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Selection of peptides that bind to the core oligosaccharide of R-form LPS from a phage-displayed heptapeptide library
FEMS Microbiology Letters 205 (2001) 349^354
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Selection of peptides that bind to the core oligosaccharide of R-form LPS from a phage-displayed heptapeptide library Ken Noda 1 , Ryohei Yamasaki
1;
*, Yumi Hironaka, Aiko Kitagawa
Department of Biochemistry and Biotechnology, Tottori University, Tottori 680-8553, Japan Received 7 June 2001 ; received in revised form 10 September 2001; accepted 21 September 2001 First published online 19 November 2001
Abstract To characterize common sites within the core oligosaccharide of the R-form lipopolysaccharide (LPS), we screened peptides from a phage-displayed heptapeptide library by using the most truncated form of R-LPS, Re-LPS (S. Typhimurium SL1165) as a ligand. After three rounds of biopanning/amplification and subsequent screening by phagemid enzyme-linked immunosorbent assay (ELISA), we selected three distinct clones that bind to the ligand LPS. We characterized the binding sites of the three clones by ELISA and thin-layer chromatography immunostaining and found that the three clones bind the two Re-LPSs (SL1165 and S. Minnesota Re595) and Rb2 -LPS. In addition, one of the clones also bound to S-form LPS (S. Enteritidis). Current data show that those clones bind to common carbohydrate structure(s) expressed in the core oligosaccharides of those LPS samples. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Lipopolysaccharide ; Core oligosaccharide; Random peptide; Phage display; Salmonella
1. Introduction Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria. LPS is composed of lipid A, core oligosaccharide and O-antigen polysaccharide [1]. Although the O-antigen is heterogeneous both in size and composition, the core oligosaccharide is highly conserved. LPS consisting of the core oligosaccharide is called R-form LPS. Re-LPS is the most truncated form of R-form LPS that consists of a dimer of 2-keto-3deoxy-mannooctulosonic acid (KDO) and lipid A (Fig. 1). Ra^Rd LPSs are produced after sequential glycosylations of the dimeric KDO moiety of Re-LPS with other neutral and N-acetyl amino sugars. Structural and immunochemical aspects of R-form LPS lacking O-antigens have been intensively studied [2^6]. Recent immunochemical studies of R-LPS have shown that some core oligosaccharides expressed on speci¢c R-LPS are immunogenic to produce cross-reactive antibodies. For example, a monoclonal antibody (MAb) raised * Corresponding author. Tel./Fax : +81 (857) 31-6751. E-mail address : [email protected] (R. Yamasaki). 1
The ¢rst two authors contributed equally to this work
against Salmonella Re mutant binds both Re-LPS and Rb2 -LPS [7], which showed not only the presence of a common carbohydrate epitope between the Re and its elongated Rb2 -LPS but also implied the possibility of existence of common epitope(s) among R-form of LPS. Although such common epitope may exist, raising antibodies speci¢c for those epitopes by immunization may have limitations since they have not yet been developed despite of attempts by several investigators [4,8]. However, speci¢c sites mimicking or resembling such epitopes could be expressed with oligo- or polypeptides as exempli¢ed by recent work on proteins [9^11] with the use of a phagedisplayed peptide library. In addition to proteins, this methodology has been applied to carbohydrates, and peptides binding to ganglioside-GM1 have been isolated [12]. Thus, recent applications using peptides that mimic proteins or carbohydrates prompted us to examine the possibility of expressing common carbohydrate structures among R-form LPS with oligopeptides. To characterize such sites, we screened peptides from a phage-displayed heptapeptide library by using Re-LPS as a ligand. We isolated three clones that bind to both the ligand LPS and Rb2 -LPS. Our current study supports that the three clones bind to sites within the carbohydrate moiety of those LPSs.
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 4 4 5 - 1
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2. Materials and methods 2.1. Reagents and general immunochemical analyses A heptapeptide library displayed on M13 ¢lamentous phage (pSKAN-HyB Library) [13] and Escherichia coli WK6VmutS were purchased from MoBiTec GmbH (Go«ttingen, Germany). MAbs (mouse IgG) speci¢c for a M13 phage and lipid A [14] were purchased form Progen Biotechnik GmbH (Heidelberg, Germany) and Sanbio (Uden, Netherlands), respectively. Both anti-mouse IgG (Q-chain speci¢c, alkaline phosphatase conjugate) and KDO (ammonium salt) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were analytical-grade reagents. Salmonella enterica serovar Typhimurium (S. Typhimurium) strain SL1165 (Re-mutant) was obtained from Salmonella Genetic Stock Centre, University Calgary (AB, Canada). SL1165 Re-LPS was extracted and puri¢ed by using a method reported by Galanos [15]. De-O-acylated Re-LPS and lipid A, respectively were prepared by hydrazinolysis [16,17] and hydrolysis [16,18] of the SL1165-LPS. Rb2 -LPS (S. Minnesota R345) was purchased from List Biological Laboratories (Campbell, CA, USA). We obtained the following LPSs from Sigma Chemical Co. (St. Louis, MO, USA) : Ra-LPS (S. Typhimurium TV119); Rc-LPS (S. Minnesota R5); Rd-LPS (S. Minnesota R7); Re-LPS (S. Minnesota Re595); S. Typhimurium S-form LPS; S. Enteritidis S-form LPS; E. coli O128:B12 LPS. Lactosyl ceramide (Lac-cer) and phosphatidylcholine (PC) were purchased from Wako Pure-Chemicals (Osaka, Japan). LPS samples were analyzed by enzyme-linked immunosorbent assay (ELISA, Fig. 2), polyacrylamide gel electrophoresis (PAGE) and thin-layer chromatography (TLC) immunostaining. Typical procedures for ELISA [17], PAGE/blot and TLC immunostaining [16,17,19] have been described previously. We used a microtiter plate from Nunc (Maxisorb loose U-16, Roskilde, Denmark) for ELISA, biopanning and phagemid ELISA. We used p-nitrophosphate for ELISA analysis and Western blue (Promega, Madison, WI, USA) for both PAGE/blot and TLC immunostaining. 2.2. Selection of clones binding to Re-LPS and characterization of their binding sites Construction of a phage-displayed heptapeptide library, biopanning, ampli¢cation and phagemid ELISA were carried out according to a supplier's protocol (pSKAN system protocols, version 2-97, MoBiTec GmbH) except for several minor modi¢cations. In addition to these modi¢cations, brief descriptions on both selection of clones and characterization of their speci¢cities will be given below. Selection of phagemid clones binding to Re-LPS (S. Typhimurium SL1165) : microtiter wells were coated by
