Core Oligosaccharides of Plesiomonas shigelloidesO54:H2 (Strain CNCTC 113/92)
2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês
10.1074/jbc.m111885200
ISSN1083-351X
AutoresTomasz Niedziela, Jolanta Łukasiewicz, Wojciech Jachymek, Monika Dzieciątkowska, Czesław Ługowski, Lennart Kenne,
Tópico(s)Aquaculture disease management and microbiota
ResumoPlesiomonas shigelloides is a Gram-negative, flagellated, rod-shaped bacterium. This ubiquitous and facultatively anaerobic organism has been isolated from such sources as freshwater, surface water, and many wild and domestic animals. The infections correlate strongly with the surface water contamination and are particularly common in tropical and subtropical habitats (1.Farmer III, J.J. Arduino M.J. Hickman-Brenner F.W. Balows A. Trüper H.G. Dworkin M. Wim H. Schleifer K.H. The Prokaryotes. 3. Springer-Verlag, New York1992: 3012-3043Google Scholar). Human infections with P. shigelloides are mostly related to drinking untreated water, eating uncooked shellfish (2.Van Houten R. Farberman D. Norton J. Ellison J. Kiehlbauch J. Morris T. Smith P. Morb. Mortal. Wkly. Rep. 1998; 47: 394-396PubMed Google Scholar, 3.Levy D.A. Bens M.S. Craun G.F. Calderon R.L. Herwaldt B.L. Morb. Mortal. Wkly. Rep. 1998; 47: 1-34PubMed Google Scholar), and visiting countries with low sanitary standards (4.Yamada S. Matsushita S. Dejsirilert S. Kudoh Y. Epidemiol. Infect. 1997; 119: 121-126Crossref PubMed Scopus (57) Google Scholar, 5.Rautelin H. Sivonen A. Kuikka A. Renkonen O.V. Valtonen V. Kosunen T.U. Scand. J. Infect. Dis. 1995; 27: 495-498Crossref PubMed Scopus (29) Google Scholar). Recent studies implicated P. shigelloides as an opportunistic pathogen in immunocompromised hosts (6.Lee A.C.W. Yuen K.Y. Ha S.Y. Chiu D.C.K. Lau Y.L. Pediatr. Hematol. Oncol. 1996; 13: 265-269Crossref PubMed Scopus (33) Google Scholar) and especially neonates (6.Lee A.C.W. Yuen K.Y. Ha S.Y. Chiu D.C.K. Lau Y.L. Pediatr. Hematol. Oncol. 1996; 13: 265-269Crossref PubMed Scopus (33) Google Scholar, 7.Riley P.A. Parasakthi N. Abdullah W.A. Clin. Infect. Dis. 1996; 23: 206-207Crossref PubMed Scopus (17) Google Scholar, 8.Clark R.B. Westby G.R. Spector H. Soricelli R.R. Young C.L. J. Infect. 1991; 23: 89-92Abstract Full Text PDF PubMed Scopus (25) Google Scholar, 9.Delforge M.L. Devriendt J. Glupczynski Y. Hansen W. Douat N. Clin. Infect. Dis. 1995; 21: 692-693Crossref PubMed Scopus (22) Google Scholar, 10.Fujita K. Shirai M. Ishioka T. Kakuya F. Acta Paediatr. Jpn. 1994; 36: 450-452Crossref PubMed Scopus (26) Google Scholar). However, it has also been associated with diarrheal illness (11.Bravo L. Monte R. Ramirez M. Garcia B. Urbaskova P. Aldova E. Cent. Eur. J. Public Health. 1998; 6: 67-70PubMed Google Scholar) and other diseases in normal hosts. P. shigelloides has been isolated from an assortment of clinical specimens, including cerebrospinal fluid, wounds, and respiratory tract. It causes gastrointestinal and localized infections originating from infected wounds, which can disseminate to other parts of the body (12.Korner R.J. Macgowan A.P. Warner B. Zentralbl. Bakteriol. 1992; 277: 334-339Crossref PubMed Scopus (13) Google Scholar). The cases of meningitis and bacteremia (10.Fujita K. Shirai M. Ishioka T. Kakuya F. Acta Paediatr. Jpn. 1994; 36: 450-452Crossref PubMed Scopus (26) Google Scholar) caused by P. shigelloides are of special interest due to their seriousness. P. shigelloides has been traditionally classified as a member of the Vibrionaceae family based on phenotypic characteristics such as polar flagella, oxidase production, and fermentation properties (1.Farmer III, J.J. Arduino M.J. Hickman-Brenner F.W. Balows A. Trüper H.G. Dworkin M. Wim H. Schleifer K.H. The Prokaryotes. 