Novel sialosphingolipids from spermatozoa of the sea urchin Anthocidaris crassispina

152

Biochimica et Biophysics Acta, 388 (1975) 152-162 @ Elsevier Scientific Publishing Company, Amsterdam

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NOVEL SIALOSPHINGOLIPIDS FROM URCHIN ANTHOCZDARZS CRASSZSPZNA”

MOTONORI

HOSHF

and YOSHITAKA

“Division of Biochemistry, Sapporo and bDepartment 35-2 Sakaecho, Itabashiku, (Received

October

NAGAIb

SPERMATOZOA

OF

THE

SEA

**

The Institute of Low Temperature Science, Hokkaido University, of Biochemistry, Tokyo Metropolitan Institute of Gerontology, Tokyo (Japan)

21st, 1974)

Summary Sialosphingolipids obtained from spermatozoa of the sea urchin, Anthocidark crassispina gave nine spots by thin-layer chromatography. Two of them were isolated by silicic acid column chromatography. The predominant one was proposed to be N-acetylneuraminyl(2 + 8) N-acetylneuraminyl(2 + G)glucopyranosyl ceramide, and the second to be Nacetylneuraminyl(2 -+ 6)glucopyranosyl ceramide.

Introduction Isono and Nagai [1,2] have reported glycosphingolipids containing glucose and sialic acid in the gametes of the sea urchin, Pseudocentrotus depressus and Hemicentrotus pulcherrimus. Kochetkov et al. [ 3,4] have found the glycolipids of the same kind in the gonads of another sea urchin, Stronglyocentrotus intermedius. Nagai and Hoshi [5] have found various sialosphingolipids in the gametes of sea urchins. The composition of these lipids is found to be characteristic for species and gamete, though there may be common carbohydrate backbone composed of glucose and sialic acid. Of the four sea urchins investigated, the sialolipids of the spermatozoa of Anthocidaris crassispina gave the most complicated pattern by thin-layer chromatography. They also studied the changes of sialolipids during early development of sea urchins [6]. This communication describes the isolation procedure for sialolipids and the structures of two sialolipids obtained from the spermatozoa of Anthoci* This this

is Part paper

Lipids, * * To

V of was

Tokyo,

whom

the

series,

presented

Biochemistry at

the

10th

of

1968.

reprint

requests

should

Lipids

Meeting

be addressed.

of

of the

Sea

Urchin

Japanese

Gametes Conference

and of

Embryos.

Part

Biochemistry

of of

153

daris crussispina.

disialo-monoglucosyl this animal.

One is monosialo-monoglucosyl ceramide and the other is ceramide, and the latter is the predominant sialolipid of

Experimental Materials

The sea urchin used in the present work was A. crassispina, collected from the sea near the Misaki Marine Biological Station. Spermatozoa shed by the injection of 0.5 M KC1 solution into the body cavity were separated from seminal plasm by centrifugation at 10 000 X g for 30 min. They were stored in 5 vols of chloroform/methanol (1 : 2, v/v) at 4°C until used. Analytical

methods

Hexose content was determined by the anthrone method [7] and the phenol-H2 SO4 method [8], hexosamine by the methods of Gatt and Berman [9] and Boas [lo] . Sialic acid was estimated by Svennerholm’s resorcinol method [ll] as modified by Miettinen and Takki-Luukkainen [12] and also by the thiobarbituric acid method [13] . Sphingosine was estimated by the method of Lauter and Trams [ 141. Lipid phosphorus was determined by the method of Bartlett [15] as modified by Isono and Nagai [l] , and ester by the method of Snyder and Stephens [16]. Thin-layer

chromatography

Throughout this study, plates (20 X 20 cm, 20 X 10 cm and 40 X 10 cm) coated with a 0.25 mm layer of silica gel G (Merck, Darmstadt) were used. They were washed with methanol/diethyl ether (3 : 1, v/v) and activated by heating at 110” C for 20 min immediately before use. The solvent systems used were: I, chloroform/methanol/2.5 M ammonia (60 : 35 : 8, v/v) [17] ; and II, chloroform/methanol/water (60 : 35 : 8, v/v) [18] for lipid analyses and III, n-butanollpyridinelwater (8 : 4 : 3, v/v) for sialic acids. The developing reagents used routinely were the Cu*+/resorcinol reagent [ll] for sialic acids and the anthrone reagent [7] for hexoses. When the material contained neither hexoses nor sialic acids, the plates were charred with 50% H2 SO,. Paper chromatography

