Hydrogen Exchange Nuclear Magnetic Resonance Spectroscopy Mapping of Antibody Epitopes on the House Dust Mite Allergen Der p 2
2001; Elsevier BV; Volume: 276; Issue: 12 Linguagem: Inglês
10.1074/jbc.m010812200
ISSN1083-351X
AutoresGeoffrey A. Mueller, Alisa M. Smith, Martin D. Chapman, Gordon S. Rule, David C. Benjamin,
Tópico(s)Toxin Mechanisms and Immunotoxins
ResumoNew strategies for allergen-specific immunotherapy have focused on reducing IgE reactivity of purified recombinant allergens while maintaining T-cell epitopes. Previously, we showed that disrupting the disulfide bonds of the major house dust mite allergen Der p 2 resulted in 10–100-fold less skin test reactivity in mite-allergic subjects but did not change in vitro T-cell proliferative responses. To provide a more complete picture of the antigenic surface of Der p 2, we report here the identification of three epitopes using hydrogen protection nuclear magnetic resonance spectroscopy. The epitopes are defined by monoclonal antibodies that are able to inhibit IgE antibody binding to the allergen. Each monoclonal antibody affected the amide exchange rate of 2–3 continuous residues in different regions of Der p 2. Based on these data, a number of other residues were predicted to belong to each epitope, and this prediction was tested for monoclonal antibody 7A1 by generating alanine point mutants. The results indicate that only a small number of residues within the predicted epitope are functionally important for antibody binding. The molecular definition of these three epitopes will enable us to target limited positions for mutagenesis and to expand our studies of hypoallergenic variants for immunotherapy. New strategies for allergen-specific immunotherapy have focused on reducing IgE reactivity of purified recombinant allergens while maintaining T-cell epitopes. Previously, we showed that disrupting the disulfide bonds of the major house dust mite allergen Der p 2 resulted in 10–100-fold less skin test reactivity in mite-allergic subjects but did not change in vitro T-cell proliferative responses. To provide a more complete picture of the antigenic surface of Der p 2, we report here the identification of three epitopes using hydrogen protection nuclear magnetic resonance spectroscopy. The epitopes are defined by monoclonal antibodies that are able to inhibit IgE antibody binding to the allergen. Each monoclonal antibody affected the amide exchange rate of 2–3 continuous residues in different regions of Der p 2. Based on these data, a number of other residues were predicted to belong to each epitope, and this prediction was tested for monoclonal antibody 7A1 by generating alanine point mutants. The results indicate that only a small number of residues within the predicted epitope are functionally important for antibody binding. The molecular definition of these three epitopes will enable us to target limited positions for mutagenesis and to expand our studies of hypoallergenic variants for immunotherapy. major group 2 allergen from D. pteronyssinus major group 2 allergen from D. farinae antibody monoclonal antibody, rDer p 2, recombinant Der p 2 with first residue altered from Asp to Ser enzyme-linked immunosorbent assay 4-morpholinepropane sulfonic acid, HSQC, heteronuclear single quantum correlation spectroscopy time of evolution in the indirectly detected time domain (15N) time of evolution in the directly detected time domain (1H) nuclear Overhauser effect spectroscopy nuclear Overhauser effect. Epidemiologic studies suggest that between 10 and 20% of the world population exhibits some form of IgE-mediated hypersensitivity, which is manifested as asthma, atopic dermatitis, or allergic rhinitis (1Platts-Mills T.A. Vervloet D. Thomas W.R. Aalberse R.C. Chapman M.D. J. Allergy Clin. Immunol. 1997; 100 (suppl.): 2-24Abstract Full Text Full Text PDF Scopus (651) Google Scholar). A number of studies have shown that sensitivity to house dust mite allergens is the most important risk factor for asthma (1Platts-Mills T.A. Vervloet D. Thomas W.R. Aalberse R.C. Chapman M.D. J. Allergy Clin. Immunol. 1997; 100 (suppl.): 2-24Abstract Full Text Full Text PDF Scopus (651) Google Scholar, 2Sporik R. Chapman M.D. Platts-Mills T.A. Clin. Exp. Allergy. 