THE JOURNAL 0 1992 by The American Society for Biochemistry

OF BIOLOGICAL CHEMISTRY and Molecular Biology, Inc

Vol. 267, No. 31, Issue of November 5, PP. 22595-22596,1992 Printed in U.S.A.

Polar Glycosphingolipids in Annelida A NOVEL SERIES OF GLYCOSPHINGOLIPIDS CONTAINING CHOLINE PHOSPHATE FROM THE EARTHWORM, PHERETIMA HILGENDORFP

(Received for publication, July 8, 1992)

Mutsumi SugitaS$, Hiroshi FujiiS, Fuyuhiko Inagakill, Minoru Suzuki(1, Chieko HayataS, and Taro Hori** From the $Department of Chemistry, Faculty of Liberal Arts and Education, Shiga University, Otsu, Shiga 520, the Departments of llMolecular Physiology and [[Membrane Biochemistry, The Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113, and **Shiga Junior College of Cultural Studies, Yokaichi, Shiga 527, Japan

A novel series of glycosphingolipids containing cho- line phosphate has been demonstrated in whole tissues of the earthworm, Pheretima hilgendorfi. The thin layer chromatographic pattern of the total polar gly- colipids revealed the presence of more than three com- ponents with positive reactions toward orcinol-sulfuric acid (sugar), molybdate (phosphate), and Dragen- dorff's (choline) spray reagents. Two of these polar glycolipids (PGL1 and PGL2) were purified by the use of successive column chromatography on QAE-Sepha- dex A-25 and silicic acid (Iatrobeads) and detected during elution by the presence of galactose-bound cho- line phosphate. The structural elucidation of the oligo- saccharide moieties was performed by compositional sugar analysis, hydrogen fluoride degradation, proton magnetic resonance spectroscopy, fast atom bombard- ment mass spectrometry, and methylation analysis. Thus, the structures of PGL, and PGLz were deduced to be as follows: cholinephosphoryl 4 6Galj31-1Cer and cholinephosphoryl~6Galj31-6Galj31-1Cer. Although the oligosaccharide structures of both PGL, and PGLz have previously been found in other organisms, the presence of a choline phosphate group as an oligosac- charide substituent is the first finding in nature. The main molecular species of the ceramide moieties were composed of beheninyl- and lignocerinyloctadecas- phingenines and their nonadecasphingenine homo- logues.

In our systematic structural analysis of glycosphingolipids of Protostomia (Mollusca and Arthropoda), the glycolipids have been found to contain quite different constituent sugars from those of Deuterostomia, e.g. mannose (1-7), glucuronic acid (8, 9), glucosamine 6-phosphate (lo), and glucose-6- monomethylaminoethylphosphonic acid (11).

This paper deals with the molecular structure of polar glycolipids from the earthworm, Pheretima hilgendorfi. The earthworm is a class of phylum Annelida, a phylogenetically

* This study was supported in part by Grant-in-Aid 4250101 for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture of Japan. Part of this work was presented at the .Japanese Conference on the Biochemistry of Lipids, June 1992, in Osaka. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ T o whom correspondence and reprint requests should be ad- dressed.

interesting organism intermediate between Mollusca and Ar- thropoda.

Our previous study paid special attention to the neutral glycolipids, and the major glycolipids found were a series of galalipids, Galpl-lCer, Galpl-GGal@l-lCer, and GalP1- GGalpl-GGal(31-1Cer (12). A search of the literature indicates that few structural studies of Annelida glycolipids have been carried out except for our laboratory (12, 13), and that the present identification of naturally occurring polar glycolipids carrying choline phosphate in nature is the first finding.

