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Occurrence and tissue distribution of c-series gangliosides in the common squid Todarodes pacificus
Comparative Biochemistry and Physiology Part B 131 (2002) 433–441
Occurrence and tissue distribution of c-series gangliosides in the common squid Todarodes pacificus Megumi Saito*, Hisayo Kitamura, Kiyoshi Sugiyama Department of Clinical Pharmacology and Therapeutics, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan Received 17 August 2001; received in revised form 12 November 2001; accepted 22 November 2001
Abstract We have recently demonstrated that the common squid Todarodes pacificus express acidic lipids that were reactive with a monoclonal antibody A2B5. In the present study, two A2B5-reactive acidic lipids were isolated from squid hepatopancreatic tissue and characterized for their structures by methods including glycolipid overlay analysis, product analysis after sialidase treatment, and electrospray ionization-mass spectrometry (ESI-MS). Accordingly, the two acidic lipid were identified as GT3 and GQ1c, respectively. Another A2B5-reactive acidic lipid in the tissue was tentatively assigned to GT2 based upon its reactivity to A2B5 and chromatographic mobility on thin-layer chromatography. The composition and concentration of c-series gangliosides significantly differed among squid tissues (i.e. hepatopancreas, cerebral ganglion, eye lens, and mantle tissue). Interestingly, the percentages of c-series gangliosides within total gangliosides of hepatopancreas and cerebral ganglion were even higher than that of cod fish brain, which is known to be highly enriched with this ganglioside species. These findings strongly support the hypothesis that c-series gangliosides in squid tissues are not derived from ganglioside-containing food intake, but biosynthesized in a tissue-specific manner. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Gangliosides; C-series gangliosides; Sialic acids; A2B5; Squid; Hepatopancreas; Cerebral ganglion; Eye lens; Mollusk; Protostomia
1. Introduction C-series gangliosides are characterized by a trisialosyl residue at the inner galactose of the hemato- or ganglio-type oligosaccharide structure (Yu and Ando, 1980). They have been detected only in the chordate among deuterostomia, though their concentrations and compositions significantly Abbreviations: Cer, ceramide: GlcCer, Glc1-19Cer; GalCer, Gal1-19Cer; LacCer, Galb1-4Glc1-19Cer; GgOse3Cer, GalNAcb1-4Galb1-4Glc1-19Cer; GgOse4Cer, Galb1-3GalNAcb1-4Galb1-4Glc1-19Cer. *Corresponding author. Tel.: q81-54-264-5763; fax: q8154-264-5764. E-mail address: [email protected] (M. Saito).
differ among animal species. C-series gangliosides are enriched in brain tissues of bony fish such as cod (Ando and Yu, 1979; Yu and Ando, 1980) and 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 brain was also reported (Waki et al., 1993a,b). Our recent study suggested that c-series gangliosides are not restricted to particular fish species, but widely distributed in bony and cartilaginous fish (Saito et al., 2001a). C-series gangliosides are also found in avian and mammalian tissues of neural and extraneural origin. The ganglioside GP1c was identified in embryonic chicken brain (Rosner et al., 1985) and
1096-4959/02/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 6 - 4 9 5 9 Ž 0 1 . 0 0 5 1 7 - 6
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Table 1 Electrospray ionization-mass spectrometry (ESI-MS) of A2B5-reactive acidic lipids a and b in squid hepatopancreas Ions observed wmyzx Lipid a
Lipid b 2q
MS
1035
wMq2Hx
CID-MS2
376 622 738 789 971 1136 1547 1676 679 705
wNeuAcqHxq wCerq2KyHxq w(NeuAc)2qHxq wGlc-CerqNaxq wGal-Glc-CerqHxq w(NeuAc)3qKxq w(NeuAc)3-Gal-Glc-Naxq w(NeuAc)2-Gal-Glc-CerqHxq w(NeuAc)3-GalqKqHx2q wNeuAc-Gal-Glc-Cerq2Kx2q
1492
wMq2Hx2q
636 651 737 871 1105 1120 1460 1768 1350 781 1110
wNeuAc-GalqKxq wCerqHxq w(NeuAc)2qHxq wGlc-CerqHxq wGal-Glc-Cerq2NayHxq w(NeuAc)3qNaxq wNeuAc-Gal-Glc-CerqKxq w(NeuAc)2-Gal-Glc-CerqHxq wGaINAc-Gal-Glc-Cerq3NayHx2q w(NeuAc)3-Gal-Glc-CerqNaqKx2q
The acidic lipids were treated with methyl iodide and anaylzed by positive ESI-MS and collision-induced dissociation (CID)-MS2. The structure corresponding to each ion peak in the spectrum is shown in the bracket.
human brain (Miller-Podraza et al., 1991), whereas GT3 was detected in hog kidney (MurakamiMurofushi et al., 1981), cat erythrocytes (Ando and Yamakawa, 1982), and human lung (Mansson et al., 1986). No evidence has been provided for the expression of c-series gangliosides in echinoderms or lower animals among deuterostomia (Hosh and Nagai, 1975; Kochetkov et al., 1976; Sugita, 1979; Prokazova et al., 1981; Smirnova et al., 1987) or animals among protostomia. Recently, we investigated acidic lipids in some protostomian animals, i.e. the common squid Todarodes pacificus and pacific octopus Octopus vulgaris, and found that some lipids were reactive with a c-series ganglioside-specific monoclonal antibody A2B5; the structures of these acidic lipids remained to be determined (Saito et al., 2001b). In the present study, we examined A2B5-reactive acidic lipids in the common squid for their structures and distribution among different tissues. 2. Materials and methods 2.1. Materials The common squid Todarodes pacificus and cod fish Gadus macrocephallus were obtained from local fish markets. Each squid tissue, i.e. hepatopancreas, cerebral ganglion, eye lenses, or mantle tissue was carefully dissected from other tissues including digestive tracts. A c-series ganglioside-
specific monoclonal antibody A2B5 was prepared as follows. A2B5-producing hybridomas (CRL 1520, American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. The culture medium containing the antibody (IgM-type) was used for experiments (Saito et al., 2001a). An anti-GM1 antibody (IgG-type) was prepared by immunizing rabbits with purified ganglioside. This antibody was successfully employed for specific detection of GM1 (Saito and Sugiyama, 2000). GlcCer, LacCer, GgOse3Cer and GgOse4Cer were purified from mild acid hydrolysates of rat brain gangliosides using a method reported previously (Svennerholm et al., 1973). Other chemicals and reagents were obtained from the following companies: Clostridium perfringens sialidase, Arthrobacter ureafaciens sialidase, goat peroxidase-conjugated antibodies against mouse IgM or rabbit IgG, peroxidaseconjugated cholera toxin B subunit, N-acetylneuraminic acid, and N-glycolylneuraminic acid (Sigma, St. Louis, MO, USA), Salmonella typhimurium sialidase (a2,3 specific, cloned from S. typhimurium LT2 and expressed in Escherichia coli) (Takara Shuzo Co., Ltd., Tokyo, Japan), high performance thin-layer chromatographic (TLC) plates (nanoplates, Merck KGaA, Darmstadt, Germany), and enhanced chemiluminescence (ECL) Western blotting detection kits (Amersham Pharmacia Biotech, Buckinghamshire, England).
