Structural analysis of deacylated lipopolysaccharide
of
Escherichia coli
strains 2513 (R4 core-type)
and F653 (R3 core-type)
Sven Mu¨ ller-Loennies, Buko Lindner and Helmut Brade
Borstel Research Center, Center for Medicine and Biosciences, Borstel, Germany
Lipopolysaccharide (LPS) of Escherichia coli strain 2513 (R4
core-type) yielded after alkaline deacylation one major
oligosaccharide by high-performance anion-exchange chro-
matography (HPAEC) which had a molecular mass of
2486.59 Da as determined by electrospray ionization mass
spectrometry. This was in accordance with the calculated
molecular mass of a tetraphosphorylated dodecasaccharide
of the composition shown below. NMR-analyses identified
the chemical structure as
where
L
-a-
D
-Hep is
L
-glycero-a-
D
-manno-heptopyranose
and Kdo is 3-deoxy-a-
D
-manno-oct-2-ulopyranosylonic
acid and all hexoses are present as
D
-pyranoses.

D
-manno-oct-2-ulosonic acid; LPS, lipopolysaccharide.
(Received 4 September 2002, accepted 22 October 2002)
Eur. J. Biochem. 269, 5982–5991 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03322.x
Lipopolysaccharide (LPS) is the major component of the
outer leaflet of the outer membrane of Gram-negative
bacteria [1]. LPS of enterobacteria consist of three
domains, namely lipid A, core-region and O-antigen [2].
Due to its exposed location, it is the major target of the
humoral immune response in mammals and the lipid A
moiety is responsible for many of the pathological effects
seen in septic shock patients. Whereas the chemical
structure of the O-antigen is highly variable, the core-
region and lipid A show only a limited structural
variability within the same species. This prompted many
investigators to attempt the isolation of antibodies
directed against the conserved regions of LPS, i.e. lipid A
andcore-region(reviewedin[3]).Itwasassumedthat
these antibodies would be both cross-reactive and cross-
protective against different Gram-negative pathogens.
Whereas a cross-protective effect was described for a
polyclonal antiserum as early as in 1966 [4], all
subsequently isolated monoclonal antibodies failed to
show cross-reactivity in vitro and cross-protectivity in vivo
[3], except one reported by DiPadova et al.(mAb
WN1 222-5). This mAb recognized LPS from all tested
clinical isolates of E. coli, Salmonella,andShigella in
Western-blots and showed cross-protective effects in vivo
against endotoxic activities of LPS [5]. The cross-reacti-
vity was attributed to a common epitope located in the

Neutral sugars, GlcN, Kdo and bound organic phosphate
were determined as described [11].
Preparation of deacylated LPS of
E. coli
2513
LPS (5 g) was de-O-acylated by mild hydrazinolysis [7]
(yield 3.84 g) and 400 mg of the latter were subjected to
alkaline de-N-acylation as described [12]. After neutraliza-
tion by addition of ion exchanger Amberlite IRA120 H
+
(Serva), 160 mg of the deacylated oligosaccharide fraction
(yield 217 mg) was subjected to high-performance anion-
exchange chromatography (HPAEC; eight runs of 20 mg
each) using a semipreparative CarboPak PA100 column
(9 · 250 mm) and a DX300 chromatography system (Dio-
nex, Germany). The main (fraction 2; oligosaccharide 1,
yield 31.44 mg) and the minor oligosaccharide (fraction 1;
oligosaccharide 2, yield 10.96 mg) were collected, neutral-
ized and desalted as described above by addition of
ion-exchanger followed by lyophilization. Conditions for
semipreparative and analytical HPAEC were as described
previously [13].
Preparation of deacylated LPS of
E. coli
F653
LPS (2.11 g) was de-O-acylated by mild hydrazinolysis
(yield 1.425 g) and 902.5 mg were further subjected to
alkaline de-N-acylation as above. The solution was neut-
ralized by addition of 8
M

31.5 p.p.m. (
13
C). All spectra were run at a temperature
of 300 K.
NMR of oligosaccharide 1 (R4 core). Two-dimensional
homonuclear
1
H,
1
H-COSY was performed with a double
quantum filter and time-proportional phase incrementation
(TPPI) (DQF-COSY). The Bruker
COSYDFTP
pulseprogram
was modified to allow water suppression with 10 Gaussian
shaped pulses of 100 ms defined by 1024 points during the
relaxation delay. Five-hundred and twelve experiments of
4096 data points each were recorded over a spectral width of
6.5 p.p.m. in each dimension. Prior to Fourier transforma-
tion F1 was zero-filled to 1024 data points.
TOCSY was performed at a spinlock field strength of
8 kHz for 75.15 ms using the Bruker
MLEVPRTP
pulse
Ó FEBS 2002 Chemical structure of E. coli R3 and R4 LPS (Eur. J. Biochem. 269) 5983
program and the same experimental parameters that were
used for TOCSY-ROESY (TORO).
A TORO-spectrum [14–17] was recorded as a two-
dimensional experiment using a fixed delay as the second
mixing time (ROESY-step). The spectrum was recorded