3. Springer-Verlag, New York1992: 3012-3043Google Scholar). However, phylogenetic analysis and assessment of the genus Plesiomonas deducted from small rRNA sequences indicate a closer relationship with members of Enterobacteriaceae (13.Ruimy R. Breittmayer V. Elbaze P. Lafay B. Boussemart O. Gauthier M. Christen R. Int. J. Syst. Bacteriol. 1994; 44: 416-426Crossref PubMed Scopus (248) Google Scholar). The serotyping scheme of P. shigelloides was proposed by Aldova, Shimada, and Sakazaki (14.Aldova E. Zentralbl. Bakteriol. 1987; 265: 253-262Google Scholar, 15.Aldova E. Syst. Appl. Microbiol. 1992; 15: 70-75Crossref Scopus (12) Google Scholar, 16.Aldova E. Benackova E. Shimada T. Danesova D. Syst. Appl. Microbiol. 1992; 15: 247-249Crossref Scopus (11) Google Scholar, 17.Shimada T. Arakawa E. Itoh K. Kosako Y. Inoue K. Zhengshi Y. Aldova E. Curr. Microbiol. 1994; 28: 351-354Crossref Scopus (15) Google Scholar, 18.Shimada T. Sakazaki R. Jpn. J. Med. Sci. Biol. 1978; 31: 135-142Crossref PubMed Scopus (39) Google Scholar, 19.Aldova E. Shimada T. Folia Microbiol. 2000; 45: 301-304Crossref PubMed Scopus (26) Google Scholar). Some O-antigens have shown cross-reactivity with antisera directed against lipopolysaccharides (LPS) 1The abbreviations used are: LPSlipopolysaccharideMALDI-TOFmatrix-assisted laser-desorption/ionization time-of-flightMSmass spectrometryPBSphosphate buffered salineGCgas chromatographyCOSYcorrelated spectroscopyTOCSYtotal correlation spectroscopyNOESYnuclear Overhauser effect spectroscopyROESYrotating frame nuclear Overhauser effect spectroscopyHMBCheteronuclear multiple bond correlationHMQCheteronuclear multiple quantum coherenceHSQCheteronuclear single quantum coherenceDEPTdistortionless enhancement by polarization transferld-Hepl-glycero-d-manno-heptoseKdo3-deoxy-d-manno-oct-2-ulosonic acidBSAbovine serum albuminOSoligosaccharideELISAenzyme-linked immunosorbent assay of Shigella sonnei, Shigella dysenteriae 1, 7 and 8, Shigella boydi 2, 9, and 13 and Shigella flexneri6 (15.Aldova E. Syst. Appl. Microbiol. 1992; 15: 70-75Crossref Scopus (12) Google Scholar, 20.Albert M.J. Ansaruzzaman M. Qadri F. Hossain A. Kibriya A. Haider K. Nahar S. Faruque S.M. Alam A.N. J. Med. Microbiol. 1993; 39: 211-217Crossref PubMed Scopus (14) Google Scholar). Two P. shigelloides strains were found to share the structure with O-antigens of S. flexneri and S. dysenteriae (20.Albert M.J. Ansaruzzaman M. Qadri F. Hossain A. Kibriya A. Haider K. Nahar S. Faruque S.M. Alam A.N. J. Med. Microbiol. 1993; 39: 211-217Crossref PubMed Scopus (14) Google Scholar, 21.Linnerborg M. Widmalm G. Weintraub A. Albert M.J. Eur. J. Biochem. 1995; 231: 839-844Crossref PubMed Scopus (21) Google Scholar). The unique structures of the O-specific polysaccharides and core oligosaccharides remain unknown, except those of O-specific polysaccharides from strains 22074, 12254 (21.Linnerborg M. Widmalm G. Weintraub A. Albert M.J. Eur. J. Biochem. 1995; 231: 839-844Crossref PubMed Scopus (21) Google Scholar), and CNCTC 113/92 (22.Czaja J. Jachymek W. Niedziela T. Lugowski C. Aldova E. Kenne L. Eur. J. Biochem. 2000; 267: 1672-1679Crossref PubMed Scopus (49) Google Scholar). The O-specific polysaccharide of strain CNCTC 113/92 LPS (serotype O54) is composed of a hexasaccharide repeating unit with the following structure: The core oligosaccharide is important for biological and physical properties of the overall lipopolysaccharide and plays a significant role in interactions with the host. Thus we now report on structural and immunochemical studies of the core oligosaccharides isolated from P. shigelloides strain CNCTC 113/92 LPS. lipopolysaccharide matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry phosphate