JOURNAL OF CHEMISTRY Vol. by U.S.A. Structure of … · 2001-07-22 · chromatography using three...

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THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 264, No. 33, Isaue of November 25, pp. 19922-19927,1989 Printed in U.S.A. Structure of Phosphonoglycosphingolipid Containing Pyruvylated Galactose in Nerve Fibers of Aplysia Izurodai” (Received for publication, May 12, 1989) Shigeko ArakiSB, Sachiko AbeS, Susumu Andoll, Kazuo KonT, Naoshi Fujiwarall, and Mei SatakeS From the $Department of Neurochemistry, Brain Research Institute and the IIDepartment of Anesthesiology, Faculty of Medicine, Niigata University, Niigata 951 and the llDepartment of Biochemistry, Tokyo Metropolitan Institute of Gerontology, Itabashiku, Tokyo 173, Japan A phosphonoglycosphingolipid, designated as FGL- IIb, was identified in nerve fibers of Aplysia kurodai by two-dimensional thin layer chromatography (Abe, S., Araki, S., and Satake, M. (1986) Biomed.Res. (Tokyo) 7, 47-51). FGL-IIb was isolated from the nervous system of A. kurodai by Iatrobeads column chromatography using three solvent systems. Pyruvic acid was identified by thin layer chromatography as its 2,4-dinitrophenylhydrazone and established by per- methylation studies to be attached as a ketal to 0-3 and 0-4 of the terminal galactose of the oligosaccha- ride chain in FGL-IIb. By sugar analysis, permethyla- tion studies, fast atom bombardment-mass spectrome- try, and proton magnetic resonance spectrometry, the structure of FGL-IIb was concluded to be [3,4-0-(1- carboxyethylidene)]GalBl+3GalNAcal+3(Fucal+ 2)(2-aminoethylphosphonyl+6)Gal~l+4Glc~l+ lceramide. Its major aliphatic components were pal- mitic acid, octadeca-4-sphingenine and anteisonona- deca-4-sphingenine. This is the first report of the oc- currence of pyruvylated galactose as a constituent of animal sphingolipid. In our laboratory, a new series of phosphonoglycosphingo- lipids have been found in tissues of Aplysiu kurodui (1). The structures of three major glycolipids isolated from the skin were determined to be as follows. 2-AEP .1 6 2 2-AEP-Galal SGL-I1 3-O-MeGalpl+3GalNAca1+3Galpl4Glc/31-+lCer f SGL-I’ 3-O-MeGalpl+3GalNAcal+3Gal~l4Glc~l~lCer 2 2-AEP+GGalal t 2-AEP 2-AEP 1 1 6 6 2 2-AEP4Galal SGL-1 4-0-MeGlcNAcal~GalNAca1+3Gal~1+4Glc~1~1Cer t * This work was supported by a Grant-in-aid 63580116 from the Ministry of Education, Science, and Culture of Japan (to S. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § To whom correspondence should be addressed. These three phosphonoglycosphingolipids (Refs. 2-4, re- spectively) have a common oligosaccharide backbone, GalNAc~v1~3[Galal+2]Galpl4Glc, and methylated sugar at the nonreducing end of the oligosaccharide chain. Two- dimensional thin layer chromatographic analyses showed that SGL-I1 and SGL-I were both present in the skin, ganglia, and nerve fibers of Aplysia, but that SGL-I’ was mainly localized in the skin. In addition, six glycolipids, tentatively named FGL-V, F-21, FGL-I, FGL-IIa, FGL-IIb, and F-9, were found only in the nervous system, and the latter three seemed to be specifically concentrated in nerve fibers (1). In the present paper, the glycolipid specifically present in nerve fibers, named FGL-IIb, was isolated and characterized. MATERIALS ANDMETHODS Isolation of Glycolipid FGL-IIb Specific to Nerve Fibers-The nerv- ous system (1 kg), consisting of nerve fibers and ganglia, was removed from live A. kurodai collected at Sad0 Island in early summer and stored at -20 “C until use. Total water-soluble glycolipid fraction (927 mg) was obtained from the acetone powder (100 g) of the nervous system of A. kurodai as described previously (2). Total glycolipid (150 mg) was dissolved in 2 ml of ch1oroform:methanol:water (60:35:8, v/v), appliedto an Iatrobeads column (6RS-8060, Iatron Lab., Tokyo, 1.6 X 36.5 cm) equilibrated with the same solvent, and then eluted with a discontinuous gradient of ch1oroform:methanol:water as de- scribed previously (2). The fractions containing FGL-IIb were lyoph- ilized and rechromatographed on another Iatrobeads column (1.0 X 45 cm), using a linear gradient of 1-propanokammonia water:water (80:5:15 to 75:5:20, v/v) in a total volume of 200 ml. Final purification of FGL-IIb was achieved by elution from an Iatrobeads column (1.0 X 95 cm) with chloroform:methanol:1.25 M ammonia water (60:40:9, v/v). The purity of the isolated FGL-IIb was examined by HPTLC’ in the following solvent systems: ch1oroform:methanol:water (55: 45:10, v/v) and chloroform:methanol:1.25 M ammonia water (60:40:9, v/v). After development for a distance of 20 cm, glycolipid was located by spraying with anthrone-H2S04 reagent (5) and then heating at 110 “C. Dephosphorylation of FGL-I16 and Permethyluted FGL-Ilb-FGL- IIb and permethylated FGL-IIb prepared as described below were treated with HF as described previously (2). The reaction mixture was neutralized with saturated LiOH solution. Then thesolution was extracted with 5 volumes of ch1oroform:methanol (2:1, v/v) and the lower chloroform layer was dried. Analysis of Chemical Composition-Colorimetric analysis of hex- osamine and phosphorus and the determinations of 2-aminoethyl- phosphonate and hexosamine in anamino acid analyzer were carried out as described previously (2). Uronic acid was analyzed with car- bazol-H2S0, reagent (6). The sialic acid was checked with resorcinol- HCl reagent on HPTLC (7). For analysis of the sugar composition, FGL-IIb and its derivatives The abbreviations used are: HPTLC, high performance thin layer chromatography; 2-AEP, 2-aminoethylphosphonate; GC-MS, gas chromatography-mass spectrometry; PMR, proton magnetic reso- nance spectroscopy; FAB-MS, fast atom bombardment-mass spec- trometry; HF, hydrogen fluoride; Cer, ceramide; -OMe, methoxy-; GLC, gas-liquid chromatography. 19922

