Báo cáo khoa học: Epitope mapping of the O-chain polysaccharide of Legionella pneumophila serogroup 1 lipopolysaccharide by saturation-transfer-difference NMR spectroscopy pot

Epitope mapping of the O-chain polysaccharide of
Legionella
pneumophila
serogroup 1 lipopolysaccharide
by saturation-transfer-difference NMR spectroscopy
Oliver Kooistra
1
, Lars Herfurth
2
, Edeltraud LuÈ neberg
3
, Matthias Frosch
3
, Thomas Peters
2
and Ulrich ZaÈ hringer
1
1
Research Center Borstel, Center for Medicine and Biosciences, Germany;
2
Institute for Chemistry, Medical University of Lu
È
beck,
Germany;
3
Institute for Hygiene and Microbiology, University of Wu
È
rzburg, Germany
Two modi®cations of 5-acetimidoylamino-7-acetamido-
3,5,7,9-tetradeoxy-
D

-glycero-
D
-galacto-non-2-ulosonic acid
(5-N-acetimidoyl-7-N-acetyllegionaminic acid) in the
O-chain polysaccharide (OPS) of the Legionella pneumophila
serogroup 1 lipopolysaccharide (LPS) concern N-methyla-
tion of the 5-N-acetimidoyl group in legionaminic acid. Both
N-methylated substituents, the (N,N-dimethylacetimidoyl)
amino and acetimidoyl(N-methyl)amino group, could be
allocated to one single legionaminic acid residue in the long-
and middle-chain OPS, respectively. Using mutants devoid
of N-methylated legionaminic acid derivatives, it could be
shown that N-methylation of legionaminic acid correlated
with the expression of the mAb 2625 epitope. In the present
study we investigated the binding of the LPS-speci®c mon-
oclonal antibody mAb 2625 to isolated OPS with surface-
plasmon-resonance biomolecular interaction analysis and
saturation-transfer-dierence (STD) NMR spec troscopy in
order to map the mAb 2625 epitope on a molecular level. It
could be demonstrated that the b inding anity of the
N-methylated legionaminic acid derivatives was indepen-
dent from the size of the isolated OPS molecular species. In
addition, STD NMR spectroscopic studies with polysac-
charide ligands with an average molecular mass of up to
14 kDa revealed that binding was mainly mediated via the
N-methylated acetimidoylamino group and via the closely
located 7-N-acetyl group of the respective legionaminic acid
residue, t hus indicating these derivatives to represent t he
major epitope of mAb 2625.
Keywords: lipopolysaccharide; Legionella pneumophila;

bioanity studies; NMR.
Legionella pneumophila is a facultative intracellular Gram-
negative bacterium and the cause of legionellosis, a pneu-
monia with a sometimes fatal progression [1]. The reservoirs
of legion ellae a re natural or man-made water systems and
their natural hosts are various amoebae species [2]. In the
human lung L. pneumophila invades and replicates within
alveolar macrophages [3]. The serogroup-speci®c antigens of
the Gram-negative legionellae reside in the lipopolysacchar-
ide (LPS) of the outer membrane [4,5].
The O-chain polysaccharide (OPS) of serogroup (Sg) 1
LPS is a homopolymer of the 5-N-acetimidoyl-7-N-acetyl
derivative of 3,5,7,9-tetradeoxy-
D
-glycero-
D
-galacto-non-2-
ulosonic acid, termed legionaminic acid (Fig. 1, structure 1)
[6,7], wh ich i s quantitatively 8-O-acetylated in strains
belonging to the Pontiac group [5,6,8], but only partially
in other Sg 1 strains of the non-Pontiac group [5,9].
In L. pneumophila Sg 1 LPS the OPS is linked to a terminal
nonreducing
L
-rhamnose (Rha
II
) of the core oligosaccharide
[10,11]. The core of the LPS lacks heptose and phosphate,
contains abundant 6-deoxy sugars and N- acetylated amino
sugars, and is highly O-acetylated [9±12].

Recently, a phase-variable expression of a virulence-
associated LPS epitope of L. pneumophila has been
described previously [13]. Chromosomal insertion and
excision of a 29-kb unstable genetic element, possibly o f
phage origin, was identi®ed as the molecular mechanism for
phase variation [ 14]. The altered LPS phenotype of the
spontaneous phase variant could be distinguished with the
aid of the LPS-speci®c mAb 2625. The reactivity of mAb
2625 w as related to the presence of N-methyl groups at the
5-N-acetimidoyl group of legionaminic acid, a modi®cation
of bacterial polysaccharid es, which is described for the ®rst
time in the accompanying paper [ 15]. The c omponents
identi®ed were the 5-N-(N,N-dimethylacetimidoyl)-7-N-
acetyl- and 5-N-acetimidoyl-5-N-methyl-7-N-acetyl- deriv-
atives of legionaminic acid (Fig. 1, structures 2 and 3,
respectively) probably located proximal to the core oligo-
saccharide of long and middle O-chain LPS from wild-type
RC1 [15]. Although serological data strongly indicate that
the N-methylated derivatives of legionaminic acid are
located close to the outer region of the core oligosaccharide,
their precise position could, unfortunately, not be deter-
mined [15]. N-Methylation was limited to one single
Correspondence to U. Za
È
hringer, Forschungszentrum Borstel,
Zentrum fu
È
r Medizin und Biowissenschaften, Parkallee 22, D-23845
Borstel, Germany. Fax: + 49 4537 188612, Tel.: + 49 4537 188462 ,
E-mail:

Abbreviations: LPS, lipopolysaccharide; O PS, O-cha in polysaccharide;
PS, polysaccharide; Sg, serogroup; GPC, gel-permeation chromato-
graphy; Kdo, 3-deoxy-
D
-manno-oct-2-ulosonicacid;Rha,
L
-rhamnose;
SPR, surface-plasmon-resonance; STD, saturation-transfer-dier-
ence; EXCY, exchange spectroscopy; FID, free induction decay.
(Received 8 August 2001, revised 13 November 2001, accepted 16
November 2001)
Eur. J. Biochem. 269, 573±582 (2002) Ó FEBS 2002
legionaminic acid residue of each polysaccharide chain
above a certain length, and was absent from short O-chain
LPSs of wild-type RC1, from the LPS of a s pontaneous
phase variant (strain 811), and an isogenic mutant ( strain
5215) [15].
In the present study we investigated the binding of the
antibody to the isolated OPS with surface-plasmon-
resonance (SPR) biomolecular interaction analyses and
saturation-transfer-difference (STD) NMR spectroscopy in
order to determine binding af®nity and the binding epitope
of the mAb 2625. Because it has not been so far possible
to depolymerize the polylegionaminic acid OPS [6] or to
deconvolute the polymers, the various legionaminic acid
derivatives could not be isolated as monomers or as
homogeneous polymers, respectively, f or separate investi-
gations. But with the aid of STD NMR spectrosc opy, a
new method for c haracterization of ligand binding [16], it
could be shown that mAb 2625 binds directly to the

N-methylated structures in the polymer. This is the ®rst
description of antibody-LPS binding examined by STD
NMR spectroscopy and shows the advantages of this
direct approach for the purpose of relatively quick and
direct epitope mapping.
MATERIALS AND METHODS
Bacterial strains, cultivation, and extraction of LPS
L. pneumophila virulent wild-type strain RC1 (Sg 1, sub-
group OLDA) is a clinical isolate described previously [13].
Avirulent strain 811 is a spontaneous phase variant derived
from wild-type RC1 [13]. Mutant strain 5215 was con-
structed by deletion of the Orf 8±12 operon required for the
biosynthesis of the mAb 2625 epitope from wild-type RC1
as described in the accompanying paper [15]. All s trains
were grown on buffered charcoal yeast extract agar with
a-growth supplement (Merck). LPS was extracted from
enzyme-digested cells by a m odi®ed pheno l/chloroform/
petroleum ether procedure as described previously [6,17].
Preparation, modi®cation, and fractionation
of PS and PS
NH4OH/HF
LPS each of wild-type RC1, mutant 5215, and phase variant
811 was degraded at 100 °Cfor2.5hwith0.1
M
NaOAc/
HOAc buffer (pH 4.4, 10 mgámL
)1
LPS), and the resultant
lipid A was removed by centrifugation (5000 g,30min).
The supernatant was lyophilized and fractionated by gel-

permeation chromatography (GPC) on a column
(2.5 ´ 120 cm; Bio-Rad) of Sephadex G-50 (S) (Pharmacia)
using 50 m
M
pyridinium/acetate buffer (pH 4.3) and mon-
itoring with a differential refractometer (Knauer). Fractions
corresponding to long- a nd short-chain polysaccharide (PS,
i.e. OPS linked to the core oligosaccharide), core oligosac-
charide, and m ono- and disaccharides contaminated with
salt were pooled and lyophilized.
The PS portion was de-O-acetylated (20%, v/v, aqueous
NH
4
OH, 37 °C, 16 h) and treated with 48% (v/v) aqueous
hydro¯uoric acid (HF, 4 °C, 168 h) in order to selectively
cleave the glycosidic linkage of 6-deoxy sugars [18] to obtain
PS
NH
4
OH/HF
as described in the accompanying paper [15].
PS
NH
4
OH/HF
was fractionated by tandem GPC to long-,
middle-, and short-chain molecular species [15].
Preparation of mAb 2625
For production of mAb 2625, the hybridoma cell line was
propagated in Dulbecco's minimal essential cell culture

medium (Biochrom) supplemented with 10% heat-inacti-
vated fetal bovine serum (Biochrom). The culture s uperna-
tant was tested for the presence of mAb 2625 in a colony blot
assay, before antibody puri®cation was carried out. Anti-
body puri®cation was performed using a HiTrap protein G
column (Pharmacia) with a GradiFrac system device
(Pharmacia). Antibodies were eluted from the protein G
resin w ith 0.1
M
glycine and eluted fractions were neutral-
ized with 1
M
Tris/HCl buffer (pH 9). Fractions were pooled
and dialysed against phosphate buffer (137.9 m
M
NaCl,
2.7 m
M
KCl, 8.1 m
M
Na
2
HPO
4
,1.5m
M
KH
2
PO
4

