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

Eur. J. Biochem. 269, 573–582 (2002) (cid:211) FEBS 2002

Epitope mapping of the O-chain polysaccharide of Legionella pneumophilaserogroup 1 lipopolysaccharide by saturation-transfer-difference NMR spectroscopy

Oliver Kooistra1, Lars Herfurth2, Edeltraud Lu¨ neberg3, Matthias Frosch3, Thomas Peters2 and Ulrich Za¨ hringer1

1Research Center Borstel, Center for Medicine and Biosciences, Germany; 2Institute for Chemistry, Medical University of Lu¨beck, Germany; 3Institute for Hygiene and Microbiology, University of Wu¨rzburg, Germany

saturation-transfer-di(cid:128)erence (STD) NMR spectroscopy in order to map the mAb 2625 epitope on a molecular level. It could be demonstrated that the binding a(cid:129)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, thus indicating these derivatives to represent the major epitope of mAb 2625.

lipopolysaccharide; Legionella pneumophila;

Keywords: bioa(cid:129)nity studies; NMR.

Two modifications 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-specific mon- oclonal antibody mAb 2625 to isolated OPS with surface- plasmon-resonance biomolecular interaction analysis and

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 (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].

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-specific 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

Recently, a phase-variable expression of a virulence- associated LPS epitope of L. pneumophila has been insertion and described previously [13]. Chromosomal excision of a 29-kb unstable genetic element, possibly of phage origin, was identified 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-specific 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 modification of bacterial polysaccharides, which is described for the first time in the accompanying paper [15]. The components identified 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¨ hringer, Forschungszentrum Borstel, Zentrum fu¨ r 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-di(cid:128)er- ence; EXCY, exchange spectroscopy; FID, free induction decay. (Received 8 August 2001, revised 13 November 2001, accepted 16 November 2001)

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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 spontaneous phase variant (strain 811), and an isogenic mutant (strain 5215) [15].

(GPC) on a

Preparation, modification, and fractionation of PS and PSNH4OH/HF LPS each of wild-type RC1, mutant 5215, and phase variant 811 was degraded at 100 (cid:176)C for 2.5 h with 0.1 M NaOAc/ HOAc buffer (pH 4.4, 10 mgÆmL)1 LPS), and the resultant lipid A was removed by centrifugation (5000 g, 30 min). The supernatant was lyophilized and fractionated by gel- permeation chromatography column (2.5 · 120 cm; Bio-Rad) of Sephadex G-50 (S) (Pharmacia) using 50 mM pyridinium/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 (cid:176)C, 16 h) and treated with 48% (v/v) aqueous hydrofluoric acid (HF, 4 (cid:176)C, 168 h) in order to selectively cleave the glycosidic linkage of 6-deoxy sugars [18] to obtain PSNH4OH/HF as described in the accompanying paper [15]. PSNH4OH/HF was fractionated by tandem GPC to long-, middle-, and short-chain molecular species [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 affinity 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 first 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.

Fig. 1. Proposed structure of Legionella pneu- mophila PSNH4OH/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 N2); 3-E and 3-Z, stereoisomers of 5-N-acetimi- doyl-7-N-acetyl-5-N-methyllegionaminic acid. The reducing RhaI residue is only present in 70% with a- and b-configuration in a ratio of approximately 5 : 1, which is also the case for free RhaII in the other 30% of the molecules. The anomeric configuration of the ketosidic linkage of the legionaminic acid residue attached to RhaII may be di(cid:128)erent and the position of the N-methylated legionaminic acid derivatives have not been confirmed. n is 40 on average for long-chain PSNH4OH/HF and 18 on average for middle-chain PSNH4OH/HF.

M A T E R I A L S A N D M E T H O D S

Preparation of mAb 2625

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 modified phenol/chloroform/ petroleum ether procedure as described previously [6,17].

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 purification was carried out. Anti- body purification was performed using a HiTrap protein G column (Pharmacia) with a GradiFrac system device (Pharmacia). Antibodies were eluted from the protein G resin with 0.1 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 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4).

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The protein concentration was determined using the bicinchoninic acid protein assay reagent kit (Pierce).

NMR spectroscopy

1D 1H 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.

was immobilized on a research grade CM5 sensor chip in 10 mM 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 mM Hepes buffer (pH 7.4) containing 150 mM NaCl and 0.005% (v/v) polysorbate 20 (BIAcore) at a flow rate of 10 lLÆmin)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 affinity (Kd) was determined by steady state affinity line- fitting 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, Kd values were determined by linear regression of Scatchard plots.

R E S U L T S

Polysaccharide samples were lyophilized three times from 2H2O and measured in 2H2O (2H, 99.996%; Cambridge Isotope Laboratories) at 27 (cid:176)C. Chemical shifts were refer- enced to external acetone (dH 2.225 p.p.m.; dC 31.45 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 1 M HCl or 1 M NaOH and recording 1D 1H NMR spectra at constant temperature (275 K). At pH 7.5 1D 1H NMR spectra were recorded at temperatures between 283 and 323 K in 10-K intervals.

