Báo cáo Y học: Chemical structure and immunoreactivity of the lipopolysaccharide of the deep rough mutant I-69 Rd–/b+ of Haemophilus influenzae

Eur. J. Biochem. 269, 1237–1242 (2002) (cid:211) FEBS 2002

Chemical structure and immunoreactivity of the lipopolysaccharide of the deep rough mutant I-69 Rd–/b+ of Haemophilusinfluenzae

Sven Mu¨ ller-Loennies, Lore Brade and Helmut Brade

Research Center Borstel, Center for Medicine and Biosciences, Borstel, Germany

anion

exchange

biol. 23, 569–577] was confirmed with neoglycoconjugates obtained by conjugation of the isolated oligosaccharides to BSA. In addition, a mAb S42-10-8 with unknown epitope specificity could be assigned using the neoglycoconjugates described herein. This mAb binds to an epitope composed of the bisphosphorylated glucosamine backbone of lipid A and Kdo-4P, whereby the latter determines the specificity strictly by the position of the phosphate group.

carbohydrate

antibody; Kdo-phosphate;

Keywords: neoglycoconjugate; serology; sugar phosphate.

From the lipopolysaccharide of the deep rough mutant I-69 Rd–/b+ of Haemophilus influenzae two oligosaccharides were obtained after de-O-acylation and separation by high-performance chromatography. Their chemical structures were determined by one- and two-dimensional 1H-, 13C- and 31P-NMR spectroscopy as aKdo-4P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P and aKdo-5P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P. The spe- cificity of mAbs S42-21 and S42-16 specific for Kdo-4P or Kdo-5P, respectively [Rozalski, A., Brade L., Kosma P., Moxon R., Kusumoto S., & Brade H. (1997). Mol. Micro-

that

Haemophilus influenzae normally colonizes the human nasopharynx but may cause severe infections, in particular meningitis, in children. A major virulence factor of this human pathogen is the type b capsule, an acidic polysac- charide composed of ribose, ribitol and phosphate and which is the basis of an effective conjugate vaccine [1]. Among other virulence factors is the lipopolysaccharide (LPS) in which we are interested for various reasons: (a) LPS is an essential component of the outer membrane in all Gram-negative bacteria; (b) LPS is the endotoxin of Gram- negative bacteria; (c) LPS is a major surface antigen leading to the induction of protective antibodies; and (d) the understanding of the biosynthesis of LPS may allow the distinct blockage of essential steps as a new strategy for the development of antibiotics [2,3].

ryl group to position 4 of the Kdo residue has also been cloned [5,6]. Coexpression of both enzymes in an Escheri- chia coli strain lacking its own Kdo transferase led to the synthesis of an LPS which contained exclusively Kdo-4P [7]. For this study mAbs were useful to identify the secondary gene products. We have reported earlier on mAb recogni- zing either the 4- or 5-phosphorylated Kdo which was chemically synthesized and conjugated to BSA [8]. In addition, we found mAb S42-10-8 which was specific for the I-69 LPS but did not react with Kdo-4P or Kdo-5P alone. Therefore, this antibody was assumed to recognize an epitope requiring, in addition to a phosphorylated Kdo residue, the phosphorylated lipid A backbone. As the LPS species containing the Kdo-4P or Kdo-5P could not be separated at time and were not yet chemically synthesized, the specificity of this mAb has not yet been elucidated. Here, we report on: (a) the successful separation of the deacylated carbohydrate backbone of I-69 LPS into two pure oligosaccharides containing either Kdo-4P or Kdo-5P; (b) the structural analysis of both oligosaccharides by NMR; and (c) the characterization of a new mAb recognizing a phosphorylated carbohydrate epitope.

The smallest LPS structure which still allows the bacter- ium to survive was found in the mutant strain I-69 Rd–/b+ of H. influenzae (referred to here as I-69) where a single phosphorylated 3-deoxy-D-manno-oct-2-ulopyrano- sonic acid (Kdo) residue is linked to the lipid A moiety. Helander et al. have shown that the I-69 LPS was composed of two molecular species with Kdo phosphorylated at either position 4 or 5 [4].

