Abstract
Buckwheat is known to cause immediate-type hypersensitivity reactions including anaphylaxis, which is mediated by specific Immunoglobulin E (IgE) antibodies. First, we analyzed the role of Cys residues in the allergenicity of the 16-kDa protein Fag e 2, one of the major allergens in buckwheat. Mutational analysis of recombinant Fag e 2 (rFag e 2) revealed that 7 out of 10 Cys mutants showed weaker IgE binding to patient's serum than wild-type rFag e 2 (rFag e 2 WT). Mutations of Cys65 and Cys66 in rFag e 2 decreased the pepsin digestibility of the protein, and an ELISA inhibition assay revealed a weaker inhibitory effect of rFag e 2 C65S than that of rFag e 2 WT. These results suggested that the Cys residues, especially Cys65, are involved in the allergenicity of rFag e 2. Then, we screened candidates for the IgE-binding epitopes on Fag e 2 using the SPOTs assay and mimotope method. We identified the peptide EGVRDLKE as a candidate for the IgE-binding epitope of Fag e 2, and Asp103 as a critical amino acid residue for the IgE-binding activity of Fag e 2. Finally, we examined the comprehensive IgE-binding profile of proteins (allergenome) in buckwheat seeds using immunoproteomic techniques. Salt-soluble proteins were extracted from buckwheat seeds. A two-dimensional immunoblot analysis using patients' sera revealed multiple spots containing known and novel IgE-binding proteins. Some spots were newly identified as 13S globulin protein subunits or isoforms. Some spots that were homologous to vicilin-like proteins indicated the presence of newly identified vicilin-like proteins in buckwheat. Our findings pave the way for the development of hypoallergens and application of more accurate diagnostic methods for buckwheat allergy.