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2.1.1. Isolation of acidic lipids Total lipids were extracted from squid tissues with 20 volumes of chloroformymethanol (1:1) and separated into neutral and acidic lipids by DEAE-Sephadex column chromatography. Acidic lipids were incubated in 0.2 M methanolic NaOH at 37 8C for 1 h, then neutralized with acetic acid. Alkali-stable acidic lipids were obtained after desalting of the neutralized mixture by Sephadex LH-20 column chromatography. 2.2. Overlay analysis Overlay analysis of acidic lipids with glycolipidspecific antibodies was carried out based upon a method reported previously (Saito et al., 1985). Acidic lipids were developed on a TLC plate. After coating with a 0.4% polyisobutylmethacrylate solution, the plate was overlaid consecutively by an anti-glycolipid antibody and peroxidaseconjugated second antibody at room temperature for 1.5 h. When lipids were analyzed with a peroxidase-conjugated cholera toxin B subunit, the step with second antibody was omitted (Wu and Ledeen, 1988). The reacted band(s) were detected on an X-ray film using the ECL method. Acidic lipids on the plate was then visualized with resorcinol-HCl reagent (Svennerholm, 1957) or orcinolH2SO4 reagent (Sewell, 1979). Densitometric analysis of chromatograms was carried out using a densitometric image analyzer (Atto Densitograph AE-6920M, Atto Co., Tokyo, Japan). 2.3. Characterization of acidic lipids using different sialidases Two A2B5-reactive acidic lipids in squid hepatopancreatic tissue were isolated by preparative TLC and LH-20 column chromatography. They were finally purified on HPLC with a size exclusion column (TSKgel a-2500, 0.8 = 30 cm, Tosoh, Tokyo, Japan) with a solvent of methanol. The purified lipid was treated with one of three different sialidases, i.e. the enzyme derived from C. perfringens, S. typhimurium, or A. ureafaciens. In the case of C. perfringens sialidase, the reaction mixture consisted of 100 mM sodium acetate (pH 4.8), acidic lipid, and 250 mUyml of the enzyme in a final volume of 0.1 ml. The mixture was incubated at 37 8C for 45 min. The reaction with S. typhimurium sialidase was carried out in a
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similar manner, except that the pH was 5.5. In both cases, the hydrolysis product(s) were isolated by LH-20 column chromatography of the reaction mixture. In another experiment, the lipid was treated with A. ureafaciens enzyme (250 mUyml, pH 4.8) in the presence of sodium deoxycholate (0.5 mgyml) at 37 8C for 45 min. The neutral lipid product(s) were purified using DEAESephadex column chromatography. 2.4. Fluorometric high performance liquid chromatographic (HPLC) analysis of sialic acids Lipid-bound sialic acids were analyzed using a fluorometric HPLC method (Hara et al., 1987). An purified acidic lipid was hydrolyzed by incubation in 25 mM sulfuric acid at 80 8C for 1 h. The hydrolysate was incubated with a 1,2-diamino4,5-methylene-dioxybenzene (DMB) reagent at 60 8C for 2.5 h. The fluorescent derivative(s) were analyzed by HPLC with an ODS column and fluorescence detector. The fluorescence excitation and emission wavelengths were at 373 and 448 nm, respectively. 2.5. Electrospray ionization-mass spectrometry (ESI-MS) of gangliosides ESI-MS of A2B5-reactive acidic lipids was carried out using an LCQ ion-trap mass spectrometer equipped with an ESI source (Finnigan MAT, USA). An acidic lipid was treated with methyl iodide using a method reported previously (Tadano-Aritomi et al., 1992). The permethylated derivative, which formed a single band on TLC, was dissolved in methanol at a concentration of 10 pmolyml, and introduced into the electrospray needle by mechanical infusion at a flow rate of 3 mlymin. The ESI capillary was kept at a voltage of q4 V at 200 8C. The tube lens offset was set at y30 V. The collision-induced dissociation (CID)-MS2 spectra were taken using helium as the collision gas. The relative collision energy scale was set at q2.5 eV. Mass spectra were averaged over ten scans. 3. Results 3.1. A2B5-reactive acidic lipids in squid hepatopancreatic tissue Acidic lipids in hepatopancreatic tissue of the common squid were developed on TLC and visu-
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alized with resorcinol-HCl reagent or immunostained with a monoclonal antibody A2B5. The tissue had a complex composition of resorcinolpositive acidic lipids (Fig. 1a, lane 2). At least three acidic lipids were reactive with A2B5; they respectively migrated near GD1b (designated as lipid a), above GT1b (lipid x), and below GQ1b on TLC (lipid b) (Fig. 1a, lane 3). Among these A2B5-reactive acidic lipids, lipids a and b were purified using methods including preparative TLC and HPLC with a size exclusion column. Lipid a formed a double band on TLC, whereas lipid b migrated as a single band (Fig. 1b). 3.2. Product analysis after sialidase treatment of A2B5-reactive acidic lipids Lipids a and b were treated with one of sialidases, and the reaction products were analyzed. The treatment of lipid a with A. ureafaciens sialidase generated an orcinol-positive neutral lipid product. This lipid was assigned to LacCer from its chromatographic mobility on TLC (Fig. 2, lane 2). Lipid b was hydrolyzed with C. perfringens sialidase, producing a resorcinol-positive acidic lipid product. This lipid was identified to be GM1 based upon the chromatographic mobility and reactivity with cholera toxin B subunit (Fig. 3, lanes 3 and 4) and with anti-GM1 antibody on TLC (data not shown). Furthermore, the overlay analysis of the reaction mixture with cholera toxin B subunit revealed another lipid product at the same
Fig. 2. Hydrolysis of lipid a by A. ureafaciens sialidase. Lipid a was incubated with A. ureafaciens sialidase (250 mUyml, at pH 4.8) at 37 8C for 45 min in the presence of sodium deoxycholate (0.5 mgyml). The neutral lipid product was developed on TLC with a solvent system of chloroformymethanolyH2 O (65:30:6) and visualized with orcinol-H2SO4 reagent. Lane 1, lipid a; lane 2, the reaction product after sialidase treatment of lipid a; and lane 3, standard neutral glycolipids.
position as GD1b on TLC (Fig. 3, lane 4). The treatment of lipid b with a2,3 specific S. typhimurium sialidase generated a single resorcinolpositive lipid product, which migrated between GT1b and GQ1b on TLC (Fig. 4, lane 3). This lipid product (designated as lipid y) was assumed to be GT1c based upon its A2B5 reactivity and chromatographic mobility on TLC (Fig. 4, lane 4).
Fig. 1. Acidic lipids in hepatopancreatic tissue of the common squid. (a) Acidic lipids were isolated from squid hepatopancreatic tissue by methods including DEAE-Sephadex column chromatography, mild base treatment and Sephadex LH-20 column chromatography. The purified acidic lipids were developed on TLC with a solvent system of chloroformymethanoly0.2% CaCl2 Ø2H2 O (45:40:10), and visualized with resorcinol-HCl reagent (lane 2) or immunostained with a monoclonal antibody A2B5 (lane 3). Lane 1, rat liver gangliosides visualized with resorcinol-HCl reagent (as reference). (b) Lipids a and b in squid hepatopancreatic tissue were purified and visualized with resorcinol-HCl reagent (lanes 2 and 3) or immunostained with A2B5 (lanes 4 and 5). Lanes 2 and 4, lipid a; and lanes 3 and 5, lipid b. Lane 1, squid hepatopancreatic acidic lipids visualized with resorcinol-HCl reagent.