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

The Kdo transferase of I-69 has been cloned and characterized and the phosphokinase adding the phospho-

Bacteria and bacterial LPS H. influenzae I-69 Rd–/b+ was cultivated as described previously [9]. Bacteria were washed with ethanol, acetone (twice), and ether, and dried. LPS was extracted from dry bacteria by the phenol/chloroform/petroleum ether method [10] in a yield of 4.4% of dry bacteria. De-O-acylated LPS was prepared after hydrazine treatment of LPS for 30 min at 37 (cid:176)C (yield: 81% based on the glucosamine content), and deacylated LPS (LPSdeac) was obtained by hydrolysis of de-O-acylated LPS in 4 M KOH as reported [11]. LPSdeac was further purified by preparative high performance anion exchange chromatography (HPAEC) using water as eluent A

Correspondence to H. Brade, Research Center Borstel, Center for Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany. Fax: + 49 4537 188419, Tel.: + 49 4537 188474, E-mail: hbrade@fz-borstel.de Abbreviations: HPAEC, high performance anion exchange chroma- tography; Kdo, 3-deoxy-D-manno-oct-2-ulopyranosonic acid; LPS, lipopolysaccharide; LPSdeac, deacylated LPS. Note: S. Mu¨ ller-Loennies and L. Brade contibuted equally to this work. (Received 8 August 2001, revised 21 December 2001, accepted 3 January 2002)

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1238 S. Mu¨ ller-Loennies et al. (Eur. J. Biochem. 269)

MAbs

and 1 M ammonium acetate as eluent B and a gradient of 1% to 99% over 80 min. Desalting was achieved by gel filtration on a column of 100 · 1.5 cm Sephadex G10 in pyridine/acetic acid/water (4 : 10 : 1000, v/v/v) at a flow rate of 1 mLÆmin)1. Fractions 1 and 2 were obtained in pure form in yields of 21.6 and 9.5%, respectively, based on the glucosamine content.

NMR spectroscopy

Monoclonal antibodies S42-16, S42-21 and S42-10-8 were obtained after immunization and selection as described [8]. Culture supernatants were prepared in at least 100 mL quantities and antibodies were purified on protein G-Sepharose (Pharmacia/LKB) according to the supplier’s instructions. Purification was ascertained by SDS/PAGE and protein concentrations were determined by the bicin- choninic acid assay (Pierce).

Serology

The deacylated LPS from H. influenzae I-69 was investi- gated by one-dimensional 1H-NMR- and 13C-NMR and spectroscopy at 600 and 150 MHz, respectively, on a Bruker DRX 600 Avance spectrometer; 31P-NMR spectra were recorded on a Bruker DPX 360 Avance spectrometer at 145 MHz. All spectra were recorded on a 0.5-mL solution of 5 mg sample in D2O. As reference served acetone 2.225 p.p.m. (1H), dioxane 67.4 p.p.m. (13C) and 85% phosphoric acid 0 p.p.m. (31P). All spectra were run at a temperature of 300 K. For 31P measurements the pD was adjusted to pD 2. Other measurements were performed at pD 6 due to the acid labile nature of the Kdo-linkage.

recorded. For

For ELISA, neoglycoconjugates were coated onto Maxi- Sorp microtiter plates (U-bottom, Nunc). Antigen solutions were adjusted to equimolar concentrations based on the amount of ligand present in the respective glycoconjugate. Unless stated otherwise, 50 lL volumes were used. Micro- titer plates were coated with the respective antigen solution in 50 mM carbonate buffer pH 9.2 at 4 (cid:176)C overnight. Plates were washed twice with distilled water; further washing was carried out in NaCl/Pi supplemented with 0.05% Tween 20 (Bio-Rad) and 0.01% thimerosal (NaCl/Pi/Tween-T). Plates were then blocked with NaCl/Pi/Tween-T supplemented with 2.5% casein (NaCl/Pi/Tween-TC) for 1 h at 37 (cid:176)C on a rocking platform followed by two washes. Appropriate antibody dilutions in NaCl/Pi/Tween-TC supplemented with 5% BSA were added and incubated for 1 h at 37 (cid:176)C. After washing, peroxidase-conjugated goat anti-(mouse IgG) Ig (heavy and light chain specific; Dianova) was added (diluted 1 : 1000) and incubation was continued for 1 h at 37 (cid:176)C. After three washes in NaCl/Pi/Tween-T, the plates were washed in substrate buffer (0.1 M sodium citrate, pH 4.5). Substrate solution was freshly prepared and was composed of azino-di-3-ethylbenzthiazolinsulfonic acid (1 mg) dissolved in substrate buffer (1 mL) with sonication in an ultrasound water bath for 3 min followed by the addition of hydrogen peroxide (25 lL of a 0.1% solution). After 30 min at 37 (cid:176)C, the reaction was stopped by the addition of 2% aqueous oxalic acid and the plates were read with a microplate reader (Dynatech MR 700) at 405 nm.