4
MLPRTP
(HMQC-TOCSY),
INDECOBIMLTPPR
(DEPT-
HMQC-TOCSY), and
INV
4
LRNDPR
(HMBC) were used.
These spectra were recorded with 4096 data points in F2
and 512 experiments in F1 over spectral widths of 10 and
120 p.p.m, respectively. Zero-filling was applied to 1024
data points in F1. For TOCSY a spinlock period of 81 ms
was applied at a field strength of 8.3 kHz. For DEPT-
HMQC-TOCSY the sweep width was reduced to 15 p.p.m.
in F1 and 3.5 p.p.m. in F2. Two-hundred and fifty-six
experiments were recorded at 2048 data points per incre-
ment and a TOCSY mixing time of 67 ms. For HMBC, F1
was enlarged to 180 p.p.m. and the delay for the evolution
of long-range couplings was set to 50 ms.
31
P spectroscopy was performed after addition of NaOD
(Sigma) until all signals appeared as sharp singuletts. The
pD was then approximated using pH paper and found to be
pD 9.
31
P,
1
H-HMQC was performed using a modified Bruker

multiplied by a shifted sine bell function and zero-filled in
F1 and F2, 4096 and 512 data points, respectively. The spin-
lock for ROE of 5.6 kHz was applied for 150 ms and
TOCSY was performed with a mixing time of 77 ms. The
field strength of the TOCSY spin-lock in this experiment
was 9.4 kHz.
HMQC, HMBC and HMQC-TOCSY were recorded as
z-gradient experiments using standard Bruker software. The
experimental setup was otherwise identical to the same
spectra recorded of oligosaccharide 1. DEPT-HMQC-
TOCSY was run as described above but the spectral width
was reduced to 30 p.p.m. in F1 and 5 p.p.m. in F2. One-
hundred and twenty-eight experiments of 64 scans with 2048
data points were recorded. The TOCSY spinlock was
applied for 90 ms.
Mass spectrometry
Mass spectra were recorded in the negative ion mode of
the mixture of oligosaccharides prior to HPAEC, of the
isolated main oligosaccharide of deacylated LPS and of
acylated purified LPS from E. coli F2513 (R4 core-type).
In addition, the deacylated minor core-oligosaccharide of
E. coli F653 (R3 core-type) was analyzed. Negative ion
electrospray ionization mass spectra were recorded on a
Fourier Transform Ion Cyclotron Resonance FT-ICR
mass spectrometer (APEX II, Bruker Daltonics, Billerica,
USA) equipped with a 7 Tesla actively shielded magnet
andanApolloionsource.Samplesweredissolvedata
concentration of  10 ngÆlL
)1
in a 50 : 50 : 0.001 (v/v/v)

AcP
219 1.1
Kdo
HCl
301 1.5
P
org
812 4.1
Glc 381 1.9
Gal 648 3.2
L
,
D
-Hep 638 3.2
C12 : 0 125 0.6
C14 : 0 166 0.9
3OH-C14 : 0 636 3.3
a
Relative to GlcN ¼ 2.0.
5984 S. Mu
¨
ller-Loennies et al. (Eur. J. Biochem. 269) Ó FEBS 2002
accordance with the common acylation pattern of E. coli
lipid A [18].
Analytical HPAEC revealed that the deacylated LPS
fraction contained one major oligosaccharide isolated by
semipreparative HPAEC. The charge deconvoluted negat-
ive ion-mode ESI-FT-ICR mass spectrum of the major
oligosaccharide fraction (Fig. 1) obtained by deacylation of
LPS revealed a prominent ion with a mass of 2486.59 m/z.

