Eur. J. Biochem.

267, 18371846 (2000) q FEBS 2000

Structural analysis of two glycosphingolipids from the lipopolysaccharide-lacking bacterium Sphingomonas capsulata
Kazuyoshi Kawahara1, Hermann Moll2, Yuriy A. Knirel2,3, Ulrich Seydel2 and Ulrich Zahringer2
1

Department of Bacteriology, The Kitasato Institute, Tokyo, Japan; 2Forschungszentrum Borstel, Zentrum fur Medizin und Biowissenschaften, Borstel, Germany; 3N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia

Two glycosphingolipids, GSL-1 and GSL-3, were isolated from Sphingomonas capsulata and studied by methylation analysis, laser desorption mass spectrometry, and 1H and 13C NMR spectroscopy, including two-dimensional 1H,1H COSY and heteronuclear 13C,1H COSY experiments. GSL-1 and GSL-3 differ in their carbohydrate part, their structures being a-d-GlcpA-(131)-Cer and a-d-Galp-(136)-a-d-GlcpN(134)-a-d-GlcpA(131)Cer, respectively. Variations occur in the ceramide of GSL-1 and GSL-3, both having the same long-chain bases, erythro-2-amino-1,3-octadecanediol (sphinganine), (13Z)-erythro-2-amino13-eicosene-1,3-diol and (13Z)-erythro-2-amino-13,14-methylene-1,3-eicosanediol, in the ratios 2.6 : 1 : 3.5 in GSL-1 and 1 : 1.2 : 1.5 in GSL-3. All bases are quantitatively substituted by amide-linked (S)-2-hydroxymyristic acid. Keywords: chemical structure; Gram-negative bacteria. glycosphingolipids; Sphingomonas capsulata; lipopolysaccharide-free

Glycosphingolipids (GSL) are amphiphilic molecules present in the plasma membrane of eukaryotic cells, where they play a role in cellcell recognition and interaction [1]. Many structural variations were reported in GSL of mammalian and other vertebrate tissues, shellfishes and insects [2], whereas in bacteria GSL are uncommon. The bacterial cell envelope is known to consist of a cytoplasmic membrane, a peptidoglycan layer, and, in Gramnegative bacteria, an outer membrane that is extremely asymmetric with respect to lipid distribution. The outer membrane is characterized by the presence of lipopolysaccharide (LPS), which is located almost exclusively on the outer leaflet [3]. No exception from this architectural principle had been reported for Gram-negative bacteria until we demonstrated that Sphingomonas paucimobilis lacks LPS [4]. This bacterium replaced LPS by two kinds of GSL. One of them had a-d-GlcA as the only sugar component and was called GSL-1. A similar GSL had been previously isolated from the bacterium Flavobacterium devorans [5], which was later transferred to a new genus Sphingomonas [6]. The other glycosphingolipid, GSL-4A, contained an a-d-Manp-(132)-ad-Galp-(136)-a-d-GlcpN-(134)-a-d-GlcpA-(13tetrasaccharide chain, thus sharing a minimal glucuronosyl ceramide structure with GSL-1. Sphingomonas capsulata GIFU 11526 [6] was deposited in the American Type Culture Collection as Flavobacterium capsulatum [7], which later transferred to the genus
Correspondence to U. Zahringer, Forschungszentrum Borstel, Zentrum fur Medizin und Biowissenschaften, Parkallee 22, D-23845 Borstel, Germany. Fax: 1 49 4537188612, Tel.: 1 49 4537188462, E-mail: uzaehringer@fz-borstel.de Abbreviations: CI, chemical ionization; EI, electron impact; GlcA, glucuronic acid; GSL, glycosphingolipid; LD, laser desorption; LPS, lipopolysaccharide; 14:0(2-OH), (S )-2-hydroxymyristic acid. (Received 20 December 1999, revised 27 January 1999, accepted 28 January 1999)