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Fig. 3. Partial hydrolysis of lipid b by C. perfringens sialidase. Lipid b was incubated with C. perfringens sialidase (250 mUyml, at pH 4.8) at 37 8C for 45 min, and the lipid products were developed on TLC. Lane 1, rat liver gangliosides: lane 2, lipid b before sialidase treatment; and lanes 3 and 4, lipid products after sialidase treatment of lipid b. Lanes 1–3, visualized with resorcinol-HCl reagent; and lane 4, reacted with cholera toxin B subunit.
N-Acetylneuraminic acid, but not N-glycolylneuraminic acid, was detected in lipids a and b using a fluorometric HPLC method (data not shown). 3.3. ESI-MS of A2B5-reactive acidic lipids Based upon the above-mentioned findings, the A2B5-reactive lipids a and b were assumed to be GT3 and GQ1c, respectively. To confirm this assumption, each lipid was permethylated and analyzed by positive ESI-MS (Table 1). The ESIMS spectrum of lipid a showed a double-charged ion of wMq2Hx2q at myz 1035, which corresponded with a hemato-type oligosaccharide structure containing three N-acetylneuraminic acids with a ceramide of the molecular mass of 534 (as the non-methylated structure) (Fig. 5a). CID-MS2 of the double-charged ion produced ion peaks that matched with the partial structures of GT3 (Fig. 5b). An ion peak corresponding with a trisialosyl structure was observed at myz 1136. The ions at myz 789 and 971 accorded with the structures of (tri-O-methyl-Glc)-Cer and (tri-O-methyl-Gal)(tri-O-methyl-Glc)-Cer. Based upon these findings, it was concluded that the trisialosyl residue was connected to the galactose of LacCer. The ESI-MS spectrum of lipid b showed a double-charged ion of wMq2Hx2q at myz 1492; this ion matched with a gangliotetraose oligosac-
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charide structure containing four N-acetylneuraminic acids with a ceramide of the molecular mass of 638 (as the non-methylated form) (Fig. 6a). The CID-MS2 spectrum of this ion showed fragment ions corresponding to the partial structures of GQ1c (Fig. 6b). The ions at myz 1120 and 1110 (double-charged ion) accorded with the structures of (NeuAc)3 and (NeuAc)3qGal-GlcCer, whereas the ions at myz 871 and 1105 matched with the structures of (tri-O-methyl-Glc)Cer and (di-O-methyl-Gal)-(tri-O-methyl-Glc)Cer, respectively. These findings indicated that a trisialosyl residue was connected to the inner galactose of the gangliotetraose structure. The fragment ion at myz 636 corresponded with the structure of (NeuAc)-(tri-O-methyl-Gal). It was thus concluded that the fourth sialic acid residue was linked to the terminal galactose of GgOse4. 3.4. The concentration and composition of c-series gangliosides in squid tissues Prior to analysis of c-series gangliosides in squid tissues, the concentration of total gangliosides was determined by quantitation of lipid-bound sialic acids using the fluorometric HPLC method. The concentrations of gangliosides in hepatopancreas (ns5), cerebral ganglion (ns5), eye lens (ns4), and mantle tissue (ns1) were 2.5"1.4, 0.069"0.030, 0.11"0.09 and 0.013 mg sialic acid
Fig. 4. Partial hydrolysis of lipid b by S. typhimurium sialidase. Lipid b was treated with S. typhimurium sialidase (250 mUyml, at pH 5.5) at 37 8C for 45 min, and the lipid product was developed on TLC. Lane 1, rat liver gangliosides; and lane 2, lipid b before sialidase treatment. Lanes 3 and 4, the reaction product after sialidase treatment of lipid b. Lanes 1–3, visualized with resorcinol-HCl reagent; and lane 4, immunostained with A2B5.
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Fig. 5. ESI-MS of lipid a. Lipid a was permethylated with methyl iodide and analyzed by positive ESI-MS (a) and CID-MS2 (b). The upper chart shows the assumed structure of lipid a and fragmentation patterns.
per g wet tissue weight, respectively. Regarding the molecular species of lipid-bound sialic acids, only N-acetylneuraminic acid was detected in all tissues. The distribution of c-series gangliosides in common squid tissues was examined using A2B5 (Fig. 7). The percentages of c-series gangliosides within total gangliosides was highest in hepatopancreatic tissue, followed by cerebral ganglion and eye lens. Interestingly, the relative enrichment of c-series gangliosides in squid hepatopancreas and cerebral ganglion was even higher than that of cod fish brain. In contrast, c-series gangliosides were not detected in squid mantle tissue in this experimental condition. The composition of c-series gangliosides differed among hepatopancreas, cerebral ganglion and eye lens. Two A2B5-reactive gangliosides above GT1b (i.e. lipid x) and below GQ1c were observed in hepatopancreatic tissue, but not in other tissues. These gangliosides were tentatively identified as GT2 and GP1c based upon
their A2B5 reactivity and chromatographic mobility on TLC. 4. Discussion In a previous study, we demonstrated that the common squid Todarodes pacificus expressed acidic lipids, which were reactive with a monoclonal antibody A2B5 (Saito et al., 2001b) This antibody was originally prepared by immunizing chicken embryonic retinal cells (Eisenbarth et al., 1979). While there has been some controversy about its reactivity, recent studies have suggested the specific binding of A2B5 to c-series gangliosides (Fenderson et al., 1987; Dubois et al., 1990; Freischutz et al., 1994; Farrer and Quarles, 1999). Very recently, we examined a structure-reactivity relationship between A2B5 and gangliosides of different structures and confirmed its strict specificity to c-series gangliosides. The epitopic structure for the antibody was assumed to include a
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Fig. 6. ESI-MS of lipid b. Lipid b was permethylated with methyl iodide and analyzed by positive ESI-MS (a) and CID-MS2 (b). The upper chart shows the assumed structure of lipid b and fragmentation patterns.
trisialosyl residue connected to the inner galactose of c-series gangliosides (Saito et al., 2001a). Thus, it is strongly suggested that A2B5-reactive acidic lipids in these molluscan species are c-series gangliosides. In the present study, we isolated two A2B5reactive acidic lipids from squid hepatopancreatic tissue and characterized their structures. The oligosaccharide core structure of these acidic lipids were determined by product analysis after sialidase treatment; lipids a and b were shown to possess the hemato-type and gangliotetraose oligosaccharide core structure, respectively. Among two reaction products from lipid b after treatment with C.
perfringens sialidase, the lower one was assumed to be GD1b because of its chromatographic mobility on TLC and reactivity to cholera toxin B subunit (Cumar et al., 1982). Sialic acid residues (including the trisialosyl structure) and their attachment to the oligosaccharide core structures were successfully identified by positive ESI-MS and CID-MS2 of the permethylated derivatives. Although the linkage of sialic acids in the trisialosyl residue remains to be determined, it is most likely that sialic acids are connected through a2,8 linkages, as observed in other polysialosyl structures of natural compounds (Schauer, 1982). Since lipid b was shown to share the same oligosacchar-
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Fig. 7. The compositions of A2B5-reactive acidic lipids in squid tissues. Acidic lipids were isolated from squid tissues, developed on TLC, and immunostained with A2B5 (lanes 2– 6). Lanes 2, hepatopancreas; 3, cerebral ganglion; 4 eye lens; and 5, mantle tissue. Lane 6, cod brain gangliosides (as reference). The amount of gangliosides per lane is equivalent to 0.1 mg sialic acid per lane. Lane 1, rat liver gangliosides visualized with resorcinol-HCl reagent.
