G-3 in bonito fish brain
 ions observed [m/z]
 G-1G-2G-3
  1. Gangliosides were permethylated with methyl iodide and analyzed by positive ESI-MS and collision-induced dissociation (CID)-MS2. The structure corresponding to each ion peak in the spectrum is shown in parenthesis.

MS1081 [M + Na + K]2+1492 [M + Na + H]2+1659 [M + 2Na]2+
CID-MS2376 [NeuAc + H]+376 [NeuAc + H]+376 [NeuAc + H]+
577 [Cer + H]+603 [NeuAc-Gal + Na]+442 [Gal-GalNAc-Gal-Glc + Na + H]2+
693 [(NeuAc)3-Gal + 3Na - H]2+649 [Cer + Na]+532 [Gal-Glc-Cer + 2K]2+
759 [(NeuAc)2 + Na]+738 [(NeuAc)2 + H]+576 [Cer + H]+
765 [(NeuAc)3-Gal-Glc + Na + H]2+847 [Glc-Cer + H]+737 [(NeuAc)2 + H]+
1041 [Gal-Glc-Cer+ K]+885 [NeuAc-Gal-GaINAc + 2Na - H]+821 [Glc-Cer + Na]+
1121 [(NeuAc)3 + Na]+1099 [(NeuAc)3-Gal-Glc-Cer + Na + K]2+996 [(NeuAc)2-Gal + K]+
1172 [(NeuAc)2-Gal-Glc + Na]+1116 [Gal-GalNAc-(NeuAc)2Gal-Glc-Cer + Na + H]2+1066 [(NeuAc)3-Gal-Glc-Cer + 2Nal2+
1402 [NeuAc-Gal-Glc-Cer + K]+1121 [(NeuAc)3 + Na]+1121 [(NeuAc)3 + Na]+
1763 [(NeuAc)2-Gal-Glc-Cer + K]+1123 [NeuAc-Gal-GaINAc-(NeuAc)Gal-Glc-Cer + Na + H]2+1371 [NeuAc-Gal-Glc-Cer + Na]+
 1862 [NeuAc-Gal-GaINAc-Gal-Glc-Cer+ H]+1733 [(NeuAc)2-Gal-Glc-Cer + Na]+
image

Figure 7.  The assumed structures of G-1, G-2 and G-3 and fragmentation patterns by ESI-MS analysis. The gangliosides G-1 (a), G-2 (b) and G-3 (c) were permethylated with methyl iodide and analyzed by ESI-MS and CID-MS2.

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The ESI-MS spectrum of G-2 demonstrated a double-charged ion of [M + Na + H]2+ at m/z 1492, which matched with a gangliotetraose oligosaccharide structure containing four sialic acids and a ceramide with the molecular mass of 614. The CID-MS2 spectrum of this ion showed fragment ions corresponding to the partial structures of GQ1c (Fig. 7b). In the ESI-MS spectrum of G-3, a double-charged ion of [M + 2Na]2+ was observed at m/z 1659. The mass of this ion accorded with a gangliotetraose structure having five sialic acids and a ceramide with the molecular mass of 566. The CID-MS2 spectrum of the double-charged ion produced fragment ions that accorded with the partial structures of GP1c (Fig. 7c).

