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Journal of Bacteriology, August 1998, p. 3891-3899, Vol. 180, No. 15
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Chemical Structure of Lipid A Isolated from
Flavobacterium meningosepticum Lipopolysaccharide
Hitomi
Kato,1
Yuji
Haishima,1
Takatoshi
Iida,1
Akira
Tanaka,2 and
Ken-ichi
Tanamoto1,*
Division of Microbiology, National Institute
of Health Sciences, Setagayaku, Tokyo 158,1
and
Department of Drug Analysis, Showa College of
Pharmaceutical Sciences, Machida, Tokyo
194,2 Japan
Received 15 December 1997/Accepted 28 May 1998
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ABSTRACT |
The chemical structure of the lipid A of the lipopolysaccharide
component isolated from Flavobacterium meningosepticum IFO 12535 was elucidated. Methylation and nuclear magnetic resonance analyses showed that two kinds of hydrophilic backbone exist in the
free lipid A: a 
(1
6)-linked
2-amino-2-deoxy-D-glucose, which is usually present
in enterobacterial lipid A's, and a
2-amino-6-O-(2,3-diamino-2,3-dideoxy--D-glucopyranosyl)-2-deoxy-D-glucose, in a molar ratio of 1.00:0.35. Both backbones were 
-glycosidically phosphorylated in position 1, and the hydroxyl groups at
positions 4, 4', and 6' were unsubstituted. Liquid secondary
ion-mass spectrometry revealed a pseudomolecular ion at
m/z 1673 [M-H]
as a major monophosphoryl
lipid A component carrying five acyl groups. Fatty acid analysis showed
that the lipid A contained 1 mol each of amide-linked
(R)-3-OH iC17:0, ester-linked
(R)-3-OH iC15:0, amide-linked
(R)-3-O-(iC15:0)-iC17:0,
and both amide- and ester-linked (R)-3-OH
C16:0. Fatty acid distribution analyses using several mass
spectrometry determinations demonstrated that the former two
constituents were distributed on positions 2 and 3 of the reducing
terminal unit of the backbones and that the latter two were attached to
the 2' and 3' positions in the nonreducing terminal residue.
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INTRODUCTION |
Lipopolysaccharide (LPS) is known to
act as an endotoxin that mediates pathophysiological changes such as
fever and shock which occur in the course of severe gram-negative
bacterial infection (5, 18, 27). The pathophysiological
activity of LPS depends on the chemical structure of the hydrophobic
portion called lipid A, the biologically active center of LPS (12,
14), which generally consists of a (16)-linked
2-amino-2-deoxy-D-glucose (GlcN) disaccharide carrying
phosphate and fatty acid residues; many fine structural variations are
observed in different bacterial families (38).
Since many of the LPSs from various gram-negative bacteria cause
similar endotoxic effects despite differences in chemical composition
and positions of substitution, the chemical structure required for the
activity does not seem to be very strict. It has been reported,
however, that several lipid A forms, isolated from the LPSs of
Porphyromonas gingivalis (16), Rhodobacter sphaeroides (22, 26) and Rhodobacter
capsulatus (20), as well as chemically synthesized
lipid A analogs (6, 12), which are structurally similar to
the active-type lipid A, exhibit dramatically low endotoxicity.
Biologically active lipid A has been found to be changed to completely
nontoxic derivatives by simple chemical modifications (28,
29). These findings indicate that the biological activity of
lipid A is controlled by the fine structural variations. The nontoxic
or low-toxicity lipid A preparations are very important for the
determination of the relationship between the chemical structure and
biological activity of lipid A, and also for the systematic development
of LPS antagonists. However, the essential structural requirements for
the complete activity or nontoxicity of lipid A are still uncertain. It
is, therefore, meaningful to study the chemical and biological
properties of naturally occurring lipid A's which possess a unique
structure.
Flavobacterium meningosepticum is an aerobic gram-negative
rod which is known to cause meningitis and septicemia in newborn infants (4, 21). Interestingly, the bacterium does not
induce Limulus gelation activity when tested with whole
cells (32), strongly suggesting that the LPS is of low
toxicity or nontoxic and that the lipid A must have a unique
structure relative to other enterobacterial lipid A's.
