Identification of a Novel Heptoglycan of α1→2-Linkedd-glycero-d-manno-Heptopyranose
1998; Elsevier BV; Volume: 273; Issue: 12 Linguagem: Inglês
10.1074/jbc.273.12.7006
ISSN1083-351X
AutoresMiriam M. Susskind, Lore Brade, Helmut Brade, Otto Holst,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoIn a preliminary investigation (Süsskind, M., Müller-Loennies, S., Nimmich, W., Brade, H., and Holst, O. (1995) Carbohydr. Res. 269, C1–C7), we identified after deacylation of lipopolysaccharides (LPS) from Klebsiella pneumoniae ssp. pneumoniaerough strain R20 (O1−:K20−) as a major fraction the oligosaccharide,where Kdo was 3-deoxy-d-manno-oct-2-ulopyranosonic acid and Hep p was manno-heptopyranose. The presence of the threo-hex-4-enuronopyranosyl residue indicated a substituent at O-4 of the second GalA residue linked to O-3 of the second l,d-Hep residue, which had been eliminated by treatment with hot alkali. We now report the complete structure of lipopolysaccharide, which was elucidated by additional characterization of isolated core oligosaccharides and analysis of the lipid A. The substituent at O-4 of the second Gal pA isd-GlcpN, which in a fraction of the LPS is substituted at O-6 by three or four residues ofd-glycero-d-manno-heptopyranose (d,d-Hepp). The complete carbohydrate backbone of the LPS is as follows,Graphical AbstractView Large Image Figure ViewerDownload (PPT)(l-glycero-d-manno-heptopyranose;l,d-Hepp), where all hexoses possess the d-configuration. Sugars marked with an asterisk are present in nonstoichiometric amounts. The structure is unique with regard to the presence of an α1→2-linkedd-glycero-d-manno-heptoglycan (oligosaccharide), which has not been described to date, and does not contain phosphate substituents in the core region. Fatty acid analysis of lipid A identified (R)-3-hydroxytetradecanoic acid as sole amide-linked fatty acid and (R)-3-hydroxytetradecanoic acid, tetradecanoic acid, small amounts of 2-hydroxytetradecanoic acid, hexadecanoic acid, and traces of dodecanoic acid as ester-linked fatty acids, substituting the carbohydrate backboned-GlcpN4Pβ1→6d-GlcpNα1P. The nonreducing GlcN carries four fatty acids, present as two 3-O-tetradecanoyltetradecanoic acid residues, one of which is amide-linked and the other ester-linked to O-3′. The reducing GlcN is substituted in a nature fraction of lipid A by two residues of (R)-3-hydroxytetradecanoic acid, one in amide and the other in ester linkage at O-3. Two minor fractions of lipid A were identified; in one, the amide-linked (R)-3-hydroxytetradecanoic acid at the reducing GlcN is esterified with hexadecanoic acid, resulting in 3-O-hexadecanoyltetradecanoic acid, and in the second, one of the 3-O-tetradecanoyltetradecanoic acid residues at the nonreducing GlcN is replaced by 3-O-dodecanoyltetradecanoic acid. Thus, the complete structure of LPS is as shown in Fig.1.After immunization of BALB/c mice, two monoclonal antibodies were obtained that were shown to be specific for the core of LPS from K. pneumoniae ssp. pneumoniae, since they did not react with LPS or whole-cell lysates of a variety of other Gram-negative species. Both monoclonal antibodies could be inhibited by LPS but not by isolated oligosaccharides and are thus considered to recognize a conformational epitope in the core region. In a preliminary investigation (Süsskind, M., Müller-Loennies, S., Nimmich, W., Brade, H., and Holst, O. (1995) Carbohydr. Res. 269, C1–C7), we identified after deacylation of lipopolysaccharides (LPS) from Klebsiella pneumoniae ssp. pneumoniaerough strain R20 (O1−:K20−) as a major fraction the oligosaccharide, where Kdo was 3-deoxy-d-manno-oct-2-ulopyranosonic acid and Hep p was manno-heptopyranose. The presence