The structure and biological characteristics of the Spirochaeta aurantia outer membrane glycolipid LGL B Evgeny Vinogradov 1 , Catherine J. Paul 2 , Jianjun Li 1 , Yuchen Zhou 2 , Elizabeth A. Lyle 3 , Richard I. Tapping 3 , Andrew M. Kropinski 2 and Malcolm B. Perry 1 1 Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada; 2 Queen’s University, Kingston, ON, Canada; 3 University of Illinois, Urbana, IL, USA In an attempt t o i solate lipopolysaccharide from Spirocha- eta aurantia, Darveau-Hancock extraction of the cell mass was performed. While no lipopolysaccharide was found, two carbohydrate-containing compounds were detected. They were resolved by size-exclusion chromatography into high molecularmass(LGL A ) and low molecular mass (LGL B ) fractions. H ere w e present the results of the a nalysis of t he glycolipid LGL B . Deacylation of LGL B with hydrazine a nd separation of the products by using anion-exchange chro- matography gave two major p roducts. Their structure w as determined by using chemical methods, NMR and mass spectrometry. All monosaccharides had the D -configuration, andasparticacidhadthe L -configuration. Intact LGL B contained two fatty groups at O-2 a nd O-3 of the glycerol residue. Nonhydroxylated C14 to C18 fatty a cids were identified, which were p redominantly unsaturated or bran- ched. LGL B was able t o gel Limulus amebocyte lysate, albeit at a lower level t han that observed f or Escherichia coli O113 lipopolysaccharide. However, even large amounts of LGL B were unable to stimulate any Toll-like receptor (TLR) examined, including TLR4 a nd TLR2, previously shown to be sensitive t o lipopolysaccharide and glycolipids from diverse bacterial origins, including other spirochetes. Keywords: glycolipid; Spirochaeta aurantia;structure. Spirochetes are a group of bacteria unified by spiral or flattened-waveform cell morphology and periplasmic endo- flagella; Spirochaeta is one of the six genera within this phylum [1]. This b acterium is a f ree-living nonpathogenic spirochete, originally isolated from pond mud and able to fix atmospheric nitrogen [2–4]. Other members of this phylum include the human pathogens Borrelia burgdorferi (Lyme disease), the Leptospira (leptospiroses), Treponema pallidum (syphilis), and T. denticola, T. brennaborense,and T. maltophilum, which are i mplicated in periodontal disease [5–7]. Although classified as Gram-negative, controversy exists over the existence of lipopolysaccharide (LPS) in the outer membranes o f spirochetes. Clear genetic a nd bio- chemical evidence exists for the presence of LPS in Leptospira [8] and for i ts absence i n T. pallidum and Borrelia [9,10]. Limited structural analysis suggests that several o ral treponemes (T. brennaborense and T. maltophilium [6], T. medium [11], and T. denticola [12]) pos sess a surface glycolipid similar to the lipotechoic acid of Gram-positive bacteria. Recently, several small surface glycolipids were identified in B. burgdorferi [13,14]. Toll-like r eceptors (TLR) a re an important component of the host response to invading bacteria, with TLR4 required for signal transduction and the inflammatory response following exposure of cells to LPS derived from Gram- negative enteric b acteria [15–17]. A lthough LPS der ived from enteric bacteria is a potent agonist for TLR4, other nonenteric bacterial L PS, such as that derived from Legionella pneumophila, Leptospira interrogans and at least one strain of Porphyromonas gingivalis can act as agonists for TLR2 [8,18,19]. The glycolipids isolated from T. denticola, T. brennabo- rense,andT. maltophilum appear to have functional similarity to LPS in that t hey possess some ability to gel Limulus amebocyte lysate (LAL) [12,20], a standard assay for endotoxin activity. In addition, while glycolipid derived from T. brennaborense stimulates immune cells through TLR4, the glycolipids from T. denticola and T. maltophilum stimulate cells through TLR2 [ 5,6,20]. T he strict correlation between t he structure of the LPS molecule wit h that of TLR specificity remains undefined but it is clear that TLR2 is capable of r ecognizing a w ider range of potential lipid A structures than TLR4 [21]. S. aurant ia has simple growth r e quirements t hat f acilitate studies otherwise limited by the amount of cell mass, a problem often limiting studies on other spirochetes [2]. We describe here the structural characterization o f the carbo- hydrate s keleton and fatty acids of one of its glycolipids, LGL B . In addition we present evidence which suggests that Correspondence to E. Vinogradov, Institute for Biological Sciences, National Research Counci l, 1 00 Sussex Dr., Ottawa, ON, Canada K1A 0R6. Fax: +1 613 952 9092, Tel.: +1 613 990 0832, E-mail: evguenii.vinogradov@nrc-cnrc.gc.ca Abbreviations: EU, endotoxin units; FAME, fatty acid methyl esters; GalNAcA, N-acetylgalactosaminuronic acid; GSL, glycosphinogo- lipids; Fuc3N, 3-ami no-3,6-dideoxygalactose; Kd o, 2-keto-3-deoxy- D -manno-oct-2-ulosonic acid; LA L, Limulus amebocyte lysate; LBP , LPS-binding protein; LPS, lipopolysaccharide; SGM, spirochaete growth medium; TLR, Toll-like receptor; TNF-a, tumour necrosis factor-a. (Received 9 August 2004, revised 30 September 2004, accepted 13 October 2004) Eur. J. Biochem. 