Immunological Characterization and Mutational Analysis of the Recombinant Protein BWp16, a Major Allergen in Buckwheat

Abstract

Buckwheat allergy is one of the most critical diseases manifested by severe and dangerous symptoms in Japan and other countries. We previously isolated the cDNA encoding protein BWp16, a member of the 2S albumin family with a conserved motif of 8 cysteine (Cys) residues. Comparison of the deduced amino acid sequences of BWp16 and related proteins in the 2S albumin family showed similarities between BWp16 and BW 8-kDa from buckwheat, Ara h 6 from peanuts and Ric c 1 from castor bean. Purified recombinant BWp16 (rBWp16) expressed in Escherichia coli was recognized by Ͼ80% of sera from patients with positive for IgE binding to buckwheat. Mutational analysis of rBWp16 revealed that 7 out of 10 mutants in the Cys residues showed weaker IgE binding to patient's serum than wild-type rBWp16 (rBWp16 WT). Mutations of Cys65 and Cys66 in rBWp16 decreased the pepsin digestibility of the protein, and an ELISA inhibition assay revealed a weaker inhibitory effect of rBWp16 C65S than that of rBWp16 WT. These results suggest that the Cys residues, especially Cys65, are involved in the allergenicity of rBWp16. Our findings provide new evidence for the role of Cys residues in 2S albumin family proteins and open the door to the production of hypoallergens and application to safe diagnostic methods and allergen-specific immunotherapy of buckwheat allergy.

Identification of an IgE-Binding Epitope of a Major Buckwheat Allergen, BWp16, by SPOTs Assay and Mimotope Screening

“…We also demonstrated that ELISA using rBWp16 could be employed for in vitro diagnosis of buckwheat allergy [12]. In addition, we showed the involvement of cysteine residues in the allergenicity of BWp16 [12]. In the present study, we have identified a peptide as the linear IgE-binding epitope of BWp16, as well as the amino acid residues critical for IgE binding in BWp16.…”

“…On the other hand, a 16-kDa protein has been reported to be resistant to pepsin digestion and to have IgE-binding activities with sera from patients with immediate hypersensitivity reaction to buckwheat [10]. Our previous studies have shown that the 16-kDa buckwheat seed storage protein (BWp16) is a strong candidate for the major buckwheat allergen involved in the 2S albumin family based on the character of the reaction between BWp16 and the sera of patients positive for IgE-binding activity to buckwheat [11,12]. …”

“…We also demonstrated that ELISA using rBWp16 could be employed for in vitro diagnosis of buckwheat allergy [12]. In addition, we showed the involvement of cysteine residues in the allergenicity of BWp16 [12].…”

Structural property of soybean protein P34 and specific IgE response to recombinant P34 in patients with soybean allergy

“…Recently, recombinant allergens have become available for diagnostic and therapeutic purposes (23,30,31). It is particularly important to evaluate T cell activation for diagnosis and specific immunotherapy using allergens.…”

Epitope mapping and identification on a 3D model built for the tartary buckwheat allergic protein TBb

“…However, buckwheat can also cause allergy [5], and results in clinical symptoms including asthma, dermatitis, eczema, and even anaphylactic shock [6]. Since the first buckwheat-allergic patient was reported in 1909, buckwheat has been recognized as one of most important food allergens manifested by severe and dangerous symptoms induced by ingestion or inhalation of a small amount of the flour or food products [7]. The largest study on buckwheat allergy has been performed among 92,680 school children of Yokohama in the 1990s [8].…”