buffered saline gas chromatography correlated spectroscopy total correlation spectroscopy nuclear Overhauser effect spectroscopy rotating frame nuclear Overhauser effect spectroscopy heteronuclear multiple bond correlation heteronuclear multiple quantum coherence heteronuclear single quantum coherence distortionless enhancement by polarization transfer l-glycero-d-manno-heptose 3-deoxy-d-manno-oct-2-ulosonic acid bovine serum albumin oligosaccharide enzyme-linked immunosorbent assay Plesiomonas shigelloides strain CNCTC 113/92, classified as serovar O54:H2 according to Aldova's antigenic scheme (14.Aldova E. Zentralbl. Bakteriol. 1987; 265: 253-262Google Scholar, 15.Aldova E. Syst. Appl. Microbiol. 1992; 15: 70-75Crossref Scopus (12) Google Scholar, 16.Aldova E. Benackova E. Shimada T. Danesova D. Syst. Appl. Microbiol. 1992; 15: 247-249Crossref Scopus (11) Google Scholar, 17.Shimada T. Arakawa E. Itoh K. Kosako Y. Inoue K. Zhengshi Y. Aldova E. Curr. Microbiol. 1994; 28: 351-354Crossref Scopus (15) Google Scholar, 19.Aldova E. Shimada T. Folia Microbiol. 2000; 45: 301-304Crossref PubMed Scopus (26) Google Scholar) and 68 different P. shigelloides O-serotypes (O1, O2, O4–O6, O9, O11–O13, O15, O17, O19, O21, O22, O24–O28, O33–O46, O48, O50, O51, O56, O58, O59, O62, O64–O68, O70–O72, O74–O77, O81–O86, O91–O98), i.e. a group representative for all currently known serotypes, were obtained from the Institute of Hygiene and Epidemiology, Prague, Czech Republic. The bacteria were grown and harvested as described previously (22.Czaja J. Jachymek W. Niedziela T. Lugowski C. Aldova E. Kenne L. Eur. J. Biochem. 2000; 267: 1672-1679Crossref PubMed Scopus (49) Google Scholar,23.Petersson C. Niedziela T. Jachymek W. Kenne L. Zarzecki P. Lugowski C. Eur. J. Biochem. 1997; 244: 580-586Crossref PubMed Scopus (50) Google Scholar). LPS was extracted from bacterial cells by the hot phenol/water method (24.Westphal O. Jann K. Methods Carbohydr. Chem. 1965; 5: 83-89Google Scholar) and purified as reported earlier (23.Petersson C. Niedziela T. Jachymek W. Kenne L. Zarzecki P. Lugowski C. Eur. J. Biochem. 1997; 244: 580-586Crossref PubMed Scopus (50) Google Scholar). The yield of LPS was 2% of the dry bacterial mass. LPS (200 mg) was degraded by treatment with 1.5% acetic acid containing 2% SDS at 100 °C for 15 min. The reaction mixture was freeze-dried, the SDS removed by extraction with 96% ethanol, and the residue suspended in water and centrifuged. The supernatant was fractionated on Bio-Gel P-10, where O-specific polysaccharide separated from shorter chains (OSIII, 33 mg) and core oligosaccharides (OSIV, 12 mg). The core oligosaccharides were further fractionated by chromatography on Bio-Gel P-2 yielding two oligosaccharides: OSIVA (8.9 mg) and OSIVB (0.9 mg). The gel permeation chromatography was performed on columns (1.6 × 100 cm) of Bio-Gel P-10 and Bio-Gel P-2, equilibrated with 0.05m pyridine/acetic acid buffer, pH 5.6. Eluates were monitored with a Knauer differential refractometer and all fractions were checked by 1H NMR spectroscopy and matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and freeze-dried. For a rapid screening of LPS from different P. shigelloides serotypes proteinase K-digested whole cell lysates were obtained by the method described earlier (25.Hitchcock P.J. Brown T.M. J. Bacteriol. 1983; 154: 269-277Crossref PubMed Google Scholar) with the following modifications. Bacteria were grown on solid medium, harvested, and suspended in PBS to a turbidity giving A600 nm = 0.6. A portion (1.5 ml) of the suspension was centrifuged, and the pellet was resuspended in 200 μl of lysing buffer (0.05 m Tris-HCl, pH 6.8, containing 4% SDS and 4% glycerol) and heated for 10 min at 100 °C. Proteinase K (EC 3.4.21.64, Sigma-Aldrich) (∼200 μg) in the lysing buffer (80 μl) was added, followed by overnight incubation at 21 °C. The digested bacterial lysate was boiled for 20 min prior to electrophoresis. The LPS was analyzed by SDS-PAGE according to the method of Laemmli (26.