271, 4685–4695 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04433.x while superfic ially resembling other spirochetal glycolipids, LGL B is a multisaccharide glycolipid and is unable to stimulate any TLR examined. Experimental procedures Bacterial strain and growth conditions The S. aurantia strain, M 1, us ed in this study, was obtained originally from E. P. Greenberg (Ohio State University, Columbus, OH, USA). It was propagated in spirochete growth medium (SGM) containing 0.4% (w/v) maltose (Sigma-Aldrich, St. Louis, MO, USA), 0.2% (w/v) tryptone and 0.2% (w/v) yeast extract (Difco), at pH 7.5. Cells were grown at 30 °C with gentle aeration ( 30 r.p.m.; orbital shaker; Forma Scientific, Marietta, O H, USA) for 24–48 h. Cell stocks were maintained in SGM in liquid nitrogen. Glycolipid isolation Isolation of LGL from S. aurantia. Bacteria were harves- ted f rom a total of 55 L of SGM and the c ombined cell pellet was extracted fo llowing the method of Darveau & Hancock [ 22]. The final product w as extracted once with cold 95 % (v/v) ethanol and twice with chloroform/meth- anol (2 : 1, v/v) to remove phospholipids and carotenoids. The r esidue was resuspended in distilled water, and contaminating protein was removed by treatment with pronase (25 lgÆmL )1 )for18hat37°C. A final extraction by chloroform/methanol (2 : 1, v/v) was followed by dialysis against distilled water using Slide-a-lyzerÒ 10K cassettes (Pierce Chemical Company, Rockford, IL, USA) and lyophilization. The overall yield was determined by comparing the mass of a white powdery material left after dialysis and lyophilization ( 547 mg), to the original d ry weight of lyophilized whole cells of S. aurantia from which that glycolipid material had been isolated (3.51 g). Column chromatography. Crude LGL (1.5 g) was dis- solved in sample buffer [20 m M Tris/HCl, pH 8; 50 m M EDTA; 10% w/v) SDS] and fractionated on a 5.5 · 40 cm column of Sephacryl S-300 HR (Sigma-Aldrich) at 25 °C [column buffer: 10 m M Tris/HCl, pH 8; 1 0 m M EDTA; 0.2 M NaCl; 0.3% (w/v) SDS]. Fractions of  2.1 mL were collected at an average fl ow rate of 1.5 mLÆmin )1 .The fractions containing the l ow m olecular mass m aterial (LGL B ), as determined by standard SDS/PAGE with silver stain [23], were pooled, precipitated with cold 0.375 M MgCl 2 in 95% (w/v) ethanol, suspended in distilled water and subjected to a second chromatography to ensure homogeneity. Material was then reprecipitated, suspended in distilled water, dialyzed, lyophilized and weighed in preparation for further analysis. Tricine–SDS/PAGE. Tricine–SDS/PAGE [15% (w/v) resolving gel; 1 0% (w/v) s pacer g el; 4.5% ( w/v) stacking gel) was u sed to e xamine the low molecular mass portions of LPS and LGL [24]. LPS from Salmonella enterica sv. typhimurium wild type , Sal. enterica sv. t yphimurium TV 119 (Ra mutant) a nd Sal. enterica sv. m innesota R5 (Rc mutant) were p urchased f rom S igma-Aldrich. Products in acryl- amide gels we re visualized by silve r staining [23]. NMR spectroscopy and general methods NMR spectra were recorded at 25 °CinD 2 OonaVarian UNITY INOVA 600 instrument using acetone as the external reference ( 1 H, 2.225 p.p.m., 13 C, 31.45 p.p.m.). Varian standard programs COSY, N OESY (mixing time of 300 ms), TOCSY (spinlock time 120 ms), HSQC, HMQCTOCSY, and gHMBC (evolution delay of 100 ms), were used. Capillary electrophoresis-electrospray mass spectrometry (CE-MS). Mass spectrometric experiments were conduc- ted by using a Q-Star Quadropole/time-of-flight instrument (Applied B iosystems/Sciex, Concord, ON, Canada). B riefly, samples were analyzed on a crystal Model 310 CE instrument (ATI Unicam, Boston, MA, USA) coupled to a Q-Star via a microIonspray interface. A sheath solution (isopropanol/methanol, 2 : 1, v/v) was delivered at a flow rate of 1 lLÆmin )1 to a low dead volume tee. The separation was obtaine d on a bare fused-silica capillary, o f  90 cm length, u sing 10 m M ammonium acetate/amm onium hydroxide in deionized water, pH 9.0, co ntaining 5% (v/v) methanol. A voltage of 25 kV was typically applied at the injection. Mass spectra were acquired w ith dwell times of 2.0 s per scan i n