incubating with the SL1165 Re-LPS (7.5 Wg) at 4³C overnight. Binding of the LPS to microtiter plates were con¢rmed by eluting the LPS bound on wells with SDS sampling bu¡er and subsequent PAGE analysis of the eluate. After coating the ligand LPS, we used 1% BSA as a blocking reagent instead of 2% skim milk. The wells were sequentially treated with PBS containing 0.5% Tween 20 (TPBS), 1.0% BSA (at room temperature for 1 h), and TPBS. After incubation with the phagemid library [100 Wl, 1011 ^1012 of colony-forming units (cfu)] in PBS at room temperature for 2.5 h, the wells were sequentially treated with T-PBS, 1% BSA (10 min), T-PBS and then with water. The bound phagemids were eluted with glycine^ HCl (100 Wl, pH 2.2), and the solution was neutralized with 2 M Tris. The titer of the phagemid solution was analyzed by infecting E. coli WK6VmutS with an aliquot (10 Wl) of the phagemid solution followed by counting the colonies survived (overnight at 37³C) on ampicillin-containing LB agar plates. The typical cfu of the phagemid solution before ampli¢cation was in the range of 103 ^105 ml31 . After checking the titer, the rest of solution was used for ampli¢cation; growing the infected E. coli in a liquid LB medium containing a M13KO7 helper phage (1010 ^1012 cfu ml31 , Amersham Pharmacia Biotech, NJ, USA) overnight at 37³C, and the phage pellets precipitated [polyethylene glycol 8000 (Sigma Chemical Co., MO, USA) and NaCl] were re-suspended in PBS. The titer of the phagemid after ampli¢cation was in the range of 1011 ^1012 ml31 . After three rounds of biopanning and ampli¢cations, selected clones were further analyzed by sequential phagemid ELISA to obtain clones with higher a¤nity. Phagemid ELISA: Wells were washed with PBS three times and 1% BSA was used for blocking. Wells were coated with LPS samples (7.5 Wg) in a similar manner as described for biopanning. After blocking (1% BSA) and washing with PBS, wells were incubated with approximately 109ÿ10 cfu of phagemids in PBS (75 Wl) for 2.5 h at room temperature. Binding of the phagemid to LPS samples was analyzed by using the anti-M13 MAb and anti-mouse IgG AP. Amino acid sequence of selected clones: Sequencing of DNA encoding peptide in a cloned phagemid was determined by using a Cy5 Thermo Sequenase dye terminator kit and an ALF express automated sequencer (both from Amersham Pharmacia Biotech, NJ, USA). Double stranded phagemid DNA was isolated from E. coli WK6VmutS infected with a cloned phagemid and used as a template. We used two primers, #2897: 5PGGAGGTCTAGATAACGAGG-3P (sense) and #1243a: 5P-ATGAATTAAGCACGGACC-3P (anti-sense) (both from Amersham Pharmacia Biotech, NJ, USA) [13]. Characterization of the binding sites of selected clones : We analyzed the following samples by phagemid ELISA : R-form LPSs [Ra, Rb2 , Rc, Rd, Re (Re595 and SL1165)] ; O-deacylated Re-LPS and lipid A (both from the SL1165
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Re-LPS); S-form LPS (S. Enteritidis, S. Typhimurium, E. coli O128:B12); and non-LPS samples (Lac-cer and PC). The ELISA procedure was similar as described earlier except for coating wells with lipid A, Lac-cer and PC. Each of three samples was dissolved in chloroform: methanol (4:1) (1 mg ml31 ) and then diluted by 10-fold with methanol. The organic solution (75 Wl) was applied to wells, and then they were evaporated to dryness. Binding of lipid A on wells was con¢rmed by ELISA using an anti-lipid A MAb [14]. Inhibition assay: The inhibitory activity of KDO on the binding of the phagemid to Re595-LPS was analyzed by the same ELISA method as described above, and glucose was also used as a negative control. After coating with the 595Re-LPS (7.5 Wg), wells were incubated with a mixture of the phagemid preparation (V1011 cfu) and KDO (10 and 100 molar equivalents of the 595-LPS). The phagemid preparation without KDO was also included in this assay. 3. Results and discussion 3.1. Selection of Re-LPS binding peptides (biopanning, screening and amino acid sequencing) To select the LPS binding peptides from a heptapeptide phagemid library, we performed three rounds of biopanning. Each biopanning consisted of the following steps: (1) immobilization of phagemids on a microtiter plate coated with Re-LPS (S. Typhimurium SL1165), (2) elimination of clones binding non-speci¢cally (sequential washing with T-PBS, 1% BSA, then with T-PBS) and (3) elution of the bound phagemids. Eluted phagemids were checked for their titers, ampli¢ed, and then used in the next round of biopanning. To eliminate non-speci¢c phagemids, the washing step, after immobilizing the phagemids, was repeated twice and three times at the second and third round of biopanning, respectively. After three sequential biopannings, we obtained 90 phagemid clones by using a limiting dilution technique. Starting from 90 clones, we selected clones showing higher a¤nity to the 1165 Re-LPS after three consecutive phagemid ELISA analyses. Fig. 1 shows the results of the ¢rst screening. Of 90 clones, we selected and ampli¢ed 16 clones whose OD values are more than 0.5 and more than double the control OD value. Sixteen clones selected were re-examined by ELISA to give ¢ve clones (45, 61, 70, 81, and 83) (data not shown). Fig. 3 shows the results of the third round of phagemid ELISA, and three clones showing higher a¤nity to the ligand LPS (45, 61 and 70) were selected. Their binding capabilities were con¢rmed after re-cloning of the three phagemids and subsequent ELISA analysis (data not shown). Of the three selected clones, clone 45 showed strongest binding to SL1165 ReLPS and clone 61 the weakest. Thus, we obtained three clones containing peptides that bind to Re-LPS.