Sphingomonas [6]. However, recent studies on its 16S rRNA sequence have shown that S. capsulata is phylogenetically far from S. paucimobilis [8,9]. Therefore, although these two Sphingomonas species are taxonomically related, they might have different GSL. Furthermore, it has been demonstrated that both GSL from S. paucimobilis are localized in the outer membrane and their carbohydrate moiety is exposed on the cell surface [10]. This and other findings indicated that Sphingomonas GSL may share structural features and possess functional and physicochemical similarities with LPS [11]. The GSL of S. capsulata might be another representative of the family of GSL from Gram-negative bacteria, which are thought to replace LPS in the outer membrane. In this paper, we report on the elucidation of the complete structure of two GSL isolated from S. capsulata. These GSL show both structural similarities with and differences from two glycosphingolipids, GSL-1 and GSL-4A, found in S. paucimobilis [4,10].

M AT E R I A L S A N D M E T H O D S
Bacterial strain and growth S. capsulata GIFU 11526 (originally F. capsulatum ATCC 14666, Type strain) was cultivated in liquid medium containing 0.5% yeast extract (Difco, Detroit, MI, USA), 0.5% casamino acids (Difco), 0.2% (NH4)2SO4, 0.2% K2HPO4, and 0.1% MgSO47H2O, using a 30 L jar fermenter at 30 8C for 24 h. The bacteria were killed by heating (100 8C, 30 min) and harvested by centrifugation (10 000 g, 10 min). Extraction and purification of GSL Bacterial cells were washed with distilled water and lyophilized. Dried cells were homogenized with chloroform/ methanol (2 : 1, v/v) to remove phospholipids, and GSL were

1838 K. Kawahara et al. (Eur. J. Biochem. 267)

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extracted twice from the residual cells with chloroform/ methanol (1 : 3, v/v) at 80 8C for 1 h. The crude GSL preparation was concentrated and applied to a column of silica gel 60 (70230 mesh, Merck, Darmstadt, Germany) for purification. Lipids were eluted using a stepwise gradient of chloroform/methanol from 4 : 1 to 1 : 3 (v/v). The elution was monitored by TLC using aluminium plates of silica gel 60 (Merck) and a solvent system of chloroform/methanol/acetic acid/water (25 : 15 : 4 : 2, v/v/v/v). Lipids were visualized by spraying with 10% sulfuric acid in ethanol and heating. GSL fractions were dialysed against dilute aqueous triethylamine pH 8.5 (0.005%, v/v) and lyophilized.

Sphingolipid analyses Fatty acids were analysed by GLC after hydrolysis with 4 m HCl (100 8C, 5 h) and esterification with ethereal diazomethane. The glycosyl moiety of GSL-3 was oxidized by stirring in aqueous triethylamine 0.005% (v/v) containing 0.025 m NaIO4 at 4 8C for 120 h in the dark. An excess of NaIO4 was destroyed with ethylene glycol, and the oxidized sugars were reduced with NaBH4 at room temperature for 3 h. After dialysis and lyophilization, the product was methanolysed (1 m HCl/methanol, 100 8C, 5 h) to liberate sphingoid and fatty acid derivatives, which were peracetylated [Ac2O/ pyridine 1 : 1 (v/v), 100 8C, 30 min] and analysed by GLC and GLC-MS. GSL-1 and GSL-3 (1 mg of each) were freed from sugars and the amide-linked fatty acid (S )-2-hydroxymyristic acid [14:0(2-OH)] by methanolysis (0.2 m HCl/methanol, 65 8C, 5 h). After neutralization and evaporation, the residue was suspended in chloroform (2 mL) and oxidized with Pb(OAc)4 (1 mg) at room temperature for 2 h. The product was extracted twice with chloroform (2 mL), the organic phases were combined, dried over Na2SO4 and evaporated. The resulting aldehyde was dissolved in diethyl ether (2 mL) and reduced to the corresponding alcohol with LiAlH4 or LiAlD4 (< 5 mg) for 1.52 h with stirring. The solvent was evaporated in a stream of nitrogen, water (1 mL) was added, and the product was extracted three times with chloroform (3 mL). The combined organic layers were dried and the residue was conventionally trimethylsilylated or esterified with freshly prepared nicotinic acid chloride in pyridine (3.5 mg in 500 ml) in a glass-tube at 85 8C for 1 h. After evaporation of pyridine, the product was extracted three times with a 1 : 1 (v/v) mixture of hexane/water (2 mL) and the organic layers were combined, dried, and analysed by GLC using a temperature gradient 150 (3 min) to 320 8C at 5 8Cmin21. Sugar and methylation analyses GlcN was determined by the method of Strominger et al. [13] after hydrolysis with 4 m HCl (100 8C, 16 h). Neutral sugars