In the present study, the chemical structure of lipid A isolated from
F. meningosepticum LPS was characterized by
compositional study, methylation analysis, mass spectrometry
(MS), and nuclear magnetic resonance (NMR) spectroscopy.
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MATERIALS AND METHODS |
Bacteria and preparation of LPS.
F. meningosepticum
IFO 12535 strain was obtained from the Institute for Fermentation
Osaka. The bacterium was cultivated in a fermentor at 30°C for
16 h in a medium consisting of 1% (wt/vol) polypeptone, 0.2%
(wt/vol) beef extract, and 0.1% (wt/vol) MgSO4 · 7H2O, at pH 7.0. The heat-killed cells were harvested by
centrifugation and washed with distilled water, acetone, ethanol, and
diethyl ether, then acetone dried (yield, 0.5 g of cells/liter).
LPS (7.1 mg/g [dry weight] of cells) was extracted from the
acetone-dried cells with a mixture of phenol-chloroform-petroleum ether
(2:5:8 [vol/vol/vol]) according to the method described by Galanos et
al. (7) and was purified by RNase and DNase (Sigma) treatments (36) and repeated ultracentrifugation
(105,000 × g, 3 h, six times).
Isolation of lipid A.
Purified LPS (1.23 g) was hydrolyzed
with 1% (vol/vol) aqueous acetic acid at 100°C for 2 h,
followed by centrifugation (14,000 × g, 10 min). The
sediment was washed with distilled water and crude lipid A was obtained
after lyophilization. The crude lipid A (708.6 mg) was purified by
Sephadex LH-20 column chromatography according to the method described
in reference 16 to yield 420 mg of purified lipid A.
Chemical modification of LPS and lipid A.
Lipid A backbone
was prepared according to the method of Hase and Rietschel
(11). Briefly, LPS (20 mg) was treated with 0.17 M NaOH
(100°C, 1 h) and 0.1 M HCl (100°C, 30 min), followed by
reduction with NaBH4 (37°C, 16 h), complete
hydrazinolysis (103°C, 40 h), and N acetylation to obtain the
reduced and N-acetylated lipid A backbone. Escherichia coli
F654 R3 LPS was treated by the same method as reference material.
De-O-acylation of LPS and lipid A was performed by treatment with
anhydrous hydrazine at 60°C for 30 min, and the de-O-acylated preparations were recovered by a method reported previously,
(13).
De-O-acylated LPS was hydrolyzed in 0.1 M HCl at 100°C for 30 min.
The centrifugal sediment recovered was treated with acetic
anhydride-pyridine (1:1 [vol/vol]) containing small amounts of
4-dimethylaminopyridine at room temperature for 16 h, followed
by
purification using silica gel column chromatography (column,
1 by 60 cm) with toluene-ethyl acetate (20:1 [vol/vol]) as the
eluent at a
flow rate of 1 ml/min. Each 1-ml volume was fractionated,
and fractions
32 to 36 were collected to yield the peracetylated
derivative of hybrid
backbone carrying three
N-acyl and no phosphate
groups.
Monophosphoryl-methylated lipid A was prepared by treating lipid A with
diazomethane according to the method of Qureshi et
al. (
23).
GLC conditions.
Gas-liquid chromatography (GLC) analysis was
performed on a model GC-14A (Shimadzu) with a HiCap-CBM5 fused silica
capillary column (25 m by 0.25 mm; GL Science) and temperature programs A (120°C for 3 min increasing to 250°C at 3°C/min), B (150°C
for 3 min, increasing to 300°C at 5°C/min), C (140°C for 3 min,
increasing to 250°C at 3°C/min), and D (180°C for 3 min,
increasing to 300°C at 5°C/min). A chemically bonded DB-5 fused
silica capillary column (DB5MS, 30 m by 0.32 mm; J & W Scientific)
was used for the determination of the absolute configurations of amino
sugars with temperature program E (180°C for 3 min, increasing to
250°C at 3°C/min). Nitrogen was used as the carrier gas.
Analytical methods.
