Structural study on lipid A and the O-specific polysaccharide of the lipopolysaccharide from a clinical isolate of Bacteroides vulgatus from a patient with Crohn’s disease Masahito Hashimoto 1,2 , Fumiko Kirikae 1 , Taeko Dohi 1 , Seizi Adachi 3 , Shoichi Kusumoto 3 , Yasuo Suda 3,4, *, Tsuyoshi Fujita 5 , Hideo Naoki 5 and Teruo Kirikae 1 1 Research Institute, International Medical Center of Japan; 2 Department of Oral Microbiology, Asahi University School of Dentistry, Japan; 3 Graduate School of Science, Osaka University, Japan; 4 Department of Bacteriology, Hyogo College of Medicine, Japan; 5 Suntory Institute for Bioorganic Research, Japan Bacteroides vulgatus has been shown to be involved in the aggravation of colitis. Previously, we separated two potent virulence factors, capsular polysaccharide (CPS) and lipo- polysaccharide (LPS), from a clinical isolate of B. vulgatus and characterized the structure of CPS. In this study, we elucidated the structures of O-antigen polysaccharide (OPS) and lipid A in the LPS. LPS was subjected to weak acid hydrolysis to produce the lipid A fraction and polysac- charide fraction. Lipid A was isolated by preparative TLC, anditsstructuredeterminedbyMSandNMRtobesimilar to that of Bacteroides fragilis except for the number of fatty acids. The polysaccharide fraction was subjected to gel- filtration chromatography to give an OPS-rich fraction. The structure of OPS was determined by chemical analysis and NMR spectroscopy to be a polysaccharide composed of the following repeating unit: [fi4)a- L -Rhap(1fi3)b- D - Manp(1fi]. Keywords: Bacteroides vulgatus; fast-atom-bombardment tandem mass spectrometry; lipopolysaccharide; MALDI- TOF-MS; NMR. Commensal flora are thought to be significantly involved in the pathogenesis of inflammatory bowel diseases, Crohn’s disease and ulcerative colitis (reviewed in [1]). As chronic intestinal inflammation in several rodent models is prevent- ed in a germ-free environment [2], efforts have been made to identify the organisms responsible for the induction or perpetuation of enterocolitis. Bacteroides are Gram-nega- tive rods and the predominant anaerobes in endogenous intestinal flora. Among these species, Bacteroides vulgatus has been shown to be involved in the aggravation of colitis. For example, immunization of guinea pigs with B. vulgatus before administration of carrageenan and feeding with viable B. vulgatus resulted in more rapid ulceration, whereas a phenotypically similar organism, Bacteroides fragilis,had no such effect [3]. HLA-B27 transgenic rats colonized with a mixture of six different obligate and facultative anaerobic bacteria including B. vulgatus developed a much more active colitis and gastritis than littermates colonized with the same mixture without B. vulgatus [4]. B27 transgenic rats monoassociated with B. vulgatus developed colitis compa- rable to that in rats colonized with the above bacterial mixture, but Escherichia coli-monoassociated rats showed no evidence of colitis [5]. Surface components of many enteric bacteria are impor- tant for their virulence. Capsular polysaccharide (CPS) and lipopolysaccharide (LPS) are two well-described virulence factors. The CPS and LPS of B. vulgatus have been suggested to play key roles in its virulence [6]. Previously, we separated CPS from a clinical isolate of B. vulgatus and characterized its structure as a novel polysaccharide composed of the following repeating unit: {fi3) b- D -Glcp(1fi6)[a- D -GalpNAc(1fi2)b- D -Galp(1fi4)]b- D - GlcpNAc(1fi3)a- D -Galp(1fi4)b- D -Manp(1fi}[7].The structure is completely different from that of the CPS prepared