positive ion detection mode. Fragment ions formed by collision activation of selected precursor ions with nitro gen in the RF-only q uadrupole c ollision ce ll, were recorded by a t ime-of-flight mass analyzer. Collision energies were typically 120 eV (laboratory f rame of refer- ence). Hydrolysis. Hydrolysis was p erformed with 4 M CF 3 CO 2 H (110 °C, 3 h), monosaccharides were conventionally con- verted into the alditol acetates and analysed by GLC on an Agilent 6850 chromatograp h equipped with a DB-17 (30 m · 0.25 mm) fused-silica column using a temperature gradient of 180 °C(2 min) fi 240 °C, at 2 °CÆmin )1 .GC- MS was performed on the Varian Saturn 2000 system with an ion-trap mass spectral detector using the same column. Gel chromatography. Gel chromatography was carried out on Sephadex G-50 (2.5 · 95 cm) and Sephadex G -15 columns (1.6 · 80 cm) in pyridinium-acetate buffer, pH 4.5 (4 mL of pyridine and 10 mL of AcOH in 1 L of water), and the eluate was monitored by a refractive index detector. Configuration experiments For determining the absolute configuration of t he mono- saccharides, product 2 (1 mg) was treated with (S)-2- butanol/AcCl (0.25 mL, 10 : 1, v/v) for 2 h at 85 °C, dried under t he stream of air, acetyl ated and t hen analysed b y GC in comparison with authentic standards, prepared from the respective monosaccharides with (S)- an d (R)-2-butanol. For determination of the configuration of N-acetylgalac- tosaminuronic acid (GalNAcA), a sample (2 mg) of LGL B was treated with 1 M HCl in methanol (100 °C, 4 h), dried, and t hen the product was peracetylated by Ac 2 O i n pyridine (0.5 + 0.5 mL, at 85 °C for 30 min) and reduced with excess NaBD 4 in96%(v/v)ethanol(1 mL)at40°C. Acetic acid (1 mL) was added, the product was dried under a 4686 E. Vinogradov et al. (Eur. J. Biochem. 271) Ó FEBS 2004 stream of air a nd then dried twice with the a ddition of 1 mL of meth anol to remove boric acid. (R)-2-BuOH ( 0.5 mL) and AcCl (0.08 mL) were added to the dry residue, the mixture was incubated for 4 h at 85 °C, filtered, dried, acetylated by Ac 2 O in p yridine ( 0.5 + 0.5 mL, at 85 °Cfor 30 min), dried and analyzed by GC-MS with the standards prepared from D -GalN and (R)- and (S)-2-BuOH. The absolute configuration of L -aspartic acid was deter- mined by chiral HPLC of the oligosaccharide hydrolysate on a Chirex D p enicillamine column (250 · 4.6 mm; Phenomenex) in 15% (v/v) methanol containing 2 m M CuSO 4 , with UV detection at 2 54 nm. Fatty acid methyl esters (FAMEs) were generated from 1 mg s amples of LGL B by the addition of 1 mL o f 3 M HCl in methanol (Alltech Associates, Inc., Deerfield, I L, USA) and incubation at 100 °C for 18 h. Following liberation of the FAMEs, the hydrolysates were neutralized with 0.46 g of silver carbonate and doped with 204.5 lg of tridecanoic acid (in n-pentanol) as an internal standard. The samples were centrifuged and the FAMEs were resolved by PerkinElmer Sigma 3 gas chromatography, equipped with a glass column [3.05 m · 2 mm internal diameter, packed with 3% (w/v) SP-2100 DOH, 100/120 Supelcoport w ith carrier gas (N 2 )], at a flow rate of 50 mlÆmin )1 . The oven was programmed as f ollows: 150 °C f or 5 min; followed by 150° to 230° Cat8° CÆmin )1 . Data analysis was conducted by using the PEAKFIT Ò v. 4.11 software package (Systat Software Inc., Richmond, CA, USA). Comparison of FAME retention times with those of a Bacterial Acid Methyl Esters CP TM mix (Matreya, Inc., Pleasant Gap, PA, USA) permitted tentative FAME identifications to be made. The latter were confirmed b y G LC-MS a nalysis (Analytical Services, Queen ’s University, K ingston, ON, Canada). LGL B was O-deacylated by hydrazinolysis, as described by Gu et al. [25]. Briefly, 40 mg o f LGL B was i ncubated with anhydrous hydrazine for 3 h at 37 °C, w ith occasion al mixing. The mixture was then chilled to )20 °Candan equal volume of chilled acetone was added dropwise. The product was recovered by centrifugation, washed once w ith chilled acetone, dried and weighed. Separation of oligosaccharides 1 a nd 2 was performed by ion-exchange chromatography on a Hitrap Q anion- exchange column containing 5 mL of Q-Sepharose Fast Flow (Amersham Pharmacia Biotech) in a gradient of water/1 M NaCl over 1 h with UV detection at 220 nm. The products were desalted by gel chromatography on a Sephadex G-15 column. Biological assays LAL a ssays were conducted by the Associates of Cape Cod, Inc. (Cape Cod, MA, USA), by using the gel-clot method, and the number of endotoxin units (EU) was compared with control standard endotoxin from Escherichia coli O113. The activation o f TLRs w as measur ed by quantifying the production of tumour necrosis factor-a (TNF-a)by whole blood cells, in response to a panel of TLR agonists, a s described by Tapping et al. [26]. Briefly, whole blood from healthy donors was collected into tubes containing heparin and d iluted 1 : 4 in RPMI 1640. Sa mples were a liquoted into 96-well plates, agonist was added, and incubatio n was carried out at 37 °C in an atmosphere of 5% carbon dioxide for 6 h. Cell supernatants were removed a nd assayed for cytokine production by standard sandwich ELISA in 96-well Immunlon plates (Dynatech L aboratories, Chant- illy, VA, USA). The TNF-a ELISA was p erformed by using mAbs 68B6A3 or 68B2B3 for capture and t he biotinylated mAb 68B3C5 (Biosource International, Camarillo, CA, USA), followed by streptavidin-conjugated horseradish peroxidase (HRP), for d etection. ELISAs were d eveloped by using o-phenylenediamine as a substrate, and the absorbance was measured at 490 nm by using a Spectramax plate reader and software (Molecular Devices, Sunnyvale, CA, USA). All values were interpolated from either a log- log or a four-parameter fit of a curve generated from appropriate standards. Agonists examined were the S. aurant ia LGL B (5 lgÆmL )1 ), zymosan (5 · 10 9 particles per mL; Molecular Probes, Eugene, OR, USA), heat-killed Staphylococcus aureus (2.5 · 10 6 particles per mL; Molecu- lar Probes), PolyIC (50 lgÆmL )1 ; Sigma Genosys, The Woodlands, TX, USA), E. coli Re595 LPS (20 ngÆmL )1 ; repurified as decribed previously [26]), R848 (1 lgÆmL )1 ; Invivogen, San Diego, CA, USA) and CpG Oligo (2 l M ; Sigma Genosys). Results Darveau-Hancock extraction o f stationary phase S. auran- tia cells gave a white powdery substance i n a yield of 15.6% based upon the cell dry wieght. T his h igh y ield is not unexpected as the surface to volume ratio of this bacterium is 13.6Ælm )1 , approximately 3 .5 times higher t han that of E. coli or Sal. enterica sv. typhimurium (3.9Ælm )1 ). The Darveau-Hancock procedure does not discriminate between high (ÔsmoothÕ)orlow(ÔroughÕ)molecularmassLPS, provides a high yield of product and should apply equally to polysaccharides or glycolipids [22]. Potential complex glycolipids were separated from previously characterized glycogen storage granules by size exclusion chromatography with examination of the fractions for carbohyd rates and hexosamines [27]. A low molecular mass carbohydrate- containing material (LGL B ) was isolated, and when examined by Tricine–SDS/PAGE [24] , demonstrated mobil- ity s imilar t o the rough LPS of a Sal. enterica sv. typhimurium TV 119 Ra mutant (Fig. 1). Another material, LGL A ,was identified as a larger glycolipid and is thought to contain O-antigen like r epeats, contributing to the b anding pattern observed in crude S. aurantia extract ( data not shown). Preliminary colorimetric analysis [28] indicated that LGL B did not contain any 2-keto-3-deoxy- D -manno-oct-2- ulosonic acid (Kdo). The material was subjected to methanolysis [29], and the fatty acid methyl esters were analyzed by GLC, revealing fi ve major acyl constituents, none of which were the characteristic hydroxylated fatty acids of LPS (Table 1). LGL B was O-deacylated by t reatment with anhyd rous hydrazine, and the oligosaccharides were separated by anion-exchange chromatography to give two main compo- nents (1 and 2), which differed by one monosaccharide residue. Their structure was determined by NMR spectros- copy, MS and chemical analysis. Monosaccharide analysis (GC of alditol acetates or acetylated products of acidic methanolysis) of both products showed that their compo- Ó FEBS 2004 Analysis of Spirochaeta aurantia glycolipid LGL B (Eur. J. Biochem. 271) 4687 sition was similar, comprising glycerol, xylose, mannose, glucose, galactose, and 3-amino-3,6-dideoxyhexose in a ratio o f 1 : 2.5 : 1 : 1.6 : 1 : 1.5, and additionally nonquan- tified glucuronic, galactosaminouronic, and galacturonic acids. The presence of excess glucose (glucitol) in alditol acetate analysis is a r esult of the reduction o f glucurono- lactone. The 3-amino-3,6-dideoxyhexose is quantified approximately b ecause of the lack of a quantitative standard compound. NMR spectra of both oligosaccharides were completely assigned by using 2 D N MR techniques ( Figs 2–4, Table 2). Monosaccharides were identified o n the basis o f vicinal proton coupling constants and 13 C NMR chemical shifts. Anomeric configurations were deduced from the J 1,2 coupling constants and chemical shifts of H-1, C-1 and C-5 signals. The position of C-6 signals of uronic acids was found from HMBC correlations to H-5 protons. Connec- tions between monosaccharides were identified on the basis of NOESY ( Fig. 3) and HMBC correlations. The following inter-residual NOEs were observed in