For paper chromatography of sugars, 2 mg of sialolipid were hydrolyzed with 2 ml of 2 M HCl at 110°C for 2 h [19]. After neutralization with Amberlite IR-4B (OH- form), the reaction mixture was concentrated to a minimum volume. The concentrated hydrolysate was subjected to paper chromatography on Whatman No. 1 paper with a solvent system of ethyl acetate/pyridine/water/ ethanol (12 : 5 : 4 : 2, v/v). Spots were detected with alkaline AgNO, [20]. Gas-liquid chromatography Sugars and fatty acids.

Gas-liquid chromatography of trimethylsilylated derivatives of methyl glycosides and of fatty acid methyl esters was carried out by the method of Yamakawa and Ueta [ 21,221. Usually 3-5 mg of lipid was methanolyzed with 5 ml of 3% methanolic HCl at 100°C for 3 h. A part of

154

fatty acid methyl esters thus obtained was hydrogenated in the presence of Adams catalyst and then analyzed by gas-liquid chromatography. The composition of fatty acid was calculated by triangulation of the peak areas. A Shimadzu GC-2C gas chromatograph equipped with a flame ionization detector was employed. A stainless steel column, 3 m X 3 mm, packed with 7% Ucon LB 550 X on Microsorb W was used at 180” C for sugar analysis. For the analysis of fatty acid methyl ester, a column of the same size, but packed with 25% diethyleneglycol succinate polyester on Celite 535 was used at 180°C. When thin-layer chromatography was used to isolate each sialolipid, methanolysis was achieved at 110°C without prior elution of lipids from scraped powder ~231.

Sialic acids. Sialic acid liberated from sialolipid was purified by column chromatography of ion-exchange resin [24]. The sialic acid was dried and dissolved in a mixture of pyridine/hexamethyldisilazane/trimethylchlorosilane (5 : 2 : 1, v/v) and kept at 60°C for several minutes. An aliquot of the mixture was injected directly onto a glass column, 3 m X 4 mm, packed with 3% SE-52 or SE-30 on Chromosorb WAW at 210” C. A Shimadzu GC-4APTF gas chromatograph equipped with flame ionization detector was used. Both N-acetylneuraminic acid and N-glycolylneuraminic acid were used as standards. In the case of periodate oxidation study, sialic acid and glucose were analysed simultaneously on a glass column, 2 m X 3 mm, packed with 1.5% OV-1 on Chromosorb WAW. A Shimadzu GC-4BMPF gas chromatograph with flame ionization detector was used. Column temperature was increased from 180 to 280” C at the programming rate of 5” C/min. Mannitol was added tg the methanolysate as an internal standard. Sphingosine bases. Sphingosine bases were determined by the method of Gaver and Sweeley [25] as modified by Taketomi and Yamakawa [26]. The trimethylsilyl derivatives of the bases were injected onto a glass column, 4 ft X l/8 inch, packed with 3.8% SE-30 on Diatoport S. An F and M Model 402 gas chromatograph was used at 180” C. Infrared spectroscopy Infrared spectra KBr pellets.

were obtained

by a Hitachi EPI-2 spectrophotometer

with

Optical i-ota tion measurement The optical rotation of sialolipid dissolved in pyridine at the wavelength of the sodium D-line was measured with a Hitachi EPU-2A spectrophotometer equipped with a Polarimetory Attachment A-2. Mass spec trome try Mass spectrometry of sialic acid was carried out with a 9000 gas chromatograph-mass spectrometer. A glass column, packed with 1% OV-1 on Chromosorb W was used at 200°C. block was maintained at 270°C and the molecular separator electron energy was 70 eV, the accelerating voltage was 3500 ing current was 60 PA.

