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The structure of the O-specific polysaccharide from Hafnia alvei strain 38 lipopolysaccharide
Carbohydrate Research, 231(1992) 51-54 Elsevier Science Publishers B.V., Amsterdam
51
The structure of the O-specific polysaccharide from Hufnia alvei strain 38 lipopolysaccharide Ewa Katzenellenbogen a, Elibieta Romanowska a, Danuta Witkowska a and Alexander S. Shashkov b a L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 12 Czerska, 53-114 Wrociaw (Poland) b N.D. Zelinsky Institute of Organic Chemistry, Academy of Sciences of the U.S.S.R., Moscow (Russia) (Received
August
3rd, 1991; accepted
October
12th, 1991)
ABSTRACT The O-specific polysaccharide of the lipopolysaccharide from H. aluei strain 38 has been established by NMR spectroscopy (13C and ‘H) and methylation analysis to have the repeating unit -+4)-p-~ManpNAc-(1 --) 4)a-D-GlcpNAc-(I --f.
INTRODUCTION
The structures of several O-specific polysaccharides from lipopolysaccharides of the Hafnia alvei strains ATCC 13337,1187,39,2, and 1211 have been elucidatedrd4. We now report the structure of the O-specific polysaccharide isolated from the lipopolysaccharide of H. alvei strain 38. RESULTS
AND DISCUSSION
Extraction of the dry bacteria gave the lipopolysaccharide (4.3%), mild acid hydrolysis of which followed by gel-filtration of the water-soluble products on Sephadex G-50 yielded a single polysaccharide fraction (PS, 39%). Acid hydrolysis of PS gave 2-amino-2-deoxyglucose and 2-amino-2-deoxymannose in the molar ratio - 1: 1 as the main components. Minute proportions of glucose and heptose, which orginated from the core, were also produced. That the hexosamine components were D was established using hexokinase5. 13C-NMR spectrum of PS (Table I) contained, inter alia, NMR spectra.-The signals for C-l at 6 100.4
0008-6215/92/$05.00
0 1992 - Elsevier
Science
Publishers
B.V. All rights reserved
and Experi-
E. Katzenellenbogen et al. / Carbohydr. Res. 231 (1992) 51-54
52
TABLE “C-NMR
I chemical
shift data for the PS from
Residue ---f4).fi-D-ManpNAc-(1
+ 4)-wD-GlcpNAc-(1
” The calculated
+
+
values6
H. alcei strain
3X (70”, DzO; acetone,
31.45 ppm) a
C-l
c-2
c-3
c-4
C-5
C-6
CHJONH
100.4 (100.5)
54.6 (54.3)
73.9 (73.5)
74.4 (74.9)
76.3 (76.5)
61.7 (61.8)
23.2 h 175.5 ‘
54.9 (55.3)
70.4 (70.6)
80.2 (80.4)
72.3 (72.0)
61.1 (61.1)
23.1 ’ 176.7 ’
99.1 (99.X)
are in parentheses.
b,c The assignments
may he interchanged.
and 54.6, for CH,CONH at 6 23.2 and 23.1, for CH,CONH at 175.5 and 176.7, and for HOCH, at 61.7 and 61.1. Computer-assisted analysis6 of the i3C-NMR spectrum revealed only one structure (1) with an acceptable S value (0.7). + 4)-@-o-MantNAc-(
B 1 + 4)-a-D-GlcpNAc-(1 -3 1
The analysis predicted the same absolute configurations for both residues because the best S value for structures with different absolute configurations was 8.5. That structure 1 was correct was indicated by the 1D and 2D ‘H-NMR data. The assignments for the ‘H-NMR spectrum (Fig. 1 and Table Ill were accomplished using 1D homonuclear double resonance in the differential mode. Connectivities were supported by the results of the NOE experiments (Fig. 21. The differential NOE spectrum with prc-irradiation of H-l of residue A is represented by signals for H-2/5 of residue A and H-4 of residue B (Fig. 2a). In the differential NOE spectrum with pre-irradiation of H-l of residue B, the signals of H-2,3 of residue B are present together with a signal for H-4 of residue A (Fig. 2bl. The spectra supported (1 + 4) linkages and the p configuration of the 2-acetamido-2deoxymannose residue. The presence of signals for H-4A in Fig. 2a and of H-3B in Fig. 2b are due to spin-diffusion in the polymer with short relaxation times. Methylution analysis.-The results of methylation analysis showed that the polymer was built from 4-substituted 2-acetamido-2-deoxyglucose and 2acetamido-2-deoxymannose residues, leading to structure 1.
