characterization of a o-fatty-acylated sulfatide from equine brain

Eur. J. Biochem.255, 2892295 (1998) FEBS1998

Characterization of a O-fatty-acylated sulfatide from equine brain

Takeshi MIKAMI 1, 2, Keiko TSUCHIHASHI1, Motoi KASHIWAGI 1, 2, Youichi YACHIDA 1, Takumi DAINO1,2, Kazuo HASHI3,Toyoaki AKINO2 and Shinsei GASA1

1 Department of Chemistry, Sapporo Medical University School of Medicine, Sapporo, Japan2 Department of Biochemistry, Sapporo Medical University School of Medicine, Sapporo, Japan3 Department of Neurosurgery, Sapporo Medical University School of Medicine, Sapporo, Japan

(Received15 January/14 April 1998) 2 EJB 98 0061/5

A sulfatide, O-fatty-acylated 3-sulfogalactosylceramide at C6-O on galactoside, was isolated fromequine brain and the chemical structure was characterized by proton NMR and MS. The O-acylation siteof the acylated sulfatide was determined by the down-field shift of protons attached to a carbon havingan O-acyl group in the NMR spectrum and by analysis of a partially methylated derivative before andafter acetalization of the intact sulfatide using GC-MS. The O-acyl chain length was determined by GLC,revealing that it exclusively had palmitoyl and stearoyl residues as the major fatty acids. The enzymaticconversion to the O-acyl sulfatide was further examined using equine brain microsomes as an enzymesource and different lipid substrates, resulting in O-acylation of 3-sulfogalactosylceramide from stearoylCoA, while 6-O-acyl galactosylceramide was not O-sulfated from phosphoadenosine phosphosulfate. Theresults were supported by the comparably different N-linked fatty acid components between two lipidsubstrates, in which the component of 6-O-acyl sulfatide was mostly similar to that of sulfatide, but notto 6-O-acyl galactosylceramide.

Keywords: O-acyl sulfatide; O-acylation; esterified sulfatide; NMR.

Of the many glycolipids, sulfated glycosphingolipids (sulfa-MATERIALS AND METHODStides) are distributed in a wide variety of mammalian tissues and

Chemicals. DEAE-Sephadex, A-25 and Sephadex, LH-20fluids, and have been reported to have several roles in bindingwere purchased from Pharmacia-LKB, silica beads (Iatrobeads)to some biologically active proteins such as laminin, thrombo-from Iatron Laboratories, TLC-plates (silica-gel 60) andspondin, von Willebrand factor and antistasin [126]. 3-O-hexadeuterodimethylsulfoxide ([2H6]Me2SO) from Merck. Stea-Sulfogalactosylceramide (Gal3S-Cer) is a major glycolipid ofroyl CoA was from Sigma (MO). 3′-[35S]Phosphoadenosine 5′-the sulfatides and localizes mainly in the central nervous systemphosphosulfate (2 Ci/mmol) and [1-14C]stearic acid (55 mCi/

and kidney, and to a minor extent in other tissues and fluidsmmol) were obtained from NEN Bioproducts. All other reagents[7, 8]. The Gal3S-Cer is biosynthesized from the precursorwere of analytical grade.

galactosylceramide (GalCer) with glycolipid sulfotransferase lo- Isolation of O-acylated sulfatide.The ratio of solvent mix-calizing in the Golgi membrane [9,10]. tures is expressed by volume. An acetone powder (104 g dry

Esterified glycolipids with long-chain fatty acids have al-mass) was obtained from whole equine brain (457 g wet mass)ready been isolated from mammalian brain, spleen and epider-by homogenization with acetone (1 g/9 ml). The glycolipidsmis [11217]. However, these modified glycolipids had an ex-were extracted three times from the powder with chloroform/clusively non-acidic glycolipid skeleton such as GalCer and glu-methanol/water, 4:8:3, at room temperature. The acidic glyco-cosylCer; where the O-acylation occurred at C2-, C3-, C4-, C6-lipid fraction was isolated from the combined and concentratedand C3,6-di-O on Gal, C3-O on Cer, and C3-O and C6-O onextracts with a DEAE-Sephadex, A-25 (acetate form) columnglucoside. Recently, O-acylated sphingomyelin at the C3-O of(2.5 cm330 cm) by elution with chloroform/methanol/water,the Cer moiety has been identified from newborn pig and infant40:60:10, containing1 M ammonium acetate after removal ofplasma [18]. In this study, we report the isolation and character-the unbound fraction from the column with chloroform/metha-ization of an O-fatty-acylated sulfatide from equine brain, andnol/water, 40:60:10. The total acidic glycolipid fraction wasfurther, the enzymatic generation of the O-esterified sulfatidecondensed, salts were removed by dialysis, and solvents evapo-whether from Gal3S-Cer or from O-acylated GalCer. rated to dryness and chromatographed on an Iatrobead column