Each temperature program is described
under "GLC conditions" above. Total fatty acids were
determined by means of GLC and GLC-MS using temperature
program A, described under "GLC conditions" above as the methyl
ester according to the method of Haeffner et al. (8).
2-Hydroxydecanoic acid and nonadecanoic acid (GL Science) were used as
internal standards. Ester- and amide-linked fatty acids were also
assigned by GLC and GLC-MS using temperature programs A and B,
respectively, according to methods described elsewhere (13, 16,
24, 37).
Neutral and amino sugars were analyzed by GLC and GLC-MS (program C) as
the alditol acetate derivatives (
9). The absolute
configurations of amino sugar constituents were determined by
GLC
analysis (program E) of the peracetylated
S-2-butylglycoside
derivatives according to the method described in reference
13.
Amino sugars present in the lipid A possessed a
D-configuration.
Colorimetric determination of GlcN was performed by using the
Morgan-Elson reaction (
25). Total phosphorus was measured
by
the method of Lowry et al. (
17).
Methylation of the lipid A backbone was performed by the method of
Hakomori (
10,
13), and the methylated products were
analyzed
by GLC-MS using temperature program D.
Protein contents were determined with an L-8500 amino acid analyzer
(Hitachi) after hydrolysis at 110°C for 24 h in 6 M HCl
containing 1% (wt/vol) phenol.
Determination of the R,S configuration of
3-hydroxy fatty acid.
Total fatty acids obtained from lipid A (10 mg) were carboxymethylated with diazomethane at 0°C for 1 min. To
determine the R,S configuration, the product (1 mg) was directly analyzed by 1H NMR spectroscopy under
the usual conditions and in the presence of a shift reagent (1 mg),
Tris-[3-(heptafluoroprolyl-hydroxymethylene)-(+)-camphorate] europium(III) derivative (2, 13). Racemic
3-hydroxyhexadecanoic acid methyl ester was used as a reference
compound.
MS.
Liquid secondary ion (LSI)-mass spectra were measured on
a ZAB-2SEQ instrument (VG Analytical) in the positive- and negative-ion modes under the conditions described previously (13). A
mixture of m-nitrobenzyl alcohol for the positive-ion mode
and m-nitrobenzyl alcohol-triethanolamine (1:1 [vol/vol])
for the negative-ion mode, each containing a small amount of Kryptofix
222 (Aldrich), was used as the matrix.
GLC-MS and fast atom bombardment-tandem MS (FAB-MS/MS) were carried out
by the methods previously reported (
16).
NMR spectroscopy.
1H NMR spectra of 3-hydroxy
fatty acid methyl ester were recorded at 400 MHz (Varian UNITY
plus-400) in CDCl3 at room temperature. Tetramethylsilane
(TMS) was used as an internal standard (0.00 ppm) of chemical shifts.
One- and two-dimensional NMR spectra of de-O-acylated and peracetylated
lipid A and monophosphoryl-methylated lipid-A derivatives
were measured
with a JEOL
-600 instrument in CDCl
3 and
C
6D
6-(CD
3)
2SO
(9:1
[vol/vol]) at 25°C. TMS (0.00 ppm) was used as an internal
standard
for
1H and
13C NMR experiments, and
31P NMR spectra were referenced to triphenyl phosphate
(
18.0 ppm)
as an external standard. Assignments were made by a field
gradient
1H,
1H-homonuclear correlation
spectroscopy (COSY) experiment and by
13C,
1H-
and
31P,
1H-heteronuclear multiple quantum
coherence (HMQC) analyses.
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RESULTS |
Chemical analysis of free lipid A.
The chemical composition of
lipid A isolated from F. meningosepticum LPS is shown in
Table 1. Fatty acid analysis of the lipid
A revealed the presence of 13-methyltetradecanoic (iC15:0), 3-hydroxy-13-methyltetradecanoic (3-OH iC15:0),
3-hydroxyhexadecanoic (3-OH C16:0), and
3-hydroxy-15-methylhexadecanoic (3-OH iC17:0) acids in a
ratio of approximately 1:1:1:2. 1H NMR analysis of the
fatty acid fraction obtained from the lipid A indicated that these
3-hydroxy fatty acids possessed the R configuration, because
the carboxymethyl proton of 3-hydroxy fatty acid methyl esters,
resonating at 3.79 ppm under usual conditions, was dose-dependently shifted to a lower field by addition of europium(III) complex shift
reagent, and the shift behavior was identical to that of authentic
(R)3-hydroxyhexadecanoic acid methyl ester.