EXPERIMENTAL PROCEDURES

Isolation of the Earthworm Polar Glycolipids-Dried tissues (450 g, commercial origin for Chinese medicine) were powdered and extracted two times with 4.5 liters of chloroform/methanol (2:1, v/v) and then with 4 liters of chloroform/methanol (l : l , v/v). The chloroform/ methanol extracts were combined and subjected to mild alkaline hydrolysis in 0.5 N KOH in methanol/water (955, v/v) a t 37 "C for 6 h. The hydrolysate was neutralized with 2 N HC1, dialyzed against tap water for 3 days, and lyophilized. The resulting 9.6 g of alkali- stable product was taken up in chloroform/methanol/water (30:60:8, v/v) and applied to a column (2.4 X 80 cm) packed with QAE- Sephadex A-25 (Pharmacia LKB Biotechnology Inc.; OH- form) equilibrated with the same solvent. The column was eluted succes- sively with the same solvent ( 5 column volumes), with pure methanol (2 volumes), and with 0.45 M ammonium acetate in methanol (5 volumes) as a polar solvent. The sample of 0.6 g from the glycolipid fraction eluted in the first solvent (chloroform/methanol/water) was applied to a column of porous silica gel (1.8 X 120 cm, Iatrobeads 6RS-8060, Iatron Laboratories, Tokyo) equilibrated with chloroform/ methanol/water (60:40:10, v/v). The column was treated with a linear gradient of chloroform/methanol/water (60:40:10, v/v, 1000 ml to 40:70:15, 1050 ml). The flow rate was 0.8 ml/min and the effluent was collected in 5-ml fractions. An aliquot from alternate tubes was analyzed by TLC, and it was found that appreciably purified glycolipid fractions were obtained as three fractions, Fraction 1 (tubes 43-54), Fraction 2 (57-68), and Fraction 3 (73-85). For removal of contami- nating minor components, final purification of each fraction was achieved by rechromatography using an Iatrobeads column (2 X 90 cm). The column was treated with a solvent mixture of 1-propanol/ water/ammonium hydroxide (75:40:5, v/v). The flow rate was 0.25 ml/min, and the effluent was collected in 3-ml fractions. Tubes 69- 102 from Fraction 1, 121-171 from Fraction 2, and 150-186 from Fraction 3 were pooled for further examination.

Analytical Procedure-Compositional sugar analysis of the isolated glycolipids, as their trimethylsilyl methyl glycosides, was carried out by GC on a capillary column of HiCap-CBPS5 (25 m; Shimadzu, Kyoto), programmed a t 2 "C/min from 140 to 230 "C. Glycolipid fatty acid and sphingoid compositions were determined by GC, using their methyl esters and trimethylsilyl derivatives, on the same capillary column programmed at 2 "C/min from 180 to 230 "C for fatty acids and at 2 "C/min from 200 to 230 "C for sphingoids. Phosphorus was quantitatively determined by the method of King (14) and sugar by the anthrone method (15) using galactose as a standard.

Thin Layer Chromatography-Silica Gel 60 precoated plates

22595

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22596 Novel Glycosphingolipids Containing Choline Phosphate (Merck) were developed with the following solvents: neutral solvents, chloroform/methanol/water (60:4010 and 30:3010, v/v); basic sol- vent, 1-propanol/water/ammonium hydroxide (75:405, v/v); and acidic solvent, 1-butanol/acetic acid/water (40:6010, v/v). Detection was performed with orcinol-H2S04 reagent for sugars, Dittmer's re- agent (molybdate) for phosphorus, Dragendorffs reagent for choline, ninhydrin reagent for the amino groups, and rhodamine 6G reagent for lipids.

Acidic Hydrolysis-Each 1 mg of sample was hydrolyzed with 6 M HC1 a t 100 "C for 3 h for estimation of the sugar, sugar phosphate, and choline. Each hydrolysate was partitioned into the two layers of a Folch system (16), and the upper aqueous layers were dried under a stream of nitrogen. The residues were subjected to TLC analysis.

Dephosphorylation with HF-Samples were treated with HF, which selectively cleaves phosphate bonds (11). In this procedure, 4 ml of 47% HF (w/v) was added at 15 "C to 2 mg of samples dissolved in 0.1 ml of dimethylsulfoxide in a polyethylene tube fitted with a cap. After incubation a t 15 "C for 20 h, the reaction mixture was dialyzed against water to remove HF, and the tube contents were lyophilized. The dephosphorylated lipid produced was purified by Iatrobeads column chromatography as described above.

Permethylation Study-Permethylation of the sample was per- formed, using 200 pg each of the isolated polar glycolipids, by the method of Ciucanu and Kerek (17). The peaks on the gas chromato- grams were identified by mass spectrometry and by comparison of the retention times with those of the following reference partially methylated alditol acetates prepared from the authentic carbohy- drate compounds: Gal,pl-6GalpJ, Galpal-$Gal,, and Gal,pl-BMeGal, (Sigma).