Two-dimensional homonuclear 1H,1H-DQF-COSY was recorded over a spectral width of 7.5 p.p.m. in both dimensions recording 512 experiments of 32 scans. Four thousand data points were recorded in F2. Zero-filling was applied in F1 to 1000 data points. Heteronuclear 1H,13C-NMR correlation spectroscopy was recorded as HMQC. Two thousand data points were recorded in F2 over a spectral width of 10 p.p.m. and 256 experiments consisting of 24 scans per increment. Phase cycling was performed using States-TPPI. Prior to Fourier transfor- mation zero-filling was applied in F1 to 512 data points. 31P-NMR spectroscopy was recorded with continuous wave decoupling during acquisition. A total of 32 scans 1H,31P-NMR COSY a HMQC was experiment was recorded consisting of 256 experiments and 32 scans each. Two thousand data points were collected over a spectral width of 10 p.p.m. in F2 and zero filling was applied in F1 to yield 512 data points. The spectral width was 10 p.p.m. in F1.

Neoglycoconjugates

The amino groups of the glucosamine residues in LPSdeac and in the oligosaccharides obtained from LPSdeac were activated with glutardialdehyde and conjugated to BSA as described [12]. The amount of ligand present in the conjugates was determined by measuring the amount of protein (Bradford assay, Bio-Rad) and glucosamine (Table 1).

For ELISA using LPS as a solid-phase antigen another protocol was used. Polyvinyl microtiter plates (Falcon 3911) were coated with various amounts of LPS dissolved in NaCl/ Pi (10 mM pH 7.3, 0.9% NaCl, 50 lL) at 4 (cid:176)C overnight or at 37 (cid:176)C for 1 h. All following steps were performed at 37 (cid:176)C with gentle agitation and all washing steps were performed four times. Coated plates were washed in NaCl/Pi, blocked for 1 h with blocking buffer (2.5% casein in NaCl/Pi) and then incubated for 1 h with mAb diluted in blocking buffer (50 lL). Plates were washed in NaCl/Pi and incubated for

Table 1. Oligosaccharides and neoglycoconjugates used in this study. For derivatization procedures see Materials and methods. Molar ratio of ligand to protein given in parentheses.

Amount of ligand (nmolÆmg)1) Chemical structure Abbreviation

33 (2.4) 16 (1.1) 15 (1.0) aKdo-4P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P aKdo-5P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P aKdo-4/5P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P-BSA aKdo-4P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P-BSA aKdo-5P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P-BSA Kdo4PGlcN2P2 Kdo5PGlcN2P2 LPSdeac-BSA Kdo4P-GlcN2P2-BSA Kdo5P-GlcN2P2-BSA

Structure and antigenicity of H. influenzae LPS (Eur. J. Biochem. 269) 1239

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1 h with peroxidase-conjugated goat anti-(mouse IgG) Ig or goat anti-(rabbit IgG) Ig (heavy and light chain specific, Dianova; diluted 1 : 1000 in blocking buffer, 50 lL). Further development of the reaction was as described above. All tests were set up in quadruplicate. Confidence values of the means were less than 10%.