Shimadzu LKB2 m X 3 mm, The ion source at 255°C. The V and the ioniz-

155

Partial hydrolysis of sialosphingolipid by dilute acid 0.5 mg of sialolipid was hydrolyzed with 4.0 ml of 25 mM H,SO, at 80” C. At intervals, aliquots were taken for analysis. The sialic acid liberated was determined by thiobarbituric acid method and glycolipids yielded upon hydrolysis were assayed by thin-layer chromatography in comparison with the original one. Permethylation analysis Sialolipid was permethylated according to Hakomori [ 271. The fully methylated lipid was methanolized with 5% methanolic HCl as described above. After the fatty acid methyl esters were extracted with light petroleum (4060” C), the methanolic phase was deacidified with Amberlite IR4B(OH- form) and concentrated to dryness. The residue was submitted to gas-liquid chromatography on a 5% neopentylglycol succinate column of 2 m at 180°C [21]. Methyl esters of fatty acids from methylated lipid were also analyzed by gasliquid chromatography as described in the section Gas-liquid chromatography. Periodate oxidation Sialolipid was oxidized with periodate according to Carter et al. [28] with a slight modification. To 750 ~1 of the lipid solution, 0.6 mg/ml in chloroform/ methanol (1 : 4, v/v), 150 ~1 of 0.2 M NaIO, was added. The reaction mixture was allowed to stand with gentle stirring at 24” C for 48 h in darkness. After the addition of 30 ~1 of ethyleneglycol, the mixture was gently stirred for 1 h. Mannitol, recrystallized, 50 pg, was added as internal standard and the organic solvent was evaporated off under a Nz stream. The residue was dried over Pz O5 and methanolyzed in 0.2 ml of 0.5 M methanolic HCl at 80°C for 18 h. Another aliquot of 750 ~1 of the lipid solution was mixed with 50 pg of mannitol and methanolyzed without oxidation. The methanolysates were dried well, silanized and submitted to gas-liquid chromatography. Isolation of sialosphingolipids Extraction and gross fractionation of lipids were carried out as reported previously [6]. For separation of sialosphingolipids from one another, four columns of silicic acid were used successively as follows. Step I. 1 g of the sialolipid mixture was dispersed in chloroform and applied onto a column of silicic acid (Mallinckrodt, 100 mesh, 40 g) mixed with Hyflosupercel (Johns-Manville, 20 g). Elution was carried out successively with chloroform, chloroform/methanol mixtures in the following proportions by volume; 9 : 1, 8 : 2, 7 : 3, 6 : 4, 4 : 6, 2 : 8 and finally methanol. Each effluent (10 ml) was monitored by the anthrone assay and thin-layer chromatography (Fig. 2). Step II. The method of Penick et al. [17] with a slight modification was used for the second step of purification. The activated silicic acid (Kant0 Chemical Co., 100 mesh, 100 g) was suspended in a mixture of chloroform/ methanol/water (65 : 30 : 5, v/v) to make a thin slurry, which was poured into a column tube (3 cm in diameter). The sialolipid fraction (Fraction VI’, 544 mg) obtained at the preceding step was dissolved in a minimum of a chloroform/methanol mixture (2 : 1, v/v) and charged on the column. The column

156

was eluted successively with chloroform/methanol/water mixture in the following proportions by volume; 65 : 30 : 5, 60 : 35 : 8 and 60 : 40 : 10, and finally with methanol. The elution profile was checked by thin-layer chromatography. Step Ill. Essentially the same procedure as Step II was repeated. For details, see the legend to Fig. 3. Step IV. At this step a long column was chosen. The eluate with a mixture of chloroform/methanol/water (60 : 35 : 8, v/v) at the preceding step was concentrated. A portion of this fraction (232 mg) was loaded on a column of Wako Gel C-100 (Wako Pure Chemical Industries, 40-100 mesh, 350 g; 2.5 X 172 cm). The column was eluted according to the same programme used in the preceding step. Each effluent was monitored by hexose assay and thin-layer chromatography. Results and Discussion Crude sialosphinolipid fraction 1.06 g, was obtained from 200 g of fresh spermatozoa. Nine sialosphingolipids, trivially named Compounds 1 to 9, a small amount of sulfonolipid [1,30] and phospholipids were detected in this fraction by thin-layer chromatography as shown in Fig. 1. The content of phospholipid calculated from the phosphorus value [31] was about 7.5%. More than 93% of sialolipid (as sialic acid) was recovered in this fraction. Though Compound 1 was purified at Step II, three more steps were required for the purification of Compound 5. Isolation of other sialolipids is in progress. Compound