Electrospray ionization-mass spectrometry of G-4

As suggested above, the ganglioside G-4 in bonito fish brain was a gangliotetraose ganglioside that had the same chromatographic mobility of G′H′ on TLC. The structure of G-4 was further analyzed by ESI-MS of the permethylated derivative (Table 2, Fig. 8). The mass spectrum of G-4 showed a double-charged ion of [M + 2Na]2+ at m/z 1840; this ion corresponded with a gangliotetraose oligosaccharide having six sialic acids and a ceramide with the molecular mass of 566. Among the fragment ions in the CID-MS2 spectrum of the double-charged ion, the ion at m/z 1104 corresponded with the structure of (NeuAc)3 + Gal-Glc-Cer. Another fragment ion at m/z 849 matched with tri-O-methyl-Glc-Cer. Thus, it was concluded that a trisialosyl residue was not connected to the glucose, but was linked to the inner galactose of the gangliotetraose oligosaccharide. As G-4 was shown to contain the same oligosaccharide structure of GM1, the innermost sialic acid of the trisialosyl residue was assumed to be connected to the galactose through an α2-3 linkage. The fragment ion at m/z 1327 corresponded with the structure of (NeuAc)3-(tri-O-methyl-hexose). This hexose could not be either the glucose or the inner galactose. Accordingly, it was deduced that another trisialosyl residue was connected to the terminal galactose of the gangliotetraose oligosaccharide structure. Based upon these findings, it was shown that G-4 had the structure of NeuAc-NeuAc-NeuAc-Galβ1-3GalNAcβ1-4(NeuAc-NeuAc- NeuAcα2-3)Galβ1-4Glcβ1-1′Cer. Accordingly, this ganglioside was assigned to GH1c.

Table 2.   ESI-MS of A2B5-reactive ganglioside G-4 in bonito fish brain
 ions observed [m/z]Corresponding structure
  1. The ganglioside was permethylated and analyzed by positive ESI-MS and CID-MS2.

MS1840[M + 2Na]2+
CID-MS2376[NeuAc + H]+
577[Cer + H]+
727[NeuAc-Gal-Glc-Cer + 2Na + 2K − 2H]2+
738[(NeuAc)2 + H]+
834[NeuAc-Gal-GaINAc + Na]+
849[Glc-Cer + 3Na − 2H]+
923[Gal-GaINAc-Gal-Glc + Na + K − H]+
996[Gal-Glc-Cer + Na]+
1103[M − 4NeuAc + 2Na]2+
1104[(NeuAc)3-Gal-Glc-Cer + 2Na + 2K − 2H]2+
1121[(NeuAc)3 + Na]+
1283[M − 3NeuAc + 2Na]2+
1327[(NeuAc)3-Gal + Na]+
1471[M − 2NeuAc + 2Na]2+
1652[M − NeuAc + 2Na]2+
1733[(NeuAc)2-Gal-Glc-Cer + Na]+
1821[M − 5NeuAc + Na]+
image

Figure 8.  The ESI-MS and CID-MS2 of G-4. The ganglioside G-4 was permethylated and analyzed by ESI-MS (a) and CID-MS2 (b). The upper chart shows the assumed structure of G-4 and fragmentation patterns.

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Specificity of A2B5 to c-series gangliosides

The reactivity of monoclonal antibody A2B5 to c-series gangliosides and other glycolipids was examined using TLC immunostaining. As shown in Table 3, A2B5 reacted with GT3, GQ1c, GP1c and GH1c, but not with GD3 and other gangliosides and neutral glycosphingolipids. In fact, A2B5 did not react with these non-c-series gangliosides or glycolipids up to the quantities of 1 nmol (data not shown). Based upon these results, it was concluded that the antigenic epitope for A2B5 included a trisialosyl residue linked to the inner galactose of the hemato- or ganglio-type oligosaccharide of c-series gangliosides. The A2B5 reactivity of c-series gangliosides decreased in order of GT3 > GQ1c > GP1c > GH1c (Fig. 9).

Table 3.   A2B5-reactivity of gangliosides and other glycolipids. A certain amount of lipid (50 pmol) was developed on TLC and immunostained with A2B5.
LipidStructureReactivity
  1. *sialylparagloboside. ** GlcCer, GalCer, LacCer, GgOse3Cer, GgOse4Cer, CTH and globoside.

