Bis-sulfoglycosphingolipid Containing a Unique 3-0-Sulfated N-Acetylgalactosamine from Rat Kidney*

A novel sulfoglycosphingolipid containing two sul- fate ester groups was isolated from the lipid extract of rat kidney. The isolation procedure involved extraction of lipids with chloroform/methanol, alkaline methanolysis, and column chromatographies with DEAE-Se- phacel and silica beads. By infrared spectroscopy, proton magnetic resonance spectroscopy, periodate oxi- dation, solvolysis, chromium trioxide oxidation, and methylation analysis of the native and partially de- graded compounds, the structure of this glycolipid is proposed to be (HS03-3)GalNAc/31-4(HS03-3)Gal/31- 4Glcfil-lCer. The presence of a unique 3-0-sulfated N-acetylgalactosamine structure was confirmed by gas chromatography-mass spectrometry. The yield of this sulfoglycolipid was 11.2 nmol/g of tissue, which was about half of that of monosulfogangliotriaosylceramide from rat kidney. Our recent studies on contained more complex galactose, glucose, and N-acetylgalactosamine. mono-sulfo-GgOse,Cer two sulfate ester groups in the molecule (6). This report describes the isolation and complete characterization of sulfoglycolipid P from rat kidney. dimethyl P-N-acetylgalactosamine, GalNAcPl-4-linked P-galactose, and GalPl-4-linked P-glucose, respectively, in GgOsesCer in

* This study was supported in part hy grants from the Ministry of Education, Science, and Culture of Japan and from the Naito Memorial Fund. 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.

MATERIALS AND METHODS'
Materials ~ Wmar rat$ were obtsyled from B commercial murce. The mixture of ecxd>c llplds from rat brain. monoaulfa-GgOse3Cer from rat kidney  Isobtim of Clycolopldr -The preparative method used mnaimfsd of lipld enraetian with chlomformlmethanoi end mild slkaane methmdyais with dessltatlon by dialysis. The acidic giycoliplds were sepBrated fmm the neutral ones by DEAE-Sephacel column chmmtography (11) and then further purification by M latmbeads mlumn. A detailed description of t h e e steps 18 @"en in the RESULTS. Specfmphotamefer (Japan Spectromopic Co., Tokyo) as described p r e w u d y ( 3 . 5).
IR Speefrum was recorded with about 150 nmol of glymlipid in a T m A-302 Infrared C a p s o t i o n Analy5n -Methyl ester8 of klty acids and methyl glycosides were Obtained by methenolyya>s of glyeolipids rnth 1.0 M anhydrous methanotic hydrogen chloride (5). Methyl ester8 of non-hydroxy and hydmxy fatty acids were Bepareled by PmpBTetive TLC developed with hexanelether (85:15. vlvl (5). GLC analyses of the trimethylsilyl derivatives Methyl ester8 of non-hydroxy fatty acids were Beparated on I column of 10% EGSS-X.
of methyl glyeoaidea end hydroxy fatty acids were performed on a column of 3% OV-101 (5).

Isolation of Sulfoglycolipid P-Kidneys
(200 g) were removed from Wistar rats (6 to 7 weeks old) of either sex, and extracted in three steps with: l ) , 19 volumes of chloroform/ methanol (2:1, v/v); 2), 10 volumes of chloroform/methanol/ 0.88% KC1 (60120:9, v/v); and 3), 10 volumes of 40 mM sodium acetate in chloroform/methanol/water (30:60:8, v/v) (5,20). The third extract was concentrated, dialyzed, and lyophilized. The lyophilized extract was combined with the first and the second extracts and concentrated to dryness. The residue was treated for 1 h a t 37 "C with 0.2 M NaOH in methanol. After neutralization with methanolic hydrogen chloride, the reaction mixture was partitioned in the Folch system (5,21,22). The clear upper phase was concentrated to 1/10 volume, dialyzed, and lyophilized. The lyophilized sample was combined with the lower phase, made up to 500 ml of chloroform/ methanol/water (5:10:1, v/v) by the addition of solvents, and applied to a column (2.0 X 50 cm) of DEAE-Sephacel (acetate form). The neutral glycolipids were eluted from the column with 3 column volumes of the same solvent mixture and the acidic lipids were separated by a linear gradient of 4.0 liters of chloroform/methanol/ammonium acetate in water (5:10:1, v/ v; 0.03 to 3.5 M). Elution of acidic lipids was monitored by TLC in Solvent System I. Sulfoglycolipid P was eluted later than monosulfo-GgOse3Cer, sulfo-GalCer, sulfo-LacCer, and gangliosides migrating similarly to GI):~ (23) and GT:~ (24), with about 2.5 to 3 M ammonium acetate. The pooled fractions of sulfoglycolipid P were concentrated, dialyzed, and lyophilized.
