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Identification of a Cross-reactive Epitope Widely Present in Lipopolysaccharide from Enterobacteria and Recognized by the Cross-protective Monoclonal Antibody WN1 222-5

2003; Elsevier BV; Volume: 278; Issue: 28 Linguagem: Inglês

10.1074/jbc.m302904200

ISSN

1083-351X

Autores

Sven Müller‐Loennies, Lore Brade, C. Roger MacKenzie, Franco E. Di Padova, Helmut Brade,

Tópico(s)

Probiotics and Fermented Foods

Resumo

Septic shock due to infections with Gram-negative bacteria is a severe disease with a high mortality rate. We report the identification of the antigenic determinants of an epitope that is present in enterobacterial lipopolysaccharide (LPS) and recognized by a cross-reactive monoclonal antibody (mAb WN1 222-5) regarded as a potential means of treatment. Using whole LPS and a panel of neoglycoconjugates containing purified LPS oligosaccharides obtained from Escherichia coli core types R1, R2, R3, and R4, Salmonella enterica, and the mutant strain E. coli J-5, we showed that mAb WN1 222-5 binds to the distal part of the inner core region and recognizes the structural element R1-α-d-Glcp-(1→3)-[l-α-d-Hepp-(1→7)]-l-α-d-Hepp 4P-(1→3)-R2 (where R1 represents additional sugars of the outer core and R2 represents additional sugars of the inner core), which is common to LPS from all E. coli, Salmonella, and Shigella. WN1 222-5 binds poorly to molecules that lack the side chain heptose or lack phosphate at the branched heptose. Also molecules that are substituted with GlcpN at the side chain heptose are poorly bound. Thus, the side chain heptose and the 4-phosphate on the branched heptose are main determinants of the epitope. We have determined the binding kinetics and affinities (K D values) of the monovalent interaction of E. coli core oligosaccharides with WN1 222-5 by surface plasmon resonance and isothermal titration microcalorimetry. Affinity constants (K D values) determined by SPR were in the range of 3.6 × 10–5 to 3.2 × 10–8m, with the highest affinity being observed for the core oligosaccharide from E. coli F576 (R2 core type) and the lowest K D values for those from E. coli J-5. Affinities of E. coli R1, R3, and R4 oligosaccharides were 5–10-fold lower, and values from the E. coli J-5 mutant were 29-fold lower than the R2 core oligosaccharide. Thus, the outer core sugars had a positive effect on binding. Septic shock due to infections with Gram-negative bacteria is a severe disease with a high mortality rate. We report the identification of the antigenic determinants of an epitope that is present in enterobacterial lipopolysaccharide (LPS) and recognized by a cross-reactive monoclonal antibody (mAb WN1 222-5) regarded as a potential means of treatment. Using whole LPS and a panel of neoglycoconjugates containing purified LPS oligosaccharides obtained from Escherichia coli core types R1, R2, R3, and R4, Salmonella enterica, and the mutant strain E. coli J-5, we showed that mAb WN1 222-5 binds to the distal part of the inner core region and recognizes the structural element R1-α-d-Glcp-(1→3)-[l-α-d-Hepp-(1→7)]-l-α-d-Hepp 4P-(1→3)-R2 (where R1 represents additional sugars of the outer core and R2 represents additional sugars of the inner core), which is common to LPS from all E. coli, Salmonella, and Shigella. WN1 222-5 binds poorly to molecules that lack the side chain heptose or lack phosphate at the branched heptose. Also molecules that are substituted with GlcpN at the side chain heptose are poorly bound. Thus, the side chain heptose and the 4-phosphate on the branched heptose are main determinants of the epitope. We have determined the binding kinetics and affinities (K D values) of the monovalent interaction of E. coli core oligosaccharides with WN1 222-5 by surface plasmon resonance and isothermal titration microcalorimetry. Affinity constants (K D values) determined by SPR were in the range of 3.6 × 10–5 to 3.2 × 10–8m, with the highest affinity being observed for the core oligosaccharide from E. coli F576 (R2 core type) and the lowest K D values for those from E. coli J-5. Affinities of E. coli R1, R3, and R4 oligosaccharides were 5–10-fold lower, and values from the E. coli J-5 mutant were 29-fold lower than the R2 core oligosaccharide. Thus, the outer core sugars had a positive effect on binding. Lipopolysaccharides (LPS 1The abbreviations used are: LPS, lipopolysaccharide; DQF-COSY, double quantum-filtered correlation spectroscopy; Glcp, glucopyranose; GlcpN, 2-amino-2-deoxy-glucopyranose, l-α-d-Hepp, l-glycero-α-d-manno-heptopyranose; HMQC, heteronuclear multiple quantum correlation; HPAEC, high performance anion exchange chromatography; ITC, isothermal titration microcalorimetry; Kdo, 3-deoxy-α-d-manno-oct-2-ulopyranosonic acid; SPR, surface plasmon resonance; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin.; endotoxin) are major surface-exposed structural components of the outer membrane of Gram-negative bacteria (1Vaara M. Brade H. Opal S.M. Vogel S.N. Morrison D.C. Endotoxin in Health and Disease. Marcel Dekker, Inc., New York1999: 31-38Google Scholar), and in enterobacteria they consist of lipid A, core region, and O-antigen in many bacteria (2Holst O. Brade H. Opal S.M. Vogel S.N. Morrison D.C. Endotoxin in Health and Disease. Marcel Dekker, Inc., New York1999: 115-154Google Scholar). The lipid A moiety is responsible for many of the pathological effects observed in septic shock, a serious condition with high mortality rates, especially among hospitalized patients in intensive care units. Septic shock is the result of an uncontrolled systemic activation of the immune system by endotoxins, leading to high levels of proinflammatory cytokines such as tumor necrosis factor-α and interleukin-1. In the fight against septicemia, therapeutic strategies are aimed at the eradication of the bacteria by antibiotics, stabilization of the circulation symptomatically, and the neutralization of endotoxic effects. For the last goal, endotoxin antagonists, antibodies against tumor necrosis factor, interleukin-1 receptor antagonists, and LPS-binding proteins (BPI (bactericidal permeability-increasing protein) and LBP (LPS-binding protein)) have been considered (3Levy O. Elsbach P. Curr. Infect. Dis. Rep. 2001; 3: 407-412Crossref PubMed Google Scholar, 4Fenton M.J. Golenbock D.T. J. Leukocyte Biol. 1998; 64: 25-32Crossref PubMed Scopus (400) Google Scholar, 5Pollack M. Ohl C.A. Rietschel E.T. Wagner H. Pathology of Sepsis and Septic Shock. Springer, Heidelberg, Germany1999: 275-297Google Scholar, 6Baumgartner J.-D. Heumann D. Glauser M.-P. Brade H. Opal S.M. Vogel S.N. Morrison D.C. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 865-876Google Scholar). Antisera against the O-antigens of endotoxins protect against homologous bacteria. However, the large number of different O-antigens in enterobacteria, the serotype-restricted specificity of such antisera, and the rapid onset of shock have prevented their introduction into clinical practice. Whereas the chemical structure of the O-antigen is highly variable, the core region and lipid A show only limited structural variability within the enterobacteria. Following the observation that antibodies against the O-antigen are protective against homologous bacteria, the search for LPS antibodies with broad cross-reactivity is a valid concept