- Email: [email protected]
Structure of the O-polysaccharide of Edwardsiella tarda PCM 1156
Carbohydrate Research 374 (2013) 45–48
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Note
Structure of the O-polysaccharide of Edwardsiella tarda PCM 1156 Ewa Katzenellenbogen a,⇑, Nina A. Kocharova b, Alexander S. Shashkov b, Sabina Górska-Fra˛czek a, Andrzej Gamian a,c, Yuriy A. Knirel b a
L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wrocław, Poland N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 47, Moscow 119991, Russian Federation c ´ skiego 10, 50-368 Wrocław, Poland Department of Medical Biochemistry, Wrocław Medical University, Chałubin b
a r t i c l e
i n f o
Article history: Received 25 January 2013 Received in revised form 22 March 2013 Accepted 24 March 2013 Available online 2 April 2013
a b s t r a c t Mild acid degradation of the lipopolysaccharide of Edwardsiella tarda PCM 1156 afforded an O-polysaccharide, which was isolated by gel-permeation chromatography on Sephadex G-50 and studied by sugar and methylation analyses along with 1H NMR and 13C NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC, and HMBC experiments. The following structure of the linear tetrasaccharide repeating unit of the O-polysaccharide was established:
Keywords: Edwardsiella tarda Lipopolysaccharide O-antigen Bacterial polysaccharide structure
Ó 2013 Elsevier Ltd. All rights reserved.
Edwardsiella tarda is a Gram-negative bacterium belonging to the Enterobacteriaceae family together with two other Edwardsiella species, Edwardsiella ictaluri and Edwardsiella hoshinae.1–3 E. tarda is the etiological agent for edwardsiellosis, a devastating fish disease responsible for severe economical losses in aquaculture industries worldwide. This bacterium inhabits and infects more than 20 species of freshwater and marine fishes as well as a broad range of other animals like reptiles, amphibians, birds, and mammals.4,5 E. tarda is the only species of the genus which can cause gastrointestinal diseases and other disorders such as myonecrosis and wound infections in humans.4,6,7 Among many different factors involved in the pathogenicity of E. tarda, there is lipopolysaccharide whose structure has been studied scarcely so far. The polysaccharide part of the lipopolysaccharide (O-antigen, O-polysaccharide, OPS) is responsible for the serospecificity of Gram-negative bacteria. A serotyping scheme for E. tarda comprising 61 O groups and 45 H antigens has been established for international use.8 Recently, we started systematic studies on E. tarda lipopolysaccharides aiming at elucidation of the structure–function relationships of these important cell surface antigens and improvement of the existing classification scheme. Earlier, unique OPS structures have been established for one strain of E. ictaluri, MT104,9 and several strains
⇑ Corresponding author. Tel.: +48 71 3371172; fax: +48 71 3709975. E-mail address: [email protected] (E. Katzenellenbogen). 0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.carres.2013.03.025
of E. tarda, including MT108,10 PCM 1153,11 and PCM 1150.12 The OPS structure of E. tarda PCM 1145, PCM 1151, and PCM 1158 was found to be related to those of Citrobacter freundii O22 and Salmonella enterica O4.11 In this work, we report on a new structure of the OPS of E. tarda PCM 1156. The lipopolysaccharide was isolated from dry bacterial cells in a yield 2% by the phenol–water method, purified from nucleic acids and degraded by mild acid hydrolysis to give a high-molecular mass OPS isolated by GPC on Sephadex G-50. Sugar analysis of the OPS by GLC of the alditol acetates derived after full acid hydrolysis with 2 M CF3CO2H revealed Fuc, Man, GlcNAc, and GalNAc in the ratios 0.9:1.0:0.8:0.9, respectively. The identity of all OPS components was confirmed by TLC of OPS hydrolyzates with 2 M CF3CO2H or 10 M HCl. Determination of the absolute configurations by GLC of the acetylated (S)-2-octyl glycosides showed that fucose is L and the other monosaccharides are D. The D configuration of GalN and GlcN was confirmed using Dgalactose oxidase (GalN content in the OPS was 15%) and hexokinase in the presence of ATP (phosphorylation of GlcN was 100%), respectively. Methylation analysis of the OPS resulted in identification of partially methylated derivatives from 3-substituted Fuc and GlcNAc, 6-substituted Man, and 4-substituted GalNAc in molar ratios 1.2:1.8:1.0:1.0 or 0.6:1.2:1.0:0.9 when 2 M CF3CO2H or 10 M HCl, respectively, was used for hydrolysis of the methylated OPS. The 1H NMR and 13C NMR (Fig. 1) spectra of the OPS showed signals for four anomeric atoms (dC 98.3–103.8; dH 4.76–5.08),
46
E. Katzenellenbogen et al. / Carbohydrate Research 374 (2013) 45–48
Figure 1. 13C NMR spectrum of the OPS of E. tarda PCM 1156. Region for CO signals is not shown. Arabic numerals refer to carbons in sugar residues denoted by letters as shown in Table 1.