,pH 7.4).
Fig. 1. Proposed structure of Legionella pneu-
mophila PS
NH
4
OH/HF
from wild-type RC1.
1,5-N-acetimidoyl-7-N-acetyllegionaminic
acid; 2-E,5-N-(N,N-dim ethylacetimidoyl)-
7-N-acetylaminolegionaminic acid ( the
descriptors cis and trans designate the posi-
tions of the N-methyl groups relative to N
2
);
3-E and 3-Z, stereoisomers of 5-N-acetimi-
doyl-7-N-acetyl-5-N-methyllegionaminic acid.
The reducin g Rha
I
residue is only present in
70% with a-andb-con®guration in a ratio of
approximately 5 : 1, which is also the case for
free Rha
II
in the other 30% of the molecules.
The anomeric con®guration of the ketosidic
linkage of the legionaminic acid residue
attached to Rha
II
may be dierent and the
position of the N-methylated legionaminic

acid derivatives have not been con®rmed. n is
40 on average for long-chain PS
NH
4
OH/HF
and
18 on average for middle-chain PS
NH
4
OH/HF
.
574 O. Kooistra et al. (Eur. J. Biochem. 269) Ó FEBS 2002
The protein concentration was determined using the
bicinchoninic acid protein assay reagent kit (Pierce).
NMR spectroscopy
1D
1
H NMR and STD spectra were recorded with a Bruker
Avance DRX-600 or DRX-500 spectrometer. Standard
Bruker software was used to acquire and process the NMR
data.
Polysaccharide samples were lyophilized three times from
2
H
2
O and measured in
2
H
2
O(

2
H, 99.996%; Cambridge
Isotope Laboratories) at 27 °C. Chemical shifts were re fer-
enced to external acetone (d
H
2.225 p.p.m.; d
C
31.45 p.p.m.).
For analysis of temperature and pH dependence of the
N-methyl signals long-chain PS
NH
4
OH/HF
from wild-type
RC1 was dissolved in 10% deuterated water, the pH was
adjusted within a range of pH 2 to pH 11 with 1
M
HCl or 1
M
NaOH and recording 1D
1
H NMR spectra at constant
temperature ( 275 K). At pH 7.5 1D
1
H NMR spectra were
recorded at temperatures between 283 and 323 K in 10-K
intervals.
MAb 2625 was ultra®ltrated 10 times using a 6-mL
10-kDa molecular mass cut-off Vivaspin centrifugal concen-
trator device (Sartorius) with deuterated phosphate buffer

composed as described above. The NMR samples were
adjusted to a mAb 2625 concentration of 16.2 l
M
based on
the U V absorption at 280 nm. A 20-fold ligand excess
(640 l
M
) over binding sites was used throughout the studies.
The time dependence of the saturation transfer was
investigated by recording STD spectra with 1 k scans and
saturation times from 0.25 s to 5 s. Relative STD values were
calculated by dividing STD signal intensities by the inten-
sities of the corresponding signals in a 1D
1
HNMRreference
spectrum of the same sample reco rded with 512 scans. STD
NMR spectra for epitope mapping were acquired using a
series of equally spaced 50 ms Gaussian shaped pulses for
saturation, with 1 ms delay betw een the pulses, and a total
saturation time of approximately 3 s. The frequency of the
protein (on-resonance) irradiation was set to the maximum
of the broad hump of overlapping protein
1
H NMR signals
in the a romatic region 7 .2 p.p.m. It was t ested t hat no a mido-
and amidino-protons (6.51 and 7.88 p.p.m., respectively, as
measured in 10% deuterated water) of the ligand were
irradiated by this setting of the on-resonance frequency. The
off-resonance irradiation frequency was set at 33.0 p.p.m.
Free induction decay values (FIDs) with on- and off-

resonance protein saturation were recorded in an alternating
fashion. Subtraction was achieved via phase cycling. A total
relaxation delay of 4.3 s and 128 dummy scans were
employed to reduce subtraction artefacts. The overall
measurement time using 6 k scans was approximately
12 h. Protein resonances were suppressed by application of
a 15-ms low power spin-lock pulse prior to acquisition.
Residual
1
H
2
HO was not suppressed.
STD NMR spectra were recorded at 300 K. In order to
assess the temperature dependence, STD NMR spectra
were also recorded a t 293 an d 315 K .
Surface-plasmon-resonance (SPR) biomolecular
interaction analyses
SPR analyses were carried out using an automated BIAcore
3000 biosensor instrument (BIAcore). mAb 2625, an IgG,
was immobilized on a research grade CM5 senso r chip in
10 m
M
sodium acetate ( pH 4.5) using the amine coupling
kit supplied by the manufacturer (BIAcore). Unreacted
moieties were blocked with ethanolamine. A control surface
with an anti-myoglobin IgG (BIAcore) was prepared in the
same manner. All measurements were performed in 10 m
M
Hepes buffer (pH 7.4) containing 150 m
M

NaCl and
0.005% (v/v) polysorbate 20 (BIAcore) at a ¯ow rate of
10 lLámin
)1
. Surfaces were regenerated by normal dissoci-
ation or with distilled water. Sensorgram data were analysed
using the BIAevaluation 3.0.2 s oftware (BIAcore). Binding
af®nity (K
d
) was determined by steady state af®nity line-
®tting based on end point values at equilibrium binding of a
series of sensorgrams generated with at least seven ligand
concentrations ranging from 1.5 l
M
to 875 l
M
and with
each concentration measured at least twice. Alternatively,
K
d
values were determined by linear regression of Scatchard
plots.
RESULTS
Preparation and characterization of ligands
LPS of L. pneumophila wild-type RC1 subjected to mild
acid hydrolysis is cleaved at the ketosidic linkage of 3-deoxy-
D
-manno-oct-2-ulosonic acid residues (Kdo
I
and Kdo

