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Toward a Better Understanding of the Basis of the Molecular Mimicry of Polysaccharide Antigens by Peptides

2005; Elsevier BV; Volume: 281; Issue: 4 Linguagem: Inglês

10.1074/jbc.m510172200

1083-351X

Marie‐Jeanne Clément, Antoine Fortuné, Armelle Phalipon, Véronique Marcel-Peyre, Catherine Simenel, Anne Imberty, Muriel Delepierre, Laurence A. Mulard,

Escherichia coli research studies

Protein conjugates of oligosaccharides or peptides that mimic complex bacterial polysaccharide antigens represent alternatives to the classical polysaccharide-based conjugate vaccines developed so far. Hence, a better understanding of the molecular basis ensuring appropriate mimicry is required in order to design efficient carbohydrate mimic-based vaccines. This study focuses on the following two unrelated sets of mimics of the Shigella flexneri 5a O-specific polysaccharide (O-SP): (i) a synthetic branched pentasaccharide known to mimic the average solution conformation of S. flexneri 5a O-SP, and (ii) three nonapeptides selected upon screening of phage-displayed peptide libraries with two protective murine monoclonal antibodies (mAbs) of the A isotype specific for S. flexneri 5a O-SP. By inducing anti-O-SP antibodies upon immunization in mice when appropriately presented to the immune system, the pentasaccharide and peptides p100c and p115, but not peptide p22, were qualified as mimotopes of the native antigen. NMR studies based on transferred NOE (trNOE) experiments revealed that both kinds of mimotopes had an average conformation when bound to the mAbs that was close to that of their free form. Most interestingly, saturation transfer difference (STD) experiments showed that the characteristic turn conformations adopted by the major conformers of p100c and p115, as well as of p22, are clearly involved in mAb binding. These latter experiments also showed that the branched glucose residue of the pentasaccharide was a key part of the determinant recognized by the protective mAbs. Finally, by using NMR-derived pentasaccharide and peptide conformations coupled to STD information, models of antigen-antibody interaction were obtained. Most interestingly, only one model was found compatible with experimental data when large O-SP fragments were docked into one of the mIgA-binding sites. This newly made available system provides a new contribution to the understanding of the molecular mimicry of complex polysaccharides by peptides and short oligosaccharides. Protein conjugates of oligosaccharides or peptides that mimic complex bacterial polysaccharide antigens represent alternatives to the classical polysaccharide-based conjugate vaccines developed so far. Hence, a better understanding of the molecular basis ensuring appropriate mimicry is required in order to design efficient carbohydrate mimic-based vaccines. This study focuses on the following two unrelated sets of mimics of the Shigella flexneri 5a O-specific polysaccharide (O-SP): (i) a synthetic branched pentasaccharide known to mimic the average solution conformation of S. flexneri 5a O-SP, and (ii) three nonapeptides selected upon screening of phage-displayed peptide libraries with two protective murine monoclonal antibodies (mAbs) of the A isotype specific for S. flexneri 5a O-SP. By inducing anti-O-SP antibodies upon immunization in mice when appropriately presented to the immune system, the pentasaccharide and peptides p100c and p115, but not peptide p22, were qualified as mimotopes of the native antigen. NMR studies based on transferred NOE (trNOE) experiments revealed that both kinds of mimotopes had an average conformation when bound to the mAbs that was close to that of their free form. Most