two nitrogen-bearing carbons of amino sugars at dC 51.2 and 56.7, one C–CH3 group of Fuc at dC 16.4 and dH 1.16, two unsubstituted C–CH2OH groups at dC 62.0 and 62.2, and one O-substituted group at dC 67.1 as well as two N-acetyl groups at dC 23.2, 23.6 (both Me), 175.9 and 176.1 (both CO); dH 2.04 and 2.05. Therefore, the OPS is regular and has a linear tetrasaccharide repeating unit containing one residue each of L-Fuc, D-Man, D-GlcNAc, and D-GalNAc. The 1H NMR and 13C NMR spectra of the OPS were assigned using 2D 1H,1H COSY, TOCSY, and 1H,13C HSQC experiments, and four sugar spin systems were identified (Table 1). A relatively large J1,2 coupling constant of 8.5 Hz indicated that GlcNAc is b-linked, whereas all other sugar residues characterized by smaller J1,2 values of <4 Hz are a-linked. The configurations of the glycosidic linkages were confirmed by H-1,H-3 and H-1,H-5 correlations for bGlcNAc and H-1,H-2 correlations for a-Man, a-Fuc, and a-GalNAc in the 2D ROESY spectrum (Fig. 2). Relatively low-field positions of the signals for C-3 of Fuc and GlcNAc, C-4 of GalNAc, and C-6 of Man at d 79.3, 81.6, 76.9, and 67.1, respectively, as compared with their positions in the corresponding non-substituted monosaccharides,13,14 defined the substitution pattern in the O-polysaccharide. The sequence of the monosaccharides was determined by 2D ROESY and 1H,13C HMBC experiments. The ROESY spectrum (Fig. 2) showed the following correlations between anomeric protons and protons at the linkage carbons: GalNAc H-1,Man H6a,6b at d 4.88/3.65, 4.02; Man H-1,Fuc H-3 at d 5.08/3.90; Fuc H-1,GlcNAc H-3 at d 5.02/3.72, and GlcNAc H-1,GalNAc H-4 at d 4.76/4.18. Accordingly, the 1H,13C HMBC spectrum (Fig. 3) showed
Table 1 H NMR and
1
13
the following correlations between anomeric protons and linkage carbons: GalNAc H-1,Man C-6 at d 4.88/67.1; Man H-1,Fuc C-3 at d 5.08/79.3; Fuc H-1,GlcNAc C-3 at d 5.02/81.6; and GlcNAc H1,GalNAc C-4 at d 4.76/76.8. The data obtained showed that the OPS of E. tarda PCM 1156 has the following structure, which, to our knowledge, is unique among the known bacterial polysaccharide structures:
1. Experimental 1.1. Bacterial strain, isolation, and degradation of the lipopolysaccharide Edwardsiella tarda PCM 1156 (Le Minor (IP) 8-65) was derived from the collection of L. Hirszfeld Institute of Immunology and Experimental Therapy (Wrocław, Poland). Bacteria were cultivated in a liquid medium with aeration at 37 °C for 24 h, harvested, and freeze-dried. The lipopolysaccharide was isolated in a yield 2.2% from dry bacterial mass by phenol–water extraction,15 recovered from water phase and purified from nucleic acids by ultracentrifugation (105,000g, 4 °C, 3 6 h). The lipopolysaccharide (300 mg) was hydrolyzed with aq 1% HOAc at 100 °C for 90 min, and, after precipitation of a lipid A
C NMR chemical shifts (d, ppm) of the OPS of E. tarda PCM 1156
Residue
?3)-b-D-GlcpNAc-(1? A ?3)-a-L-Fucp-(1? B ?6)-a-D-Manp-(1? C ?4)-a-D-GalpNAc-(1? D
Sugar
N-Acetyl group
C-1 H-1
C-2 H-2
C-3 H-3
C-4 H-4
C-5 H-5
C-6 H-6a, 6b
C-1
C-2 H-2
102.7 4.76 101.1 5.02 103.8 5.08 98.3 4.88
56.7 3.88 68.4 3.84 71.2 4.09 51.2 4.17
81.6 3.72 79.3 3.90 71.8 3.91 69.5 4.04
70.1 3.54 72.9 3.89 67.6 3.84 76.9 4.18
76.8 3.48 68.0 4.34 72.9 3.92 71.6 4.01
62.2 3.77, 3.96 16.4 1.16 67.1 3.65, 4.02 62.0 3.75, 3.83
175.9