R E S U L T S

and compound 2

Isolation and structural analysis of the phosphorylated carbohydrate backbone of I-69 LPS

Table 4). Both compounds contained one glycosidic phos- phate linked to the a-GlcN (A) of the lipid A backbone leading to a splitting of the signal of its anomeric proton and another phosphate linked to the 4-position of the b-config- ured GlcN (B). The far downfield position of the chemical shifts of proton H-4 and carbon C-4 of the Kdo-residue (C) of compound 1 and the downfield shift to the same frequencies of proton H-5 and carbon C-5 of the Kdo-resi- due (C) of compound 2 identified compound 1 as Kdo-4P- GlcN2-P2 as Kdo-5P-GlcN2-P2 (Tables 2–4). The correct position of phosphates was finally determined by 1H,31P-HMQC spectroscopy.

Serology

The LPS of H. influenzae I-69 was successively de-O-acylated and de-N-acylated with hydrazine and potassium hydrox- ide, respectively, leading to two major products as revealed by HPAEC (Fig. 1). The two peaks, compounds 1 and 2, could be separated from each other by preparative HPAEC with yields of 11.6 mg (21.6% of LPS) and 5.1 mg (9.5% of LPS) for Kdo-4P-GlcN2-P2 and Kdo-5P-GlcN2-P2, respectively.

Both compounds were identified by one- and two- dimensional NMR spectroscopy. Spectra of both contained characteristic signals of a single a-Kdo-residue, one b-linked GlcN and one a-configured GlcN [7]. In addition, three phosphate-residues were identified by 31P-NMR spectro- scopy (Fig. 2). With respect to the carbohydrate and phosphate composition the two compounds were identical and was reflected by almost identical one-dimensional 1H-NMR spectra (Fig. 3, Table 2). As expected the com- pounds differed in their phosphate substitution (Fig. 3,

Both oligosaccharides were activated with glutardialdehyde and conjugated to BSA as described [12]. Chemical analyses indicated a molar ratio of protein to ligand of 1 : 1.1 and 1 : 1.0 for Kdo-4P-GlcN2-P2-BSA and Kdo-5P-GlcN2- P2-BSA, respectively. Both neoglycoconjugates were used in ELISA to determine the epitope specificities of mAb. LPS and LPSdeac-BSA were used for comparison, whereby the latter contained a mixture of 4- and 5-phosphorylated Kdo in the ratio as it occurs in natural LPS. Clone S42-16 and S42-21 were confirmed to be specific for Kdo-5P and Kdo-4P, respectively. As seen in Fig. 4B clone S42-16 bound over a wide range of antigen coating concentrations to Kdo-5P-GlcN2-P2-BSA at (10–0.08 pmol per well) antibody concentrations as low as 1 ngÆmL)1. No binding of this antibody was observed with Kdo-4P-GlcN2-P2-BSA even at highest antigen concentration (10 pmol per well) and antibody concentration (10 lgÆmL)1) (Fig. 4A). The mAb S42-21 bound only to Kdo-4P-GlcN2-P2-BSA (Fig. 4C) but not to Kdo-5P-GlcN2-P2-BSA (Fig. 4D) The affinity of mAb S42-21 was approximately 200 times lower than that of mAb S42-16 for the homologous epitope.

Fig. 2. 31P-NMR spectrum of aKdo-4P-(2 fi 6)-bGlcN-4P-(1 fi 6)- aGlcN-1P (top) and aKdo-5P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P (bottom). Fig. 1. HPAEC chromatogram of deacylated LPS from H. influenzae I-69. Shown is the analytical separation of the crude mixture (A) and the analytical chromatography of the isolated species (B and C). Peaks 1 and 2 represent aKdo-4P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P and aKdo-5P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P, respectively.

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The generation of clone S42-10-8 has been reported previously [8] but its epitope specificity could not be determined so far. Binding of this antibody was tested in

ELISA using various concentrations of Kdo-4P-GlcN2- P2-BSA and Kdo-5P-GlcN2-P2-BSA, LPS or LPSdeac- BSA.

Fig. 3. 1H-NMR spectra of aKdo-4P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P (top) and aKdo-5P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P (bottom). The asterisk indicates signals of tryethylamine.