NMR spectroscopy Spectra (360 MHz 1H-NMR and 90.5 MHz 13C-NMR) were recorded on an AM-360 spectrometer (Bruker, Germany) at 32 8C. Samples were dissolved in a mixture of CDCl3 : CD3OD : D2O 2 : 2 : 0.2 (v/v). Chemical shifts are referenced to internal chloroform (dH 7.26 p.p.m., dC 77.0 p.p.m.). Standard Bruker software (disnmr, version 8911 01.0) was used to run two-dimensional 1H,1H COSY and heteronuclear 13C,1H COSY experiments.

GLC, GLC-MS and laser-desorption MS GLC was performed on a GC-14 A instrument (Shimadzu, Kyoto, Japan) equipped with a chemically bonded capillary column (25 m 0.2 mm) of CBP-1 or CBP-10 (Shimadzu). GLC-MS was performed on a Hewlett-Packard HP 5985 instrument (Palo Alto, CA) equipped with a chemically bonded fused silica capillary column (25 m 0.32 mm) of SE-54 (Weeke, Muhlheim, Germany). Electron impact (EI) mass spectra were recorded at 70 eV. Ammonia was used as a reactant gas in chemical ionization (CI) MS. Laser-desorption (LD-MS) was performed on a LAMMA 500 instrument (Leybold-Heraeus, Koln, Germany) under conditions described previously [12]. CsI and NaI were used as cationization reagents.

Fig. 1. Negative ion mode LD mass spectrum of GSL-3 of S. capsulata. M1, M2 and M3 refer to the molecules with sphinganine, eicosasphing-13-enine, and 13,14-methyleneeicosasphinganine, respectively.

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Glycosphingolipids from Sphingomonas capsulata (Eur. J. Biochem. 267) 1839

were determined as alditol acetates by GLC after acid hydrolysis (0.1 m HCl, 100 8C, 48 h), reduction with NaBH4 and peracetylation. Determination of uronic acid was performed by the carbazole-sulfuric acid method [14] after hydrolysis with 1 m H2SO4 (100 8C, 5 h). In addition, GlcA was identified by GLC after carboxyl-reduction, hydrolysis (1 m CF3COOH, 120 8C, 2 h), carbonylreduction (NaBH4) and peracetylation. GSL-3 was N-acetylated (1.6% Ac2O in methanol, v/v) and permethylated by the method of Hakomori [15] with some modifications [16,17]. The product was hydrolysed with 1 m CF3COOH at 120 8C for 2 h, and liberated sugars were conventionally reduced with NaBH4, peracetylated (Ac2O/ pyridine) and analysed by GLC-MS.