Preparation and characterization of ligands

MAb 2625 was ultrafiltrated 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 lM based 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 1D 1H 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 protein 1H NMR signals in the aromatic region 7.2 p.p.m. It was tested that no amido- 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 1H2HO was not suppressed.

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 (KdoI and 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 fi 8)-KdoII disaccharide, 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 PSNH4OH/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 PSNH4OH/HF contained only Rha linked as fi 3)-a-L-RhaII- (1 fi 3)-L-RhaI disaccharide (70%) or as fi 3)-L-RhaII monosaccharide (30%) to polylegionaminic acid [15] (Fig. 1). Only the long- and middle-chain PS (as well as PSNH4OH/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.

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

Long-, middle-, and short-chain PSNH4OH/HF from wild- type RC1 were investigated by 1D 1H NMR spectroscopy and signal integration was performed to calculate the average chain-length of the PSNH4OH/HF and the distribution of N-methylated legionaminic acid derivatives [15]. Integra- tion of the signals of 1D 1H NMR spectra indicated that the

SPR analyses were carried out using an automated BIAcore 3000 biosensor instrument (BIAcore). mAb 2625, an IgG,

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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 PSNH4OH/HF from wild-type RC1, obtained with less data points, generated Kd values in the same range: 43 lM (Scatchard: 43 lM) for long-chain PSNH4OH/HF and 30 lM (Scatchard: 31 lM) for middle-chain PSNH4OH/HF. The sensorgrams for long-chain PSNH4OH/HF had a similar square pulse form as that for short-chain PSNH4OH/HF. With the long- and middle-chain PSNH4OH/HF from mutant 5215 and phase variant 811, no measurable affinity could be determined. Resonance units were not higher than those obtained with buffer as control experiments.

average chain-length of long-, middle-, and short-chain PSNH4OH/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, 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-acetyl deriva- tives of legionaminic acid was 1 : 1 in long-chain and 1 : 2 in middle-chain PSNH4OH/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 specific length. The proposed structure of PSNH4OH/HF from wild-type RC1, which was used for SPR analyses and STD NMR spectroscopy experiments is presented in Fig. 1. The PSNH4OH/HF from mutant 5215 and phase variant 811 had the same chain- length as that from wild-type RC1 [15].

STD NMR experiments of middle-chain PSNH4OH/HF from wild-type RC1 in the presence of mAb 2625

SPR studies with immobilized mAb 2625

To describe the epitope responsible for the binding inter- action of the polysaccharide chain with mAb 2625 at atomic resolution, both PSNH4OH/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 PSNH4OH/HF and were completed with long-chain PS (see below) in order to study the influence of the carbohydrate polymer chain on binding.

In order to investigate the binding behaviour of mAb 2625, binding affinity (Kd) was determined by SPR for the binding to immobilized mAb 2625 of isolated long- and middle- chain PSNH4OH/HF from L. pneumophila wild-type RC1, mutant 5215, and phase variant 811. The PSNH4OH/HF from wild-type RC1 bound to mAb 2625 with a rapid association and dissociation to and from the antibody, typical for low– affinity interaction like antibody-carbohydrate binding [19]. The Kd value for middle-chain PSNH4OH/HF from wild-type RC1 determined at equilibrium binding was 26 lM (Fig. 2,

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/HF from mutant 5215 lacking the N-methyl groups also with and without antibody. The latter PSNH4OH/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 sufficient for efficient 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 significant sat- uration transfer from the protein to the ligand in the STD spectra (Fig. 4B). Comparison of the STD spectrum with the corresponding 1D 1H 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 (cid:25) 15%. The maximum values for 3-E and 3-Z were considerably lower, (cid:25) 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 1H 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 PSNH4OH/HF from wild-type RC1 with immobilized mAb 2625. Steady-state a(cid:129)nity line-fitting based on end point values at equilibrium binding obtained with 14 ligand concentrations between 1.5 lM and 292 lM (A). Scat- chard analysis based on the same data (B).

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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 ((cid:25) 3 and 2%; Fig. 3B,C) and, furthermore, these signals were also detected as the only signals in the STD spectrum the middle-chain PSNH4OH/HF from mutant 5215 of (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 PSNH4OH/HF could not be confirmed by our experiments.

The 1D 1H NMR spectra of mAb 2625 together with middle-chain PSNH4OH/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 filters used during the prepa- ration of the samples or mAb 2625.

Temperature and pH dependence of 1D 1H 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], 1D 1H NMR spectra of long-chain PSNH4OH/HF from 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 influenced 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 (cid:25) 7 and beginning with pH (cid:25) 8 the lower-field pair of signals of 2-E [dH 3.30 (trans) and dH 3.19 (cis)] broadened and began to coalesce, so that from pH (cid:25) 9 only one sharp signal was detected (Fig. 6A–F). The higher-field 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 (cid:25) 2) it seemed that the ratio of the signals, balanced at neutral pH, was slightly changed towards the 3-E isomer.