interestingly, saturation transfer difference (STD) experiments showed that the characteristic turn conformations adopted by the major conformers of p100c and p115, as well as of p22, are clearly involved in mAb binding. These latter experiments also showed that the branched glucose residue of the pentasaccharide was a key part of the determinant recognized by the protective mAbs. Finally, by using NMR-derived pentasaccharide and peptide conformations coupled to STD information, models of antigen-antibody interaction were obtained. Most interestingly, only one model was found compatible with experimental data when large O-SP fragments were docked into one of the mIgA-binding sites. This newly made available system provides a new contribution to the understanding of the molecular mimicry of complex polysaccharides by peptides and short oligosaccharides. Bacterial capsular polysaccharides (CPS) 2The abbreviations used are: CPS, capsular polysaccharides; NOE, nuclear Overhauser effect; tr, transferred; ROESY, rotating frame nuclear Overhauser enhancement spectroscopy; NOESY, nuclear Overhauser effect spectroscopy; ELISA, enzyme-linked immunosorbent assay; Ab, antibody; mAb, monoclonal Ab; STD, saturation transfer difference; O-SP, O-specific polysaccharide; LPS, lipopolysaccharides; PBS, phosphate-buffered saline; BSA, bovine serum albumin; TOCSY, total correlation spectroscopy. and lipopolysaccharides (LPS) are known to be important virulence factors and major targets of the protective immune response of the host (1MacLeod C.M. Hodges R.G. Heidelberg M. Bernhard W.G. J. Exp. Med. 1945; 82: 445-465Crossref Scopus (365) Google Scholar). Several polysaccharide vaccines such as those targeting Streptococcus pneumoniae, Neisseria meningitidis, or Salmonella typhi were proven efficient in adults and are thus commercially available. Their ineffectiveness in infants has been successfully circumvented with the licensing of polysaccharide:protein conjugates such as those targeting Haemophilus influenzae b, S. pneumoniae, and N. meningitidis group C infections (2Roy R. Drug Discovery Today: Technologies. 2004; 1: 327-336Crossref PubMed Scopus (130) Google Scholar). A possible alternative may derive from the use of accurate synthetic mimics of the bacterial polysaccharide antigens. This innovative approach has been mostly developed along two lines, including the use of either synthetic oligosaccharides or peptides mimicking the carbohydrate determinants recognized by anti-carbohydrate monoclonal antibodies (mAb) conferring protection in experimental models of infection. Indeed, semi-synthetic glycoconjugates incorporating oligosaccharides mimicking fragments of bacterial polysaccharide antigens were shown to be highly immunogenic in mice (3Goebel H.H. Ikeda K. Schulz F. Burck U. Kohlschutter A. Acta Neuropathol. 1981; 55: 247-249Crossref PubMed Scopus (11) Google Scholar, 4Pozsgay V. Chu C. Pannell L. Wolfe J. Robbins J.B. Schneerson R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5194-5197Crossref PubMed Scopus (178) Google Scholar, 5Peeters C.C. Evenberg D. Hoogerhout P. Kayhty H. Saarinen L. van Boeckel C.A. van der Marel G.A. van Boom J.H. Poolman J.T. Infect. Immun. 1992; 60: 1826-1833Crossref PubMed Google Scholar). The "proof of concept" was recently demonstrated in humans with the efficacy of such a semi-synthetic glycoconjugate in protecting against H. influenzae b infection (6Verez-Bencomo V. Fernandez-Santana V. Hardy E. Toledo M.E. Rodriguez M.C. Heynngnezz L. Rodriguez A. Baly A. Herrera L. Izquierdo M. Villar A. Valdes Y. Cosme K. Deler M.L. Montane M. Garcia E. Ramos A. Aguilar A. Medina E. Torano G. Sosa I. Hernandez I. Martinez R. Muzachio A. Carmenates A. Costa L. Cardoso F. Campa C. Diaz M. Roy R. Science. 