oligosaccharides 1 and2:P1G4(in1),C1G4(in2),andG1A2,G1A1,A1G5, A1L4, A1L3, L1E4, E 1F4, F1I4, I1D4, D1N3, D1N4, N1Q1, B1K4, K1E3, O1I3, and M1D3. These correlations include several contacts to nontransglycosidic protons next to the linkage position, and between H-1 o f a monosac- charide a nd H-5 of a glycosylating residue in the e vent of an a-(1–2)-linkage. R espective H MBC c orrelations between H-1 and a carbon at the transglycosidic position were identified for all linkages. Amide linkage between C-6 of residue E and an amino group of the aspartic acid was identified on t he basis of the HMBC correlation b etween H-2 of aspartic acid and C-6 of the GalA E, thu s showing that aspartic acid is amide linked through i ts amino group to C-6 of galacturonic acid E (Fig. 4). Absolute configuration o f the monosaccharides was determined by GC analysis of acety lated 2-butyl glycosides. For 3-amino-3,6-dideoxygalactose (Fuc3N), the O-specific polysaccharide from Proteus penneri 16wasusedasa source of reference D -Fuc3N, where its D -configuration was determined earlier [30]. For determining the configuration of GalNAcA, an LGL B sample was treated with HCl in 1 5 4 3 2 [ppm] 2 Fig. 2. 1 H NMR spectra of oligosaccharides 1and2. 15 234 Fig. 1. Visualization of LGL B by Tricine–SDS/PAGE and silver staining rev eals that this material c o-electrophoreses with the Ra form of lipopolysaccharide (LPS) from Salmonella enterica sv. typhimurium. Lane 1, wild-type Sal. enterica sv. typhimurium LPS, 30 lg; lane 2, Spirochaeta aurantia crude LGL, 20 lg; lane 3, S. aurantia LGL B , 15 lg; lane 4, Sal. enterica sv. typhimurium TV119 ( Ra mutant), 2 lg; lane 5, Sal. enterica sv. minnesota R5 (Rc m u tant), 2 lg. Table 1. Fatty acid methyl e ster (FAME) analysis, GLC and GLC-MS indicated that the majority of fatty acids contained in Spirochaeta aurantia LGL B are either branched or unsaturated. Values stated a re the average n molÆmg )1 with standard deviations (±) obtained from quantifying and averaging areas under specific peaks from GLC analysis of four separate samples of LGL B . Identity of fatty acid LGL B (nmolÆmg )1 ) Tetradecanoic acid (C14:0) 34.9 ± 1.3 13-Methyltetradecanoic acid (iC15:0) 224.1 ± 9.0 15-Methylpentadecanoic acid (iC16:0) 117.6 ± 12.4 9-Hexadecenoic acid (C16:1 9 ) 155.3 ± 10.5 9-Octadecenoic acid (C18:1 9 ) 58.3 ± 5.6 4688 E. Vinogradov et al. (Eur. J. Biochem. 271) Ó FEBS 2004 methanol, a nd the product w as peracetylated in order to reacetylate free amino groups; it was checked by GC-MS for the presence of methyl ester of m ethyl G alNAc. The product w as reduced with NaBD 4 in 96% (v/v) ethanol at 40 °C,treatedwithHCl-(R)-2-BuOH, acetylated and ana- lysed by GC-MS. A total ion chromatogram showed no well pronounced peaks; however, a fragmentogram for t he expected glycosyl cation of m/z 332 contained peaks with the same retention time as that obtained f rom D -GalN with (R)-2-BuOH; mass spectra of the products obtained from LGL B wereshiftedtohighmassbytwounitsowingto a double deuteration at C-6. Thus, G alNA had the D -configuration. These data, taken together, allow u s to p ropose the structures of oligosaccharides 1 and 2, as presented in Scheme 1. The o ligosaccharides were further analyzed by CE-MS and by CE-MS/MS (Figs 5 and 6). The mass spectra obtained in positive ion detection mode for oligosaccharide 1 showed a m ajor do ubly charged ion at m/z 1180.25 (observed molecular mass: 2358.50 Da; calculated exact molecular mass for C 85 H 130 O 72 N 4 : 2358.6633 Da). The MS for oligosaccharide 2 showed a m olecular mass of 2375.56 Da (calculated exact mass for C 85 H 129 O 74 N 3 : 2375.6422). In addition, an ammonium adduct of com- pound 2 with m/z 1197.25 was observed as the most abundant ions (observed molecular mass: 2392.50 Da). The composition details, as well as some sequence information of those two major c omponents w ith m/z 1180.24 and 1197.25, were further characterized by tandem mass spectrometry (MS/MS). The fragmentation of cationic oligosaccharides typically proceeds by cleavage at the glycosidic bonds, which provides sequence and branching information [31]. The charge state of a fragment ion is then identified by u sing the isotope profile, owing to the high resolution provided by the TOF mass analyser. The product-ion s pectrum ( MS/MS s pectrum), obtained from a doubly charged ion a t m/z 1197.25, is illustrated in Fig. 6. This spectrum revealed two major doubly charged ions at m/z 503.63 and 672.67, which corresponds to the single charged ion at m/z 1006.27 and 1344.37, respectively. In addition, a series of single charged ions was generated via the formation of complementary fragment pair of B and Y ions. A s i ndicated i n