II
)and
in some molecules at the ketosidic linkage between the
legionaminic acid of the OPS a nd Rha of the core
oligosaccharide, to release lipid A, a lateral a-
D
-man-
nose
II
-(1 ® 8)-Kdo
II
disaccharide, a major heptasaccharide
core fragment, OPS, and PS (i.e. O PS linked t o the core
heptasaccharide), respectively [9±11]. In the majority o f the
molecules, OPS was attached to the core heptasaccharide.
The PS was fractionated by G PC to long- and short-chain
molecular species, the latter containing also middle-chain
molecular species. Part of the long-chain PS was used
without further treatment for STD NMR spectroscopy
experiments (see below). The rest of the PS was de-O-
acetylated to remove abundant O-acetyl groups in the
linkage region between the core o ligosaccharide and the
OPS [9,10], subsequently subjected to HF-treatment to
cleave the glycosidic linkage of the 6-deoxy sugars, e.g.
L
-rhamnose, between the core oligosaccharide and the OPS
[15,18], and fractionated by tandem GPC. By this procedure
long-chain, a low amount of middle-chain, and short-chain
PS
NH

4
OH/HF
devoid of most core sugars were isolated [15].
LPS from mutant 5215 and phase variant 811 was
degraded by the same procedure. As described, the isolated
PS
NH
4
OH/HF
contained only Rha linked as ® 3)-a-
L
-Rha
II
-
(1 ® 3)-
L
-Rha
I
disaccharide (70%) or as ® 3)-
L
-Rha
II
monosaccharide (30%) to polylegionaminic acid [15]
(Fig. 1 ). Only the long- a nd middle-chain PS (as well as
PS
NH
4
OH/HF
) from wild-type RC1 contained legionaminic
acid derivatives N-methylated at the 5-acetimidoylamino

group, which were absent from short-chain OPS of wild-
type RC1, the entire OPS of mutant 5215, and only found in
traces in the OPS of phase variant 811.
Long-, middle-, a nd short-chain PS
NH
4
OH/HF
from wild-
type RC1 were investigated by 1D
1
H NMR spectroscopy
and signal integration was performed to calculate the
average chain-length of the PS
NH
4
OH/HF
and the distribution
of N-methylated legionaminic acid derivatives [15]. Integra-
tion of the signals of 1D
1
H NMR spectra indicated that the
Ó FEBS 2002 Epitope mapping with mAb 2625 (Eur. J. Biochem. 269) 575
average chain-length of long-, middle-, and short-chain
PS
NH
4
OH/HF
is about 40, 18, and 10 legionaminic acid res-
idues [15], resulting in a calculated average molecular mass
of approximately 12.9 kDa, 6.0 kDa, an d 3.4 kDa, respec-

tively. The ratio of the 5-N-(N,N-dimethylacetimidoyl)-7-N-
acetyl and 5-N-ace timidoyl-5-N-methyl-7-N-acetyl deriva-
tives of legionaminic acid was 1 : 1 in long-chain and 1 : 2 in
middle-chain PS
NH
4
OH/HF
, respectively. Based on the rela-
tive intensities of the proton signals it was concluded that
only one legionaminic acid residue is N-methylated in each
polysaccharide chain above a speci®c length. The proposed
structure of PS
NH
4
OH/HF
from wild -type RC1, which was
used for SPR analyses and STD NMR spectroscopy
experiments i s presented in Fig. 1. The PS
NH
4
OH/HF
from
mutant 5215 and phase variant 811 had the same chain-
length as that from wild-type RC1 [15].
SPR studies with immobilized mAb 2625
In order to investigate the binding behaviour of mAb 2625,
binding af®nity (K
d
) was determined by SPR for the binding
to immobilize d mAb 2625 of isolated long- and middle-

chain PS
NH
4
OH/HF
from L. pneumophila wild-type RC1,
mutant 5215, and phase variant 811. The PS
NH
4
OH/HF
from
wild-type RC1 bound to mAb 2625 with a rapid association
and dissociation to and from the antibody, typical for low±
af®nity interaction like antibody-carbohydrate binding [19].
The K
d
value for middle-chain PS
NH
4
OH/HF
from wild-type
RC1 determined at equilibrium binding was 26 l
M
(Fig. 2 ,
top panel), determination by linear regression analysis of
Scatchard plot gave a value of 21 l
M
(Fig. 2, bottom panel).
Direct comparison of long- and middle-chain PS
NH
4

OH/HF
from wild-type RC1, obtained with less data points,
generated K
d
values in the same range: 43 l
M
(Scatchard:
43 l
M
) for long-chain PS
NH
4
OH/HF
and 30 l
M
(Scatchard:
31 l
M
) for middle-chain PS
NH
4
OH/HF
. The sensorgrams for
long-chain PS
NH
4
OH/HF
had a similar square pulse form as
that for short-chain PS
NH

4
OH/HF
. With the long- and
middle-chain PS
NH
4
OH/HF
from mutant 5215 and phase
variant 811, n o measurable af®nity could be determined.
Resonance units were not higher than those obtained with
buffer as control experiments.
STD NMR experiments of middle-chain PS
NH
4
OH/HF
from wild-type RC1 in the presence of mAb 2625
To describe the epitope responsible for the binding inter-
action of the polysaccharide chain with mAb 2625 at atomic
resolution, both PS
NH
4
OH/HF
and PS were investigated by
STD NMR experiments. Because of the complexity of the
ligand molecules, initial investigations were done using the
smaller, apparently less complex middle-chain PS
NH
4
OH/HF
and were completed with long-chain PS (see below) in order

to study the in¯uence of the carbohydrate polymer chain on
binding.
Four samples were prepared, two contained t he binding
middle-chain PS
NH
4
OH/HF
from wild-type RC1 wit h and
without mAb 2625 and two contained the middle-chain
PS
NH
4
OH/HF
from mutant 5215 lacking the N-methyl groups
also with and without antibody. The latter PS
NH
4
OH/HF
did
not show binding activity in SPR experiments. Optimization
of the experimental set-up for STD NMR spectroscopy was
achieved using samples without any mAb present. In that
case, STD spectra did not contain ligand signals, because
saturation transfer does not occur without the protein (data
not shown). Investigation of the time dependence of the
saturation transfer with saturation times from 0.25 s to 5 s
showed that 3 s was suf®cient for ef®cient transfer of
saturation from the protein to the ligand protons (Fig. 3).
The signals of all N- and C-linked methyl groups present in
STD spectra showed similar behaviour.