from B. fragilis [8]. However, the structure of the LPS of B. vulgatus has not been fully defined; only SDS/ PAGE profiles [9,10] and the immunochemical character- ization [11] of LPS have been reported. In this paper, we describe the structural elucidation of possible virulent factors, O-antigen polysaccharide (OPS) and lipid A moiety, in LPS prepared from B. vulgatus. MATERIALS AND METHODS Bacteria and LPS B. vulgatus IMCJ 1204 was isolated from the feces of a patient with Crohn’s diseases at the International Medical Center of Japan. LPS was separated as described previously [7]. Briefly, bacterial cells grown in GAM broth under anaerobic conditions were extracted with phenol/water. The Correspondence to T. Kirikae, Research Institute, International Medical Center of Japan, Shinjuku, Tokyo 162-8655, Japan. Fax: + 81 3 3202 7364, Tel.: + 81 3 3202 7181 (ext. 2838), E-mail: tkirikae@ri.imcj.go.jp Abbreviations: CPS, capsular polysaccharide; FAB-MS/MS, fast atom bombardment-tandem mass spectrometry; HMBC, heteronuclear multiple bond connectivity; LPS, lipopolysaccharide; OPS, O-antigen polysaccharide. *Present address: Department of Nanostructure and Advanced Materials, Graduate School of Science and Engineering, Kagoshima University, Japan. (Received 25 March 2002, revised 5 June 2002, accepted 20 June 2002) Eur. J. Biochem. 269, 3715–3721 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03062.x extract was subjected to enzymatic digestion with DNase and RNase followed by proteinase K, and then phenol/ water extraction again to yield the crude LPS preparation. LPS was separated by hydrophobic interaction chroma- tography [12]. The preparation was subjected to stepwise separation on octyl-Sepharose using 0.1 M acetate buffer (pH 4.5) containing 15% propan-1-ol and the same acetate buffer containing 60% propan-1-ol to give the pass-through (OS-P) and retained (OS-R) fractions, respectively. The OS- R fraction contained LPS from the SDS/PAGE analysis as described in Results and discussion. LPS from B. fragilis NCTC 10581 was prepared by a procedure similar to that described above. LPS from E. coli O111:B4 was purchased from Sigma (St Louis, MO, USA). Chemical degradation and separation LPS was hydrolyzed with 0.6% acetic acid at 105 °Cfor 2.5 h, and the reaction mixture was partitioned with chloroform/methanol/water (2 : 1 : 3, v/v/v). The hydro- phobic products were separated by TLC (No. 5715; Merck, Darmstadt, Germany) using the solvent system chloroform/ methanol/water/triethylamine (300 : 120 : 20 : 1, v/v/v/v) and visualized with anisaldehyde/sulfuric acid reagent. Lipid A was isolated by preparative TLC. The hydrophilic products were subjected to gel-filtration chromatography on Sephacryl S-200 HR (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Fractions of 2.5 mL were collected and monitored by measuring phosphorus and hexose contents. The eluates were combined, dialyzed and lyo- philized. The combined fraction was used as an OPS-rich fraction and analyzed by the following procedures. Analytical procedures Phosphorus content was determined by the method of Bartlett [13]. Hexose content was measured by the anthrone/ sulfuric acid method [14]. The sugar constituents of a sample were analysed by the alditol acetate method [15]. Methylation analysis was carried out using NaOH as described by Ciucanu & Kerek [16]. Absolute configurations of sugars were determined using R-(+)-butan-2-ol [8]. Fatty acids were analyzed by the method of Ikemoto et al. [17]. Alditol acetate, partially methylated alditol acetate, acetylated butyl glycoside and fatty acid methyl ester