signals originating from 3-deoxy protons of Kdo-residues
were present. Full assignment of proton and carbon
chemical shifts and determination of
3
J
H,H-
coupling con-
stant values identified two pyranosidic Kdo-residues. Their
a-configuration was evident from the resonance frequencies
of the deoxy-protons (equatorial H3 > 2.4 p.p.m. for
b-Kdop) and the chemical shift values of the H-5 protons
[19]. All sugars were present as pyranoses which was
deduced from their C-4 carbon chemical shifts (above
80 p.p.m. for furanoses, Table 3). Correlation signals from
anomeric protons to intraresidue C-5 in HMBC corrobor-
ated the pyranose-configuration of sugar residues. All other
sugars except two were also a-configurated which was
determined by the analysis of J
C-1,H-1
-coupling constants
(> 172 Hz) from a HMQC spectrum recorded without
decoupling during acquisition. Signals of anomeric carbons
at 99.75 p.p.m. and 103.20 p.p.m. were assigned to a b-
GlcpN (164 Hz, residue B) and b-Galp (164 Hz, residue M),
respectively. Their b-configuration was confirmed by their
3
J
H,H-
coupling constants ( 8 Hz) and their intraresidual
NOE connectivities between H-1, H-3 and H-5. Three

tion spectrum indicated its chemical shift at 4.025 p.p.m., in
agreement with the chemical shift of the same proton in
previously analyzed oligosaccharides from E. coli J-5 [13].
Further residues were identified as a-Glcp (residues G and
I), a-Galp (residues K and L), and a-GlcpN(A).
The analysis of an HMBC spectrum showing intraresid-
ual cross-correlation signals from anomeric protons to
carbons C-3 and C-5 was important for the assignment of
spin-systems and chemical shifts of carbon. Additionally,
long-range correlations between protons of adjacent sugar
residues across the glycosidic bond established their
sequence; this was confirmed by the analysis of a NOESY
spectrum and a 2D-TOCSY-ROESY (TORO) [14–17]
spectrum (Fig. 3). This latter experiment facilitated the
assignment because all protons connected by scalar cou-
plings and part of a spin system detectable by TOCSY show
connectivities to protons close in space to any of these
protons. Therefore, more correlation signals are observed in
the region of anomeric protons that resolves signal overlap,
the identification of ROE signals is simplified and corro-
borated by further correlation signals within the adjacent
residue. Furthermore, due to the asymmetry of the experi-
ment with respect to the magnetization transfer mechanism
the pulse sequence generates signals in the vertical plane
Fig. 1. Charge deconvoluted ESI-FT-ICR mass spectrum of the deac-
ylated LPS of E. coli strain 2513 (R4 core). Shown are the spectra of
the mixture prior to separation (A) and of the isolated oligosaccharide
1(B).
Fig. 2.
1

-a-
D
-Hepp-(1 fi 7)-
L
-a-
D
-Hepp-(1 fi 3)-
L
-a-
D
-Hepp which
was connected to the inner Kdo (residue C) in position 5.
The residues G, I, K, L, and M were those of the outer core
and the NMR analyses confirmed the results obtained by
methylation analysis [9]. Long-range NOEs were observed
between H-3a (strong) and H-3e (very weak) of a-Kdop
(residue C) and H-1 of
L
,
D
-Hepp (residue H) and between
the anomeric proton of the inner
L
,
D
-Hepp residue (E) and
the equatorial H-3 of the side-chain a-Kdop (residue D).
Four phosphate residues were identified (Fig. 4A, Table
4) that were shown by HMQC to be linked to protons A1
(a-GlcpN), B4 (b-GlcpN), E4 (