1992; 22: 897-906Crossref PubMed Scopus (258) Google Scholar). More than 10 mite allergens have been defined, and the 14-kDa Group 2 allergens (Der p 21 and Der f 2) are considered major allergens because of the fact that 80–90% of patients have specific IgE Ab to these allergens (3Heymann P.W. Chapman M.D. Aalberse R.C. Fox J.W. Platts-Mills T.A. J. Allergy Clin. Immunol. 1989; 83: 1055-1067Abstract Full Text PDF PubMed Scopus (253) Google Scholar). Previously we reported that Der p 2 is structurally a member of the immunoglobulin superfamily, although the function of the allergen remains unknown (4Mueller G.A. Benjamin D.C. Rule G.S. Biochemistry. 1998; 37: 12707-12714Crossref PubMed Scopus (82) Google Scholar). Therapy for allergic disease includes allergen avoidance, pharmacotherapy, and allergen-specific immunotherapy. Recently, new strategies for immunotherapy have been proposed with the aim of improving efficacy, patient compliance, and associated risks (5Smith A.M. Chapman M.D. Bousquet J. Yssel H. Immunotherapy in Asthma. Marcel Dekker, Inc., New York1999Google Scholar). Our studies have focused on the generation of hypoallergenic variants; the underlying hypothesis is that reducing IgE reactivity will reduce IgE-mediated side effects (6Smith A.M. Chapman M.D. Mol. Immunol. 1996; 33: 399-405Crossref PubMed Scopus (156) Google Scholar). The mapping of epitopes on Der p 2 and Der f 2 is an important step toward the development of hypoallergenic variants. Using murine mAb and sera from mite-allergic subjects, we have shown that the epitopes on the Group 2 allergens are conformational and that the three disulfide bonds stabilize this structure (6Smith A.M. Chapman M.D. Mol. Immunol. 1996; 33: 399-405Crossref PubMed Scopus (156) Google Scholar, 7Lombardero M. Heymann P.W. Platts-Mills T.A.E. Fox J.W. Chapman M.D. J. Immunol. 1990; 144: 1353-1360PubMed Google Scholar). Mutational analysis of surface residues found that substitution of threonine for lysine at position 100 had reduced avidity for mAb 7A1, and mutations of residues 44–46 affected the avidity of a second mAb, αDpX (8Smith A.M. Chapman M.D. Clin. Exp. Allergy. 1997; 27: 593-599Crossref PubMed Scopus (39) Google Scholar). A third mAb, 6D6, belongs to a group of mAb that recognize the different naturally occurring isoforms of Der p 2 at residue 114 2Smith, A. M. Benjamin, D. C., Hozic, N., Derewenda, U., Smith, W., Thomas, W. R., Gafvelin, G., van Hage-Hamsten M., and Chapman, M. D. (2001) J. Allergy Clin. Immunol., in press.(9Mueller G.A. Smith A.M. Williams D.C. Hakkaart G.A. Aalberse R.C. Chapman M.D. Rule G.S. Benjamin D.C. J. Biol. Chem. 1997; 272: 26893-26898Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 10Haakart G.A.J. Aalberse R.C. Chapman M.D. van Ree R. Clin. Exp. Allergy. 1998; 28: 169-174Crossref PubMed Scopus (36) Google Scholar). Although the above studies have been informative, they provide an incomplete map of the epitopes and do not necessarily identify those residues on Der p 2 that contribute significantly to the binding energy. Epitopes can also be mapped by measuring changes in amide hydrogen exchange rates of the antigen that occur as a result of the formation of an immune complex (11Patterson Y. Englander S.W. Roder H. Science. 1990; 249: 755-759Crossref PubMed Scopus (183) Google Scholar, 12Benjamin D.C. Williams D.C. Smith-Gill S.J. Rule G.S. Biochemistry. 1992; 31: 9539-9545Crossref PubMed Scopus (67) Google Scholar, 13Williams D.C. Benjamin D.C. Poljak R.J. Rule G.S. J. Mol. Biol. 1996; 257: 866-876Crossref PubMed Scopus (63) Google Scholar). These rates can be conveniently measured using NMR techniques. This method was first used by Pattersonet al. (11Patterson Y. Englander S.W. Roder H. Science. 1990; 249: 755-759Crossref PubMed Scopus (183) Google Scholar) to localize the epitope of an anti-cytochromec mAb. We have subsequently measured similar effects in a number of anti-lysozyme antibodies (13Williams D.C. Benjamin D.C. Poljak R.J. Rule G.S. J. Mol. Biol. 1996; 257: 866-876Crossref PubMed Scopus (63) Google Scholar). In general, those residues that are in the structural epitope (those that either contact the Ab or are buried by it) show the largest reduction in exchange rates. Once the location of the epitope has been obtained using amide exchange measurements the residues, which are important for mAb binding, can be systematically identified using scanning alanine mutagenesis (14Jin L. Fendly B.M. Wells J.A. J. Mol. Biol. 1992; 226: 851-865Crossref PubMed Scopus (199) Google Scholar, 15Dall'Acqua W. Goldman E.R. Lin W. Teng C. Tsuchiya D. Li H. Ysern X. Braden B.C. Li Y. Smith-Gill S.J. Mariuzza R.A. Biochemistry. 