ide structure with GM1, it was concluded that the innermost sialic acid of the trisialosyl residue was connected to the inner galactose through a a2,3 linkage. Lipid b was shown to contain a sialic acid residue that was susceptible to the action of a2.3 specific sialidase. This sialic acid was assumed to be the one that was connected to the terminal galactose of the gangliotetraose oligosaccharide structure. Based upon these findings and specific reactivity to A2B5, lipids a and b were identified to be GT3 and GQ1c, respectively. Regarding the structures of sialic acids in GT3 and GQ1c, N-acetylneuraminic acid was detected as the sole molecular species. However, the ganglioside samples were prepared by procedures including mild base treatment. Thus, it is possible that these c-series gangliosides or parts of them may originally exist as alkali-labile O-acetyl derivatives of N-acetylneuraminic acid, as reported in cod fish brain (Waki et al., 1993a,b). Lipid x was tentatively assigned to GT2 based upon the reactivity with A2B5 and chromatographic behavior on TLC (Yu and Ando, 1980). Lipid y was supposed to be GT1c that is produced from GQ1c by the removal of the sialic acid residue at the terminal galactose. C-series gangliosides were found in various tissues of the common squid. As for the origin of these gangliosides, one may claim that gangliosides in squid tissues may be derived from contam-
ination by ganglioside-containing foods in the digestive tract. However, there are several reasons against this possibility. The hepatopancreas constitutes a part of the digestive system, but is clearly demarcated from digestive tracts and therefore, can be dissected without contamination by gut contents. Regarding the cerebral ganglion, eye lenses, and mantle tissues, there is no risk of possible contamination by food in digestive tracts, because of their anatomical location. Accordingly, the presence of gangliosides in these tissues strongly suggests that gangliosides are intrinsic components of squid tissues. This assumption is further supported by the fact that the concentration and composition of c-series gangliosides significantly differ among squid tissues. It has been accepted that c-series gangliosides are expressed only in the chordate among deuterostomia. C-series gangliosides are enriched in lower animals such as adult fish brain (Ando and Yu, 1979; Yu and Ando, 1980; Waki et al., 1993a,b; Nakamura et al., 1997, 2000; Saito et al., 2001a). In avian and mammalian brain, they and their O-acetyl derivatives are temporarily expressed at certain embryonic stages but hardly detected at adult ages (Dubois et al., 1986, 1990; Rosner et al., 1988, 1993; Hirabayashi et al., 1988, 1989; Letinic et al., 1998). In contrast, c-series gangliosides have never been detected in echinoderms among deuterostomia. The present study provides solid evidence against the prevailing view that c-series gangliosides are limited to the chordate among deuterostomia. References Ando, N., Yamakawa, T., 1982. On the minor gangliosides of erythrocyte membranes of Japanese cats. J. Biochem. (Tokyo) 91, 873–881. Ando, S., Yu, R.K., 1979. Isolation and characterization of two isomers of brain tetrasialogangliosides. J. Biol. Chem. 254, 12224–12229. Cumar, F.A., Maggio, B., Caputto, R., 1982. Gangliosidecholera toxin interactions: a binding and lipid monolayer study. Mol. Cell. Biochem. 46, 155–160. Dubois, C., Magnani, J.L., Grunwald, G.B., et al., 1986. Monoclonal antibody 18B8, which detects synapse-associated antigens, binds to ganglioside GT3 (II3 (NeuAc)3LacCer). J. Biol. Chem. 261, 3826–3830. Dubois, C., Manuguerra, J.-C., Hauttecoeur, B., Maze, J., 1990. Monoclonal antibody A2B5, which detects cell surface antigens, binds to ganglioside GT3 (II3(NeuAc)3LacCer) and to its 9-O-acetylated derivative. J. Biol. Chem. 265, 2797–2803.
M. Saito et al. / Comparative Biochemistry and Physiology Part B 131 (2002) 433–441 Eisenbarth, G.S., Shimizu, K., Bowring, M.A., Wells, S., 1979. Monoclonal antibody to a plasma membrane antigen of neurons. Proc. Natl. Acad. Sci. USA 76, 4913–4917. Farrer, R.G., Quarles, R.H., 1999. GT3 and its O-acetylated derivative are the principal A2B5-reactive gangliosides in cultured O2A lineage cells and are down-regulated along with O-acetyl GD3 during differentiation to oligodendrocytes. J. Neurosci. Res. 57, 371–380. Fenderson, B.A., Andrews, P.W., Nudelman, E., Clausen, H., Hakomori, S., 1987. Glycolipid core structure switching from globo- to lacto- and ganglio-series during retinoic acidinduced differentiation of TERA-2-derived human embryonal carcinoma cells. Dev. Biol. 122, 21–34. Freischutz, B., Saito, M., Rahmann, H., Yu, R.K., 1994. Activities of five different sialyltransferases in fish and rat brains. J. Neurochem. 62, 1965–1973. Hara, S., Takemori, Y., Yamaguchi, M., Nakamura, M., Ohkura, Y., 1987. Fluorometric high-performance liquid chromatography of N-acetyl- and N-glycolylneuraminic acids and its application to their microdetermination in human and animal sera, glycoproteins and glycolipids. Anal. Biochem. 164, 138–145. Hirabayashi, Y., Hirota, M., Matsumoto, M., Tanaka, H., Obata, K., Ando, S., 1988. Developmental changes of c-series polysialogangliosides in chick brains revealed by mouse monoclonal antibodies M6704 and M7103 with different epitope specificities. J. Biochem. (Tokyo) 104, 973–979. Hirabayashi, Y., Hirota, M., Suzuki, Y., Matsumoto, M., Obata, K., Ando, S., 1989. Developmentally expressed O-acetyl ganglioside GT3 in fetal rat cerebral cortex. Neurosci. Lett. 106, 193–198. Hosh, M., Nagai, Y., 1975. Novel sialosphingolipids from spermatozoa of the sea urchin Anthocidaris grassispina. Biochim. Biophys. Acta 388, 152–162. Kochetkov, N.K., Smirnov, G.P., Chekareva, N.V., 1976. Isolation and structural studies of sulfated sialosphingolipid from the sea urchin Echinocardium cordatum. Biochim. Biophys. Acta 424, 274–283. Letinic, K., Heffer-Lauc, M., Rosner, H., Kostovic, I., 1998. C-pathway polysialogangliosides are transiently expressed in the human cerebrum during fetal development. Neurosci. 86, 1–5. Mansson, J.-E., Mo, H., Egge, H., Svennerholm, L., 1986. Trisialosyllactosylceramide (GT3) is a ganglioside of human lung. FEBS Lett. 196, 259–262. Miller-Podraza, H., Mansson, J.-E., Svennerholm, L., 1991. Pentasialogangliosides of human brain. FEBS Lett. 288, 212–214. Murakami-Murofushi, K., Tadano, K., Koyama, I., Ishizuka, I., 1981. A trisialosylganglioside GT3 of hog kidney. Structure and biosynthesis in vitro. J. Biochem. (Tokyo) 90, 1817–1820. Nakamura, K., Tamai, Y., Kasama, T., 1997. Ganglioside of dogfish (SQUALUS ACANTHIAS) brain. Neurochem. Int. 30, 593–604. Nakamura, K., Kojima, H., Suzuki, M., Suzuki, A., Tamai, Y., 2000. Novel polysialogangliosides of skate brain. Structure determination of tetra, penta and hexasialogangliosides with a NeuAc-GalNAc linkage. Eur. J. Biochem. 267, 5198–5208. Prokazova, N.V., Mikhailov, A.T., Kocharov, S.L., et al., 1981. Unusual gangliosides of eggs and embryos of the sea urchin