1H-Chemical shift (p.p.m.) and coupling constants (Hz) for proton

Table 2. 1H-NMR chemical shift data of compounds 1 and 2. NR, not resolved.

Compound Residue H-1 H-2 H-3ax H-3eq H-4 H-5 H-6a H-6b H-7 H-8a H-8b

4.123 1 A fi 6aGlcN1P

3.873 10 3.859 3.448 10 3.859 3.740 3.852 12; 9 3.495 4.248 4 3.698 B fi 6bGlcN4P 5.659 4; 8a 4.908 8 3.380 10 3.072 10 C aKdo4P 4.179 3.926 3.652 2.149 6 3.918 12; NR 6 4.124 2 A fi 6aGlcN1P

a 3JH-1,P.

4.514 6 3.426 10 3.840 3.727 3.766 9 3.861 12; 9 3.471 4.229 4 3.714 B fi 6bGlcN4P 5.654 4; 7a 4.902 8 3.364 11 3.074 10 C aKdo5P 4.141 4.507 3.907 3.649 1.925 )12; 12 3.889 10 3.854 10 1.919 )12; 12 2.142 5 3.736 9 3.941 13; NR

Structure and antigenicity of H. influenzae LPS (Eur. J. Biochem. 269) 1241

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13C-Chemical shift ( p.p.m.) of carbon

Table 3. 13C-NMR chemical shift data of compounds 1 and 2. ND, not determined.

C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 Compound Residue

1

69.77 63.47 2

A fi 6aGlcN1P B fi 6bGlcN4P C aKdo4P A fi 6aGlcN1P B fi 6bGlcN4P C aKdo5P 91.10 99.14 ND 91.16 99.02 ND 54.45 55.74 ND 54.45 55.76 ND 69.76 72.08 33.77 69.61 72.02 34.92 70.07 74.44 70.98 70.12 74.52 65.99 72.69 74.20 65.88 72.68 74.09 71.00 69.36 62.40 71.68 69.20 62.60 71.64 69.65 63.07

31P-Chemical shift (p.p.m.) for compound

Table 4. 31P-NMR chemical shifts of compounds 1 and 2.

1 2 Residue

A B C )1.41 0.58 0.68 )1.64 )0.01 1.65

Kdo-4P linked to the bisphosphorylated glucosamine backbone of the LPS of H. influenzae I-69.

Although, Kdo-4P alone is not bound to the antibody, the position of the phosphate group strictly determines the specificity of the epitope as no binding was observed with antigens containing Kdo-5P instead of Kdo-4P or with antigens containing nonphosphorylated Kdo.

Fig. 5. Binding curve of mAb S42-10-8. The ligands were I-69 LPS (A) and LPSdeac-BSA (B). The coating concentrations used were 400 (d), 200 (m), 100 (j) 50 (r), 25 (s), 12.5 (n), 6.3 (h) and 3.2 (e) pmolÆml)1 for LPSdeac-BSA. Due to the poor coating e(cid:129)ciency of LPS 2000 (d), 1000 (m), 500 (j) 250 (r), 125 (s), 63 (n), 32 (h) and 16 (e) pmolÆml)1 were used for the immobilization of LPS. Both were reacted with mAb concentrations indicated on the abscissa. Values are the mean of quadruplicates with confidence values not exceeding 10%.

Fig. 4. Binding curves of mAbs S42-16 (A and B), S42-21 (C and D), and S42-10-8 (E and F) to Kdo4P-GlcN2P2-BSA (A, C and E) and Kdo5P-GlcN2P2-BSA (B, D and F). ELISA plates were coated with 200 (d), 100 (m), 50 (j) 25 (r), 12.5 (s), 6.3 (n), 3.2 (h) and 1.6 (e) pmol ligandÆml)1 and reacted with the mAb concentrations indicated on the abscissa. Values are the mean of quadruplicates with confidence values not exceeding 10%.

D I S C U S S I O N

As seen in Fig. 4E, mAb S42-10-8 bound to Kdo-4P- GlcN2-P2-BSA and with comparable affinity to LPS (Fig. 5A) or LPSdeac-BSA (Fig. 5B) as solid phase antigen. No binding was observed with Kdo-5P-GlcN2-P2-BSA (Fig. 4F).