The lipid A also contained 467.0 nmol of total phosphate/mg, 416.7 nmol
of GlcN/mg, and 86.5 nmol of
2,3-diamino-2,3-dideoxy-
D-glucose
(GlcN3N)/mg the latter
being identified by GLC-MS and NMR analyses.
In the electron impact
(EI)-mass spectrum of the alditol acetate
derivative of GlcN3N,
characteristic fragment ions that originated
from the cleavage between
C-2 and C-3 were detected at
m/z 144
(C-1 to C-2 [C1-C2]
fragment) and
m/z 288 (C3-C6 fragment), as
shown in Fig.
1. Several daughter ions were observed at
m/z 228,
169, 168, 126, 102, and 84, caused by elimination
of acetic acid
(
60) or an
N-acetyl group (
42) from C3-C6
and C1-C2 fragment
ions. Ions at
m/z 156, 155, 114, and 113 were assigned as daughter
ions that originated from the C1-C3 fragment.
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FIG. 1.
EI-mass spectrum and fragmentation pattern of the
peracetylated derivative of GlcN3N found in F. meningosepticum lipid A.
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The protein content of the lipid A determined by amino acid analysis
was 0.36% (wt/wt).
Determination of linkage type of fatty acid.
Ester-linked
fatty acids were investigated by trans-esterification of the
lipid A with 0.25 M sodium methylate treatment. One mole each of
iC15:0 and (R)-3-OH iC15:0,
and a part of (R)-3-OH C16:0 methyl
esters, were detected as O-acyl residues, and no free fatty
acid was detected in the methanolysates by GLC analysis, indicating
that iC15:0, (R)-3-OH iC15:0, and a
part of (R)-3-OH C16:0 exist as ester-linked
residues in F. meningosepticum and that the hydroxyl groups
of 3-hydroxy fatty acids bound directly to the hydroxyl groups of lipid
A backbone are not esterified by the second acyl residue. Two moles of
(R)-3-OH iC17:0 and a significant amount of
(R)-3-OH C16:0 were quantitatively recovered as
amide-linked fatty acids from the residual de-O-acylated lipid A.
In the analysis of amide-linked 3-acyloxyacyl residues, an acyloxyacyl
group was detected on GLC (retention time, 29 min)
and was identified
as
(
R)-3-
O-(iC
15:0)-iC
17:0
methyl ester by
GLC-MS. In the EI-mass spectrum (Fig.
2), characteristic fragment
ions
generated from the cleavage of the second acyl residue of
the methyl
ester were observed at
m/z 225, 241, and 299. The molecular
mass of the derivative was confirmed to be 524 Da (
m/z
542 [M+NH
4]
+) in chemical ionization-MS.
These results indicate that ester-linked
iC
15:0 exists as a
second acyl residue of 1 mol of amide-linked
(
R)-3-OH
iC
17:0 in the lipid A.
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FIG. 2.
EI-mass spectrum and fragmentation pattern of methyl
ester derivatives of
(R)-3-O-(13-methyltetradecanoyl)-15-methylhexadecanoic
acid found in F. meningosepticum lipid A.
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Analysis of lipid A backbone.