2004; 305: 522-525Crossref PubMed Scopus (427) Google Scholar). However, access to the required carbohydrate haptens is often a roadblock. Therefore, besides the investigation of anti-idiotype antibody (7Stein H. Gatter K.C. Heryet A. Mason D.Y. Lancet. 1984; 2: 71-73Abstract PubMed Scopus (22) Google Scholar), expanding the concept of mimicry led in the recent past to extensive exploring of the potential mimicking of polysaccharide and/or complex oligosaccharide antigens by peptides (8Pirofski L.A. Trends Microbiol. 2001; 9: 445-451Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 9Johnson M.A. Rotondo A. Pinto B.M. Biochemistry. 2002; 41: 2149-2157Crossref PubMed Scopus (27) Google Scholar, 10Monzavi-Karbassi B. Cunto-Amesty G. Luo P. Kieber-Emmons T. Trends Biotechnol. 2002; 20: 207-214Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). These peptide mimotopes, i.e. peptide mimics inducing an anti-carbohydrate antibody response upon immunization, have been proposed as potential surrogate antigens of carbohydrates in vaccine development (10Monzavi-Karbassi B. Cunto-Amesty G. Luo P. Kieber-Emmons T. Trends Biotechnol. 2002; 20: 207-214Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Indeed, because of their ease of manufacture and their intrinsic immunogenic properties, peptide mimotopes may have greater advantage over complex carbohydrate haptens issued from bacterial cell cultures or low yielding multistep syntheses. However, not all peptide mimics of carbohydrate antigens behave as mimotopes. Despite the large number of known peptide mimics, only few peptide mimotope-based experimental vaccines have been reported so far (11Luo P. Agadjanyan M. Qiu J. Westerink M.A. Steplewski Z. Kieber-Emmons T. Mol. Immunol. 1998; 35: 865-879Crossref PubMed Scopus (41) Google Scholar, 12Cunto-Amesty G. Dam T.K. Luo P. Monzavi-Karbassi B. Brewer C.F. Van Cott T.C. Kieber-Emmons T. J. Biol. Chem. 2001; 276: 30490-30498Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 13Fleuridor R. Lees A. Pirofski L. J. Immunol. 2001; 166: 1087-1096Crossref PubMed Scopus (102) Google Scholar, 14Lesinski G.B. Westerink M.A. J. Microbiol. Methods. 2001; 47: 135-149Crossref PubMed Scopus (110) Google Scholar, 15Maitta R.W. Datta K. Lees A. Belouski S.S. Pirofski L.A. Infect. Immun. 2004; 72: 196-208Crossref PubMed Scopus (49) Google Scholar, 16Buchwald U.K. Lees A. Steinitz M. Pirofski L.A. Infect. Immun. 2005; 73: 325-333Crossref PubMed Scopus (48) Google Scholar). It is believed that a better understanding of the molecular basis of peptide-carbohydrate mimicry could help the rational design of potent peptide mimotope-based vaccines. In particular, whether mimicry is structural, functional, or both remains an unsolved question (9Johnson M.A. Rotondo A. Pinto B.M. Biochemistry. 2002; 41: 2149-2157Crossref PubMed Scopus (27) Google Scholar, 17Harris S.L. Craig L. Mehroke J.S. Rashed M. Zwick M.B. Kenar K. Toone E.J. Greenspan N. Auzanneau F.I. Marino-Albernas J.R. Pinto B.M. Scott J.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2454-2459Crossref PubMed Scopus (121) Google Scholar, 18Valadon P. Nussbaum G. Oh J. Scharff M.D. J. Immunol. 1998; 161: 1829-1836PubMed Google Scholar, 19Cunto-Amesty G. Luo P. Monzavi-Karbassi B. Lees A. Kieber-Emmons T. Vaccine. 2001; 19: 2361-2368Crossref PubMed Scopus (25) Google Scholar). Available x-ray data of carbohydrate-protein and of the corresponding peptide mimotope-protein complexes along with information on the thermodynamics of peptide mimic-protein binding are somewhat scarce (17Harris S.L. Craig L. Mehroke J.S. Rashed M. Zwick M.B. Kenar K. Toone E.J. Greenspan N. Auzanneau F.I. Marino-Albernas J.R. Pinto B.M. Scott J.