Dephosphorylation of the Permethylated Polar Glycolipids-The sugar attachment site of choline phosphate was determined by meth- ylation analysis followed by dephosphorylation with H F (9, 11). Permethylation of the polar glycolipids (about 2 mg each) was carried out as described above, and the permethylated product was purified on a silicic acid column equilibrated with chloroform as usual. The purified permethylated polar glycolipid recovered from the column with 1 volume of chloroform/methanol (9010, v/v) was dissolved in a small amount of dimethylsulfoxide and treated with HF under the same conditions as described above. The dephosphorylated compound was subjected to acetolysis, hydrolysis, reduction with sodium boro- hydride, and acetylation. The partially methylated alditol acetates thus obtained were analyzed by GC and GC-MS. Identification of peaks was performed by comparing retention times on GC and confirmed by comparison of their physicochemical data with those reported by Bjorndal et al. (18) and Jansson et al. (19).

Positive Ion FAH-MS Spectrometry (FAB-MS)-FAB-MS of the polar glycolipids was performed using a JEOL HX-110 mass spec- trometer equipped with a JMA-DA 5000 computer system. m-Nitro- benzylalcohol was used as the matrix. The accelerating voltage was 8.0 kV, and the primary beam for the bombardment was 6.0 keV of xenon.

'H NMR Spectroscopy-NMR spectra were obtained with a JEOL JNM-GX500 spectrometer at 60 "C. One milligram of sample was dissolved in 0.4 ml of dimethylsulfoxide-ds containing 2% D20, and tetramethylsilane was used as the internal standard as to chemical

a b

- - 1 2 3 4 1 2 3 4

FIG. 1. Thin layer chromatograms of P. hilgendorfi polar glycolipids. Plates were developed (a) with chloroform/methanol/ water (60:40:10, v/v) or ( b ) with 1-propanol/water/ammonium hy- droxide (75:405, v/v). Lune I, polar glycolipid fraction, still contain- ing neutral glycolipid contaminants, fractionated by QAE-Sephadex column chromatography; lanes 2-4, isolated PGL, to PGL3. Spots were visualized with orcinol-H2S04 reagent.

FIG. 2. Thin layer chromatogram of the products obtained from polar glycolipids, PGL, to PGLs, by treatment with HF for dephosphorylation and the infrared spectrum of PGL,. Lane I, a mixture of the neutral glycolipids from the earthworm, P. hilgendorfi (NGL], Galpl-Cer; NGL2, Galpl-6Galpl-Cer; NGLa, Ga1~1-6Ga1~1-6Ga1~1-Cer) (12); lanes 2, 4, and 6, intact polar glyco- lipids, PGL, to PGL,; lanes 3 ,5 , and 7, dephosphorylated products of PGL, to PGL3. The plate was developed with chloroform/methanol/ water (604010, v/v) and the spots were visualized with orcinol- H2S04 reagent. In the infrared spectrum of PGL,, the band marked with an arrow a t around 960 cm" indicates the presence of the choline residue.

a QQ. 0 '

""

1 2 3 4

- a "

1 2

FIG. 3. Thin layer chromatograms of the acidic hydroly- sates of PGLl. Panel a: lane 1, intact PGL,; lane 2, authentic galactose; lane 3, authentic galactose 6-phosphate; lune 4, acidic hydrolysate of PGL,. Panel b: lane 1, authentic choline; lune 2, acidic hydrolysate of PGL,. The plates were developed with chloroform/ methanol/water (30:30:10, v/v) for a and with 1-butanol/acetic acid/ water (40:6010, v/v) for b. The spots were visualized with orcinol- H2S04 reagent for a and with Dragendorff s reagent for b.

shift. All NMR spectra were recorded with 16K data points and with a spectral width of 4,000 Hz.

RESULTS AND DISCUSSION

Isolation of Three Polar Glycolipids (PGLl to PGL)' Lipids extracted from whole tissues of the earthworm, P.

hilgendorfi, were submitted to mild alkaline hydrolysis, and the resulting alkali-stable substances (9.6 g) were applied to a QAE-Sephadex column. The polar glycolipids with the

' The abbreviations used are: PGL, polar glycolipid; NGL, neutral glycolipid; lGal, 2,3,4,6-tetra-0-methyl-1,5-di-O-acetylgalactitol; 1,6Gal, 2,3,4-tri-0-methyl-1,5,6-tri-O-acetylgalactitol.