were analyzed by GC or GC-MS as described previously [7]. SDS/PAGE was performed using 15% polyacrylamide gels by the method of Laemmli [18]. The gel was partially oxidized with periodic acid and then visualized by the silver staining method [19]. NMR spectroscopy and MS 1 H- and 13 C-NMR spectra were recorded on a JMN-LA500 spectrometer (JEOL, Tokyo, Japan) equipped with an indirect detection gradient probe, IDG500-5VJ (Nanorac Cryogenics, Martinez, CA, USA) at 500 and 126 MHz, respectively. Spectra of lipid A were obtained at 327 K at a concentration of 0.6 mgÆmL )1 in CDCl 3 /CD 3 OD (2 : 1, v/v). The chemical shifts are expressed as d values using chloroform (d ¼ 7.2 p.p.m.) for 1 H spectra. The spectra of the OPS-rich fraction were recorded at 303 K at a concentration of 6 mgÆmL )1 in D 2 O. The chemical shifts are expressed as d values using water (d ¼ 4.7 p.p.m.) for 1 H-NMR spectra and benzene (d ¼ 128 p.p.m.) as an external standard for 13 C-NMR spectra. 1D DANTE, DQF-COSY, TOCSY, ROESY, HMQC and heteronuclear multiple bond connectivity (HMBC) spectra were obtained as described previously [7]. MALDI-TOF-MS was performed with a Voyager-DE STR (PerSeptive Biosystems, Framingham, MA, USA) instrument. Samples were dissolved in dichloromethane/ methanol (2 : 1, v/v), combined with sinapic acid as a matrix, and placed on a sample plate. Spectra were obtained using the RDE2000 method. FAB-MS/MS was carried out with a JMS-HX/HX110A tandem mass spectrometer (JEOL) in the negative ion mode. Nitrobenzyl alcohol was used as a matrix. The sample was ionized with 6 KeV Xe atoms, and the ions were accelerated through 10 KeV. Argon was used as the collision gas. RESULTS AND DISCUSSION Analysis of LPS As shown in Fig. 1, a ladder-like pattern was observed in the SDS/PAGE profiles of LPS from B. vulgatus IMCJ 1204, indicating that the LPS contains OPS. The repeating unit of the OPS was shorter than that from E. coli. Breeling et al. [9] analyzed LPS from various strains of B. vulgatus using SDS/PAGE and showed that four of eight strains Fig. 1. SDS/PAGE profile of LPS. 3716 M. Hashimoto et al.(Eur. J. Biochem. 269) Ó FEBS 2002 contained OPS. They demonstrated that the LPS with OPS tended to be associated with immune enhancement of colitis. On the other hand, a closely related LPS from B. fragilis did not possess OPS (Fig. 1) as described previously [10]. B. fragilis has been reported to have less ability for immune enhancement of ulcerative colitis than B. vulgatus [3]. The OPS portion of B. vulgatus LPS is therefore probably important in colitis. The chemical composition of the LPS is summarized in Table 1. It contains sugars, amino sugar, fatty acids and phosphate. The fatty acid components were similar to those of B. fragilis [20,21], suggesting structural similarity in the lipid A moiety. The sugar components were different from those from B. fragilis, which lacks OPS [21], and those from B. vulgatus ATCC 8482 [11]. We previously reported that the sugar components of CPS from B. vulgatus IMCJ 1204 were also different from those of B. vulgatus ATCC 8482 [7]. These results indicate that the structural variation in surface glycoconjugates among the strains of B. vulgatus is great, and structural differences may affect virulence [6]. Structure of lipid A moiety in LPS LPSwassubjectedtoweakacidhydrolysistogive hydrophilic and hydrophobic products. The chemical compositions of the hydrophobic products are summarized in Table 1. GlcN, fatty acids and phosphate were present in the molar proportions 2 : 3.3 : 1.4. Absolute configuration analysis confirmed that GlcN has a D configuration. On