1998; 37: 7981-7991Crossref PubMed Scopus (146) Google Scholar, 16Benjamin D.C. Perdue S.S. Methods ( Orlando ). 1996; 9: 508-515Crossref PubMed Scopus (63) Google Scholar). The mutation to alanine reduces the side chain to as small as possible without substantially altering the secondary structure. Usually, glycine and proline are not altered, because both of these residues strongly influence the configurational entropy of the peptide chain. This approach has been used to map B-cell epitopes in a number of different model systems. Jin et al. (14Jin L. Fendly B.M. Wells J.A. J. Mol. Biol. 1992; 226: 851-865Crossref PubMed Scopus (199) Google Scholar) studied 43 different mutants of human growth hormone in combination with 21 different monoclonal antibodies. A study by Dall'Acqua et al. (15Dall'Acqua W. Goldman E.R. Lin W. Teng C. Tsuchiya D. Li H. Ysern X. Braden B.C. Li Y. Smith-Gill S.J. Mariuzza R.A. Biochemistry. 1998; 37: 7981-7991Crossref PubMed Scopus (146) Google Scholar) introduced alanine mutations into both the antibody and antigen (lysozyme) and reported the structure of a complex between the antibody and one of the lysozyme mutants. Benjamin and Perdue (16Benjamin D.C. Perdue S.S. Methods ( Orlando ). 1996; 9: 508-515Crossref PubMed Scopus (63) Google Scholar) characterized the interaction of 70 mutants of staphylococcal nuclease with 10 different mAb. All of these studies found that an average of 3 to 4 residues were energetically important to mAb binding. In this study, a residue was classified as being energetically or functionally important if the free energy of binding of the mutant to mAb binding was 1.0 kcal/mol or greater. The studies of growth hormone and staphylococcal nuclease examined virtually the entire surface of the respective proteins, and the study of lysozyme involved residues known to be in contact with Ab from crystallographic studies. In this study, the hydrogen exchange data were used to provide a starting point for a more focal analysis of the epitopes on Der p 2. Recombinant Der p 2 (rDer p 2) was expressed and purified from Escherichia coli cultures as previously described (9Mueller G.A. Smith A.M. Williams D.C. Hakkaart G.A. Aalberse R.C. Chapman M.D. Rule G.S. Benjamin D.C. J. Biol. Chem. 1997; 272: 26893-26898Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Briefly, the protein was recovered from the insoluble fraction of the cell sonicate and resolubilized with 6m guanidine (one-fifth the original cell culture volume), and after dialysis against buffer (10 mm Trizma (Tris base), 1 mm EDTA, pH 8.5), the protein was purified by mAb affinity chromatography. 15N-Labeled protein was obtained by growing the bacteria on a minimal medium with15N-ammonium sulfate as the sole nitrogen source. The murine mAb used in this study were produced at the University of Virginia Lymphocyte Culture Center and have been described in detail elsewhere (17Ovsyannikova I.G. Vailes L.D. Li Y. Heymann P.W. Chapman M.D. J. Allergy Clin. Immunol. 1994; 94: 537-546Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The mAb αDpX was produced by Dr. Rob Aalberse and colleagues (18van der Zee J. Van Swieten P. Jansen H.M. Aalberse R.C. J. Allergy Clin. Immunol. 1988; 81: 884-896Abstract Full Text PDF PubMed Scopus (117) Google Scholar). Radioimmunoassay and ELISA have shown that this panel of 8 mAb defines three antigenic regions on Der p 2 (17Ovsyannikova I.G. Vailes L.D. Li Y. Heymann P.W. Chapman M.D. J. Allergy Clin. Immunol. 