(2.5 cm350 cm) by stepwise elution with chloroform/methanol/Correspondence toS. Gasa, Department of Chemistry, Sapporowater using1000 ml each of 90:10:0.5, 80:20:2, 70:30:3,

Medical University School of Medicine, Chuo-ku S1 W17, Sapporo, 60:40:4, 50:50:5 and 40:60:6. The fractions containing a gly-Japan 060 colipid migrating faster than Gal3S-Cer on TLC and containing

Abbreviations.Cer, ceramide; FAB-MS, fast-atom-bombardment2Gal3S-Cer were further separately chromatographed on an Iatro-mass spectrometry ; GalCer, galactosylceramide; Gal3S-Cer, 3-O-bead column with a smaller size (1 cm340 cm) for the formersulfogalactosylceramide; Me2SO, dimethylsulfoxide. Fatty acids are ab-fraction and with a different size (1.5 cm350 cm) for the latterbreviated as C(carbon chain length):[saturation (0) or unsaturationby stepwise elution with chloroform/methanol/water with(1)]:[hydroxy (h) if present].

Enzyme.AcylCoA:cholesterol acyltransferase (EC 2.3.1.26). 300 ml each of 98:2:0.1, 96:4:0.2, 94:6:0.3, 92:8 :0.4 and

290 Mikami et al. (Eur. J. Biochem. 255)

90:10:0.5, and the chromatographies were repeated to obtain Faculty of Agriculture of Hokkaido University, as reported pre-viously [24, 25].a homogenous glycolipid. The faster-migrating glycolipid and

Gal3S-Cer were obtained with yields of 5 mg and 355 mg, re- Measurement of NMR spectrum. One-dimensional andtwo-dimensional proton-NMR spectra of the purified glycolipidspectively. The purified glycolipids were chromatographed on a

TLC plate, developed with chloroform/methanol/water, 80:20:2 (approximately1 mg) in 0.3 ml of [2H6]Me2SO containing 2%2H2O were measured at 90°C in the Fourier-transform mode onor 90:10:0.5 and visualized by staining with orcinol/sulfuric

acid reagent under heating or with Azure A reagent [19]. a Varian JNM-GX500 spectrometer equipped with a JEC-980Bcomputer at the above laboratory, as described previously [24,The total neutral glycolipid fraction obtained after passage

of the above extracts through a DEAE-column was chromato- 25]. The chemical shift was measured using tetramethylsilane asan internal standard.graphed on an Iatrobead column in the same manner as de-

scribed above, to isolate O-acylated GalCer species (the detailedMeasurement of FAB-MS spectrum. Negative fast-atombombardment (FAB)-MS was performed on a JEOL JMS-procedure and results will be published elsewhere). From the

neutral lipid fraction, 6-O-acyl GalCer was obtained with a yield HX100 mass spectrometer equipped with a JMA-DA500 data-lizer in the above NMR-MS laboratory. The sample was bom-of 8 mg, and the acylated lipid was employed as a reference

glycolipid for NMR study and as a substrate for enzymatic con- barded by Xe gas at 6 kV (20 mA) in a matrix of triethanol-amine, and the fragments were accelerated at 5 kV, as describedversion toO-acyl Gal3S-Cer.