Only the sample containing mAb 2625 and middle-chain
PS
NH
4
OH/HF
from wild-type RC1 showed signi®cant sat-
uration transfer from the protein to the ligand in the STD
spectra (Fig. 4B). Comparison of the STD spectrum with
the corresponding 1D
1
H N MR spectrum (Fig. 4A) c learly
demonstrated the involvement of the N-methyl groups of
the N-methylated legionaminic acid derivatives 2, 3-E and
3-Z in binding. Investigation of the time dependence
revealed that saturation transfer to the two N-methyl groups
in 2 was identical and reached a maximum STD of  15%.
The maximum values for 3-E and 3-Z were considerably
lower,  7 and 10%, respectively (Fig. 3A). Therefore, the
N-methyl groups in 2 showed a twofold more effective
saturation transfer compared to the ones in 3-E and 3- Z.
Similar effects were observed for
1
HNMRsignalsofthe
C-methyl groups of the N-acetimidoyl a nd N-acetyl groups
in 2, 3-E and 3-Z (Fig. 5). The signals were partially
superimposed by the i ntense resonances of the correspond-
ing methyl groups of the major component in the mixture,
legionaminic acid 1. Signals for the C-methyl group of the
N-acetimidoyl group in 2, 3-E and 3-Z reached a maximum
Fig. 2. Surface-plasmon-reson ance a na lysis o f middle-chain PS

NH
4
OH/HF
from wild-type RC1 with immobilized mAb 2625. Steady-state anity
line-®tting based on end point values at equilibrium binding obtained
with 14 ligand concentrations between 1.5 l
M
and 292 l
M
(A). Scat-
chard analysis based on the same d ata (B).
576 O. Kooistra et al. (Eur. J. Biochem. 269) Ó FEBS 2002
STD effect o f  9, 6.6, and 7.5%, respectively (Fig. 3B).
Signals for the C-methyl group of the N-acetyl group
reached a maximum STD effect of  9% in 2,and6%in
3-E (Fig. 3C). The assignment of the N-acetyl group of 2
was solely based on the STD NMR experiments. Further-
more, one signal of the N-acetyl group of 3-Z could not be
identi®ed unambiguously, either in the 1D
1
HNMR
spectrum or in t he STD NMR spectrum, and one signal
(d
H
2.22) in the STD NMR spectrum showing signi®cant
saturation transfer ( 6% maximum STD effect) could not
be assigned at all. Proton signals from t he pyranose ring or
the side chain of the N-methylated legionaminic acid
derivatives could not be assigned unequivocally due to
noise. For the major nonmethylated legionaminic acid (1),

only a signal for H9 and for the other two groups with the
most intense signals in the 1D
1
H NMR spectrum, i.e.
the C-methyl groups of the N-acetimidoyl group and the
N-acetyl group, respectively, were observed. However,
maximum STD e ffects for these signals were rather lo w
( 3 and 2%; Fig. 3 B,C) and, furthermore, these signals
were also de tected as the only signals in the STD spectrum
of the middle-chain PS
NH
4
OH/HF
from mutant 5215
(Fig. 4 D). Most probably, the STD NMR signals of the
C-methyl groups of the N-acetimidoyl group and the
N-acetyl group of 1 were due to relaxation artefacts.
Signals of the Rha protons were not present in the STD
spectra, which is most obvious for the signals of the
anomeric proton s and the methyl protons of the 6-deoxy
groups because these signals are well separated in the
corresponding 1D
1
H NMR spectra. Therefore, partici-
pation in binding of these residues located at the reducing
end of PS
NH
4
OH/HF
could not be con®rmed by our

experiments.
The 1D
1
H NMR spectra of mAb 2625 together with
middle-chain PS
NH
4
OH/HF
from both strains (Fig. 4A,C)
showed signals belonging most likely to glycerol. They were
not present in the corresponding STD spectra (Fig. 4B,D)
because glycerol does not bind to the mAb. Glycerol
probably originated from the membrane of the centrifugal
concentrator device or from ®lters used during the prepa-
ration of the samples or mAb 2625.
Temperature and pH dependence of 1D
1
H NMR
signals of N-methyl groups
To measure the temperature and pH dependence of the
signals o f the N-methyl groups in 2 and 3 due to
chemical exchange [20], 1D
1
H NMR spectra of long-chain
PS
NH
4
OH/HF
from strain RC1 were recorded under various
conditions. It was observed that both changes of the pH at

constant temperature and changes o f the temperature at
appropriate constant pH in¯uenced the form of the signals
in a similar manner. Lowering the pH had a similar effect as
a decrease in temperature and vice versa, although the latter
couldbebettermonitoredinsmallsteps.
At constant temperature (275 K), the four separated
N-methylsignalscouldbeobserveduptopH 7and
beginning with pH  8 the lower-®eld pair of signals of 2-E
[d
H
3.30 (trans)andd
H
3.19 (cis)] broadened and began to
coalesce, so that from pH  9 only one sharp signal was
detected (Fig. 6A±F). The higher-®eld pair o f N-methyl
signals [d
H
3.03 (3-Z)andd
H
2.95 (3-E)] remained
unchanged at high pH, although at l ow pH (pH  2) it
seemed that the ratio of the signals, balanced at neutral pH,
was slightly changed towards the 3-E isomer.
On the other hand, at constant pH 7.5 the increase of the
temperature from 283 K in 1 0-K steps to 323 K showed
that the two separated signals for the N-me thyl groups of 2
broadened, coalesced, and ®nally were observed as one
sharp signal with an average chemical shift ( Fig. 6G±M).
The two separated signals of the N -methyl group of 3-E
and 3-Z did not signi®cantly change within this range

