Studies on the Glycosphingolipids of the Starfish, Asterina Pectinifera III

Home , Arabinose

J. Biochem. 86, 765-772 (1979)

Studies on the Glycosphingolipids of the Starfish , Asterina pectinifera1

‡V. Isolation and Structural Studies of Two Novel Gangliosides

Containing Internal Sialic Acid Residues

Mutsumi SUGITA

Department of Chemistry, Faculty of Liberal Arts and Education, Shiga University, Otsu, Shiga 520

Received for publication, March 30, 1979

Two gangliosides, provisionally named Gangliosides ‡T and 2 in previous studies, were isolated

from starfish, Asterina pectinifera by silicic acid, DEAE-Sephadex, and Iatrobeads column

chromatography procedures, and preparative thin-layer chromatography, and their structures

were established. On the basis of the results of partial acid hydrolysis, methylation and

oxidation with chromium trioxide, Gangliosides 1 and 2 were proposed to be ArapƒÀ(16)-

GalpƒÀ(1 4)8-O-McNeuGc(2 3)GalpƒÀ(1 4)GlcpƒÀ(11)-ceramide and ArapƒÀ(16)-

GalpƒÀ(14)NeuGc(23)GalpƒÀ(1 4)GlcpƒÀ(11)-ceramide, respectively.

The ceramide moieties of both gangliosides had similar phytosphingosine and 2-hydroxy fatty acid compositions, and both gangliosides were structurally related to the previously

described Ganglioside 3.

In studies on the composition of glycosphingolipids as Araf,p(16)GalpƒÀ(14)[GalpƒÀ(18)]Neu- of the starfish, a new class of ganglioside which Gc(23)Galp,ƒÀ(14)GlcpƒÀ(11)-ceramide (2). contains internal sialic acid residues in its sugar This paper reports studies on the chemical chain has been found in Asterina pectinifera (1). structures of the two gangliosides with the highest

The ganglioside fraction of the starfish was sepa (Ganglioside 1) and the intermediate (Ganglioside rated into at least three components by thin-layer 2) rates of migration on thin-layer chromatog chromatography. The slowest migrating com raphy. ponent, tentatively termed Ganglioside 3, was the most abundant, and its structure was characterized MATERIALS AND METHODS

1 This study was supported in part by a grant from the General-The type of starfish used as a Ministry of Education, Science and Culture of Japan. source of gangliosides, A. pectinifera, was essen For part 11 of this series, see Ref. (2). tially the same as in the previous study (2). Methods Abbreviations: Ara, arabinose; Gal, galactose; Glc, of thin-layer chromatography, paper chromatog glucose; NeuAc, N-acetylneuraminic acid; NeuGc, N- raphy, gas-liquid chromatography, and gas chro glycolylneuraminic acid; 8-O-MeNeuGc, 8-0-methyl- N-glycolylneuraminic acid; CMH, ceramide mono matograph-mass spectrometry were also essen hexoside; CDH, ceramide dihexoside. tially as described previously (2). Hexose content

Vol. 86, No. 3, 1979 765 766 M. SUGITA

was determined by the anthrone method (3) and (yield, 250 mg), was isolated at this column frac pentose by the method of Dische and Borenfreund tionation step and its structure has been established

(4). Sialic acid was estimated by Svennerholm's (2). For the isolation of Gangliosides 1 and 2, resorcinol method (5). preparative thin-layer chromatography was em Isolation and Purification of Gangliosides 1 ployed; Gangliosides 1 and 2 were obtained in pure and 2-The isolation procedure consisted of form in yields of 35 mg and 80 mg, respectively extraction with a mixture of chloroform and (Fig. 1). methanol, Folch partition, and separation of gan The infrared spectra of Gangliosides 1 and 2

gliosides from the upper phase, using three types showed strong acid amide bond absorptions at of column chromatography successively, as de 1540 and 1645 cm 1, and a free hydroxy group

scribed earlier (2). Final purification to obtain at 3300 cm 1, but no absorption at 1730 cm-1

single components was achieved by preparative due to ester carbonyl.