Saponification of glycolipids. The purified glycolipid (ap- previously [24, 25].Enzymatic conversion toO-acyl Gal3S-Cer.Equine brainproximately 0.1 mg) or a radioactive band due to the glycolipid

extracted from an autoradiographed TLC plate after enzymatic (250 g wet mass) was homogenized with a Polytron in 25 mMTris/HCl, pH 7.4, containing 0.25 M sucrose,1 mM EDTA andO-acylation was treated with1ml of 1% sodium methoxide in

methanol for 2 h at room temperature. After neutralization with1mM phenylmethylsulfonyl fluoride. The homogenates werecentrifuged at10003g for 10 min, and the supernatant was fur-acetic acid, the mixture was concentrated and applied to an LH-

20 column (0.5 cm310 cm) with chloroform/methanol/water, ther spun at1.231043g for 20 min. The microsomes were ob-tained after centrifugation of the1.231043g supernatant at60:30:4.5 for desalting. The passed-through glycolipid from the

column was chromatographed on a TLC plate as above. 1.0531053g for 1 h, suspended in150 mM sodium phosphate,pH 7.4, and stored at280°C until use. Protein amount was mea-Analysis of lipid moiety. The components of O-linked and

N-linked fatty acids of the glycolipid were separately analyzed sured using the BCA protein assay reagent (Pierce) and bovineserum albumin as a standard. Enzymatic O-acylation of Gal3S-as their methyl esters according to the method of Tamai et al.

[20] with a slight modification. Briefly, approximately 0.5 mg Cer was performed according to the method of Kritchevsky andKothari [27], using liposomes composed of lecithin and14C-la-of O-acylated glycolipid was stirred under a nitrogen atmosphere

in 0.1 M sodium methoxide in methanol at room temperature. beled substrate lipids (cholesterol as a control and Gal3S-Cer)at a molar ratio of10:1. The solvents of a mixture containingThe de-O-acylation reaction was completed within1 h by moni-

toring with TLC. The liberated fatty acid methyl ester (O-linked egg yolk lecithin (0.20µmol, Sigma) and [14C]cholesterol(5.73104 dpm/20 nmol, NEN products) or [14C]Gal3S-Cerfatty acid) was extracted with1 ml of n-hexane three times, fol-

lowed by washing the combined extracts with1ml of water and (73104 dpm/20 nmol, prepared as described below) were evapo-rated to dryness under a stream of nitrogen atmosphere at roomevaporation of the solvent to dryness. The residual methanolic

solution was neutralized with1M HCl, and the solvent was temperature. The lipid mixture was completely dried in a desic-catorin vacuofor more than 2 h.10 µl of 1.5 M potassium phos-evaporated to dryness. The residue containing de-O-acylated

glycolipid was further methanolyzed with1M HCl in methanol phate buffer, pH 7.4, and 20µl of water were added to the lipidmixture and mixed well for1min, followed by incubation atat 80°C for 16 h, and the liberated N-linked fatty acid methyl

esters were extracted as described above. The O-linked and 40°C for 30 min. Liposomes with a translucent lipid dispersionwere then obtained by sonication (25 W,1 min, UR-200P, TomyN-linked fatty acid methyl esters were separately analyzed by

GLC (Shimadzu GC-14 A) using a capillary column Seiko Co., Ltd) of the mixture. The liposomes were incubatedwith the above microsome protein (390µg) in a final volume of(0.25 mm350 m) coated with 0.1% of DB-5 with temperatures

programmed from160°C to 280°C at 5°C/min. The long-chain 90µl at 37°C for 90 min. The O-acylation reaction was thenstarted by adding stearoyl CoA (50 nmol/10 µl 0.5 M potassiumbase of the purified glycolipid was analyzed by GLC under the

same conditions as above according to the method of Gaver and phosphate, pH 7.4) for 30 min at 37°C. The lipids were isolatedfrom the incubation mixture by adding 0.5 ml of chloroform/Sweeley [21].

Analysis of O-acylated position.The methylation analysis methanol, 2:1, and the resultant lower layer was collected andevaporated to dryness. The residual materials were dissolved inof the purified glycolipid was performed before and after acetali-

zation, to determine the O-acylation site as reported previously10 µl of chloroform/methanol, 2:1, and chromatographed on aTLC plate (10 cm310 cm), developed with petroleum ether/di-[22225]. Briefly, 1mg of the glycolipid was first acetalized

with 1.0 ml of methyl vinyl ether in1ml of Me2SO solution ethyl ether/acetic acid, 80:30:1, for the product from choles-terol, and chloroform/methanol/water, 65:25:4, for the productcontaining1.0 mg ofp-toluene sulfonic acid at 0°C for 6 h. The

reaction mixture was concentrated and applied to an LH-20 col- from Gal3S-Cer. The radioactive bands were visualized andquantified with a Bioimaging Analyzer (BAS2000, Fuji) afterumn (1 cm330 cm) in chloroform to remove acid and reaction

