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Immunological characterization and mutational analysis of the recombinant protein BWp16, a major allergen in buckwheat.
- Source :
-
Biological & pharmaceutical bulletin [Biol Pharm Bull] 2008 Jun; Vol. 31 (6), pp. 1079-85. - Publication Year :
- 2008
-
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 >80% 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.
- Subjects :
- Adolescent
Adult
Amino Acid Sequence
Blotting, Western
Child
Child, Preschool
Cysteine genetics
DNA Mutational Analysis
DNA, Complementary biosynthesis
DNA, Complementary genetics
Enzyme-Linked Immunosorbent Assay
Escherichia coli genetics
Female
Humans
Hydrolysis
Immunoglobulin E metabolism
Infant
Male
Molecular Sequence Data
Mutagenesis, Site-Directed
Pepsin A chemistry
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins immunology
Antigens, Plant genetics
Antigens, Plant immunology
Fagopyrum genetics
Fagopyrum immunology
Subjects
Details
- Language :
- English
- ISSN :
- 0918-6158
- Volume :
- 31
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- Biological & pharmaceutical bulletin
- Publication Type :
- Academic Journal
- Accession number :
- 18520034
- Full Text :
- https://doi.org/10.1248/bpb.31.1079