The final purification of sulfoglycolipid P was achieved by column chromatography using Iatrobeads (0.6 X 70 cm) and a linear gradient with a total of 800 ml of chloroform/methanol/water, 75:25:1.5 to 40:60:3 (v/v). Isolated sulfoglycolipid P was examined for purity by TLC in Solvent Systems I, 11, and 111, and found to be a homogeneous band which stained with orcinol, but not with acid molybdate or resorcinol, indicating the presence of hexose and the absence of phosphate or sialic acid in the molecule. In the neutral and acidic solvent systems (I and HI), sulfoglycolipid P migrated slower than GgOseCer and monosulfo-GgOse:rCer (Fig. 1, A and B). In the basic solvent system (11). sulfoglvcolipid P migrated slower than monosulfo-GgOse:iCer, but similar1.v to GgOse.,Cer ( Fig. I('). The yield of sulfoglycolipid I' on galactose basis was determined to be 11.2 nmol/g of tissue by GLC.
Infrared and Proton Magnetic Resonnnce Spectroscop- The IR spectrum of sulfoglycolipid P (Fig. 2  obtained when spectra were recorded at 60 "C. When these values were compared to those obtained in chloroform/methanol, the GalNAc proton shifted to a higher magnetic field by 0.34 to 0.35 ppm and the Gal proton shifted by 0.20 ppm. These values in dimethyl sulfoxide were close to those of P-Nacetylgalactosamine, GalNAcPl-4-linked P-galactose, and GalPl-4-linked P-glucose, respectively, in GgOsesCer recorded in dimethyl sulfoxide at 110 "C (25).
Composition Analysis-GLC of the trimethylsilylated methylglycosides, formed from the carbohydrate portion of sulfoglycolipid P, established the presence of equimolar amounts of galactose, glucose, and N-acetylgalactosamine. The colorimetric analyses showed the presence of 2 mol of sulfate ester groups and 1 mol of sphingoid base (Table I).
The result of the analysis of sulfate was in good agreement with the much larger absorption of the sulfate ester group (1240 and 820 cm") in the IR spectrum (Fig. 2 of Ref. 6). The compositions of fatty acids and sphingoid bases in sulfoglycolipid P resembled those in monosulfo-GgOse3Cer from rat kidney (5). The major nonhydroxy fatty acids were 24:O and 22:0, and the proportion of 2-hydroxy fatty acids was only 3.2% of total fatty acids. The peaks of nonhydroxy and 2hydroxy fatty acids were identified by comparing their retention times with those of standards, and by mass chromatograms. 4-Hydroxysphinganine accounted for 81.7% of the total sphingoid bases present in Sulfoglycolipid P (Table 11). The mass spectrum of the trimethylsilyl derivative of 4-hydroxysphinganine in sulfoglycolipid P was close to that of 4-hydroxysphinganine in ganglioside Ghla from bovine kidney (19). These data, together with those of IR and PMR spectroscopy, were consistent with a triglycosylceramide containing two sulfate ester groups.
Methylation and Periodate Oxidation of Native Sulfogycolipid P-It has been established that sulfate esters survive the methylation procedure (5). Also with sulfoglycolipid P, it was confirmed that methylation was complete and the desulfation during the process was minimal as judged on TLC compared with permethylated monosulfo-GgOse3Cer as well as GgOseaCer. The permethylated native sulfoglycolipid P, The methyl glycosides were obtained by the anhydrous methanolysis of the glycolipids and analyzed by GLC, as described in the text. For the conditions of periodate oxidation, solvolysis, and chromium trioxide oxidation, see the text. Colorimetric procedures were used for sulfate (12) and sphingoid base (13) determination. The molar ratio was calculated on glucose as 1.0, except for chromium trioxide oxidation, where the molar ratio was calculated on glucose without oxidation as 1.0 with myo-inositol as the internal standard.  The values are given as weight per cent. The nonhydroxy fatty acid methyl esters were run on a 10% EGSS-X column. The hydroxy fatty acid and sphingoid base were run as their trimethylsilyl derivatives on a 3% OV-101 column. Peaks were identified by comparing their retention times with those of standards and also bv GC-MS.