solvents. The sugar-positive fractions excluded from the column exposure for 8 h. The substrate [14C]Gal3S-Cer was preparedfrom lysoGal3S-Cer [28] and [1-14C]stearoyl chloride, the latterwere collected and evaporated to dryness. The acetalized or in-

tact glycolipid was methylated with methyliodide and methyl- of which was prepared from [1-14C]stearic acid and thionyl chlo-ride as reported previously [29].sulfinyl carbanion, followed by acetolysis/hydrolysis, reduction

with NaBH4 and peracetylation, according to the method of Ha- Enzymatic O-sulfation of 6-O-acyl GalCer was carried outin 20 mM Tris/HCl, pH 7.2, containing 20 nmol of 6-O-acylkomori [26]. The liberated, partially methylated galactitol ace-

tate was analyzed by GC-MS using a JMS-HX100 mass spec- GalCer,100 nmol of 3′-phosphoadenosine-5′ [35S]phosphosul-fate (53104 dpm) and1 mg of microsome protein, as reportedtrometer equipped with a 25-m capillary column (i.e., 0.25 mm)

coated with 1% OV-1 with temperatures programmed from earlier [30]. The extraction and chromatography on a TLC plateof the lipid product were performed as described above.150°C to 280°C at 5°C/min at the NMR-MS Laboratory of the

291Mikami et al. (Eur. J. Biochem. 255)

Table 1. Lipid composition of theglycolipid, Gal3S-Cer and 6-O-acyl-GalCer. The glycolipids were methanolyzed as described in Materialsand Methods. The liberated O-linked (O-Acyl) and N-linked (N-Acyl)fatty acid methyl esters were separately analyzed by GLC. The long-chain base was analyzed by GLC as trimethylsilyl ether derivative aftermethanolization [21].

Fatty acida Composition of

glycolipid studied 3-sulfo- 6-O-acylGalCerGalCer

O-Acyl N-Acyl N-Acyl O-Acyl N-Acyl

%Fig. 1. TLC of an O-acylated glycolipid from equine brain beforeand after saponification. The O-acylated or non-acylated glycolipid

16:0 31 14from equine brain was chromatographed on a TLC-plate, before (A) and16:1 1after (B) saponification. In (A) and (B) lane1 indicates GalCer, lane 2,17:0 1the glycolipid studied, lane 3, Gal3S-Cer, and O, origin. (A) and (B)18:0 39 2 1 35 4were developed with chloroform/methanol/water, 90:10:0.5 and18:1 17 880:20:2, respectively, and visualized by staining with orcinol/sulfuric20:0 2 2 1 5 2acid reagent.20:1 2 222:0 1 2 1 5 222:0:h trb

RESULTS 22:1 223:0 5 2 2 3Isolation of O-acylated sulfatides.The purified acidic glyco-23:0:h tr 5

lipid that migrated faster than Gal3S-Cer and the copurified23:1 1Gal3S-Cer were chromatographed on a TLC plate (Fig.1). Posi- 24:0 2 20 22 7 11tive staining of these glycolipids with Azure A reagent (data not24:0:h 4 3 28shown), a specific reagent for a sulfated compound [19], con- 24:1 1 23 31 2 5firmed the glycolipid to be sulfatide. The glycolipid further mi-24:1 :h 10 8 12

25:0 3grated to a position identical to that of Gal3S-Cer on TLC after25:0:h 2 2 7saponification (Fig.1), suggesting that it linked to some alkali-25:1 7 9 5labile group in the molecule. The equine brain 6-O-acyl GalCer,26:0 1 4 4 8 4a glycolipid similar to that already isolated and characterized26:0:h 1 3 3from other mammalian brains, was identified by NMR and FAB-26:1 1 6 9 3 3

