Identification of an IgE-binding epitope of a major buckwheat allergen, BWp16, by SPOTs assay and mimotope screening.
BACKGROUND
The buckwheat 16-kDa protein (BWp16), as reported in our previous study, is a major allergen in buckwheat; however, the IgE-binding epitopes of BWp16 have not as yet been identified.


METHODS
We screened candidates for IgE-binding epitopes on BWp16 by using arrays of overlapping peptides synthesized on activated cellulose membranes (SPOTs membrane). The mimotope method was also used to analyze IgE-binding epitopes of BWp16. Nine single alanine (Ala) mutants of BWp16 expressed in Escherichia coli were used to confirm the epitopes of BWp16. The IgE-binding activity of single Ala mutants of BWp16 was determined by ELISA with mouse anti-BWp16 polyclonal antiserum or ELISA inhibition with sera from buckwheat allergic patients.


RESULTS
The SPOTs assay identified amino acid residues 99-110, i.e. EGVRDLKELPSK, as a candidate for the linear IgE-binding epitope of BWp16. The mimotope method indicated that peptides similar to EGVRDLKE were candidate sequences for epitopes of BWp16. Ala scanning of rBWp16 revealed that all EGVRDLKE peptides containing a single amino acid mutation had weaker IgE-binding activity than rBWp16 WT. An ELISA inhibition assay for rBWp16 WT revealed the inhibitory effect of rBWp16 D103A to be less than that of rBWp16 WT.


CONCLUSIONS
We identified the peptide EGVRDLKE as a very likely candidate for the IgE-binding epitope of BWp16, and Asp103 as the critical amino acid in BWp16. This is the first report on the identification of IgE-binding epitopes of BWp16. Our findings will contribute to the production of BWp16 hypoallergens, and to allergen-specific immunotherapy for buckwheat allergy.
Satoh
Rie
R
Division of Novel Foods and Immunochemistry, National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan.
Koyano
Satoru
S
Takagi
Kayoko
K
Nakamura
Rika
R
Teshima
Reiko
R
D000293:Adolescent; D000328:Adult; D000485:Allergens / Q000032:analysis* / Q000737:chemistry; D000595:Amino Acid Sequence; D002648:Child; D002675:Child, Preschool; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D019612:Fagopyrum / Q000276:immunology*; D005260:Female; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D008297:Male; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019151:Peptide Library
D000485:Allergens; D019151:Peptide Library; D007073:Immunoglobulin E