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Strongylocentrotus intermedius. Eur. J. Biochem. 115, 671–677. Rosner, H., Rahmann, H., Reuter, G., Schauer, R., PeterKatalinic, J., Egge, H., 1985. Mass spectrometric identification of the pentasialoganglioside GP1c of embryonic chicken brain. Biol. Chem. Hoppe-Seyler 366, 1177–1181. Rosner, H., Greis, C., Henkle-Fahle, S., 1988. Developmental expression in embryonic rat and chicken brain of a polysialoganglioside-antigen reacting with the monoclonal antibody Q211. Dev. Brain Res. 42, 161–171. Rosner, H., Sonnentag, U., Meiri, K., 1993. Developmental changes of growth cone gangliosides of the postnatal rat cerebrum. Neuroreport 4, 1207–1210. Saito, M., Yu, R.K., Cheung, N.-K.V., 1985. Ganglioside GD2 specificity of monoclonal antibodies to human neuroblastoma cell. Biochem. Biophys. Res. Commun. 127, 1–7. Saito, M., Sugiyama, K., 2000. A distinct ganglioside composition of rat pancreatic islets. Arch. Biochem. Biophys. 376, 371–376. Saito, M., Kitamura, H., Sugiyama, K., 2001. The specificity of monoclonal antibody A2B5 to c-series gangliosides. J. Neurochem. 78, 1–11. Saito, M., Kitamura, H., Sugiyama, K., 2001. Occurrence of gangliosides in the common squid and pacific octopus among protostomia. Biochim. Biophys. Acta 1511, 271–280. Schauer, R., 1982. Chemistry, metabolism and biological function of sialic acid. Adv. Carbohyd. Chem. Biochem. 40, 131–234. Sewell, A.C., 1979. An improved thin-layer chromatographic method for urinary oligosaccharide screening. Clin. Chim. Acta 92, 411–414. Smirnova, G.P., Kochetkov, N.K., Sadovskaya, V.L., 1987. Gangliosides of the starfish Aphelasterias japonica, evidence for a new linkage between two N-glycolylneuraminic acid residues through the hydroxy group of the glycolic acid residue. Biochim. Biophys. Acta 920, 47–55. Sugita, M., 1979. Studies on the glycosphingolipid of the starfish, Asterina pectinifera. J. Biochem. 86, 289–300. Svennerholm, L., 1957. Quantitative estimation of sialic acids. II. a colorimetric resorcinol-hydrochloric acid method. Biochim. Biophys. Acta 24, 604–611. Svennerholm, L., Mansson, J.E., Li, Y.T., 1973. Isolation and structural determination of a novel ganglioside, a disialosylpentahexosylceramide from human brain. J. Biol. Chem. 248, 740–742. Tadano-Aritomi, K., Kasama, T., Handa, S., Ishizuka, I., 1992. Isolation and structural characterization of a mono-sulfated isoglobotetraosyl-ceramide, the first sulfoglycosphingolipid of the isoglobo-series, from rat kidney. Eur. J. Biochem. 209, 305–313. Waki, H., Murata, A., Kon, K., Maruyama, K., Kimura, S., Ogura, H., Ando, S., 1993. Isolation and characterization of a trisialyllactosylceramide, GT3, containing an O-acetylated sialic acid in cod fish brain. J. Biochem. 113, 502–507. Waki, H., Masuzawa, A., Kon, K., Ando, S., 1993. A new Oacetylated trisialoganglioside, 9-O-acetyl GT2, in cod brain. J. Biochem. (Tokyo) 114, 459–462. Wu, G., Ledeen, G.R., 1988. Quantification of gangliotetraose gangliosides with cholera toxin. Anal. Biochem. 173, 368–375. Yu, R.K., Ando, S., 1980. Structures of some new complex gangliosides of fish brain. Adv. Exp. Med. Biol. 125, 33–45.
Occurrence and tissue distribution of c-series gangliosides in the common squid Todarodes pacificus Megumi Saito*, Hisayo Kitamura, Kiyoshi Sugiyama Department of Clinical Pharmacology and Therapeutics, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan Received 17 August 2001; received in revised form 12 November 2001; accepted 22 November 2001
Abstract We have recently demonstrated that the common squid Todarodes pacificus express acidic lipids that were reactive with a monoclonal antibody A2B5. In the present study, two A2B5-reactive acidic lipids were isolated from squid hepatopancreatic tissue and characterized for their structures by methods including glycolipid overlay analysis, product analysis after sialidase treatment, and electrospray ionization-mass spectrometry (ESI-MS). Accordingly, the two acidic lipid were identified as GT3 and GQ1c, respectively. Another A2B5-reactive acidic lipid in the tissue was tentatively assigned to GT2 based upon its reactivity to A2B5 and chromatographic mobility on thin-layer chromatography. The composition and concentration of c-series gangliosides significantly differed among squid tissues (i.e. hepatopancreas, cerebral ganglion, eye lens, and mantle tissue). Interestingly, the percentages of c-series gangliosides within total gangliosides of hepatopancreas and cerebral ganglion were even higher than that of cod fish brain, which is known to be highly enriched with this ganglioside species. These findings strongly support the hypothesis that c-series gangliosides in squid tissues are not derived from ganglioside-containing food intake, but biosynthesized in a tissue-specific manner. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Gangliosides; C-series gangliosides; Sialic acids; A2B5; Squid; Hepatopancreas; Cerebral ganglion; Eye lens; Mollusk; Protostomia
1. Introduction C-series gangliosides are characterized by a trisialosyl residue at the inner galactose of the hemato- or ganglio-type oligosaccharide structure (Yu and Ando, 1980). They have been detected only in the chordate among deuterostomia, though their concentrations and compositions significantly Abbreviations: Cer, ceramide: GlcCer, Glc1-19Cer; GalCer, Gal1-19Cer; LacCer, Galb1-4Glc1-19Cer; GgOse3Cer, GalNAcb1-4Galb1-4Glc1-19Cer; GgOse4Cer, Galb1-3GalNAcb1-4Galb1-4Glc1-19Cer. *Corresponding author. Tel.: q81-54-264-5763; fax: q8154-264-5764. E-mail address: [email protected] (M. Saito).
differ among animal species. C-series gangliosides are enriched in brain tissues of bony fish such as cod (Ando and Yu, 1979; Yu and Ando, 1980) and 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 brain was also reported (Waki et al., 1993a,b). Our recent study suggested that c-series gangliosides are not restricted to particular fish species, but widely distributed in bony and cartilaginous fish (Saito et al., 2001a). C-series gangliosides are also found in avian and mammalian tissues of neural and extraneural origin. The ganglioside GP1c was identified in embryonic chicken brain (Rosner et al., 1985) and
1096-4959/02/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 6 - 4 9 5 9 Ž 0 1 . 0 0 5 1 7 - 6
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Table 1 Electrospray ionization-mass spectrometry (ESI-MS) of A2B5-reactive acidic lipids a and b in squid hepatopancreas Ions observed wmyzx Lipid a
Lipid b 2q
MS
1035
wMq2Hx
CID-MS2
376 622 738 789 971 1136 1547 1676 679 705
wNeuAcqHxq wCerq2KyHxq w(NeuAc)2qHxq wGlc-CerqNaxq wGal-Glc-CerqHxq w(NeuAc)3qKxq w(NeuAc)3-Gal-Glc-Naxq w(NeuAc)2-Gal-Glc-CerqHxq w(NeuAc)3-GalqKqHx2q wNeuAc-Gal-Glc-Cerq2Kx2q
1492
wMq2Hx2q
636 651 737 871 1105 1120 1460 1768 1350 781 1110
wNeuAc-GalqKxq wCerqHxq w(NeuAc)2qHxq wGlc-CerqHxq wGal-Glc-Cerq2NayHxq w(NeuAc)3qNaxq wNeuAc-Gal-Glc-CerqKxq w(NeuAc)2-Gal-Glc-CerqHxq wGaINAc-Gal-Glc-Cerq3NayHx2q w(NeuAc)3-Gal-Glc-CerqNaqKx2q
The acidic lipids were treated with methyl iodide and anaylzed by positive ESI-MS and collision-induced dissociation (CID)-MS2. The structure corresponding to each ion peak in the spectrum is shown in the bracket.