thin-layer chromatography as follows. The Sugar Compositions of Gangliosides ‡T and 2-

gangliosides obtained by column chromatography Gas-liquid chromatography of the N-acetyl-O- were applied (approximately 5 to 10 mg per plate) trimethylsilyl ether derivatives of the methyl

to Silica gel H thin-layer plates (2.5 mM thick, glycosides which were obtained from the individual

20•~20 cm, Nakarai Chemical Co., Kyoto). The components of Gangliosides 1 and 2 revealed the

plates were developed in 1-propanol-2.5 M ammonia presence of arabinose, glucose, galactose, and

(7 : 3, v/v). The gangliosides contained in the sialic acid (or 8-O-methylsialic acid) in a molar silica gel scrapings were each extracted with

chloroform-methanol-water (40 :60 : 10, by vol.). Partial Acid Hydrolysis of Gangliosides-

Partial acid hydrolysis of ganglioside was per

formed in 0.1 M HC1 for 90 min at 80•Ž. The

hydrolysates were partitioned by the addition of chloroform and methanol to give a final solvent

mixture of chloroform-methanol-water (10 : 5 :3,

by vol.) which separated into a lower organic

phase and an upper aqueous methanolic phase. The glycolipid fragments in the organic phase

were identified by comparison of their migration

on Merck plates (Silica gel 60, E. Merck, Darms

tadt) with the migration of ceramide hexoside

isolated previously (6), as well as by gas-liquid

chromatography. The aqueous methanolic phase Fig. 1. Thin-layer chromatogram on silica gel of the was evaporated to dryness and the residual mate gangliosides isolated from the lipid extract of the starfish, rial was subjected to Dowex 1•~8 (acetate form, A. pectinifera. Solvent systems: (A) 1-propanol-2.5 M 200 to 400 mesh) column chromatography by the ammonia (7 :3, v/v) and (B) chloroform-methanol- method of Hoshino et al. (7). water (60 : 25 : 4, by vol.). Detection with: (A) re sorcinol and (B) orcinol-sulfuric acid spray reagents. 1, Ganglioside fraction obtained by partition, dialysis, RESULTS AND DISCUSSION and Mallinckrodt silicic acid and DEAE-Sephadex Extraction with a mixture of chloroform and column chromatographies from total lipid extract of methanol followed by Folch partition yielded A. pectinifera. 2 and 3, Gangliosides ‡T and 2 (the sub stances described in this paper) isolated by Iatrobeads 5.6 g of a crude ganglioside fraction, starting from column chromatography and preparative thin-layer 7 kg of the starfish, A. pectinifera. The crude chromatography. 4, Ganglioside 3 (2). 5 and 6, ganglioside fraction was subjected to three types Neutral glycosphingolipids derived from Gangliosides of column fractionation. Three gangliosides, 1 and 2 by partial acid hydrolysis. 7, Authentic provisionally named Gangliosides 1, 2, and 3, were ceramide glucoside (upper) and ceramide lactoside detected. The most abundant, Ganglioside 3 (lower) isolated from A. pectinifera (6).

J. Biochem. GANGLIOSIDES OF STARFISH 767

ratio of approximately 1 : 1 : 2 : 1 (Fig. 2). Table side was butanolyzed and analyzed as described I shows the results of chemical analysis of Gan previously (2); each was found to consist ex gliosides ‡T and 2. For the determination of acetyl clusively of N-glycolylneuraminic acid.

or glycolyl residues of sialic acids, each ganglio- Presence of 8-O-Methyl Sialic Acid in Gan

glioside 1-To confirm the structure of sialic acids in Gangliosides ‡T and 2, Peaks 4 and 5 of Fig. 2 were analyzed by gas chromatography-mass

spectrometry. The mass spectrum of Peak 5,

shown in Fig. 3B, was identical with that of the

trimethylsilyl ether (N-acetyl) derivative of the

methyl ester methyl glycoside of N-acetylneuraminic

acid (8). In the spectrum of Peak 4 (Fig. 3A),

peaks at m/e 552 (M-15) (cf. m/e 610 for the N- acetylneuraminic acid derivative), at m/e 508

