Epitope mapping of the O-chain polysaccharide of Legionellaby saturation-transfer-difference NMR spectroscopy Oliver Kooistra1, Lars Herfurth2, Edeltraud LuÈneberg3, Matthias Frosch3, Th
Trang 1Epitope mapping of the O-chain polysaccharide of Legionella
by saturation-transfer-difference NMR spectroscopy
Oliver Kooistra1, Lars Herfurth2, Edeltraud LuÈneberg3, Matthias Frosch3, Thomas Peters2
and Ulrich ZaÈhringer1
1 Research Center Borstel, Center for Medicine and Biosciences, Germany; 2 Institute for Chemistry, Medical University of LuÈbeck, Germany; 3 Institute for Hygiene and Microbiology, University of WuÈrzburg, 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-dierence (STD) NMR spectroscopy in order to map the mAb 2625 epitope on a molecular level It could be demonstrated that the binding anity 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, thus indicating these derivatives to represent the major epitope of mAb 2625
Keywords: lipopolysaccharide; Legionella pneumophila; bioanity 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 legionellae are 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], which is 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 nonreducingL-rhamnose (RhaII) 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 of 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 was related to the presence of N-methyl groups at the 5-N-acetimidoyl group of legionaminic acid, a modi®cation
of bacterial polysaccharides, which is described for the ®rst time in the accompanying paper [15] The components identi®ed were the 5-N-(N,N-dimethylacetimidoyl)-7-N-acetyl- and 5-N-acetimidoyl-5-N-methyl-7-N-5-N-(N,N-dimethylacetimidoyl)-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 ZaÈhringer, Forschungszentrum Borstel,
Zentrum fuÈr Medizin und Biowissenschaften, Parkallee 22, D-23845
Borstel, Germany Fax: + 49 4537 188612, Tel.: + 49 4537 188462,
E-mail: uzaehr@fz-borstel.de
Abbreviations: LPS, lipopolysaccharide; OPS, O-chain 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-dier-ence; EXCY, exchange spectroscopy; FID, free induction decay.
(Received 8 August 2001, revised 13 November 2001, accepted 16
November 2001)
Trang 2legionaminic 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 spontaneous
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, for separate
investi-gations But with the aid of STD NMR spectroscopy, a
new method for characterization 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
M A T E R I A L S A N D M E T H O D S
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 strains
were grown on buffered charcoal yeast extract agar with
a-growth supplement (Merck) LPS was extracted from
enzyme-digested cells by a modi®ed phenol/chloroform/
petroleum ether procedure as described previously [6,17]
Preparation, modi®cation, and fractionation
of PS and PSNH4OH/HF
LPS each of wild-type RC1, mutant 5215, and phase variant
811 was degraded at 100 °C for 2.5 h with 0.1MNaOAc/ HOAc buffer (pH 4.4, 10 mgámL)1LPS), and the resultant lipid A was removed by centrifugation (5000 g, 30 min) 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 mMpyridinium/acetate buffer (pH 4.3) and mon-itoring with a differential refractometer (Knauer) Fractions corresponding to long- and short-chain polysaccharide (PS, i.e OPS linked to the core oligosaccharide), core oligosac-charide, and mono- and disaccharides contaminated with salt were pooled and lyophilized
The PS portion was de-O-acetylated (20%, v/v, aqueous
NH4OH, 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
PSNH4OH/HFas described in the accompanying paper [15]
PSNH 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 superna-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 with 0.1Mglycine and eluted fractions were neutral-ized with 1MTris/HCl buffer (pH 9) Fractions were pooled and dialysed against phosphate buffer (137.9 mM NaCl, 2.7 mMKCl, 8.1 mMNa2HPO4, 1.5 mMKH2PO4, 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-dimethylacetimidoyl)-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 reducing Rha I residue is only present in 70% with a- and b-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 dierent 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 NH4OH/HF
Trang 3The protein concentration was determined using the
bicinchoninic acid protein assay reagent kit (Pierce)
NMR spectroscopy
1D1H 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
2H2O and measured in 2H2O (2H, 99.996%; Cambridge
Isotope Laboratories) at 27 °C Chemical shifts were
refer-enced to external acetone (dH2.225 p.p.m.; dC31.45 p.p.m.)