F ig. 6 , t he fragment ion a t m/z 18 76.56 corresponded to the loss of the nonreducing end C-G-A unit and ammonium from the molecular ion. Further fragmentation gave the fragment ion at m/z 1436.42, owing to the loss o f the branching xylose residues M and O, a nd of GlcA residue L. The remaining linear sequence, consisting of K-B-F-I -D-N-Q, was confirmed by t he observation of 5.6 5.4 5.2 5.0 4.8 4.6 ppm 5.6 5.4 5.2 5.0 4.8 4.6 ppm 5.2 4.7 4.2 3.7 ppm TOCSY NOESY A12 B12,14 B13 C12 C13 C15 C14 D12,13 D15 D14 E15 E14 E12 E13 F12 F13 G15 F14 I12 I13 I14 E45 G12 G14 G13 K12 K13 K14,15 L12 L13,15 L14 F45 I45 D45 I24 E34 E24 I34 D34 N14 I23 F34 N12 N13 M15 O15 M14 M13 M12 M15 O12 O15 O13 O14 F24 A1:G1 A1:G5 A12 A1:L4 A1:L3 B12 B1:K4 C1:G4 C12 D12 D1:N4 D1:N3 G12 F12 E12 G1:A2 E35 E5:F2 E1:F4 F1:I4 E45 I1:D4 I12 K1:E3 K15 K12 F45 O1:I3 M1:D3 I34 I35 F34 N15 N13 N1:Q1 M15 O15 O13 Fig. 3. Fragments of TOCSY (left) an d NOESY (right) s pectra o f oligosaccharide 2. Intraresidual correlations are labeled with a letter designation o f the m o nosaccharide residue and numbers o f the c orrelating protons. Inte r-residual correlations a re labeled withlettersforbothmonosaccharides. Ó FEBS 2004 Analysis of Spirochaeta aurantia glycolipid LGL B (Eur. J. Biochem. 271) 4689 fragment ions at m/z 292.06, 468.09, 613.1 6, 789.21, 1006.27, 1182.24, and 1344.37, respectively. The fragment ion at m/z 556.15 corresponds to the unit I-D-N, which might result from the loss of Q from I-D-N-Q (m/z 648.20) o r from the loss of F f rom F -I-D-N (m/z 732 .18). However, many o ther combinations of fragments are also possible, because of the existence of branches i n t he molecule. Similarly, the t andem MS was conducted for the doubly charged ion at m/z 1180.25 (data not shown) and the mass spectral data f ully agree with the sequence determined by NMR. Knowledge of the deacylated oligosaccharide structures allowed analysis of intact glycolipids by NMR. Spectra of reasonable quality were obtained at 60 °C in the presence of 5% fully deuterated SDS. All monosaccharides present in the products 1 and 2 were identified, a nd the ratio of structures 1 a nd 2 w as c lose to 1 : 1. All chemical shifts remained m ostly unchanged with the e xception of H/C-2 and H /C-3 of the glycerol residue. P roton signals were strongly downfield shifted owing to acylation ( Table 2); 13 C signals also experienced downfield substitution effects. No data regarding attachment of particular acyl groups at O-2 and O-3 of the glycerol residue was obtained. Several attempts to obtain a mass spectrum of the LGL B by using CE-MS, ESI-MS and MALDI were unsuccessful, prob- ably because this compound is not soluble without a detergent. These results show that no additional acylation, except at the glycerol residue, is present in the oligosac- charides. LGL B does not activate any Toll-like receptor The gelation of LAL is a standard assay based on the nonspecific immune response of the horseshoe crab, and is used to assess the endotoxic potential of various substances [32]. LGL B displayed a 100- fold less endot oxic potential, registering 2.5 · 10 5 EUÆmg )1 when compared to an E. coli O113 LPS control (1 · 10 7 EUÆmg )1 ) in a LAL gel clot assay. LGL B was also examined for its ability to act as a TLR agonist. Attempts to measure a reaction f rom cells trans- fected specifically with human TLR2 or TLR4 were unsuccessful, regardless of the concentration of LGL B examined (data not shown). T he whole blood assay u ses fresh human blood (which contains a variety of Toll receptors) and measures the total r elease of TNF-a by ELISA [26]. Cells were stimulated with defined TLR agonists (zymosan a nd heat-killed Staph. aureus for TLR2; PolyIC for TLR3; E. coli Re595 LPS for TLR4; R848 for TLR7; and CpG O ligo ( 2 l M ) for TLR9), and the production of TNF-a was q uantified (Fig. 7). Even when 5.5 5.0 4.5 4.0 3.5 3.0 100 80 60 40 178 174 170 Asp2 Asp3 Asp13;Asp34 E56 Asp2:E6 Asp12;Asp24 HSQC HMBC E5 Fig. 4. Fragments of HSQC and HMBC spectra of compound 1. Labels illustrate assignment of the amide linkage between the amino group of Asp and the carboxyl group of GalA residue E. Scheme 1. The structures of o ligosaccharides 1 and 2. Oligosaccharide 1, R ¼ a-Fuc3N (P); o ligosaccharide 2, R ¼ a-Glc (C). 4690 E. Vinogradov et al. (Eur. J. Biochem. 