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) with modifications as described previously (27.Niedziela T. Petersson C. Helander A. Jachymek W. Kenne L. Lugowski C. Eur. J. Biochem. 1996; 237: 635-641Crossref PubMed Scopus (29) Google Scholar). The LPS bands were visualized by the silver staining method (28.Tsai C.M. Frasch C.E. Anal. Biochem. 1982; 119: 115-119Crossref PubMed Scopus (2317) Google Scholar). Sugars were analyzed as their alditol acetates by GC-MS (23.Petersson C. Niedziela T. Jachymek W. Kenne L. Zarzecki P. Lugowski C. Eur. J. Biochem. 1997; 244: 580-586Crossref PubMed Scopus (50) Google Scholar, 29.Sawardeker J.S. Sloneker J.H. Jeanes A.R. Anal. Chem. 1965; 37: 1602-1604Crossref Scopus (1484) Google Scholar). The absolute configurations of the sugars were determined as described by Gerwig et al. (30.Gerwig G.J. Kamerling J.P. Vliegenthart J.F.G. Carbohydr. Res. 1978; 62: 349-357Crossref Scopus (800) Google Scholar, 31.Gerwig G.J. Kamerling J.P. Vliegenthart J.F. Carbohydr. Res. 1979; 77: 10-17Crossref PubMed Scopus (549) Google Scholar) using (−)-2-butanol for the formation of 2-butyl glycosides. The trimethylsilylated butyl glycosides were then identified by comparison with authentic samples (produced from re-spective sugar and (−)-2-butanol) on GC-MS. Carboxyl reduction of the native oligosaccharide was carried out according to the method of Taylor et al. (32.Taylor R.L. Shively J.E. Conrad H.E. Methods. Carbohydr. Chem. 1976; 7: 149-151Google Scholar) as described previously (23.Petersson C. Niedziela T. Jachymek W. Kenne L. Zarzecki P. Lugowski C. Eur. J. Biochem. 1997; 244: 580-586Crossref PubMed Scopus (50) Google Scholar). Methylations were performed both on N-acetylated and carboxyl-reduced oligosaccharides and only N-acetylated oligosaccharides according to the method of Hakomori (33.Hakomori S. J. Biochem. (Tokyo). 1964; 55: 205-208PubMed Google Scholar). The methyl ester groups of the latter methylated oligosaccharides were reduced with Superdeuteride (LiB(C2H5)32H) as described by Bhat et al. (34.Bhat U.R. Krishnaiah B.S. Carlson R.W. Carbohydr. Res. 1991; 220: 219-227Crossref PubMed Scopus (45) Google Scholar). The methylated sugars were analyzed as partially methylated alditol acetates by GC-MS as previously described (23.Petersson C. Niedziela T. Jachymek W. Kenne L. Zarzecki P. Lugowski C. Eur. J. Biochem. 1997; 244: 580-586Crossref PubMed Scopus (50) Google Scholar). GC-MS was carried out with a Hewlett-Packard 5971A system using an HP-1 fused-silica capillary column (0.2 mm × 12 m) and a temperature program 150 → 270 °C at 8 °C min−1. Amino acid analysis was carried out as described (35.MacKenzie S.L. Tenaschuk D. J. Chromatogr. 1974; 97: 19-24Crossref PubMed Scopus (101) Google Scholar, 36.MacKenzie S.L. Hogge L.R. J. Chromatogr. 1977; 132: 485-493Crossref PubMed Scopus (31) Google Scholar). The core oligosaccharide (1 mg) was hydrolyzed with 6m hydrochloric acid at 100 °C for 24 h and concentrated to dryness. Subsequently, n-butanol (0.5 ml) and acetyl chloride (50 μl) were added, and the reaction was carried out at 120 °C for 20 min, followed by the evaporation to dryness. Heptafluorobutyric anhydride (100 μl) was added, and the mixture was heated for 5 min at 150 °C. The N-heptafluorobutyryl n-butyl ester derivative of amino acid was analyzed by GC-MS on the same system as described above, but a temperature program 100 → 270 °C at 5 °C min−1. Oligosaccharide