EXPERIMENTAL
Isolation and analysis of the O-specific polysaccharide. -Hafnia aluei strain 38 was obtained from the collection of the Institute of Immunology and Experimental Therapy (Wroclaw). The lipopolysaccharide and the O-specific polysaccharide (PSI were prepared as described’. The O-specific polysaccharide was obtained after acid hydrolysis (aque-
E. Katzenellenbogen et al. /Carbohydr.
5.0
4.5
Res. 231 (1992) 51-54
4.0
3.5
53
3.0
2.5
2.0
1.5
P.p.m
Fig. 1.360-MHz ‘H-NMR spectrum of the PS from H. alvei strain 38 (70”, D,O; internal acetone, 2.225 ppm).
“-4A
H-2A
M. -XT
,~‘~~1”~~1”“1~~“J~~“I~‘~‘~~‘~‘1~~~’1~’~(1”~~1’~~‘J”~‘~“~’
4.10
4.05
4.00
3.95
3.90
3.05
3.80
3.75
P-P m.
3.70
3.65
3.60
3.55
3.50
Fig. 2. Expanded ‘H-NMR spectrum of the PS from H. alvei strain 38, and differential NOE spectra with pre-irradiation of H-l of residue A (a) and residue B (b).
Katzenellenbopm et al. / Carbohydr. Rex 231 (1992) 5I-54
54
TABLE ‘H-NMR
II data (8 in ppm. J in Hz) for the PS of H. ahei strain
38 (70”, D20;
internal
acetone.
2.225
PPm) Residue
H-l
+ 4)-P-o-ManpNAc-(I
+ 4)-cu-o-GlcpNAc-(I
+
+
H-3
H-2
H-4
H-5
4.88
4.51
4.04
3.68
3.54
J,.? 1.4
J,,, 4.4
J,,, 0.1
-I,,, 9.1
Jvm 2.2 .l5.m4.2
5.25 J,,, 3.9
3.82
3.73
3.84
J,,,
Jx.4 9.2
J4.5 9.2
3.94 10.5
ous 1% HOAc, loo”, 2.5 h) of the lipopolysaccharide followed by purification on a column (1.6 X 100 cm) of Sephadex G-50. Sugar and methylation analyses were performed as described’ and the absolute configuration of each hexosamine was determined by a modified enzymic method”. General.-GLC-MS was carried out with a Hewlett-Packard 5971A system, using an HP-1 glass capillary column (0.2 mm X 12 m) and temperature program 150 + 270” at 8”/min. All NMR spectra were obtained using a Bruker AM-360 spectrometer at 70” on solutions in DZO (internal acetone; 2.225 ppm for ‘H and 31.45 ppm for ‘%I, ACKNOWLEDGMENT
We thank spectrometer
Professor J. Dabrowski for permission to use the Bruker AM-360 (Max-Planck-Institut fur Medizinische Forschung, Heidelberg).
REFERENCES A. Gamian, E. Romanowska, H.J. Opferkuch, M. Hauck, and J. Dabrowski, Eur. X Eioclzem., 186 (1989) 611-620. E. Katzenellenbogen, E. Romanowska, N.A. Kocharova, A.S. Shashkov, G.M. Lipkind, Y.A. Knirel. and N.K. Kochetkov, Curbohydr. Res., 203 (1990) 219-227. A. Gamian, E. Romanowska, U. Dabrowski, and J. Dabrowski. Biochemisrry. 30 (1991) 5032-5038. E. Katzenellenbogen, E. Romanowska, U. Dabrowski. and J. Dabrowski, Eur. J. Biochem.. 200 (1991) 40 I-407. D.H. Brown, Methods Enzymol., 3 (1957) 15X-162. G.M. Lipkind, AS. Shashkov, Y.A. Knirel, E.V. Vinogradov, and N.K. Kochetkov, Carbohydr. Res., 175 (1988) 59-75. E. Romanowska, A. Romanowska, C. tugowski, and E. Katzenellenbogen, Eur. J. Biochem.. 121 (1981) 119-123. E. Romanowska, A. Gamian, and J. Dabrowski, Eur. J. Biochem., 161 (1986) 557-564.
