Molecular cloning, recombinant expression and IgE-binding epitope of omega-5 gliadin, a major allergen in wheat-dependent exercise-induced anaphylaxis.
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.
Matsuo
Hiroaki
H
Department of Dermatology, Shimane University School of Medicine, Izumo, Japan. hmatsuo@med.shimane-u.ac.jp
Kohno
Kunie
K
Morita
Eishin
E
D000485:Allergens / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D000595:Amino Acid Sequence; D000707:Anaphylaxis / Q000209:etiology / Q000235:genetics* / Q000276:immunology*; D052179:Antigens, Plant; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D018744:DNA, Plant / Q000235:genetics; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015444:Exercise; D017343:Genes, Plant; D005903:Gliadin / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014908:Triticum / Q000235:genetics* / Q000276:immunology*; D021182:Wheat Hypersensitivity / Q000150:complications
D000485:Allergens; D052179:Antigens, Plant; D018744:DNA, Plant; D000939:Epitopes; D011994:Recombinant Proteins; C475180:omega-5 gliadin, wheat; D007073:Immunoglobulin E; D005903:Gliadin