5

Compound 5 weighing 87 mg was isolated in a homogeneous form on thin-layer chromatography at the purification step IV, although most of this lipid was still found in other fractions. This compound displayed typical absorption peaks of glycosphingolipid but not any at 1730 cm-‘, where sharp absorption by ester carbonyl was known, by infrared spectrometry (Fig. 4). This lipid was optically active ([a] 6” = - 24.8”; C = 1.2, in pyridine). Table I shows the chemical analysis of this lipid. Carbohydrate moiety. Paper chromatography of reducing sugar components revealed only one spot identical with authentic glucose. This finding was further confirmed by gas-liquid chromatography. Sialic acid was released completely from this lipid with 25 mM H2S04 at 80” C within 90 min. Two hydrolysis products were found, as shown in Fig. 5, the final asialosphingolipid and the intermediate sialosphingolipid. While the latter was identical with Compound 1 on thin-layer chromatography, the former was identified as glucocerebroside. Recently, glucofuranoside was reported as a constituent of sea urchin sialosphingolipid [ 321. Glucose of the lipid here studied, however, is not in furanose but in pyranose form because of its stability to weak acid. The permethylation study described below agrees with this finding. Sialic acids of the sialolipids were proved to be N-acetylneuraminic acids by thin-layer chromatography and also by mass spectrometry combined with gas-liquid chromatography. For mass spectrometry, sialic acids were derivatized to O-trimethylsilylated sialic acid trimethylsilyl ester trimethylsilyl ketosides. The spectrum

157

0

m

(ttright PWPl@)

Fig. 1. Thin-layer chromatogram of sialosphingolipids obtained from sea urchin gametes. Thin-layer chromatography was performed on silica gel G plate (10 X 40 cm) with solvent system I; chloroform/methanol/2.5 M ammonia (60 : 35 : 8, v/v). Sialolipids were located by a resorcinol spray. The gametes were presented by abbreviations as follows: AS, sperm of A. crassispina: AE, egg of A. crassispina; PS, sperm of P. depressus; PE, egg of P. depressus. The laboratory notation system is illustrated. The pink spots are contaminating sulfonolipids.

of silanized sialic acid of the lipid resembled to that of silanized N-acetylneuraminic acid. Especially in the high mass region, they were practically identical. The following ions were assigned as indicated in parentheses in this re726(M’--CHs ), 698(M’-COCH3 ), 636(M’-CHx ), gion. m/e = 741(M+), -TMSOH), 624(M’-COOTMS, 536(M’-CHOTMS - CH, OTMS), 446(M’356(M’-CHOTMS . CH, OTMS, -2 CHOTMS * CH2 OTMS,-TMSOH), TMSOH). Compound 5 was hydrolyzed by sialidases from Clostridium perfringens but not by those from Influenza Virus (Lee) or Streptomyces (MB223-Ml, MB 395-A5 and MB 600-Ak2). The enzymes from a strain of Streptomyces (MB503Cl) and New Castle Disease Virus (Narashino) hydrolyzed this lipid to

Fig. 2. An elution

profile

of crude siatosphingalipids obtained from spermatozoa of the sea urchin A. was applied onto a column of silicic acid (M~l~c~odt, 100 mesh, 40 g) mixed with 20 g of Hyflo Super Gel. Elution was carried out successively with chloroform, chloroform/ methanol mixtures and methanol. The ratios of solvents are presented at the bottom of the figure, An aliquot (0.3 ml) of each effluent (10 ml) was assayed by the anthrone method. Circles indicate the mobility of lipids on thin-layer chromatography with solvent system I, detected by anthrone spray and resorcinol spray. Fraction VI and VII were combined (Fraction VI’; 544 mg) and rechromatographed.