23.6 2.04
176.1
23.2 2.05
E. Katzenellenbogen et al. / Carbohydrate Research 374 (2013) 45–48
47
Figure 2. Part of a ROESY spectrum of the OPS of E. tarda PCM 1156. The corresponding parts of the 1H NMR spectrum are shown along the axes. Arabic numerals refer to protons in sugar residues denoted by letters as shown in Table 1.
Figure 3. Part of a 1H,13C HMBC spectrum of the OPS of E. tarda PCM 1156. The corresponding parts of the 1H NMR and 13C NMR spectra are shown along the horizontal and vertical axes, respectively. Arabic numerals refer to H,C cross-peaks in sugar residues denoted by letters as shown in Table 1.
sediment, the carbohydrate-containing material (59% of the lipopolysaccharide mass) was fractionated by GPC on a column (2.0 100 cm) of Sephadex G-50 Fine to give an OPS in a yield 10.5% of the total carbohydrate material eluted from the column. 1.2. Chemical analyses Sugar and methylation analyses were performed as described.16,17 For sugar analysis, the OPS was hydrolyzed with 2 M CF3CO2H (120 °C, 2 h). Sugars were converted into the alditol acetates and analyzed by GLC–MS16 using a Thermo Focus ITQ 700
instrument equipped with an TR-5 ms glass capillary column (0.2 mm 12 m) and a temperature program from 150 to 270 °C at 12 °C min 1. For GlcN and GalN determination, the OPS was hydrolyzed with 4 M HCl (105 °C, 18 h). Determination of the absolute configurations by GLC of the acetylated (S)-2-octyl glycosides was performed as described.18 The absolute configuration of GalN was determined with D-galactose oxidase,19 and that of GlcN using hexokinase in the presence of ATP20 after hydrolysis of OPS with 4 M HCl (18 h, 105 °C). The degree of GlcN phosphorylation was checked by GLC analysis of the enzyme-treated, NaHB4-reduced, and peracetylated OPS hydrolyzate. The strong acidic conditions
48
E. Katzenellenbogen et al. / Carbohydrate Research 374 (2013) 45–48
of hydrolysis ensure quantitative release of hexosamines from polysaccharide. The OPS was hydrolyzed with 10 M HCl or 2 M CF3CO2H as above and subjected to TLC on DC-Fertigplatten Kieselgel plates in a solvent system of EtOAc/pyridine/HOAc/water (5:5:1:3, v/v/v/v). The resolved and reference sugars were stained with the molybdate/H2SO4 reagent.21 Methylation of the OPS was carried out using the Gunnarsson procedure.17 The methylated OPS was recovered by extraction with chloroform/water (1:1, v/v), hydrolyzed with 2 M CF3CO2H (120 °C, 2 h) or 10 M HCl (80 °C, 30 min), and the partially methylated monosaccharides were converted into alditol acetates and analyzed by GLC–MS as above. 1.3. NMR spectroscopy An OPS sample was freeze-dried twice from 99.9% D2O and dissolved in 99.95% D2O. 1H NMR and 13C NMR spectra were recorded on a Bruker Avance II 600 MHz spectrometer at 40 °C. Chemical shifts are reported with internal sodium 3-(trimethylsilyl)propanoate-2,2,3,3-d4 (dH 0) and acetone (dC 31.45) as references. The 2D TOCSY and ROESY spectra were recorded with a 150-ms duration of MLEV-17 spin-lock and a 200-ms mixing time, respectively. The 1H,13C HMBC spectrum was recorded with a 60-ms delay for the evolution of long-range spin couplings. Acknowledgment This work was supported by the Federal Targeted Program for Research and Development in Priority Areas of Russia’s Science and Technology Complex for 2007–2013 (State contract No.