Transcript of JOURNAL OF CHEMISTRY Vol. by U.S.A. Structure of … · 2001-07-22 · chromatography using three...

Page 1: JOURNAL OF CHEMISTRY Vol. by U.S.A. Structure of … · 2001-07-22 · chromatography using three solvent systems. Pyruvic acid was identified by thin layer chromatography as its

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 33, Isaue of November 25, pp. 19922-19927,1989 Printed in U.S.A.

Structure of Phosphonoglycosphingolipid Containing Pyruvylated Galactose in Nerve Fibers of Aplysia Izurodai”

(Received for publication, May 12, 1989)

Shigeko ArakiSB, Sachiko AbeS, Susumu Andoll, Kazuo KonT, Naoshi Fujiwarall, and Mei SatakeS From the $Department of Neurochemistry, Brain Research Institute and the IIDepartment of Anesthesiology, Faculty of Medicine, Niigata University, Niigata 951 and the llDepartment of Biochemistry, Tokyo Metropolitan Institute of Gerontology, Itabashiku, Tokyo 173, Japan

A phosphonoglycosphingolipid, designated as FGL- IIb, was identified in nerve fibers of Aplysia kurodai by two-dimensional thin layer chromatography (Abe, S., Araki, S., and Satake, M. (1986) Biomed. Res. (Tokyo) 7, 47-51). FGL-IIb was isolated from the nervous system of A. kurodai by Iatrobeads column chromatography using three solvent systems. Pyruvic acid was identified by thin layer chromatography as its 2,4-dinitrophenylhydrazone and established by per- methylation studies to be attached as a ketal to 0-3 and 0-4 of the terminal galactose of the oligosaccha- ride chain in FGL-IIb. By sugar analysis, permethyla- tion studies, fast atom bombardment-mass spectrome- try, and proton magnetic resonance spectrometry, the structure of FGL-IIb was concluded to be [3,4-0-(1- carboxyethylidene)]GalBl+3GalNAcal+3(Fucal+ 2)(2-aminoethylphosphonyl+6)Gal~l+4Glc~l+ lceramide. Its major aliphatic components were pal- mitic acid, octadeca-4-sphingenine and anteisonona- deca-4-sphingenine. This is the first report of the oc- currence of pyruvylated galactose as a constituent of animal sphingolipid.

In our laboratory, a new series of phosphonoglycosphingo- lipids have been found in tissues of Aplysiu kurodui (1). The structures of three major glycolipids isolated from the skin were determined to be as follows.

2-AEP .1 6

2

2-AEP-Galal

SGL-I1 3-O-MeGalpl+3GalNAca1+3Galpl4Glc/31-+lCer

f

SGL-I’ 3-O-MeGalpl+3GalNAcal+3Gal~l4Glc~l~lCer 2

2-AEP+GGalal t

2-AEP 2-AEP 1 1 6 6

2

2-AEP4Galal

SGL-1 4-0-MeGlcNAcal~GalNAca1+3Gal~1+4Glc~1~1Cer

t

* This work was supported by a Grant-in-aid 63580116 from the Ministry of Education, Science, and Culture of Japan (to S. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed.

These three phosphonoglycosphingolipids (Refs. 2-4, re- spectively) have a common oligosaccharide backbone, GalNAc~v1~3[Galal+2]Galpl4Glc, and methylated sugar at the nonreducing end of the oligosaccharide chain. Two- dimensional thin layer chromatographic analyses showed that SGL-I1 and SGL-I were both present in the skin, ganglia, and nerve fibers of Aplysia, but that SGL-I’ was mainly localized in the skin. In addition, six glycolipids, tentatively named FGL-V, F-21, FGL-I, FGL-IIa, FGL-IIb, and F-9, were found only in the nervous system, and the latter three seemed to be specifically concentrated in nerve fibers (1).