(M-59) (cf. m/e 566), at m/e 315 (M-CH2OTMS- TMSOH-NH5Ac) (cf. m/e 373), and at m/e 284

(M-CH2OTMS-2TMSOH) (cf. m/e 342) were observed. The mass difference of 58 m.u. indicates

that the sialic acid (Peak 4) in Ganglioside 1 con

tains an 0-methyl group instead of an O-trimethylsilyl group.

Next, to determine the position of the 0-

methyl substituent in the sialic acid residues,

Ganglioside 1 was subjected to methanolysis in

1.0 tit methanolic HCl for 17 h at 75•Ž. The

resulting methyl glycosides were peracetylated in Fig. 2. Gas chromatograms of trimethylsilyl ether pyridine-acetic anhydride (3 : 2, v/v) for 12 h at (N-acetyl) derivatives of the individual sugar components room temperature, then the peracetylated deriva of Gangliosides 1 (A) and 2 (B). 1, Arabinose; 2, tives were analyzed by gas chromatography-mass galactose; 3, glucose; 4, 8-O-methyl-N-acetylneuraminic acid (the occurrence of this form of sialic acid in starfish spectrometry. Figure 4 shows the mass spectrum

gangliosides is described in the text); 5, N-acetyl- of the peracetylated derivative of the methyl ester neuraminic acid. These analyses were performed on methyl glycoside of sialic acid obtained from a column of 3% OV-1. The column temperature was Ganelioside 1. This mass spectrum shows an

programmed from 140•Ž to 230•Ž at 2•Ž/min.

TABLE I. Chemical analysis of Gangliosides 1 and 2 isolated from the starfish, A. pectinifera.

a The theoretical percent composition was calculated for a molecule containing 2-hydroxy-C22:0 acid and Cl7- phytosphingosine. b Resorcinol assay value as N-glycolylneuraminic acid. c Anthrone assay value as a mixture of glucose and galactose, 1 : 2. d As arabinose.

Vol. 86, No. 3, 1979 768 M. SUGITA

Fig. 3. Gas chromatograph-mass spectrometric patterns of the N-acetyl-trimethylsilylated methyl ester methyl glycosides of the sialic acids from Gangliosides 1 (A) and 2 (B). (A) and (B) correspond to Peaks 4 and 5 in Fig. 2, respectively. Analysis was performed on an LKB 9000 instrument.

The intense and important peak at m/e 117 corre sponds to the fragment CH2OAcCHOCH3, which is evidently formed by cleavage between C-7 and C-8, and suggests that the 0-methyl group must be attached to C-8 (9). Studies of the distribution in nature of sialic acids have thus far disclosed the presence of 8-O-methyl sialic acids (N-glycolyl- or N-acetylneuraminic acids) only in starfish (9, 10). 8-O- Fig. 4. Gas chromatograph-mass spectrometric pat- Methyl-N-glycolylneuraminic acid has been iso tern of the peracetylated methyl ester methyl glycoside lated from the starfish, Asterias forbesi by Warren, of the sialic acid from Ganglioside 1. The fragment at and its structure was determined by periodate m/e 117 is a diagnostic ion for 8-O-methyl-N-acetyl- oxidation (10). 8-O-Methyl-N-acetylneuraminic neuraminic acid derivatives (9). acid has been found in the starfish, Distolasterias nipon by Zhukova et al. and its structure was intense peak for M at m/e 418 (cf. m/e 446 for the elucidated by gas chromatography-mass spec corresponding derivative of N-acetylneuraminie trometry and by alkaline degradation (9, 11). acid), and the mass difference of 28 m.u. indicates Chemical Degradation Products of Gangliosides the presence of an O-methyl group instead of an 1 and 2-Partial acid hydrolysis of the two gan O-acetyl group. All the fragments formed from gliosides liberated, from each, ceramide lactoside the molecular ion (m/e 418) by elimination of one (Fig. 1) and water-soluble degradation products. to three molecules of acetic acid and ketene are The water-soluble degradation products were shifted by 28 m.u. towards smaller mass compared separated by Dowex 1-acetate column chromatog to the derivative of N-acetylneuraminic acid. raphy (Fig. 5). The materials obtained from each

