Hydrogen/deuterium exchange memory NMR reveals structural epitopes involved in IgE cross-reactivity of allergenic lipid transfer proteins
2020; Elsevier BV; Volume: 295; Issue: 51 Linguagem: Inglês
10.1074/jbc.ra120.014243
ISSN1083-351X
AutoresMartina Di Muzio, Sabrina Wildner, Sara Huber, Michael Hauser, Eva Vejvar, Werner Auzinger, Christof Regl, Josef Laimer, Danila Zennaro, Nicole Wopfer, Christian G. Huber, Ronald van Ree, Adriano Mari, Peter Lackner, Fátima Ferreira, Mario Schubert, Gabriele Gadermaier,
Tópico(s)Allergic Rhinitis and Sensitization
ResumoIdentification of antibody-binding epitopes is crucial to understand immunological mechanisms. It is of particular interest for allergenic proteins with high cross-reactivity as observed in the lipid transfer protein (LTP) syndrome, which is characterized by severe allergic reactions. Art v 3, a pollen LTP from mugwort, is frequently involved in this cross-reactivity, but no antibody-binding epitopes have been determined so far. To reveal human IgE-binding regions of Art v 3, we produced three murine high-affinity mAbs, which showed 70–90% coverage of the allergenic epitopes from mugwort pollen–allergic patients. As reliable methods to determine structural epitopes with tightly interacting intact antibodies under native conditions are lacking, we developed a straightforward NMR approach termed hydrogen/deuterium exchange memory (HDXMEM). It relies on the slow exchange between the invisible antigen-mAb complex and the free 15N-labeled antigen whose 1H-15N correlations are detected. Due to a memory effect, changes of NH protection during antibody binding are measured. Differences in H/D exchange rates and analyses of mAb reactivity to homologous LTPs revealed three structural epitopes: two partially cross-reactive regions around α-helices 2 and 4 as well as a novel Art v 3–specific epitope at the C terminus. Protein variants with exchanged epitope residues confirmed the antibody-binding sites and revealed strongly reduced IgE reactivity. Using the novel HDXMEM for NMR epitope mapping allowed identification of the first structural epitopes of an allergenic pollen LTP. This knowledge enables improved cross-reactivity prediction for patients suffering from LTP allergy and facilitates design of therapeutics. Identification of antibody-binding epitopes is crucial to understand immunological mechanisms. It is of particular interest for allergenic proteins with high cross-reactivity as observed in the lipid transfer protein (LTP) syndrome, which is characterized by severe allergic reactions. Art v 3, a pollen LTP from mugwort, is frequently involved in this cross-reactivity, but no antibody-binding epitopes have been determined so far. To reveal human IgE-binding regions of Art v 3, we produced three murine high-affinity mAbs, which showed 70–90% coverage of the allergenic epitopes from mugwort pollen–allergic patients. As reliable methods to determine structural epitopes with tightly interacting intact antibodies under native conditions are lacking, we developed a straightforward NMR approach termed hydrogen/deuterium exchange memory (HDXMEM). It relies on the slow exchange between the invisible antigen-mAb complex and the free 15N-labeled antigen whose 1H-15N correlations are detected. Due to a memory effect, changes of NH protection during antibody binding are measured. Differences in H/D exchange rates and analyses of mAb reactivity to homologous LTPs revealed three structural epitopes: two partially cross-reactive regions around α-helices 2 and 4 as well as a novel Art v 3–specific epitope at the C terminus. Protein variants with exchanged epitope residues confirmed the antibody-binding sites and revealed strongly reduced IgE reactivity. Using the novel HDXMEM for NMR epitope mapping allowed identification of the first structural epitopes of an allergenic pollen LTP. This knowledge enables improved cross-reactivity prediction for patients suffering from LTP allergy and facilitates design of therapeutics. Mapping invisible epitopes by NMR spectroscopyJournal of Biological ChemistryVol. 295Issue 51PreviewDefining discontinuous antigenic epitopes remains a substantial challenge, as exemplified by the case of lipid transfer polyproteins, which are common pollen allergens. Hydrogen/deuterium exchange monitored by NMR can be used to map epitopes onto folded protein surfaces, but only if the complex rapidly dissociates. Modifying the standard NMR-exchange measurement to detect substoichiometric complexes overcomes this time scale limitation and provides new insights into recognition of lipid transfer polyprotein by antibodies. Full-Text PDF Open Access Interaction of antibodies with their antigen-binding sites (epitopes) is vital to maintain health but may also contribute to immunological diseases. To understand these interactions, determination of binding specificities and precise epitope localization is a crucial but not trivial task (1Abbott W.M. Damschroder M.M. Lowe D.C. Current approaches to fine mapping of antigen-antibody interactions.Immunology. 2014; 142 (24635566): 526-53510.1111/imm.12284Crossref PubMed Scopus (90) Google Scholar). A reliable technique to investigate binding of an antigen with its respective antibody is X-ray crystallography. This, however, typically requires production and extensive testing of truncated antibody fragments that need to generate high-quality (co-)crystals (2Mirza O. Henriksen A. Ipsen H. Larsen J.N. Wissenbach M. Spangfort M.D. Gajhede M. Dominant epitopes and allergic cross-reactivity: complex formation between a Fab fragment of a monoclonal murine IgG antibody and the major allergen from birch pollen Bet v 1.J. Immunol. 2000; 165 (10861069): 331-33810.4049/jimmunol.165.1.331Crossref PubMed Scopus (181) Google Scholar, 3Padavattan S. Flicker S. Schirmer T. Madritsch C. Randow S. Reese G. Vieths S. Lupinek C. Ebner C. Valenta R. Markovic-Housley Z. High-affinity IgE recognition of a conformational epitope of the major respiratory allergen Phl p 2 as revealed by X-ray crystallography.J. Immunol. 2009; 182 (19201867): 2141-215110.4049/jimmunol.0803018Crossref PubMed Scopus (99) Google Scholar). 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Our recent study revealed that patients' IgE and murine IgG recognize exclusively conformational antibody-binding epitopes (25Wildner S. Griessner I. Stemeseder T. Regl C. Soh W.T. Stock L.G. Völker T. Alessandri C. Mari A. Huber C.G. Stutz H. Brandstetter H. Gadermaier G. Boiling down the cysteine-stabilized LTP fold—loss of structural and immunological integrity of allergenic Art v 3 and Pru p 3 as a consequence of irreversible lanthionine formation.Mol. Immunol. 2019; 116 (31654938): 140-15010.1016/j.molimm.2019.10.012Crossref PubMed Scopus (8) Google Scholar), suggesting a folded protein during allergic sensitization (37Pomés A. Chruszcz M. Gustchina A. Wlodawer A. Interfaces between allergen structure and diagnosis: know your epitopes.Curr. Allergy Asthma Rep. 2015; 15 (25750181): 50610.1007/s11882-014-0506-9Crossref PubMed Scopus (12) Google Scholar, 38Sanchez-Trincado J.L. Gomez-Perosanz M. Reche P.A. Fundamentals and methods for T- and B-cell epitope prediction.J. Immunol. Res. 2017; 2017 (29445754)268016010.1155/2017/2680160Crossref PubMed Scopus (282) Google Scholar). So far, no information on the localization of antibody-binding sites accounting for the IgE (cross)-reactive epitopes of Art v 3 is available. Thus, Art v 3 serves as an excellent representative to study structural epitopes using high-affinity antibodies. For this purpose, we developed a novel NMR-based approach termed hydrogen/deuterium-exchange memory (HDXMEM). This method utilizes the equilibrium of free and mAb-bound protein, which results in reduced H/D exchange rates of antibody-bound regions due to a memory effect. To reveal antigen-antibody interaction at the residue level, murine monoclonal antibodies that covered relevant human IgE-binding epitopes of Art v 3 were used for mapping. Together with reduced antibody reactivity to Art v 3 epitope variants, distinct residues relevant for IgE binding and LTP cross-reactivity were determined. To generate mAbs, mice were immunized with recombinant Art v 3.0201 (for simplicity termed Art v 3). We produced the nontagged allergen using Escherichia coli Rosetta-gami cells enabling disulfide bond formation relevant for the LTP fold (25Wildner S. Griessner I. Stemeseder T. Regl C. Soh W.T. Stock L.G. Völker T. Alessandri C. Mari A. Huber C.G. Stutz H. Brandstetter H. Gadermaier G. Boiling down the cysteine-stabilized LTP fold—loss of structural and immunological integrity of allergenic Art v 3 and Pru p 3 as a consequence of irreversible lanthionine formation.Mol. Immunol. 2019; 116 (31654938): 140-15010.1016/j.molimm.2019.10.012Crossref PubMed Scopus (8) Google Scholar). The protein showed the correct identity and high purity (>98%) as verified by gel electrophoresis and MS (Fig. S1, A and B). Recombinant Art v 3 was subcutaneously administered six times, and murine spleens were harvested after 56 days (Fig. S1C). After generating hybridoma cells, single B cells were obtained by limiting dilution. Three clones with high affinity to Art v 3 were selected, and mAbs were purified using protein G columns (Fig. S1D). We finally obtained 10–12 mg of each Art v 3–specific IgG1 antibody (i.e. mAb I, mAb II, and mAb III). All mAbs efficiently bound the native protein in ELISA, whereas no reactivity was observed when Art v 3 was reduced and alkylated (Fig. 1A). This suggests that the obtained mAbs recognize structural epitopes on the disulfide bond–stabilized structure. To characterize the binding affinity between Art v 3 and the mAbs, we used isothermal titration calorimetry (ITC) and surface acoustic wave (SAW) measurements. Highly comparable binding affinities ranging from 5.5 to 75.6 nm were observed with both techniques (Fig. 2). Recognition by mAb I generated a large enthalpy gain, but the binding had to overcome an entropic barrier (positive −TΔS). Interaction with mAb II again generated a smaller enthalpy gain than entropy gain, resulting in a very similar ΔG and thus KD compared with mAb I. Interaction with mAb III showed a slightly lower affinity and was mainly entropy-driven. These results suggest that despite similar affinities, the antibody-binding mechanisms on the molecular level must be quite different. Interestingly, the SAW data revealed extremely slow binding kinetics (Fig. S2A). Dissociation rate constants (koff) extracted from fitting the decay phases were in the range of 10−5 to 10−4 s−1 for the three mAbs, corresponding to half-lives on the order of several hours (Fig. S2B). To determine whether purified murine mAbs cover relevant human IgE-binding epitopes, we performed ELISAs using sera of 21 mugwort pollen–allergic patients. All sera were positive to Art v 3, whereas IgE binding to homologous LTPs revealed diverse and patient-specific sensitization patterns (Table S1). In ELISA, preincubation with mAb I reduced on average 90% (range 29–98%) of IgE binding. Both, mAb II and mAb III led to a decrease in IgE reactivity of around 70%. The specificity of the assay was verified using an unrelated mAb as control (Fig. 1B). Based on these data, our mAbs qualify as surrogate antibodies for mapping structural IgE-binding regions of Art v 3. To verify whether Art v 3 adopts the typical LTP fold in solution and to subsequently map epitopes on the amino acid level, we used NMR spectroscopy to sequence-specifically assign the backbone chemical shifts. This was achieved using recombinant 13C15N-labeled Art v 3 (Fig. S1, A and B) and standard triple-resonance experiments HNCA, HNCACB, CBCA(CO)NH, HNCO, and HN(CA)CO (39Sattler M. Schleucher J. Griesinger C. Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients.Prog. Nucl. Magn. Reson. Spectrosc. 1999; 34: 93-15810.1016/S0079-6565(98)00025-9Abstract Full Text Full Text PDF Scopus (1386) Google Scholar) at 278 and 298 K. An extensive buffer exchange to phosphate buffer, pH 6.0, was required to obtain a homogeneous protein preparation, as judged from the 15N HSQC spectrum (Fig. S3) providing a protein fingerprint, in which every amino acid is represented by one signal. The chemical shift assignment determined at 298 K was used to predict the secondary structure of Art v 3 in solution (Fig. S4). Localization of α-helices is in agreement with regions determined in the crystal structure (PDB entry 6FRR), thus confirming the correct folding of this Art v 3 preparation in solution. To evaluate whether traditional NMR titration studies are feasible to map binding epitopes, as suggested by Razzera et al. (40Razzera G. Gadermaier G. de Paula V. Almeida M.S. Egger M. Jahn-Schmid B. Almeida F.C. Ferreira F. Valente A.P. Mapping the interactions between a major pollen allergen and human IgE antibodies.Structure. 2010; 18 (20696401): 1011-102110.1016/j.str.2010.05.012Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), a series of 15N HSQC spectra was used to monitor the binding of the individual mAbs to the 15N-labeled allergen. This resulted in a gradual disappearance of Art v 3 signals, indicative of a slow exchange regime and a formed complex that was beyond the size limit of NMR spectroscopy, resulting in the disappearance of all NMR signals. Neither changes in peak positions nor changes in line widths were observed (Fig. S5), which could have been used for epitope mapping. Instead, the signal intensity of all NH signals decreased uniformly for all residues with increasing mAb concentration. In summary, interactions of Art v 3 with mAbs could not be mapped by traditional two-dimensional NMR titrations because no chemical shift perturbations or line broadening effects were observed. To map the functional epitopes of Art v 3 with the three intact mAbs in situ despite the tight binding and extremely slow koff rates, we developed an innovative approach based on H/D exchange. This overcomes the limitations of conventional H/D exchange measurements because such tight interactions between antigens and intact mAbs that are not directly detectable by NMR spectroscopy due to their large size, or rely on a tedious protocol of quenching the exchange and separating the antigen from the mAb for each time point (21Paterson Y. Englander S.W. Roder H. An antibody binding site on cytochrome c defined by hydrogen exchange and two-dimensional NMR.Science. 1990; 249 (1697101): 755-75910.1126/science.1697101Crossref PubMed Scopus (183) Google Scholar). Our novel approach, the HDXMEM, uses the memory of the H/D exchange in the bound, invisible form and the exchange between free and bound allergen. We therefore measured H/D exchange times of the free allergen in t
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