In the present paper, the glycolipid specifically present in nerve fibers, named FGL-IIb, was isolated and characterized.

MATERIALS AND METHODS

Isolation of Glycolipid FGL-IIb Specific to Nerve Fibers-The nerv- ous system (1 kg), consisting of nerve fibers and ganglia, was removed from live A. kurodai collected at Sad0 Island in early summer and stored at -20 “C until use. Total water-soluble glycolipid fraction (927 mg) was obtained from the acetone powder (100 g) of the nervous system of A. kurodai as described previously (2). Total glycolipid (150 mg) was dissolved in 2 ml of ch1oroform:methanol:water (60:35:8, v/v), applied to an Iatrobeads column (6RS-8060, Iatron Lab., Tokyo, 1.6 X 36.5 cm) equilibrated with the same solvent, and then eluted with a discontinuous gradient of ch1oroform:methanol:water as de- scribed previously (2). The fractions containing FGL-IIb were lyoph- ilized and rechromatographed on another Iatrobeads column (1.0 X 45 cm), using a linear gradient of 1-propanokammonia water:water (80:5:15 to 75:5:20, v/v) in a total volume of 200 ml. Final purification of FGL-IIb was achieved by elution from an Iatrobeads column (1.0 X 95 cm) with chloroform:methanol:1.25 M ammonia water (60:40:9, v/v). The purity of the isolated FGL-IIb was examined by HPTLC’ in the following solvent systems: ch1oroform:methanol:water (55: 45:10, v/v) and chloroform:methanol:1.25 M ammonia water (60:40:9, v/v). After development for a distance of 20 cm, glycolipid was located by spraying with anthrone-H2S04 reagent (5) and then heating at 110 “C.

Dephosphorylation of FGL-I16 and Permethyluted FGL-Ilb-FGL- IIb and permethylated FGL-IIb prepared as described below were treated with HF as described previously (2). The reaction mixture was neutralized with saturated LiOH solution. Then the solution was extracted with 5 volumes of ch1oroform:methanol (2:1, v/v) and the lower chloroform layer was dried.

Analysis of Chemical Composition-Colorimetric analysis of hex- osamine and phosphorus and the determinations of 2-aminoethyl- phosphonate and hexosamine in an amino acid analyzer were carried out as described previously (2). Uronic acid was analyzed with car- bazol-H2S0, reagent (6). The sialic acid was checked with resorcinol- HCl reagent on HPTLC (7).

For analysis of the sugar composition, FGL-IIb and its derivatives

The abbreviations used are: HPTLC, high performance thin layer chromatography; 2-AEP, 2-aminoethylphosphonate; GC-MS, gas chromatography-mass spectrometry; PMR, proton magnetic reso- nance spectroscopy; FAB-MS, fast atom bombardment-mass spec- trometry; HF, hydrogen fluoride; Cer, ceramide; -OMe, methoxy-; GLC, gas-liquid chromatography.

19922

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Phosphonoglycosphingolipid Containing Pyruvylated Galactose 19923

were methanolyzed in 5% anhydrous methanolic HC1 at 100 "C for 3 h and trimethylsilyl or trimethylsilyl N-acetyl derivatives of methyl- glycoside were analyzed by GLC as described previously (2). Methyl esters of fatty acids were extracted from the methanolysate with petroleum ether and analyzed by GLC using a 2% OV-1 column maintained at 220 "C (2). Long chain bases were obtained by hydrol- ysis of FGL-IIb in 2 N aqueous methanolic HCl at 85 "C for 3 h and seoarated as their aldehvde derivatives by GLC on a column of 2% Oir-1 at 180 "C (2).

Chemical Modification of FGL-llb-FGL-IIb (300 pg) was treated with 1 ml of 0.05 M HC1 in methanol a t room temperature for 5 h, and then reduced using 0.3 ml of 10% NaBH4 at room temperature overnight, or incubated with 1 ml of 0.2 M NaOH in 90% methanol a t 37 "C for 2 h. After treatment with acetic acid and desalting by Sephadex LH-20 column chromatography, the glycolipid products were analyzed by HPTLC using the solvent system, chloro- form:methanol:water (55:45:10, v/v) or their sugar compositions were determined by GLC as described above.

Determination of Pyruvic Acid-Pyruvic acid was identified by TLC as its 2,4-dinitrophenylhydrazone. FGL-IIb (500 pg) was hydro- lyzed with 0.5 ml of 2 N HCl at 100 'C for 3 h, and the 2,4- dinitrophenylhydrazone derivative was prepared by treating the hy- drolyzate with 0.2 ml of a solution of 0.1% 2,4-dinitrophenylhydrazine in 2 N HC1 (8). This derivative was extracted with ethyl acetate and compared by HPTLC with the 2,4-dinitrophenylhydrazone of an authentic sample prepared in the same manner. The cellulose HPTLC plates pretreated with 0.1 N NaHC03 solution were developed with tert-amyl a1cohol:ethanol:O.l N NaHC03 (50:10:40, v/v), and spots were detected under ultraviolet illumination.