J. Biochem. GANGLIOSIDES OF STARFISH 769

Fig. 6. Paper chromatograms of the sialic acid-containing oligosaccharide fractions obtained by Dowex 1-acetate column chromatography. Paper [‡T] was

developed with a 1-butanol-pyridine-water (6 : 4 :3, by vol.) system and Paper [‡U] with an ethyl acetate-

pyridine-acetic acid-water (5 : 5 : 1 : 3, by vol.) system. 1, Oligosaccharide A (obtained from Fr. I in Fig. 5); 2, Oligosaccharide B (Fr. ‡U); 3, Oligosaccharide C

Fig. 5. Fractionation on a column of Dowex 1•~8 (Fr. ‡V); 4, Oligosaccharide D (Fr. ‡W): 5, authentic (1.0•~40 cm, acetate form, 200 to 400 mesh) of the N-glycolylneuraminic acid; 6, authentic N-acetyl- water-soluble materials obtained from partial acid hy neuraminic acid. drolysates of Gangliosides I (A) and 2 (B). Elution was performed first with 500 ml of water and then with

a gradient formed from 300 ml each of water and 0.5 M charide C; arabinose, galactose, and sialic acid,

acetic acid. Sialic acid content (•ü), hexose content 1 : 1 : 1. Oligosaccharide D; galactose and sialic

(•œ), and pentose content (•~) were determined as de acid, 1 : 1. scribed in " MATERIALS AND METHODS." Sialic Acid Positioned at the Reducing End of Oligosaccharides-To identify sialic acid residues fraction (Frs. ‡T, ‡U, ‡V, and ‡W in Fig. 5) were positioned at the reducing end, Oligosaccharides provisionally named Oligosaccharides A, B, C, A, B, C, and D were reduced with NaBH4 and and D, and they were each shown to be homo analyzed by gas-liquid chromatography as their

geneous by paper chromatography with 1-butanol- trimethylsilyl derivatives after methanolysis and

pyridine-water (6:4:3, by vol.) and ethyl N-acetylation. As shown in Table ‡U, gas chro acetate-pyridine-acetic acid-water (5:5:1:3, by matographic analysis indicated the presence of the

vol.) as solvent systems (Fig. 6 and Table ‡U). The following components. Oligosaccharide A;

yields are listed in Table ‡U. Gas chromatographic arabinose, galactose, and 8-O-methylneuraminitol analysis of the oligosaccharides indicated the in a molar ratio of approximately 1 :1 :1.

presence of the following components (Table ‡U). Oligosaccharide B; galactose and 8-O-methyl- Oligosaccharide A; arabinose, galactose, and neuraminitol, 1 : 1. Oligosaccharide C; arabinose,

8-0-methyl sialic acid in a molar ratio of approxi galactose, and neuraminitol, 1 : 1 : 1. Oligosac- mately 1 :1 :1. Oligosaccharide B; galactose charide D; galactose and neuraminitol, 1 : 1.

and 8-0-methyl sialic acid, 1 : 1. Oligosac- These results suggest that all four oligosaccharides

Vol. 86, No. 3, 1979 770 M. SUGITA

TABLE ‡U. Properties of oligosaccharides obtained from partial acid hydrolysates of Gangliosides 1 and 2.

a Amounts of starting material were 25 mg of Ganglioside ‡T and 50 mg of Ganglioside 2. b Ascending on Toyo