351
Fig. 1. Schematic structures of Salmonella LPS. GlcNAc, N-acetyl glucosamine ; Glc, glucose; Gal, galactose; KDO, 2-keto-D-manno-3-deoxyoctulosonic acid; Hep, L-glycero-D-manno-heptose; GlcN, glucosamine.
To determine the identities of three clones (45, 61, and 70), we analyzed the amino acid sequences of the heptapeptides. The double stranded phagemid DNA was extracted from E. coli infected with each of the phagemid clones and the DNA sequences encoding the randomized region were determined using a conventional dideoxy termination method. Table 1 shows the amino acid sequences of three heptapeptides. The three peptides did not have any common amino acid sequences among them, which showed that the selected three clones carry distinct amino acid sequences. 3.2. The three peptides are suggested to bind the oligosaccharide moiety of Re-LPS We examined the binding of the three peptides to Rform LPSs (Ra to Re), S-form LPSs (S. Enteritidis, S. Typhimurium, E. coli O128) by using phagemid ELISA (Fig. 4). In addition to SL1165 Re-LPS (ligand LPS), the Re595-LPS (S. Minnesota) was included for the analysis. We also prepared de-O-acylated LPS by hydrazinolysis [16,17] of SL1165 Re-LPS and lipid A prepared after hydrolysis [16,18] of the same Re-LPS. Except for S. Typhimurim Re-LPS, the core oligosaccharide structures of the LPS used in this experiment have been characterized [1,4,6] and their schematic structures are shown in Fig. 1. As Fig. 4 shows, in addition to the 1165 Re-LPS, the three peptides (45, 61, 70) bound the Re595-LPS. Both clones 45 and 70 bound to the 1165 Re-LPS even after the de-O-acylation of the lipid A, whereas clone 61 showed slightly lower binding to the de-O-acylated LPS. Similarly, removal of the KDO residues from the 1165-LPS also a¡ected the binding, and the three peptides showed much lower binding to the lipid A than to the intact LPS. Among the other R-form LPSs, the three peptides bound only to Rb2 -LPS but not to Ra-, Rc-, and RdLPSs. In contrast to the R-form LPS, the peptides showed di¡erent binding patterns to the S-form LPS tested. Clone 45 bound strongly to S. Enteritidis LPS. However, it showed lower a¤nity to both S. Typhimurium LPS and E. coli O-128 LPS. The peptide 61 showed a slightly higher binding to three S-form LPSs than to Ra-, Rc-, Rd-LPSs,
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Fig. 2. Phagemid ELISA analysis of 90 clones isolated after three rounds of biopanning. Each well coated with SL1165 Re-LPS (7.5 Wg) was incubated with V109 ^1010 cfu of each phagemid and sequentially treated with an anti-M13 MAb and anti-mouse IgG (alkaline phosphatase conjugate). p-Nitrophosphate was used as a substrate. Results are expressed as mean values (OD 405 nm at 60 min) of duplicate analyses.
whereas clone 70 showed only residual bindings to the Sform LPSs tested. The bindings of the three peptides to Lac-cer and PC were slightly higher than those to Ra, Rc, Rd-LPSs. TLC staining analysis (data not shown) also con¢rmed positive binding of the three peptides to ReLPSs (1165 and 595) and Rb2 -LPS. Based on published structures of the LPS samples used in this study, the results obtained with the ELISA (Fig. 4) and TLC immunostaining showed the following: (1) the three peptides do not bind to the lipid A portion or fatty acids present in the lipid A possibly common to all the LPSs examined, which was supported by their stronger binding to the two Re-LPSs and Rb2 -LPSs and also by the fact that the binding to the 1165 Re-LPS was not abolished even after O-linked fatty acids of its lipid A moiety were removed by hydrazinolysis. (2) In addition to the results described above, lower binding of the three
clones to the1165 lipid A than the intact LPS suggests that the peptides bind presumably to the carbohydrate moiety, a dimer of KDO of the two Re-LPS. Alternatively, an area including the linkage between KDO and lipid A moiety may be the site for their binding. (3) Preferential binding of the three peptides to Rb2 -LPS shows that the binding sites described above are also expressed in this LPS. It is not clear why those sites are expressed in the Rb2 -LPS whose carbohydrate moiety is larger than those of Rcand Rd-LPSs (Fig. 1). However, similar to our results, a MAb raised against S. Minnesota Re595 has been reported to bind to both Re- and Rb2 -LPSs, but not to other R-form LPSs [7]. (4) Binding of peptide 45 to S. Enteritidis LPS shows that its binding site is also expressed in this S-form LPS. (5) Slightly higher bindings of the three clones to Lac-cer and PC than those to Ra-, Rcand Rd-LPSs suggest that the peptides have non-speci¢c a¤nity to their hydrophobic sites which are not present in the LPS samples. To further elucidate the binding sites of the three clones, we examined the inhibitory activities of a KDO monomer on the binding of the three clones to Re-LPS (Re595) [20]. We used 10 and 100 molar equivalents of the KDO, and the bindings of 45 and 61 to the LPS were not inhibited even with the 100 equivalents of KDO. However, 6% and Table 1 The amino acid sequencesa of the selected phagemid clones
Fig. 3. Phagemid ELISA analysis of ¢ve clones selected. Five phagemids selected (V109 ^1010 cfu per well) were re-examined for their binding to Re-LPS (7.5 Wg per well). The bound phagemids were detected as described in Fig. 2. Results are expressed as mean values (OD 405 nm at 60 min) of duplicate analyses
Phagemid clones
Amino acid sequences
45 61 70
Arg Val Val Lys Glu Ser Arg Tyr Ser Ala Leu Glu Glu Gly Met Met Gly Val Gly Thr Ser
a Amino acid sequences were deduced from the DNA sequence data for random peptide coding region of the double stranded phagemid DNA extracted from E. coli infected with each of the clones.