Removal of Pyruvic Acid-For removal of pyruvic acid, FGL-IIb (500 pg) was treated with 1 ml of 0.01 N HCl for 6 h a t 100 "C. The solution was then neutralized, dialyzed, and freeze-dried (9).

Methylation Analysis-Permethylation experiments on FGL-IIb, HF-treated FGL-IIb, and FGL-IIb after removal of pyruvic acid were carried out by a modification (10) of the method of Imanari and Tamura (11). The methylated samples were acetolyzed with 0.6 N HCI in 80% acetic acid at 80 "C for 18 h, reduced with NaBH4, and then acetylated with acetic anhydride:pyridine (l : l , v/v) a t 100 "C for

analyzed by GLC and GC-MS on a 2% OV-1 column at 170 "C. 10 min? The partially methylated alditol acetates obtained were

Moss Spectrometry-Partially methylated alditol acetates were analyzed by GC-MS in a JEOL JMS-DX300 spectrometer with an ionization voltage of 70 eV and an ion accelerating voltage of 3.0 kV. Negative ion fast atom bombardment-mass spectrometry of FGL-IIb was performed in a JEOL DX 304/DX 304 mass spectrometer equipped with a JMA-DA5000 computer system. A mixture of trieth- anolamine and 15-crown ether-5 (2:1, v/v) was used as matrix. The target was bombarded with xenon atoms, and the spectrum was

kV. recorded in the negative ion mode at an accelerating voltage of 3.0

Proton Magnetic Resonance Spectroscopy-Proton magnetic reso- nance spectra were recorded with a Varian (XL-400, Varian Associ- ates Inc.) instrument a t 400 MHz in a Fourier transform mode at a probe temperature of 70 "C. The sample was dissolved in dimethyl sulfoxide-ds containing 10% deuterium oxide (12).

Preparation of Anti-FGL-llb Antiserum-The procedure used was essentially as reported (13), but a lower dose of purified FGL-IIb (200 pg) with 0.3 ml of pertussis vaccine (Takeda Chemical Industries, Ltd.) was used for primary immunization of the rabbit. Two booster injections of FGL-IIb (100 pg) were given, and anti-FGL-IIb anti- serum was obtained 2 weeks after the last injection. The anti-FGL- IIb antiserum obtained had a titer of 4096 by peroxidase enzyme- linked immunosorbent assay (14).

Enzyme Immunostaining of Glycolipids-Total glycolipid (45 pg) extracted from the nerve fibers of Aplysia was applied to two plastic TLC plates (10 X 20 cm, Polygram Si1 G/UV254, Macherey-Nagel, Federal Republic of Germany). The plates were developed with chlo- roform:methanol:0.2% aqueous CaClz (55:45:10, v/v) in the first di-

v/v) in the second dimension (1). The lipid antigens on these two- mension, and then with 1-propano1:ammonia water:water (75:5:25,

dimensional thin layer chromatograms were enzyme-immunostained by the method of Higashi et al. (15). The anti-FGL-IIb antiserum was diluted 50 times with 10 mM phosphate-buffered saline (pH 7.5) supplemented with 1% bovine serum albumin, 1% polyvinylpyrroli-

* M. Ohashi, K. Uchida, and T. Yamakawa presented at 26th Annual Meeting of the Japanese Conference on the Biochemistry of Lipids, Niigata, July 14, 1984.

done, and 0.02% NaN3 before use. Horseradish peroxidase-labeled goat IgG antibody to rabbit IgG (Cappel Laboratories) was used as a secondary antibody at 500-fold dilution with phosphate-buffered sa- line supplemented with 3% polyvinylpyrrolidone. The other plate was sprayed with anthrone reagent for identification of individual glyco- lipids.

RESULTS

Isolation of FGL-ZZb-FGL-IIb was purified from the total water-soluble glycolipid fraction of the nervous system of A. kurodai by Iatrobeads column chromatography using three solvent systems as described under "Materials and Methods." The isolated FGL-1% migrated as a single spot on HPTLC in two different solvent systems and stained with anthrone and Dittmer-Lester's reagents (16), but not with resorcinol re- agent for sialic acid. The yield of FGL-IIb was 8.4 mg/100 g, dry weight, of the nervous system.

Composition of FGL-ZZb-The results of colorimetric anal- yses and identification of the ninhydrin-positive compounds in an amino acid analyzer showed that FGL-IIb contained 1 mol each of galactosamine, phosphorus, and 2-aminoethyl- phosphonate (Table I). The fatty acids of FGL-IIb consisted predominantly of palmitic acid, and the long chain bases were octadeca-4-sphingenine (41%), anteisononadeca-4-sphingen- ine (46%), and others (13%) (Table I). GLC showed that FGL-IIb contained equimolar amounts of glucose, fucose, and N-acetylgalactosamine and, in addition, an unknown sugar, overlapping the first peak of methylglucoside (Table I and Fig. LA). Furthermore, methanolysis of FGL-IIb pretreated with HF, which selectively cleaves phosphate ester bonds (17), yielded 1 mol of galactose besides the methylglycosides ob- tained from intact FGL-IIb (Table 11). These results showed that 2-aminoethylphosphonate is attached to the galactose residue, because the linkage between sugar and 2-amino- ethylphosphonate cannot be cleaved on usual methanolysis (2). Proton magnetic resonance data revealed the presence of five anomeric protons, indicating the presence of five mono- saccharides in intact FGL-IIb (Fig. 5). Thus, FGL-IIb con- tained fucose, glucose, N-acetylgalactosamine, and galactose in a molar ratio of 1:l:l:l and an unidentified sugar.