No. 51 paper. Solvent systems: ‡T, 1-butanol-pyridine-water (6 : 4 : 3, by vol.) and ‡U, ethyl acetate-pyridine-acetic acid-water (5 : 5 : 1 : 3, by vol.). These data correspond to Fig. 6. o,d 8-O-Methyl-N-acetylneuraminic acid and 8-O-methylneuraminitol. e, f N-Acetylneuraminic acid and N-acetylneuraminitol.

contain sialic acid at the reducing end of their TABLE ‡V. Partially methylated alditol acetates.

sugar chains. Glycosidic Linkages of Gangliosides 1 and 2-

Methylation studies: The linkages of glycosides

were determined by permethylation and gas

chromatograph-mass spectrometry on the basis

of their retention times relative to that of 2,3,4,6-

tetra-O-methyl-l,5-di-O-acetylglucitol as an internal

standard, and from mass spectrometric data on alditol acetates of the partially methylated sugars

(12, 13). The results obtained from studies of the partially methylated alditol acetates of intact Gangliosides 1 and 2, as well as ceramide dihex a Ceramide dihexoside isolated from partial acid hydrol osides and Oligosaccharides A and C isolated from ysates of Gangliosides 1 and 2. b Oligosaccharides A partial acid hydrolysates of Gangliosides 1 and 2, and C isolated from partial acid hydrolysates of Gangli are summarized in Table ‡V. Next, in order to osides 1 and 2, respectively.

Fig. 7. Fatty acid composition of the glycosphingolipids isolated from the starfish, A. pectinifera. a, Carbon number (chain length):number of double bonds. b, Both CMH and CDH contain nonhydroxy fatty acids to the extent of about 35% of the total fatty acids (6). In this figure, each hydroxy fatty acid is expressed as a percentage of the total hydroxy fatty acid component. c, The fatty acids were entirely 2-hydroxy fatty acids, as confirmed by gas chromatograph-mass spectrometry. d, Data from ref. (2).

J. Biochem. •y GANGLIOSIDES OF STARFISH 771

Fig. 8. Long-chain base composition of the glycosphingolipids isolated from the starfish, A. pectinifera . Analyses were performed by gas-liquid chromatography on the fatty acid methyl esters derived from long-chain bases by oxidation (15). The amounts found are given as percentages of the total area on gas chromatograms . u, The long-chain bases were solely phytosphingosines. b, Data from ref. (6). Data from ref. (2).

examine the linkages of the terminal sugar and of

sialic acid, the reduced Oligosaccharides A and C

were permethylated, methanolyzed and per

acetylated. Both of the resulting partially methy

lated neuraminitol acetate methyl esters were analyzed by gas chromatograph-mass spectrometry.

The mass spectra were the same, and showed

intense ions at m/e 262 and 274, indicating that

C-4 of these sialic acids was substituted (2).

Anomeric configurations: After oxidation of

Gangliosides 1 and 2 with chromium trioxide,

5.3% (from Ganglioside 1) and 6.2% (from

Ganglioside 2) of glucose, 8.1% and 8.6% of

galactose, and 2.7% and 3.2% of arabinose re mained unoxidized. Since the four neutral residues of Gangliosides 1 and 2 are almost com

pletely oxidized, they thus appear to be ƒÀ-glyco- sidically linked.

Based on the evidence described above, the

structures of Gangliosides 1 and 2 were deter-

mined to be ArapƒÀ(16)GalpƒÀ(14)8-O-Me-

NeuGe(23)GalpƒÀ(14)GlepƒÀ(11)-ceramide and ArapƒÀ(16)GalpƒÀ(14)NeuGc(23)Galp-

ƒÀ(14)GlepƒÀ(11)-ceramide, respectively. Fatty Acids and Long-Chain Bases-The fatty

acids and the long-chain bases of Gangliosides 1

and 2 are summarized in Figs. 7 and 8. The fatty

acids were entirely 2-hydroxy fatty acids ranging

in length from C16 to C24, among which C22, C23,

and C24 acids were predominant. The long-chain

bases consisted exclusively of C16, C17, and C18

phytosphingosines of iso and ƒ¿nteiso types. These Fig. 9. Structures of the five glycosphingolipids iso two ceramide moieties are similar to those of lated from A. pectinifera. The anomeric configurations

