Volume 4 Supplement 2

5th International Symposium on Molecular Allergology (ISMA 2013)

Open Access

Find the match! A tool for residue-specific analysis of epitopes in Bet v 1-like allergens

  • Dirk Schiller1,
  • Hanna Berkner2,
  • Maximilian Hartl2,
  • Christian Seutter von Loetzen2,
  • Michaela Gubesch1,
  • Felix Husslik1,
  • Daniela Weigand1,
  • Iris Lauer1,
  • Andreas Reuter1,
  • Jonas Lidholm3,
  • Barbara Ballmer-Weber4,
  • Stefan Vieths1,
  • Paul Rösch2 and
  • Dirk Schiller1
Clinical and Translational Allergy20144(Suppl 2):P9

DOI: 10.1186/2045-7022-4-S2-P9

Published: 17 March 2014

Background

Birch pollen-allergic subjects often develop Bet v 1-specific IgE that cross-reacts with homologous food allergens. Bet v 1 and its homologs in pollen and food display exclusively conformational epitopes. We established a system to specifically analyze epitope cross-reactivity of Bet v 1-related allergens. The enzyme norcoclaurine synthase (NCS) from Thalictrum flavum is structurally homologous to Bet v 1 but does not bind IgE reacting with Bet v 1-like allergens. By substituting amino acids in a variant of NCS, the impact of individual residues of Bet v 1-like allergens in IgE binding can be studied.

Methods

Residues potentially involved in IgE binding of Bet v 1 were inserted into NCS. NCS variants were purified and protein secondary and tertiary structures were evaluated by CD and NMR spectroscopy. Sera of birch pollen allergic patients were analyzed for IgE binding to NCS variants and Bet v 1-like food allergens. Cross-reactivity of patients' IgE was assessed by competitive binding assays using variants of NCS and Bet v 1 as well as Cor a 1, Dau c 1, Api g 1, Pru av 1, and Gly m 4, respectively.

Results

CD spectra and 1H15N-HSQC NMR indicated Bet v 1-like structure of NCS variants. sIgE binding increased significantly with the number of amino acids substituted in NCS from ≤ 0.35 kUA/L in 94% (65/69) of sera to CAP values up to 17.4 kUA/L for NCSN57/I58E/D60N/V63P/D68K (NCS_5x) in 68% (47/69) of sera. IgE interaction of NCS_5x could be competitively inhibited with Bet v 1, Cor a 1, and Gly m 4, whereas strongly reduced inhibition was observed with Bet v 1N43A/E45S/N47A/K55A, Api g 1, Pru av 1, and Dau c 1, respectively. Structural analysis of the IgE-binding site of NCS_5x showed high conformational similarity with Bet v 1, Cor a 1 and Gly m 4 and low similarity with Pru av 1, Api g 1, and Dau c 1, respectively. Murine monoclonal antibody BV16 abolished Bet v 1-specific inhibition of IgE binding to NCS_5x indicating an overlap between an IgE and IgG epitope.

Conclusions

Five residues critical for IgE cross-reactivity in a subgroup of Bet v 1-like allergens were identified. NCS_5x bound IgE that cross-reacted only with Bet v 1-related allergens sharing an epitope structure similar to that of NCS_5x. Thus non-allergenic NCS with its Bet v 1-homologous structure is a powerful system for the residue-specific analysis of epitopes of Bet v 1-related allergens.

Authors’ Affiliations

(1)
Paul-Ehrlich-Institut, Divison of Allergology
(2)
Department of Biopolymers, University of Bayreuth
(3)
Thermo Fisher Scientific, ImmunoDiagnostics Division
(4)
University Hospital, Allergy Unit

Copyright

© Schiller et al; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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