51
The structure of the O-specific polysaccharide from Hufnia alvei strain 38 lipopolysaccharide Ewa Katzenellenbogen a, Elibieta Romanowska a, Danuta Witkowska a and Alexander S. Shashkov b a L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 12 Czerska, 53-114 Wrociaw (Poland) b N.D. Zelinsky Institute of Organic Chemistry, Academy of Sciences of the U.S.S.R., Moscow (Russia) (Received
August
3rd, 1991; accepted
October
12th, 1991)
ABSTRACT The O-specific polysaccharide of the lipopolysaccharide from H. aluei strain 38 has been established by NMR spectroscopy (13C and ‘H) and methylation analysis to have the repeating unit -+4)-p-~ManpNAc-(1 --) 4)a-D-GlcpNAc-(I --f.
INTRODUCTION
The structures of several O-specific polysaccharides from lipopolysaccharides of the Hafnia alvei strains ATCC 13337,1187,39,2, and 1211 have been elucidatedrd4. We now report the structure of the O-specific polysaccharide isolated from the lipopolysaccharide of H. alvei strain 38. RESULTS
AND DISCUSSION
Extraction of the dry bacteria gave the lipopolysaccharide (4.3%), mild acid hydrolysis of which followed by gel-filtration of the water-soluble products on Sephadex G-50 yielded a single polysaccharide fraction (PS, 39%). Acid hydrolysis of PS gave 2-amino-2-deoxyglucose and 2-amino-2-deoxymannose in the molar ratio - 1: 1 as the main components. Minute proportions of glucose and heptose, which orginated from the core, were also produced. That the hexosamine components were D was established using hexokinase5. 13C-NMR spectrum of PS (Table I) contained, inter alia, NMR spectra.-The signals for C-l at 6 100.4
0008-6215/92/$05.00
0 1992 - Elsevier
Science
Publishers
B.V. All rights reserved
and Experi-
E. Katzenellenbogen et al. / Carbohydr. Res. 231 (1992) 51-54
52
TABLE “C-NMR
I chemical
shift data for the PS from
Residue ---f4).fi-D-ManpNAc-(1
+ 4)-wD-GlcpNAc-(1
” The calculated
+
+
values6
H. alcei strain
3X (70”, DzO; acetone,
31.45 ppm) a
C-l
c-2
c-3
c-4
C-5
C-6
CHJONH
100.4 (100.5)
54.6 (54.3)
73.9 (73.5)
74.4 (74.9)
76.3 (76.5)
61.7 (61.8)
23.2 h 175.5 ‘
54.9 (55.3)
70.4 (70.6)
80.2 (80.4)
72.3 (72.0)
61.1 (61.1)
23.1 ’ 176.7 ’
99.1 (99.X)
are in parentheses.
b,c The assignments
may he interchanged.
and 54.6, for CH,CONH at 6 23.2 and 23.1, for CH,CONH at 175.5 and 176.7, and for HOCH, at 61.7 and 61.1. Computer-assisted analysis6 of the i3C-NMR spectrum revealed only one structure (1) with an acceptable S value (0.7). + 4)-@-o-MantNAc-(
B 1 + 4)-a-D-GlcpNAc-(1 -3 1
The analysis predicted the same absolute configurations for both residues because the best S value for structures with different absolute configurations was 8.5. That structure 1 was correct was indicated by the 1D and 2D ‘H-NMR data. The assignments for the ‘H-NMR spectrum (Fig. 1 and Table Ill were accomplished using 1D homonuclear double resonance in the differential mode. Connectivities were supported by the results of the NOE experiments (Fig. 21. The differential NOE spectrum with prc-irradiation of H-l of residue A is represented by signals for H-2/5 of residue A and H-4 of residue B (Fig. 2a). In the differential NOE spectrum with pre-irradiation of H-l of residue B, the signals of H-2,3 of residue B are present together with a signal for H-4 of residue A (Fig. 2bl. The spectra supported (1 + 4) linkages and the p configuration of the 2-acetamido-2deoxymannose residue. The presence of signals for H-4A in Fig. 2a and of H-3B in Fig. 2b are due to spin-diffusion in the polymer with short relaxation times. Methylution analysis.-The results of methylation analysis showed that the polymer was built from 4-substituted 2-acetamido-2-deoxyglucose and 2acetamido-2-deoxymannose residues, leading to structure 1.