271) Ó FEBS 2004 high concentrations of LGL B were added, no production of TNF-a was detected, showing t hat this large glycolipid cannot stimulate TLR2, -3, -4, -7 or -9. Discussion Although s ome s tructural i nformation has been obtained from other spirochetes, the complete elucidation o f t he LGL B from S. aurantia represents the first complete structure o f a large glycolipid from these bacteria. The dodecasaccharide LGL B is anchored by a diacyl glycerol. A glycolipid containing a single sugar, BbGL-II, and also anchored on a g lycerol, has been identified in B. burg- dorferi [13]. I t i s s urface localized, and antibodies to this molecule were detected in patients with Lyme disease. A diacyl glycerol anchor has also been purposed for the glycolipids of T. denticola, T. maltophilum,andT. brenn- aborense [6,12]. A glycolipid identified in T. pectinovorum contained glycerol, and the majority of fatty acids were branched, although on the basis of detection of Kdo in this material, the authors designated it LPS. A diacyl glycerol anchor may substitute for lipid A, an observa- tion supported by the absence of any homologs to genes involved in lipid A biosynthesis in the completed ge- nomes of B. burgdorferi, T. pallidum or T. denticola [9,10,33]. All o f t he treponemal g lycolipids i dentified have either fully saturated o r branched fatty acids , in contrast to the Table 2. NMR data for compounds LGL B , 1 and 2. Data refer to both compounds, except where indicated. N-Acetyl at I2: H-2/C-2 2.12/23.8, C-1176.2 p.p.m. Unit, compound 1 2 3 4 5 (5eq) 6 (5ax) 6b A, a-Man 1 H 5.71 4.12 3.99 3.92 3.72 3.90 3.90 13 C 99.8 81.2 71.5 67.7 74.1 61.5 B, a-Fuc3N 1 H 5.58 4.01 3.68 4.02 4.18 1.27 13 C 98.6 66.3 53.6 69.4 67.7 16.6 P, a-Fuc3N (1) 1 H 5.58 3.99 3.65 4.01 4.16 1.25 13 C 98.6 66.3 53.6 69.4 67.7 16.6 C, a-Glc (2) 1 H 5.55 3.59 3.76 3.47 3.78 3.86 3.86 13 C 99.6 73.0 74.0 70.5 73.0 62.4 D, a-GalA 1 H 5.32 4.22 4.23 4.69 4.65 13 C 96.5 68.7 79.3 79.4 72.4 175.6 E, a-GalA6Asp 1 H 5.20 4.12 4.20 4.70 5.05 13 C 100.8 68.8 79.4 79.4 72.2 170.6 F, a-GalA 1 H 5.18 3.89 4.21 4.50 4.79 13 C 99.3 68.9 69.6 80.3 72.8 176.0 G, a-GlcA (1) 1 H 5.19 3.69 4.06 3.83 4.30 13 C 101.6 73.1 75.0 77.3 73.7 177.2 G, a-GlcA (2) 1 H 5.19 3.69 4.06 3.82 4.30 13 C 101.6 73.1 75.0 77.8 73.7 177.2 I, a-GalNAcA 1 H 5.11 4.46 4.10 4.69 4.76 13 C 99.3 49.8 77.3 77.6 72.9 176.0 K, b-GlcA 1 H 4.80 3.50 3.86 3.91 3.90 13 C 104.8 74.7 77.4 77.4 78.0 176.3 L, b-GlcA (1) 1 H 4.81 3.34 3.75 3.83 3.77 13 C 104.4 74.9 77.5 77.4 78.1 176.4 L, b-GlcA (2) 1 H 4.81 3.34 3.75 3.83 3.74 13 C 104.4 74.9 77.5 77.4 78.1 176.4 M, b-Xyl 1 H 4.62 3.39 3.46 3.72 3.33 3.98 13 C 106.5 74.4 77.3 70.5 66.4 N, b-Gal 1 H 4.55 3.72 3.91 4.29 3.80 3.88 3.88 13 C 104.1 70.4 78.6 66.4 76.2 62.3 O, b-Xyl 1 H 4.47 3.28 3.42 3.69 3.33 3.98 13 C 106.6 73.9 77.1 70.5 66.4 Asp 1 H 4.42 2.93 2.93 13 C 178.8 52.3 39.1 178.8 Q, Gro, 1 and 2 1 H 3.85 4.03 3.68 4.00 3.76 13 C 72.1 71.7 63.7 Q, Gro, LGL B 1 H 3.84 5.32 4.15 3.84 4.50 13 C 69.8 72.0 64.8 Ó FEBS 2004 Analysis of Spirochaeta aurantia glycolipid LGL B (Eur. J. Biochem. 271) 4691 1000 1100 1200 1300 1400 m/z 1197.25 1189.28 1180.30 1116.27 1131.28 1000 1 100 1200 1300 1400 1180.25 1114.29 1 2 Fig. 5. CE-MS spectra of oligosaccharides 1 and 2. 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z 613.16 556.15 468.09 672.67 146.08 503.63 732.18 648.20 218.06 789.21 714.18 292.06 824.24 321.08 1006.27 339.09 1199.29 663.67 807.22 1023.26 376.09 1344.37 1744.531436.42 1182.24 1876.56 K B F I 1612.48 D NQL OM C-G-A,NH 3 Fig. 6. MS/MS spectrum obtained from a doubly charged ion at m/z 1197.25 of o ligosaccharide 2. 4692 E. Vinogradov et al. (Eur. J. Biochem. 271) Ó FEBS 2004 unsaturated acyl group of BbGL-II. Schultz et al. indicated that the presence of fatty acid branching in T. denticola is analogous to adap tations in Gram-positive bacteria to alter membrane fluidity [12]. Gram-negative bacteria are known to m odify the d egree of saturation in their fatty acids to modulate membrane fluidity [34,35]. LGL B contained both unsaturated and branched fatty acids (i.e. C14:0, iC15:0, C16:1), t he only spirochete glycolipid identified, to date, with both of these modi- fications, suggesting LGL B may form highly fluid mem- branes. S. aurant ia LGL B comprises 15% lipid by mass, corres- ponding well with the p roportion of fatty acids in the glycolipid OML521 (10.7%) from T. denticola, a glycolipid that is also estima ted t o be similar in size to Ra LPS [12]. Ra LPS is t he minimum L PS unit required for efficient and proper folding, and functioning, of porin [36]. T. denticola and S. aurantia possess two of the largest porins yet discovered in Gram-negative bacteria: given the absence of LPS i n t hese bacteria, OML251 and LGL B may function in place of Ra LPS, and contribute to the folding or stabilization of porin [37,38]. While S. aurantia stains Gram -negative and possesses an outer membrane containing porin, phylogenetically it is not closely related to the bacterial phylum (Proteobacte- ria) that contains the typical Gram-negative cells, such