MS (data not shown). 26:1 :h 2 1 3Unknown 1 7 3 6 1

Analysis of lipid moiety. The analysis of fatty acids liberatedLong chainfrom the glycolipid at the different linkages revealed that C16 Baseand C18 acids were a major components as O-acyl residues andd18:0c 5 4 2C24 acid, with saturation and unsaturation was a major compo-d18:1 d 88 89 95nent as an N-acyl group, as summarized in Table1. The constitu- d20:1e 7 7 3tional ratio of the O-acyl residue in the glycolipid was compara-

a Abbreviated as (carbon chain) :[saturation (0) or unsaturationble to that of 6-O-acyl GalCer, and that of the N-acyl group in(1)]:[hydroxy (h)].the glycolipid was mostly similar to that of Gal3S-Cer rather

b Trace (,1%).than that of 6-O-acyl GalCer. The similarity of the N-acyl com-c C18 Sphinganine.ponent between the glycolipid and Gal3S-Cer led us to consider d C18 Sphingenine.

a biosynthetic generation of new glycolipid from Gal3S-Cer, e C20 Sphingenine.rather than from 6-O-acyl GalCer. C18 Sphingenine was detectedfrom the glycolipid as a major long-chain base similar to thosefrom Gal3S-Cer and 6-O-acyl GalCer (Table1). together with upper field shifts of protons atδ 4.167 (I6a in

Fig. 2) and 4.110 (I6b) (data not shown). Hence, the triplet signalAnalysis of O-acylated position. The GC-MS analysis of the was assigned toA-methylene protons on the O-fatty acyl residue.partially methylated alditol acetates indicated that the glycolipidThe other protons attached to the carbons of sugar and lipidprovided a1,3,5-tri -O-Ac 2,4,6-tri -O-Me galactitol and a 6-O- moieties of the new glycolipid were assigned by two-dimen-Me 1,2,3,4,5-penta-O-Ac galactitol (data not shown), before andsional chemical-shift-correlated spectroscopy (Fig. 3) and the as-after acetalization, respectively. These MS spectra were identicalsignments are summarized in Table 2, together with those ofto those derived from suitable authentic glycolipids. JudgingGal3S-Cer and 6-O-acyl GalCer as the reference. The galacto-from the observation of these galactose derivatives, the glyco-side H6 (C6 protons; I6a and I6b in Fig. 3) in the glycolipid werelipid was found to be substituted at C3-O of the galactose withassigned with the cross-peaks (from I1-2 to I6a-6b) in the two-an alkaline-stable group such as sulfate and at C6-O with andimensional spectrum, and their chemical shifts were markedlyalkali-labile residue like that of an O-acyl ester. lower compared with those of Gal3S-Cer, suggesting an acyl

group binding to the C6-O on the sugar moiety. In addition,galactoside H3 (δ 4.009) of the glycolipid shifted to a lowerNMR study. The one-dimensional proton-NMR spectrum of the

glycolipid revealed a signal atδ 2.273 ppm with a triplet, com- field than that (δ 3.319) of 6-O-acyl GalCer, being responsiblefor binding to C3-O with sulfate [31], and resonated in a fieldpared with that of Gal3S-Cer (Fig. 2). The triplet peak disap-

peared from the glycolipid after saponification of the glycolipid, similar to that (δ 3.975) of Gal3S-Cer (Table 2). Regarding these


Fig.3. Two-dimensional NMR spectrum of the glycolipid.The homo-Fig. 2. One-dimensional proton NMR spectrum of the glycolipid.Thenuclear two-dimensional chemical shift-correlated spectrum wasNMR spectrum of the purified new glycolipid was measured at 90°Cobtained as indicated in the text. Cross connectivity between protonsusing 500-MHz NMR in a solution of [2H6]Me2SO-2H2O (DMSO). Theis demonstrated as, e.g., I6a,6b between H6a and H6b on Gal in theprotons on Gal, the long-chain base, fatty acid and amide in the spectrumspectrum.are abbreviated as I, L, FA and NH, respectively. The numbering of the

protons is indicated in Fig. 5.

ions of the glycolipid were responsible for the different chaindown-field shifts of H6 and H3 of the glycolipid, as well as thelengths of the fatty acids. As assigned in the figure, ions with adata of TLC analysis with saponification, the structure of thecombination of N-linked and O-linked fatty acyl chains wereglycolipid was suggested to be esterified with an additional al-observed in the pseudomolecular ion field. The ions atm/z 1126kali-labile residue, probably an O-acyl group, to C6-O on theandm/z 1154 were observed as the major signals, and they weregalactoside of Gal3S-Cer. assigned to C24:1 as the N-acyl chain and C16:0 as the O-acyl,