of the threo-hex-4-enuronopyranosyl residue indicated a substituent at O-4 of the second GalA residue linked to O-3 of the second l,d-Hep residue, which had been eliminated by treatment with hot alkali. We now report the complete structure of lipopolysaccharide, which was elucidated by additional characterization of isolated core oligosaccharides and analysis of the lipid A. The substituent at O-4 of the second Gal pA isd-GlcpN, which in a fraction of the LPS is substituted at O-6 by three or four residues ofd-glycero-d-manno-heptopyranose (d,d-Hepp). The complete carbohydrate backbone of the LPS is as follows, (l-glycero-d-manno-heptopyranose;l,d-Hepp), where all hexoses possess the d-configuration. Sugars marked with an asterisk are present in nonstoichiometric amounts. The structure is unique with regard to the presence of an α1→2-linkedd-glycero-d-manno-heptoglycan (oligosaccharide), which has not been described to date, and does not contain phosphate substituents in the core region. Fatty acid analysis of lipid A identified (R)-3-hydroxytetradecanoic acid as sole amide-linked fatty acid and (R)-3-hydroxytetradecanoic acid, tetradecanoic acid, small amounts of 2-hydroxytetradecanoic acid, hexadecanoic acid, and traces of dodecanoic acid as ester-linked fatty acids, substituting the carbohydrate backboned-GlcpN4Pβ1→6d-GlcpNα1P. The nonreducing GlcN carries four fatty acids, present as two 3-O-tetradecanoyltetradecanoic acid residues, one of which is amide-linked and the other ester-linked to O-3′. The reducing GlcN is substituted in a nature fraction of lipid A by two residues of (R)-3-hydroxytetradecanoic acid, one in amide and the other in ester linkage at O-3. Two minor fractions of lipid A were identified; in one, the amide-linked (R)-3-hydroxytetradecanoic acid at the reducing GlcN is esterified with hexadecanoic acid, resulting in 3-O-hexadecanoyltetradecanoic acid, and in the second, one of the 3-O-tetradecanoyltetradecanoic acid residues at the nonreducing GlcN is replaced by 3-O-dodecanoyltetradecanoic acid. Thus, the complete structure of LPS is as shown in Fig.1. After immunization of BALB/c mice, two monoclonal antibodies were obtained that were shown to be specific for the core of LPS from K. pneumoniae ssp. pneumoniae, since they did not react with LPS or whole-cell lysates of a variety of other Gram-negative species. Both monoclonal antibodies could be inhibited by LPS but not by isolated oligosaccharides and are thus considered to recognize a conformational epitope in the core region. Klebsiella pneumoniae is an important Gram-negative pathogenic bacterium associated with nosocomial infections (1Montgomerie J.Z. Rev. Infect. Dis. 1979; 1: 736-753Crossref PubMed Scopus (135) Google Scholar). It represents a major cause of mortality in hospital-acquired infections (2Williams P. Tomàs J.M. Rev. Med. Microbiol. 1990; 1: 196-204Google Scholar) and is (following Escherichia coli) the second most frequent microorganism isolated from patients with Gram-negative septicemia. Capsular polysaccharides and LPS 1The abbreviations used are: LPS, lipopolysaccharide; Kdo, 3-deoxy-d-manno-oct-2-ulopyranosonic acid;d,d-Hep,d-glycero-d-manno-heptose;l,d-Hep,l-glycero-d-manno-heptose; HPAE, high performance anion exchange chromatography; GLC, gas-liquid chromatography; PAGE, polyacrylamide gel electrophoresis; LD, laser desorption; FAB, fast atom bombardment; MS, mass spectrometry; COSY, correlated spectroscopy; NOE, nuclear Overhauser enhancement; NOESY, nuclear Overhauser enhancement spectroscopy; TOCSY, total correlation spectroscopy; HMQC, heteronuclear multiple quantum coherence; EIA, enzyme immunoassay. are important virulence factors of K. pneumoniae (1Montgomerie J.Z. Rev. Infect. Dis. 1979; 1: 736-753Crossref PubMed Scopus (135) Google Scholar, 2Williams P. Tomàs J.M. Rev. Med. Microbiol. 