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Novel Glycosphingolipids Containing Choline Phosphate 22597

TABLE I Glycosidic linkage analysis (substituted positions of sugars) of PGL,

and PGL, Method A, alditol acetates obtained from intact PGLs and NGL,;

Method B. from the dephosphorylated PGLs; Method C, from the perrnethylated and dephosphorylated PGLs.

Methylation method PGL, PGL, NGL,"

A NDb 1,6'Gal 1,6Gal

B lGal 1,6Gal

C 1,6Gal 1,6Gal

1Gal

lGal

" NGL, is a reference neutral glycolipid (Gal,L?l-6Gal/3l-Cer) (12).

' Numbers express the substituted positions of sugar residues. Not detected.

200 400 600 800 i 000 I e00 t r2 : i ? /Z

P

PGLl ( A ) and PGL, ( B ) . FIG. 4. Positive-mode FAB-MS spectra of polar glycolipids,

4.0 3.5 PPrn 3.0

the 'H NMR spectra of polar glycolipids, PGLl ( A ) and PGLz FIG. 5. Anomeric proton and choline phosphate regions of

( B ) .

TABLE I1 Chemical compositions of the ceramide portions of polar glycolipids,

PGL, and PGL, Composition PGL, PGL,

%

Fatty acid 160 2.6 1.5 18:O 5.9 5.8 20:o 5.5 4.9 21:o 3.6 3.7 22:o 49.3 56.0 23:O 8.1 7.1 24:O 25.0 20.9

br ,dl8l 54.7 52.5 d18:l 24.5 18.7 br,dl9:1 20.8 28.8

Long chain base"

br, branched d, dihydroxy long chain base.

neutral glycolipid fraction (4.2 g) were recovered from the QAE-Sephadex column with chloroform/methanol/water and could be separated into three subfractions by Iatrobeads gra- dient elution column chromatography with chloroform/meth- anol/water. Contaminating amounts of minor components in each fraction necessitated further purification by Iatrobeads column chromatography using a single elution with l-pro- panol/water/ammonium hydroxide. The yields of the purified polar glycolipid components were: PGLl, 156 mg; PGLZ, 41 mg; and PGL3, <4 mg. Fig. 1 shows the TLC pattern of these purified polar glycolipids. They were stained by orcinol-HzS04 and molybdate spray reagents and, in addition, surprisingly reacted with Dragendorffs reagent on TLC plates. These observations indicated the presence of the choline phosphate group within the glycolipid molecules. The infrared spectrum of PGLl shows a sharp absorption band at around 960 cm", which is in part attributable to choline (Fig. 2). Dephospho- rylation of the three polar glycolipids with HF yielded prod- ucts that no longer stained with molybdate or Dragendorffs reagents and which migrated to different positions on TLC from the parent glycolipids (Fig. 2). The new positions cor- responded to those of three neutral glycolipids from the present organism already reported on (12). The molar ratios of phosphorus/sugar of the three polar glycolipids were 1.00:0.92 for PGL1, 1.00:2.01 for PGL2, and 1.00:3.00 for PGL3.

Carbohydrate Constituent Analysis

Monosaccharide constituents were analyzed quantitatively as methyl glycoside-TMS derivatives, after acid hydrolysis of the dephosphorylated products obtained from PGL1, PGL,, and PGLs. The dephosphorylation was required because the residue substituted with choline phosphate could not be de- termined as the free neutral sugar under the conditions of acid hydrolysis employed here (5% methanolic HCl at 100 "C for 3 h). The following appropriate molar ratios were deter- mined; PGL, contained 1 mol of Gal, PGL, contained 2 mol of Gal, and PGL, contained Gal:Man, 2.3:l. Due to the small quantities of PGL3 remaining after this analysis, it was not possible to further characterize this lipid.

Linkage Position of the Choline Phosphate in Polar Glycolipids

Acid Hydrolysis-PGL1 was hydrolyzed with 6 N HCl at 100 "C for 3 h, yielding galactose 6-phosphate, galactose, and choline as shown by TLC (Fig. 3); it should be noted that production of some galactose is expected from galactose 6- phosphate under these hydrolytic conditions. Hydrolysis of PGL, yielded the same three products (results not shown).

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22598 Novel Glycosphingolipids Containing Choline Phosphate

Exoglycosidase Analysis (12)"The polar glycolipids were not acted upon by any exoglycosidases tested. However, after dephosphorylation of PGL, and PGL, by HF, the neutral glycolipids recovered were labile toward jack bean P-galacto- sidase (Seikagaku Kogyo Co., Ltd., Tokyo), both of them yielding a product with the TLC migration behavior of cer- amide.