as E. coli. Other nonproteobacterial organisms in which glycolipids replace LPS include Chloroflexus aurantiacus and Fibrobacter succinogenes. The former bacterium is thought to contain outer membrane galactolipids [39], while the latter c ontains a low molecular mass glycolipid with glycerol anchor and man y charged groups in the oligosaccharide p art, which makes it, in overall d esign, similar t o S. aurantia LGL B . I nterestingly F. succinogenes also has a capsular polysaccharide with a lipid anchor [40]. Even within the Proteobacteria, one finds examples where LPS has been replaced by glycolipids. Sphingomonas pau- cimobilis and N ovosphingobium capsulatum contain glyco- sphinogolipids (GSLs) [41–43] in lieu of LPS. Although there is no similarity at a structural level to LPS, studies investigating treponemal glycolipids have shown that most are able to gel LAL and stimulate Toll-like r eceptors [12,20], suggesting t hat a t a functional level they possess some similarity. LGL B was able to gel LAL, but did not stimulate any TLR examined: this is an unusual situation, paralleled in the spirochete litera- ture only by the inability of the Borrelia glycolipids to activate TLR2 o r -4 [13]. TNF-a release was measured following the e xposure o f h uman mononuclear cells to two diffe rent GSLs from S. paucimobilis: t he mono- glycosylated GSL-1, and the tetraglycosylated GSL-4A. GSL-1 was unable to activate the release of monokines, in contrast to the larger GSL-4A, although induction was still 10 000-fold below that of the LPS standard [44]. While this appears to be s imilar to the situation with the monoglycosylated BbGL-II, the inability of LGL B to stimulate TNF-a release precludes size as the only explanation for the difference in biological activity observed with GSLs. Another oral spirochete implicated in periodontal dis- ease, T. medium, contains the glycolipid, Tm-Gp, which abrogates TLR activation through interactions with LPS-binding protein (LBP) and CD14, two important components of TLR-mediated innate immunity [45]. The blocking by Tm-Gp was dependent on the lipid portion of the molecule, but whether S. aurantia LGL B would block a TLR response is unknown. Structural studies of Tm-Gp have focused o n a tetrasaccharide repeating unit, likened by Asai and colleagues to the repeating unit of the LPS O-antigen [11]. The c haracteristic laddering pattern on SDS/ PAGE suggests that Tm-Gp is different from LGL B , although t hey both contain an a spartic a cid residue. T he structures of the bioactive portion of Tm-Gp, and of t he other treponemal glycolipid TLR agonists, need to be elucidated to begin to identify possible motifs involved in modulating TLR activity. T his i s especially interesting w hen one realizes that the existing literature does not contain any direct demonstration of a ligand-type interaction between a TLR and any glycoconjugate, LPS or otherwise. LPS has been shown, however, to bind LBP [46]. Interestingly, a decrease in the fluidity of Re LPS, instigated by a Zn 2+ - induced increase in acyl chain order, elevated the production of TNF-a from human monocytes. The increase in acyl chain order increased the bond strength between Re LPS and LBP, and was thought to increase the transport o f the LPS to t he target membrane. LBP is an important precursor in the TLR-dependent release of TNF-a and h as been shown to interact with both the T. maltophilum and T. brennaborense glycolipids to enhance their ability to stimulate TLRs [6]. It is tempting to speculate that the highly disordered acyl chains of LGL B could abrogate the interaction with LBP and prevent any release of TNF-a in the whole blood assay for TLR activation. Specific struc- tural e ntities of LPS, producing certain biological effects, have been extensively studied given the central role of this molecule in pathogenesis and vaccine development. Char- acterization of any biological activity o f spirochete glyco- lipids is important for similar reasons, especially in the case of B. burgdorferi BbGL-II, given the difficulties in develop- ing an effective proteinaceous vaccine targeting this organ- ism [13,47]. 0 5 10 15 20 25 30 35 40 TNF-α (ng/mL) No Zymosan HKSA PolyIC Re LPS R848 CpG LGL B Fig. 7. Tumour necrosis factor-a (TNF-a) production through activation of Toll-like receptors (TLR) in the presence of different agonists. 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The structure and biological characteristics of the Spirochaeta aurantia outer membrane glycolipid LGL B Evgeny Vinogradov 1 , Catherine J C-6 of residue E and an amino group of the aspartic acid was identified on t he basis of the HMBC correlation b etween H-2 of aspartic acid and C-6 of the