and C24:1 as the N-acyl and C18:0 as the O-acyl and/or C24:0and C18:1 and/or C26:1 and C16:0, respectively, regardingFAB-MS study. The negative FAB-MS spectrum of the glyco-

lipid showed two subsets of fragment ions, a larger ion group fatty acid data from GLC analysis as described above. The ionsof Gal3S-Cer were observed atm/z 888, 890, 902, 904 and 916due to pseudomolecular ions of Gal3S-Cer having an additional

acyl chain, and a smaller one due to the ions of Gal3S-Cer as the major signals due to C24:1, C24:0, C25:1, C24:1 havinga hydroxyl group, and C26:1 for all of the N-acyl chains, re-(Fig. 4). Since the long-chain base of the glycolipid was com-

posed of C18 sphingenine as a major base (Table1), the fragment spectively.

Table 2. Chemical shifts in the NMR spectra (500 MHz, 90°C) of GL-1 and reference glycolipids.Proton NMR spectra of the glycolipids in[2H]Me2SO containing 2%2H2O were measured at 90°C on a 500-MHz spectrometer.

Glycolipid Chemical shift,δ of

H1a H1b H2 H3 H4 H5 H6a H6b -OCOCH2-

ppm

Studied glycolipid(6-O-Acyl Gal3S-Cera)

LCB (L)b 3.898 3.541 3.804 3.991 5.410 5.578 1.983 2 2.272Gal (I)c 4.200 2 3.517 4.009 3.946 3.634 4.167 4.110

6-O-Acyl GalCer

LCB 3.865 3.563 3.828 4.036 5.416 5.605 2 2 2.265Gal 4.123 2 3.355 3.319 3.636 3.588 4.173 4.123

Gal3S-CerLCB 3.931 3.559 3.798 3.964 5.394 5.562 1.977 2 2Gal 4.166 2 3.515 3.975 3.982 3.379 3.525 3.498 2

a 6-O-Acyl I 3SO3-GalCer.b Long-chain base (abbreviated as ‘L’ in the Figs 2 and 3).c Abbreviated as ‘I’ in the Figs 2 and 3.


Fig. 4. Negative FAB-MS spectrum of the glycolipid.The partial FAB-MS spectrum was measured as indicated in the text. The numbers inparentheses in the fragment region of Cer and HSO3-Hex-Cer (e.g., 24:1) and of acyl-HSO3-Hex-Cer (e.g., 24:1116:0) mean C24 fatty acid withunsaturation for the former and C24 N-linked fatty acid with unsaturation plus C16 O-linked fatty acid with saturation for the latter.

Fig. 5. Structure of the glycolipid studied.

[14C]Gal3S-Cer and lecithin instead of cholesterol for the former,revealing a new band with anRf identical to that of the authenticglycolipid (Fig. 6). The radioactive degradation products such asCer and fatty acid from [14C]Gal3S-Cer were not detectedthrough the reaction. In the enzymatic reaction, Gal3S-Cer wasesterified at 94 pmol · h21 · mg protein21, whereas cholesterolwas esterified at12 pmol · h21 · mg21. The newly appeared bandidentical to that of the glycolipid was converted to the positionof Gal3S-Cer on TLC analysis after saponification (data notshown), indicating addition of a fatty acyl group to Gal3S-Certhrough the enzymatic reaction, though the acylation site wasnot identified. No bands including the glycolipid appeared when

Fig. 6. Enzymatic conversion to the glycolipid.The liposomes com- a mitochondrial fraction and cytosolic fraction of equine brainposed of lecithin and 3-sulfo[14C]GalCer were incubated with equine were employed as enzyme sources in the above assay system.brain microsomes, and the reaction was then started by addition of stea-However, an assay of sulfotransferase toward 6-O-acyl GalCerroyl CoA. The lipids were extracted and subjected to TLC analysis,with 3′-phosphoadenosine 5′-[35S]phosphosulfate and braindeveloped with chloroform/methanol/water, 80:20:2. Lane1 shows microsomes or other subcellular fractions did not provide anyauthentic glycolipid; 2, [14C]C18-Cer (N-[14C]stearoyl sphingenine); 3,

products (data not shown).[14C]stearic acid; 4, the lipids from the above reaction; 5, the lipids fromthe reaction without microsomes. Lane1 is stained by orcinol/sulfuricacid reagent and lanes 2 to 5 are autoradiograms.