OSIII (5 mg) was dissolved in saturated NaHCO3 (2 ml) at 0 °C and treated with acetic anhydride (3 × 100 μl, with 10-min intervals). Reaction mixture was stored for additional 30 min at 0 °C, the product purified on a column (1.6 × 100 cm) of Bio-Gel P-2 and the N-acetylated OSIII oligosaccharide examined by NMR spectroscopy and MALDI-TOF MS. MALDI MS of the investigated oligosaccharides, in positive or negative mode, was run on a Bruker Reflex III time-of-flight instrument. Conjugates of core oligosaccharides with BSA were analyzed using a Kratos Kompact-SEQ instrument. 2,5-Dihydroxybenzoic acid and sinapinic acid were used as matrices for analyses of oligosaccharides and glycoconjugates, respectively. NMR spectra of the oligosaccharides were obtained for 2H2O solutions and H2O solutions containing 10% of 2H2O, at 35 °C on Bruker DRX 400 and DRX 600 spectrometers. All spectra were obtained using acetone (δH 2.225, δC 31.05) as internal reference. The core oligosaccharide fractions were repeatedly exchanged with 2H2O with intermediate lyophilization. The data were acquired and processed using standard Bruker software. The processed spectra were assigned with the help of the SPARKY program (37.Goddard T.D. Kneller D.G. SPARKY. 3rd Ed. University of California, San Francisco2001Google Scholar). The signals were assigned by one- and two-dimensional experiments (COSY, clean-TOCSY, NOESY, ROESY, HMBC, HSQC-DEPT, and HSQC with and without carbon decoupling). In the clean-TOCSY experiments the mixing times used were 30, 60, and 100 ms. The delay time in the HMBC was 60 ms and the mixing times in the NOESY and ROESY experiments were 200 ms. The core oligosaccharide (OSIVA) was isolated and purified as described above. The conjugation was carried out as described previously (38.Boratynski J. Roy R. Glycoconj. J. 1998; 15: 131-138Crossref PubMed Scopus (52) Google Scholar). Briefly, core oligosaccharide OSIVA (2.5 mg) solutions in H2O (100 μl) was mixed with an equal volume of BSA (1 mg) solution in H2O. Dimethylformamide was added to a final concentration of 2%, and the mixture was freeze-dried. Dry preparation was heated at 110 °C for 30 min, dissolved in PBS (1 ml), and dialyzed against PBS (3 × 1 liter). The products were analyzed by MALDI-TOF MS, and their antigenic properties were determined in the immunoblotting test, using polyclonal anti-P. shigelloides CNCTC 113/92 antibodies. Rabbits were immunized with the P. shigelloides core oligosaccharide-BSA conjugate, suspended in a complete Freund adjuvant, and polyclonal antibodies against the conjugates were obtained by the procedures previously described (39.Lugowski C. Romanowska E. Eur. J. Biochem. 1978; 91: 89-97Crossref PubMed Scopus (35) Google Scholar). Enzyme-linked immunosorbent assay (ELISA), using LPS as solid-phase antigen, was performed by a modification (40.Jennings H.J. Lugowski C. J. Immunol. 1981; 127: 1011-1018PubMed Google Scholar) of the method described by Voller et al.(41.Voller A. Draper C. Bidwell D.E. Bartlett A. Lancet. 1975; 1: 426-428Abstract PubMed Scopus (157) Google Scholar). Immunoblotting was done as previously described (23.Petersson C. Niedziela T. Jachymek W. Kenne L. Zarzecki P. Lugowski C. Eur. J. Biochem. 1997; 244: 580-586Crossref PubMed Scopus (50) Google Scholar). A goat anti-rabbit IgG conjugated with alkaline phosphatase (Bio-Rad) was used as the second antibody and p-nitrophenyl phosphate and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium were applied as detection systems for ELISA and immunoblotting, respectively. The LPS of P. shigelloides CNCTC 113/92 was isolated by conventional methods and analyzed by SDS-PAGE, showing fractions consisting of core oligosaccharide substituted with different numbers of oligosaccharide repeating units as well as unsubstituted core oligosaccharides. The O-specific polysaccharide and core oligosaccharides were liberated by mild acidic hydrolysis of the LPS and isolated by gel filtration on Bio-Gel P-10. In addition to the polysaccharide fraction, which was analyzed previously (22.Czaja J. Jachymek W. Niedziela T. Lugowski C. Aldova E. Kenne L. Eur. J. Biochem. 