TABLE I Chemical compositions of FGL-llb from the nervous system of

A. kurodai Hexosamine (as galactosamine)" 1.1 mol Phosphorus (by King's method)" 1.0 mol 2-Aminoethylph~sphonate~ 0.9 mol Galactosamineb 1.0 mol Fucose' 0.9 mol Galactose' - Glucose"' 1.0 mol N-Acetylgalactosamine' 0.9 mol Unknown sugar' -+ Fatty acid composition

16:O 94.2% 180 5.8%

Octadeca-4-sphingenine 41% Anteisononadeca-4-sphingenine 46% Uncharacterized 13%

Sphingosine base composition

Determined by colorimetric methods. bDetermined in an amino acid analyzer (Hitachi 835) after hy-

drolysis of FGL-IIb with 4 N HC1 at 100 "C for 8 h. The sugar composition and molar ratios were determined by GLC

as trimethylsilyl or trimethylsilyl N-acetyl derivatives of methylgly- cosides.

dThe peak of the methylglycoside of unknown sugar overlapped the first peak of methylglucoside, as described in the text, and the amount of glucose was estimated on the basis of the area of the second peak of methylglucosides.

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19924 Phosphonoglycosphingolipid Containing Pyruvylated Galactose

OaINAc

I

0 5 IO 15 20 25 min

FIG. 1. Gas chromatograms of trimethylsilyl derivatives of methylglycosides obtained from FGL-IIh and reduced FGL- IJb. FGL-IIb (A), reduced FGL-IIb ( B ) , and authentic standards (C) were methanolyzed in 5% anhydrous methanolic HC1 at 100 "C for 3 h. The methylglycosides obtained were trimethylsilylated and ana- lyzed on a column of 2% OV-l at 170 "C. Xylitol was added as an internal standard.

Characterization of the Unknown Sugar-After treatment with 0.05 M HC1 in methanol for 5 h at room temperature, acid methanol-treated FGL-IIb migrated faster than the orig- inal FGL-IIb on HPTLC (Fig. 2, lane 2). FGL-IIb after acid methanol treatment and then reduction with NaBH4 migrated between the untreated FGL-IIb and the acid methanol-treated FGL-IIb (Fig. 2, lane 3). When the acid methanol-treated FGL-IIb was further treated with 0.2 M NaOH in 90% meth- anol at 37 "C for 2 h, the reaction product showed the same RF value as the original FGL-IIb (Fig. 2, lane 4 ) . These results suggested the formation of a methyl ester of the carboxyl group during acid methanol treatment and reduction by NaBH4 of the methyl ester of the carboxyl group. The peak of the methylglycoside of the unknown sugar overlapped the first peak of methylglucoside, because in gas chromatograms

of the trimethylsilylated methanolysate of FGL-IIb the ratio of the first to the second peak of methylglucoside was high compared with that of the authentic methylglucoside (Fig. 1, A and C ) . This phenomenon was not seen in the gas chro- matogram of methylglycoside of reduced FGL-IIb, which showed the peaks for 1 mol of methylgalactoside (Table 11, Fig. 1B). These results suggested that the unknown galactose derivative with a free carboxyl group is present in FGL-IIb and that after reduction of the carboxyl group this galactose derivative could be converted to methylgalactoside by usual methanolysis.

No sialic acid or uronic acid could be detected. From observations by FAB-MS (Fig. 4), we assumed the

presence of a carboxyl compound of M, = 88 linked to a galactose residue.

A thin layer chromatogram of 2,4-dinitrophenylhydrazone derivatives obtained from the hydrolysate of FGL-IIb with 2 N HCl at 100 "C for 3 h showed the presence of pyruvic acid in FGL-IIb (Fig. 3).

Characterization of Depyruvylated FGL-IIb-FGL-IIb was treated with 0.01 N HCl for 6 h at 100 "C. The solution was then neutralized, dialyzed, and freeze-dried. The material recovered migrated slower than the original FGL-IIb as two spots on HPTLC and stained with anthrone and Dittmer- Lester's reagent. The two components of depyruvylated FGL- IIb were separated by preparative HPTLC developed for a distance of 20 cm with the solvent system, chloro- form:methanol:water (55:45:10, v/v). On gas chromatograms of the methanolysate of the lower spot, the peak of methyl- glycoside of pyruvylated galactose disappeared, and the peaks of 1 mol of methylgalactoside emerged (Table 11) beside peaks of 1 mol each of fucose, N-acetylgalactosamine, and glucose. These results showed that the pyruvic acid residue was re- moved from FGL-IIb by hydrolysis with 0.01 N HC1 for 6 h at 100 "C, but that other glycoside bonds in the lower spot were not affected by this treatment. The upper spot was depyruvylated and defucosylated FGL-IIb (data not shown).