1994; 94: 537-546Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), and the mAb 7A1, αDpX, and 6D6 were selected as representative of these regions. For this study, mAb were purified from ascites fluid by precipitation with (NH4)2SO4 followed by affinity chromatography using an rDer p 2 antigen affinity column. Protein concentrations were calculated using extinction coefficients of E0.1% = 1.42 and E0.1% = 0.72 for murine IgG and rDer p 2, respectively. The resins used for the mAb purification and the amide proton exchange rate protection studies (described below) were constructed using Affi-Gel-10 (Bio-Rad Laboratories, Hercules, CA) according to manufacturer protocols. Briefly, 25 mg of protein was coupled per 1 ml of gel bed in a buffer of 0.1 m MOPS, 80 mm NaCl, pH 7.5. Typically greater than 95% of the protein coupled to the matrix. The capacities of the columns were as follows: rDer p 2 column, 27 mg of mAb; mAb 6D6 column, 5 mg of rDer p 2; mAb 7A1 column, 10 mg of rDer p 2; and mAb αDpX column, 13 mg of rDer p 2. Increasing concentrations of mAb were used to inhibit IgE binding to rDer p 2 in a modified enzyme immunoassay. The antigen was bound directly to the microtiter plate or presented by mAb αDpX. Sera were added along with increasing concentrations of mAb so that the final concentration of serum was 1:4 or 1:8, and the concentration of mAb ranged from 0.01 to 100 μg/ml. IgE binding was detected using biotinylated goat anti-human IgE (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and streptavidin horseradish peroxidase (Sigma). The percent inhibition was calculated from theA405 of IgE Ab binding to rDer p 2 in the absence of inhibitor mAb. The positive control experiment used the anti-Der p 2 chimeric mAb 2B12-IgE developed by Schurmann et al. (19Schurmann J. Perdok G.J. Lourens T.E. Parren P. Chapman M.D. Aalberse R.C. J. Allergy Clin. Immunol. 1997; 99: 545-550Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Sera from 4 mite-allergic subjects (radioallergosorbent assay positive to Dermatophagoides pteronyssinusextract), pooled sera from 7 additional subjects, and 1 radioallergosorbent assay negative control subject were tested. The amide exchange kinetics of the mAb·rDer p 2 complex was measured using the method described by Williams et al. (13Williams D.C. Benjamin D.C. Poljak R.J. Rule G.S. J. Mol. Biol. 1996; 257: 866-876Crossref PubMed Scopus (63) Google Scholar). Briefly15N-rDer p 2 was loaded on the mAb column in phosphate-buffered saline (10 mm sodium phosphate, pH 7.4, 100 mm NaCl) and washed with three column-volumes of phosphate-buffered saline. This was followed with three more column-volumes of NMR sample buffer (10 mm sodium phosphate, 200 mm K2SO4, 10 mm NaCl, 1 m EDTA, pH 6.6) made with 99% D2O. Time 0 was considered the time at which the second volume of buffer was added. The columns were left at 4 °C for 48 h and then rinsed with 3 volumes of D2O before elution with 0.2 m acetic acid. The eluted protein was titrated to pH 3.2 using NaOH to quench the amide exchange. The 48-h control sample was made by concentrating and diluting the protein 3 times with the deuterated exchange buffer. The exchange was considered initiated after the first dilution, because the sample was greater than 94% D2O at this point. After a 48-h incubation at 4 °C the sample was titrated to pH 3.2 with HCl to quench the reaction. The protein was then placed into column elution buffer by 3 rounds of concentration and dilution. The control sample for the 0-h time point and the sample used for the assignment of the spectra were in H2O containing 0.2 m acetic acid. All NMR experiments were carried out on a Varian Unity Plus spectrometer operating at a proton frequency of 500 MHz. Amide peak intensities were obtained from two-dimensional HSQC spectra acquired using 256 t1 points and 1024 t2 points and with 64 transients (20Bodenhausen G. Ruben D.G. Chem. Phys. Lett. 1993; 69: 185-189Crossref Scopus (2434) Google Scholar). The three-dimensional HSQC-NOESY spectra (21Marion D. Driscoll P.C. Kay L.E. Wingfield P.T. Bax A. Gronenborn A.M. Clore G.M. Biochemistry. 1989; 28: 6150-6156Crossref PubMed Scopus (935) Google Scholar) were obtained as previously described (9Mueller G.A. Smith A.M. Williams D.C. Hakkaart G.A. Aalberse R.C. Chapman M.D. Rule G.S. Benjamin D.C. J. Biol. Chem. 1997; 272: 26893-26898Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). All spectra were transformed and analyzed using Felix 95.0 (Biosym/Molecular Simulations, San Diego, CA) with standard protocols. Residues potentially within a given epitope were predicted by taking the midpoint between the two protected residues that were furthest apart and determining which residues had atoms that were less than 12.5 Å from this point. A 12.5-Å radius circle would have an area of 490 Å2 whereas a sphere of this radius would have a surface area of 1,960 Å2 (22Davies D.R. Padlan E.A. Annu. Rev. Biochem. 