2000; 267: 1672-1679Crossref PubMed Scopus (49) Google Scholar), two fractions with lower molecular mass components were obtained, i.e. OSIII (yield, 16.5% of LPS) and OSIV (yield, 6% of LPS). The fraction OSIV was further separated on Bio-Gel P-2 giving the two main oligosaccharides OSIVA (yield, 4.5% of LPS) and OSIVB (yield, 0.5% of LPS). Because the initial NMR investigation indicated the presence of uronic acid, Kdo, and one non-acetylated glucosamine residue in the oligosaccharides, all subsequent sugar and methylation analyses were done on N-acetylated and carboxyl-reduced oligosaccharides to detect these residues. Composition analysis of the carboxyl-reduced and N-acetylated oligosaccharide OSIVB together with determination of the absolute configuration revealed the presence of ld-Hep, d-Glc, d-Gal, and d-GlcN (relative proportions of 2.7:1.8:2.9:0.8) in the carboxyl-reduced OSIVB oligosaccharide. Methylation analysis was performed on this carboxyl-reduced and N-acetylated OSIVB but also on only N-acetylated OSIVB. The methyl esters of the latter methylated material were reduced with Superdeuteride generating two deuterium on C-6 of the former uronic acid. These analyses showed the presence of 2,3,7-trisubstitutedld-Hep p, 3,4-disubstitutedld-Hep p, terminalld-Hep p, 4-substitutedd-Glc pN, terminal d-Glc p, terminal d-Gal p, 4-substitutedd-Gal pA, and 5-substituted Kdo (relative proportions 0.8:1.1:1.0:0.7:0.9:1.8:0.6:0.7) in the original core oligosaccharide OSIVB. In oligosaccharide OSIVA the ratio of terminald-Glc p was twice as high as in OSIVB and 4,6-disubstituted d-Glc pN was identified instead of 4-substituted d-Glc pN. All other components and ratios were found to be the same as in OSIVB. The substitution positions and the ring forms were supported by NMR data (see below). The MALDI-TOF mass spectra of the oligosaccharides (Fig. 1, A and B) showed main ions at m/z 1660.55 [M+Na]+, 1638.53 [M+H]+, and 1642.53 [M-H2O+Na]+for OSIVB and m/z 1822.76 [M+Na]+, 1844.75 [M-H+2Na]+, and 1804.74 [M-H2O+Na]+ for OSIVA. This suggests a nonasaccharide in OSIVB and a decasaccharide in OSIVA differing only in one hexose unit (162.21Da difference). The nine sugars, two Gal, one Glc, three Hep, one GalA, one GlcN, and one Kdo, give together a monoisotopic mass of 1637.52 and an average mass of 1638.41. The mass spectrum of the isolated OSIII (Fig. 1C) component showed main ions at m/z 2865.94 [M+H]+, 2887.91 [M+Na]+, and 2847.93 [M-H2O+H]+. The mass difference of 1065.15 between OSIII and OSIVA can be explained by one repeating unit of the O-specific polysaccharide substituting the core. The mass of OSIII thus supports a hexadecasaccharide structure with one repeating unit linked to the core oligosaccharide. The 1H (Fig. 2A) and HSQC-DEPT (Fig. 3) NMR spectra of the core oligosaccharide OSIVB contained main signals for eight anomeric protons and carbons, and in addition a Kdo spin system confirming a nonasaccharide (the sugar residues are indicated by capital letters as shown in the structure below, and these letters refer to the corresponding sugars through the entire text, tables, and figures). The 1H (Fig. 2B) and HSQC-DEPT NMR spectra of the core oligosaccharide OSIVA contained main signals for nine anomeric protons and carbons and a Kdo spin system, thus confirming a decasaccharide