Identification of an IgE-binding epitope of a major buckwheat allergen, BWp16, by SPOTs assay and mimotope screening.

Abstract

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.

• Authors: Rie Satoh, Satoru Koyano, Kayoko Takagi, Rika Nakamura, Reiko Teshima
• Journal: International archives of allergy and immunology (Int Arch Allergy Immunol) Vol. 153 Issue 2 Pg. 133-40 ( 2010) ISSN: 1423-0097 [Electronic] Switzerland
• PMID: 20407269 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2010 S. Karger AG, Basel.

Chemical References

• Allergens
• Peptide Library
• Immunoglobulin E

Topics

• Adolescent
• Adult
• Allergens (analysis, chemistry)
• Amino Acid Sequence
• Child
• Child, Preschool
• Enzyme-Linked Immunosorbent Assay
• Epitope Mapping
• Fagopyrum (immunology)
• Female
• Humans
• Immunoglobulin E (immunology)
• Male
• Molecular Sequence Data
• Mutagenesis
• Peptide Library