cmssispina.1 g of crude sialolipid

Fig. 3. Silicic acid column ehromatogram of the Compound B-rich fraction obtained at Step II. After dissolved in a minimum of chloroform/methanol mixture (2 : 1. v/v), 414 mg of material was loaded on a silicic acid column (Kanto Chemical Co., 6(t80 mesh, 120 g) and eluted with mixtures of chloroform/ methanol/water in the following proportions by volume: 65 : 30 : 5: 60 : 35 : 8; 60 : 40 : 10 and finally with methanol. An aliquot (0.1 ml) of each effluent was subjected to sugar assay by the phenol-HzSOq method and another aliquot of 10 ~1 was analyzed by thin-layer chromatography with solvent system I (detection: resorcinol reagent). The eluates with a mixture of chlorofo~/methanol/water (60 : 35 : 8, v/v) were combined and further purified at the following step (see the text).

159

1

I

4oco

3cco

I

I

2cco

I

I

1800

I

I

1600

Wave Fig.

4. Infrared

TABLE

SPectrum

of Compound

I

number 5.

I

1400

I

I

1200

I

I

1000

I

I

800

I

I

600

I

400

(cm+)

Infrared spectrum

was obtained

with KBr pellet.

I

COMPOSITION

OF COMPOUND

5 % composition

Sphingosine Glucose N-Acetylneuraminic Hexosamine Phosphorus

acid

Molar ratio

Tbeoretical*

Determined

Theoretical

Determined

22.0 13.2

20.2 12.5** 12.5*** 46.4? 38.2tt 1.7 nil

1.00 1.00

0.97 1.00 1.00 2.17 1.78 0.14

45.3 -

* The theoretical percent composition the sole fatty acid component. * * Anthrone assay value. * * * Phenol-HzSOq assay value. t Resorcinol assay value. tt Thiobarbituric acid assay value.

was calculated

2.00 -

on a molecule

containing

a docosenoic

acid as

Fig. 5. Thin-layer chromatograms of the products by partial hydrolysis of Compound 5. Compound 5 was hydrolyzed with 25 mM HzS04 at 80°C. The glycolipid yielded upon hydrolysis was assayed by thin-layer chromatography with solvent system I. Detection: a, resorcinol reagent: b, anthrone reagent. x: These spots were yeUow. Lanes 1, 5 and 7, crude sialolipids of A. crassispina spermatozoa; Lanes 2 and 8, purified Compound 5; Lanes 3 and 9, 15-min hydrolysate; Lanes 4 and 10, 30-min hydrolysate; Lane 11, l-h hydrolysate; Lane 12, 2-h hydrolysate: Lane 13, 4-h hydrolysate and Lanes 6 and 14, bovine brain cerebrosides.

160

some extent (Aoyagi, T., Hoshi, M. and Nagai, Y., unpublished). Susceptibility of Compound 5 to the microbial and viral sialidases suggests a-configuration of the C-2 ketosidic linkage of the ~-acetyl~leuraminic acid residue. When Compound 5 was methylated and subjected to gas-liquid chromatography, only two peaks, corresponding to methyl /3- and a-2,3,4 tri-O-methyl glucoside, were found. This indicates that only one N-acetylneuraminic acid is linked to glucopyranose at C-6. By periodate oxidation, the content of sialic acid in Compound 5 was decreased to 48.4% of the original value, while only 12.0% of glucose was recovered. Thus, one sialic acid residue in this lipid was not affected upon periodate oxidation, so it has a substituent at C-8. Fatty acids. As shown in Table II, 86% of fatty acids of Compound 5 was docosenoic acid. After hydrogenation, 91% was docosanoic acid. Fatty acid composition was not changed by methylation of Compound 5, therefore no hydroxy acid may exist in this lipid. Throughout the purification procedure, Compound 5 may be segregated according to the difference of fatty acid moiety. The “native” Compound 5 was isolated by preparative thin-layer chromatography of crude sialolipid mixture. Docosenoic acid and stearic acid are predominant in the “native” one (Table II). Sphingosine bases. Sphingosine bases of Compound 5 were revealed to be C, ~-sphingosine as a major component, concomitant with a small amount of C, ~-dihydrosphingosine. Phytosphingosine was not detected, though it was found in a sialolipid of sea urchin by Kochetkov et al. [4]. From the evidence described above, Compound 5, the predominant sialosphingolipid of spermatozoa of A. crassispina, is concluded to be N-acetylTABLE

