Food and Drug Reactions and Anaphylaxis
Transglutaminase-mediated cross-linking of a peptic fraction of ω-5 gliadin enhances IgE reactivity in wheat-dependent, exercise-induced anaphylaxis,☆☆

https://doi.org/10.1067/mai.2003.1498Get rights and content

Abstract

Background: Patients with wheat-dependent, exercise-induced anaphylaxis (WDEIA) experience recurrent anaphylactic reactions when exercising after ingestion of wheat products. We have identified ω-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 ω-5 gliadin peptides in WDEIA. Methods: Purified ω-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 ω-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 ω-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 ω-5 gliadin. Conclusions: ω-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. (J Allergy Clin Immunol 2003;111:1386-92.)

Section snippets

Patients

Twenty patients with WDEIA (7 female and 13 male patients; mean age, 43 years; range, 25-70 years) took part in the study. All patients had convincing clinical histories of FDEIA after ingestion of wheat products and positive skin prick test reactions to wheat (1:10 wt/vol 0.9% NaCl), gliadin (1 mg/mL in 20% ethanol; crude, Sigma Chemical Co, St Louis, Mo), and purified ω-5 gliadin (50 μg/mL in 20% ethanol-PBS) prepared as previously described.3 ω-5 Gliadin-specific IgE levels ranged from 0.1

tTG treatment of digested ω-5 gliadin forms high-molecular-weight complexes showing strong IgE binding in immunoblotting

In immunoblotting pooled sera from 10 patients with WDEIA showed clear IgE binding to the peptides derived from pepsin digestion of ω-5 gliadin (Fig 1).

. IgE immunoblotting of pooled patient sera (n = 10) to 3 μg of intact, pepsin-digested, and pepsin-trypsin-digested ω-5 gliadin treated with 9, 3, and 1 μg of tTG. Lane A, Intact ω-5 gliadin; lane B, peptic digest; lane C, peptic digest plus tTG (3:1); lane D, peptic digest plus tTG (1:1); lane E, peptic digest plus tTG (1:3); lane F,

Discussion

We show that the IgE-binding ability of ω-5 gliadin, a major allergen in WDEIA, is retained after pepsin and trypsin digestion and that digested peptides are cross-linked by tTG into high-molecular-weight complexes. Cross-linking of a dominant peptic fraction of ω-5 gliadin considerably increases IgE binding both in vitro and in vivo in patients with WDEIA. We hypothesize that tTG activation during physical exercise could create large allergen complexes capable of eliciting anaphylactic

Acknowledgements

We thank Mrs Leena Petman for expert skin prick testing.

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    Supported by grants from the Finnish Cultural Foundation, the Helsinki University Hospital Research Fund, and the Finnish Society of Dermatology.

    ☆☆

    Reprint requests: Kati Palosuo, MD, Laboratory of Immunotoxicology, Finnish Institute of Occupational Health, Topeliuksenkatu 41 b, 00250 Helsinki, Finland.

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