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Fig. 4. Characterization of the speci¢cities of the three peptides (45, 61, 70) selected by phagemid ELISA. Wells coated with each ligand (7.5 Wg) were incubated with each of the three phagemids (V109ÿ10 cfu) and treated in a similar manner as described in Fig. 2. R-LPS: R-form LPSs; S-LPS: Sform LPSs; De-O-acyl Re: de-O-acylated Re-LPS (SL1165); Re-lipid A: lipid A prepared from Re-LPS (SL1165); Lac-cer : lactosyl ceramide, PC: phosphatidylcholine. The strain numbers of the bacteria producing R-LPS are shown in parenthesis. Results are expressed as mean values (OD 405 nm at 20 min) of duplicate analyses.
57% of the binding of clone 70 was inhibited with 10 and 100 equivalents of KDO, respectively (data not shown). We used 100 molar equivalents of glucose as a negative control, and this neutral sugar showed no inhibitory activity under the conditions used. The results of the above inhibition assay together with the di¡erence in binding of the three clones to the S-form LPSs (Fig. 4) suggest that their binding sites are di¡erent. This could be re£ected in their di¡erent amino acid sequences (Table 1). Inhibition of the binding of peptide 70 with KDO shows that its binding site may include the structure of a monomeric KDO. Peptides 45 and 61 may recognize sites expressed in a KDO dimer or close to the KDO-lipid A linkage (Fig. 1) and these possible sites could be conformational. Although we were not able to use a dimeric or trimeric forms of KDO which are not commercially available, the use of such oligomers will provide further insights on the possible binding sites within the Re-LPSs, as well as Rb2 LPS which contain the trimeric KDO as a partial structure [1] (Fig. 1). Our current data indicate that three phagemids bind to a common carbohydrate structure within the two Re-LPSs (SL1165 and Re595) although we were not be able to identify their exact binding sites. In addition, the same carbohydrate structure within the two LPSs is also expressed in Rb2 -LPS. To further characterize those binding sites, we would need to screen out oligopeptides of higher a¤nity to the target LPS. By using such oligopeptides, we are able to di¡erentiate non-speci¢c bindings from speci¢c bindings, which would facilitate characterization of their binding sites. Further, determination of the overall structures of the R-form LPS would be helpful for us to understand why the clones selected bound to Rb2 -LPS but not to other R-LPS samples. In summary, for the ¢rst time we isolated three distinct phagemids that bind to two Re-LPSs (SL1165 and Re595)
and Rb2 -LPS from a phage-displayed heptapeptide library. In addition to these R-form LPSs, one of the clones bound to S-form LPS (S. Enteritidis). Our current data show that the three phagemids bind to common carbohydrate structure(s) within the core oligosaccharides of those LPSs. Acknowledgements We thank Drs. T. Taki, D. Ishikawa, Y. Yamano and T. Kawano for their valuable suggestions and comments. We also thank Dr. M. Muramatsu for providing reagents for the DNA sequencing and T. Saito for his technical assistance for the sequence analysis. This study was supported in part by Grants-in-Aid (09660298 and 10306022) for Scienti¢c Research from the Ministry of Education, Science and Technology, Japan.
References [1] Rick, P.D. (1987) Lipopolysaccharide biosynthesis. In: Escherichia coli and Salmonella typhimurium : Cellular and Molecular Biology (Neidhardt, F.C., Ed.), pp. 648^662. American Society for Microbiology, Washington, DC. [2] Bennett-Guerrero, E., McIntosh, T.J., Barclay, G.R., Snyder, D.S., Gibbs, R.J., Mythen, M.G. and Poxton, I.R. (2000) Preparation and preclinical evaluation of a novel liposomal complete-core lipopolysaccharide vaccine. Infect. Immun. 68, 6202^6208. [3] Stanislavsky, E.S., Makarenko, T.A., Kholodkova, E.V. and Lugowski, C. (1997) R-form lipopolysaccharides (LPS) of Gram-negative bacteria as possible vaccine antigens. FEMS Immunol. Med. Microbiol. 18, 139^145. [4] Pollack, M., Chia, J.K., Koles, N.L., Miller, M. and Guelde, G. (1989) Speci¢city and cross-reactivity of monoclonal antibodies reactive with the core and lipid A regions of bacterial lipopolysaccharide. J. Infect. Dis. 159, 168^188.
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[5] DiPadova, F., Gram, H., Barclay, G.R., Poxton, I.R., Liehl, E. and Rietschel, E.T. (1996) Monoclonal antibodies to endotoxin core as a new approach in endotoxemia therapy. In: Novel therapeutics strategies in the treatment of sepsis (Morrison, D.C. and Ryan, J.L., Eds.), pp. 13^31. Marcel Dekker, New York. [6] Holst, O. and Brade, H. (1992) Chemical structure of the core region of lipopolysaccharides in bacterial endotoxic Lipopolysaccharides. In: Molecular Biochemistry and Celluar Biology (Morrison, D.C. and Ryan, J.L., Eds.), Vol. 1, pp. 135^170. CRC Press, Boca Raton, FL. [7] de Jongh-Leuvenink, J., Bouter, A.S., Marcelis, J.H., Schellekens, J. and Verhoef, J. (1986) Cross-reactivity of monoclonal antibodies against lipopolysaccharides of Gram-negative bacteria. Eur. J. Clin. Microbiol. 5, 148^151. [8] Nnalue, N.A., Khan, G.N. and Mustafa, N. (1999) Cross-reactivity between six Enterobacteriaceae complete lipopolysaccharide core chemotypes. J. Med. Microbiol. 48, 433^441. [9] Phalipon, A., Folgori, A., Arondel, J., Sgaramella, G., Fortugno, P., Cortese, R., Sansonetti, P.J. and Felici, F. (1997) Induction of anticarbohydrate antibodies by phage library-selected peptide mimics. Eur. J. Immunol. 27, 2620^2625. [10] Cwirla, S.E., Balasubramanian, B., Du¤n, D.J., Wagstrom, C.R., Gates, C.M., Singer, S.C., Davis, A.M., Tansik, R.L., Mattheakis, L.C., Boytos, C.M., Schatz, P.J., Baccanari, D.P., Wrighton, C.M., Barrett, R.W. and Dower, W.J. (1997) Peptide agonist of the thrombopoietin receptor as potent as the natural cytokine. Science 276, 1696^1699. [11] Oldenburg, K.R., Loganathan, D., Goldstein, I.J., Schultz, P.G. and Gallop, M.A. (1992) Peptide ligands for a sugar-binding protein isolated form a random peptide library. Proc. Natl. Acad. Sci. USA 89, 5393^5397. [12] Matsubara, T., Ishikawa, D., Taki, T., Okahata, Y. and Sato, T. (1999) Selection of ganglioside GM1-binding peptides by using a phage library. FEBS Lett. 456, 253^256.