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2454-2459Crossref PubMed Scopus (121) Google Scholar, 20Young A.C. Valadon P. Casadevall A. Scharff M.D. Sacchettini J.C. J. Mol. Biol. 1997; 274: 622-634Crossref PubMed Scopus (75) Google Scholar, 21Vyas N.K. Vyas M.N. Chervenak M.C. Bundle D.R. Pinto B.M. Quiocho F.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15023-15028Crossref PubMed Scopus (67) Google Scholar). Thus to date, although analysis of the topography of ligand-receptor complementarity may be performed by a variety of methods, available knowledge on the molecular features of peptide-carbohydrate mimicry mostly relies on data obtained from NMR and molecular modeling studies as reviewed recently (22Johnson M.A. Pinto B.M. Carbohydr. Res. 2004; 339: 907-928Crossref PubMed Scopus (52) Google Scholar). By aiming to prevent S. flexneri bacterial infections, during the past few years we investigated the development of synthetic mimics of the major protective S. flexneri antigen. S. flexneri, a Gram-negative bacillus, is responsible for the endemic form of shigellosis, a human dysenteric syndrome causing a high mortality rate in infants, particularly in developing countries (23Kotloff K.L. Winickoff J.P. Ivanoff B. Clemens J.D. Swerdlow D.L. Sansonetti P.J. Adak G.K. Levine M.M. Bull. W.H.O. 1999; 77: 651-666PubMed Google Scholar). The disease, characterized by bacterial invasion of the human colonic mucosa, leads to acute inflammation and subsequent massive tissue destruction (24Phalipon A. Sansonetti P.J. Crit. Rev. Immunol. 2003; 23: 371-401Crossref PubMed Scopus (55) Google Scholar). Protection induced upon infection is serotype-specific (24Phalipon A. Sansonetti P.J. Crit. Rev. Immunol. 2003; 23: 371-401Crossref PubMed Scopus (55) Google Scholar), pointing to the O-specific polysaccharide moiety (O-SP) of the bacterial LPS as the major target for protective immunity. In line with the success of the CPS-protein conjugate vaccines, protein conjugates of detoxified S. flexneri 2a LPS, the prevalent serotype in humans, were shown to be safe and immunogenic both in adults and young children (25Ashkenazi S. Passwell J.H. Harlev E. Miron D. Dagan R. Farzan N. Ramon R. Majadly F. Bryla D.A. Karpas A.B. Robbins J.B. Schneerson R. J. Infect. Dis. 1999; 179: 1565-1568Crossref PubMed Scopus (71) Google Scholar). More recently, we developed fully synthetic glycoconjugates as well as promising neoglycoproteins exposing well designed synthetic saccharidic haptens mimicking S. flexneri 2a O-SP as potential vaccines against the homologous infection (26Wright K. Guerreiro C. Laurent I. Baleux F. Mulard L.A. Org. Biomol. Chem. 2004; 2: 1518-1527Crossref PubMed Google Scholar, 27Belot F. Guerreiro C. Baleux F. Mulard L.A. Chemistry. 2005; 11: 1625-1635Crossref PubMed Scopus (61) Google Scholar). 3Phalipon, A., Costachel, C., Grandjean, C., Thuizat, A., Guerreiro, C., Tanguy, M., Nato, F., Vulliez-Le Normand, B., Bélot, F., Wright, K., Marcel-Peyre, V., Snsonetti, P. J., and Mulard, L. (2005) J. Immunol., in press. Alternatively, we also investigated the potential of peptide mimotopes, and we reported the first example of immunogenic mimicry of carbohydrates by peptides identified by screening of phage-displayed nonapeptide libraries with two protective mAbs of the A isotype (mIgA) specific for S. flexneri serotype 5a, mIgA C5, and mIgA I3 (28Phalipon A. Folgori A. Arondel J. Sgaramella G. Fortugno P. Cortese R. Sansonetti P.J. Felici F. Eur. J. Immunol. 1997; 27: 2620-2625Crossref PubMed Scopus (115) Google Scholar). Among the 19 peptide sequences selected upon screening with mIgA I3, p100c (YKPLGALTH) and p115 (KVPPWARTA, also interacting with mIgA C5) only induce anti-O-SP antibodies in mice upon immunization with the corresponding phage particles. Most interestingly, the mimotopes share no obvious consensus sequence and do not cross-react with one another. However, as often reported by others (29Pincus S.H. Lepage S.R. Jung R.F. Massey J.G. Jaseja M. Int. Rev. Immunol. 