S. paucimobilis [4,10], whereas GSL-3 with Rf 0.18 migrated faster than GSL4A from S. paucimobilis having Rf 0.11 [4,10]. Negative ion mode LD-MS showed a heterogeneity in the sphingolipid moiety of GSL-1 and GSL-3. The latter was characterized by the molecular masses of 1026, 1052 and 1066 Da (Fig. 1). Fragment ions with m/z 527, 553 and 567, which were derived from the ceramide moiety, showed the same heterogeneity pattern. LD-MS analysis of GSL-1 revealed the molecular masses of 703, 729 and 743 Da and the same fragment ions as in GSL-3. Therefore, the heterogeneity occurs in the sphingolipid moiety and is similar in both GSL. These data showed also that GSL-1 and GSL-3 have monosaccharide and trisaccharide carbohydrate moieties, respectively. Studies on the sphingolipid moiety GLC analysis of the methyl ester revealed the presence of 14:0(2OH) as the only fatty acid in both GSL-1 and GSL-3. GLC of the l-phenylethylamide derivative showed that 14:0 (2-OH) has the (S)-configuration. NMR spectroscopy showed the presence of cyclopropanecontaining and unsaturated sphingoids in both GSL studied. The 1H-NMR spectra of GSL-1 and GSL-3 (Fig. 2) contained characteristic signals of a cyclopropane ring (d 20.45 and 0.47

R E S U LT S
Isolation and characterization of GSL The crude extract from S. capsulata contained two kinds of GSL having Rf 0.48 and 0.18 in TLC and also significant amounts of contaminating phospholipids. Purification by silica gel column chromatography afforded two GSL, one with a monosaccharide and the other with a trisaccharide carbohydrate chain (GSL-1 and GSL-3, respectively). GSL-1 from S. capsulata with Rf 0.48 was identical to GSL-1 from

Fig. 2. The 360 MHz 1H-NMR spectrum of GSL-3 of S. capsulata. Arabic numerals refer to protons in sphingoid and 14:0(2-OH) moieties denoted as s and h, respectively.

1840 K. Kawahara et al. (Eur. J. Biochem. 267)

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for the methylene protons and d 0.56 for the methine protons in GSL-3). Similar signals were present in the spectra of GSL from S. paucimobilis, containing (13Z )-erythro-2-amino-13, 14-methylene-1,3-octadecanediol [4]. In addition, the spectra of GSL-1 and GSL-3 showed two coupled multiplets for the olefinic and allylic protons of another, monounsaturated sphingoid (d 5.23 and 1.93, respectively; data from the 1H,1H COSY spectrum of GSL-3). The same minor signals were present also in the 1H-NMR spectrum of GSL-4A from S. paucimobilis, although the corresponding unsaturated base has not been identified in the course of chemical studies [4] owing to its low content. The 13C-NMR spectrum of GSL-3 (Fig. 3) confirmed the presence of a cyclopropyl ring (signals at d 10.3 for CH2 and d 15.2 for two CH groups; data from the DEPT spectrum) and a double bond (signals at d 129.3 and 129.4). The position of the signals for allylic carbons at d 26.6 and 26.7 (data from the 13C,1H COSY spectrum) indicated the cis configuration of the olefinic group (the signals for the trans isomer would appear at d 3233) [18]. Methanolysis of GSL-3 afforded only small amounts of sphingoids, most likely owing to the attachment of GlcA. When GSL-3 was oxidized by periodate prior to methanolysis, three long-chain bases were released smoothly and analysed by GLC-MS in the EI and CI modes as peracetylated derivatives. They had respective relative retention times of 1, 1.14 and 1.22,