[13] Ro«ttgen, P. and Collins, J. (1995) A human pancreatic secretory trypsin inhibitor presenting a hypervariable highly constrained epitope via monovalent phagemid display. Gene 164, 243^250. [14] Erich, T., Schellekens, J., Bouter, A., Van Kranen, J., Brouwer, E. and Verhoef, J. (1989) Binding characteristics and cross-reactivity of three di¡erent antilipid A monoclonal antibodies. J. Immunol. 143, 4053^4060. [15] Galanos, C. and Luderitz, O. (1993) Isolation and puri¢cation of Rform lipopolysaccharides: Further applications of the PCP method. Methods Carbohydr. Chem. 9, 11^18. [16] Yamasaki, R., Schneider, H., Gri¤ss, J.M. and Mandrell, R. (1988) Epitope expression of gonococcal lipooligosaccharide (LOS) : Importance of the lipoidal moiety for expression of an epitope that exists in the oligosaccharide moiety of LOS. Mol. Immunol. 25, 799^809. [17] Yamasaki, R., Koshino, H., Kurono, S., Nishinaka, Y., McQuillen, D.P., Kume, A., Gulati, S. and Rice, P.A. (1999) Structural and immunochemical characterization of a Neisseria gonorrhoeae epitope de¢ned by a monoclonal antibody 2C7: The antibody recognizes a conserved epitope on speci¢c lipo-oligosaccharides in spite of the presence of human carbohydrate epitopes. J. Biol. Chem. 274, 36550^36558. [18] Yamasaki, R., Bacon, B.E., Schneider, H. and Gri¤ss, J.M. (1991) Structural determination of oligosaccharides derived from lipooligosaccharide F62 of Neisseria gonorrhoeae by chemical, enzymatic and two-dimensional NMR methods. Biochemistry 30, 10566^10575. [19] Yamasaki, R., Kerwood, D.E., Schneider, H., Quinn, K.P., Gri¤ss, J.M. and Mandrell, R.E. (1994) The structure of lipooligosaccharide produced by Neisseria gonorrhoeae, strain 15253, isolated from a patient with disseminated infection : Evidence for a new glycosylation pathway of the gonococcal lipooligosaccharide. J. Biol. Chem. 269, 30345^30351. [20] Aurell, C.A. and Wistrom, A.O. (1998) Critical aggregation concentrations of Gram-negative bacterial lipopolysaccharides (LPS). Biochem. Biophys. Res. Commun. 253, 119^123.
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Selection of peptides that bind to the core oligosaccharide of R-form LPS from a phage-displayed heptapeptide library Ken Noda 1 , Ryohei Yamasaki
1;
*, Yumi Hironaka, Aiko Kitagawa
Department of Biochemistry and Biotechnology, Tottori University, Tottori 680-8553, Japan Received 7 June 2001 ; received in revised form 10 September 2001; accepted 21 September 2001 First published online 19 November 2001
Abstract To characterize common sites within the core oligosaccharide of the R-form lipopolysaccharide (LPS), we screened peptides from a phage-displayed heptapeptide library by using the most truncated form of R-LPS, Re-LPS (S. Typhimurium SL1165) as a ligand. After three rounds of biopanning/amplification and subsequent screening by phagemid enzyme-linked immunosorbent assay (ELISA), we selected three distinct clones that bind to the ligand LPS. We characterized the binding sites of the three clones by ELISA and thin-layer chromatography immunostaining and found that the three clones bind the two Re-LPSs (SL1165 and S. Minnesota Re595) and Rb2 -LPS. In addition, one of the clones also bound to S-form LPS (S. Enteritidis). Current data show that those clones bind to common carbohydrate structure(s) expressed in the core oligosaccharides of those LPS samples. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Lipopolysaccharide ; Core oligosaccharide; Random peptide; Phage display; Salmonella
1. Introduction Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria. LPS is composed of lipid A, core oligosaccharide and O-antigen polysaccharide [1]. Although the O-antigen is heterogeneous both in size and composition, the core oligosaccharide is highly conserved. LPS consisting of the core oligosaccharide is called R-form LPS. Re-LPS is the most truncated form of R-form LPS that consists of a dimer of 2-keto-3deoxy-mannooctulosonic acid (KDO) and lipid A (Fig. 1). Ra^Rd LPSs are produced after sequential glycosylations of the dimeric KDO moiety of Re-LPS with other neutral and N-acetyl amino sugars. Structural and immunochemical aspects of R-form LPS lacking O-antigens have been intensively studied [2^6]. Recent immunochemical studies of R-LPS have shown that some core oligosaccharides expressed on speci¢c R-LPS are immunogenic to produce cross-reactive antibodies. For example, a monoclonal antibody (MAb) raised * Corresponding author. Tel./Fax : +81 (857) 31-6751. E-mail address : [email protected] (R. Yamasaki). 1
The ¢rst two authors contributed equally to this work
against Salmonella Re mutant binds both Re-LPS and Rb2 -LPS [7], which showed not only the presence of a common carbohydrate epitope between the Re and its elongated Rb2 -LPS but also implied the possibility of existence of common epitope(s) among R-form of LPS. Although such common epitope may exist, raising antibodies speci¢c for those epitopes by immunization may have limitations since they have not yet been developed despite of attempts by several investigators [4,8]. However, speci¢c sites mimicking or resembling such epitopes could be expressed with oligo- or polypeptides as exempli¢ed by recent work on proteins [9^11] with the use of a phagedisplayed peptide library. In addition to proteins, this methodology has been applied to carbohydrates, and peptides binding to ganglioside-GM1 have been isolated [12]. Thus, recent applications using peptides that mimic proteins or carbohydrates prompted us to examine the possibility of expressing common carbohydrate structures among R-form LPS with oligopeptides. To characterize such sites, we screened peptides from a phage-displayed heptapeptide library by using Re-LPS as a ligand. We isolated three clones that bind to both the ligand LPS and Rb2 -LPS. Our current study supports that the three clones bind to sites within the carbohydrate moiety of those LPSs.