(Fig. 6 G±M). T he N-methyl signals of 3 did not change
even under the drastic conditions pH  11 and 323 K, a pH
Fig. 3. Time dependence of magnetization transfer for selected saturated
signals of methyl groups of the legionaminic acid derivatives. Th e time
dependence for the N-methyl groups (A) and the C-linked methyl
groups of the a cetimidoylamin o (B) and the acetamido (C) substitu-
ents, respectively, of 2 (s), 3-E (n), 3-Z (e), and 1 (h) are shown. The
two signals of the N-methyl groups of 2 showed identical behaviour. A
signal of the C-methyl group of the acetamido group in 3-Z could not
be identi®ed unambiguously, and one signal (´) could not be assigned
to any proton. Magnetization transfer for the C-methyl groups of 1
probably accounts for relaxation artefacts.
Ó FEBS 2002 Epitope mapping with mAb 2625 (Eur. J. Biochem. 269) 577
at which t he N-methyl signals of 2 already coalesced at low
temperature (283 K; Fig. 6F). The signals of the major
nonmethylated legionaminic acid (1) were not signi®cantly
changed apart from better resolution at low pH or high
temperature.
STD NMR experiments with mAb 2625 together
with long-chain PS from wild-type RC1
at different temperatures
STD NMR spectroscopy experiments with t he long-chain
PS from strain RC1 were performed for several reasons.
After mild acid hydrolysis of the LPS without further
degradation, it is dif®cult to obtain middle-chain PS, which
can only be i solated as a mixture with short-chain PS [9],
which in contrast t o long- and middle-chain PS, does not
contain N-methylated l egionaminic acid derivatives [15].
Long-chain PS on the other hand, which quantitatively
contains one N-methylated legionaminic acid derivative,

could be isolated as a well-separated fraction. Furthermore,
Fig. 4. 1D
1
HNMR(AandC)andSTD
NMR (B and D) spectra of middle-chain
PS
NH
4
OH/HF
from wild-type RC1 (A an B) and
mutant 5215 (C and D) in the presence of mAb
2625. Low intensity signals in the spectrum in
(D) are probably due to subtraction artefacts
of the originally most intense proton signals of
the N-acetimidoyl and N-acetyl grou ps in 1,
respectively. Spectra were recorded at 300 K.
Bold numbers refer to structures shown in
Fig. 1. NMe
cis
and NMe
trans
,N-methyl
groups of 2-E;NMe,N-methylgroupofthe
isomers of 3; NAm
CH
3
and NAc
CH
3
,C-methyl

group of the acetimidoylamino and acetamido
substituents, respectively.
Fig. 5. Detail of the STD NMR spectrum of the middle-chain
PS
NH
4
OH/HF
from wild-type RC1 in the presence of mAb 2625 showing
the resonance region of the C-linked methyl groups. Signals of 1 are
probably d ue to subtraction artefacts of the originally most intense
proton signals of the N-acetimidoyl and the N-acetyl groups, respect-
ively. The sign al marke d by ´ co uld not be assigned to any proton.
Bold numbers refer to structures sh own in Fig. 1. F or abb reviatio ns
see legend to Fig. 4.
578 O. Kooistra et al. (Eur. J. Biochem. 269) Ó FEBS 2002
it was the aim to investigate the native PS molecule, i.e. with
the complete O-acetylated core heptasaccharide, and also to
measure ligands with a considerably high molecular mass
(11±17 kDa). The molecular mass of the ligand is a sensitive
factor in STD NMR spectroscopy, becau se the higher the
mass of the ligand, the slower its motion, and the more
effective i s spin diffusion. STD spectra with temperature
variations were recorded to investigate if the method is
applicable to epitopes such as 2, which are subjected to
chemical exchange (see above).
In the STD spectrum of the sample containing mAb 2625
together with the l ong-chain PS from strain RC1 with an
average molecular mass of 14 kDa, signi®cant saturation
transfer f rom the protein to the liga n d p rotons a t 2 93 K c ould
be detected mainly for t he sign als o f the N- methyl groups in 2

(Fig. 7B) as was observed with PS
NH
4
OH/HF
(see above). The
STD spectrum of the long-chain PS from strain RC1
containing no protein (Fig. 7C) was performed as reference
experiment and showed that direct irradiation of ligand
resonances could not be avoided under these experimental
conditions, d espite a r elaxation delay of 4.3 s and a satu ration
time of 3 s. Nevertheless, saturation transfer to the N-methyl
groups was not observed under these conditions. A satura-
tion transfer was observed for the polysaccharide. The
experiment also shows that the molecular mass of a ligand in
STD NMR experiments may well exceed a few kDa.
Interestingly, saturation transfer could also be detected
under conditions, where the two signals o f the N-methyl
groups in 2 were coalesced, i.e. at elevated temperatures (315
K; Fig. 8D). Although there is probably no chemical
exchange in the bound state, only the single broad proton
signal arising from chemical exchange in the free state was
observed.
DISCUSSION
Structural studies aiming at an exact description of the
epitope of monoclonal antibodies are time-intensive and
laborious. F or example, the epitopes of two anti-L. pneu-
mophila LPS antibodies have been described by a series of
Fig. 6. Dependence of proton signals of the
N-methyl groups in 2 and 3 from pH and tem-
perature. 1D