3 1.780 2.156 4.108 4.051 3.663 3.999 3.751 3.957
4 1.787 2.163 4.133 4.044 3.649 3.998 3.742 3.985
1Efi 3-aHep 4P 5.289 4.061 4.128 4.415 4.218 4.097 3.917 3.766
3 5.287 4.067 4.136 4.412 4.223 4.102 3.903 3.784
4 5.293 4.084 4.168 4.430 4.205 4.129 3.934 3.795
1Ffi 3,7-aHep 4P 5.089 4.402 4.115 4.405 3.849 4.245 3.698 3.698
3 5.092 4.385 4.093 4.354 3.826 4.260 3.696 3.696
4 5.176 4.380 4.059 4.012 3.732 4.146 3.674 3.752
1Gfi 3-aGlc 5.195 3.638 4.073 3.732 3.885 3.891 3.785
3 5.198 3.595 4.185 3.776 3.878 3.863 3.729
4 5.289 3.705 4.116 3.787 3.880 3.926 3.749
1HaHep 4.934 3.964 3.857 3.846 3.638 4.042 3.739 3.739
3 4.943 3.968 3.866 3.848 3.648 4.003 3.673 3.758
4 fi 7-aHep 4.944 3.993 3.874 3.849 3.621 4.233 3.909 3.730
1Ifi 2,4-aGlc 5.754 3.743 4.034 3.727 4.189 4.014 3.816
3 fi 2,3-aGal 5.938 4.189 4.342 4.319 4.313 3.770 3.770
4 5.903 4.225 4.321 4.358 4.299 3.800 3.769
1Kfi 2-aGal 5.542 3.990 4.097 3.857 4.072 3.779 3.734
3 fi 2-aGlc 5.512 3.735 3.885 3.470 3.765 3.922 3.746
4 5.550 3.746 3.910 3.495 3.765 3.931 3.771
1LaGal 5.247 3.840 3.943 3.983 4.138 3.700–3.777 3.700–3.777
3 aGlc 5.222 3.552 3.752 3.453 3.907 3.929 3.771
4 5.208 3.566 3.756 3.450 3.935 3.933 3.840
1MbGal 4.451 3.538 3.652 3.913 3.708 3.522 3.618
3 aGlcN 5.415 3.382 3.902 3.556 4.057 3.950 3.814
4 5.427 3.404 3.926 3.571 4.079 3.957 3.810
4NaGlcN 5.224 3.352 3.944 3.494 3.763 3.778 3.778
5986 S. Mu
¨
ller-Loennies et al. (Eur. J. Biochem. 269) Ó FEBS 2002

-Hepp (E, F, H), three Glcp (G,
K, L) residues and one Galp (I) residue. All sugars except
one were present as a-pyranoses as evident from
3
J
H-1,H-2
and J
C-1,H-1
coupling constants and
13
C chemical shifts. The
chemical shift and the
3
J
H-1,H-2
coupling constant of the
anomeric proton (8.5 Hz) of one GlcpN (residue B) as well
as NOE correlation signals between H-1, -3 and -5
confirmed its b-configuration. In addition, four phosphate
residues were present (Fig. 4B, Table 4) which were located
at positions 1 and 4¢ of the lipid A backbone (GlcpN-
disaccharide at the reducing end, residues A and B) and at
positions 4 of both
L
,
D
-Hepp-residues E and F as deter-
mined by
31
P,

-
Glcp1 fi 2-[a-
D
-GlcpN1 fi 3]-a-
D
-Galp1 fi 3-a-
D
-Glcp.
These residues thus represented the outer core and con-
firmed the results previously obtained by methylation
analyses [6,7,9,21]. The sequence deduced from observed
NOEs was corroborated by cross-correlation signals in
HMBC across the glycosidic bonds.
In comparison to the major oligosaccharide 2,
1
H- (Fig. 6B) and
13
C-NMR-spectra of the minor core
oligosaccharide 3 contained an additional set of signals
originating from an a-
D
-GlcpN-residue which was there-
fore a tridecasaccharide. NOE NMR-spectra, ROTO and
HMBC confirmed that this residue (N, Fig. 7) was located
at position 7 of the side chain
L
,
D
-Hepp (residue H)
leading to downfield shifts of protons F7a and F7b as well

phosphate residue. This compound, however, was present
only in minute amounts and was not seen in HPAEC. It is
known that in enterobacterial LPS the first heptose (residue
E) is substituted in position 4 with 2-aminoethanol diphos-
phate instead of a monophosphate [2]. The origin of this
oligosaccharide can thus be explained by the possibility that
the strong alkaline treatment did not hydrolyze the 2-
aminoethanol diphosphodiester between phosphate groups
but to a small extent between the 2-aminoethanol and
phosphate leading to a diphosphate at this position. A mass
spectrum of purified LPS (not shown) recorded to explain
the origin of this molecular ion and of additional molecular
ions with a lower mass of m/z 18, which were present in the
spectra of alkali treated LPS (Fig. 1), only contained
molecular ions with a mass difference of m/z 123 (2-
aminoethanol monophosphate) but not with additional m/z
80 (phosphate). Therefore an additional monophosphate
substitution at a different location may be excluded.
Furthermore, the spectrum did not contain molecular ions
with a lower mass of Dm/z 18 which thus were artefacts.
Similar artefacts have been described previously by Olstho-
orn et al. [22].
There was no heterogeneity with respect to the
substitution by additional sugar residues attached to the
side chain-heptose as observed in other LPS-core struc-
tures (E. coli F470, E. coli F653, Shigella sonnei, Shigella
flexneri, Erwinia carotovora FERM P-7576, Proteus
mirabilis R110/1959, Citrobacter freundii O23) [2].
Notably, the phosphate substitution at position 4 of the
second heptose (residue F) is quantitative and no smaller