DISCUSSION

Here we isolated an acidic glycolipid, migrating faster thanGal3S-Cer on TLC, from equine brain and identified its structureFrom the combined data of the above analyses, the structure

of the glycolipid was identified as 6-O-acyl 3-O-sulfo GalCer, by NMR, MS and chemical techniques, as 6-O-acyl 3-O-sulfoGalCer. The most important assignment of the glycolipid wasas illustrated in Fig. 5, having C16:0 and C18:0 as a major O-

linked fatty acid and C24: 0 and C24:1 as a major N-linked fatty the acylation site, which was identified first of all by down-fieldshifts of C6 methylene protons of the Gal in the NMR spectrum,acid.though the Gal H3 was also shifted down-field compared withthat of 6-O-acyl GalCer (Table1). The acylation site was con-Enzymatic conversion to O-acyl Gal3S-Cer.The enzymatic

synthesis of 6-O-acyl Gal3S-Cer was examined using equine clusively assigned by methylation analysis of the glycolipidusing GC-MS before and after blocking with acetalization underbrain microsomes as an enzyme source, 6-O-acyl GalCer or

Gal3S-Cer as an acceptor and stearoyl CoA as a fatty acid donor. low pH [22, 23]. From the methylation analysis before and afteracetalization, a 3-O-acetyl galactitol derivative in the former andAn assay of acyl CoA-cholesterol-O-acyltransferase was applied

to the O-acylation of Gal3S-Cer in liposomes containing 6-O-methyl galactitol derivative in the latter were observed, in-


7. Karlsson, K. A., Samuelsson, B. E. & Steen, G. O. (1968) Structuredicating an alkali-stable group, which survived exposure underand function of sphinolipids.1. Differences in sphingolipid long-high pH to the methylation reaction; as a sulfate residue waschain base pattern between kidney cortex, medulla, and papillae,attached to C3-O of the Gal, an alkali-labile group, it decom-Acta Chem. Scand. 22, 136121363.posed under the methylation base but survived the acetalization

8. Karlsson, K. A., Samuelsson, B. E. & Steen, G. O. (1968) StructurepH, like the O-acyl residue bound to the C6-O of the Gal. The and function of sphingolipids. 2. Differences in sphingolipid con-presence of sulfate and O-acyl residues as the additional groups centration, especially concerning sulfatides, between some re-on the GalCer skeleton as well as the chain length of the fatty gions of bovine kidney,Acta Chem. Scand. 22, 272322724.acid were estimated by FAB-MS and GC-MS analyses. The9. Honke, K., Yamane, M., Ishii, A., Kobayashi, T. & Makita, A.

(1996) Purification and characterization of 3′-phosphoadenosine-chain length of the O-linked fatty acid of the 6-O-acyl Gal3S-5′-phosphosulfate:GalCer sulfotransferase from human renal can-Cer was mostly similar to that of other O-acylated GalCer spe-cer cells,J. Biochem.(Tokyo) 119, 4212427.cies reported previously [11214, 16, 17]. Although the biologi-

10. Honke, K., Tsuda, M., Hirahara, Y., Ishii, A., Makita, A. & Wada,cal meaning of the exclusive composition of palmitoyl and stear-Y. (1997) Molecular cloning and expression of cDNA encodingoyl residues as O-linked fatty acid, clearly different from that ofhuman 3′-phosphoadenylylsulfate:galactosylceramide 3′-sulfo-the N-linked acid, in O-acylated glycolipids, including 6-O-acyl transferase,J. Biol. Chem. 272, 486424868.

Gal3S-Cer is not clear, it is of interest that the chain length of11. Norton, W. T. & Brotz, M. (1963) New glycolipids of brain: a mo-O-linked fatty acid in O-acylated glycolipids is mostly similar noalkyl-monoacyl glyceryl galactoside and cerbroside fatty acidto that of O-linked fatty aldehydes, C16 and C18 aldehydes in esters,Biochem. Biophys. Res. Commun. 12, 1982203.

12. Klenk, E. & Lohr, J. P. (1967) On the ester cerebrosides of brain,plasmaloGalCer [32] and plasmalopsychosine [33].Hoppe-Seylers Z. Phys. Chem. 348, 171221714.With respect to enzymatic conversion to 6-O-acyl Gal3S-

13. Kishimoto, Y., Wajda, M. & Radin, N. S. (1968) 6-acyl galactosylCer, O-acylation of Gal3S-Cer occurred when using equine brainceramides of pig brain: structure and fatty acid composition,J.microsomes and stearoyl CoA rather than sulfation of 6-O-acylLipid Res. 9, 27233.