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 4 4 5 - 1
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2. Materials and methods 2.1. Reagents and general immunochemical analyses A heptapeptide library displayed on M13 ¢lamentous phage (pSKAN-HyB Library) [13] and Escherichia coli WK6VmutS were purchased from MoBiTec GmbH (Go«ttingen, Germany). MAbs (mouse IgG) speci¢c for a M13 phage and lipid A [14] were purchased form Progen Biotechnik GmbH (Heidelberg, Germany) and Sanbio (Uden, Netherlands), respectively. Both anti-mouse IgG (Q-chain speci¢c, alkaline phosphatase conjugate) and KDO (ammonium salt) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were analytical-grade reagents. Salmonella enterica serovar Typhimurium (S. Typhimurium) strain SL1165 (Re-mutant) was obtained from Salmonella Genetic Stock Centre, University Calgary (AB, Canada). SL1165 Re-LPS was extracted and puri¢ed by using a method reported by Galanos [15]. De-O-acylated Re-LPS and lipid A, respectively were prepared by hydrazinolysis [16,17] and hydrolysis [16,18] of the SL1165-LPS. Rb2 -LPS (S. Minnesota R345) was purchased from List Biological Laboratories (Campbell, CA, USA). We obtained the following LPSs from Sigma Chemical Co. (St. Louis, MO, USA) : Ra-LPS (S. Typhimurium TV119); Rc-LPS (S. Minnesota R5); Rd-LPS (S. Minnesota R7); Re-LPS (S. Minnesota Re595); S. Typhimurium S-form LPS; S. Enteritidis S-form LPS; E. coli O128:B12 LPS. Lactosyl ceramide (Lac-cer) and phosphatidylcholine (PC) were purchased from Wako Pure-Chemicals (Osaka, Japan). LPS samples were analyzed by enzyme-linked immunosorbent assay (ELISA, Fig. 2), polyacrylamide gel electrophoresis (PAGE) and thin-layer chromatography (TLC) immunostaining. Typical procedures for ELISA [17], PAGE/blot and TLC immunostaining [16,17,19] have been described previously. We used a microtiter plate from Nunc (Maxisorb loose U-16, Roskilde, Denmark) for ELISA, biopanning and phagemid ELISA. We used p-nitrophosphate for ELISA analysis and Western blue (Promega, Madison, WI, USA) for both PAGE/blot and TLC immunostaining. 2.2. Selection of clones binding to Re-LPS and characterization of their binding sites Construction of a phage-displayed heptapeptide library, biopanning, ampli¢cation and phagemid ELISA were carried out according to a supplier's protocol (pSKAN system protocols, version 2-97, MoBiTec GmbH) except for several minor modi¢cations. In addition to these modi¢cations, brief descriptions on both selection of clones and characterization of their speci¢cities will be given below. Selection of phagemid clones binding to Re-LPS (S. Typhimurium SL1165) : microtiter wells were coated by
incubating with the SL1165 Re-LPS (7.5 Wg) at 4³C overnight. Binding of the LPS to microtiter plates were con¢rmed by eluting the LPS bound on wells with SDS sampling bu¡er and subsequent PAGE analysis of the eluate. After coating the ligand LPS, we used 1% BSA as a blocking reagent instead of 2% skim milk. The wells were sequentially treated with PBS containing 0.5% Tween 20 (TPBS), 1.0% BSA (at room temperature for 1 h), and TPBS. After incubation with the phagemid library [100 Wl, 1011 ^1012 of colony-forming units (cfu)] in PBS at room temperature for 2.5 h, the wells were sequentially treated with T-PBS, 1% BSA (10 min), T-PBS and then with water. The bound phagemids were eluted with glycine^ HCl (100 Wl, pH 2.2), and the solution was neutralized with 2 M Tris. The titer of the phagemid solution was analyzed by infecting E. coli WK6VmutS with an aliquot (10 Wl) of the phagemid solution followed by counting the colonies survived (overnight at 37³C) on ampicillin-containing LB agar plates. The typical cfu of the phagemid solution before ampli¢cation was in the range of 103 ^105 ml31 . After checking the titer, the rest of solution was used for ampli¢cation; growing the infected E. coli in a liquid LB medium containing a M13KO7 helper phage (1010 ^1012 cfu ml31 , Amersham Pharmacia Biotech, NJ, USA) overnight at 37³C, and the phage pellets precipitated [polyethylene glycol 8000 (Sigma Chemical Co., MO, USA) and NaCl] were re-suspended in PBS. The titer of the phagemid after ampli¢cation was in the range of 1011 ^1012 ml31 . After three rounds of biopanning and ampli¢cations, selected clones were further analyzed by sequential phagemid ELISA to obtain clones with higher a¤nity. Phagemid ELISA: Wells were washed with PBS three times and 1% BSA was used for blocking. Wells were coated with LPS samples (7.5 Wg) in a similar manner as described for biopanning. After blocking (1% BSA) and washing with PBS, wells were incubated with approximately 109ÿ10 cfu of phagemids in PBS (75 Wl) for 2.5 h at room temperature. Binding of the phagemid to LPS samples was analyzed by using the anti-M13 MAb and anti-mouse IgG AP. Amino acid sequence of selected clones: Sequencing of DNA encoding peptide in a cloned phagemid was determined by using a Cy5 Thermo Sequenase dye terminator kit and an ALF express automated sequencer (both from Amersham Pharmacia Biotech, NJ, USA). Double stranded phagemid DNA was isolated from E. coli WK6VmutS infected with a cloned phagemid and used as a template. We used two primers, #2897: 5PGGAGGTCTAGATAACGAGG-3P (sense) and #1243a: 5P-ATGAATTAAGCACGGACC-3P (anti-sense) (both from Amersham Pharmacia Biotech, NJ, USA) [13]. Characterization of the binding sites of selected clones : We analyzed the following samples by phagemid ELISA : R-form LPSs [Ra, Rb2 , Rc, Rd, Re (Re595 and SL1165)] ; O-deacylated Re-LPS and lipid A (both from the SL1165