human brain (Miller-Podraza et al., 1991), whereas GT3 was detected in hog kidney (MurakamiMurofushi et al., 1981), cat erythrocytes (Ando and Yamakawa, 1982), and human lung (Mansson et al., 1986). No evidence has been provided for the expression of c-series gangliosides in echinoderms or lower animals among deuterostomia (Hosh and Nagai, 1975; Kochetkov et al., 1976; Sugita, 1979; Prokazova et al., 1981; Smirnova et al., 1987) or animals among protostomia. Recently, we investigated acidic lipids in some protostomian animals, i.e. the common squid Todarodes pacificus and pacific octopus Octopus vulgaris, and found that some lipids were reactive with a c-series ganglioside-specific monoclonal antibody A2B5; the structures of these acidic lipids remained to be determined (Saito et al., 2001b). In the present study, we examined A2B5-reactive acidic lipids in the common squid for their structures and distribution among different tissues. 2. Materials and methods 2.1. Materials The common squid Todarodes pacificus and cod fish Gadus macrocephallus were obtained from local fish markets. Each squid tissue, i.e. hepatopancreas, cerebral ganglion, eye lenses, or mantle tissue was carefully dissected from other tissues including digestive tracts. A c-series ganglioside-
specific monoclonal antibody A2B5 was prepared as follows. A2B5-producing hybridomas (CRL 1520, American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. The culture medium containing the antibody (IgM-type) was used for experiments (Saito et al., 2001a). An anti-GM1 antibody (IgG-type) was prepared by immunizing rabbits with purified ganglioside. This antibody was successfully employed for specific detection of GM1 (Saito and Sugiyama, 2000). GlcCer, LacCer, GgOse3Cer and GgOse4Cer were purified from mild acid hydrolysates of rat brain gangliosides using a method reported previously (Svennerholm et al., 1973). Other chemicals and reagents were obtained from the following companies: Clostridium perfringens sialidase, Arthrobacter ureafaciens sialidase, goat peroxidase-conjugated antibodies against mouse IgM or rabbit IgG, peroxidaseconjugated cholera toxin B subunit, N-acetylneuraminic acid, and N-glycolylneuraminic acid (Sigma, St. Louis, MO, USA), Salmonella typhimurium sialidase (a2,3 specific, cloned from S. typhimurium LT2 and expressed in Escherichia coli) (Takara Shuzo Co., Ltd., Tokyo, Japan), high performance thin-layer chromatographic (TLC) plates (nanoplates, Merck KGaA, Darmstadt, Germany), and enhanced chemiluminescence (ECL) Western blotting detection kits (Amersham Pharmacia Biotech, Buckinghamshire, England).
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2.1.1. Isolation of acidic lipids Total lipids were extracted from squid tissues with 20 volumes of chloroformymethanol (1:1) and separated into neutral and acidic lipids by DEAE-Sephadex column chromatography. Acidic lipids were incubated in 0.2 M methanolic NaOH at 37 8C for 1 h, then neutralized with acetic acid. Alkali-stable acidic lipids were obtained after desalting of the neutralized mixture by Sephadex LH-20 column chromatography. 2.2. Overlay analysis Overlay analysis of acidic lipids with glycolipidspecific antibodies was carried out based upon a method reported previously (Saito et al., 1985). Acidic lipids were developed on a TLC plate. After coating with a 0.4% polyisobutylmethacrylate solution, the plate was overlaid consecutively by an anti-glycolipid antibody and peroxidaseconjugated second antibody at room temperature for 1.5 h. When lipids were analyzed with a peroxidase-conjugated cholera toxin B subunit, the step with second antibody was omitted (Wu and Ledeen, 1988). The reacted band(s) were detected on an X-ray film using the ECL method. Acidic lipids on the plate was then visualized with resorcinol-HCl reagent (Svennerholm, 1957) or orcinolH2SO4 reagent (Sewell, 1979). Densitometric analysis of chromatograms was carried out using a densitometric image analyzer (Atto Densitograph AE-6920M, Atto Co., Tokyo, Japan). 2.3. Characterization of acidic lipids using different sialidases Two A2B5-reactive acidic lipids in squid hepatopancreatic tissue were isolated by preparative TLC and LH-20 column chromatography. They were finally purified on HPLC with a size exclusion column (TSKgel a-2500, 0.8 = 30 cm, Tosoh, Tokyo, Japan) with a solvent of methanol. The purified lipid was treated with one of three different sialidases, i.e. the enzyme derived from C. perfringens, S. typhimurium, or A. ureafaciens. In the case of C. perfringens sialidase, the reaction mixture consisted of 100 mM sodium acetate (pH 4.8), acidic lipid, and 250 mUyml of the enzyme in a final volume of 0.1 ml. The mixture was incubated at 37 8C for 45 min. The reaction with S. typhimurium sialidase was carried out in a
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similar manner, except that the pH was 5.5. In both cases, the hydrolysis product(s) were isolated by LH-20 column chromatography of the reaction mixture. In another experiment, the lipid was treated with A. ureafaciens enzyme (250 mUyml, pH 4.8) in the presence of sodium deoxycholate (0.5 mgyml) at 37 8C for 45 min. The neutral lipid product(s) were purified using DEAESephadex column chromatography. 2.4. Fluorometric high performance liquid chromatographic (HPLC) analysis of sialic acids Lipid-bound sialic acids were analyzed using a fluorometric HPLC method (Hara et al., 1987). An purified acidic lipid was hydrolyzed by incubation in 25 mM sulfuric acid at 80 8C for 1 h. The hydrolysate was incubated with a 1,2-diamino4,5-methylene-dioxybenzene (DMB) reagent at 60 8C for 2.5 h. The fluorescent derivative(s) were analyzed by HPLC with an ODS column and fluorescence detector. The fluorescence excitation and emission wavelengths were at 373 and 448 nm, respectively. 2.5. Electrospray ionization-mass spectrometry (ESI-MS) of gangliosides ESI-MS of A2B5-reactive acidic lipids was carried out using an LCQ ion-trap mass spectrometer equipped with an ESI source (Finnigan MAT, USA). An acidic lipid was treated with methyl iodide using a method reported previously (Tadano-Aritomi et al., 1992). The permethylated derivative, which formed a single band on TLC, was dissolved in methanol at a concentration of 10 pmolyml, and introduced into the electrospray needle by mechanical infusion at a flow rate of 3 mlymin. The ESI capillary was kept at a voltage of q4 V at 200 8C. The tube lens offset was set at y30 V. The collision-induced dissociation (CID)-MS2 spectra were taken using helium as the collision gas. The relative collision energy scale was set at q2.5 eV. Mass spectra were averaged over ten scans. 3. Results 3.1. A2B5-reactive acidic lipids in squid hepatopancreatic tissue Acidic lipids in hepatopancreatic tissue of the common squid were developed on TLC and visu-