16.552.11.7050) using the equipment of the Center for Collective Use EBEE of the FGBOU VPO MarGTU. References 1. Ewing, W. H.; McWhorter, A. C.; Escobar, M. R.; Lubin, A. H. Int. Bull. Bacteriol. Nomencl. Taxon 1965, 15, 33–38. 2. Grimont, P. A. D.; Grimont, F.; Richard, C.; Sakazaki, R. Curr. Microbiol. 1980, 4, 347–351. 3. Hawke, J. P.; McWhorter, A. C.; Steigerwalt, A. G.; Brenner, D. J. Int. J. Syst. Bacteriol. 1981, 31, 396–400. 4. Janda, J. M.; Abbott, S. L. Clin. Infect. Dis. 1993, 17, 742–748. 5. Mohanty, B. R.; Sahoo, P. K. J. Biosci. 2007, 32, 1331–1344. 6. Abbott, S. L.; Janda, J. M. Prokaryotes 2006, 6, 72–89. 7. Slaven, E. M.; Lopez, F. A.; Hart, S. M.; Sanders, C. V. Clin. Infect. Dis. 2001, 32, 1430–1433. 8. Tamura, K.; Sakazaki, R.; McWhorter, A. C.; Kosako, Y. J. Clin. Microbiol. 1988, 26, 2343–2346. 9. Vinogradov, E.; Nossova, L.; Perry, M. B.; Kay, W. W. Carbohydr. Res. 2005, 340, 1509–1513. 10. Vinogradov, E.; Nossova, L.; Perry, M. B.; Kay, W. W. Carbohydr. Res. 2005, 340, 85–90. 11. Katzenellenbogen, E.; Kocharova, N. A.; Toukach, P. V.; Gorska, S.; Bogulska, M.; Gamian, A.; Knirel, Y. A. Carbohydr. Res. 2012, 347, 186–188. 12. Katzenellenbogen, E.; Kocharova, N. A.; Shashkov, A. S.; Górska-Fra˛czek, S.; Bogulska, M.; Gamian, A.; Knirel, Y. A. Carbohydr. Res. 2013, 368, 84–88. 13. Bock, K.; Pedersen, C. Adv. Carbohydr. Chem. Biochem. 1983, 41, 27–66. 14. Lipkind, G. M.; Shashkov, A. S.; Knirel, Y. A.; Vinogradov, E. V.; Kochetkov, N. K. Carbohydr. Res. 1988, 175, 59–75. 15. Westphal, O.; Jann, K. Methods Carbohydr. Chem. 1965, 5, 83–91. 16. Sawardeker, J. S.; Sloneker, J. H.; Jeans, A. Anal. Chem. 1965, 37, 1602–1604. 17. Gunnarsson, A. Glycoconj. J. 1987, 4, 239–245. 18. Leontein, K.; Lönngren, J. Methods Carbohydr. Chem. 1993, 9, 87–89. 19. Fischer, W.; Zapf, J. Hoppe Seylers Z. Physiol. Chem. 1964, 337, 186–195. 20. Sturgeon, R. J. Methods Carbohydr. Chem. 1980, 8, 135–137. 21. Katzenellenbogen, E.; Kocharova, N. A.; Zatonsky, G. V.; Witkowska, D.; Bogulska, M.; Shashkov, A. S.; Gamian, A.; Knirel, Y. A. Eur. J. Biochem. 2003, 270, 2732–2738.