structure. Because all the 1H NMR spectra were complex and contained overlapping signals, the major signals and spin systems were assigned by COSY, TOCSY with different mixing times, and HSQC experiments. By comparing the chemical shifts with previously published NMR data for respective monosaccharides (42.Gorin P.A. Mazurek M. Can. J. Chem. 1975; 53: 1212-1227Crossref Google Scholar, 43.Jansson P.E. Kenne L. Widmalm G. Carbohydr. Res. 1989; 188: 169-191Crossref PubMed Scopus (502) Google Scholar, 44.Susskind M. Brade L. Brade H. Holst O. J. Biol. Chem. 1998; 273: 7006-7017Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) and considering the 3 JH, Hvalues for the coupling between ring protons, estimated from the cross-peaks in the two-dimensional spectra, the sugars could be identified and their anomeric configuration determined.Figure 3Selected 1 JH,C- and 3 JH,C-connectivities in HSQC-DEPT and HMBC spectra of the core oligosaccharide OSIVB of P. shigelloides O54. The spectra were obtained for H2O/2H2O solutions at 600 MHz and 35 °C. The cross-peaks are labeled as explained in the legend to Fig. 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Starting with the signal for the anomeric proton, H-1, the COSY spectrum identified the H-2 signal and the TOCSY spectra with different mixing times the H-3 to H-7 signals. The H-7 signals of heptose residues were identified in the TOCSY experiments starting with the assigned H-3 and H-4 signals. The HSQC-TOCSY experiments were used for unambiguous assignment of overlapping signals. From the assigned 1H signals and the one-bond C–H connectivities, the carbon signals were assigned in the gradient-enhanced HSQC-DEPT spectrum (Fig. 3), and the linkage positions were determined from the high chemical shifts of the signals from the substituted carbons. The CH2 carbon signals were readily identified in the HSQC-DEPT experiment from negative cross-peaks. An unequivocal identification of the H-7, C-7 as a negative cross-peak in the HSQC-DEPT experiment was further confirmed in the HSQC-TOCSY experiment. By these procedures all the spin systems comprising 1H and 13C resonances were determined (Table I).Table I1H and 13C NMR chemical shifts of the P. shigelloides O54 (strain CNCTC 113/92) core oligosaccharidesResidueOligosaccharideChemical shiftOS IIIOS IVAOS IVBH1 C1H2(H3ax) C2H3(H3eq) C3H4 C4H5 C5H6, H6′ C6H7, H7′ C7H8, H8′ C8 CH3COppmA→5)-Kdo***1.852.164.104.133.823.713.55, 3.85174.4096.8034.7066.7374.9772.5770.1364.77B→3,4)-l-glycero-α-d-manno-Hep p-(1→***5.114.054.144.194.164.113.64, 3.71101.2771.0075.8074.7372.5369.6363.93Cβ-d-Gal p-(1→***4.443.543.623.883.643.73, 3.77aAssignment of resonance can be interchanged.104.2072.2073.6769.6376.2062.47D→2,3,7)-l-glycero-α-d-manno-Hep p-(1→***5.254.134.104.033.634.153.59, 3.75100.1379.1077.4067.6373.6368.9070.83Eβ-d-Glc p-(1→***4.553.233.483.433.563.77, 3.85103.7774.2376.4070.3076.5061.67F l-glycero-α-d-manno-Hep p-(1→***4.923.953.833.843.584.023.63, 3.71101.3771.1771.7767.2072.4069.8564.07G→4)-α-d-Gal pA-(1→***5.453.894.134.434.52101.4369.6069.5379.9072.10175.77H→4)-α-d-Glc pN-(1→*5.173.323.993.744.233.81, 3.8996.855.269.578.972.360.5HbCorresponding residues present in a different environment.→4,6)-α-d-Glc pN-(1→**5.183.354.003.894.394.00, 4.1696.9054.9069.4578.1071.0068.00Iβ-d-Glc p-(1→*4.493.283.423.373.483.7, 3.88103.974.277.070.976.961.7IbCorresponding residues present in a different environment.