TLC analysis, two major and several minor spots were detected among the hydrophobic products (Fig. 2). The negative-ion mode MALDI-TOF mass spectrum revealed the presence of a monophosphoryl lipid A (Fig. 3A). The molecular mass heterogeneity can be explained by the degree of acylation and the chain length of the fatty acid. The ions at m/z 1688.4, 1674.4, 1660.4, and 1646.3 represented a monophosphoryl lipid A bearing five fatty acids, e.g. m/z 1660.4 contains 12 (or 13)-Me-14 : 0, 15 : 0 (3-OH), 16 : 0 (3-OH) and 17 : 0 (3-OH) in the molar proportions 1 : 1 : 2 : 1. The ions at m/z 1432.2, 1420.2, 1406.2 and 1392.2 corresponded to a monophosphoryl lipid A bearing four fatty acids, e.g. m/z 1420.2 consists of 12 (or 13)-Me-14 : 0, two 16 : 0 (3-OH) and 17 : 0 (3-OH). A monophosphoryl lipid A bearing three fatty acids was detected at 1180.0, 1166.0, 1151.9 and 1137.9, e.g. m/z 1160.6 includes 12 (or 13)-Me-14 : 0, 16 : 0 (3-OH) and 17 : 0 (3-OH). Diphosphoryl lipid A could not be detected. The major components of the hydrophobic products were isolated by preparative TLC and analyzed by MALDI- TOF-MS (data not shown). The negative-ion mode spec- trum of the less hydrophobic component (R f 0.5) revealed monophosphoryl lipid A containing four fatty acids. The positive-ion mode spectrum of the more hydrophobic component (R f 0.8) showed a lipid A structure with four fatty acids but no phosphate. The latter component may be a byproduct of the hydrolysis reaction or a natural contaminant. These results indicate that the LPS from B. vulgatus mainly contains lipid A carrying four fatty acids and one phosphate. Thus, the component with R f 0.5 was further analyzed as the main component of lipid A. The structure of the lipid A component was established by NMR and MS. The 1 H NMR signals of the isolated lipid A were assigned using DQF-COSY and TOCSY, and the data are summarized in Table 2. Two sets of sugar signals were observed. The coupling constants of the signals revealed a glucopyranosyl configuration. As only D -GlcN was observed in the compositional analysis, the sugars were determined as GlcN and designated GlcN I and GlcN II in order of the 1 H chemical shift of the anomeric proton (H1). The downfield shift (d ¼ 5.34 p.p.m.) and the coupling constant (6.7 Hz for J H,P ) of H1-GlcN I showed a phosphate substitution at the 1-position of GlcN I . The coupling constant (3.0 Hz for 3 J 1,2 ) confirmed the a configuration. The coupling constant (8.2 Hz for 3 J 1,2 ) for H1-GlcN II showed a b configuration. These results indicate that lipid A possesses a common diglucosamine backbone, and GlcN I is located at the reducing end. No downfield shift of H4-GlcN II (d ¼ 3.16 p.p.m.) revealed a free hydroxy group at O4-GlcN II and a monophosphate structure. The down- field shift of H3-GlcN I (d ¼ 4.94 p.p.m.) indicated an acyl substitution at O3-GlcN I , whereas the signal of H3-GlcN II Fig. 2. TLC profile of the hydrophobic products from the acetic acid hydrolysate of LPS. Table 1. Chemical composition of the LPS from B. vulgatus IMCJ1204. nd, Not detected. Component Amount (lmolÆmg )1 ) LPS Hydrophobic products OPS-rich fraction Sugars 2.22 0.81 3.28 Rha 0.71 ND 1.32 Fuc 0.19 ND ND Man 0.43 ND 1.38 Gal 0.41 ND 0.37 Glc 0.37 ND 0.21 GlcN 0.11 0.81 ND Fatty acids 0.70 1.33 ND 12-Me-13 : 0 0.03 0.04 14 : 0 0.01 0.02 13-Me-14 : 0 0.07 0.11 12-Me-14 : 0 0.06 0.13 15 : 0 0.01 0.02 15 : 0 (3-OH) 0.10 0.15 16 : 0 (3-OH) 0.29 0.57 15-Me-16 : 0 (3-OH) 0.05 0.15 17 : 0 (3-OH) 0.08 0.13 Phosphate 0.26 0.56 ND Ó FEBS 2002 Lipopolysaccharide of B. vulgatus (Eur. J. Biochem. 