The chemical structure of the
lipid A backbone was determined by methylation analysis. Two major
peaks were observed at 17.48 and 22.1 min on GLC at an intensity ratio
of 1.00:0.35. The former was identified as
6-O- [2-deoxy-3,4,6-tri-O-methyl-2-(N-methylacetamido)--D- glucopyranosyl)-2-deoxy-1,3,4,5-tetra-O-methyl-2-(N-methyl- acetamido)-D-glucitol, which originated from the usual GlcN disaccharide backbone, because the
EI-mass spectrum and retention time were identical to those of the same
derivative prepared from E. coli R3 LPS possessing a
(16)-linked GlcN disaccharide lipid A backbone. On the other hand, the latter peak was identified as
6-O-[2,3-dideoxy-4,6-di-O-methyl- 2,3-di-(N-methylacetamido)-D-glucopyranosyl]-2-deoxy- 1,3,4,5-tetra-O-methyl-2-(N-methylacetamido)-D-glucitol. In the
EI-mass spectrum (Fig. 3), fragment ions
representing cleavage of the glycosidic linkage of the hybrid
disaccharide were recognized at m/z 276 and 301. A
significant fragment ion at m/z 218, corresponding to
cleavage of the C-4-C-5 bond, which was characteristic of the derivative of (16)-linked GlcN disaccharide (3), was
also detected in the spectrum. As shown in Fig. 3, other characteristic fragment ions were obtained at m/z 130 (C1-C2 fragment) and
m/z 174 (C1-C3 fragment), as well as several daughter ions
based on the loss of methanol (32), acetic acid (60), or an
N-acetyl group (42). This EI-mass spectrum was
almost identical to that of the same derivative of
(16)-linked GlcN3N-GlcN disaccharide reported by Moran
et al. (19). These results indicate that two kinds of lipid
A backbones exist in the F. meningosepticum lipid A: one
(16)-linked GlcN disaccharide backbone that is normally present in enterobacterial lipid A's and a (16)-linked
GlcN3N-GlcN disaccharide, the GlcN3N residue of which was found to
possess a configuration by NMR analysis as described below.
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FIG. 3.
EI-mass spectrum and fragmentation pattern of the
permethylated
2-acetamido-6-O-(2,3-diacetamido-2,3-dideoxy-D-glucopyranosyl)-2-deoxy-D-glucitol
derivative originating from the hybrid backbone present in
F. meningosepticum lipid A.
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Distribution of fatty acid and phosphate residues.
The
distribution pattern of fatty acid and phosphate residues on the
F. meningosepticum lipid A backbone was determined by LSI-MS and FAB-MS/MS. The LSI-mass spectrum of F. meningosepticum lipid A in the negative-ion mode is shown in Fig.
4. A predominant ion observed at
m/z 1673 [M-H] corresponds to monophosphoryl
lipid A species carrying 1 mol each of (R)-3-OH
iC15:0, (R)-3-OH C16:0,
(R)-3-OH iC17:0, and (R)-3-O-(iC15:0)-iC17:0
on the lipid A backbone. Characteristic ions originating from the
reducing terminal unit of the lipid A were also detected at
m/z 767 and 795, which arise from the cleavage of the
glycosidic linkage and C-1'-C-2'---C-1'-O bond, respectively (Fig.
4). The fragment ion at m/z 749 corresponds to the daughter
ion caused by elimination of H2O from the ion at
m/z 767. These fragment ions indicate that the reducing
terminal unit of F. meningosepticum lipid A consists of
monophosphoryl GlcN replaced by 1 mol each of
(R)-iC15:0 and (R)-3-OH
iC17:0 and that a nonreducing terminal residue contains 1 mol each of GlcN (or GlcN3N), (R)-3-OH
C16:0, and
(R)-3-O-(iC15:0)-iC17:0, respectively. These results also showed that the hydroxyl groups at positions 4 and 6 in the nonreducing terminal unit of the lipid A
species having a GlcN3N-GlcN hybrid backbone exist in the free form,
because the amide-linked (R)-3-OH C16:0 and
(R)-3-O-(iC15:0)-iC17:0 are attached to positions 3 and 2 of the GlcN3N residue, respectively. Several species based on the loss of acyl and phosphate residues were usually observed in LSI-mass spectra in most of the lipid A
preparations, but F. meningosepticum lipid A appears to
be extremely homogeneous.
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FIG. 4.
LSI-mass spectrum of F. meningosepticum
lipid A in the negative-ion mode. A mixture of triethanolamine and
3-nitrobenzyl alcohol (1:1 [vol/vol]) containing a small amount of
Kryptofix 222 was used as the matrix.
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Since the difference in molecular mass between a GlcN disaccharide
backbone and an identically acylated hybrid backbone is
only 1 Da,
which is within the uncertainty of mass scale calibration,
it was
necessary to degrade the material further to differentiate
between the two backbone disaccharides. As shown in Fig.