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alized with resorcinol-HCl reagent or immunostained with a monoclonal antibody A2B5. The tissue had a complex composition of resorcinolpositive acidic lipids (Fig. 1a, lane 2). At least three acidic lipids were reactive with A2B5; they respectively migrated near GD1b (designated as lipid a), above GT1b (lipid x), and below GQ1b on TLC (lipid b) (Fig. 1a, lane 3). Among these A2B5-reactive acidic lipids, lipids a and b were purified using methods including preparative TLC and HPLC with a size exclusion column. Lipid a formed a double band on TLC, whereas lipid b migrated as a single band (Fig. 1b). 3.2. Product analysis after sialidase treatment of A2B5-reactive acidic lipids Lipids a and b were treated with one of sialidases, and the reaction products were analyzed. The treatment of lipid a with A. ureafaciens sialidase generated an orcinol-positive neutral lipid product. This lipid was assigned to LacCer from its chromatographic mobility on TLC (Fig. 2, lane 2). Lipid b was hydrolyzed with C. perfringens sialidase, producing a resorcinol-positive acidic lipid product. This lipid was identified to be GM1 based upon the chromatographic mobility and reactivity with cholera toxin B subunit (Fig. 3, lanes 3 and 4) and with anti-GM1 antibody on TLC (data not shown). Furthermore, the overlay analysis of the reaction mixture with cholera toxin B subunit revealed another lipid product at the same
Fig. 2. Hydrolysis of lipid a by A. ureafaciens sialidase. Lipid a was incubated with A. ureafaciens sialidase (250 mUyml, at pH 4.8) at 37 8C for 45 min in the presence of sodium deoxycholate (0.5 mgyml). The neutral lipid product was developed on TLC with a solvent system of chloroformymethanolyH2 O (65:30:6) and visualized with orcinol-H2SO4 reagent. Lane 1, lipid a; lane 2, the reaction product after sialidase treatment of lipid a; and lane 3, standard neutral glycolipids.
position as GD1b on TLC (Fig. 3, lane 4). The treatment of lipid b with a2,3 specific S. typhimurium sialidase generated a single resorcinolpositive lipid product, which migrated between GT1b and GQ1b on TLC (Fig. 4, lane 3). This lipid product (designated as lipid y) was assumed to be GT1c based upon its A2B5 reactivity and chromatographic mobility on TLC (Fig. 4, lane 4).
Fig. 1. Acidic lipids in hepatopancreatic tissue of the common squid. (a) Acidic lipids were isolated from squid hepatopancreatic tissue by methods including DEAE-Sephadex column chromatography, mild base treatment and Sephadex LH-20 column chromatography. The purified acidic lipids were developed on TLC with a solvent system of chloroformymethanoly0.2% CaCl2 Ø2H2 O (45:40:10), and visualized with resorcinol-HCl reagent (lane 2) or immunostained with a monoclonal antibody A2B5 (lane 3). Lane 1, rat liver gangliosides visualized with resorcinol-HCl reagent (as reference). (b) Lipids a and b in squid hepatopancreatic tissue were purified and visualized with resorcinol-HCl reagent (lanes 2 and 3) or immunostained with A2B5 (lanes 4 and 5). Lanes 2 and 4, lipid a; and lanes 3 and 5, lipid b. Lane 1, squid hepatopancreatic acidic lipids visualized with resorcinol-HCl reagent.
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Fig. 3. Partial hydrolysis of lipid b by C. perfringens sialidase. Lipid b was incubated with C. perfringens sialidase (250 mUyml, at pH 4.8) at 37 8C for 45 min, and the lipid products were developed on TLC. Lane 1, rat liver gangliosides: lane 2, lipid b before sialidase treatment; and lanes 3 and 4, lipid products after sialidase treatment of lipid b. Lanes 1–3, visualized with resorcinol-HCl reagent; and lane 4, reacted with cholera toxin B subunit.
N-Acetylneuraminic acid, but not N-glycolylneuraminic acid, was detected in lipids a and b using a fluorometric HPLC method (data not shown). 3.3. ESI-MS of A2B5-reactive acidic lipids Based upon the above-mentioned findings, the A2B5-reactive lipids a and b were assumed to be GT3 and GQ1c, respectively. To confirm this assumption, each lipid was permethylated and analyzed by positive ESI-MS (Table 1). The ESIMS spectrum of lipid a showed a double-charged ion of wMq2Hx2q at myz 1035, which corresponded with a hemato-type oligosaccharide structure containing three N-acetylneuraminic acids with a ceramide of the molecular mass of 534 (as the non-methylated structure) (Fig. 5a). CID-MS2 of the double-charged ion produced ion peaks that matched with the partial structures of GT3 (Fig. 5b). An ion peak corresponding with a trisialosyl structure was observed at myz 1136. The ions at myz 789 and 971 accorded with the structures of (tri-O-methyl-Glc)-Cer and (tri-O-methyl-Gal)(tri-O-methyl-Glc)-Cer. Based upon these findings, it was concluded that the trisialosyl residue was connected to the galactose of LacCer. The ESI-MS spectrum of lipid b showed a double-charged ion of wMq2Hx2q at myz 1492; this ion matched with a gangliotetraose oligosac-
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charide structure containing four N-acetylneuraminic acids with a ceramide of the molecular mass of 638 (as the non-methylated form) (Fig. 6a). The CID-MS2 spectrum of this ion showed fragment ions corresponding to the partial structures of GQ1c (Fig. 6b). The ions at myz 1120 and 1110 (double-charged ion) accorded with the structures of (NeuAc)3 and (NeuAc)3qGal-GlcCer, whereas the ions at myz 871 and 1105 matched with the structures of (tri-O-methyl-Glc)Cer and (di-O-methyl-Gal)-(tri-O-methyl-Glc)Cer, respectively. These findings indicated that a trisialosyl residue was connected to the inner galactose of the gangliotetraose structure. The fragment ion at myz 636 corresponded with the structure of (NeuAc)-(tri-O-methyl-Gal). It was thus concluded that the fourth sialic acid residue was linked to the terminal galactose of GgOse4. 3.4. The concentration and composition of c-series gangliosides in squid tissues Prior to analysis of c-series gangliosides in squid tissues, the concentration of total gangliosides was determined by quantitation of lipid-bound sialic acids using the fluorometric HPLC method. The concentrations of gangliosides in hepatopancreas (ns5), cerebral ganglion (ns5), eye lens (ns4), and mantle tissue (ns1) were 2.5"1.4, 0.069"0.030, 0.11"0.09 and 0.013 mg sialic acid
Fig. 4. Partial hydrolysis of lipid b by S. typhimurium sialidase. Lipid b was treated with S. typhimurium sialidase (250 mUyml, at pH 5.5) at 37 8C for 45 min, and the lipid product was developed on TLC. Lane 1, rat liver gangliosides; and lane 2, lipid b before sialidase treatment. Lanes 3 and 4, the reaction product after sialidase treatment of lipid b. Lanes 1–3, visualized with resorcinol-HCl reagent; and lane 4, immunostained with A2B5.