269) 3717 (d ¼ 3.28 p.p.m.) did not shift to a lower field, confirming no acylation at O3-GlcN II . The downfield shift of the proton signal for the b-position of fatty acid III (HbIII) at d ¼ 4.99 p.p.m. revealed acylation at this position. Further characterization was achieved by FAB-MS/MS. The frag- mentation patterns of the parent ion at m/z 1420 indicated the fatty acid distribution as shown in Fig 3B, e.g. cleavage A showed a 3-hydroxy fatty acid (17 : 0 or 16 : 0) substitution of N2-GlcN I , while cleavage B–E showed two 3-hydroxy fatty acid (16 : 0 and 17 : 0, or 16 : 0 · 2) substitutions of GlcN I and an acyoxyacyl substitution at N2-GlcN II . Cleavage F indicated the chain length of the fatty acid on the acyoxyacyl group to be mainly 15, confirming the result of the compositional analysis. In the minor triacylated lipid A, the fragmentation patterns of the parent ion at m/z 1166 suggested a lack of fatty acid at O3-GlcNI (data not shown). This result agrees with previous studies [21–23]. The lipid A from B. fragilis NCTC 9343 has previously been isolated and characterized as having a penta-acyl and monophosphoryl structure [21]. The lipid A from a closely related bacterium, Porphyromonas gingivalis, has been reported to mainly contain one phosphate and three (P. gingivalis 381) [22] or four (P. gingivalis SU63) [23] fatty acids. These observations indicate that the fundamental structure of lipid A from Bacteroidaceae is similar but the number of acyl substituents is variable. The LPS showed significantly less activity than E. coli LPS in inducing production of tumor necrosis factor in human peripheral whole blood cells, with a dose–response curve that shifted to Table 2. 1 H-NMR data for isolated lipid A. The spectra were mea- suredat297KinCDCl 3 /CD 3 OD (2 : 1, v/v). The chemical shifts are expressed as d values (p.p.m.). The coupling constants are shown in parentheses. Proton Chemical shift (coupling constant) GlcN I H1 5.34 ( 3 J 1,2 3.0, J P,H 6.7) H2 3.99 ( 3 J 2,3 11.0, J P,H 3.2) H3 4.94 ( 3 J 3,4 9.3) H4 3.37 ( 3 J 4,5 10.3) H5 3.83 H6 3.60 ( 3 J 5,6 6.0, 2 J 6,6 12.1) GlcN II H1¢ 4.36 ( 3 J 1,2 8.2) H2¢ 3.37 ( 3 J 2,3 9.9) H3¢ 3.28 ( 3 J 3,4 8.9) H4¢ 3.16 ( 3 J 4,5 9.4) H5¢ 3.07 H6¢ 3.52 ( 3 J 5,6 6.0, 2 J 6,6 11.9) 3.66 ( 3 J 5,6 2.3) Fatty acids HaI 2.00, 2.10 HbI 3.70 HaII 2.19, 2.29 HbII 3.76 HaIII 2.29 HbIII 4.99 HaIV 2.09 HbIV 1.38 Fig. 3. MALDI-TOF-MS spectrum of hydro- phobic products from the acetic acid hydroly- sate of LPS (A), and FAB-MS/MS spectrum of the parent ion at m/z 1420 (B). 3718 M. Hashimoto et al.(Eur. J. Biochem. 269) Ó FEBS 2002 an  10 3 -fold higher concentration (data not shown). Lipid A is an active moiety of LPS in the induction of cytokines, including tumor necrosis factor, and the phos- phate residue at the 4¢-position is a critical site for the activity [24]. Therefore, the monophosphoryl lipid A in the LPS must be responsible for this weak activity. Structure of OPS moiety in LPS To analyze the structure of the OPS moiety, the hydrophilic products from the acetic acid hydrolysate of LPS were separated by gel-filtration chromatography to give the high- molecular-mass OPS-rich fraction (30%). Mainly two sugars, Rha and Man, were detected in the OPS-rich fraction on analysis of the sugar constituents (Table 1). The approximate molar ratio of Rha to Man was 1 : 1. Abso- lute configuration analysis demonstrated that Man has a D configuration and Rha an L configuration. On methylation analysis, 2,3,4-tri-O-methyl-6-deoxyhexose, 2,3-di-O- methyl-6-deoxyhexose and 2,4,6-tri-O-methyl-hexose were mainly observed. The 1 H- and 13 C-NMR spectra of the OPS-rich