1
H NMR spectra of the long-
chain PS
NH
4
OH/HF
from wild-type RC1 were
recorded in 10% deuterated water at constant
temperature (275 K) with p
1
H/
2
H values of
 2,  7,  8,  9, and  11 (A±F), and with
constant pH (p
1
H/
2
H7.5)attemperatures
between 323 and 283 K raised in 10-K inter-
vals (G±M), respectively. Only the resonance
region of the N-methyl groups (2.8±
3.4 p.p.m.) is shown.
Ó FEBS 2002 Epitope mapping with mAb 2625 (Eur. J. Biochem. 269) 579
extensive e xperiments; the epitope of mAb 3/1 is associated
with quantitative 8-O-acetylation of polylegionaminic acid
[9,21,22] and mAb LPS-1 recognizes the highly O-acetylated
region interve ning t he core oligosaccharide and the OPS of
Sg 1 strains [9,13,23].
Investigations of crystal structures of monoclonal anti-

bodies in complex with carbohydrate antigens have shown
that a small antigenic determinant can dictate a highly
speci®c immune response [24]. The OPS of the LPS from the
two Vibrio cholerae serotypes Inaba and Ogawa is a homo-
polymer o f a-(1 ® 2)-linked N-(3-deoxy-
L
-glycero-tetronyl)-
D
-perosamine [25,26] differing only by the presence of a
single residue of 2-O-methy l-N-(3-deoxy-
L
-glycero-tetronyl)-
D
-perosamine as nonreducing terminal unit in the OPS of
serotype Ogawa [27,28]. The crystal structure of a F ab
fragment from mAb S-20-4 in complex with synthetic OPS
fragments as antigen showed that the t erminal 2-O-met hyl-
N-(3-deoxy-
L
-glycero-tetronyl)-
D
-perosamine r esidue is the
primary antigenic determinant [24].
STD NMR spectroscopy [16] offers an ef®cient alterna-
tive approach to identify the residues or substructures
involved in binding to monoclonal antibodies or other
receptor proteins. A p rerequisite for S TD NMR s pectro-
scopy is that the ligand is reversibly bound to the protein.
Fig. 7. 1D
1

H NMR (A) and STD NMR (B
and C) spectra of long-chain PS from strain
RC1inthepresence(AandB)andabsence(C)
of mAb 2625. Rather strong unspeci®c irradi-
ation of ligand signals in the spectrum in (C) is
observed despite the absence o f protein, but
not of the signals of the N-methyl groups as in
the spectrum in (B) reco rd ed in the pres ence of
mAb 2625. Spectra were recorded at 300 K.
Bold numbers refer to structures shown in
Fig. 1. For abbreviations see legend to Fig. 4.
Fig. 8. 1D
1
HNMR(AandC)andSTD
NMR (B and D) spectra of long-chain PS from
strain RC1 in the p resence of mAb 2625
recorded with p
2
H 7.4 at 293 K (A and B) and
315 K (C and D). Only the resonance region of
the N-methyl groups ( 2.8±3.4 p.p.m.) is
shown.
580 O. Kooistra et al. (Eur. J. Biochem. 269) Ó FEBS 2002
The binding af®nity (K
d
) should b e in the range of 1 m
M
to
 10 n
M

. Stronger binding often suffers from off-rates being
too low. This usually prevents suf®cient amounts of
saturated ligand that can be detected in the unbound state.
The bound ligand cannot be detected because line widths
are far too large for a complex of this size. The binding
af®nity can be measured by surface-plasmon-resonance
biomolecular interaction analyses [29].
The 5-N-(N,N-dimethylacetimidoyl)-7-N-acetyl (2)and
5-N-acetimidoyl-5-N-methyl-7-N-acetyl (3) derivatives of
legionaminic acid were identi®ed as being responsible for
phase variation of the epitope of the LPS-speci®c mAb 2625
[15]. In order to determine t he binding af®nity of isolated
PS
NH
4
OH/HF
molecular s pecies of different size, SPR ana-
lyses were performed with immobilized mAb 2625. It could
be shown that wild-type but not mutant middle- and long-
chain PS
NH
4
OH/HF
bound with signi®can t af®nity, which
proved the epitope still to be present in the degraded
PS
NH
4
OH/HF
. The binding af®nity was low, in the range of

approximately 30 l
M
, which could also be seen from the
rapid association and dissociation to and from the antibody,
typically observed for low-af®nity interaction [19]. Mixtures
of PS
NH
4
OH/HF
molecular species of different size containing
different ratios of the N-methylated legionaminic acid
derivates bound with similar af®nities. Nevertheless, the
observed low af®nity allowed to perform STD NMR
spectroscopy with the aim of more precisely describing its
epitope. STD spectra unequivocally demonstrated both
types of N-methyl groups (2, 3-E and 3-Z) of one single
legionaminic acid derivative in the polymer to be involved in
binding. Only appropriate material from wild-type RC1
interacted with mAb 2625 and material from mutant 5215
was not interacting with mAb 2625. Although middle-chain
PS
NH
4
OH/HF
from wild-type RC1 that was used for STD
experiments was a heterogeneous mixture with respect to
chain-length, number of Rha residues at the reducing end,
and the content of different derivatives of N-methylated
legionaminic acid, the method could be used to show a
preference for binding of mAb 2625 to only these