2001; 20: 221-227Crossref PubMed Scopus (2) Google Scholar, 30Prinz D.M. Smithson S.L. Westerink M.A. J. Immunol. Methods. 2004; 285: 1-14Crossref PubMed Scopus (30) Google Scholar), their amino acid sequences contain aromatic and hydrophobic residues but also amino acids having cyclic side chains, including at least one proline. Besides, based on a combination of NMR and molecular modeling studies, we proposed a conformational model for the S. flexneri 5a O-SP whose biological repeating unit is the branched pentasaccharide I (Structure 1) (31Lindberg A.A. Cam P.D. Chan N. Phu L.K. Trach D.D. Lindberg G. Karlsson K. Karnell A. Ekwall E. Rev. Infect. Dis. 1991; 13: 231-237Crossref Scopus (11) Google Scholar). Study of both the antigenicity and the conformation of the four synthetic frame-shifted pentasaccharides corresponding to pentasaccharide I (32Mulard L.A. Ughetto-Monfrin J. J. Carbohydr. Chem. 2000; 19: 193-220Crossref Scopus (14) Google Scholar) suggested that the DA(E)BC sequence is the structure that best mimics the native O-SP antigen (33Clement M.J. Imberty A. Phalipon A. Perez S. Simenel C. Mulard L.A. Delepierre M. J. Biol. Chem. 2003; 278: 47928-47936Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). More recently, the pentasaccharide DA(E)BC was shown to act as a mimotope. 4L. Mulard and A. Phalipon, unpublished results. Here we report the antigenicity and the NMR findings on the preferred conformation of p100c and p115 peptide mimotopes both in their free and mIgA-bound forms. Analysis was also performed using peptide p22 (KRHFLSQRQ, mIgA C5- and mIgA I3-specific), one of the 17 nonimmunogenic peptides selected during the original screening (28Phalipon A. Folgori A. Arondel J. Sgaramella G. Fortugno P. Cortese R. Sansonetti P.J. Felici F. Eur. J. Immunol. 1997; 27: 2620-2625Crossref PubMed Scopus (115) Google Scholar). Antibody-bound conformations and epitope mapping were derived from transferred NOE (trNOE) (34Clore G.M. Gronenborn A.M. J. Magn. Reson. 1982; 48: 402-417Google Scholar, 35Clore G.M. Gronenborn A.M. J. Magn. Reson. 1983; 53: 423-442Google Scholar) and saturation transfer difference (STD) experiments (36Mayer M. Meyer B. Angew. Chem. Int. Ed. 1999; 38: 1784-1788Crossref PubMed Scopus (1366) Google Scholar), respectively. The conformational preferences observed for the peptides were tentatively related to those derived from NMR and molecular modeling analysis of the DA(E)BC-mIgA complexes that led to a theoretical model of the recognition of S. flexneri 5a O-SP by mIgA I3. This contribution adds to the few reports investigating molecular mimicry by analyzing both peptide mimic-mAb and carbohydrate-mAb recognition features (37Johnson M.A. Pinto B.M. J. Am. Chem. Soc. 2002; 124: 15368-15374Crossref PubMed Scopus (50) Google Scholar, 38Johnson M.A. Jaseja M. Zou W. Jennings H.J. Copie V. Pinto B.M. Pincus S.H. J. Biol. Chem. 2003; 278: 24740-24752Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 39Johnson M.A. Pinto B.M. Bioorg. Med. Chem. 2004; 12: 295-300Crossref PubMed Scopus (32) Google Scholar). Material—Selected nonapeptides p100c (YKPLGALTH), p115 (KVPPWARTA), and p22 (KRHFLSQRQ) were purchased from Synthem (Saint-Christol-Les-Alès, France). The peptide p100c was further cyclized in the Unité de Chimie Organique at the Pasteur Institute. Pentasaccharide DA(E)BC was used in its methyl glycoside form DA(E)BC-OMe (40Mulard L.A. Clément M.-J. Segat-Dioury F. Delepierre M. Tetrahedron. 