1990; 59: 439-473Crossref PubMed Scopus (693) Google Scholar). Considering that the surface of protein antigen epitopes are convoluted, the area predicted should exceed the 600–900-Å2 surface area of known protein epitopes (22Davies D.R. Padlan E.A. Annu. Rev. Biochem. 1990; 59: 439-473Crossref PubMed Scopus (693) Google Scholar) and thus over-predict the size of the epitope. The accessible surface area was determined using a 9.0-Å probe (23Novotny J. Handschumacher E. Haber E. Bruccoleri R.E. Carlson W.B. Fanning E.W. Smith J.A. Rose G.D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 226-230Crossref PubMed Scopus (268) Google Scholar). Residues with accessible side chains (see Table II) were changed to alanine using the QuickChange kit from Stratagene (La Jolla, CA). Plasmid DNA was isolated using Wizard Plus minipreps (Promega, Madison, WI) and sequenced using automated methods. Because a polymerase chain reaction-based method was used to create the mutations, the entire coding sequence of each mutant was sequenced to ensure that no unintended changes were introduced.Table IIMutants of Der p 2 and the change in ΔΔG of binding mAb 7A1ResidueIC50 NativeIC50 MutantrelKAΔΔGΔGΔGNative/mutantkcal/molHis-302.365.640.420.52 2-aMutants showing ΔΔG greater than 0.50 kcal/mol.Arg-312.3630.000.081.51 2-aMutants showing ΔΔG greater than 0.50 kcal/mol.Lys-332.3610.820.220.93 2-aMutants showing ΔΔG greater than 0.50 kcal/mol.Ser-572.3910.650.230.88 2-aMutants showing ΔΔG greater than 0.50 kcal/mol.Asp-592.632.171.21−0.11Leu-612.632.660.990.01Val-632.753.930.700.21Asn-931.8011.040.161.07 2-bThe mutant N93A failed to interact with the αDpX control mAb.Lys-962.7513.510.200.94 2-aMutants showing ΔΔG greater than 0.50 kcal/mol.Ile-972.6112.790.200.94 2-aMutants showing ΔΔG greater than 0.50 kcal/mol.Lys-1002.472.710.910.06Ser-1012.472.830.870.08Glu-1022.756.430.430.50 2-aMutants showing ΔΔG greater than 0.50 kcal/mol.Asn-1032.472.960.840.11Val-1052.612.421.08−0.05Thr-1232.122.101.01−0.07His-1241.802.390.750.17Lys-1261.802.530.710.20Ile-1272.392.690.890.07Arg-1282.394.290.560.35Asp-1292.614.200.620.282-a Mutants showing ΔΔG greater than 0.50 kcal/mol.2-b The mutant N93A failed to interact with the αDpX control mAb. Open table in a new tab The ability of rDer p 2 and the various alanine mutants to inhibit mAb binding to solid phase rDer p 2 was measured using a goat anti-mouse IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD) conjugated to horseradish peroxidase (9Mueller G.A. Smith A.M. Williams D.C. Hakkaart G.A. Aalberse R.C. Chapman M.D. Rule G.S. Benjamin D.C. J. Biol. Chem. 1997; 272: 26893-26898Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 24Smith A.M. Woodward M.P. Hershey C.W. Hershey E.D. Benjamin D.C. J. Immunol. 1991; 146: 1254-1258PubMed Google Scholar). The results were calculated as the percentage inhibition using the value of maximum inhibition by rDer p 2 as 100% inhibition. The curves were fit to a sigmoidal function using Sigma Plot (SPSS Science, Chicago, IL) to determine the concentration of 50% inhibition (IC50). Relative binding constants were calculated, according to Equation 1, as follows. rel KA=IC50putive /IC50 mutant(Eq. 1) The change in free energy of the binding reaction as a result of the mutation was calculated from Equation 2 (13Williams D.C. Benjamin D.C. Poljak R.J. Rule G.S. J. Mol. Biol. 1996; 257: 866-876Crossref PubMed Scopus (63) Google Scholar),ΔΔG=−RTlnrelKA(Eq. 2) where the standard error in the determination of the ΔΔG is ∼0.1 kcal/mol. Consequently, a ΔΔG greater than 0.5 kcal/mol was considered significant. The mAb used in this study have been characterized by a variety of techniques including inhibition radioimmunoassay and ELISA and have been shown to define three antigenic regions on Der p 2 (17Ovsyannikova I.G. Vailes L.D. Li Y. Heymann P.W. Chapman M.D. J. Allergy Clin. Immunol. 