TABLE I11
Partially methylated alditol acetates derived from the native and modified sulfoglycolipid P Peaks were identified by gas chromatography and gas chromatography-mass spectrometry, as described in the text. Gas chromatography was performed on columns of 3% SP-2340, 3% OV-17, and 3% OV-101. The molar ratio was calculated on 2,3,6-tri-O-methylglucitol acetate as 1.0. after acetolysis, reduction, and acetylation, gave three major peaks on GLC, which co-chromatographed with the acetates of 2,3,6-tri-O-methylglucitol, 2,6-di-O-methylgalactitol, and 4,6-di-O-methyl-2-N-methylacetamidogalactitol, in approximately equimolar proportion (Table 111). Retention times of these three peaks were identical to those of standards by GLC using three different columns, SP-2340, OV-101, and OV-17. These peaks were also analyzed by mass spectrometry using a combined gas chromatograph-mass spectrometer. Fig. 2A shows the mass chromatogram of partially methylated alditol acetates obtained from Forssman glycolipid. Although the acetates of 2,3,6-tri-O-methylgalactitol, 2,3,6-tri-O-methylglucitol, and 2,4,6-tri-O-methylgalactitol could not be separated completely on the column of OV-101, these peaks could be distinguished on the mass chromatogram using m / z 161 and 162 (inset in Fig. 2 A ) . Because NaB2H4 was used in the reduction step instead of N a B a , t h e characteristic ion at m/ z 161, which was derived from the structure containing C-1 to C-3 of the 4-substituted hexose, shifted to m/z 162 (16). The ion at m / z 161, which was derived from the structure containing C-4 to C-6 of the 3-substituted hexose, was present in  Fig. 2.4. By comparing the retention times and mass chromatograms of the two peaks of 4-substituted hexitol acetates (Peaks 1 and 2 in Fig. 2 A ) from Forssman glycolipid with that of 2,3,6-tri-O-methylglucitol acetate from oligosaccharide of GTlb, Peaks 1 and 2 from Forssman glycolipid were identified as 2,3,6-tri-O-methylgalactitoi acetate and 2,3,6-tri-0-methylglucitol acetate, respectively. The presence of m/z 162 in Peak 2 of native sulfoglycolipid P (Fig. 2B) and desulfated product (Fig. 2C, see below) indicated the presence of 4substituted hexose in native sulfoglycolipid P. The mass spectra obtained from Peaks 2 and 4 in native sulfoglycolipid P were also identical to acetates of 2,3,6-O-methylglucitol and 2,6-di-O-methylgalactitol, respectively, prepared from the oligosaccharide of GTlb and Forssman glycolipid.
The fragment ions derived from partially methylated hex-  Fig. 2 A ) , were also observed in Peak 6 of native sulfoglycolipid P (Fig. 2B). The mass spectrum of Peak 6 of sulfDglycolipid P (Fig. 3A) (Table I). This was in contrast to the case of monosulfo-GgOse3Cer, where N-acetylgalactosamine was completely destroyed under similar conditions (5). The resistance of 4-substituted glucose suggested that glucose was linked to a sphingoid base ( 5 ) . Therefore, the possible attachment of the sulfate ester group to C-4 of glucose was ruled out. The survival of galactose and N-acetylgalactosamine indicated that the C-3 hydroxyls of both galactose and N-acetylgalactosamine were occupied. These results of periodate oxidation were in agreement with those of the permethylation study. Since the possible attachment of the sulfate ester at C-4 of galactose was ruled out by the result of IR spectroscopy (the absence of an axial sulfate ester group), it was suggested that the sulfate ester groups were attached to the C-3 hydroxyls of both N-acetylgalactosamine and galactose. Accordingly, it was reasonable to conclude that the C-4 hydroxyl of galactose was substituted by N-acetylgalactosamine and that C-4 of glucose was substituted by galactose.
From these results, the structure of sulfoglycolipid P was tentatively assigned as GalNAcl-4Gall-4GlcCer sulfated at C-3 of both N-acetylgalactosamine and galactose.
Analyses of Partially Degraded Sulfoglycolipid P-To c o n f i i the point of attachment of the sulfate ester groups, sulfoglycolipid P was subjected to desulfation by solvolysis in dimethyl sulfoxide/methanol (9:1, v/v) containing 8 mM H,SO, ( 5 ) . TLC of the products, after reaction at 80 "C for 10 min, showed that about 50% of sulfoglycolipid P was converted to a compound (desulfated sulfoglycolipid P-I, DSP-I) which migrated similarly to monosulfo-GgOse3Cer. After 30 min, intact sulfoglycolipid P was not detected and only the band which migrated similarly to monosulfo-Gg0se3Cer was observed on TLC.
GLC of the methylglycosides showed that DSP-I and DSP-I1 contained equimolar amounts of galactose, glucose, and Nacetylgalactosamine. DSP-I11 contained only galactose and glucose, indicating that a part of sulfoglycolipid P was converted to a GalNAc-free glycolipid composed of galactose and glucose in the molar ratio of 1:l (Table I). These results established the presence of N-acetylgalactosamine on the nonreducing end.
From these results, the structures of the desulfated products were proposed to be GalNAcl-4(HS03-3)Gall-4GlcCer (DSP-I), GalNAcl-4Gall-4GlcCer (DSP-II), and Gall-4GlcCer (DSP-111). It was confiied that the subterminal galactose of native sulfoglycolipid P was substituted at C-3 by a sulfate ester group and at C-4 by a N-acetylgalactosamine, and that the terminal N-acetylgalactosamine was substituted at C-3 by another sulfate ester group.