II

FATTY

ACID

URCHIN

COMPOSITION

OF

THE

SIALOSPHINGOLIPID,

SPERMATOZOA I__-..____

Fatty

Percentage

of total

fatty

acid

acids Compound

“Native” Compound

16

0

6.4

-

16

1

0.4

-

18

0

30.5

-

18

1

19 19

0

1.8 1.3

-

1

0.7

-

20

0

20

1

6.1 4.1

-

21

0

1.4

-

21

1

0.9

-

22

0

22

1

1.3 45.1

85.5

22

2

trace

5.8

23

0

trace

4.4

23

1

23

2

24

0

* Isolated * * Isolated

5% *

5*

3.2

trace

trace

-

by preparative by

successive

thin-layer column

chromatography.

chromatography.

COMPOUND

5 ISOLATED

FROM

SEA

161

Fig. 6. The proposed structure for the main sialosphingolipid crassispina. C l- and 1 C-conformation were tentatively assigned

neuraminyl( 2 + 8) N-acetylneuraminyl( shown in Fig. 6. Compound

(Compound 5) of spermatozoa of A. for glucose and sialic acid. respectively.

2 -+ 6) glucopyranosyl

ceramide,

as

1

Compound 1 was isolated in a practically homogeneous form (yield; 26 mg). A trace of sulfonolipid was the sole contaminant. Compound 1 contained glucose and sialic acid but no hexosamine. The molar ratio of glucose to sialic acid was 1.00 : 1.13. Moreover, the sialosphingolipid formed by partial hydrolysis of Compound 5 was identical with this lipid on thin-layer chromatography. Thus Compound 1 may be N-acetylneuraminyl(2 -+ 6)glucopyranosyl ceramide. Both Compounds 5 and 1 are novel sialosphingolipids which have a direct bonding between glucose and sialic acid. Another novel lipid, monosialodiglucosyl ceramide has been isolated as the major sialosphingolipid of eggs of the same species. The study of its precise structure is in progress. Although many sialolipids were found on thin-layer chromatography, their neutral sugar composition was very simple, virtually glucose only [ 51. Very recently, Kochetkov et al. [33] isolated disialodiglucosyl ceramide from gonads of sea urchin, S. intermedius, though the precise structure was not revealed. Since they collected the animals in the breeding season, some, if not all, of this lipid may be present in the gametes. Though Heckers and Stoffel [34] suggested the presence of diglucosyl sialyl ceramide in pig platelets, the sialosphingolipids of sea urchin gametes may be characterized by the carbohydrate chain composed of glucose and sialic acid. Ishizuka et al. [ 351 found a novel sulfated galactoglycerolipid in mammalian testes and spermatozoa. This glycolipid was not detected in sea urchin gametes, while they contain sulfonolipid found by Nagai and collaborators [ 1,30,36,37] and cholesteryl sulfate [ 381. Cell surface saccharides were proved recently to play an important role in mammalian fertilization [ 39-411. It has been found in this laboratory that some particular sialosphingolipids are located at the cell surface of spermatozoa (Ohsawa, T. and Nagai, Y., reference 42). The biological function of sialosphingolipids as surface determinant in the process of fertilization and early development is now under investigation. Acknowledgements The authors

wish to express their heartful

gratitude

to Professor

T. Yama-

162

kawa, of the Faculty of Medicine, The IJniversity of Tokyo and Professor H. Sugawara, of the Department of Biology, College of General Education. The University of Tokyo, for their advice and criticism throughout this work. They must also thank Drs. T. Murata and S. Takahashi of the Central Research Institute, Shimadzu Co., Kyoto, who performed mass spectrometry. Thanks are also due to the Director and Staff of Misaki Marine Biological Station, The University of Tokyo, where a part of the present study was carried out. This work was supported by a grant from the Ministry of Education, Japan. References 1

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