1994; 94: 537-546Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). To demonstrate that these antigenic regions also represent IgE-binding regions, we determined the ability of each mAb to inhibit the binding of IgE from allergic sera. Fig.1, panel A shows the control experiment using the murine-human chimeric IgE mAb that contains the 2B12 variable region. The data in panel A clearly show that inhibition of binding of the 2B12-IgE hybrid occurs only when the inhibiting mAb bind to the same antigenic region as does the 2B12 mAb itself. For example, only mAbs 1D8, 6D6, 4G7, and 2B12, which were previously shown by classical competitive inhibition studies to bind to the same antigenic regions on Der p 2 (17Ovsyannikova I.G. Vailes L.D. Li Y. Heymann P.W. Chapman M.D. J. Allergy Clin. Immunol. 1994; 94: 537-546Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), are capable of inhibiting the binding of the 2B12-IgE chimeric mAb in a dose-dependent manner. In contrast, mAb 7A1 and αDpX, each of which binds to one of two other nonoverlapping regions, do not inhibit. These results are in complete agreement with a more detailed study on the competitive inhibition of these and other mAb for binding to Der p 2 (17Ovsyannikova I.G. Vailes L.D. Li Y. Heymann P.W. Chapman M.D. J. Allergy Clin. Immunol. 1994; 94: 537-546Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Panels B and C show the ability of mAb to competitively inhibit binding of IgE in sera from two house dust mite-allergic subjects, and panel D shows results using pooled sera from 7 additional mite-allergic subjects. Each serum gave unique curves, and the maximum inhibition at 100 μg/ml ranged from 10 to 50% for any given mAb. Thus, the ability of the mAb to inhibit IgE binding to rDer p 2 suggests that these mAb are appropriate markers for IgE epitopes. Fig.2 shows sections of HSQC spectra of rDer p 2 for experiments conducted with mAb 7A1. Peaks for residues 73, 94, 97, and 101 are shown. The panels show spectra acquired in H2O (left), after 48 h in D2O (center), and after 48 h in D2O of a complex of Der p 2 and mAb 7A1 (right). These spectra clearly show that residues 94 and 101 were strongly protected from amide proton exchange while complexed with mAb. In contrast residue 97 was weakly protected, and residue 73 was not protected at all. A similar analysis of spectra obtained for rDer p 2 complexed with mAb αDpX and mAb 6D6 (data not shown) showed that residues 72, 73, and 75 were protected by mAb αDpX, and residues 111 and 116 were protected by mAb 6D6. The protected residues are highlighted on the structure of rDer p 2 in Fig. 3. Also displayed in Fig. 3 are the accessible surfaces of those residues that were predicted to be involved in the full structural epitope. The amino acid residues predicted to be in the epitope for each mAb are listed in Table I.Figure 3Epitopes of mAb 7A1, mAb αDpX, and mAb 6D6. Residues that were found to have altered exchange rates are mapped on the structure of Der p 2. For each epitope those residues that were found to be protected from amide-proton exchange by mAb are colored red. The molecular surface for the residues predicted to be within each epitope is displayed with cyan-colored dots. The residues that form the αDpX epitope reside primarily on the upper β-sheet whereas those that form the 6D6 epitope reside primarily on the bottom β-sheet.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IAmino acid residues of Der p 2 within predicted epitopesmAb 7A1mAb αDpXmAb 6D618–2047–527–1327–3768–8141–5556–6384–9170–7392–106110–11276, 80123–129107–120 Open table in a new tab A number of mutants were constructed to test the contribution to binding of different residues in the predicted epitope for mAb 7A1 (TableII). We selected mAb 7A1, because previous studies had shown that Lys-100 was important for binding (8Smith A.M. Chapman M.D. Clin. Exp. Allergy. 1997; 27: 593-599Crossref PubMed Scopus (39) Google Scholar). Only those amino acids with side chain exposure to solvent were analyzed. For example, although Trp-92 and Val-94 are both within the predicted epitope, they do not show any side chain exposure to solvent. Also systematically excluded from mutagenesis were glycine and proline residues. The binding of mAb 7A1 to these mutants was measured using competitive inhibition ELISA, and the calculated IC50values for these mutants are shown in Table II. A total of 8 residues (amino acids 30, 31, 33, 57, 93, 96, 97, and 102) showed a ΔΔG value greater than 0.5 kcal/mol, strongly suggesting that each of these residues contribute significantly to the binding affinity (Fig.4). The reduction in binding affinity, which occurred as a result of the mutation, was likely to be caused by changes in the direct interaction of the mAb with the altered residue. Alternatively, the mutation may have affected the secondary or tertiary structure of Der p 2. As a control, the alanine mutants were used to inhibit mAb αDpX binding to rDer p 2. The only mutant that did not interact with mAb αDpX in a manner similar to the native protein was N93A (data not shown). Thus the effect of this mutation cannot be definitively attributed to direct interactions of residue Asn-93 with mAb 7A1. Although the control experiments with mAb αDpX could identify gross changes in the structure, it was possible that more subtle changes occurred in the mutant proteins. Consequently, a detailed analysis of the K33A mutant was performed to assess the magnitude of the changes in the structure of Der p 2 due to this mutation. The HSQC spectrum of K33A was very similar to rDer p 2, and a portion of this spectrum is shown in Fig. 5 A. Theboxes overlaid on this section of the spectrum indicate the position of peaks in the HSQC spectrum of rDer p 2. One peak disappeared (at 120 ppm nitrogen and 7.32 ppm proton), this peak corresponded to Lys-33. Two new peaks from Ala-33 appeared that were not in the spectrum of rDer p 2. The presence of two peaks indicated that this residue exchanged slowly between two different conformations. However, the structure of these two conformations must be similar based on the similar pattern of NOEs for Ala-33 shown in Fig. 5 C. In an analysis of the full HSQC spectra, the only other peaks that shifted in the spectrum of K33A were Gly-32 and Val-94. The HSQC spectrum shown in Fig. 5 A suggests that the replacement of Lys-33 by Ala was not totally benign. To show that the changes in chemical shifts were not indicative of a significant structural rearrangement of the K33A protein, a three-dimensional15N1H HSQC-NOESY was acquired to detect changes in interproton distances. A section of this spectrum, corresponding to the amide proton of Ala-33, is shown in Fig. 5 C. For comparison the region of the same spectrum for residue Lys-33 in rDer p 2 is shown in Fig. 5 B. Indicated on these spectra are the NOE assignments from the previous structure determination. The same sequential and long range NOEs were found in the spectra of rDer p 2 and the spectrum of the K33A mutant. The NOEs from residue Ala-33 that are similar to those from Lys-33 are the sequential NOEs to the alpha protons of residues 32 and 34 and the long range peaks to the methyl protons of Val-94. Examining the NOEs from the new amide peak indicated that the differences in the spectra were the lack of intraresidue peaks due to the Lys-33 side chain protons and a new peak that was likely the methyl of Ala-33. These changes would be expected because of replacement of Lys by Ala. An examination of the NOEs from Val-94 and Gly-32 showed similar results; the pattern of NOEs did not change as a result of the K33A mutation (data not shown). These results indicate that there are no large structural changes induced by the K33A mutant; thus, this residue is likely to be in direct contact with mAb 7A1. To date, no data exist describing IgE Ab binding sites for any allergen at the molecular level. The lack of allergen-specific monoclonal IgE Ab precludes epitope mapping using crystallographic analysis of allergen-antibody complexes. The NMR methods used here and in other studies (11Patterson Y. Englander S.W. Roder H. Science. 1990; 249: 755-759Crossref PubMed Scopus (183) Google Scholar, 12Benjamin D.C. Williams D.C. Smith-Gill S.J. Rule G.S. Biochemistry. 1992; 31: 9539-9545Crossref PubMed Scopus (67) Google Scholar, 13Williams D.C. Benjamin D.C. Poljak R.J. Rule G.S. J. Mol. Biol. 1996; 257: 866-876Crossref PubMed Scopus (63) Google Scholar) have clearly shown that it is possible to
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