2002; 58: 2593-2604Crossref Scopus (18) Google Scholar). mAb mIgA C5 and mIgA I3 were prepared as described previously (41Phalipon A. Kaufmann M. Michetti P. Cavaillon J.M. Huerre M. Sansonetti P. Kraehenbuhl J.P. J. Exp. Med. 1995; 182: 769-778Crossref PubMed Scopus (143) Google Scholar). Inhibition ELISA—Characterization of the oligosaccharide determinant recognized by the mIgA was performed by measuring the mIgA-oligosaccharide interaction as follows. First of all, a standard curve was established for each mIgA tested. Different concentrations of the mAb were incubated overnight at 4 °C on microtiter plates coated with purified S. flexneri 5a LPS at a concentration of 5 μg/ml in carbonate buffer, pH 9.6, and subsequently incubated with 1% PBS/BSA for 30 min at 4 °C. After washing with PBS/Tween 20 (0.05%), alkaline phosphatase-conjugated anti-mouse IgA was added at a dilution of 1:5,000 (Sigma) for 1 h at 37 °C. After washing with PBS/Tween 20 (0.05%), the substrate was added (12 mg of p-nitrophenyl phosphate in 1.2 ml of 1 m Tris-HCl buffer, pH 8.8, and 10.8 ml of 5 m NaCl). Once the color developed, the plate was read at 405 nm (Dynatech MR 4000 microplate reader). A standard curve A = f([Ab]) was fitted to the quadratic equation Y = aX2 + bX + c, where Y is the absorbance and X is the Ab concentration. Correlation factor (r2) of 0.99 was routinely obtained. Then the amount of oligosaccharides giving 50% inhibition of mIgA binding to LPS (IC50) was determined as follows. Each mIgA at a given concentration (chosen as the minimal concentration of Ab which gives the maximal absorbance on the standard curve) was incubated overnight at 4 °C with various concentrations of each of the oligosaccharides to be tested, in 1% PBS/BSA. Measurement of unbound mIgA was performed as described above using microtiter plates coated with purified LPS from S. flexneri 5a, and the mAb concentration was deduced from the standard curve. The recognition capacity of anti-LPS mIgA for LPS was determined as described above using various concentrations of LPS that were incubated in solution overnight at 4 °C with the predefined concentration of each mIgA. IC50 was defined as the concentration of oligosaccharides required to inhibit 50% of mIgA binding to LPS. NMR Spectroscopy—All 1H NMR experiments were recorded at 298 K on a Varian Unity Inova spectrometer operating at 1H frequencies of 500 MHz. 1H chemical shifts were given relative to an external standard of 4,4-dimethyl-4-silapentane sodium sulfonate at 0 ppm. Free Peptides—The samples were prepared in 90% H2O and 10% D2O at pH 5 for p115 and p100c and at pH 6.5 for p22. The solution concentrations were about 10, 3, and 8 mm for p115, p100c, and p22, respectively. DQF-COSY (42Rance M. Sorensen O.W. Bodenhausen G. Wagner G. Ernst R.R. Wuthrich K. Biochem. Biophys. Res. Commun. 1983; 117: 479-485Crossref PubMed Scopus (2596) Google Scholar), TOCSY (43Griesinger C. Otting G. Wüthrich K. Ernst R.R. J. Am. Chem. Soc. 1988; 110: 7870-7872Crossref Scopus (1198) Google Scholar), and ROESY (44Kessler H. Griesinger C. Kerssebaum R. Wagner K. Ernst R.R. J. Am. Chem. Soc. 1987; 109: 607-609Crossref Scopus (421) Google Scholar) experiments were recorded with 512 increments and 16 scans at 298 K. The TOCSY and ROESY experiments were acquired using a mixing time of 80 and 400 ms, respectively. Water suppression was performed using the WATERGATE pulse sequence (45Piotto M. Saudek V. Sklenar V. J. Biomol. NMR. 1992; 2: 661-665Crossref PubMed Scopus (3538) Google Scholar). All NMR spectra were collected in the phase-sensitive mode using the States-Haberkorn method (46States D.J. Haberkorn R.A. Ruben D.J. J. Magn. Reson. 