Sugar Linkage and Sequence-Table I11 summarizes the results of methylation analyses of FGL-IIb, HF-treated FGL- IIb, and depyruvylated FGL-IIb. The presence of 2,3,4-tri-0- methylfucitol in FGL-IIb shows that the fucose residue is located at its terminal. The peak of 4,6-di-O-methylgalactitol, which was detected as a trace peak in the intact FGL-IIb, was found among the partially methylated alditol acetates from HF-treated FGL-IIb. This result shows that FGL-IIb branches at the position of 2-aminoethylphosphonate-Gal, because no partially methylated alditol acetate of the 2- aminoethylphosphonate-sugar complex could be found (2,18). The presence of 2,6-di-O-methylgalactitol in the intact FGL- IIb and the formation of 2,3,4,6-tetra-O-methylgalactitol with concomitant disappearance of 2,6-di-O-methylgalactitol from permethylated alditol acetates derived from the depyruvylated

TABLE I1 Molar ratios of the carbohydrate constituents of FGL-Ilb, HF-treated FGL-Ilb, reduced FGL-Ilb, and

depyruvyrated FGL-IIb Molar ratio"

Fucose Galactose Glucoseb Unknown N-Acetyl- sugar galactosamine

FGL-IIb 0.9 - 1.0 + 0.9 HF-treated FGL-IIb 0.8 1.0 1.0 + 0.7 Reduced FGL-IIb 1.1 1.1 1.0 - 0.9 DeDvruvslated FGL-IIb (lower mot) 1.0 1.1 1.0 - 1 .o

a The sugar composition and molar ratios were determined by GLC as trimethylsilyl or trimetylsilyl N-acetyl

* Determined on the basis of the area of the second peak of methylglucoside. derivatives of methylglycosides after methanolysis of the glycolipids.

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Phosphonoglycosphingolipid Containing Pyruvylated Galactose 19925

FIG. 2. Thin layer chromatogram of FGL-IIb, acid metha- nol-treated FGL-IIb, reduced FGL-IIb, and FGL-IIb after mild alkali treatment. Lane I, the total water-soluble glycolipid fraction of the nervous system; lane 2, FGL-IIb, lane 3, FGL-1% after acid methanol treatment; lane 4, FGL-IIb after acid methanol treat- ment and then reduction; lane 5, FGL-IIb after acid methanol treat- ment and then mild alkaline hydrolysis. Spots developed with chlo- roform:methanol:water (55:45:10, v/v) and detected with anthrone- sulfuric acid reagent.

FIG. 3. Thin layer chromatogram of 2,4-dinitrophenylhy- drazone derivatives. FGL-IIb was hydrolyzed with 2 N HCI at 100 “C for 3 h, and the hydrolysate was treated with 0.1% 2,4- dinitrophenylhydrazine in 2 N HCI. The 2,4-dinitrophenylhydrazone extracted with ethyl acetate was applied to a cellulose HPTLC plate pretreated with 0.1 N NaHC03 solution, developed with tert-amyl alcoho1:ethanol:O.l N NaHC03 (50:10:40, v/v) and detected under ultraviolet illumination. Lane I, authentic pyruvic acid lane 2, FGL- IIb.

FGL-IIb (lower spot) showed that pyruvic acid is linked as a ketal form to 0-3 and 0-4 of the terminal galactose of the oligosaccharide chain in FGL-IIb.

Fig. 4 shows the negative ion FAB-MS spectrum and fragmentation diagram of FGL-IIb. Molecular ion species, [M - HI-, were detected a t m/z 1562 and 1548, which corre- spond to the molecular weight of FGL-IIb with hexadecanoy- loctadecasphingenine and hexadecanoylanteisononadecas- phingenine, respectively. In addition, the ions originating from the following sequential cleavage of the carbohydrate residue were also detected. Fragment ions due to the loss of nonreducing terminal pyruvylated galactose were detected a t

m/z 1316 and 1330. The ions resulting from cleavage at the position between N-acetylgalactosamine and inside galactose were obtained at m/z 1113 and 1127, which are due to [Fuc+ (2-AEP+Gal)+Glc+Cer]. In addition, the ions at m/z 698 and 712 are due to [Glcxe r ] and t he ions at m/z 536 and 550 are derived from Cer. Thus, it became evident that the sequence of FGL-IIb is (pyruvylated-Gal)+GalNAc+(Fuc) (2-AEP)Gal+Glc+Cer. Loss of the carboxyl group of pyruvic acid from FGL-IIb produced m/z 1504 and 1518 [M - 451. Furthermore, the ions a t m/z 1444 and 1458 [M - 1051 should be due to loss of [pyruvic acid + OH] from FGL-IIb. Loss of [Glc-Cer] from the reducing terminal of FGL-IIb produced m/z 867. These results confirmed the above sequence of FGL- IIb.