for the immunotherapy of Gram-negative sepsis. Many investigators attempted the isolation of antibodies that are directed against the conserved regions of LPS (i.e. the lipid A and core region (reviewed in Ref. 7Müller-Loennies S. Di Padova F.E. Brade L. Heumann D. Rietschel E.T. Andrew P.W. Oyston P. Smith G.L. Stewart-Tull D.E. Fighting Infection in the 21st Century. Blackwell Science Ltd., Oxford2000: 143-178Crossref Scopus (4) Google Scholar)). Such antibodies have been presumed to be cross-reactive and cross-protective against different Gram-negative pathogens. Such a cross-protective effect was described for a polyclonal antiserum by Braude and Douglas (8Braude A.I. Douglas H. J. Immunol. 1972; 108: 505-512PubMed Google Scholar); however, all subsequently isolated LPS-specific monoclonal antibodies failed to show cross-reactivity in vitro and cross-protectivity in vivo (7Müller-Loennies S. Di Padova F.E. Brade L. Heumann D. Rietschel E.T. Andrew P.W. Oyston P. Smith G.L. Stewart-Tull D.E. Fighting Infection in the 21st Century. Blackwell Science Ltd., Oxford2000: 143-178Crossref Scopus (4) Google Scholar), with the exception of mAb WN1 222-5 (9Di 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. Infect. Immun. 1993; 61: 3863-3872Crossref PubMed Google Scholar). This mAb bound to LPS from all tested clinical isolates of Escherichia coli, Salmonella, and Shigella in Western blots and ELISA and showed cross-protective effects in vivo against the endotoxic activities of LPS (9Di 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. Infect. Immun. 1993; 61: 3863-3872Crossref PubMed Google Scholar). The smallest LPS structure bound by WN1 222-5 was found to be present in LPS from E. coli J-5. Due to the lack of a functional UDP-galactose-4-epimerase (ΔgalE mutant) (10Elbein A.D. Heath E.C. J. Biol. Chem. 1965; 240: 1919-1925Abstract Full Text PDF PubMed Google Scholar), this strain is unable to incorporate galactose into its LPS and therefore produces a truncated LPS consisting of several glycoforms (Fig. 1). The cross-reactivity was therefore attributed to a common epitope located in the inner core region of these LPS (9Di 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. Infect. Immun. 1993; 61: 3863-3872Crossref PubMed Google Scholar). LPS preparations are heterogenous and there are no methods available for the separation of acylated LPS into homogeneous compounds due to their amphiphilic nature. For this reason, the exact epitope of WN1 222-5 could not be determined using LPS. We have therefore developed methods for the deacylation of these molecules under conditions that do not cleave glycosidic bonds (11Holst O. Müller-Loennies S. Lindner B. Brade H. Eur. J. Biochem. 1993; 214: 695-701Crossref PubMed Scopus (46) Google Scholar) and the purification of LPS oligosaccharides. These oligosaccharides are then amenable to a detailed structural characterization and conjugation to proteins (12Brade L. Holst O. Brade H. Infect. Immun. 1993; 61: 4514-4517Crossref PubMed Google Scholar, 13Müller-Loennies S. Grimmecke D. Brade L. Lindner B. Kosma P. Brade H. J. Endotoxin Res. 2002; 8: 295-305Crossref PubMed Google Scholar). For J-5 LPS, five different oligosaccharides, which differ in their carbohydrate structures and phosphate substitution (Fig. 1), were obtained by deacylation under strong alkaline conditions (14Müller-Loennies S. Holst O. Lindner B. Brade H. Eur. J. Biochem. 1999; 260: 235-249Crossref PubMed Scopus (31) Google Scholar). The chemical structures of five different E. coli core types (R1 to R4 and K-12) and two chemically distinct core oligosaccharides of S. enterica (2Holst O. Brade H. Opal S.M. Vogel S.N. Morrison D.C. Endotoxin in Health and Disease. Marcel Dekker, Inc., New York1999: 115-154Google Scholar, 15Vinogradov E.V. Van Der D.K. Thomas-Oates J.E. Meshkov S. Brade H. Holst O. Eur. J. Biochem. 