STD effect of (cid:25) 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 (cid:25) 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 identified unambiguously, either in the 1D 1H NMR spectrum or in the STD NMR spectrum, and one signal (dH 2.22) in the STD NMR spectrum showing significant saturation transfer ((cid:25) 6% maximum STD effect) could not be assigned at all. Proton signals from the pyranose ring or

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 finally 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 significantly change within this range (Fig. 6G–M). The N-methyl signals of 3 did not change even under the drastic conditions pH (cid:25) 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 identified 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.

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at 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 significantly 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 difficult 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 1H NMR (A and C) and STD NMR (B and D) spectra of middle-chain PSNH4OH/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. NMecis and NMetrans, N-methyl groups of 2-E; NMe, N-methyl group of the isomers of 3; NAmCH3 and NAcCH3, C-methyl group of the acetimidoylamino and acetamido substituents, respectively.

Fig. 5. Detail of the middle-chain the STD NMR spectrum of PSNH4OH/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.

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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.

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, 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).

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.

Fig. 6. Dependence of proton signals of the N-methyl groups in 2 and 3 from pH and tem- perature. 1D 1H NMR spectra of the long- chain PSNH4OH/HF from wild-type RC1 were recorded in 10% deuterated water at constant temperature (275 K) with p1H/2H values of (cid:25) 2, (cid:25) 7, (cid:25) 8, (cid:25) 9, and (cid:25) 11 (A–F), and with constant pH (p1H/2H 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.

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

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, significant 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

580 O. Kooistra et al. (Eur. J. Biochem. 269)

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Fig. 7. 1D 1H 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 unspecific 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.

extensive 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].

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].

Investigations of crystal structures of monoclonal anti- bodies in complex with carbohydrate antigens have shown that a small antigenic determinant can dictate a highly specific immune response [24]. The OPS of the LPS from the two Vibrio cholerae serotypes Inaba and Ogawa is a homo- polymer of a-(1 fi 2)-linked N-(3-deoxy-L-glycero-tetronyl)- D-perosamine [25,26] differing only by the presence of a

STD NMR spectroscopy [16] offers an efficient 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. 8. 1D 1H 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 p2H 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.

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Epitope mapping with mAb 2625 (Eur. J. Biochem. 269) 581

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 affinity to mAb 2625 (data not shown).

The binding affinity (Kd) should be in the range of 1 mM to (cid:25) 10 nM. Stronger binding often suffers from off-rates being too low. This usually prevents sufficient 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 affinity can be measured by surface-plasmon-resonance biomolecular interaction analyses [29].

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 of the N-methyl group of the 5-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- nation of the group at high pH, which destabilizes the double- 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 confirmed 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).

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 identified as being responsible for phase variation of the epitope of the LPS-specific mAb 2625 [15]. In order to determine the binding affinity 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 significant affinity, which proved the epitope still to be present in the degraded PSNH4OH/HF. The binding affinity 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-affinity interaction [19]. Mixtures of PSNH4OH/HF molecular species of different size containing different ratios of the N-methylated legionaminic acid derivates bound with similar affinities. Nevertheless, the observed low affinity 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 PSNH4OH/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.

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 modification of the OPS, a sufficient magnetization transfer was observed, showing that in specific cases the molecular mass limit of the ligand for STD NMR spectroscopy can be extended.

This is the first 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 purified to absolute homogeneity.

A C K N O W L E D G E M E N T S

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 affinity) 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 affinity. The question of whether the N-methylated legionaminic acid derivative binds with different affinity to mAb 2625 can only be answered by experiments with homogeneous and structurally defined

We thank Dr C. Roll for help with temperature dependence NMR spectroscopy experiments, and Dr T. Weimar for help with SPR

582 O. Kooistra et al. (Eur. J. Biochem. 269)

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7-acetamido-3,5,7,9-tetradeoxynon-2-ulosonic acid in the O-chain polysaccharide. Eur. J. Biochem. 269, 560–572. analysis. This work was financially supported by grants from the Deutsche Forschungsgemeinschaft, LU 514/2-2 (E. L. and M. F.) and ZA 149/3-2 (U. Z.).

16. Mayer, M. & Meyer, B. (1999) Characterization of ligand binding by saturation transfer di(cid:128)erence NMR spectroscopy. Angew. Chem. International Ed. 38, 1784–1788.

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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

The following material is available from http://www. blackwell-science.com/products/journals/suppmat/ejb/ ejb2684/ejb2684sm.htm

15. Kooistra, O., Lu¨ neberg, E., Knirel, Y.A., Frosch, M. & Za¨ hrin- ger, U. (2001) N-Methylation in polylegionaminic acid is associ- ated with the phase-variable epitope of Legionella pneumophila serogroup 1 lipopolysaccharide. Identification of 5-(N,N-dime- thylacetimidoyl) amino- and 5-acetimidoyl (N-methyl) amino-