1990; 1: 196-204Google Scholar), and structures of the former (3Roberts I.S. Annu. Rev. Microbiol. 1996; 50: 285-315Crossref PubMed Scopus (527) Google Scholar) and of O-antigens (see Ref. 4Süsskind M. Müller-Loennies S. Nimmich W. Brade H. Holst O. Carbohydr. Res. 1995; 269: C1-C7Crossref PubMed Scopus (35) Google Scholar for references) from LPS have been investigated extensively. A structural investigation of the LPS core region began only recently (4Süsskind M. Müller-Loennies S. Nimmich W. Brade H. Holst O. Carbohydr. Res. 1995; 269: C1-C7Crossref PubMed Scopus (35) Google Scholar, 5Severn W.B. Kelly R.F. Richards J.C. Whitfield C. J. Bacteriol. 1996; 178: 1731-1741Crossref PubMed Google Scholar). In a preliminary investigation (4Süsskind M. Müller-Loennies S. Nimmich W. Brade H. Holst O. Carbohydr. Res. 1995; 269: C1-C7Crossref PubMed Scopus (35) Google Scholar) of LPS from the rough mutant K. pneumoniae ssp. pneumoniae R20 (O1−:K20−), we isolated from deacylated LPS the major fraction of the carbohydrate backbone and characterized its structure. It possessed a terminal threo-hex-4-enuronic acid residue, which resulted from β-elimination under the alkaline conditions used, indicating a further substitution at position O-4 of a second GalA residue. In another investigation (5Severn W.B. Kelly R.F. Richards J.C. Whitfield C. J. Bacteriol. 1996; 178: 1731-1741Crossref PubMed Google Scholar), the structure of the core region of LPS from serotype O8 was found to be similar to that of LPS of serotype O1. In this case, the eliminated substituent was α-d-Glcp linked at O-4 of the second GalA residue. Both core regions lack phosphate residues, which is so far unique in enterobacterial LPS. Several attempts have been made to develop anti-LPS antibodies as therapeutic agents in septic patients. All three regions of LPS, i.e. the O-specific polysaccharide, the core region, and the lipid A, can act as immunogen; however, the O-antigen expresses high structural variability, even within one species, and lipid A antigenicity is cryptic in LPS and exposed as a neoantigen only after removal of the lipid A-distal saccharide moiety (6Brade L. Engel R. Christ W.J. Brade H. Infect. Immun. 1997; 65: 3961-3965Crossref PubMed Google Scholar). The core region has been identified as a suitable target for the induction of antibodies with broad cross-reactivity among all E. colistrains. One antibody, named WN1 222–5 (7Di Padova F.E. Brade H. Barclay R. Poxton I.R. Liehl E. Schütze E. Kocher H.P. Ramsay G. Schreier M.H. McClelland D.B.L. Rietschel E.T. Infect. Immun. 1993; 61: 3863-3872Crossref PubMed Google Scholar, 8Di Padova F.E. Gram H. Barclay R. Poxton I.R. Liehl E. Rietschel E.T. Morisson D.C. Ryan J.L. Novel Therapeutic Strategies in the Treatment of Sepsis. Marcel Dekker Inc., New York1995: 13-31Google Scholar), recognizes an epitope present in all core types of E. coli, Salmonella enterica, and Shigella, which is primarily constituted by sugars of the inner core region. The outer core oligosaccharide and phosphate substituents seem to stabilize a given conformation also required for binding. Since our preliminary investigation of the Klebsiella core had indicated a similar structure of the heptose-Kdo region as in E. coli, except for the absence of phosphate groups, we investigated details of the chemical structure and its relation to immunoreactivity in Klebsiella. K. pneumoniae ssp. pneumoniae rough strain R20 (O1−:K20−) was obtained from W. Nimmich (Institute of Medical Microbiology, University of Rostock, Germany). Its cultivation and the isolation of LPS from dried