Identification Of Epitope Fragments And Critical Amino Acid For Allergen TBb In Tartary Buckwheat

Buckwheat is one kind of crop that has high nutritional and medical values. It is rich in mineral, prandial fibre, protein, vitamine, multi-hydroxybenzene compound and other bioactive substances. These substances have shown significant efficiency in preventing and curing the modern civilization diseases of cardiovascular diseases and rheumarthritis etc. However, buckwheat can also cause allergy, which is induced by contacting or eating buckwheat food, and results in clinical symptoms including asthma, dermatitis, eczema and even anaphylactic shock. It is merely the antigens determinants in part of the protein, which resulted in hypersensitivity that can stimulate the creation of antibody, and it is called epitope. As a result, the demand of identifying the allergen molecule and clarifying the mechanism of buckwheat allergy is severely urgent, for development of the accurate diagnosis and safer immunotherapy.Our previous studies have received the full-length tartary buckwheat allergenic protein cDNA sequence named TBt, and it was registered on GenBank in 2006 (Accession No. DQ849083). According to the result of sequence analysis, TBt contains two structural domains, the C-terminal domain (TBa) and the N-terminal domain (TBb). An allergic protein TBb(35kD) as reported in our previous study, is a major allergen in tartary buckwheat; however, the IgE-binding epitope and critical amino acid of TBb have not been identified.According to the result of epitope prediction, eleven epitopes of allergic protein TBb was divided into four epitope fragments and they were constructed using segmental expression method. Four epitope fragments were expressed in Escherichia coli to confirm the epitopes of TBb, respectively. According to the results of sequence analysis and molecular modeling, three mutants (R141A,R139A and D144A) of the epitope fragment (F2) were constructed using site-directed mutagenesis. Following purification by affinity chromatography, immunological assays were performed for determining the IgE-binding activity. After the recombinant proteins were purified, SDS-PAGE analysis showed that the purity was>95%. Results of ELISA and dot blot identified that F2 as an immunodominant epitope fragment of TBb with the greatest binding activity to serum IgE from patients. The sequence analysis and molecular modeling indicated that the predicted epitopes 6 (IFRVREGDV) in F2 contain identical or structurally homologous amino acids. Immunological assays revealed that mutant R141A comparing with R139A and D144A, had the weakest IgE-binding activity to patient sera than wild-type F2.We identified the epitope fragment F2 contain the linear IgE-binding epitope of TBb after sequence analysis and molecular modeling. Using site-directed mutagenesis and immunological assays, we found Argl41 is the critical amino acid while Arg139 and Asp144 might be involved in the allergic activity of TBb. This is the first report on the IgE-binding epitope and critical amino acid of TBb. These findings will contribute to the production of TBb hypoallergens, and to allergen-specific immunotherapy for tartary buckwheat allergy.