1999; 261: 629-639Crossref PubMed Scopus (51) Google Scholar, 16Müller-Loennies S. Lindner B. Brade H. Eur. J. Biochem. 2002; 269: 5982-5991Crossref PubMed Scopus (48) Google Scholar, 17Olsthoorn M.M. Petersen B.O. Schlecht S. Haverkamp J. Bock K. Thomas-Oates J.E. Holst O. J. Biol. Chem. 1998; 273: 3817-3829Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) are known, and all possess identical inner core structures (Fig. 2). Minor differences in the inner core structures relate to the substitution of the side chain heptose with GlcpN and the concomitant lack of phosphate on the branched heptose residue. Major structural differences between the core types are observed in the outer core region. Identical inner core structures have been described for Shigella species (2Holst O. Brade H. Opal S.M. Vogel S.N. Morrison D.C. Endotoxin in Health and Disease. Marcel Dekker, Inc., New York1999: 115-154Google Scholar). Using a panel of highly purified oligosaccharides from Salmonella enterica sv. Minnesota (R1), E. coli F470 (R1 core-type), E. coli F576 (R2 core-type), E. coli F653 (R3 core-type), E. coli F2513 (R4 core-type), and E. coli J-5, shown in Figs. 1 and 2, we have determined the minimal epitope that is recognized by mAb WN1 222-5 by ELISA, ELISA inhibition, isothermal titration microcalorimetry (ITC), and surface plasmon resonance (SPR) and studied the influence of the outer core on the binding of WN1 222-5 to enterobacterial LPS. Bacteria, Extraction of LPS, and Isolation of Oligosaccharides— E. coli F470 (R1 core-type) (15Vinogradov E.V. Van Der D.K. Thomas-Oates J.E. Meshkov S. Brade H. Holst O. Eur. J. Biochem. 1999; 261: 629-639Crossref PubMed Scopus (51) Google Scholar), E. coli F576 (R2 core-type) (15Vinogradov E.V. Van Der D.K. Thomas-Oates J.E. Meshkov S. Brade H. Holst O. Eur. J. Biochem. 1999; 261: 629-639Crossref PubMed Scopus (51) Google Scholar), E. coli F653 (R3 core type) (16Müller-Loennies S. Lindner B. Brade H. Eur. J. Biochem. 2002; 269: 5982-5991Crossref PubMed Scopus (48) Google Scholar), E. coli F2513 (R4 core type) (16Müller-Loennies S. Lindner B. Brade H. Eur. J. Biochem. 2002; 269: 5982-5991Crossref PubMed Scopus (48) Google Scholar), Salmonella enterica sv. Minnesota (Salmonella R1 core type) (17Olsthoorn M.M. Petersen B.O. Schlecht S. Haverkamp J. Bock K. Thomas-Oates J.E. Holst O. J. Biol. Chem. 1998; 273: 3817-3829Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), and the E. coli strain J-5 (ΔgalE mutant) (14Müller-Loennies S. Holst O. Lindner B. Brade H. Eur. J. Biochem. 1999; 260: 235-249Crossref PubMed Scopus (31) Google Scholar) were cultivated, and LPS was extracted from each. Briefly, LPS was isolated by phenol/chloroform/petrolether extraction (18Galanos C. Lüderitz O. Westphal O. Eur. J. Biochem. 1969; 9: 245-249Crossref PubMed Scopus (1369) Google Scholar) and de-O-acylated by mild hydrazinolysis, followed by de-N-acylation under strong alkaline conditions (4 m KOH, 120 °C, 16 h) (11Holst O. Müller-Loennies S. Lindner B. Brade H. Eur. J. Biochem. 1993; 214: 695-701Crossref PubMed Scopus (46) Google Scholar). After 3-fold extraction with chloroform, the mixture of deacylated oligosaccharides was separated by high performance anion exchange chromatography (HPAEC) as reported (14Müller-Loennies S. Holst O. Lindner B. Brade H. Eur. J. Biochem. 1999; 260: 235-249Crossref PubMed Scopus (31) Google Scholar, 15Vinogradov E.V. Van Der D.K. Thomas-Oates J.E. Meshkov S. Brade H. Holst O. Eur. J. Biochem. 