bacteria have been reported earlier (4Süsskind M. Müller-Loennies S. Nimmich W. Brade H. Holst O. Carbohydr. Res. 1995; 269: C1-C7Crossref PubMed Scopus (35) Google Scholar). LPS from E. coli with core types R1, R2, R3, R4, and K-12 strain W3100, and S. enterica sv. Minnesota R60 were obtained from our LPS collection. Clinical isolates of Enterobacter, Serratia, Hafnia, Citrobacter, Proteus vulgaris, Morganella morganii, Neisseria sicca, S. enterica sv. Panama, Providencia alcaligenes, Pseudomonas aeruginosa, Shigella sonnei, Hemophilus influenzae, and Bacteroides fragilis were kindly provided by R. Podschun (Department of Medical Microbiology and Virology, University of Kiel, Germany). The LPS (208 mg) was hydrolyzed in 1% acetic acid (100 °C, 45 min), and the precipitate was removed by centrifugation (2,500 × g, 1 h) and lyophilized to give lipid A (94 mg, 45% of LPS). The supernatant was evaporated to dryness, dissolved in water, centrifuged (100,000 × g, 4 °C, 2 h) and separated by gel permeation chromatography on TSK HW40 (S). The main fraction (52 mg, 25% of LPS) was separated by high performance anion exchange chromatography (HPAE), from which, after desalting by gel permeation chromatography, oligosaccharides 1–5 could be isolated in pure form: heptasaccharide 1 (3.7 mg, 1.8% of the LPS), octasaccharide 2 (13.4 mg, 6.4% of the LPS), decasaccharide 3 (4 mg, 1.9% of the LPS), undecasaccharide 4 (4.5 mg, 2.2% of the LPS), and dodecasaccharide 5 (1.8 mg, 0.9% of the LPS). The LPS was dephosphorylated (48% HF, 4 °C, 48 h; yield was 76% of the LPS) and hydrolyzed (1% acetic acid, 100 °C, 90 min), and the dephosphorylated lipid A was extracted with CH2Cl2, dried, and subjected to laser desorption mass spectrometry (LD-MS). Compositional analysis, gel permeation chromatography on TSK HW40 (S), and the determination of the absolute configuration of Glc and GlcN were performed as described (9Müller-Loennies S. Holst O. Brade H. Eur. J. Biochem. 1994; 224: 751-760Crossref PubMed Scopus (48) Google Scholar). For the determination of the absolute configuration of GalA, LPS was methanolyzed (0.5 m methanolic HCl, 85 °C, 40 min) and centrifuged, and the supernatant was carboxyl-reduced (NaBH4, 4 °C, 24 h). The content of the resultingd-Gal was measured using d-galactose oxidase (Sigma) and a peroxidase assay (Boehringer Mannheim), according to the supplier's instructions. The absolute configuration of the hydroxy fatty acids was determined by GLC of the phenylethylamide derivatives (10Rietschel E.T. Eur. J. Biochem. 1976; 64: 423-428Crossref PubMed Scopus (111) Google Scholar). Analyses for ester- and amide-linked fatty acids and ester-linked acyloxyacyl groups were performed as described (11Wollenweber H.-W. Rietschel E.T. J. Microbiol. Methods. 1990; 11: 195-211Crossref Scopus (149) Google Scholar), as was SDS-PAGE (10 and 18% acrylamide) (12Vinogradov E.V. Pantophlet R. Dijkshoorn L. Brade L. Holst O. Brade H. Eur. J. Biochem. 1996; 239: 602-610Crossref PubMed Scopus (51) Google Scholar), gels of which were stained with silver nitrate for the detection of LPS (13Tsai C.M. Frasch C.E. Anal. Biochem. 1982; 119: 115-119Crossref PubMed Scopus (2315) Google Scholar) or with Coomassie Brilliant Blue for the detection of proteins. Analytical and semipreparative HPAE were performed as described (14Holst O. Bock K. Brade L. Brade H. Eur. J. Biochem. 