relative peak areas of 1 : 1.2 : 1.5 and molecular masses of 427, 453 and 467 Da (CI MS data). The same compounds in the ratios 2.6 : 1 : 3.5 were derived also from GSL-1. One of them, having the lowest molecular mass, was identified as peracetylated erythro-2-amino-octadecane-1,3-diol (sphinganine). The compound with the highest molecular mass was indistinguishable by the retention time and the EI mass spectrum from the derivative of (13Z )-erythro-2-amino-13,14-methylene-eicosane1,3-diol, which has been identified earlier in GSL-4A from S. paucimobilis [4]. The molecular mass of 453 Da for the third compound pointed to an eicosasphingenine, which was identified as (13Z )-erythro-2-amino-13-eicosene-1,3-diol. Long-chain alcohols were prepared from GSL-3 by methanolysis and oxidation with Pb(OAc)4 followed by reduction with LiAlH4 and analysed by GLC-MS as trimethylsilyl derivatives. As a result, the following compounds were identified: hexadecanol (from sphinganine) (11Z )-11,12methyleneoctadecanol [from (13Z )-erythro-2-amino-13,14methylene-1,3-eicosanediol], and (11Z )-11-octadecenol [from (13Z )-erythro-2-amino-13-eicosene-1,3-diol]. The last compound had the same retention time as the authentic sample prepared by carboxyl reduction of (11Z)-11-octadecenoic acid and differed from the (11E )-isomer. In order to confirm the position of the cyclopropyl ring and the double bond, the long-chain alcohols were converted to

Fig. 3. The 90 MHz 13C-NMR spectrum of GSL-3 of S. capsulata. Arabic numerals refer to carbons in sphingoid and 14:0(2-OH) residues denoted as s and h, respectively.

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Glycosphingolipids from Sphingomonas capsulata (Eur. J. Biochem. 267) 1841

Fig. 4. EI mass spectrum of (11Z)-11-octadecenylnicotinate derived from GSL-3 of S. capsulata.

nicotinates. GLC-MS analysis of the 11,12-methyleneoctadecanol derivative with the molecular mass of 387 Da showed a series of fragments differing by 14 units, which was interrupted after m/z 262 (the C1C10 fragment) and continued from m/z 302 (the C1C12 fragment) (Fig. 4). Thus, the cyclopropyl ring is located between C11 and C12. A similar pattern for the 11-octadecenol derivative with the molecular mass of 373 Da, characterized by the interruption of the homolog series between m/z 262 and 288, confirmed the location of the double bond between the same carbons. Studies on the carbohydrate moiety GSL-1 was found to contain GlcA and no other sugar component. Sugar analysis of GSL-3 showed that it contained d-Gal, d-GlcN, and d-GlcA. Methylation analysis of GSL-3 revealed 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-d-galactitol and 1,5,6-tri-O-acetyl-2-deoxy-3,4-di-O-methyl-2-(N-methylacetamido)-d-glucitol, indicating that Gal was at the terminal end and GlcN was substituted at position 6. When GSL-3 was hydrolysed, N-acetylated, borohydride reduced and permethylated, a disaccharide derivative was identified by GLC-MS, in addition to methylated monosaccharide alditols. It had a molecular mass of 526 Da determined by CI-MS and gave characteristic fragment ions in EI-MS at m/z 250 and 2603228, corresponding to the reducing (GlcA-ol) and nonreducing (GlcNAc) sugar moieties, respectively. This

derivative had the identical retention time and the identical MS fragmentation pattern to those of the carbonyl-reduced and permethylated GlcNAc-(134)-GlcA-ol disaccharide derived from GSL-4A of S. paucimobilis [4]. The 1 H-NMR and 13 C-NMR spectra of GSL-3 were assigned using two-dimensional 1H,1H COSY (Fig. 5) and heteronuclear 13 C,1 H COSY (Fig. 6) experiments (Table 1). When the unambiguous assignment was complicated by close position of proton signals in the 1H-NMR spectrum (e.g. as in Gal), the 13 C-NMR chemical shift data of the corresponding monosaccharides [19] and the glycosylation effects [20], as well as data of a related model compound, GSL-4A from S. paucimobilis with known structure [4], were used. Relatively small J1,2 coupling constant values of 3.2 3.8 Hz for the signals of the anomeric protons at d 4.76, 4.85 and 5.67 indicated that all three sugar constituents in GSL-3 are a-linked. The 13C-NMR chemical shift data of GSL-3 demonstrated substitution of GlcA at position 4 and GlcN at position 6, the latter finding being consistent with the methylation analysis data. Indeed, the signals for C4 of GlcA and C6 of GlcN were shifted downfield to d 74.9 and 67.1, respectively, as compared to their position in the spectra of the corresponding nonsubstituted monosaccharides [19]. The chemical shifts for C2C6 of Gal were similar to those in nonsubstituted a-galactopyranose, thus confirming the terminal position of this monosaccharide in GSL-3.