→4)-β-d-Glc p-(1→*4.533.343.663.583.533.64, 3.83103.273.475.380.375.361.0Kβ-d-Gal p-(1→***4.483.523.643.903.683.70, 3.78aAssignment of resonance can be interchanged.103.8771.9773.7069.7076.4062.47L→3)-β-d-Glc p NAc-(1→*4.623.853.623.523.513.74, 3.932.08101.656.482.270.175.961.422.8, 175.1M→4)-α-l-Rha p-(1→*4.853.773.813.633.991.26102.171.871.281.868.217.7N→3)-2-O-Ac-6d-β-d-manno-Hep p-(1→*5.025.614.073.523.511.75, 2.18∼3.792.17100.570.578.269.373.334.458.821.1, 174.4O→3)-d-glycero-β-d-manno-Hep p-(1→*4.763.983.683.773.504.073.75, 3.7797.967.478.166.4577.872.462.0P→4)-α-l-Rha p-(1→*4.953.983.863.613.981.2997.371.571.679.168.217.9Qβ-d-Gal f-(1→*5.304.134.084.013.833.67, 3.71109.382.477.583.771.563.8Spectra were obtained for 2H2O solutions at 35 °C. Acetone (δH 2.225, δC 31.05) was used as internal reference. The presence of a residue in the respective oligosaccharide is marked with an asterisk. The chemical shifts are given as averaged values for the residues in the same environment.a Assignment of resonance can be interchanged.b Corresponding residues present in a different environment. Open table in a new tab Spectra were obtained for 2H2O solutions at 35 °C. Acetone (δH 2.225, δC 31.05) was used as internal reference. The presence of a residue in the respective oligosaccharide is marked with an asterisk. The chemical shifts are given as averaged values for the residues in the same environment. Residue G with the H-1/C-1 signals at δ 5.44/101.5 ppm and non-resolved JH-1,H-2 coupling, was assigned as the 4-substituted α-d-Gal pA residue based on the characteristic five proton spin system, the high chemical shifts of the H-5 (δ 4.52), H-4 (δ 4.43), H-3 (δ 4.12), and C-4 (δ 79.9) signals, the large vicinal couplings between H-2 and H-3 and small vicinal coupling between H-3, H-4, and H-5. ResidueD with the H-1/C-1 signals at δ 5.23/100.3 ppm, JH-1,H-2 < 2 Hz was recognized as the 2,3,7-substitutedl-glycero-α-d-manno-Hep p residue from the 1H and 13C chemical shifts, small vicinal couplings between H-1, H-2, and H-3, and the relatively high chemical shifts of the C-2 (δ 79.2), C-3 (δ 77.4), and C-7 (δ 70.9) signals. Residue H with the H-1/C-1 signals at δ 5.17/96.8 ppm, JH-1,H-2 3.6 Hz was assigned as the 4-substituted α-d-Glc pN residue based on the low chemical shifts of the C-2 signal (δ 55.2), the relative high chemical shift of the C-4 signal (δ 78.9) and the large vicinal couplings between all ring protons. Residue B with the H-1/C-1 signals at δ 5.09/101.3 ppm, JH-1,H-2< 2 Hz was recognized as the 3,4-disubstitutedl-glycero-α-d-manno-Hep p residue on the basis of the small vicinal couplings between H-1, H-2, and H-3 and the relatively high chemical shifts of the C-3 (δ 75.8) and C-4 (δ 74.8.) signals. Residue F with the H-1/C-1 signals at δ 4.91/101.4 ppm, JH-1,H-2 < 2 Hz was recognized as the terminall-glycero-α-d-manno-Hep p residue due to the small vicinal couplings between H-1, H-2, and H-3 and similar chemical shifts as those of the monosaccharidel-α-d-Hep p. Residue E with the H-1/C-1 signals at δ 4.53/103.9 ppm, JH-1,H-2 7.8 Hz was recognized as terminal β-d-Glc p from the similarity of the 1H and 13C chemical shifts with those of β-d-Glc p and the large vicinal couplings between all protons in the sugar ring. Residue K with the H-1/C-1 signals at δ 4.43/104.1 ppm, JH-1,H-27.8 Hz as well as residue C with the H-1/C-1 signals at δ 4.42/104.3 ppm, JH-1,H-2 7.8 Hz were assigned as terminal β-d-Gal p residues due to the large coupling between H-1, H-2, and H-3 and the small vicinal coupling between H-3, H-4, and H-5, and chemical shifts similar to those of β-d-Gal p. Residue A was identified as a 5-substituted Kdo on the basis of characteristic deoxy proton signals, found at δ 1.86 ppm (H-3ax) and δ 2.16 ppm (H-3eq), and a high chemical shift of the C-5 signal (δ 75.0 ppm). In OSIVA an additional terminal β-d-Glc p (residue I), δ 4.49/103.9 ppm, JH-1,H-2 7.8 Hz, was found and residue H with the H-1/C-1 signals at δ 5.16/
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