1
H-NMR spectra of E. coli F653 major (oligosaccharide 3, A)
and minor (oligosaccharide 4, B) oligosaccharide.
5988 S. Mu
¨
ller-Loennies et al. (Eur. J. Biochem. 269) Ó FEBS 2002
phosphorylase and that both reactions occur simultaneously
since no molecules are detected which contain both, GlcpN
and phosphate, or no GlcpN and no phosphate at this
position. The presence of phosphate at this position in
oligosaccharides, which possess a side-chain heptose residue
is explained by the fact that the activity of heptosytrans-
ferase III (WaaQ) is dependent on the phosphate at this
position [23]. Thus, it may be that the reaction of this
GlcpN-transferase is energetically driven by the removal of
the phosphate. It may also be that two different enzymatic
activities are active and the lack of the side-chain GlcpN
substitution in the R4 core oligosaccharide can be either due
to the lack of the responsible GlcpN-transferase or by the
lack of a phosphatase if the phosphate has to be removed
prior to glycosylation.
The reactivity of E. coli 2513 LPS with mAb WN1 222-5
can be explained by an inner-core structure identical to
those found in LPS from all core-types of E. coli and
Salmonella. Investigation of the inter-residual NOE con-
nectivities revealed that no long range NOEs indicative of a
backfolding of the outer core were found. Therefore, it is
apparent that the outer core and the inner core form two
structural confined domains, which may be a prerequisite
for the accessibility of the inner core sugars for the binding

1HaHep 101.31 70.44 71.65 67.54 72.74 70.28 64.19
3 101.40 71.19 71.89 67.53 72.79 70.40 64.20
4 fi 7-aHep 101.60 71.15 71.71 67.36 73.04 69.04 71.46
1Ifi 2,4-aGlc 95.86 74.96 70.18 79.17 71.33 60.30
3 fi 2,3-aGal 95.14 69.37 72.31 65.55 71.09 61.78
4 95.54 69.31 72.22 65.44 71.03 62.06
1Kfi 2-aGal 93.50 73.43 69.18 71.06 72.24 62.30
3 fi 2-aGlc 92.57 76.27 72.81 70.93 75.43 61.89
4 92.81 76.73 72.83 70.77 75.19 62.05
1LaGal 96.20 68.54 69.72 70.79 71.55 61.59
3 aGlc 97.10 72.74 74.36 70.56 73.34 62.06
4 97.40 72.74 74.38 70.57 73.35 61.65
1MbGal 103.20 71.26 72.84 68.99 75.68 63.76
3 aGlcN 91.87 55.03 71.08 70.87 73.55 61.59
4 91.87 55.03 71.17 70.91 73.59 61.68
4NaGlcN 97.42 55.33 71.08 70.84 73.50 61.67
Table 4.
31
P-NMR chemical shifts (p.p.m.) of deacylated LPS of E. co li
strain 2513 (R4 core-type, 1) and F653 (R3 core-type, 3 major and 4
minor).
Residue
Compound
134
A fi 6-aGlcN 1P 3.16 3.20 3.19
B fi 6-bGlcN 4P 4.38 4.40 4.41
E fi 3-aHep 4P 5.06 5.10 4.96
F fi 3,7-aHep 4P 5.00 5.05
Ó FEBS 2002 Chemical structure of E. coli R3 and R4 LPS (Eur. J. Biochem. 269) 5989
computational calculation of a partial oligosaccharide

With respect to the recognition of these structures by
antibodies and the structural characterization of epitopes
leading to cross-reactivity, the full assignment of carbon
and, in particular, proton resonances now provides the
basis for detailed NMR-based conformational analysis of
the inner core region of enterobacterial LPS. Furthermore,
it will allow the interpretation of saturation transfer
difference measurements [24] aiming at further character-
ization of the epitope recognized by the cross-reactive
mAb WN1 222-5.
ACKNOWLEDGEMENTS
We greatfully acknowledge the technical assistance of V. Susott, and
Helga Lu
¨
thje as well as the kind gift of the minor core oligosaccharide
from E. coli rough mutant F470 by Drs O. Holst and E. Vinogradov.
This research was financially supported by the Deutsche Forschungsg-
emeinschaft Grants DFG L1-448 (BL).
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Ó FEBS 2002 Chemical structure of E. coli R3 and R4 LPS (Eur. J. Biochem. 269) 5991