1842 K. Kawahara et al. (Eur. J. Biochem. 267)

Table 1. 1H-NMR and Residue
13

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C-NMR chemical shifts for the carbohydrate moiety of GSL-3 (p.p.m.). Chemical shift H1 H2 3.72 3.06 3.46 C2 68.4 54.2 71.2 H3 3.75 3.72 3.80 C3 69.3 70.2 73.6 H4 3.85 3.20 3.71 C4 69.1 69.7 74.9 H5 3.81 3.83 3.96 C5 70.4 70.8 71.7 H6a 3.63 3.63 C6 60.8 67.1 175.0 H6b 3.72 3.83

a-d-Galp-(13 36)-d-GlcpN-(13 34)-d-GlcpA-(13

4.85 5.67 4.76 C1

a-d-Galp-(13 36)-d-GlcpN-(13 34)-d-GlcpA-(13

97.9 94.7 98.6

Fig. 5. Part of a 360 MHz 1H,1H-COSY spectrum of GSL-3 of S. capsulata. The corresponding part of the 1H-NMR spectrum is displayed along the horizontal axis. Arabic numerals refer to protons in the sugar residues denoted as follows: I, GlcA; II, GlcN; III, Gal.

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Glycosphingolipids from Sphingomonas capsulata (Eur. J. Biochem. 267) 1843

Fig. 6. Part of a 90 MHz 13C,1H-COSY spectrum of GSL-3 of S. capsulata. The corresponding part of the 1H-NMR and 13C-NMR spectra are displayed along the vertical and horizontal axes, respectively. Arabic numerals refer to atoms in the residues denoted as follows: I, GlcA; II, GlcN; III, Gal; s, sphingoid.

Combining the MS and NMR data obtained, it could be concluded that the carbohydrate moiety of GSL-3 has the structure of a a-d-Galp-(136)-a-d-GlcpN-(134)-a-d-GlcpA trisaccharide.

DISCUSSION
As a result of this work, we propose the chemical structures for two GSL isolated from S. capsulata (Fig. 7). Both GSL-1 and GSL-3 represent a mixture of three molecules, differing in the nature of the long-chain base. Two of the sphingoids present in S. capsulata have been previously reported as components of GSL from S. paucimobilis [4], in particular of GSL-4A (Fig. 7). The third long-chain base, (13Z )-erythro-2-amino13-eicosene-1,3-diol, has been characterized for the first time in S. capsulata as in GSL from S. paucimobilis it is present only as a minor component. Thus, the molar ratios of erythro-2-amino-1,3-octadecanediol (13Z )-erythro-2-amino13-eicosene-1,3-diol and (13Z )-erythro-2-amino-13,14-methylene-1,3-eicosanediol were estimated as 1 : 1.2 : 1.5 and 12.5 : 1 : 11.5, respectively, in GSL-3 from S. capsulata and GSL-4A from S. paucimobilis reinvestigated in this work. All sphingolipids from both species contain (S )-2-hydroxymyristic acid as the only fatty acid component. GSL-3 from S. capsulata has a truncated carbohydrate chain lacking the terminal Man residue as compared to that of S. paucimobilis [4]. With respect to the carbohydrate moiety, the monoglycosyl GSL-1 from S. capsulata is identical to GSL-1 from S. paucimobilis [4]. These data show that both glycosyl and sphingolipid parts of GSL are closely related by structure in the two Sphingomonas species studied. GSL from some other species of Sphingomonas may vary not only in the length of the carbohydrate chain, but also in the sugar sequence [21]. As is the case for S. paucimobilis [4], S. capsulata is devoid of LPS and is thus the second Gram-negative bacterium, that replaces LPS in the outer membrane by GSL. Although GSL