5, two
peaks were predominantly detected
in the LSI-mass spectrum of
the de-O-acylated lipid A of
F. meningosepticum. A molecular ion
was observed at
m/z 1209 [M-H]
; it corresponds to the
monophosphoryl lipid A species carrying
three
N-acyl
residues, 1 mol of (
R)-3-OH C
16:0, and 2 mol of
(
R)-3-OH
iC
17:0 on the
(1
6)-linked
GlcN3N-GlcN hybrid backbone. Another
ion at
m/z 956 [M-H]
was identified as a monophosphoryl GlcN
disaccharide replaced
by 2 mol of amide-linked
(
R)-3-OH iC
17:0. Thus, the presence of
a
(1
6)-linked GlcN3N-GlcN hybrid backbone in addition to a GlcN
disaccharide backbone was again recognized in the LSI-mass spectrum
of
the de-O-acylated lipid A.
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FIG. 5.
LSI-mass spectrum of F. meningosepticum
de-O-acylated lipid A in the negative-ion mode. A mixture of
triethanolamine and 3-nitrobenzyl alcohol (1:1 [vol/vol]) containing
a small amount of Kryptofix 222 was used as the matrix.
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The pattern of distribution of acyl residues was also confirmed
by FAB-MS/MS of the molecular ions at
m/z 1209 [M-H]
and 956 [M-H]
, detected in the
LSI-mass spectrum of the de-O-acylated lipid
A (Fig.
5). The tandem
spectrum of the ion at
m/z 1209 [M-H]
is
shown in Fig.
6. A fragment ion at
m/z 406.2 caused by the
cleavage of the C-1-O---C-2-C-3
bond of the reducing terminal unit
of the backbone revealed that a
phosphate residue was linked to
position 1 of the backbone and that an
amino group at position
2 was N acylated with (
R)-3-OH
iC
17:0 (Fig.
6). Other characteristic
fragment ions were
observed at
m/z 508.3 and 524.3 and
m526.3
z and 554.3; they were generated by the
cleavage of the glycosidic
linkage and C-1'-C-2'---C-5'-O bond,
respectively (Fig.
6). The
tandem spectrum of the ion at
m/z
956 [M-H]
was almost identical to that of the ion at
m/z 1209 [M-H]
(data not shown).
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FIG. 6.
Negative-ion mode FAB-MS/MS of the molecular ion at
m/z 1209 [M-H] detected in the LSI-mass
spectrum of F. meningosepticum de-O-acylated
lipid A.
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Structure of the peracetylated derivative of de-O-acylated lipid A
having a hybrid backbone.
LSI-MS and NMR analyses of the
peracetylated derivative of the hybrid backbone carrying three
N-acyl and no phosphate residues were performed. In
positive-ion mode LSI-MS, a molecular ion was detected at
m/z 1467 [M+H]+, and a fragment ion
originating from the nonreducing terminal unit was also observed at
m/z 853, indicating that 1 mol each of (R)-3-OH
C16:0 and (R)-3-OH iC17:0 was
distributed on the nonreducing terminal unit and the remaining 1 mol of
(R)-3-OH iC17:0 was present in the reducing
terminal residue. The 1H and 13C NMR data are
shown in Tables 2 and 3. In the
1H NMR analysis (Table 2), the J2,3 (10.4 Hz),
J3,4 (9.9 Hz), and J4,5 (9.9 Hz) values of the
nonreducing terminal unit indicated that this sugar constituent
possesses the gluco-conformation, and the J1,2 value (8.4 Hz) revealed the -configuration. Two amide protons were assigned at
6.48 ppm and 6.37 ppm, and they were cross-coupled with H-2 (3.96 ppm) and H-3 (4.09 ppm), respectively, indicating that this sugar
unit possesses two amino groups at positions 2 and 3. On the other
hand, the - and -anomers of the reducing terminal unit, which
were produced during acetylation, were assigned. In both anomers, the
J2,3, J3,4, and J4,5 values indicated the gluco-configuration, and an amino group exists at position 2, because the amide proton cross-coupled with H-2 was detected at 6.04 ppm for the -anomer and 6.34 ppm for the
-anomer. H-6a and H-6b were slightly shifted to a higher field,
because the nonreducing terminal unit was linked to position 6. These findings were also supported by the 13C NMR data shown in
Table 3. These results clearly showed
that the hybrid backbone present in F. meningosepticum
lipid A consists of a (16)-linked GlcN3N-GlcN hybrid
disaccharide.