For location of 2-aminoethylphosphonate, the permethyl- ated FGL-IIb was treated with HF, which selectively cleaves phosphate ester bonds (17), and then subjected to acetolysis. HF treatment of the permethylated FGL-IIb produced 4-0- methylgalactitol, indicating substitution by 2-aminoethyl- phosphonate at C-6 of internal galactose. The presence of the small peak of 4,6-di-O-methylgalactitol in permethylation studies of FGL-IIb, depyruvylated FGL-IIb, and permethyl- ated and then HF-treated FGL-IIb was due to partial dephos- phorylation during permethylation under alkaline conditions (2, 18). From the results of the permethylation studies and the fragmentation diagram of the FAB-MS spectrum, the sugar sequence and linkage of FGL-IIb was concluded to be [3,4-0- (l-carboxyethylidene)]Gall+3GalNAcl+3(Fucl+2) (2-aminoethylphosphonyl4)Gall+4Glcl+lceramide.

Proton Magnetic Resonance Spectra of FGL-ZZb-The pro- ton magnetic resonance spectrum of FGL-IIb revealed three 0- and two a-protons in the anomeric region, as shown in Fig. 5. The three anomeric peaks are easily assigned to p-glucose (4.17 ppm, J = 7.69 Hz), internal &galactose (4.44 ppm, J = 7.69 Hz), and a-N-acetylgalactosamine (5.03 ppm, J = 3.29 Hz) by comparison with other structurally identified phos- phonoglycosphingolipids and their derivatives such as SGL- 11, HF-treated SGL-I1 (2), and SGL-I’ (3). The doublet a t 5.22 ppm ( J = 4.03 Hz) was assigned to the anomeric proton of a-fucose, because the chemical shift of H-5 at 4.13 ppm that was connected to the C-6 methyl group of fucose at 1.09 ppm was established by two-dimensional correlated spectros- copy (two-dimensional COSY) (data not shown) (19-21). The H-5 resonance at 4.13 ppm also show that the fucosyl residue is attached to the internal galactosyl residue through a1+2 linkage. Consequently, the signal a t 4.39 ppm ( J = 7.69 Hz) could be assigned to terminal pyruvylated galactose (Table IV). The peaks at 1.86 and 1.46 ppm show the protons of the N-acetylmethyl residue of N-acetylgalactosamine and of the methyl group of pyruvic acid, respectively (9,22) (Fig. 5).

Thus, FGL-IIb was concluded to be: 2-AEP

HOOC,, , k 4 1 6 G a l ~ 1 ~ 3 G a l N A c a l ~ G a l ~ l ~ ~ l c ~ l ~ ~ c e r

HsC . , , ~ \ 0 / 3 2 t

Fucal

Zmmunochemical Findings-Total water-soluble glycolipids of nerve fibers were separated two-dimensionally on plastic TLC plates, and the immunoreactivities of individual lipids with anti-FGL-IIb antiserum were examined by enzyme im- munostaining as described under “Materials and Methods.” The other plate was sprayed with anthrone reagent. Fig. 6 shows that the glycolipids designated as FGL-I, FGL-IIa, FGL-IIb, FGL-V, and F-9 reacted with anti-FGL-IIb anti- serum on a TLC plate. We could show that the epitope for

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19926 Phosphonoglycosphingolipid Containing Pyruvyhted Galactose TABLE 111

Partially methylated alditol acetates derived from FGL-llb, HF-treated FGL-llb, and depyruvyluted FGL-llb Peaks were identified by GLC and GC-MS on a 2% OV-1 column at 170 "C as described in the text.

Hexitol and hexosaminitol

acetates

2,3,4-Tri-O-methylfucitol 2,3,4,6-Tetra-O-methylgalactitol 2,3,6-Tri-0-methylglucitol 2,6-Di-O-methylgalactitol 4,6-Di-O-methylgalactitol 4-0-Methylgalactitol 4.6-Di-0-methvl-2-N-methvlacetamidoealactitol

Permethylation then acetolysis

FGL-IIb HF-treated FGL-IIb

Depyruvylated FGL-IIb

+ - + + (+)" - +

Permethylation, HF-treatment,

then acetolysis

FGL-IIb

+ - + +

(+) + +

a (+) indicates the presence of a small peak.

1316 1330

2-AE

FIG. 4. Negative ion FAB mass spectrum and fragmentation diagram of FGL-IIb.

I

,4 pyruvateGa1 I Me*%

I "-"" ' ' ' ' ' ' 5.0 4s II

I

nance spectra of FGL-IIb. FGL-IIb FIG. 5. Proton magnetic reso-

was dissolved in dimethyl sulfoxide-de containing 10% deuterium oxide. The spectrum was measured at 400 MHz in the Fourier transform mode at a probe temperature a t 70 'C. Figure at the left

the anomeric region of FGL-IIb. Me,SO, upper corner is an expanded spectrum of

dimethyl sulfoxide.