N-methylated legionaminic acid derivatives in the polymer.
Moreover, i t could be shown that not only the N-methyl
groups of the respective N-acetimidoyl groups but also
other groups in close proximity were involved in binding.
The signal of the C-methyl group of the same N-acetimidoyl
groups was observed as was the signal of the C-methyl
group of the N-acetyl group linked to C7 in the side chain of
the respective legionaminic acid derivative.
The N-methyl groups in 2 showed a twofold more
effective saturation transfer compared to those in 3-E and
3-Z. If the explanation for more intensive saturation
transfer is a larger fraction bound due to stronger binding
(i.e. higher af®nity) or shorter distances between protons of
ligand and protein cannot be distinguished at the moment.
However, b oth cases would suggest 5-N-(N,N-dimethylace-
timidoyl)-7-N-acetyllegionaminic acid (2) to be a preferred
epitope of mAb 2625. The lower saturation transfer to the
N-methyl group in 3-E or 3-Z on the other hand, might be
explained by a lower off-rate of the ligand resulting in a
lower amount of free but saturated ligand, which would i n
turn indicate a higher af®nity. The question of whether the
N-methylated legionaminic acid derivative binds with
different af®nity to mAb 2625 can only be answered
by experiments with homogeneous and structurally de®ned
ligands, which to date are not available. However, as the
three different N-methylated acetimidoylamino groups (i.e.
in 2, 3-E,and3-Z) share great structural similarities it is still
possible that they bind with equal af®nity to mAb 2625
(data not shown).
The proton signals of the N-methyl groups of 5-N-

(N,N-dimethylacetimidoyl)-7-N-acetyllegionaminic acid (2)
showed a temperature- and pH-dependent behaviour typical
for a rotatio n process a t a partial double bond [20]. The
proton sign als o f t he N -methyl group of the 5 -N-acetimidoyl-
5-N-methyl-7-N-acetyllegionaminic acid (3) d id not show
such a behaviour, although a partial double bond character
was observed; no chemical exchange could be observed
under the conditions applied. From these data it is conclud ed
that under certain conditions it is possible to observe the
interconversion of the cis and trans N-methyl groups in 2 at
the partial double bond between the dimethylated n itrogen
and the nonprotonated carbon, i.e. the chemical exchange
process. Rotation is fast on the NMR timescale o r, more
precisely, more frequent [20] and, thus, only one proton
signal for both N-methyl groups with the average chemical
shift can be observed. The reason could either be deproto-
nation of the group at high pH,which destabilize s thedouble-
bonded transition s tate, or due to a lowered activation energy
of the r otational barrier at high temperature [20], or a
combination of both. In contrast, the isomers 3-E and 3-Z
were not observed to undergo chemical exchange. This can
probably be ascribed mainly to steric hindrance of the bulky
group of the polymer -linked derivatives of legionaminic a cid,
so that chemical exchange is slow on the NMR timescale,
thus indicating that it rarely occurs. Nevertheless, at the
moment both isomers o f 3 were present in approximately
equimolar ratio. However, at low pH, protonation of both
nitrogens of the acetimidoyl(N-methyl)amino group could
be the reason for preponderance of one of the isomers (3-E ).
The observed chemical exchange for 2, but not for 3,was

con®rmed by 2D EXSY experiments [30] at 300 K and 343 K
of samples at neutral pH, where o nly cross-correlations for
the proton signals of the exchanging N-methyl groups in 2
could be detected (data not shown).
Interestingly, recording of STD spectra under conditions
were the (N,N-dimethylacetimidoyl)amino group is under-
going chemical exc hange, i.e. a t elevated tempe ratures was
also possible. Although there is probably no chemical
exchange in the bound state, only the single (coalesced)
proton signal arising from chemical e xchange in t he free
state is observed. Despite the high average molecular mass
of the ligand (11±17 kDa) and the epitope being just a minor
modi®cation of the OPS, a suf®cient magnetization transfer
was observed, showing that in speci®c cases the molecular
mass limit of the ligand for STD NMR spectroscopy can be
extended.
This is the ® rst d escription of an application o f STD NMR
spectroscopy to identify the LPS epitope of a monoclonal
antibody showing the advantages of this direct approach for
the purpose of r elatively quick and direct epitope determi-
nation with relatively small amounts of protein and ligands,
which do not need to be puri®ed to absolute homogeneity.
ACKNOWLEDGEMENTS
We thank Dr C. Roll for help with temperature dependence NMR
spectroscopy expe riments, and Dr T. W eimar for help with SPR
Ó FEBS 2002 Epitope mapping with mAb 2625 (Eur. J. Biochem. 269) 581
analysis. T his work was ®nancially s upported by grants from the
Deutsche Forschungsgemeinschaft, LU 514/2-2 (E. L. and M. F.) and
ZA 149/3-2 (U. Z.).
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SUPPLEMENTARY MATERIAL
The following material is available from http://www.
blackwell-science.com/products/journals/suppmat/ejb/
ejb2684/ejb2684sm.htm
582 O. Kooistra et al. (Eur. J. Biochem. 269) Ó FEBS 2002