Molecular basis of pollen-related food allergy: identification of a second cross-reactive IgE epitope on Pru av 1, the major cherry ( Prunus avium ) allergen

Division of Allergology, Paul-Ehrlich-Institut, Paul-Ehrlich-Strasse 51-59, 63225 Langen, Germany.
Biochemical Journal (Impact Factor: 4.4). 02/2005; 385(Pt 1):319-27. DOI: 10.1042/BJ20040842
Source: PubMed


Birch (Betula verrucosa) pollen-associated food allergy is a well-characterized syndrome, which is due to the cross-reactivity of IgE antibodies to homologous allergens in various foods. One crossreacting area on the major birch pollen allergen Bet v 1 and its homologue in cherry (Prunus avium) Pru av 1 has already been identified. This is the so-called 'P-loop' region, which encompasses amino acid residues around position 45 and is found on the two virtually identical tertiary protein structures. We tried to determine an additional IgE cross-reacting patch on Pru av 1 and Bet v 1. The putative IgE-binding region on Pru av 1 was localized with a mAb (monoclonal antibody) that was generated against Bet v 1, and cross-reacts with several Bet v 1 homologues in food and inhibits the binding of patients' IgE to Pru av 1. mAb reactivity pattern was analysed and amino acid positions 28 and 108 of Pru av 1 were selected and mutated by site-directed mutagenesis. The Pru av 1 mutants were produced as recombinant proteins and characterized for their folding, mAb- and IgE-binding capacity and allergenic potency with a cellular assay using the humanized rat basophilic leukaemia cell line RBL-25/30. Amino acid position 28 is involved in a second major IgE-binding region on Pru av 1 and probably on Bet v 1. The identification of this second major IgE-binding region is an essential prerequisite to understand the phenomenon of cross-reactivity and its clinical consequences, and to produce hypoallergenic proteins for an improved immunotherapy of type I allergy.

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Available from: Andreas Hoffmann, Oct 04, 2015
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    • "Guanidine treatment also caused a shift in the CD spectrum, consistent with a relative decrease in α-helical structure and gain of random structure. Together these findings suggest that αhelices are likely candidates for one or more conformational epitopes in Jun a 1. IgE epitopes in other allergens have been found in α-helices (Mine et al., 2003; Rafnar et al., 1998; Vrtala et al., 1999; Wiche et al., 2005). When Jun a 1 was reduced and alkylated in the presence of guanidine, there was no further decrease in IgE binding, suggesting that neither disulfide bond nor the free sulfhydryl group are required for any conformational epitopes in Jun a 1. "
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    ABSTRACT: We have previously identified four linear IgE epitopes on Jun a 1, the dominant allergen in mountain cedar pollen, and mapped these to the surfaces of a molecular model and to the crystal structure of this glycoprotein. The aim of the present study was to determine if Jun a 1 also displays conformational IgE epitopes. Jun a 1 was denatured by heating at 75 degrees C for 1h, exposure to 6M guanidine or by reductive alkylation in the presence and absence of guanidine. The effects of these manipulations on the binding to IgE from patients with mountain cedar hypersensitivity was evaluated by an ImmunoCAP inhibition assay, using Jun a 1-specific caps. Treatment-associated changes in the 3D-structure were assessed by dynamic light scattering and CD spectroscopy. IgE binding to native Jun a 1 was inhibited 92+/-9% by soluble native protein and 92+/-9% by reduced and alkylated Jun a 1. However, the capacity of Jun a 1 to inhibit the binding of IgE antibodies was significantly diminished upon denaturation by heat, guanidine alone, or reduction and alkylation in guanidine, compared to native Jun a 1. Reductive alkylation treatment under denaturing conditions also increased the Stoke's radius, suggesting that the protein was partially unfolded. Analysis of the circular dichroism (CD) spectra suggested that heating and treatment with guanidine caused a loss of alpha-helical structure. Guanidine also caused an increase in random coil structure. Thus, at least a portion of the anti-Jun a 1 IgE antibodies produced by allergic humans recognize conformational epitopes and it is likely that some of these epitopes reside in alpha-helical structures of Jun a 1.
    Molecular Immunology 05/2007; 44(10):2781-5. DOI:10.1016/j.molimm.2005.12.001 · 2.97 Impact Factor
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    • "Due to the polyclonal nature of the IgE response, it is difficult to analyse directly the structure of immune complexes between IgE and allergens. Therefore, most studies on IgE epitopes of Bet v 1 related allergens have focused on site-directed mutagenesis (Ferreira et al., 1998; Scheurer et al., 1999; Neudecker et al., 2003; Wiche et al., 2005; Ma et al., 2006) or, more recently on direct affinity selection and enrichment of IgE binding surface structures utilising libraries of peptide mimics (Mittag et al., 2006). In the present study we focused on the " P-loop " which has been suggested as a major epitope area on Bet v 1 (Mirza et al., 2000) and homologous food allergens (Holm et al., 2001; Neudecker et al., 2003; Mittag et al., 2006). "
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    ABSTRACT: For better understanding the cross-reactivity between the major birch pollen and celery allergens, Bet v 1 and Api g 1, respectively, putative epitope areas and structurally important positions for IgE-binding of the isoforms Api g 1.01 and Api g 1.02 were point mutated. The IgE binding capacities were measured in ELISA, the IgE cross-reactivity between the isoforms, mutants and Bet v 1 investigated by ELISA-inhibition experiments with serum pools from patients with confirmed celery allergy (DBPCFC). Api g 1.01 displayed a clearly higher frequency and capacity of IgE binding than Api g 1.02. In Api g 1.01, substitution of lysine against glutamic acid at amino acid position 44, a key residue of the Bet v 1 "P-loop", increased the IgE-binding properties. Structural instability due to proline insertion at position 111/112 resulted in loss of IgE binding of Api g 1.01, but not of Api g 1.02. Between Api g 1.01 and Api g 1.02 only partial cross-reactivity was seen. The data suggest that the IgE epitopes of the two isoforms are distinct and that in contrast to Api g 1.01, the "P-loop" region plays an important role for IgE binding of celery allergic subjects to Api g 1.02. Understanding and investigation of the molecular mechanisms in celery allergy is an important step to generate hypoallergenic proteins for safe and efficacious immunotherapy of food allergy.
    Molecular Immunology 05/2007; 44(10):2518-27. DOI:10.1016/j.molimm.2006.12.023 · 2.97 Impact Factor
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    ABSTRACT: Hypersensitivity reactions can be differentiated into IgE- and non-IgE-mediated allergic and also non- allergic reactions. In this article we explore currently available tests used to distinguish non-IgE condi- tions. Testing involves not only estimation of the dif- ferent antibody types but also cellular activation and inflammatory markers.
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