Kdo is a common constituent of LPS and its presence is essential for the survival of Gram-negative bacteria. Ac- cording to our present knowledge of the Kdo-lipid A region one Kdo residue is linked to position 6¢ of the glucosamine disaccharide backbone of lipid A and is substituted at position 5 by another sugar and at position 4 by another sugar or phosphate [13]. The LPS of the deep rough mutant I-69 of H. influenzae is unique in being composed of only one

The data show, together with those published earlier [8], that mAb S42-10-8 binds to a complex epitope composed of

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1242 S. Mu¨ ller-Loennies et al. (Eur. J. Biochem. 269)

2. Raetz, C.R.H. (1990) Biochemistry of endotoxins. Ann. Rev. Biochem. 59, 129–170.

3. Rietschel, E.T., Kirikae, T., Schade, F.U., Mamat, U., Schmidt, G., Loppnow, H., Ulmer, A.J., Za¨ hringer, U., Seydel, U., Di Padova, F., Schreier, M. & Brade, H. (1994) Bacterial endotoxin: Molecular relationships of structure to activity and function. FASEB J. 8, 217–225.

4. Helander, I., Lindner, B., Brade, H., Altmann, K., Lindberg, A.A., Rietschel, E.Th & Za¨ hringer, U. (1988) Chemical structure of the lipopolysaccharide of Haemophilus influenzae strain I-69 Rd–/b+. Eur. J. Biochem. 177, 483–492.

5. White, K.A., Kalashov, I.A., Cotter, R.J. & Raetz, C.R.H. (1997) A mono-functional 3-deoxy-D-manno-octulosonic acid (Kdo) transferase and a Kdo kinase in extracts of Haemophilus influen- zae. J. Biol. Chem. 272, 16555–16563.

phosphorylated Kdo residue in addition to lipid A whereby the Kdo is phosphorylated either at position 4 or 5. There was some uncertainty in the beginning whether the Kdo-5P was the result of phosphate migration [4], however, when mAbs specific for the 4- or 5-P became available it could be shown that both antibodies bound to native bacteria [8]. The final proof that both phosphates are made by the bacterium was provided recently when we coexpressed the monofunc- tional Kdo transferase and a phosphokinase of H. influenzae in E. coli resulting in LPS which contained exclusively Kdo- 4P [7]. As the LPS obtained from this recombinant strain was deacylated by the same protocol as used in this study it is apparent that the appearance of the 5P is not the result of phosphate migration. Therefore, we conclude that H. influenzae possesses two independent phosphokinases attaching phosphate to position 4 or 5 whereby the 5-kinase has not yet been identified. With the results presented here the complete structures of the phosphorylated carbohydrate backbones of both LPS species made by H. influenzae I-69 are uniquivocally established and we have presented a for preparing these two oligosaccharides in protocol sufficient quantities.

6. White, K.A., Lin, S., Cotter, R.J. & Raetz, C.R.H. (1999) A Haemophilus influenzae gene that encodes a membrane bound 3-deoxy-D-manno-octulosonic Acid (Kdo) kinase. J. Biol. Chem. 274, 31391–31400.

7. Brabetz, W., Mu¨ ller-Loennies, S. & Brade, H. (2000) 3-Deoxy- D-manno-oct-2-ulosonic acid (Kdo) transferase (WaaA) and kdo kinase (KdkA) of Haemophilus influenzae are both required to complement a waaA knockout mutation of Escherichia coli. J. Biol. Chem. 275, 34954–34962.

8. Rozalski, A., Brade, L., Kosma, P., Moxon, R., Kusumoto, S. & Brade, H. (1997) Characterization of monoclonal antibodies recognizing three distinct, phosphorylated carbohydrate epitopes in the lipopolysaccharide of the deep rough mutant I-69 Rd-/b+ of Haemophilus influenzae. Mol. Microbiol. 23, 569–577.