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Note
Structure of the O-polysaccharide of Edwardsiella tarda PCM 1156 Ewa Katzenellenbogen a,⇑, Nina A. Kocharova b, Alexander S. Shashkov b, Sabina Górska-Fra˛czek a, Andrzej Gamian a,c, Yuriy A. Knirel b a
L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wrocław, Poland N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 47, Moscow 119991, Russian Federation c ´ skiego 10, 50-368 Wrocław, Poland Department of Medical Biochemistry, Wrocław Medical University, Chałubin b
a r t i c l e
i n f o
Article history: Received 25 January 2013 Received in revised form 22 March 2013 Accepted 24 March 2013 Available online 2 April 2013
a b s t r a c t Mild acid degradation of the lipopolysaccharide of Edwardsiella tarda PCM 1156 afforded an O-polysaccharide, which was isolated by gel-permeation chromatography on Sephadex G-50 and studied by sugar and methylation analyses along with 1H NMR and 13C NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC, and HMBC experiments. The following structure of the linear tetrasaccharide repeating unit of the O-polysaccharide was established:
Keywords: Edwardsiella tarda Lipopolysaccharide O-antigen Bacterial polysaccharide structure
Ó 2013 Elsevier Ltd. All rights reserved.
Edwardsiella tarda is a Gram-negative bacterium belonging to the Enterobacteriaceae family together with two other Edwardsiella species, Edwardsiella ictaluri and Edwardsiella hoshinae.1–3 E. tarda is the etiological agent for edwardsiellosis, a devastating fish disease responsible for severe economical losses in aquaculture industries worldwide. This bacterium inhabits and infects more than 20 species of freshwater and marine fishes as well as a broad range of other animals like reptiles, amphibians, birds, and mammals.4,5 E. tarda is the only species of the genus which can cause gastrointestinal diseases and other disorders such as myonecrosis and wound infections in humans.4,6,7 Among many different factors involved in the pathogenicity of E. tarda, there is lipopolysaccharide whose structure has been studied scarcely so far. The polysaccharide part of the lipopolysaccharide (O-antigen, O-polysaccharide, OPS) is responsible for the serospecificity of Gram-negative bacteria. A serotyping scheme for E. tarda comprising 61 O groups and 45 H antigens has been established for international use.8 Recently, we started systematic studies on E. tarda lipopolysaccharides aiming at elucidation of the structure–function relationships of these important cell surface antigens and improvement of the existing classification scheme. Earlier, unique OPS structures have been established for one strain of E. ictaluri, MT104,9 and several strains
⇑ Corresponding author. Tel.: +48 71 3371172; fax: +48 71 3709975. E-mail address: [email protected] (E. Katzenellenbogen). 0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.carres.2013.03.025
of E. tarda, including MT108,10 PCM 1153,11 and PCM 1150.12 The OPS structure of E. tarda PCM 1145, PCM 1151, and PCM 1158 was found to be related to those of Citrobacter freundii O22 and Salmonella enterica O4.11 In this work, we report on a new structure of the OPS of E. tarda PCM 1156. The lipopolysaccharide was isolated from dry bacterial cells in a yield 2% by the phenol–water method, purified from nucleic acids and degraded by mild acid hydrolysis to give a high-molecular mass OPS isolated by GPC on Sephadex G-50. Sugar analysis of the OPS by GLC of the alditol acetates derived after full acid hydrolysis with 2 M CF3CO2H revealed Fuc, Man, GlcNAc, and GalNAc in the ratios 0.9:1.0:0.8:0.9, respectively. The identity of all OPS components was confirmed by TLC of OPS hydrolyzates with 2 M CF3CO2H or 10 M HCl. Determination of the absolute configurations by GLC of the acetylated (S)-2-octyl glycosides showed that fucose is L and the other monosaccharides are D. The D configuration of GalN and GlcN was confirmed using Dgalactose oxidase (GalN content in the OPS was 15%) and hexokinase in the presence of ATP (phosphorylation of GlcN was 100%), respectively. Methylation analysis of the OPS resulted in identification of partially methylated derivatives from 3-substituted Fuc and GlcNAc, 6-substituted Man, and 4-substituted GalNAc in molar ratios 1.2:1.8:1.0:1.0 or 0.6:1.2:1.0:0.9 when 2 M CF3CO2H or 10 M HCl, respectively, was used for hydrolysis of the methylated OPS. The 1H NMR and 13C NMR (Fig. 1) spectra of the OPS showed signals for four anomeric atoms (dC 98.3–103.8; dH 4.76–5.08),
46
E. Katzenellenbogen et al. / Carbohydrate Research 374 (2013) 45–48
Figure 1. 13C NMR spectrum of the OPS of E. tarda PCM 1156. Region for CO signals is not shown. Arabic numerals refer to carbons in sugar residues denoted by letters as shown in Table 1.
