Molecular cloning, recombinant expression and IgE-binding epitope of omega-5 gliadin, a major allergen in wheat-dependent exercise-induced anaphylaxis.

Abstract

Wheat omega-5 gliadin has been identified as a major allergen in wheat-dependent exercise-induced anaphylaxis. We have detected seven IgE-binding epitopes in primary sequence of the protein. We newly identified four additional IgE-binding epitope sequences, QQFHQQQ, QSPEQQQ, YQQYPQQ and QQPPQQ, in three patients with wheat-dependent exercise-induced anaphylaxis in this study. Diagnosis and therapy of food allergy would benefit from the availability of defined recombinant allergens. However, because omega-5 gliadin gene has not been cloned, recombinant protein is currently unavailable. We sought to clone the omega-5 gliadin gene and produce the homogeneous recombinant protein for use in an in vitro diagnostic tool. Using a PCR-based strategy we isolated two full-length omega-5 gliadin genes, designated omega-5 and omega-5b, from wheat genomic DNA and determined the nucleotide sequences. The protein encoded by omega-5a was predicted to be 439 amino acids long with a calculated mass of 53 kDa; the omega-5b gene would encode a 393 amino acid, but it contains two stop codons indicating that omega-5b is pseudogene. The C-terminal half (178 amino acids) of the omega-5a gliadin protein, including all 11 IgE-binding epitope sequences, was expressed in Escherichia coli by means of the pET system and purified using RP-HPLC. Western blot analysis and dot blot inhibition assay of recombinant and native omega-5 gliadin purified from wheat flour demonstrated that recombinant protein had IgE-binding ability. Our results suggest that the recombinant protein can be a useful tool for identifying patients with wheat-dependent exercise-induced anaphylaxis in vitro.

• Authors: Hiroaki Matsuo, Kunie Kohno, Eishin Morita
• Journal: The FEBS journal (FEBS J) Vol. 272 Issue 17 Pg. 4431-8 (Sep 2005) ISSN: 1742-464X [Print] England
• PMID: 16128812 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Allergens
• Antigens, Plant
• DNA, Plant
• Epitopes
• Recombinant Proteins
• omega-5 gliadin, wheat
• Immunoglobulin E
• Gliadin

Topics

• Allergens (chemistry, genetics, immunology, metabolism)
• Amino Acid Sequence
• Anaphylaxis (etiology, genetics, immunology)
• Antigens, Plant
• Base Sequence
• Binding Sites (genetics)
• Cloning, Molecular
• DNA, Plant (genetics)
• Epitopes (chemistry, genetics, metabolism)
• Exercise
• Genes, Plant
• Gliadin (chemistry, genetics, immunology, metabolism)
• Humans
• Immunoglobulin E (metabolism)
• In Vitro Techniques
• Molecular Sequence Data
• Recombinant Proteins (chemistry, genetics, immunology, metabolism)
• Triticum (genetics, immunology)
• Wheat Hypersensitivity (complications)