1999; 261: 629-639Crossref PubMed Scopus (51) Google Scholar, 16Müller-Loennies S. Lindner B. Brade H. Eur. J. Biochem. 2002; 269: 5982-5991Crossref PubMed Scopus (48) Google Scholar). Pure oligosaccharides were then desalted by gel chromatography on Sephadex G-10 in 10 mm NH4HCO3, followed by lyophilization. Deamination of E. coli J-5 LPS—LPS isolated from E. coli J-5 was subjected to a deamination reaction as described by Vinogradov et al. (19Vinogradov E. Cedzynski M. Ziolkowski A. Swierzko A. Eur. J. Biochem. 2001; 268: 1722-1729Crossref PubMed Scopus (30) Google Scholar). One ml of acetic acid and 200 mg of NaNO2 were added to 200 mg of LPS in 10 ml of water, and after a 12-h incubation at ambient temperature, the deaminated LPS was collected by centrifugation (4 h, 4 °C, 120,000 × g). The precipitate was dissolved in water and dialyzed against deionized water (3× 1 liter, 4 °C) and lyophilized (yield: 149 mg). An aliquot (50 mg) was then de-O- and de-N-acylated, yielding four oligosaccharides, which were isolated by semipreparative HPAEC and gel filtration as described above (oligosaccharide (OS) 1, 4.2 mg; OS 2, 1.8 mg; OS 3, 2.3 mg; OS 4, 0.8 mg). Neoglycoconjugates—Neoglycoconjugates of deacylated oligosaccharides were prepared as described (20Brade L. Brunnemann H. Ernst M. Fu Y. Holst O. Kosma P. Naher H. Persson K. Brade H. FEMS Immunol. Med. Microbiol. 1994; 8: 27-41Crossref PubMed Google Scholar). Briefly, ligands (2.5 mg) were dissolved in 200 μl of 50 mm carbonate buffer, pH 9.2; glutardialdehyde (25%, electron microscopy grade; Merck) was added (1% final concentration); and the sample was stirred for 4 h at 25 °C under N2 atmosphere. Excess glutardialdehyde was removed by lyophilization, and the samples were redissolved in 200 μl of water. BSA (2.5 mg) was added from a 10 mg ml–1 solution in 50 mm carbonate buffer, pH 9.2, and the mixture was incubated overnight at 25 °C. Finally, 250 μg of NaBH4 was added, and the samples were incubated for 1 h at 4 °C in the dark followed by dialysis against water once and three times against PBS, pH 7.2. mAb WN1 222-5—The generation and selection of mAb WN1 222-5 has been described in detail previously (9Di 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. Infect. Immun. 1993; 61: 3863-3872Crossref PubMed Google Scholar). Stock solutions of affinity purified mAb were kept at–20 °C in aliquots (1 mg ml–1). ELISA—Binding of mAb WN1 222-5 to the neoglycoconjugates was determined by ELISA. Varying amounts of glycoconjugates were coated onto 96-well microtiter plates (Nunc, Maxisorb) and tested against serial dilutions of antibody. Antibody binding was detected with enzyme-conjugated anti-mouse IgG and substrate and measured photometrically at 405 nm. Experiments were done in quadruplicate, and mean values were calculated. Confidence values did not exceed 10%. Binding of the mAb WN1 222-5 to fully acylated LPS was determined using LPS as a solid phase antigen instead of neoglycoconjugates (21Müller-Loennies S. Brade L. Brade H. Eur. J. Biochem. 2002; 269: 1237-1242Crossref PubMed Scopus (10) Google Scholar). For ELISA inhibition, serial dilutions of inhibitor in PBS-Tween 20/casein/BSA (30 μl) were mixed in V-shaped microtiter plates (NUNC) with an equal volume of antibody diluted in the same buffer to give an A 405 of 1.0 without the addition of inhibitor. After incubation (15 min, 37 °C), 50 μl of the mixture were added to antigen-coated ELISA plates. Further steps were as described above. All measurements were done at least twice in duplicate with confidence values not exceeding 20%. Surface Plasmon Resonance—Analyses were performed with a BIA-CORE 3000 instrument (Biacore, Inc.). WN1 222-5 was immobilized on a CM5 sensor chip (Biacore) at a surface density of ∼20,000 RU using the amine coupling kit from Biacore. Analyses were carried out at 25 °C in 10 mm HEPES, pH 7.4, containing 3 mm EDTA, 0.005% P-20, and 150 mm or 300 mm NaCl. Surface regeneration was not necessary. Data were evaluated using the BIAevaluation 3.0 software (Biacore). Isothermal Titration Microcalorimetry—Microcalorimetric experiments were performed on an MCS isothermal titration calorimeter (Microcal Inc., Northampton, MA). mAb WN1 222-5 was dialyzed against PBS, pH 7.2, and the concentration was determined by UV measurement (1 mg ml–1 = A 280 of 1.35). The mAb concentration was adjusted to 7.55 μm, assuming a molecular mass of 150 kDa, and the microcalorimeter cell was filled with the antibody solution (volume = 1.3 ml). Purified and desalted deacylated LPS oligosaccharide were dissolved at a concentration of 0.35 mm in dialysis buffer and loaded into the syringe of the microcalorimeter. Both solutions were thoroughly degassed prior to loading. After temperature equilibration, the ligand was injected into the cell in 5-μl portions, and the evolved heat was measured with the first injection not considered for data analysis. A total of 20 injections were performed with 5-min equilibration times between injections. Data were corrected for heat of dilution by measuring the same number of buffer injections and subtraction from the sample data set. Dissociation constants were determined using the MicroCal Origin version 2.9 analysis software and the model of 1 set of binding sites. The antibody concentration in the cell was corrected after the curve fitting as described in the ITC Data Analysis in the Origin Version 2.9 manual provided by the manufacturer. Nuclear Magnetic Resonance— 1H (600.12 MHz), 13C (150.13 MHz), and 31P (242.13 MHz) NMR spectra were recorded with a Bruker DRX Avance spectrometer with a 4-mg sample in 0.5 ml of D2O. Acetone (2.225 ppm) (1H) and dioxane (67.4 ppm) (13C) served as references. All spectra were run at a temperature of 300 K. One-dimensional 1H, 13C, and 31P and two-dimensional homonuclear 1H,1H (DQF-COSY, NOESY, TOCSY), heteronuclear 1H,13C, and 1H,31P NMR correlation spectra (HMQC) were recorded using Bruker standard pulse programs and analyzed with Bruker Xwinnmr software. Oligosaccharide Preparation—Deacylation of LPS from E. coli strains F470 (15Vinogradov E.V. Van Der D.K. Thomas-Oates J.E. Meshkov S. Brade H. Holst O. Eur. J. Biochem. 1999; 261: 629-639Crossref PubMed Scopus (51) Google Scholar), F576 (15Vinogradov E.V. Van Der D.K. Thomas-Oates J.E. Meshkov S. Brade H. Holst O. Eur. J. Biochem. 1999; 261: 629-639Crossref PubMed Scopus (51) Google Scholar), F563 (16Müller-Loennies S. Lindner B. Brade H. Eur. J. Biochem. 2002; 269: 5982-5991Crossref PubMed Scopus (48) Google Scholar), and F2513 (16Müller-Loennies S. Lindner B. Brade H. Eur. J. Biochem. 2002; 269: 5982-5991Crossref PubMed Scopus (48) Google Scholar), purification of oligosaccharides, and characterization of the chemical structures has been described in the respective publications. S. enterica sv. Minnesota LPS was isolated as described (17Olsthoorn M.M. Petersen B.O. Schlecht S. Haverkamp J. Bock K. Thomas-Oates J.E. Holst O. J. Biol. Chem. 1998; 273: 3817-3829Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) and treated according to the same procedure as the other LPS. The chemical structures are depicted in Fig. 2. Deacylation of LPS from E. coli J-5 yielded five oligosaccharides (Fig. 1), which differed in their carbohydrate structure and phosphate substitution (14Müller-Loennies S. Holst O. Lindner B. Brade H. Eur. J. Biochem. 