GalCer. This selectivity utilizing a specific precursor glycolipid14. Tamai, Y. (1968) Further study on the faster running glycolipid inwas regarded as a reasonable phenomenon from comparison of brain,Jpn. J. Exp. Med. 38, 65273.the N-linked fatty acid composition between Gal3S-Cer and 6-15. Gray, G. M., White, R. J. & Majer, J. R. (1978)1-(3′-O-Acyl)-beta-O-acyl GalCer; the composition of 6-O-acyl Gal3S-Cer was glucosyl-N-dihydroxypentatriacontadienoylsphingosine, a major

component of the glucosulceramides of pig and human epidermis,mostly similar to that of Gal3S-Cer rather than that of 6-O-acylBiochim. Biophys. Acta 528, 1272137.GalCer (Table1), the latter of which had hydroxylated C24 acids

16. Yasugi, E., Saito, E., Kasama, T., Kojima, H. & Yamakawa, T.in a higher amount compared with that of the former. The associ-(1982) Occurrence of 2-O-acyl galactosyl ceramide in whaleation of O-acylation with the fatty acid composition of thebrain,J. Biochem.(Tokyo) 91, 112121127.

Gal3S-Cer suggested that an O-acylation enzyme might recog-17. Yasugi, E., Kasama, T., Kojima, H. & Yamakawa, T. (1983) Occur-nize the N-linked fatty acid moiety of the substrate. The assay rence of 2-O-acyl galactosyl ceramide in human and bovinemethod for the O-acylation of Gal3S-Cer was applied with con- brains,J. Biochem.(Tokyo) 93, 159521599.ditions suitable for an O-acylation of cholesterol, since maximal18. Kramer, J. K., Blackwell, B. A., Dugan, M. E. & Sauer, F. D. (1996)

Identification of a new sphingolipid 3-O-acyl-d-erythro-sphingo-conditions for lecithin-cholesterol acyltransferase and other O-myelin in newborn pig and infant plasma,Biochim. Biophys. Actaacyltransferases forming acylglycerol did not esterify the Gal3S-1303, 47255.Cer (data not shown). It is not clear, and we have no reports

19. Iida, N., Toida, T., Kushi, Y., Handa, S., Fredman, P., Svennerholm,on, whether acyl CoA-cholesterol acyltransferase or other O-L. & Ishizuka, I. (1989) A sulfated glucosylceramide from ratacyltransferase(s) specifically catalyze the glycolipid, if present, kidney, J. Biol. Chem. 264, 597425980.

or whether they both act on the O-acylation of Gal3S-Cer or20. Tamai, Y., Nakamura, K., Takayama Abe, K., Uchida, K., Kasama,GalCer. Thus, further investigation for characterization of the O- T. & Kobatake, H. (1993) Less polar glycolipids in Alaskan pol-acylation enzyme for the glycolipid is required. lack brain : isolation and characterization of acyl galactosyl di-

acylglycerol, acyl galactosyl ceramide, and acyl glucosyl ceram-We are deeply indebted to Mr Kim Barrymore, for his help in the ide, J. Lipid Res. 34, 6012608.

preparation of this article, Drs Masahiko Chiba and Yoshihiro Maeda,21. Gaver, R. C. & Sweeley, C. C. (1965) Methods for methanolysis offor their technical help in the preparation of glycolipids, and the Hok- sphingolipids and direct determination of long-chain bases by gaskaido Ebetsu Meat Inspection Office, for donation of equine brains. This chromatography,J. Am. Oil Chem. Soc. 42, 2942298.work was supported in part by Grant-in-Aid for Scientific Research on22. Hakomori, S. & Saito, T. (1969) Isolation and characterization of aPriority Areas No.10142712 from the Ministry of Education, Science glycosphingolipid having a new sialic acid,Biochemistry 8,and Culture, Japan. 508225088.

23. Gasa, S., Makita, A. & Kinoshita, Y. (1983) Further study of thechemical structure of the equine erythrocyte hematoside contain-REFERENCESing O-acetyl ester,J. Biol. Chem. 258, 8762881.

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