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Re-LPS); S-form LPS (S. Enteritidis, S. Typhimurium, E. coli O128:B12); and non-LPS samples (Lac-cer and PC). The ELISA procedure was similar as described earlier except for coating wells with lipid A, Lac-cer and PC. Each of three samples was dissolved in chloroform: methanol (4:1) (1 mg ml31 ) and then diluted by 10-fold with methanol. The organic solution (75 Wl) was applied to wells, and then they were evaporated to dryness. Binding of lipid A on wells was con¢rmed by ELISA using an anti-lipid A MAb [14]. Inhibition assay: The inhibitory activity of KDO on the binding of the phagemid to Re595-LPS was analyzed by the same ELISA method as described above, and glucose was also used as a negative control. After coating with the 595Re-LPS (7.5 Wg), wells were incubated with a mixture of the phagemid preparation (V1011 cfu) and KDO (10 and 100 molar equivalents of the 595-LPS). The phagemid preparation without KDO was also included in this assay. 3. Results and discussion 3.1. Selection of Re-LPS binding peptides (biopanning, screening and amino acid sequencing) To select the LPS binding peptides from a heptapeptide phagemid library, we performed three rounds of biopanning. Each biopanning consisted of the following steps: (1) immobilization of phagemids on a microtiter plate coated with Re-LPS (S. Typhimurium SL1165), (2) elimination of clones binding non-speci¢cally (sequential washing with T-PBS, 1% BSA, then with T-PBS) and (3) elution of the bound phagemids. Eluted phagemids were checked for their titers, ampli¢ed, and then used in the next round of biopanning. To eliminate non-speci¢c phagemids, the washing step, after immobilizing the phagemids, was repeated twice and three times at the second and third round of biopanning, respectively. After three sequential biopannings, we obtained 90 phagemid clones by using a limiting dilution technique. Starting from 90 clones, we selected clones showing higher a¤nity to the 1165 Re-LPS after three consecutive phagemid ELISA analyses. Fig. 1 shows the results of the ¢rst screening. Of 90 clones, we selected and ampli¢ed 16 clones whose OD values are more than 0.5 and more than double the control OD value. Sixteen clones selected were re-examined by ELISA to give ¢ve clones (45, 61, 70, 81, and 83) (data not shown). Fig. 3 shows the results of the third round of phagemid ELISA, and three clones showing higher a¤nity to the ligand LPS (45, 61 and 70) were selected. Their binding capabilities were con¢rmed after re-cloning of the three phagemids and subsequent ELISA analysis (data not shown). Of the three selected clones, clone 45 showed strongest binding to SL1165 ReLPS and clone 61 the weakest. Thus, we obtained three clones containing peptides that bind to Re-LPS.
351
Fig. 1. Schematic structures of Salmonella LPS. GlcNAc, N-acetyl glucosamine ; Glc, glucose; Gal, galactose; KDO, 2-keto-D-manno-3-deoxyoctulosonic acid; Hep, L-glycero-D-manno-heptose; GlcN, glucosamine.
To determine the identities of three clones (45, 61, and 70), we analyzed the amino acid sequences of the heptapeptides. The double stranded phagemid DNA was extracted from E. coli infected with each of the phagemid clones and the DNA sequences encoding the randomized region were determined using a conventional dideoxy termination method. Table 1 shows the amino acid sequences of three heptapeptides. The three peptides did not have any common amino acid sequences among them, which showed that the selected three clones carry distinct amino acid sequences. 3.2. The three peptides are suggested to bind the oligosaccharide moiety of Re-LPS We examined the binding of the three peptides to Rform LPSs (Ra to Re), S-form LPSs (S. Enteritidis, S. Typhimurium, E. coli O128) by using phagemid ELISA (Fig. 4). In addition to SL1165 Re-LPS (ligand LPS), the Re595-LPS (S. Minnesota) was included for the analysis. We also prepared de-O-acylated LPS by hydrazinolysis [16,17] of SL1165 Re-LPS and lipid A prepared after hydrolysis [16,18] of the same Re-LPS. Except for S. Typhimurim Re-LPS, the core oligosaccharide structures of the LPS used in this experiment have been characterized [1,4,6] and their schematic structures are shown in Fig. 1. As Fig. 4 shows, in addition to the 1165 Re-LPS, the three peptides (45, 61, 70) bound the Re595-LPS. Both clones 45 and 70 bound to the 1165 Re-LPS even after the de-O-acylation of the lipid A, whereas clone 61 showed slightly lower binding to the de-O-acylated LPS. Similarly, removal of the KDO residues from the 1165-LPS also a¡ected the binding, and the three peptides showed much lower binding to the lipid A than to the intact LPS. Among the other R-form LPSs, the three peptides bound only to Rb2 -LPS but not to Ra-, Rc-, and RdLPSs. In contrast to the R-form LPS, the peptides showed di¡erent binding patterns to the S-form LPS tested. Clone 45 bound strongly to S. Enteritidis LPS. However, it showed lower a¤nity to both S. Typhimurium LPS and E. coli O-128 LPS. The peptide 61 showed a slightly higher binding to three S-form LPSs than to Ra-, Rc-, Rd-LPSs,
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Fig. 2. Phagemid ELISA analysis of 90 clones isolated after three rounds of biopanning. Each well coated with SL1165 Re-LPS (7.5 Wg) was incubated with V109 ^1010 cfu of each phagemid and sequentially treated with an anti-M13 MAb and anti-mouse IgG (alkaline phosphatase conjugate). p-Nitrophosphate was used as a substrate. Results are expressed as mean values (OD 405 nm at 60 min) of duplicate analyses.