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Fig. 5. ESI-MS of lipid a. Lipid a was permethylated with methyl iodide and analyzed by positive ESI-MS (a) and CID-MS2 (b). The upper chart shows the assumed structure of lipid a and fragmentation patterns.
per g wet tissue weight, respectively. Regarding the molecular species of lipid-bound sialic acids, only N-acetylneuraminic acid was detected in all tissues. The distribution of c-series gangliosides in common squid tissues was examined using A2B5 (Fig. 7). The percentages of c-series gangliosides within total gangliosides was highest in hepatopancreatic tissue, followed by cerebral ganglion and eye lens. Interestingly, the relative enrichment of c-series gangliosides in squid hepatopancreas and cerebral ganglion was even higher than that of cod fish brain. In contrast, c-series gangliosides were not detected in squid mantle tissue in this experimental condition. The composition of c-series gangliosides differed among hepatopancreas, cerebral ganglion and eye lens. Two A2B5-reactive gangliosides above GT1b (i.e. lipid x) and below GQ1c were observed in hepatopancreatic tissue, but not in other tissues. These gangliosides were tentatively identified as GT2 and GP1c based upon
their A2B5 reactivity and chromatographic mobility on TLC. 4. Discussion In a previous study, we demonstrated that the common squid Todarodes pacificus expressed acidic lipids, which were reactive with a monoclonal antibody A2B5 (Saito et al., 2001b) This antibody was originally prepared by immunizing chicken embryonic retinal cells (Eisenbarth et al., 1979). While there has been some controversy about its reactivity, recent studies have suggested the specific binding of A2B5 to c-series gangliosides (Fenderson et al., 1987; Dubois et al., 1990; Freischutz et al., 1994; Farrer and Quarles, 1999). Very recently, we examined a structure-reactivity relationship between A2B5 and gangliosides of different structures and confirmed its strict specificity to c-series gangliosides. The epitopic structure for the antibody was assumed to include a
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Fig. 6. ESI-MS of lipid b. Lipid b was permethylated with methyl iodide and analyzed by positive ESI-MS (a) and CID-MS2 (b). The upper chart shows the assumed structure of lipid b and fragmentation patterns.
trisialosyl residue connected to the inner galactose of c-series gangliosides (Saito et al., 2001a). Thus, it is strongly suggested that A2B5-reactive acidic lipids in these molluscan species are c-series gangliosides. In the present study, we isolated two A2B5reactive acidic lipids from squid hepatopancreatic tissue and characterized their structures. The oligosaccharide core structure of these acidic lipids were determined by product analysis after sialidase treatment; lipids a and b were shown to possess the hemato-type and gangliotetraose oligosaccharide core structure, respectively. Among two reaction products from lipid b after treatment with C.
perfringens sialidase, the lower one was assumed to be GD1b because of its chromatographic mobility on TLC and reactivity to cholera toxin B subunit (Cumar et al., 1982). Sialic acid residues (including the trisialosyl structure) and their attachment to the oligosaccharide core structures were successfully identified by positive ESI-MS and CID-MS2 of the permethylated derivatives. Although the linkage of sialic acids in the trisialosyl residue remains to be determined, it is most likely that sialic acids are connected through a2,8 linkages, as observed in other polysialosyl structures of natural compounds (Schauer, 1982). Since lipid b was shown to share the same oligosacchar-
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Fig. 7. The compositions of A2B5-reactive acidic lipids in squid tissues. Acidic lipids were isolated from squid tissues, developed on TLC, and immunostained with A2B5 (lanes 2– 6). Lanes 2, hepatopancreas; 3, cerebral ganglion; 4 eye lens; and 5, mantle tissue. Lane 6, cod brain gangliosides (as reference). The amount of gangliosides per lane is equivalent to 0.1 mg sialic acid per lane. Lane 1, rat liver gangliosides visualized with resorcinol-HCl reagent.
ide structure with GM1, it was concluded that the innermost sialic acid of the trisialosyl residue was connected to the inner galactose through a a2,3 linkage. Lipid b was shown to contain a sialic acid residue that was susceptible to the action of a2.3 specific sialidase. This sialic acid was assumed to be the one that was connected to the terminal galactose of the gangliotetraose oligosaccharide structure. Based upon these findings and specific reactivity to A2B5, lipids a and b were identified to be GT3 and GQ1c, respectively. Regarding the structures of sialic acids in GT3 and GQ1c, N-acetylneuraminic acid was detected as the sole molecular species. However, the ganglioside samples were prepared by procedures including mild base treatment. Thus, it is possible that these c-series gangliosides or parts of them may originally exist as alkali-labile O-acetyl derivatives of N-acetylneuraminic acid, as reported in cod fish brain (Waki et al., 1993a,b). Lipid x was tentatively assigned to GT2 based upon the reactivity with A2B5 and chromatographic behavior on TLC (Yu and Ando, 1980). Lipid y was supposed to be GT1c that is produced from GQ1c by the removal of the sialic acid residue at the terminal galactose. C-series gangliosides were found in various tissues of the common squid. As for the origin of these gangliosides, one may claim that gangliosides in squid tissues may be derived from contam-
ination by ganglioside-containing foods in the digestive tract. However, there are several reasons against this possibility. The hepatopancreas constitutes a part of the digestive system, but is clearly demarcated from digestive tracts and therefore, can be dissected without contamination by gut contents. Regarding the cerebral ganglion, eye lenses, and mantle tissues, there is no risk of possible contamination by food in digestive tracts, because of their anatomical location. Accordingly, the presence of gangliosides in these tissues strongly suggests that gangliosides are intrinsic components of squid tissues. This assumption is further supported by the fact that the concentration and composition of c-series gangliosides significantly differ among squid tissues. It has been accepted that c-series gangliosides are expressed only in the chordate among deuterostomia. C-series gangliosides are enriched in lower animals such as adult fish brain (Ando and Yu, 1979; Yu and Ando, 1980; Waki et al., 1993a,b; Nakamura et al., 1997, 2000; Saito et al., 2001a). In avian and mammalian brain, they and their O-acetyl derivatives are temporarily expressed at certain embryonic stages but hardly detected at adult ages (Dubois et al., 1986, 1990; Rosner et al., 1988, 1993; Hirabayashi et al., 1988, 1989; Letinic et al., 1998). In contrast, c-series gangliosides have never been detected in echinoderms among deuterostomia. The present study provides solid evidence against the prevailing view that c-series gangliosides are limited to the chordate among deuterostomia. References Ando, N., Yamakawa, T., 1982. On the minor gangliosides of erythrocyte membranes of Japanese cats. J. Biochem. (Tokyo) 91, 873–881. Ando, S., Yu, R.K., 1979. Isolation and characterization of two isomers of brain tetrasialogangliosides. J. Biol. Chem. 254, 12224–12229. Cumar, F.A., Maggio, B., Caputto, R., 1982. Gangliosidecholera toxin interactions: a binding and lipid monolayer study. Mol. Cell. Biochem. 46, 155–160. Dubois, C., Magnani, J.L., Grunwald, G.B., et al., 1986. Monoclonal antibody 18B8, which detects synapse-associated antigens, binds to ganglioside GT3 (II3 (NeuAc)3LacCer). J. Biol. Chem. 261, 3826–3830. Dubois, C., Manuguerra, J.-C., Hauttecoeur, B., Maze, J., 1990. Monoclonal antibody A2B5, which detects cell surface antigens, binds to ganglioside GT3 (II3(NeuAc)3LacCer) and to its 9-O-acetylated derivative. J. Biol. Chem. 265, 2797–2803.
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