GM3NeuAcα2-3Galβ1-4Glcβ1-1′Cer0
GM1Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1′Cer0
3 | 
NeuAcα2 
GD3NeuAcα2-8NeuAcα2-3Galβ1-4GIcβ1-1′Cer0
GD1aNeuAcα2-3Galβ1-3GaINAcβ1-4Galβ1-4Glcβ1-1′Cer0
3 | 
NeuAcα2 
GD1bGalβ1-3GaINAcβ1-4Galβ1-4GIcβ1-1′Cer 0
3 | 
NeuAcα2-8NeuAcα2 
GT3NeuAcα2-8NeuAcα2-8NeuAcα2-3Galβ1-4Glcβ1-1′Cer100
GT1bNeuAcα2-3Galβ1-3GaINAcβ1-4Galβ1-4Glcβ1- 1′Cer0
3 | 
NeuAcα2-8NeuAcα2 
GQ1bNeuAcα2-8NeuAcα2-3Galβ1-3GaINAcβ1-4Galβ1-4Glcβ1-1′Cer0
3 | 
NeuAcα2-8NeuAcα2 
GQlcNeuAcα2-3Galβ1-3GaINAcβ1-4Galβ1-4Glcβ1- 1′Cer59
3 | 
NeuAcα2-8NeuAcα2-8NeuAcα2 
GP1cNeuAcα2-8NeuAcα2-3Galβ1-3GaINAcβ1-4Galβ1-4Glcβ1-1′Cer49
3 | 
NeuAcα2-8NeuAcα2-8NeuAcα2 
GH1cNeuAcα2-8NeuAcα2-8NeuAcα2-3Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1′Cer36
3 | 
NeuAcα2-8NeuAcα2-8NeuAcα2 
Fuc-GM1Fucα1-2Galβ1-3GaINAcβ1-4Galβ1-4Glcβ1-1′Cer0
3 | 
NeuAcα2 
LM1*NeuAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer0
Othersneutral glycosphingolipids**0
image

Figure 9.  The A2B5 reactivity of c-series gangliosides. (a) Different amounts of c-series gangliosides were developed on TLC and immunostained with A2B5. (b) The reactive bands were quantitated by densitometric analysis. ○, GT3; ●, GQ1c; □, GP1c; ▪, GH1c.

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Phylogenetic profiles of major and c-series gangliosides in brain tissue

The phylogeny of c-series gangliosides in brain tissue was examined using the A2B5 preparation. As shown in Fig. 10(a), the composition of brain gangliosides significantly changed along the phylogenetic line; the proportions of polysialo gangliosides decreased concomitantly with increasing ratios of less-polar gangliosides in higher animals. c-Series gangliosides in brain tissue showed a distinct phylogenetic pattern; they were enriched in lower animals including different bony fish, but hardly detected in mammalian species (Fig. 10b).

image

Figure 10.  Phylogenetic profiles of the compositions of major and c-series gangliosides in brain tissue. Gangliosides were isolated from brain tissue of different animals of adult age and visualized with resorcinol-HCl reagent (a) or immunostained with A2B5 (b). In both panels: lane 1, pacific mackerel; lane 2, bonito; lane 3, bluefin tuna; lane 4, red sea bream; lane 5, bullfrog; lane 6, terrapin; lane 7, chicken; lane 8, rat; lane 9, pig. The amount of gangliosides per lane is equivalent to 1 µg (a) or 50 ng sialic acid (b).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

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.

The phylogeny of major gangliosides in brain tissue has been well documented (Iwamori and Nagai 1978; Nagai and Iwamori 1980; Irwin 1984; Hilbig 1984; Hilbig and Rahmann 1987; Kappel et al. 1993; Irvine and Seyfried 1994). The proportion of polysialo species tends to decrease concomitantly with increasing proportions of less-polar species along the phylogenetic line, though some exceptions have been observed (Irvine and Seyfried 1994). The present study not only confirmed these phylogenetic trends of major gangliosides, but also demonstrated a distinct phylogenetic pattern of c-series gangliosides in brain tissue. It is known that c-series gangliosides are expressed in cartilaginous fish brain (Nakamura et al. 1997; Nakamura et al. 2000). Thus, it is assumed that c-series gangliosides are not restricted to certain fish species, but more widely distributed in bony and cartilaginous fish than expected previously.

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors would like to thank Drs Susumu Ando, Kazuo Kon and Hatsue Waki (Tokyo Metropolitan Institute of Gerontology, Japan) for their valuable comments and discussion on this study.

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  3. Materials and methods
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  5. Discussion
  6. Acknowledgements
  7. References
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