(1987)

9. Zamze, S.E., Ferguson, M.A.J., Moxon, E.R., Dwek, R.A. & Rademacher, T.W. Identification of phosphorylated 3-deoxy-manno-octulosonic acid as a component of Haemophilus influenzae lipopolysaccharide. Biochem. J. 245, 583–587.

10. Galanos, C., Lu¨ deritz, O. & Westphal, O. (1969) A new method for the extraction of R. lipopolysaccharides. Eur. J. Biochem. 9, 245–249.

11. Holst, O., Broer, W., Thomas-Oates, J.E., Mamat, U. & Brade, H. (1993) Structural analysis of two oligosaccharide biphosphates isolated from the lipopolysaccharide of a recombinant strain of Escherichia coli F515 (Re chemotype) expressing the genus-specific epitope of Chlamydia lipopolysaccharide. Eur. J. Biochem. 214, 703–710.

12. Brade, L., Holst, O. & Brade, H. (1993) An artificial glycoconju- gate containing the bisphosphorylated glucosamine disaccharide backbone of lipid A binds lipid A monoclonal antibodies. Infect. Immun. 61, 4514–4517.

We have performed this study not only to definitely identify the two differently phosphorylated LPS species but also to learn more about the recognition of charged carbohydrate epitopes by antibodies. We are interested in this aspect to better understand protein–carbohydrate inter- actions in general and the binding of antibodies against bacterial LPS in particular, as some of them are able to neutralize the endotoxic activities of LPS which are embed- ded in the phosphorylated lipid A moiety [14]. We have already characterized antibodies against the isolated lipid A moiety [15] or against Kdo [16] or Kdo-P [8]. In this context mAb S42-10-8 against I-69 LPS was of specific interest for us as it binds to an epitope composed of Kdo-P and lipid A; however, its detailed epitope specificity could not be inves- tigated so far due to the lack of appropriately defined antigens. The successful separation of these oligosaccharides described here together with a previously described conju- gation protocol [14] allowed the characterization of the epitope specificity of mAb S42-10-8. The binding data obtained in ELISA unequivocally proved that this mAb the trisaccharide aKdo-4P-(2–6)-bGlcN-4P- recognizes (1–6)-aGlcN-1P; it does not bind to Kdo, Kdo-4P, Kdo- 5P or aKdo-(2–4)-aKdo-(2–6)-bGlcN-4P-(1–6)-aGlcN-1P. The availability of both oligosaccharides as free ligands and as neoglycoconjugates now enables us to investigate further this antibody by NMR and crystallography.

13. Holst, O. (1999) Chemical structure of the core region of lipo- polysaccharides. In Endotoxin in Health and Disease. (Brade, H., Opal, S.M., Vogel, S.N. & Morrison, D.C., eds), pp. 115–154. Marcel Dekker Inc., New York.

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

We thank R. Moxon (Oxford, UK) for strain I-69 and V. Susott and S. Cohrs for technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (grant SFB470/C1 to L. B.). 14. Di Padova, F.E., Brade, H., Barclay, G.R., Poxton, I.R., Liehl, E., Schuetze, E., Kocher, H.P., Ramsay, G., Schreier, M.H., McClelland, D.B.L. & Rietschel, E.T. (1993) A broadly cross- protective monoclonal antibody binding to Escherichia coli and Salmonella lipopolysaccharide. Infect. Immun. 61, 3863–3872. 15. Kuhn, H.-M., Brade, L., Appelmelk, B.J., Kusumoto, S., Riets- chel, E.T. & Brade, H. (1992) Characterization of the epitope specificity of murine monoclonal antibodies directed against lipid A. Infect. Immun. 60, 2201–2210.

R E F E R E N C E S

16. Brade, H., Brabetz, W., Brade, L., Holst, O., Lo¨ bau, S., Lucakova, M., Mamat, U., Rozalski, A., Zych, K. & Kosma, P. lipopolysaccharide. J. Endotoxin Res. 4, (1997) Chlamydial 67–84. 1. Moxon, E.R. (1992) Session III: Pathogenesis of invasive Hae- mophilus influenzae disease. Molecular basis of invasive Haemo- philus influenzae type b disease. J. Infect. Dis. 165, S77–S81.