two nitrogen-bearing carbons of amino sugars at dC 51.2 and 56.7, one C–CH3 group of Fuc at dC 16.4 and dH 1.16, two unsubstituted C–CH2OH groups at dC 62.0 and 62.2, and one O-substituted group at dC 67.1 as well as two N-acetyl groups at dC 23.2, 23.6 (both Me), 175.9 and 176.1 (both CO); dH 2.04 and 2.05. Therefore, the OPS is regular and has a linear tetrasaccharide repeating unit containing one residue each of L-Fuc, D-Man, D-GlcNAc, and D-GalNAc. The 1H NMR and 13C NMR spectra of the OPS were assigned using 2D 1H,1H COSY, TOCSY, and 1H,13C HSQC experiments, and four sugar spin systems were identified (Table 1). A relatively large J1,2 coupling constant of 8.5 Hz indicated that GlcNAc is b-linked, whereas all other sugar residues characterized by smaller J1,2 values of <4 Hz are a-linked. The configurations of the glycosidic linkages were confirmed by H-1,H-3 and H-1,H-5 correlations for bGlcNAc and H-1,H-2 correlations for a-Man, a-Fuc, and a-GalNAc in the 2D ROESY spectrum (Fig. 2). Relatively low-field positions of the signals for C-3 of Fuc and GlcNAc, C-4 of GalNAc, and C-6 of Man at d 79.3, 81.6, 76.9, and 67.1, respectively, as compared with their positions in the corresponding non-substituted monosaccharides,13,14 defined the substitution pattern in the O-polysaccharide. The sequence of the monosaccharides was determined by 2D ROESY and 1H,13C HMBC experiments. The ROESY spectrum (Fig. 2) showed the following correlations between anomeric protons and protons at the linkage carbons: GalNAc H-1,Man H6a,6b at d 4.88/3.65, 4.02; Man H-1,Fuc H-3 at d 5.08/3.90; Fuc H-1,GlcNAc H-3 at d 5.02/3.72, and GlcNAc H-1,GalNAc H-4 at d 4.76/4.18. Accordingly, the 1H,13C HMBC spectrum (Fig. 3) showed
Table 1 H NMR and
1
13
the following correlations between anomeric protons and linkage carbons: GalNAc H-1,Man C-6 at d 4.88/67.1; Man H-1,Fuc C-3 at d 5.08/79.3; Fuc H-1,GlcNAc C-3 at d 5.02/81.6; and GlcNAc H1,GalNAc C-4 at d 4.76/76.8. The data obtained showed that the OPS of E. tarda PCM 1156 has the following structure, which, to our knowledge, is unique among the known bacterial polysaccharide structures:
1. Experimental 1.1. Bacterial strain, isolation, and degradation of the lipopolysaccharide Edwardsiella tarda PCM 1156 (Le Minor (IP) 8-65) was derived from the collection of L. Hirszfeld Institute of Immunology and Experimental Therapy (Wrocław, Poland). Bacteria were cultivated in a liquid medium with aeration at 37 °C for 24 h, harvested, and freeze-dried. The lipopolysaccharide was isolated in a yield 2.2% from dry bacterial mass by phenol–water extraction,15 recovered from water phase and purified from nucleic acids by ultracentrifugation (105,000g, 4 °C, 3 6 h). The lipopolysaccharide (300 mg) was hydrolyzed with aq 1% HOAc at 100 °C for 90 min, and, after precipitation of a lipid A
C NMR chemical shifts (d, ppm) of the OPS of E. tarda PCM 1156
Residue
?3)-b-D-GlcpNAc-(1? A ?3)-a-L-Fucp-(1? B ?6)-a-D-Manp-(1? C ?4)-a-D-GalpNAc-(1? D
Sugar
N-Acetyl group
C-1 H-1
C-2 H-2
C-3 H-3
C-4 H-4
C-5 H-5
C-6 H-6a, 6b
C-1
C-2 H-2
102.7 4.76 101.1 5.02 103.8 5.08 98.3 4.88
56.7 3.88 68.4 3.84 71.2 4.09 51.2 4.17
81.6 3.72 79.3 3.90 71.8 3.91 69.5 4.04
70.1 3.54 72.9 3.89 67.6 3.84 76.9 4.18
76.8 3.48 68.0 4.34 72.9 3.92 71.6 4.01
62.2 3.77, 3.96 16.4 1.16 67.1 3.65, 4.02 62.0 3.75, 3.83
175.9
23.6 2.04
176.1
23.2 2.05
E. Katzenellenbogen et al. / Carbohydrate Research 374 (2013) 45–48
47
Figure 2. Part of a ROESY spectrum of the OPS of E. tarda PCM 1156. The corresponding parts of the 1H NMR spectrum are shown along the axes. Arabic numerals refer to protons in sugar residues denoted by letters as shown in Table 1.