1982; 48: 286-292Google Scholar). Ligand-Antibody Interactions—Shigemi tubes were used for all samples. In order to prepare NMR samples of pentasaccharide DA(E)BC-OMe in the presence of the antibodies mIgA C5 and mIgA I3, mAbs were concentrated after repeated cycles of exchange with D2O buffer (50 mm deuterated sodium phosphate, 100 mm NaCl, pH 6.5) in Amicon Centriprep-10 concentrators. trNOE experiments (34Clore G.M. Gronenborn A.M. J. Magn. Reson. 1982; 48: 402-417Google Scholar, 35Clore G.M. Gronenborn A.M. J. Magn. Reson. 1983; 53: 423-442Google Scholar, 47Jimenez-Barbero J. Peters T. NMR Spectroscopy of Glycoconjugates. Wiley-VCH, New York2002: 289-307Crossref Google Scholar) performed on different pentasaccharide:binding site ratios (5:1, 10:1, 15:1, 20:1, and 30:1) showed that the most favorable ratio for trNOE was 20:1. So the final samples were prepared with 3.75 μm antibody and 0.3 mm pentasaccharide in 380 μl of the above mentioned D2O buffer. trNOE, trROE, and STD experiments (36Mayer M. Meyer B. Angew. Chem. Int. Ed. 1999; 38: 1784-1788Crossref PubMed Scopus (1366) Google Scholar) on pentasaccharide DA(E)BC-OMe in the presence of mIgA C5 and mIgA I3 were recorded at 500 and 600 MHz, respectively. trNOE experiments were performed with mixing times of 100, 150, 250, 300, and 400 ms at 303 K to obtain build-up curves and trROE with a mixing time of 400 ms. The conformation of the free peptides was studied at pH 5. However, with this pH value being close to the isoelectric points of the mIgAs, a study of peptides in their bound conformation was performed at pH 6.5 to avoid precipitation of the mAb. Similarly to the DA(E)BC-mIgA complexes, a peptide:antibody-binding site ratio of 20:1 was used (0.3 mm:3.75 μm). trNOE experiments were performed with mixing times of 100, 150, 250, 300, and 400 ms. To be sure that the observed negative cross-peaks were real trNOEs, NOESY spectra were recorded under the same pH, temperature, and concentration values with the peptides alone. Furthermore, to discard any impact on NOE effects of viscosity increase as a result of the mAb presence, a NOESY spectrum (τm = 200 ms) of p115 was registered in the presence of BSA at the same concentration ratio as that used with the mIgAs. Because no negative NOE cross-peaks were observed in either case, it was assumed that the negative NOEs observed in the presence of mIgA were trNOEs. Selective saturation of antibody resonances were performed for all STD-NMR experiments at 0.3 ppm (30 ppm for reference spectra) using a series of 40 gaussian-shaped pulses (50- and 10-ms delay between pulses, excitation width γB1/2π, approximately 50 Hz) for a total saturation time of 2.4 s. The one-dimensional STD spectra were recorded with 4096 scans at 288 and 298 K for the pentasaccharide and the peptides, respectively. Subtraction of saturated spectra from reference spectra was obtained by phase cycling (36Mayer M. Meyer B. Angew. Chem. Int. Ed. 1999; 38: 1784-1788Crossref PubMed Scopus (1366) Google Scholar). For DA(E)BC-OMe, two STD-TOCSY experiments (48Herfurth L. Weimar T. Peters T. Angew. Chem. Int. Ed. Engl. 2000; 39: 2097-2099Crossref PubMed Scopus (14) Google Scholar) were recorded with selective saturation at 0.3 and 30 ppm, respectively. Differences between the two spectra were performed using the VNMR software. No attempt here was made to quantify STD-NMR intensities, as it is known that these exhibit a complex dependence on relaxation times, correlation times, exchange rates, and on binding site proton density. Indeed, only when short saturation times are used, i.e. less than 1 s, can intensities reflect ligand proton-protein proton distances (37Johnson M.A. Pinto B.M. J. Am. Chem. Soc. 2002; 124: 15368-15374Crossref PubMed Scopus (50) Google Scholar). Here the saturation time of 2.4 s prevented us from quantitative analysis. Distance and Angle Constraints—The cross-peak volumes from trNOESY and trROESY experiments of the pentasaccharide in the presence of mIgAs were measured with the VNMR software. Distances between neighboring protons were calculated by the usual 1/r6 NOE/distance relationship (49Baleja J. Moult J. Sykes B.D. J. Magn. Reson. 