Abstract
BackgroundRecently, a protein homologous to glutathione-S-transferases (GST) was detected in prominent amounts in birch pollen by proteomic profiling. As members of the GST family are relevant allergens in mites, cockroach and fungi we investigated the allergenic relevance of GST from birch (bGST).MethodologybGST was expressed in Escherichia coli, purified and characterized by mass spectrometry. Sera from 217 birch pollen-allergic patients were tested for IgE-reactivity to bGST by ELISA. The mediator-releasing activity of bGST was analysed with IgE-loaded rat basophil leukaemia cells (RBL) expressing human FcεRI. BALB/c mice were immunized with bGST or Bet v 1. Antibody and T cell responses to either protein were assessed. IgE-cross-reactivity between bGST with GST from house dust mite, Der p 8, was studied with murine and human sera in ELISA. The release kinetics of bGST and Bet v 1 from birch pollen were assessed in water, simulated lung fluid, 0.9% NaCl and PBS. Eluted proteins were quantified by ELISA and analysed by immunoblotting.Principle findingsOnly 13% of 217 birch pollen-allergic patients showed IgE-reactivity to bGST. In RBL assays bGST induced mediator release. Immunization of mice with bGST induced specific IgE and a Th2-dominated cellular immune response comparably to immunization with Bet v 1. bGST did not cross-react with Der p 8. In contrast to Bet v 1, only low amounts of bGST were released from pollen grains upon incubation in water and the different physiological solutions.Conclusion/SignificanceAlthough bGST is abundant in birch pollen, immunogenic in mice and able to induce mediator release from effector cells passively loaded with specific IgE, it is a minor allergen for birch pollen-allergic patients. We refer this discrepancy to its limited release from hydrated pollen. Hence, bGST is an example demonstrating that allergenicity depends mainly on rapid elution from airborne particles.
Highlights
Pollen from birch (Betula verrucosa) represents an important source of inhalant allergens in Central and Northern Europe, North America, and Japan [1]
To confirm the validity of the cloned sequence, birch pollen extract was separated by SDS-PAGE, the band corresponding to natural GST was excised and in-gel digested with trypsin
Proteomic profiling has revealed that birch pollen (BP) contains substantial amounts of a 27 kDa protein belonging to the GST family [12]
Summary
Pollen from birch (Betula verrucosa) represents an important source of inhalant allergens in Central and Northern Europe, North America, and Japan [1]. The single major allergen Bet v 1 belongs to the family of pathogenesis-related (PR)-10 proteins and displays a characteristic structure termed the Bet v 1-fold [2]. Five additional allergens have been identified in birch pollen (BP) which are all minor allergens. Bet v 3 and Bet v 4 are Ca2+-binding proteins [5,6]. Bet v 6 was identified as an isoflavone reductase-related protein [7] and Bet v 7 was reported to be a member of the cyclophilin family. As members of the GST family are relevant allergens in mites, cockroach and fungi we investigated the allergenic relevance of GST from birch (bGST)
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