A. pectinifera ceramide glucoside, ceramide lacto- of sialic acids are tentative assignments.

Vol. 86, No. 3, 1979 772 M. SUGITA

side, and Ganglioside 3, which have previously been characterized (2, 6). REFERENCES Five glycosphingolipids, two neutral and three 1. Sugita, M. & Hori, T. (1976) J. Biochem. 80, 637-640 acidic lipids, were isolated from the starfish, A. 2. Sugita, M. (1979) J. Biochem. 86, 289-300 pectinifera (Fig. 9). The three acidic glyco 3. Roe, J.H. (1955) J. Biol. Chem. 212, 335-343 sphingolipids form an interesting ganglioside 4. Dische, Z. & Borenfreund, E. (1957) Biochim. Bio

series. Indeed, the gangliosides of A. pectinifera phys. Acta 23, 639-642 are quite distinct from those studied so far in other 5. Svennerholm, L. (1957) Biochim. Biophys. Acta 24, animals as regards their sugar composition (14, 601-611 15) in that these gangliosides each contain an 6. Sugita, M. (1977) J. Biochem. 82, 1307-1312 arabinose residue and an internal sialic acid 7. Hoshino, 0., Zehavi, U., Sinay, P., & Jeanloz, R.W. residue in the sugar chain. The gangliosides are (1972) J. Biol. Chem. 247, 381-390 8. Kamerling, J.P., Vliegenthart, J.F.G., & Vink, J. also interesting in that their sialic acids consist (1974) Carbohyd. Res. 33, 297-306 solely of N-glycolylneuraminic acids. Finally, 9. Kochetkov, N.K., Chizhov, O.S., Kadentsev, V.I., Ganglioside I contains 8-O-methyl-N-glycolyl- Smirnova, G.P., & Zhukova, LG. (1973) Carbohyd. neuraminic acid; this is the first time that this acid Res. 27, 5-10 has been identified in glycolipids. 10. Warren, L. (1964) Biochim. Biophys. Acta 83, Among the five A. pectinifera glycosphingo 129-132 lipids studied in this laboratory (2, 6), the fatty 11. Zhukova, I.G., Smirnova, G.P., Bodanovskaya, acid and the long-chain base compositions are T.A. & Chekareva, N.K. (1972) Abstr. Papers, 5th All-Union Conference on Chemistry and Biochemistry virtually identical (Figs. 7 and 8). Thus, and in of Carbohydrates, "Nauka" p. 65 view of the oligosaccharide structures of these 12. Bjorndal, H., Hellerqvist, G.G., Lindberg, B., &

glycosphingolipids, it seems probable that a Svensson, S. (1970) Angew. Chem. 82, 643-652 biosynthetic relationship links CMH, CDH, and 13. Jansson, P., Kenne, L., Lindgren, H., Lindberg, B., Gangliosides 1, 2, and 3 in a single metabolic & Lonngren, J. (1976) A Practical Guide to the pathway. Methylation Analysis of Carbohydrates in Chem. Commun. (Stockholm) No. 8 (Oct. 15), pp. 1-75 The author would like to acknowledge the valuable dis 14. Laine, R.A., Stellner, K., & Hakomori, S. (1974) in cussions and suggestions of Prof. T. Hori of this labor Methods in Membrane Biology (Korn, E.D., ed.) atory and Prof. H. Katsuki of Kyoto University, during Vol. 2, pp. 205-244, Plenum Press, New York the course of these investigations. The author also 15. Hakomori, S. & Ishizuka, ‡T. (1975) in Handbook of wishes to thank Dr. J.T. Dulaney of the University of Biochemistry and Molecular Biology "Lipids, Carbo Tennessee, U.S.A., for helpful comments on the manu hydrates, Steroids" (Fasman, G.D., ed.) 3rd ed., script. pp. 41625, CRC Press, Cleveland

J. Biochem.