1990; 87: 375-384Google Scholar). NOE-derived and trROE distances were obtained from initial NOE build-up rates, which were calculated by NOE volumes fitting during different mixing times. The intra-residue distance of 2.52 Å between the H-1 and H-2 protons of the α-l-rhamnopyranosyl unit B was used as a reference for distance calibration. Distance constraints of free peptides were obtained from the ROESY spectrum run at 298 K with a 400-ms mixing time. For peptides in the presence of mIgA, distance constraints were obtained from the trNOESY spectra run at 298 K with a 200-ms mixing time. NOE intensities were evaluated from the height of the cross-peaks. For structure calculations, upper limit distances of 2.8, 3.5, and 5 Å were used for strong, medium, and weak NOEs, respectively (50Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986Crossref Google Scholar). The 3JNH-Hα values were used to restrain Φ angles as follows: for 3J >9 Hz, -155° < Φ < -85°; for 8 Hz < 3J <9 Hz, -175° < Φ < -65°; for 5 Hz < 3J <7 Hz, -105° < Φ < -55°; for 3J <5 Hz, -90° < Φ < -40° (51Pardi A. Billeter M. Wuthrich K. J. Mol. Biol. 1984; 180: 741-751Crossref PubMed Scopus (940) Google Scholar). Structure Calculations—Structure calculations of free and bound peptides were run on a Silicon Graphics work station using the standard protocol of the DYANA program (52Guntert P. Mumenthaler C. Wuthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2558) Google Scholar). A total of 100 structures were calculated using the torsion angle dynamics protocol. The structures were sorted according to the final value of the target function, and the 20 best structures were analyzed in terms of distance and angle violations. Of these 20 structures, the 10 best structures were visualized by using MOLMOL (53Koradi R. Billeter M. Wuthrich K. J. Mol. Graphics. 1996; 14: 51-55Crossref PubMed Scopus (6498) Google Scholar). Homology Modeling of the IgA I3 Fab Fragment and Docking—The search for structures with sequences similarities was performed with Blast (54Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60216) Google Scholar) on sequences of all proteins with known three-dimensional structure in the Protein Data Bank (55Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27906) Google Scholar). Five structures of interest were downloaded and used as template by the Composer program for the building of VL and VH chains of IgA I3 (56Blundell T. Carney D. Gardner S. Hayes F. Howlin B. Hubbard T. Overington J. Singh D.A. Sibanda B.L. Sutcliffe M. Eur. J. Biochem. 1988; 172: 513-520Crossref PubMed Scopus (250) Google Scholar). The Tripos force field (57Clark M. Cramer R.D.I. van den Opdenbosch N. J. Comput. Chem. 1989; 10: 982-1012Crossref Scopus (2832) Google Scholar) option of the Sybyl program (SYBYL) was used to minimize the energy of the resulting model whose stereochemical features were validated with the PROCHECK program (58Laskowski R. MacArthur M. Moss D. Thornton J. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). The Autodock3 program (59Morris G.M. Goodsell D.S. Halliday R.S. Huey R. Hart W.E. Belew R.K. Olson A.J. J. Comput. Chem. 1998; 19: 1639-1662Crossref Scopus (9300) Google Scholar) was used for docking oligosaccharides and peptides in the binding site of modeled IgA I3 Fab. Because the goal was to model the behavior of the O-SP, calculations were performed on the largest possible fragment co