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TABLE 2.
1H NMR data for the peracetylated derivative
of F. meningosepticum de-O-acylated lipid A-HCl
containing a hybrid backbonea
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TABLE 3.
13C NMR data for the peracetylated derivative
of F. meningosepticum de-O-acylated lipid
A-HCl containing a hybrid backbonea
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NMR analysis of monophosphoryl-methylated lipid A.
In order to
determine the positions of free-hydroxyl groups and the attachment site
of phosphate residue, one- and two-dimensional 1H NMR
analyses of the monophosphoryl-methylated derivative of F. meningosepticum lipid A were performed (Table
4). The H-1 signal (6.01 ppm;
J1,2 = 3.12 Hz) of the reducing terminal unit of the lipid
A was shifted at 0.55 ppm to a lower field in comparison to the
unsubstituted H-1 signal (5.46 ppm) (23), indicating that
the phosphate residue was -glycosidically linked to position 1. Since direct J coupling of the hydroxyl proton with H-4 (3.98 ppm) was
detected at 5.86 ppm, the hydroxyl group at position 4 of the reducing
terminal unit was identified as being free form. Furthermore, an amide
proton was assigned at 8.12 ppm (2-NH; JNH,2 = 9.34 Hz),
and it was cross-coupled with the proton at position 2 (4.68 ppm). The
other signals that originated from the unit were detected at 4.10 ppm
(H-6b), 4.19 ppm (H-5), 4.39 ppm (H-6a), and 5.64 ppm (H-3). An H-1'
proton was detected at 5.05 ppm (J1',2' = 7.51 Hz) as a
signal originating from the nonreducing terminal unit of the lipid A. The resonation was shifted to a higher field (0.41 ppm) by
glycosidical substitution, and the J1',2' value (7.51 Hz) revealed a -configuration. However, no other signals were
able to be clearly assigned because of the heterogeneity.
The location of phosphate groups was directly determined by
31P NMR analysis. One signal was predominantly observed at
0.669
ppm in the
31P NMR spectrum. This signal was
cross-coupled with the H-1 proton
in
31P,
1H-HMQC analysis, proving that the
phosphate residues are attached
to position 1 of the lipid A backbone.
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DISCUSSION |
The proposed chemical structure of F. meningosepticum lipid A, determined by chemical and
physicochemical analysis in the present study, is shown in Fig.
7. It was noted that the hydrophilic backbone consisted of an unusual hybrid backbone identified as a
(16)-linked GlcN3N-GlcN disaccharide in addition to a
(16)-linked GlcN disaccharide backbone, which is widely
distributed in many LPS molecules (38). Both backbones are
1-O--glycosidically phosphorylated, and
(R)-3-OH iC15:0 and (R)-3-OH
iC17:0 attached to the reducing terminal unit are linked to
positions 3 and 2, respectively. The hydroxyl group at position 4 of
the unit exists in the free form in the lipid A molecule, and position
6 is the site to which the nonreducing terminal unit is linked.
Although the exact attachment sites of (R)-3-OH
C16:0 and
(R)-3-O-(iC15:0)-iC17:0 on the nonreducing terminal unit were not determined, they are assumed
to link to positions 3' and 2' of the unit based on the following
results: (i) all
(R)-3-O-(iC15:0)-iC17:0
existed as an amide-linked acyl residue, while (R)-3-OH
C16:0 was detected as both amide- and ester-linked
residues; (ii) monophosphoryl GlcN disaccharide carrying 2 mol of
amide-linked (R)-3-OH iC17:0 at positions 2 and
2' was determined by LSI-MS to be a single component of de-O-acylated
lipid A; and (iii) the hydroxyl groups at positions 4' and 6' of lipid
GlcA species having N3N-GlcN hybrid backbones were identified as free
form by LSI-MS.