For analysis of temperature and pH dependence of the
N-methyl signals long-chain PSNH4OH/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 1MHCl or 1
MNaOH and recording 1D1H NMR spectra at constant
temperature (275 K) At pH 7.5 1D1H 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 lMbased on
the UV absorption at 280 nm A 20-fold ligand excess
(640 lM) 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 1D1H NMR reference
spectrum of the same sample recorded 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 between 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 protein1H NMR signals
in the aromatic region 7.2 p.p.m It was tested that no
amido-and amidino-protons (6.51 amido-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
Residual1H2HO was not suppressed
STD NMR spectra were recorded at 300 K In order to
assess the temperature dependence, STD NMR spectra
were also recorded at 293 and 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 sensor chip in
10 mMsodium 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 mM
Hepes buffer (pH 7.4) containing 150 mM 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 software (BIAcore) Binding af®nity (Kd) 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 lM to 875 lM and with each concentration measured at least twice Alternatively,
Kdvalues were determined by linear regression of Scatchard plots
R E S U L T S
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 (KdoIand KdoII) and
in some molecules at the ketosidic linkage between the legionaminic acid of the OPS and Rha of the core oligosaccharide, to release lipid A, a lateral a-D -man-noseII-(1 ® 8)-KdoIIdisaccharide, a major heptasaccharide core fragment, OPS, and PS (i.e OPS linked to the core heptasaccharide), respectively [9±11] In the majority of the molecules, OPS was attached to the core heptasaccharide The PS was fractionated by GPC 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 oligosaccharide 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
PSNH 4 OH/HFdevoid 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
PSNH 4 OH/HFcontained only Rha linked as ® 3)-a-L-RhaII -(1 ® 3)-L-RhaI disaccharide (70%) or as ® 3)-L-RhaII
monosaccharide (30%) to polylegionaminic acid [15] (Fig 1) Only the long- and middle-chain PS (as well as
PSNH 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-, and short-chain PSNH4OH/HFfrom wild-type RC1 were investigated by 1D1H NMR spectroscopy and signal integration was performed to calculate the average chain-length of the PSNH 4 OH/HFand the distribution
of N-methylated legionaminic acid derivatives [15] Integra-tion of the signals of 1D1H NMR spectra indicated that the
Trang 4average chain-length of long-, middle-, and short-chain
PSNH 4 OH/HFis 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, and 3.4 kDa,
respec-tively The ratio of the
5-N-(N,N-dimethylacetimidoyl)-7-N-acetyl and 5-N-acetimidoyl-5-N-methyl-7-N-5-N-(N,N-dimethylacetimidoyl)-7-N-acetyl
deriva-tives of legionaminic acid was 1 : 1 in long-chain and 1 : 2 in
middle-chain PSNH 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 PSNH4OH/HFfrom wild-type RC1, which was
used for SPR analyses and STD NMR spectroscopy
experiments is presented in Fig 1 The PSNH4OH/HFfrom
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 (Kd) was determined by SPR for the binding
to immobilized mAb 2625 of isolated long- and
middle-chain PSNH 4 OH/HF from L pneumophila wild-type RC1,
mutant 5215, and phase variant 811 The PSNH 4 OH/HFfrom
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 Kdvalue for middle-chain PSNH4OH/HFfrom wild-type
RC1 determined at equilibrium binding was 26 lM(Fig 2,
top panel), determination by linear regression analysis of Scatchard plot gave a value of 21 lM(Fig 2, bottom panel) Direct comparison of long- and middle-chain PSNH 4 OH/HF
from wild-type RC1, obtained with less data points, generated Kdvalues in the same range: 43 lM(Scatchard:
43 lM) for long-chain PSNH 4 OH/HFand 30 lM(Scatchard:
31 lM) for middle-chain PSNH 4 OH/HF The sensorgrams for long-chain PSNH 4 OH/HFhad a similar square pulse form as that for short-chain PSNH 4 OH/HF With the long- and middle-chain PSNH 4 OH/HF from mutant 5215 and phase variant 811, no 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 PSNH4OH/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 PSNH 4 OH/HFand 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 PSNH 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 the binding middle-chain PSNH4OH/HF from wild-type RC1 with and without mAb 2625 and two contained the middle-chain
PSNH4OH/HFfrom mutant 5215 lacking the N-methyl groups also with and without antibody The latter PSNH4OH/HFdid 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
PSNH4OH/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 1D1H NMR spectrum (Fig 4A) clearly 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 for1H NMR signals of the C-methyl groups of the N-acetimidoyl and N-acetyl groups
in 2, 3-E and 3-Z (Fig 5) The signals were partially superimposed by the intense 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-resonance analysis of middle-chain PS NH 4 OH/HF
from wild-type RC1 with immobilized mAb 2625 Steady-state anity
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 data (B).