1995; 229: 194-200Crossref PubMed Scopus (51) Google Scholar), with the modification that in semipreparative HPAE the CarboPac PA1 column was eluted at 3 ml min−1, using a gradient program of isocratically 1% B (0–10 min), then linearly to 2% B over 20 min, linearly to 3% B over 10 min, linearly to 5% B over 10 min, and linearly to 7% over 10 min (A, deionized water; B, 1 m sodium acetate, pH 6.0). Methylation of carboxyl-reduced oligosaccharide 1 (2 mg) was carried out according to Ciucanu and Kerek (15Ciucanu I. Kerek F. Carbohydr. Res. 1984; 131: 209-217Crossref Scopus (3205) Google Scholar). The methylated sample was hydrolyzed (2 mtrifluoroacetic acid, 100 °C, 2 h), reduced (NaB2H4), acetylated, and analyzed using GLC-MS. Methylation analysis of the Kdo region was performed on dephosphorylated LPS (6 mg) as published (16Tacken A. Rietschel E.T. Brade H. Carbohydr. Res. 1986; 149: 279-291Crossref PubMed Scopus (60) Google Scholar). GLC and GLC/MS were carried out as described (9Müller-Loennies S. Holst O. Brade H. Eur. J. Biochem. 1994; 224: 751-760Crossref PubMed Scopus (48) Google Scholar). The temperature program in GLC was as follows: 110 °C for 3 min and then 3 °C min−1 to 270 °C. LD-MS was performed on a laser microprobe mass analyzer 500 (Leibold-Heraeus, Cologne, Germany), equipped with a microprobe and a time-of-flight mass analyzer. The intensity of the laser beam was 1012 watts cm−1. Between 5 and 20 pg of the sample were analyzed per laser pulse. For fast atom bombardment (FAB)-MS, LPS preparations were dissolved in water at a final concentration of 10 mg ml−1. Positive and negative ion mode FAB-MS were carried out using the first two sectors of a Jeol JMS-SX/SX 102A tandem mass spectrometer (Department of Mass Spectrometry, Utrecht University, The Netherlands) operating at 10 kV (mass range, m/z 20–2400) accelerating voltage. The FAB gun was operated at an emission current of 10 mA with xenon as the bombarding gas. Spectra were scanned at a speed of 30 s for the full mass range specified by the accelerating voltage used and were recorded and processed on a Hewlett-Packard HP 9000 data system. In all experiments, the matrix used was thioglycerol (2–3 μl), and the probe was loaded with 1 μl of sample solution. For structural assignments, NMR spectra were recorded on solutions (0.5 ml) of oligosaccharides 1, 3, and 4 (2–3 mg each), 2 (13 mg), and 5 (1.5 mg) in2H2O with a Bruker AMX 600 spectrometer (1H NMR, 600.13 MHz, 13C NMR 125.77 MHz) at 27 °C. The resonances were measured relative to internal acetone ((CH3)2CO δH 2.225; δC 31.07). One-dimensional 1H,13C, and 31P NMR spectra were recorded with a Bruker AM 360 L spectrometer (1H, 360 MHz; 13C, 90.6 MHz; 31P, 145.8 MHz) at 23 °C (CH3CN, δH 1.95 ppm; δC 1.70 ppm) to confirm purity. Coupling constants (±0.5 Hz) were determined on a first order basis. The phase-sensitive COSY experiments were performed using double quantum filtering (17Rance M. Sørensen O.W. Bodenhausen G. Wagner G. Ernst R.R. Wüthrich K. Biochem. Biophys. Res. Commun. 1983; 117: 479-485Crossref PubMed Scopus (2597) Google Scholar, 18Piantini U. Sørensen O.W. Ernst R.R. J. Am. Chem. Soc. 1982; 104: 6800-6801Crossref Scopus (1887) Google Scholar) with the Bruker COSYPHDQ microprogram, using 1,024 t1 increments and a sweep width of 2.5 KHz and collecting 4,096 data points in the F2dimension. The data matrix was zero-filled in the F1 dimension to give a matrix of 4 × 2,048 points and was resolution-enhanced in both dimensions by a shifted sine-bell function before Fourier transformation. The total correlation spectroscopy (TOCSY) (19Braunschweiler L. Ernst R.R. J. Magn. Reson. 1983; 53: 521-528Crossref Scopus (3108) Google Scholar) and the nuclear Overhauser enhancement spectroscopy (NOESY) (20Jeener J. Meier B.H. Bachmann P. Ernst R.R. J. Chem. Phys. 1979; 71: 4546-4553Crossref Scopus (4838) Google Scholar) experiments were performed according to the method of States et al. (21States D.J. Haberkorn R.A. Ruben D.J. J. Magn. Reson. 