To confwm the anomeric configuration, desulfated sulfoglycolipid P (DSP-11) was subjected to chromium trioxide oxidation. Table I shows that all carbohydrate units of DSP-I1 were almost completely oxidized by CrOs, although the parallel experiment using GbOsesCer indicated that 1 mol of galactose survived. Considering the above results, together with those of PMR, it was established that all the sugar residues in sulfoglycolipid P are linked /?-glycosidically.

DISCUSSION
Recent studies on the structure and distribution of sulfoglycolipids indicate that the number of sulfoglycolipids is increasing to form a group of acidic glycolipids comparable to gangliosides (23,28,29). The N-acetylglucosamine-containing sulfoglycolipids were reported in hog gastric mucosa (20), and sialic acid-containing sulfoglycolipids were found in sea urchin (30,31) and in bovine gastric mucosa (32,33). We discovered a sulfoglycosphingolipid containing N-acetylgalactosamine from rat kidney (3)(4)(5). However, there has been no report SO far of a sulfoglycolipid which contains more than one sulfate ester group in the molecule. On the basis of the experimental FIG. 4. The structure of sulfoglycolipid P. The ceramide moiety was shown as N-(n-tetracosanoyl)-4-hydrox-results described in this paper, the structure of the new sulfoglycolipid (sulfoglycolipid P) is a bis-sulfated triglycosylceramide with the following structure: (HS03-3)GalNAcPl-4(HSO3-3)Gal/3l-4Glc~l-1Cer (Fig. 4). Thus, in the later part of this report, sulfoglycolipid P wiU be referred to as bissulfogangliotriaosylceramide (bis-sulfo-GgOse3Cer). The yield of bis-sulfo-GgOseaCer was 11.2 nmol/g of tissue, which was about 6 and 50 mol %, respectively, of sulfo-GalCer and monosulfo-GgOse3Cer from rat kidney (5).
The chemical shift values of bis-sulfo-GgOsesCer recorded in dimethyl sulfoxide at 60 "C were close to those of GgOseaCer obtained in dimethyl sulfoxide at 110 "C (25). In contrast, it was reported that the values of GalNAc proton markedly shifted down field in gangliosides G M~ and GMI due to the attachment of sialic acid at the C-3 hydroxyl of neighboring galactose (25). It was suggested that the effect of a 3sulfate ester group at the galactose for the resonance of the anomeric proton of the N-acetylgalactosamine in bis-sulfo-GgOsesCer was negligible in comparison with the effect of sialic acid in gangliosides.
The desulfation profiie of bis-sulfo-GgOseaCer in dimethyl sulfoxide showed that the sulfate ester group attached to the C-3 hydroxyl of the terminal N-acetylgalactosamine was much more labile than the sulfate ester group at C-3 of the internal galactose. It was suggested that the juxtapositioning of the sulfate to the proton-donating amide at C-2 of N-acetylgalactosamine may be responsible for the resistance of the internal sulfate ester group to solvolysis. The partial cleavage of the terminal N-acetylgalactosamine also occurred similar to the case with monosulfo-GgOseaCer (5).
Since the presence of 3-0-sulfated N-acetylgalactosamine is unusual, we used several methods, such as IR spectroscopy, solvolysis, periodate oxidation, and permethylation, to establish the position of the sulfate ester group. The peaks of partidy methylated alditol acetates were identified by comparing their retention times as well as the mass spectra and the mass chromatograms with those of standard compounds prepared from Forssman glycolipid and the oligosaccharide of ganglioside G~lb. All these data confirmed the presence of 3substituted N-acetylgalactosamine. Gangliosides containing NeuAcP-3-linked N-acetylgalactosamine and NeuAc2-6 linked N-acetylgalactosamine have been found in human erythrocytes (34) and in frog brain (35), respectively. However, in the sulfoglycolipids described to date, the sulfate ester group is situated at C-3 of galactose or C-6 of N-acetylglucosamine, and there has been no report about the occurrence of sulfated N-acetylgalactosamine. To our knowledge, mucopolysaccharides or sulfated glycoproteins which contain a 3-0-sulfated N-acetylgalactosamine have never been reported, although recently, a 3-0, N-disulfated glucosamine was demonstrated to be a unique component of heparin (36).
Chlorosulfolipids containing two sulfate ester groups were reported in a golden-brown alga, Ochromonas danica (37). These lipids were detected in fresh water species but not in any marine species (38). Although the chemical structure of bis-sulfo-GgOse3Cer is quite different from that of chlorosulfolipids, both of them have two sulfate ester groups in the molecule. It is speculated that these two types of bis-sulfolipids may have some common functions in membranes.