Enlarging the toolbox for allergen epitope definition with an allergen-type model protein.

BACKGROUND: Birch pollen-allergic subjects produce polyclonal cross-reactive IgE antibodies that mediate pollen-associated food allergies. The major allergen Bet v 1 and its homologs in plant foods bind IgE in their native protein conformation. Information on location, number and clinical relevance of IgE epitopes is limited. We addressed the use of an allergen-related protein model to identify amino acids critical for IgE binding of PR-10 allergens. METHOD: Norcoclaurine synthase (NCS) from meadow rue is structurally homologous to Bet v 1 but does not bind Bet v 1-reactive IgE. NCS was used as the template for epitope grafting. NCS variants were tested with sera from 70 birch pollen allergic subjects and with monoclonal antibody BV16 reported to compete with IgE binding to Bet v 1. RESULTS: We generated an NCS variant (Δ29NCSN57/I58E/D60N/V63P/D68K) harboring an IgE epitope of Bet v 1. Bet v 1-type protein folding of the NCS variant was evaluated by 1H-15N-HSQC NMR spectroscopy. BV16 bound the NCS variant and 71% (50/70 sera) of our study population showed significant IgE binding. We observed IgE and BV16 cross-reactivity to the epitope presented by the NCS variant in a subgroup of Bet v 1-related allergens. Moreover BV16 blocked IgE binding to the NCS variant. Antibody cross-reactivity depended on a defined orientation of amino acids within the Bet v 1-type conformation. CONCLUSION: Our system allows the evaluation of patient-specific epitope profiles and will facilitate both the identification of clinically relevant epitopes as biomarkers and the monitoring of therapeutic outcomes to improve diagnosis, prognosis, and therapy of allergies caused by PR-10 proteins.

Serum testing of genetically modified soybeans with special emphasis on potential allergenicity of the heterologous protein CP4 EPSPS.

Roundup Ready soy contains the CP4-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) protein. Serum IgE from two distinct populations of soy-allergic patients were recruited to determine their IgE-binding specificity. One population consisted of 10 adult patients from Europe, whose primary diagnosis was soy food allergy with some also having mite allergy. In addition, 6 primarily mite-allergic, 6 food-allergic (celery, carrot, milk, shrimp, walnut, and apple), and 5 non-allergic patients were tested. Another population consisted of 13 children from Korea, whose primary diagnosis was atopic dermatitis and secondarily soy and egg sensitization. In addition, 11 non-allergic patients were tested. Each patient population was extensively characterized with respect to clinical symptoms, specific IgE (CAP) scores, and total IgE. Immunoblots and ELISA assays were developed using serum IgE from these patients and soy extracts, CP4 EPSPS, rice extract, ovalbumin, rubisco, purified major peanut allergen Ara h 2, the putative soy allergen Gly m Bd 30k and mite allergen Der f 2 proteins as the intended targets. Immunoblot results indicated that soy-allergic patients bound soy extracts but did not specifically bind rubisco or CP4 EPSPS. ELISA results were in general agreement with the immunoblot results except that rubisco bound significant quantities of serum IgE from some patients. These results indicate that the CP4 EPSPS protein does not bind significant quantities of IgE from two geographically distinct sensitive populations and there is no evidence for an increased allergenic potential of this biotech protein.

Enantioselective analysis of monoterpenes in different grape varieties during berry ripening using stir bar sorptive extraction- and solid phase extraction-enantioselective-multidimensional gas chromatography-mass spectrometry.

The stereoisomeric ratios of various genuine metabolites of linalool (furanoid and pyranoid linalool oxides, hotrienol) and citronellol (cis- and trans-rose oxide) were determined in grape berries by means of enantioselective-multidimensional gas chromatography-mass spectrometry. Stereoisomers of the metabolites could be separated on a chiral column with a modified cyclodextrin as stationary phase. The detailed stereoselective analysis of the furanoid and pyranoid linalool oxides in the cv. Morio-Muskat during berry ripening is giving evidence that furanoid linalool oxides are generated via two different reaction pathways. Additionally, stereoselective analysis of rose oxide in different varieties that have attained commercial maturity has been performed demonstrating that cis-(2S,4R)-rose oxide is the main stereoisomer in all varieties. (3S)-Hotrienol was the main stereoisomer in all varieties with enantiomeric purities that were always higher than 90%.

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.

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.