Trang 5STD effect of 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, and 6% 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 1H NMR
spectrum or in the STD NMR spectrum, and one signal
(dH2.22) in the STD NMR spectrum showing signi®cant
saturation transfer ( 6% maximum STD effect) could not
be assigned at all Proton signals from the 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 1H NMR spectrum, i.e the C-methyl groups of the N-acetimidoyl group and the N-acetyl group, respectively, were observed However, maximum STD effects for these signals were rather low ( 3 and 2%; Fig 3B,C) and, furthermore, these signals were also detected as the only signals in the STD spectrum
of the middle-chain PSNH 4 OH/HF from mutant 5215 (Fig 4D) 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 protons and the methyl protons of the 6-deoxy groups because these signals are well separated in the corresponding 1D 1H NMR spectra Therefore, partici-pation in binding of these residues located at the reducing end of PSNH 4 OH/HF could not be con®rmed by our experiments
The 1D 1H NMR spectra of mAb 2625 together with middle-chain PSNH 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 1D1H NMR signals of N-methyl groups
To measure the temperature and pH dependence of the signals of the N-methyl groups in 2 and 3 due to chemical exchange [20], 1D1H NMR spectra of long-chain
PSNH 4 OH/HFfrom strain RC1 were recorded under various conditions It was observed that both changes of the pH at constant temperature and changes of 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 could be better monitored in small steps
At constant temperature (275 K), the four separated N-methyl signals could be observed up to pH 7 and beginning with pH 8 the lower-®eld pair of signals of 2-E [dH3.30 (trans) and dH3.19 (cis)] broadened and began to coalesce, so that from pH 9 only one sharp signal was detected (Fig 6A±F) The higher-®eld pair of N-methyl signals [dH 3.03 (3-Z) and dH 2.95 (3-E)] remained unchanged at high pH, although at low 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 10-K steps to 323 K showed that the two separated signals for the N-methyl 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 6G±M) The 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 The time
dependence for the N-methyl groups (A) and the C-linked methyl
groups of the acetimidoylamino (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.
Trang 6at which the 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 the 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 isolated as a mixture with short-chain PS [9], which in contrast to long- and middle-chain PS, does not contain N-methylated legionaminic 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 H NMR (A and C) and STD 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 groups 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-methyl group of the 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 due to subtraction artefacts of the originally most intense
proton signals of the N-acetimidoyl and the N-acetyl groups,
respect-ively The signal marked by ´ could not be assigned to any proton.
Bold numbers refer to structures shown in Fig 1 For abbreviations
see legend to Fig 4.
Trang 7it 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, because the higher the
mass of the ligand, the slower its motion, and the more
effective is 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 long-chain PS from strain RC1 with an
average molecular mass of 14 kDa, signi®cant saturation
transfer from the protein to the ligand protons at 293 K could
be detected mainly for the signals of the N-methyl groups in 2
(Fig 7B) as was observed with PSNH4OH/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, despite a relaxation delay of 4.3 s and a saturation 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 of 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
D I S C U S S I O N
Structural studies aiming at an exact description of the epitope of monoclonal antibodies are time-intensive and laborious For 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 NH4OH/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 H 7.5) at temperatures
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.
Trang 8extensive experiments; 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 intervening the 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 of 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-methyl-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 Fab fragment from mAb S-20-4 in complex with synthetic OPS fragments as antigen showed that the terminal 2-O-methyl-N-(3-deoxy-L-glycero-tetronyl)-D-perosamine residue 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 prerequisite for STD NMR spectro-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 RC1 in the presence (A and B) and absence (C)
of mAb 2625 Rather strong unspeci®c irradi-ation of ligand signals in the spectrum in (C) is observed despite the absence of protein, but not of the signals of the N-methyl groups as in the spectrum in (B) recorded in the presence 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 H NMR (A and C) and STD NMR (B and D) spectra of long-chain PS from strain RC1 in the presence 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.
Trang 9The binding af®nity (Kd) should be in the range of 1 mMto
10 nM 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 the binding af®nity of isolated
PSNH4OH/HF molecular species 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 PSNH4OH/HF bound with signi®cant af®nity, which
proved the epitope still to be present in the degraded
PSNH 4 OH/HF The binding af®nity was low, in the range of
approximately 30 lM, 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 PSNH 4 OH/HFmolecular 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
PSNH 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, it 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, both 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 in
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, and 3-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 rotation process at a partial double bond [20] The proton signals oftheN-methyl group ofthe5-N-acetimidoyl-5-N-methyl-7-N-acetyllegionaminic acid (3) did 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 concluded 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 nitrogen and the nonprotonated carbon, i.e the chemical exchange process Rotation is fast on the NMR timescale or, 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- nationofthegroupathighpH,whichdestabilizesthedouble-bonded transition state, or due to a lowered activation energy
of the rotational 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 acid,
so that chemical exchange is slow on the NMR timescale, thus indicating that it rarely occurs Nevertheless, at the moment both isomers of 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 only 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 exchange, i.e at elevated temperatures was also possible Although there is probably no chemical exchange in the bound state, only the single (coalesced) proton signal arising from chemical exchange in the 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 description of an application of STD NMR spectroscopy to identify the LPS epitope of a monoclonal antibody showing the advantages of this direct approach for the purpose of relatively 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
A C K N O W L E D G E M E N T S
We thank Dr C Roll for help with temperature dependence NMR spectroscopy experiments, and Dr T Weimar for help with SPR
Trang 10analysis This work was ®nancially supported 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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S U P P L E M E N T A R Y M A T E R I A L
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