1982; 48: 286-292Google Scholar) in the phase-sensitive mode. A decoupling in the presence of scalar interactions (DIPSI-2) (22Rucker S.P. Shaka A.J. Mol. Phys. 1989; 68: 509-517Crossref Scopus (266) Google Scholar) spin lock pulse sequence was used in the TOCSY experiment with a mixing time of 125 ms and a spin lock power of 7,300 Hz. The NOESY experiments were performed with a mixing time of 200 ms. The intensities of NOESY cross-peaks were classified as strong, medium, or weak, using cross-peaks from intraring proton-proton contacts for calibration. The13C,1H COSY spectra were measured in the1H detected mode via multiple quantum coherence (HMQC) (23Bax A. Morris G. J. Magn. Reson. 1981; 42: 501-505Google Scholar), using a GARP sequence (24Shaka A.J. Barker P.B. Freeman R. J. Magn. Reson. 1985; 67: 547-552Google Scholar) to decouple 13C couplings during acquisition. The experiments were carried out in the phase-sensitive mode by the States time-proportional phase incrementation phase-cycling method (25Marion D. Ikura M. Tschudin R. Bax A. J. Magn. Reson. 1989; 85: 393-399Google Scholar) acquiring a total of 4,096 points over a sweep width of 4,545 Hz in F2 and 1,024 in F1 over 13,500 Hz. Processing was performed using standard Bruker software after zero-filling to 2,048 in F1. The COSY, TOCSY, NOESY, and HMQC spectra were assigned using the computer program PRONTO (26Kjaer M. Andersen K.V. Poulsen F.M. Methods Enzymol. 1994; 289: 288-307Crossref Scopus (164) Google Scholar), which allows the simultaneous display of different two-dimensional spectra and the individual labeling of cross-peaks. Monoclonal antibodies were prepared by conventional protocols after immunization of BALB/c mice with heat-killed bacteria (K. pneumoniae ssp. pneumoniae rough strain R20) by intravenous injection on days 0, 7, 14, and 21 with 20, 20, 60, and 120 μg, respectively. On days 90, 91, and 92, mice received a booster injection of 200 μg each; the first injection was intravenous, and the last two were intraperitoneal. Fusion was performed on day 94. Cell culture, media, and the fusion protocol were described previously (27Fu Y. Baumann M. Kosma P. Brade L. Brade H. Infect. Immun. 1992; 60: 1314-1321Crossref PubMed Google Scholar). Primary hybridomas were screened by Western blot with bacterial whole-cell lysates as antigen, and relevant hybridomas were cloned at least three times by limiting dilution, isotyped using an isotyper kit (Bio-Rad), and adapted to serum-free medium supplement with Ultroser (Life Technologies, Inc.). Culture supernatants were prepared in at least 100-ml quantities, and antibodies were purified on protein G-Sepharose (Pharmacia Biotech Inc.) according to the supplier's instructions. Purification was ascertained by SDS-PAGE, and protein concentrations were determined by using the bicinchoninic acid assay (Pierce). mAb WN1 222–5 (IgG2a) binding to the core region of all E. coli strains has been described earlier (7Di Padova F.E. Brade H. Barclay R. Poxton I.R. Liehl E. Schütze E. Kocher H.P. Ramsay G. Schreier M.H. McClelland D.B.L. Rietschel E.T. Infect. Immun. 1993; 61: 3863-3872Crossref PubMed Google Scholar, 8Di Padova F.E. Gram H. Barclay R. Poxton I.R. Liehl E. Rietschel E.T. Morisson D.C. Ryan J.L. Novel Therapeutic Strategies in the Treatment of Sepsis. Marcel Dekker Inc., New York1995: 13-31Google Scholar). All serological methods such as enzyme immunoassay (EIA), using LPS as solid phase antigen, EIA inhibition, hemagglutination and inhibition of hemagglutination, and the Western blot technique were described previously (27Fu Y. Baumann M. Kosma P. Brade L. Brade H. Infect. Immun. 1992; 60: 1314-1321Crossref PubMed Google Scholar, 28Rozalski A. Brade L. Kosma P. Moxon R. Kusumoto S. Brade H. Mol. Microbiol. 