fraction are shown in Fig. 4. Two anomeric signals were mainly observed, and the corresponding sugars were designated as a andbinorderof 1 H chemical shift. The 1 H signals were assigned using DQF-COSY, TOCSY and ROESY spectra, and the 13 C signals were assigned using HMQC and HMBC spectra. Some of the coupling constants that were not determined from 1D spectra were estimated using DQF- COSY spectra; 9–10 Hz for 3 J 3,4 of residue a and 1–2 Hz for 3 J 1,2 of residue b. The data are summarized in Table 3. Residue a was assigned as a- L -rhamnopyranose (a- L -Rhap). The manno-type configuration was clearly revealed by the characteristic singlet-like signals of H1-a and coupling of signals H1 to H4. Intraresidual correlation between H1-a and C5-a in HMBC spectra (Fig. 5A) confirmed the pyranosyl configuration. The chemical shift of 1.31 p.p.m. for H6-a and 17.8 p.p.m. for C6-a was indicative of a 6-deoxy structure and confirmed this residue to be rhamno- pyranose. The 1 J C,H value for the anomeric position of residue a was determined to be 173 Hz from the nondecou- pling DEPT spectrum indicating the a configuration [25]. The downfield shift of C4-a showed that a glycoside is attached at O4 of residue a [26]. Residue b was assigned as b- D -mannopyranose (b- D -Manp). The mannopyranosyl configuration was clearly revealed by the characteristic singlet-like signals of H1-b and H2-b, coupling of signals H2 to H4, and intraresidual correlation between H1-b and C5-b in HMBC spectra (Fig. 5A). Intraresidual correlations between H1 and H5 in the ROESY spectrum revealed the b configuration (Fig. 5B). The 1 J C,H value (164 Hz) con- firmed the anomeric configuration. The downfield shift of C3-b indicated a 3-O-substituted structure. Some minor signals (designated as a¢) were observed in the 1 Hand 13 C spectra and assigned as L -Rhap (Table 3). No downfield shift was observed in 13 C-NMR spectra, indicating a nonsubsti- tuted Rha. The signal of H4-a¢ was approximately one third the intensity of that of H1-a or H1-b, indicating its ratio. Fig. 4. 1 H(A)and 13 C (B) NMR spectra of the OPS-rich fraction. Table 3. NMR data for OPS. The spectra were measured at 303 K in D 2 O. The chemical shifts are expressed as d values (p.p.m.). The coupling constants are in parentheses. nd, Not determined; a¢ is estimated to be the nonsubstituted Rha located at the nonreducing terminus of the OPS chain. Carbohydrate residues H1 ( 3 J 1,2 ) C1 H2 ( 3 J 2,3 ) C2 H3 ( 3 J 3,4 ) C3 H4 ( 3 J 4,5 ) C4 H5 C5 H6 ( 3 J 5,6 ) C6 ( 3 J 5,6 , 2 J 6,6 ) a(a-Rhap) 4.95 3.97 3.96 3.68 3.98 1.31 (1.2) (3.4) (9–10) (9.5) (6.2) 97.1 71.2 71.6 80.6 68.4 17.8 a¢ (a-Rhap) 4.95 3.96 3.83 3.44 3.93 1.25 (nd) (3.5) (9.7) (9.6) (6.3) nd 71.2 71.1 72.9 69.6 17.5 b(b-Manp) 4.86 4.25 3.66 3.64 3.37 3.91 3.75 (nd) (3.2) (9.6) (9.3) (2.2) (6.0, 12.4) 101.4 67.6 77.9 66.0 77.1 61.9 Ó FEBS 2002 Lipopolysaccharide of B. vulgatus (Eur. J. Biochem. 269) 3719 The glycosidic linkages were established by the HMBC experiment (Fig. 5A). Long-range coupling from H1-a to C3-b showed that residue a was linked to O3 of residue b. Coupling from H1-b to C4-a indicated that residue b was linked to O4 of residue a. Interresidual cross-peaks in ROESY could not be assigned because of the overlapping of signal, except for the cross-peak between H1-a and H2-b (Fig. 5B). The cross-peak may support the above linkage. These glycosidic linkages are consistent with the methyla- tion analysis. As no other O-substituted sugar was observed in the methylation analysis, OPS had a linear structure. Thus, the nonsubstituted Rha was estimated to be located at the