EXPERIMENTAL
Isolation and analysis of the O-specific polysaccharide. -Hafnia aluei strain 38 was obtained from the collection of the Institute of Immunology and Experimental Therapy (Wroclaw). The lipopolysaccharide and the O-specific polysaccharide (PSI were prepared as described’. The O-specific polysaccharide was obtained after acid hydrolysis (aque-
E. Katzenellenbogen et al. /Carbohydr.
5.0
4.5
Res. 231 (1992) 51-54
4.0
3.5
53
3.0
2.5
2.0
1.5
P.p.m
Fig. 1.360-MHz ‘H-NMR spectrum of the PS from H. alvei strain 38 (70”, D,O; internal acetone, 2.225 ppm).
“-4A
H-2A
M. -XT
,~‘~~1”~~1”“1~~“J~~“I~‘~‘~~‘~‘1~~~’1~’~(1”~~1’~~‘J”~‘~“~’
4.10
4.05
4.00
3.95
3.90
3.05
3.80
3.75
P-P m.
3.70
3.65
3.60
3.55
3.50
Fig. 2. Expanded ‘H-NMR spectrum of the PS from H. alvei strain 38, and differential NOE spectra with pre-irradiation of H-l of residue A (a) and residue B (b).
Katzenellenbopm et al. / Carbohydr. Rex 231 (1992) 5I-54
54
TABLE ‘H-NMR
II data (8 in ppm. J in Hz) for the PS of H. ahei strain
38 (70”, D20;
internal
acetone.
2.225
PPm) Residue
H-l
+ 4)-P-o-ManpNAc-(I
+ 4)-cu-o-GlcpNAc-(I
+
+
H-3
H-2
H-4
H-5
4.88
4.51
4.04
3.68
3.54
J,.? 1.4
J,,, 4.4
J,,, 0.1
-I,,, 9.1
Jvm 2.2 .l5.m4.2
5.25 J,,, 3.9
3.82
3.73
3.84
J,,,
Jx.4 9.2
J4.5 9.2
3.94 10.5
ous 1% HOAc, loo”, 2.5 h) of the lipopolysaccharide followed by purification on a column (1.6 X 100 cm) of Sephadex G-50. Sugar and methylation analyses were performed as described’ and the absolute configuration of each hexosamine was determined by a modified enzymic method”. General.-GLC-MS was carried out with a Hewlett-Packard 5971A system, using an HP-1 glass capillary column (0.2 mm X 12 m) and temperature program 150 + 270” at 8”/min. All NMR spectra were obtained using a Bruker AM-360 spectrometer at 70” on solutions in DZO (internal acetone; 2.225 ppm for ‘H and 31.45 ppm for ‘%I, ACKNOWLEDGMENT
We thank spectrometer
Professor J. Dabrowski for permission to use the Bruker AM-360 (Max-Planck-Institut fur Medizinische Forschung, Heidelberg).
REFERENCES A. Gamian, E. Romanowska, H.J. Opferkuch, M. Hauck, and J. Dabrowski, Eur. X Eioclzem., 186 (1989) 611-620. E. Katzenellenbogen, E. Romanowska, N.A. Kocharova, A.S. Shashkov, G.M. Lipkind, Y.A. Knirel. and N.K. Kochetkov, Curbohydr. Res., 203 (1990) 219-227. A. Gamian, E. Romanowska, U. Dabrowski, and J. Dabrowski. Biochemisrry. 30 (1991) 5032-5038. E. Katzenellenbogen, E. Romanowska, U. Dabrowski. and J. Dabrowski, Eur. J. Biochem.. 200 (1991) 40 I-407. D.H. Brown, Methods Enzymol., 3 (1957) 15X-162. G.M. Lipkind, AS. Shashkov, Y.A. Knirel, E.V. Vinogradov, and N.K. Kochetkov, Carbohydr. Res., 175 (1988) 59-75. E. Romanowska, A. Romanowska, C. tugowski, and E. Katzenellenbogen, Eur. J. Biochem.. 121 (1981) 119-123. E. Romanowska, A. Gamian, and J. Dabrowski, Eur. J. Biochem., 161 (1986) 557-564.