These hydrolytic and enzymatic analyses suggest that the phosphate group is carried on the terminal galactose of both polar glycolipids, and that all galactoses are linked via P- glycosidic bonds.

Methylation Analysis-Further evidence concerning the at- tachment site of choline phosphate to the polar glycolipids was gained by studying the partially methylated alditol ace- tates obtained from intact glycolipids, as well as from glyco- lipids subjected either to prior dephosphorylation or to per- methylation followed by dephosphorylation. The products were analyzed by GC and GC-MS and are listed in Table I, as are also, for comparison, those derived from NGL2, the nonphosphorylated analogue of PGL,. The product alditol acetates observed during the course of these studies were 2,3,4,6-tera-O-methyl-1,5-di-O-acetylgalactitol (1Gal) and 2,3,4-tri-0-methyl-1,5,6-tri-O-acetylgalactitol (1,6Gal).

Application to the intact polar glycolipids (Method A in Table I) yields no detectable galactitol derivative from either PGLl or the terminal galactose of PGL,, since both of the expected galactitol products remain phosphorylated and are hence not observable under the conditions used here for GC analysis. The only product observed from PGLz is therefore 1,6Gal derived from the internal galactose. Dephosphoryla- tion with HF prior to methylation (Method B) yields 1,6Gal from the internal galactose of PGL, and lGal from the ter- minal galactose of both PGL, and PGL,. However, if the polar glycolipids are first permethylated and products, still substi- tuted with choline phosphate, are then dephosphorylated (Method C), subsequent acetylation produces 1,6Gal from both PGL, and PGLz.

These results indicate that choline phosphate is attached to the 6-hydroxy group of the terminal galactose in PGLl and PGL,.

FAB-MS Study Positive-ion mode FAB mass spectra of PGLl and PGLz

shown in Fig. 4 demonstrated clearly the presence of the molecular ions at m/z 935, 949, 963, and 977 in PGLl and at m/z 1097, 1111, 1125, and 1139 in PGL,. In addition, the fragmentation patterns showed the sugar sequences to be as indicated in the figure (Fig. 3, a and b ) . Fragments at m/z 328 and 344 exhibited by PGLl and at m/z 328,344,490, and 506 by PGLz suggest the presence of a choline phosphate group located on a hexose. The increased size of the molecular ions obtained from PGL, as compared with PGL1, i.e. 162 mass numbers, is attributable to the presence of a second hexose residue in the former compound. The major molecular ion

species of the ceramide moieties of PGL, and PGL, which were detected at m/z 949 and 1111 are assigned as C22:O fatty acid and C18:l sphingoid.

NMR Study The above structures suggested for PGLl and PGLz were

further supported by the results of 'H NMR spectroscopy, as shown in Fig. 5. In the anomeric region of each spectrum, the presence of one anomeric proton for PGLl (chemical shift, 4.061 ppm) and two protons for PGLz (chemical shifts, 4.074 and 4.178 ppm) was demonstrated by the coupling constant values (6.83-6.84 Hz), indicating that the sugar residues of both polar glycolipids occur in the p-anomers. Also, in both spectra, two distinctive signals attributable to methylene pro- tons of the choline phosphate were observed, whose chemical shifts were 3.535 and 4.073 ppm, respectively.

Aliphatic Components The aliphatic components of the polar glycolipids were

determined by GC and GC-MS (Table 11). The main fatty acids were C22:O and C24:0, and the sphingoids were com- posed of octadeca- and nonadeca-4-sphingenines, in both glycolipids. Considering the similarity of the aliphatic com- ponents in these polar glycolipids compared with their neutral counterparts (12), it would appear likely that polar glycolipids in the earthworm are derived from the neutral glycolipids which were reported earlier in this organism.

Acknowledgment-We thank Dr. A. Suzuki of the Tokyo Metro- politan Institute of Medical Science and Dr. J. T. Dulaney of the University of Tennessee for their helpful comments.

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M Sugita, H Fujii, F Inagaki, M Suzuki, C Hayata and T Horicontaining choline phosphate from the earthworm, Pheretima hilgendorf.Polar glycosphingolipids in annelida. A novel series of glycosphingolipids

1992, 267:22595-22598.J. Biol. Chem. 

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