nonreducing terminus of the OPS chain. Taking these observations into account, the structure of the OPS moiety was deduced to be that shown in Fig. 5C. In the Bacteroides group, the structure of the polysac- charide part of LPS from B. fragilis NCTC 9343 has been studied [27]. It was shown to lack the OPS moiety but to contain the Gal-rich core saccharide. On the other hand, we demonstrated that B. vulgatus IMCJ 1204 has a short OPS consisting of Rha and Man. Although we have not studied the structure of the core saccharide, it would be made up of Gal and Glc. The results of this study showed that the polysaccharide region of LPS from Bacteroides has wide structural variation. As the structure of the lipid A moiety is similar to that of B. fragilis but the polysaccharide part is completely different, the difference in structure of the polysaccharide region may reflect the virulence of LPS in inflammatory bowel diseases. Recently, Ogura et al.[28] demonstrated that a frameshift mutation in NOD2 was associated with susceptibility to Crohn’s disease. NOD2 seems to function as a receptor for LPS with the leucine-rich repeat motif [29]. The structure of LPS responsible for the recognition of NOD2 is so far unknown, but it may recognize the polysaccharide region of LPS. In summary, we found the structure of lipid A and the OPS moiety in LPS from a clinical isolate of B. vulgatus, IMCJ 1204, to be a GlcN 2 backbone with a phosphate and mainly four fatty acids for lipid A, and [fi4)a- L -Rhap (1fi3)a- D -Manp(1fi]fortheOPSmoiety. ACKNOWLEDGEMENTS This study was supported in part by a grant from the Ministry of Education, Science and Culture of Japan (13670289 to T. K.), grants and contracts from International Health Cooperation Research Fig. 5. HMBC (A) and ROESY (B) spectra, and proposed chemical structure of the OPS moiety (C). 3720 M. Hashimoto et al.(Eur. J. Biochem. 269) Ó FEBS 2002 (11A-1) from the Ministry of Health and Welfare of Japan, and ÔResearch for the FutureÕ program no. 97L00502 from the Japan Society for the Promotion of Science. REFERENCES 1. Sartor, R.B. (1997) Pathogenesis and immune mechanisms of chronic inflammatory bowel diseases. Am.J.Gastroenterol.92, 5S–11S. 2. Elson, C.O., Sartor, R.B., Tennyson, G.S. & Riddell, R.H. 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(1985) Structural studies of the polysaccharide part of the cell wall lipopoly- saccharide from Bacteroides fragilis NCTC 9343. Eur. J. Biochem. 151, 657–661. 28. Ogura, Y., Bonen, D.K., Inohara, N., Nicolae, D.L., Chen, F.F., Ramos, R., Britton, H., Moran, T., Karaliuskas, R., Duerr, R.H., Achkar, J.P., Brant, S.R., Bayless, T.M., Kirschner, B.S., Hanauer, S.B., Nunez, G. & Cho, J.H. (2001) A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease. Nature 411, 603–606. 29. Inohara, N., Ogura, Y., Chen, F.F., Muto, A. & Nunez, G. (2001) Human Nod1 confers responsiveness to bacterial lipopoly- saccharides. J. Biol. Chem. 276, 2551–2554. Ó FEBS 2002 Lipopolysaccharide of B. vulgatus (Eur. J. Biochem. 269) 3721 . Structural study on lipid A and the O-specific polysaccharide of the lipopolysaccharide from a clinical isolate of Bacteroides vulgatus from a patient with Crohn’s disease Masahito Hashimoto 1,2 ,. OPS-rich fraction on analysis of the sugar constituents (Table 1). The approximate molar ratio of Rha to Man was 1 : 1. Abso- lute configuration analysis demonstrated that Man has a D configuration and. the fatty acid on the acyoxyacyl group to be mainly 15, confirming the result of the compositional analysis. In the minor triacylated lipid A, the fragmentation patterns of the parent ion at m/z