title: Transglutaminase-mediated cross-linking of a peptic fraction of omega-5 gliadin enhances IgE reactivity in wheat-dependent, exercise-induced anaphylaxis.
abstract: BACKGROUND
  Patients with wheat-dependent, exercise-induced anaphylaxis (WDEIA) experience recurrent anaphylactic reactions when exercising after ingestion of wheat products. We have identified omega-5 gliadin (Tri a 19) as a major allergen in WDEIA, but the role of exercise in eliciting the symptoms remains obscure.
  
  
  OBJECTIVE
  The aim was to examine whether tissue transglutaminase (tTG)-mediated cross-linking could be involved in modulating the IgE-binding ability and in vivo reactivity of digested omega-5 gliadin peptides in WDEIA.
  
  
  METHODS
  Purified omega-5 gliadin was digested with pepsin or with pepsin and trypsin and treated with tTG. The binding of IgE antibodies in pooled sera from 10 patients with WDEIA was studied by means of immunoblotting before and after tTG treatment of the digested peptides. The peptides derived from pepsin digestion were separated by means of gel-filtration chromatography, and IgE reactivity of 4 different peptide fractions was studied by immunoblotting before and after tTG treatment. The fraction showing the greatest degree of cross-linking by tTG was further studied by means of IgE ELISA, ELISA inhibition, and skin prick testing.
  
  
  RESULTS
  The IgE-binding ability of omega-5 gliadin was retained after pepsin and pepsin-trypsin digestion. tTG treatment of the whole peptic digest formed large peptide complexes, with molecular weights ranging from 40 to greater than 200 kd. These cross-linked aggregates bound IgE antibodies in immunoblotting more intensely than untreated, pepsin-digested, or pepsin-trypsin-digested omega-5 gliadin. A gel-filtration fraction of the whole peptic digest corresponding to the highest peak of the chromatogram and showing the greatest degree of tTG-mediated cross-linking showed an increase in serum IgE reactivity in ELISA after tTG treatment, as well as a shift of reactivity to cross-linked complexes. In the 20 patients with WDEIA, the mean skin prick test wheal elicited by this tTG-treated peptic fraction was 77% larger (P <.001) than that elicited by the untreated peptic fraction and 56% larger (P <.01) than that elicited by intact omega-5 gliadin.
  
  
  CONCLUSIONS
  Omega-5 gliadin-derived peptides are cross-linked by tTG, which causes a marked increase in IgE binding both in vitro and in vivo. Activation of tTG during exercise in the intestinal mucosa of patients with WDEIA could lead to the formation of large allergen complexes capable of eliciting anaphylactic reactions.
authors.affiliation: Department of Dermatology, Hospital for Skin and Allergic Diseases, University of Helsinki, Helsinki, Finland.
authors.forename: Kati
authors.initials: K
authors.lastname: Palosuo
authors.forename: Elina
authors.initials: E
authors.lastname: Varjonen
authors.forename: Jenni
authors.initials: J
authors.lastname: Nurkkala
authors.forename: Nisse
authors.initials: N
authors.lastname: Kalkkinen
authors.forename: Rauno
authors.initials: R
authors.lastname: Harvima
authors.forename: Timo
authors.initials: T
authors.lastname: Reunala
authors.forename: Harri
authors.initials: H
authors.lastname: Alenius
mesh_terms: D000328:Adult; D000368:Aged; D000485:Allergens / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D000707:Anaphylaxis / Q000209:etiology / Q000276:immunology*; D052179:Antigens, Plant; D001667:Binding, Competitive; D003432:Cross-Linking Reagents / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D015444:Exercise; D005260:Female; D005512:Food Hypersensitivity / Q000276:immunology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005903:Gliadin / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000276:immunology*; D046911:Macromolecular Substances; D008297:Male; D008875:Middle Aged; D010455:Peptides / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D012882:Skin Tests; D011503:Transglutaminases / Q000378:metabolism*; D014908:Triticum / Q000276:immunology*
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D010455:Peptides; C475180:omega-5 gliadin, wheat; D007073:Immunoglobulin E; D005903:Gliadin; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins