High-Affinity IgE Recognition of a Conformational Epitope of the Major Respiratory Allergen Phl p 2 As Revealed by X-Ray Crystallography This information is current as of September 28, 2021. Sivaraman Padavattan, Sabine Flicker, Tilman Schirmer, Christoph Madritsch, Stefanie Randow, Gerald Reese, Stefan Vieths, Christian Lupinek, Christof Ebner, Rudolf Valenta and Zora Markovic-Housley J Immunol 2009; 182:2141-2151; ; Downloaded from doi: 10.4049/jimmunol.0803018 http://www.jimmunol.org/content/182/4/2141 http://www.jimmunol.org/ References This article cites 62 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/182/4/2141.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology High-Affinity IgE Recognition of a Conformational Epitope of the Major Respiratory Allergen Phlp2AsRevealed by X-Ray Crystallography1 Sivaraman Padavattan,2* Sabine Flicker,2† Tilman Schirmer,* Christoph Madritsch,† Stefanie Randow,‡ Gerald Reese,‡ Stefan Vieths,‡ Christian Lupinek,† Christof Ebner,§ Rudolf Valenta,3,4†¶ and Zora Markovic-Housley3* We report the three-dimensional structure of the complex between the major respiratory grass pollen allergen Phl p 2 and its specific human IgE-derived Fab. The Phl p 2-specific human IgE Fab has been isolated from a combinatorial library constructed from lymphocytes of a pollen allergic patient. When the variable domains of the IgE Fab were grafted onto human IgG1, the resulting Ab (huMab2) inhibited strongly the binding of allergic patients’ IgE to Phlp2aswell as allergen-induced basophil Downloaded from degranulation. Analysis of the binding of the allergen to the Ab by surface plasmon resonance yielded a very low dissociation ؋ ؊10 ␧ ؍ constant (KD 1.1 10 M), which is similar to that between IgE and Fc RI. The structure of the Phl p 2/IgE Fab complex was determined by x-ray crystallography to 1.9 Å resolution revealing a conformational epitope (876 Å2) comprised of the planar surface of the four-stranded anti-parallel ␤-sheet of Phl p 2. The IgE-defined dominant epitope is discontinuous and formed by 21 residues located mostly within the ␤ strands. Of the 21 residues, 9 interact directly with 5 of the 6 CDRs (L1, L3, H1, H2, H3) of the IgE Fab predominantly by hydrogen bonding and van der Waals interactions. Our results indicate that IgE Abs recognize http://www.jimmunol.org/ conformational epitopes with high affinity and provide a structural basis for the highly efficient effector cell activation by allergen/ IgE immune complexes. The Journal of Immunology, 2009, 182: 2141–2151. he hallmark of type I hypersensitivity diseases, e.g., al- series of inflammatory processes through the binding to and cross- lergic asthma, rhinitis, skin inflammation, food allergy, linking of the high-affinity receptor for IgE (Fc␧RI) on mast cells, T anaphylactic shock, which affect more than 25% of the basophils, eosinophils, and professional APCs such as dendritic population, is the formation of IgE Abs against per se harmless cells (4–7). In contrast, IgE-allergen immune complexes can reg- Ags, i.e., allergens (1). IgE Abs represent the least abundant class ulate IgE production and T cell activation via the low-affinity re- by guest on September 28, 2021 of immunoglobulins occurring at approximately 1000-fold lower ceptor for IgE (Fc␧RII, i.e., CD23) (8–11). Allergen recognition concentrations in serum and other body fluids compared with IgG also directly stimulates the IgE production in IgEϩ B cells leading and other Ig classes (2, 3). Nevertheless, minute amounts of im- to increased levels of serum IgE in patients after allergen contact mune complexes consisting of IgE Abs and allergens can trigger a (12, 13). Interestingly, mucosal contact with tiny allergen amounts *Division of Structural Biology, Biozentrum, University of Basel, Basel, Switzerland; strongly boosts allergen-specific IgE Ab production but only †Division of Immunopathology, Department of Pathophysiology, Center for Physi- weakly induces rises of allergen-specific IgG or IgA production ology and Pathophysiology, Medical University of Vienna, Vienna, Austria; ‡Paul Ehrlich Institut, Department of Allergology, Langen, Germany; §Allergie-Ambulato- (13). rium Reumannplatz, Vienna, Austria; ¶Christian Doppler Laboratory for Allergy Re- Moreover, extremely low concentrations (ng/ml) of allergens search, Department of Pathophysiology, Center for Physiology and Pathophysiology, are sufficient to induce rapid and strong inflammatory responses Medical University of Vienna, 1090 Vienna, Austria through degranulation of mast cells and basophils as well as Received for publication September 12, 2008. Accepted for publication November 28, 2008. through the activation of allergen-specific T cells (6, 14, 15). One The costs of publication of this article were defrayed in part by the payment of page prerequisite for this efficient activation of immune cells is a high- charges. This article must therefore be hereby marked advertisement in accordance affinity binding of allergen-IgE immune complexes to Fc␧RI on with 18 U.S.C. Section 1734 solely to indicate this fact. effector cells. In fact, IgE binds primarily with its C␧2 domain with 1 This project was supported by the Swiss National Foundation Grant 31-116804 (to extremely high affinity (K ϳ 10Ϫ9 M) to the ␣-chain of Fc␧RI Z.M.-H.), by Grant 813003 of the Austrian Research Promotion Agency and BIOMAY, D Vienna, Austria to (S.F.), by Grant F1815 of the Austrian Science Fund (to R.V.), by a (16). research grant from BIOMAY, Vienna, Austria, and the Christian Doppler Research Blood and body fluids of allergic patients contain relatively high Association, Vienna, Austria. levels of allergen-specific IgG Abs (17). However, only after a Coordinates and structure factors have been deposited in the Protein Data Bank with more than 100-fold increase of allergen-specific IgG during aller- accession numbers 2vxq and r2vxqsf, respectively. gen-specific immune therapy are these IgG Abs able to compete 2 S.P. and S.F. contributed equally to this study. with allergen-specific IgE Abs and to prevent mast cell degran- 3 R.V. and Z.M.-H. contributed equally to this study. ulation and T cell activation (18). One possibility for the effi- 4 Address correspondence and reprint requests to Prof. Rudolf Valenta, Christian cient recognition of allergen by IgE Abs is that IgE recognizes Doppler Laboratory for Allergy Research, Division of Immunopathology, Department of Pathophysiology, Center for Physiology and Pathophysiology, Vienna General different epitopes on allergens than IgG Abs. Evidence for dif- Hospital, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, ferent epitope recognition comes from epitope mapping studies Austria. E-mail: [email protected] performed with allergen fragments and the analysis of the bind- Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 ing specificities of defined allergen-specific human IgG Abs www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803018 2142 X-RAY CRYSTAL STRUCTURE OF AN ALLERGEN/IgE COMPLEX 0.3nM 40 0.6nM 1.25nM 2.5nM 5.0nM FIGURE 1. Sensor chip-based 10nM studies of the interaction between rPhl 30 20nM p 2 and huMab2. rPhl p 2 was injected at 2-fold increasing concentrations from 0.3 to 20 nM (curves bottom to 20 top) into the flow cell containing im- mobilized huMab2 rPhl p 2-specific RU Ab. Recorded curves (colored) and 10 calculated curves (black), which repre- sent a fitting of the response data to a 1:1 interaction model were superim- 0 posed onto each other. The signal inten- sity (RU) is shown on the y-axis whereas the x-axis displays the time (s). -10 0 500 1000 1500 2000 2500 Time [s] Downloaded from (19, 20). Studies performed with allergen-specific serum IgE vs Determination of the affinity and kinetics of the interaction serum IgG report controversial results regarding possible between rPhl p 2 and huMab2 differences between the binding strength of allergen-specific The surface of a CM5 sensor chip (BIACore AB) was activated by the injec- http://www.jimmunol.org/ IgE and IgG Abs (21, 22). tion of a 1:1 mixture of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Many three-dimensional structures of important allergens have hydrochloride) and NHS at a flow rate of 5 ␮l/min for 7 min. The purified been solved but only few allergen-Ab complexes have been char- huMab2 (c ϭ 10 ␮g/ml) which had been diluted in 10 mM sodium acetate (pH acterized so far (23–26). We have previously isolated human IgE Fabs specific for major respiratory grass pollen allergens from a Table I. Inhibition of patients’ IgE binding to rPhlp2bytherPhl p combinatorial library constructed from lymphocytes of a pollen a allergic patient (27–29). 2-specific antibodies This is the first report of the three-dimensional structure of the ␣ ␣ Phlp5 huMab2 % r Phl p r Phl p % by guest on September 28, 2021 complex between an IgE-derived Fab with a major respiratory al- Patient ab (OD) (OD) Inhibition 5 (OD) 2 (OD) Inhibition lergen, the grass pollen allergen Phl p 2, which is recognized by 1 1.510 0.634 58.0 n.d.