Anomeric proton H-1 (DDm) 4.39 5.03 5.22 4.44 4.17 J 1 , z (Hz)

_. . I

7.69 3.29 4.03 7.69 7.69

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Phosphonoglycosphingolipid Containing Pyruvylated Galactose 19927

- K i L V

*

FGL-I i

L. . 'KiLllb

-F-16

FIG. 6. Enzyme immunostaining of anti-FGL-IIb antiserum wi th total glycolipid isolated from nerve fibers of A. kurodai. Total glycolipid of nerve fibers (45 pg) was separated on plastic TLC plates with chloroform:methanol:0.2% aqueous CaCI2 (55:45:10, v/v) in the first dimension and 1-propano1:ammonia water:water (75:5:25, v/v) in the second dimension. A, enzyme imrnunostaining; B, an- throne reagent.

anti-FGL-IIb antibodies involved the free carboxyl group of the pyruvic acid linked to galactose at the nonreducing end of FGL-IIb? Therefore, these glycolipids also seem to contain pyruvic acid linked to galactose a t their nonreducing end.

DISCUSSION

In the past 10 years, the mollusc Aplysia has become at- tractive animal models for study of the cellular and molecular mechanisms of neurobiological functions of the brain. We have found a novel group of glycolipids in tissues of A. kurodai (1) and studied the structures (2-4) and cellular localizations of these acidic phosphonoglycosphingolipids to obtain infor- mation on their biological functions (13, 14). In the present study, we isolated a phosphonoglycosphingolipid, designated as FGL-IIb, that seemed to be specifically concentrated in nerve fibers of A. kurodai (1) and characterized it as [3,4- 0-(l-carboxyethylidene)]Gal/31+3GalNAcal+3(Fucal+2) (2-aminoethylphosphonyl+6)Gal~1+4Glc/3l+lceramide.

We raised a polyclonal antibody against FGL-IIb and ex- amined the antigens that reacted with this anti-FGL-IIb antiserum by enzyme immunostaining on TLC plates. The antiserum reacted with FGL-IIb, FGL-I, FGL-IIa, FGL-V, and F-9, which were identified in ganglia and nerve fibers on TLC plates. Esterification or reduction of the pyruvic acid moiety of FGL-IIb resulted in loss of its antigenicity against the antiserum, and saponification of the acid methanol- treated FGL-IIb restored the rea~tivity.~ These results indi- cate that the epitope for the anti-FGL-IIb antibodies includes the free carboxyl group of pyruvic acid in FGL-IIb. As a first step in elucidating the biological functions of

these acidic glycolipids, including FGL-IIb, we examined the localization of antigens in the tissues of A. kurodai immuno- histochemically. With this antiserum, only nerve bundles were stained distinctly. From histochemical findings in cryo- stat sections pretreated with ch1oroform:methanol (2:1, v/v) and from results by Western blot analysis of the nervous system, the staining was concluded to be due to glycolipid antigens. These results showed that these phosphonoglyco- sphingolipids, including FGL-IIb, are localized in nerve bun- dles."

~~

' Watanabe, Y., Abe, S., Araki, S., Kumanishi, T., and Satake, M., (1989) J. Biochern. (Tokyo), in press.

We speculate from this observation that these phosphono- glycosphingolipids containing pyruvylated galactose may play some role in neurite extension and fasciculation. However, their precise neurobiological functions in the nervous system of Aplysia remain to be determined.

Pyruvic acid is known to be present as a ketal form attached to various saccharide residues in extracellular and cell wall acidic polysaccharides of different bacteria (9,23). In addition, pyruvic acid has also been found in mycobacterial glycolipids (24, 25), and it is proposed that these glycolipids are species- specific surface antigens of the microorganisms (24, 25). But to our knowledge, this is the first report of 3,4-O-(l-carboxy- ethy1idene)galactose as a constituent of animal sphingolipid.

A common oligosaccharide core structure, GalNAcal+ 3Gal/31+4Glc@, is present in the three major phosphonogly- cosphingolipids of the skin (2-4) and FGL-IIb. There are very few known glycolipids possessing the same backbone structure compared with the number of glycolipids with gangliotriaose (GalNAcpl+4Gal/314Glc/3) as their backbone structure. A blood group A active glycolipid, GalNAcal+3(Fucal+2) Gal/31+4Glc/31+1Cer, in rat small intestine (26) and the main glycolipid of abalone, Haliotis japonica (shellfish), Fuccul+3GalNAca1-+3(Fucal+2)Gal~l+4Glc/3l+lCer(27) have been reported. A systematic structural analysis of FGL-I, FGL-IIa, FGL-

V, and F-9 that reacted with the anti-FGL-IIb antibody and F-21 is now in progress to obtain more information on specific glycolipids in the nervous system of Aplysia.

Acknowledgments-We are grateful to Dr. Y. Honrna and K. Iwami for collecting A. kurodai, S. Yamada for performing amino acid analyses, Dr. A. Hayashi, Dr. T. Yamagata, Dr. S. Gasa, Dr. Y. Uda, Dr. M. Hiraiwa, N. Fujii, Dr. T. Ohno and Dr. K. Shimoji for helpful advice, and S. Oyanagi and Y. Hoshino for excellent technical assist- ance.

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