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

FEBS J. 2005 Sep;272(17):4431-8. doi: 10.1111/j.1742-4658.2005.04858.x.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

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