whereas clone 70 showed only residual bindings to the Sform LPSs tested. The bindings of the three peptides to Lac-cer and PC were slightly higher than those to Ra, Rc, Rd-LPSs. TLC staining analysis (data not shown) also con¢rmed positive binding of the three peptides to ReLPSs (1165 and 595) and Rb2 -LPS. Based on published structures of the LPS samples used in this study, the results obtained with the ELISA (Fig. 4) and TLC immunostaining showed the following: (1) the three peptides do not bind to the lipid A portion or fatty acids present in the lipid A possibly common to all the LPSs examined, which was supported by their stronger binding to the two Re-LPSs and Rb2 -LPSs and also by the fact that the binding to the 1165 Re-LPS was not abolished even after O-linked fatty acids of its lipid A moiety were removed by hydrazinolysis. (2) In addition to the results described above, lower binding of the three
clones to the1165 lipid A than the intact LPS suggests that the peptides bind presumably to the carbohydrate moiety, a dimer of KDO of the two Re-LPS. Alternatively, an area including the linkage between KDO and lipid A moiety may be the site for their binding. (3) Preferential binding of the three peptides to Rb2 -LPS shows that the binding sites described above are also expressed in this LPS. It is not clear why those sites are expressed in the Rb2 -LPS whose carbohydrate moiety is larger than those of Rcand Rd-LPSs (Fig. 1). However, similar to our results, a MAb raised against S. Minnesota Re595 has been reported to bind to both Re- and Rb2 -LPSs, but not to other R-form LPSs [7]. (4) Binding of peptide 45 to S. Enteritidis LPS shows that its binding site is also expressed in this S-form LPS. (5) Slightly higher bindings of the three clones to Lac-cer and PC than those to Ra-, Rcand Rd-LPSs suggest that the peptides have non-speci¢c a¤nity to their hydrophobic sites which are not present in the LPS samples. To further elucidate the binding sites of the three clones, we examined the inhibitory activities of a KDO monomer on the binding of the three clones to Re-LPS (Re595) [20]. We used 10 and 100 molar equivalents of the KDO, and the bindings of 45 and 61 to the LPS were not inhibited even with the 100 equivalents of KDO. However, 6% and Table 1 The amino acid sequencesa of the selected phagemid clones
Fig. 3. Phagemid ELISA analysis of ¢ve clones selected. Five phagemids selected (V109 ^1010 cfu per well) were re-examined for their binding to Re-LPS (7.5 Wg per well). The bound phagemids were detected as described in Fig. 2. Results are expressed as mean values (OD 405 nm at 60 min) of duplicate analyses
Phagemid clones
Amino acid sequences
45 61 70
Arg Val Val Lys Glu Ser Arg Tyr Ser Ala Leu Glu Glu Gly Met Met Gly Val Gly Thr Ser
a Amino acid sequences were deduced from the DNA sequence data for random peptide coding region of the double stranded phagemid DNA extracted from E. coli infected with each of the clones.
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Fig. 4. Characterization of the speci¢cities of the three peptides (45, 61, 70) selected by phagemid ELISA. Wells coated with each ligand (7.5 Wg) were incubated with each of the three phagemids (V109ÿ10 cfu) and treated in a similar manner as described in Fig. 2. R-LPS: R-form LPSs; S-LPS: Sform LPSs; De-O-acyl Re: de-O-acylated Re-LPS (SL1165); Re-lipid A: lipid A prepared from Re-LPS (SL1165); Lac-cer : lactosyl ceramide, PC: phosphatidylcholine. The strain numbers of the bacteria producing R-LPS are shown in parenthesis. Results are expressed as mean values (OD 405 nm at 20 min) of duplicate analyses.
57% of the binding of clone 70 was inhibited with 10 and 100 equivalents of KDO, respectively (data not shown). We used 100 molar equivalents of glucose as a negative control, and this neutral sugar showed no inhibitory activity under the conditions used. The results of the above inhibition assay together with the di¡erence in binding of the three clones to the S-form LPSs (Fig. 4) suggest that their binding sites are di¡erent. This could be re£ected in their di¡erent amino acid sequences (Table 1). Inhibition of the binding of peptide 70 with KDO shows that its binding site may include the structure of a monomeric KDO. Peptides 45 and 61 may recognize sites expressed in a KDO dimer or close to the KDO-lipid A linkage (Fig. 1) and these possible sites could be conformational. Although we were not able to use a dimeric or trimeric forms of KDO which are not commercially available, the use of such oligomers will provide further insights on the possible binding sites within the Re-LPSs, as well as Rb2 LPS which contain the trimeric KDO as a partial structure [1] (Fig. 1). Our current data indicate that three phagemids bind to a common carbohydrate structure within the two Re-LPSs (SL1165 and Re595) although we were not be able to identify their exact binding sites. In addition, the same carbohydrate structure within the two LPSs is also expressed in Rb2 -LPS. To further characterize those binding sites, we would need to screen out oligopeptides of higher a¤nity to the target LPS. By using such oligopeptides, we are able to di¡erentiate non-speci¢c bindings from speci¢c bindings, which would facilitate characterization of their binding sites. Further, determination of the overall structures of the R-form LPS would be helpful for us to understand why the clones selected bound to Rb2 -LPS but not to other R-LPS samples. In summary, for the ¢rst time we isolated three distinct phagemids that bind to two Re-LPSs (SL1165 and Re595)
and Rb2 -LPS from a phage-displayed heptapeptide library. In addition to these R-form LPSs, one of the clones bound to S-form LPS (S. Enteritidis). Our current data show that the three phagemids bind to common carbohydrate structure(s) within the core oligosaccharides of those LPSs. Acknowledgements We thank Drs. T. Taki, D. Ishikawa, Y. Yamano and T. Kawano for their valuable suggestions and comments. We also thank Dr. M. Muramatsu for providing reagents for the DNA sequencing and T. Saito for his technical assistance for the sequence analysis. This study was supported in part by Grants-in-Aid (09660298 and 10306022) for Scienti¢c Research from the Ministry of Education, Science and Technology, Japan.
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