Characterization and expression of cross-reacting allergens responsible for pollen-associated food allergies

Hertha Firnberg Position T 70 Characterization of pollen-associated food allergens Karin HOFFMANN- SOMMERGRUBER 29.06.1999 Food allergies represent a health problem of increasing relevance for industrialized countries. Nuts, peanuts, fruits and vegetables are important sources for those allergens. Frequently, pollen allergic patients display symptoms when eating plant food (pollen-associated food allergen). The symptoms comprise local reactions such as swelling and itching of lips and throat (oral allergy syndrome, OAS). After sensibilization against pollen allergens, immunoglobulins are raised which cross-react with homologous structures of plant food. Usually, these cross- reactive allergens are highly conserved proteins even found in distantly related species. One aim of this project is the characterization of cross-reactive allergens in fruits and vegetables. Especially, celery allergens of 40 to 60 kDa are of great importance for defining the "cefery-mugwort-birch-spice-syndrome". There are no data of these proteins available. Individual allergens will be characterized by proteinchemical methods. N-terminal sequences will be determined. Subsequently, allergen-coding sequences will be cloned. The expression of these allergens will be performed in E. coli. Furthermore, we plan to express food allergens in the yeast Pichia pastoris and in transgenic tobacco plants. Allergen-specific recombinant antibodies will be isolated from a phage display library. With the use of purified single chain antibodies and recombinant allergens we will establish immunoassays. These tools will improve the diagnosis of food allergy. Furthermore, specific allergen content in plant food can be determined and may help to set up dietary recommendations.

Pollen-associated food allergies are defined as Type 1 allergic reactions directed against homologous proteins present in tree and grass pollens as well as in various plant foods. The symptoms comprise swelling, itching and reddening of the lips, tongue and soft palate and are defined as "oral allergy syndrome". During sensitisation pollen allergic patients develop specific IgE-antibodies directed against a number of pollen allergens. If these IgE- antibodies react with homologous proteins present in plant foods pollen-associated food allergies can result. The relevant allergens are highly conserved proteins found in even botanically distantly related species. A considerable number of the already identified plant allergens belongs to one of the 14 families of pathogenesis-related proteins, proteins which are induced upon stress, wounding or pathogen attack. In the recent project cross-reactive allergens from vegetables have been identified and characterised. Profilin- homologous proteins have been identified as food allergens from bell pepper and tomato. These proteins have been produced as recombinant proteins in E. coli. Bet v 1, the major birch pollen allergen is a member of family 10 of the pathogenesis-related proteins. Members of this allergen family have been identified from European Chestnut and various isoforms from the homologous protein from celery. From bell pepper the osmotin-like protein, a member of the pathogenesis-related protein family 5, has been isolated and identified as a food allergen. Celery, represents a relevant food allergen source in Central Europe. It is also in part causing the birch-celery-spice syndrome. A new allergen, Api g 5 (two variants with 56 kDa and 58 kDa), has been isolated from celery protein extracts. Partial amino acid sequences display homologies to the group of "berberine-bridge forming enzymes", another protein family involved in reactions upon pathogen attack and stress stimuli. Api g 5 represents the first identified higher molecular weight allergen of the birch-celery-spice syndrome. The N-glycans from Api g 5 are responsible for binding of specific IgE-antibodies. These plant glycans are cross-reactive and are also present on other proteins from celery, birch pollen and mugwort pollen. These N-glycans play an important role in the IgE- dependent cell reactions and the clinical relevance of these carbohydrate structures will be investigated in future experiments. These new identified and characterised pollen associated food allergens may contribute to the improvement of already existing immunoassays and to the development of new assays. In turn these assays represent a valuable tool for optimised diagnosis as well as for the screening of plant foods for their allergen levels.