1999; 260: 235-249Crossref PubMed Scopus (31) Google Scholar). We have prepared an additional oligosaccharide from E. coli J-5 by deamination of LPS and subsequent deacylation under strong alkaline conditions that lacked phosphate at the branched heptose and was devoid of GlcpN on the side chain heptose. E. coli J-5 does not naturally produce such an oligosaccharide. Analysis by analytical HPAEC (Fig. 3A) revealed retention times of 25.7, 27.7, 35.0, and 37.3 min for oligosaccharides 1–4, respectively. Oligosaccharides 2–4 possessed identical retention times to those of deacylated E. coli J-5 LPS without deamination (Fig. 3B). From previous studies, it was known that these are heptasaccharide P 3 (Fig. 3, OS 2), octasaccharide P 4 (OS 3), and heptasaccharide P 4 (OS 4). This was confirmed by 1H NMR spectroscopy of the purified oligosaccharides. There was no signal at a retention time of nonasaccharide P 3 (18.7 min, OS 5) after deamination, and a new peak appeared at 25.7 min (OS 1). A 1H NMR spectrum of the latter compound (Fig. 4) contained six signals of anomeric protons and two pairs of signals of deoxyprotons, indicating the presence of two Kdo residues. Comparison with the 1H NMR spectrum of nonasaccharide P 3 of deacylated E. coli J-5 LPS indicated that the side chain GlcpN was missing. This was finally proven by the assignment of all 1H and 13C NMR chemical shifts (Table I), determination of coupling constants by 1H,1H DQF-COSY, and nuclear Overhauser effect NMR experiments. Thus, this oligosaccharide had the same carbohydrate structure as octasaccharide P 4 (Fig. 1). However, the upfield resonance frequencies of proton H-4 and carbon C-4 of the second heptose residue indicated that this position was not substituted with phosphate (Fig. 1). The 31P NMR spectrum accordingly contained only three resonances (Fig. 5) with chemical shifts of –1.41, 0.54, and 1.60 ppm. Therefore, this oligosaccharide, designated octasaccharide 1 P 3, contained only three phosphate residues, and a 1H,31P HMQC-COSY NMR experiment proved that the phosphate at the 4-position of the second heptose (residue F in Fig. 4) was absent as in nonasaccharide P 3.Fig. 4A, 1H NMR spectrum of octasaccharide 1 P3 obtained after deamination and deacylation of E. coli J-5 LPS. Six signals of anomeric protons originating from anomeric protons as indicated (labeling of residues as in the structure depicted) and two pairs of signals from deoxyprotons of Kdo residues (residues C and D) identified the isolated oligosaccharide as an octasaccharide. B, the region of anomeric protons of nonasaccharide P 3 (top) in comparison with octasaccharide 1 P 3 (bottom) did contain a signal of an additional GlcpN residue (marked by an arrow), which was absent in octasaccharide P 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table I1H and 13C NMR chemical shift data for octasaccharide 1 P3 obtained by deamination and deacylation of E. coli J-5 LPSResidueChemical shiftH-1H-2H-3axH-3eqH-4H-5H-6aH-6bH-7aH-7bH-8aH-8bC-1C-2C-3C-4C-5C-6C-7C-8PPMA →6-αGlcN 1P5.6853.4303.9063.6124.1163.7684.29091.6654.7469.8469.9272.9670.03B →6-βGlcN 4P4.8403.0983.8713.8153.7433.4533.67599.7255.9772.3374.8274.2962.78C →4,5-αKdo1.9342.1254.1264.2583.6963.8443.8973.605NDaND, not determined.ND34.7870.9070.0372.6970.0364.14D αKdo1.7772.1254.1074.0293.6493.9933.9473.746NDND35.2866.3467.0072.6970.4363.63E →3-αHep 4P5.2824.0644.1574.4274.2044.0963.6723.73199.5571.2777.2870.6672.8069.4863.43F →3,7-αHep5.1274.3554.0164.0183.6924.1693.72-3.753.72-3.75102.7069.9578.9766.1072.6768.6470.76G →3-αGlc5.2403.5313.7803.3973.8473.9033.746100.7772.2173.3470.1072.5560.88H →7αHep4.9163.9853.8553.8603.6394.0103.6723.725101.4370.4171.0066.5871.9869.4063.51a ND, not determined. Open table in a new tab Fig. 531P NMR spectrum of octasaccharide 1 P 3 obtained after deamination and deacylation of E. coli J-5 LPS. Three signals of 31P nuclei proved the presence of three phosphate groups that were substituting positions 1 and 4′ of the lipid A GlcpN residues and position 4 of l-α-d-Hep (residue E; for structure see inset in Fig. 4).View Large Image Figure ViewerDownload Hi-res image Download (PPT) ELISA with LPS and Complete Core Structures—It was previously shown that WN1 222-5 binds to whole cells of E. coli and S. enterica and to the LPS of these bacteria in Western blots and passive immunohemolysis (9Di 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. Infect. Immun. 1993; 61: 3863-3872Crossref PubMed Google Scholar). The minimal LPS structure bound by WN1 222-5 was the LPS of the rough mutant strain E. coli J-5. To verify this reactivity by ELISA, we first immobilized LPS of E. coli core types R1 to R4 and S. enterica sv. Minnesota on microtiter plates and investigated their reactivity with mAb WN1 222-5 (Fig. 6A). The antibody reacted with all of these LPS. We then investigated whether the lipid A was important for the binding and studied the inhibitory activities of deacylated LPS oligosaccharides by ELISA inhibition. When LPS was treated with mild acid, the mixture of E. coli R3 deacylated LPS oligosaccharides did not show any inhibitory activity up to the concentration tested (5 μg/well; see Table III). On the contrary, oligosaccharides from the same LPS obtained by deacylation under alkaline conditions, which retained the lipid A backbone sugars and the side chain Kdo substitution, possessed inhibitory activity (50% inhibition at 20 ng/well). Therefore, fatty acids did not influence the binding and were not part of the WN1 222-5 epitope. As can be seen in Fig. 6B, mAb WN1 222-5 bound to all tested BSA-neoglycoconjugates of E. coli LPS obtained after alkaline deacylation to the same extent as to LPS. All further experiments were therefore done with oligosaccharides obtained after alkaline deacylation.Table IIIDetermination of inhibitory concentrations of deacylated enterobacterial LPS oligosaccharides with WN1 222–5 by ELISA inhibitionInhibitoraFor structures of inhibitors see Figs. 1 and 2; E. coli R3 OS 1 and OS 2 refer to the core oligosaccharides without and with GlcpN on the side chain heptose, respectively. E. coli R3 (1% HAc) and (KOH) are mixtures of oligosaccharides obtained after mild acid and alkaline degradation, respectively.Amount of inhibitor yielding 50% ELISA inhibitionE. coli R3-BSA solid phase antigensbSolid phase antigens were prepared from E. coli R3 LPS after deacylation under alkaline conditions and conjugation to BSA. E. coli R3-BSA contains the mixture of LPS oligosaccharides, whereas E. coli R3 OS1-BSA refers to the conjugated purified oligosaccharide.E. coli R3 OS1-BSA solid phase antigensbSolid phase antigens were prepared from E. coli R3 LPS after deacylation under alkaline conditions and conjugation to BSA. E. coli R3-BSA contains the mixture of LPS oligosaccharides, whereas E. coli R3 OS1-BSA refers to the conjugated purified oligosaccharide.ng/wellμMng/wellμME. coli R3 (1% HAc)5000NDcND, not determined.NDNDE. coli J-5 (1% HAc)5000NDNDNDE. coli R3 (KOH)20NDNDNDE. coli R3 (OS 1)100.1190.2E. coli R3GlcN (OS 2)12509.7500039.0Heptasaccharide P3NDND250031.9Heptasaccharide P41561.9125015.0Octasaccharide 1 P3NDND3123.6Octasaccharide P450.1100.1Nonasaccharide P3125012.7500051.0a For structures of inhibitors see Figs. 1 and 2; E. coli R3 OS 1 and OS 2 re

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