1997; 23: 569-577Crossref PubMed Scopus (14) Google Scholar). Compositional analysis of the LPS (structure shown in Fig.1) revealed the presence ofd-Glc; d-GlcN; d-GalA;l,d-Hep; d,d-Hep; Kdo; (R)-3-hydroxytetradecanoic acid; tetradecanoic acid; small amounts of 2-hydroxytetradecanoic acid, hexadecanoic acid, and dodecanoic acid; and phosphate. In an earlier report (4Süsskind M. Müller-Loennies S. Nimmich W. Brade H. Holst O. Carbohydr. Res. 1995; 269: C1-C7Crossref PubMed Scopus (35) Google Scholar) on the structure of the core region from LPS of K. pneumoniae ssp. pneumoniae strain R20, we presented as a major product obtained from deacylated LPS one oligosaccharide representing the carbohydrate backbone of lipid A and part of the core region (Structure 1). This oligosaccharide possessed a terminal β-threo-hex-4-enuronopyranosyl residue, indicating the loss of at least one substituent at O-4 of a GalA residue by β-elimination due to treatment with hot KOH. To elucidate the complete structure of the core region, we treated the LPS with 1% acetic acid to cleave the ketosidic Kdo-lipid A linkage and isolated the resulting core oligosaccharides. In a first step, two fractions were obtained from gel permeation chromatography of the hydrolysate, the second of which contained mainly Kdo and was thus not further investigated. The major fraction was separated by HPAE at pH 6.0 (Fig.2), yielding oligosaccharides 1–5 (Fig.3). Monosaccharide analysis of the oligosaccharides revealed that they all contained d-Glc,l,d-Hep, Kdo, and d-GlcN. In oligosaccharides 3–5, d,d-Hep was additionally identified.Figure 3Structures of oligosaccharides M3, M4, and 1–5. R 1, β-GalA J in M3, M4, 2, 4, and 5;R 2, d,d-Hep residues L, M, N, and O (beginning from the reducing terminus) in 3–5. Except where indicated, hexoses are d-configured.View Large Image Figure ViewerDownload (PPT) The structures of the isolated oligosaccharides were characterized by NMR spectroscopy and are presented in Fig. 3. Chemical shifts of1H and 13C NMR spectra were assigned using COSY, TOCSY, NOESY, and HMQC experiments and the computer program PRONTO (26Kjaer M. Andersen K.V. Poulsen F.M. Methods Enzymol. 1994; 289: 288-307Crossref Scopus (164) Google Scholar). The data are given in Tables Table I, Table II, Table III. Nine signals were identified in the anomeric region of the 1H NMR spectrum of decasaccharide 3 (Figs. 4 and5, TableI), six of which (residues E, F, G, L, M, N, O; compare Fig. 1) were assigned to manno-configured heptose residues as established by small JH-1,H-2 coupling constants (≤2 Hz, except in the case of residue E, which possessed JH-1,H-22.5 Hz) and by the coupling constants of the other ring protons. Three other anomeric signals were attributed to two α- (residues H and K) and one β-linked (I) hexoses possessing an axial H-2, as characterized by the chemical shifts and JH-1, H-2 coupling constants (5.322 ppm (3.8 Hz), residue H; 5.127 ppm (4.0 Hz), residue K; 4.551 ppm (7.8 Hz), residue I). Characteristic high field signals of deoxy protons at 1.810 ppm (H-3ax) and 2.119 ppm (H-3eq) identified α-linked Kdo as the 10th residue. Residue K was established as α-linked GlcN by characteristic high field shifts of H-2 (3.233 ppm) and C-2 (54.5 ppm). The 13C NMR spectrum was assigned by an HMQC experiment (Fig. 6, TableII). It contained eight signals in the anomeric region, one of which (at 100.7 ppm) consisted of three nonresolved resonances. Low field shifted signals indicated the substitution at O-2 of residues L and M (C-2 at 78.8 ppm, nonresolved), at O-4 of H (C-4 at 79.6 ppm), at O-5 of C (C-5 at 74.6 ppm), at O-6 of K (C-6 at 65.2 ppm), at O-3 (C-3 at 75.8 ppm) and O-4 (C-4 at 74.1 ppm) of E, and at O-3 (C-3 at 