Figure 3. Part of a 1H,13C HMBC spectrum of the OPS of E. tarda PCM 1156. The corresponding parts of the 1H NMR and 13C NMR spectra are shown along the horizontal and vertical axes, respectively. Arabic numerals refer to H,C cross-peaks in sugar residues denoted by letters as shown in Table 1.
sediment, the carbohydrate-containing material (59% of the lipopolysaccharide mass) was fractionated by GPC on a column (2.0 100 cm) of Sephadex G-50 Fine to give an OPS in a yield 10.5% of the total carbohydrate material eluted from the column. 1.2. Chemical analyses Sugar and methylation analyses were performed as described.16,17 For sugar analysis, the OPS was hydrolyzed with 2 M CF3CO2H (120 °C, 2 h). Sugars were converted into the alditol acetates and analyzed by GLC–MS16 using a Thermo Focus ITQ 700
instrument equipped with an TR-5 ms glass capillary column (0.2 mm 12 m) and a temperature program from 150 to 270 °C at 12 °C min 1. For GlcN and GalN determination, the OPS was hydrolyzed with 4 M HCl (105 °C, 18 h). Determination of the absolute configurations by GLC of the acetylated (S)-2-octyl glycosides was performed as described.18 The absolute configuration of GalN was determined with D-galactose oxidase,19 and that of GlcN using hexokinase in the presence of ATP20 after hydrolysis of OPS with 4 M HCl (18 h, 105 °C). The degree of GlcN phosphorylation was checked by GLC analysis of the enzyme-treated, NaHB4-reduced, and peracetylated OPS hydrolyzate. The strong acidic conditions
48
E. Katzenellenbogen et al. / Carbohydrate Research 374 (2013) 45–48
of hydrolysis ensure quantitative release of hexosamines from polysaccharide. The OPS was hydrolyzed with 10 M HCl or 2 M CF3CO2H as above and subjected to TLC on DC-Fertigplatten Kieselgel plates in a solvent system of EtOAc/pyridine/HOAc/water (5:5:1:3, v/v/v/v). The resolved and reference sugars were stained with the molybdate/H2SO4 reagent.21 Methylation of the OPS was carried out using the Gunnarsson procedure.17 The methylated OPS was recovered by extraction with chloroform/water (1:1, v/v), hydrolyzed with 2 M CF3CO2H (120 °C, 2 h) or 10 M HCl (80 °C, 30 min), and the partially methylated monosaccharides were converted into alditol acetates and analyzed by GLC–MS as above. 1.3. NMR spectroscopy An OPS sample was freeze-dried twice from 99.9% D2O and dissolved in 99.95% D2O. 1H NMR and 13C NMR spectra were recorded on a Bruker Avance II 600 MHz spectrometer at 40 °C. Chemical shifts are reported with internal sodium 3-(trimethylsilyl)propanoate-2,2,3,3-d4 (dH 0) and acetone (dC 31.45) as references. The 2D TOCSY and ROESY spectra were recorded with a 150-ms duration of MLEV-17 spin-lock and a 200-ms mixing time, respectively. The 1H,13C HMBC spectrum was recorded with a 60-ms delay for the evolution of long-range spin couplings. Acknowledgment This work was supported by the Federal Targeted Program for Research and Development in Priority Areas of Russia’s Science and Technology Complex for 2007–2013 (State contract No.
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