have recently been found also in Chlorobium limicola [22] and Zymomonas mobilis [23], they cannot be considered as common components of bacterial cells. It is worthy of noting that some other bacteria, such as Bacteroides [2426], Bdellovibrio [27], Acetobacter [28], Flavobacterium (Sphingobacterium) [29], and the aquatic bacteria Arcocella aquatica NO-502 and Flectobacillus major FM [30] contain sphingolipids as cell constituents. Biosynthetic pathways of GSL in bacteria remain unknown. In yeast [31] and animal tissues [32], sphinganine is synthesized from palmitoyl-CoA and l-Ser. As palmitic acid and (11Z )-11-octadecenoic acid (cis-vaccenic acid) are the main cellular fatty acids of Sphingomonas [6], and the pathway of the cyclopropyl ring formation from the corresponding unsaturated fatty acid is well known in Gram-negative bacteria [33], it is reasonable to hypothesize that sphingoids found in Sphingomonas GSL are synthesized through pathways similar to those in eukaryotic cells, using fatty acyl-CoA available in bacterial cells. However, sphingosine, a 4,5-dehydrogenated sphinganine, which is common in eukaryotic cells, has not yet been found in bacteria. Remarkably, like enterobacterial LPS GSL-4A from S. paucimobilis can induce interleukin-1 and interleukin-6 in, and release tumour necrosis factor-a from, human monocytes, although the doses needed to reach the inducing potency of LPS (1 ngmL21) were 100010 000-fold higher [34]. However, studies using mAbs against CD14 (MEK 18) have demonstrated that the stimulation of monocytes by GSL is not mediated by CD14 and the mechanism of the GSL action is thus different from LPS [35]. An alternative way of tumour necrosis factor-a release and interleukin-1 stimulation was hypothesized by Kolesnick and colleagues [36,37]. They suggested also that a structural similarity between lipid A and ceramide may account for the ability of lipid A to stimulate ceramidase in monocytes [37,38]. Furthermore, comparison of physicochemical properties of Re LPS and GSL-1

1844 K. Kawahara et al. (Eur. J. Biochem. 267)

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Fig. 7. Chemical structures of GSL-1 and GSL-3 of S. capsulata and GSL-4A of S. paucimobilis.

demonstrated that in asymmetric membranes composed of phospholipids and either LPS or GSL, the mechanism of complement activation is similar [11,38]. Therefore, the supramolecular organization of LPS and GSL and their functioning in the bacterial membrane are likely to be similar as well. As a result of these properties, bacterial GSL may be of great interest in studies of proteins involved in LPS-mediated stimulation of immunocompetent cells.

We also thank Mrs. M. Lohs for preparing figures and Mrs. G. Stegelmann Muller for photographic work. Financial support of Deutsche Forschungsgemeinschaft (SFB 470, project B5), Kibun Foods Inc., and Kibun Food Chemifa Co. is acknowledged.

REFERENCES
1. Merrill, A.H. Jr, Hannun, Y.A. & Bell, R.M. (1993) Sphingolipids and their metabolites in cell regulation. In Advances in Lipid Research, Vol. 25 (Bell, R.M., Hannun, Y.A. & Merrill, A.H. Jr, eds), pp. 124. Academic Press, San Diego, USA. 2. Kundu, S.K. (1992) Glycolipids: structure, synthesis, functions. In Glycoconjugates: Composition, Structure, and Function (Allen, H.J.

ACKNOWLEDGEMENTS
We are grateful to Dr B. Lindner (Forschungszentrum Borstel) for help with LD-MS, and Mrs. K. Jakob and I. Mizuta for excellent technical assistant.

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