79.6 ppm) and O-7 (C-7 at 69.9 ppm) of F. Residues G, I, and N are terminal residues.Figure 5TOCSY spectrum (anomeric region) of oligosaccharide 3. The spectrum was recorded at 600 MHz and 27 °C. The cross-peaks are labeled as explained in the legend to Fig. 4.View Large Image Figure ViewerDownload (PPT)Table I1H NMR chemical shift data for oligosaccharides derived from K. pneumoniae R20 lipopolysaccharideSugar residueAtomδ in oligosaccharide12345ppmC (α-Kdo)H-3ax1.8101.8101.802H-3eq2.1262.1192.114H-44.064.0364.0684.0554.054H-54.1124.1134.1124.1064.126H-63.7983.7973.7993.7923.800H-73.7213.7293.7193.7263.751H-8a3.5533.5473.5543.543.554H-8b3.8273.8303.8253.8223.827E (l,d-Hep)H-15.0785.0815.0745.0735.081H-24.0224.0274.0264.0234.028H-34.1804.1464.1684.1434.186H-44.1964.2094.1814.2054.186H-54.1284.1464.1264.1394.132H-64.0734.0544.0584.0424.081H-7a3.6743.6713.6733.6633.665H-7b3.7053.7083.7053.7023.705I (β-Glc)H-14.5634.5604.5514.5504.593H-23.2613.2653.2633.2593.268H-33.4923.4823.4773.4713.501H-43.3343.4473.3323.4343.471H-53.3793.5353.3853.5283.532H-6a3.7033.9003.7103.8893.856H-6b3.8684.1833.8644.1764.178J (β-GalA)H-14.4824.4734.456H-23.5383.5313.549H-33.7023.6943.688H-44.1474.151-aNonresolved.4.157H-54.0564.0454.054F (l,d-Hep)H-15.2125.1145.2055.1055.125H-24.1264.1164.1324.1144.066H-33.9733.9103.9723.9023.949H-43.9713.9843.9703.9763.997H-53.6683.7123.6723.7073.735H-64.1094.1044.1044.0984.107H-7a3.6723.681-aNonresolved.3.6673.7743.685H-7b3.7103.681-aNonresolved.3.7133.6843.685G (l,d-Hep)H-14.9044.8884.8874.8804.889H-23.9703.9723.9653.9673.977H-33.8373.8353.8353.8303.840H-43.8123.8083.8143.7973.816H-53.5813.5803.5813.5753.584H-63.9953.9953.9953.9883.998H-7a3.6283.6303.6233.6253.637H-7b3.6973.6983.6973.6943.703H (α-GalA)H-15.3035.2725.3225.2625.300H-23.8593.8953.8643.8733.840H-34.0594.1034.0184.1004.071H-44.3574.3694.3614.3424.327H-54.4184.4584.4664.4634.407K (α-GlcN)H-14.9405.1645.1275.1434.917H-22.6703.2673.2333.2652.666H-33.5723.9023.8273.8793.557H-43.3833.4913.5643.5523.446H-54.0104.1314.2834.2774.182H-6a3.761-aNonresolved.3.7603.6053.6053.971H-6b3.761-aNonresolved.3.7944.0084.0023.597L (d,d-Hep)H-15.1085.0985.103H-23.9393.9323.963H-33.8733.8653.911H-43.7453.7343.732H-53.6703.6623.670H-63.9923.9883.995H-7a3.6973.6653.674H-7b3.7793.6793.775ppmM (d,d-Hep)H-15.2695.2625.268H-24.0194.0104.011H-33.8603.8523.864H-43.7443.7373.744H-53.7443.7373.750H-63.9993.9904.003H-7a3.6903.6823.796H-7b3.7991-aNonresolved.3.7893.695N (d,d-Hep)H-14.9714.9645.272H-24.0053.9974.015H-33.7543.7433.854H-43.7343.7143.741H-53.7453.7363.744H-63.9893.9884.003H-7a3.6903.8053.796H-7b3.7991-aNonresolved.3.6823.695O (d,d-Hep)H-14.972H-24.008H-33.757H-43.736H-53.744H-64.001-aNonresolved.H-7a3.801-aNonresolved.H-7b3.701-aNonresolved.1-a Nonresolved. Open table in a new tab Figure 6Part of the 1H,13C HMQC spectrum of oligosaccharide 3. The letters refer to the carbohydrate residues as shown in Fig. 1, and the Arabic numerals refer to the proton/carbon in the respective residue.View Large Image Figure ViewerDownload (PPT)Table II13C NMR chemical shift data for oligosaccharides derived from K. pneumoniae R20 lipopolysaccharideSugar residueAtomδ in oligosaccharide12345ppmC (α-Kdo)C-1175.5a175.4b175.6c175.4d175.4eC-2100.5100.5100.4100.7100.7C-3ND2-fND, not determined.34.634.534.5NDC-466.266.466.266.366.3C-574.674.674.674.474.4C-671.671.771.671.571.7C-769.569.569.569.369.5C-863.963.963.963.863.9E (l,d-Hep)C-1100.9100.7100.7100.6100.5C-270.570.570.370.570.4C-376.177.475.877.477.5C-474.373.674.174.1C-571.672.071.472.072.2C-669.169.269.069.269.3C-763.463.363.263.363.3I (β-Glc)C-1102.8102.5102.6102.4102.8C-274.374.374.174.074.3C-375.775.875.775.775.8C-470.170.069.969.869.8
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