F. meningosepticum lipid A has a number of
chemically unique characteristics compared to other
enterobacterial lipid A's (12, 14, 38). F. meningosepticum lipid A mainly contains relatively longer-chain
and isoform fatty acids, in contrast to the enterobacterial lipid A's,
which contain (R)-3-hydroxytetradecanoic acid as the main
constituent of acyl residues. Regarding the location of fatty acids,
F. meningosepticum lipid A contained only 1 mol of an
acyloxyacyl group at position 2', while two or sometimes three
acyloxyacyl residues are present at positions 2' and 3', or
additionally at position 2 (in the case of Salmonella), of
the nonreducing terminal in enterobacterial lipid A's. A pattern of
distribution of phosphate groups different from that of enterobacterial
lipid A's was also recognized in F. meningosepticum
lipid A, which completely lacked an ester-linked phosphate residue
attached to position 4' of the lipid-A backbone. Interestingly, a quite
similar structure has been found in the lipid A isolated from oral
anaerobic gram-negative bacteria such as Bacteroides
fragilis (34) and P. gingivalis (16). These lipid A's have the same fatty acid
composition and phosphate group distribution pattern as
F. meningosepticum lipid A, although a small amount of
ester-linked phosphate group was recognized in P. gingivalis
lipid A. However, F. meningosepticum lipid A could be
obviously distinguished chemically from these oral bacterial lipid A's
based on its hybrid backbone consisting of (16)-linked
GlcN3N-GlcN disaccharide in addition to the usual (16)-linked
GlcN disaccharide. Such an unusual GlcN3N has been found to be a
component of the lipid A backbone in Brevundimonas (Pseudomonas) diminuta, Brevundimonas
vesicularis, Rhodopseudomonas viridis,
Rhodopseudomonas sulfoviridis, Rhodopseudomonas
palustris, Legionella pneumophila, and
Campylobacter jejuni (1, 15, 19, 33, 35). The
backbones of R. diminuta and B. vesicularis consist of the disaccharide of the diamino sugar, in which position 1 may be replaced by D-glucuronic acid. R. viridis, R. sulfoviridis, and R. palustris
lipid A's have unique backbones consisting of the diamino
monosaccharide only. The backbone of C. jejuni lipid A is
similar to that of F. meningosepticum, which contains a
(16)-linked GlcN3N-GlcN disaccharide forming the lipid A backbone
in addition to a (16)-linked GlcN disaccharide and a GlcN3N
disaccharide. With the exception of these structural similarities,
F. meningosepticum lipid A has a unique fatty acid
composition, phosphate distribution, and hybrid backbone. Moreover, the
lipid A preparation seems to contain unknown compounds in the backbone
structure, because the total amount of GlcN3N recovered by the
compositional analysis does not match the theoretical quantity obtained
from the other structural analysis, which indicates the existence of a
hybrid backbone in the lipid A. Recovery was not increased by any
hydrolysis or degradation procedures tested. The reason for this is not
clear, but it may be based on the tight linkage of the unknown
compounds to the GlcN3N residue, which may make the detection of the
amino sugar impossible.
We have recently proposed the complete lipid A structure of P. gingivalis (16). Using the lipid A, we demonstrated
that the lipid A moiety of P. gingivalis LPS, which
exhibited relatively lower activity in LPS-responsive mice than lipid A
moieties from enteric bacteria, specifically mediates the activation of
LPS-unresponsive C3H/HeJ mice (30, 31). Since the chemical
structure of F. meningosepticum has similarities
to that of P. gingivalis, as found in the present
study, the biological properties of this lipid A are of especially
great interest, and studies are currently in progress in our
laboratory.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Microbiology, National Institute of Health Sciences, 1-18-1 Kamiyoga,
Setagayaku, Tokyo 158, Japan. Phone: 81-3-3700-1141, ext. 274. Fax:
81-3-3707-6950. E-mail: tanamoto{at}nihs.go.jp.
| |
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Journal of Bacteriology, August 1998, p. 3891-3899, Vol. 180, No. 15
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
