Molecular characterization, expression in Escherichia coli, and epitope analysis of a two EF-hand calcium-binding birch pollen allergen, Bet v 4

Birch pollen belongs to the most potent elicitors of Type I allergic reactions in early spring. Using serum IgE from a birch pollen allergic patient, two cDNA clones (clone 6 and clone 13) were isolated hom a birch pollen expression cDNA library constructed in phage lambda gt11. Clone 6 encoded a 9.3 kD two EF-hand calcium-binding protein, designated Bet v 4, with significant end to end sequence homology to EF-hand calcium-binding allergens from weed and grass pollen. Recombinant Bet v 4, expressed as beta-galactosidase fusion protein, reacted with serum IgE from approximately 20% of pollen allergic individuals. Depletion of allergen-bound calcium by EGTA treatment lead to a substantial reduction of IgE-binding to Bet v 4, indicating that protein-bound calcium is necessary for the maintenance of IgE-epitopes. The greatly reduced IgE-binding capacity of clone 13, a Bet v 4 fragment that lacked the 16 N-terminal amino acids, indicated that the N-terminus contributes significantly to the proteins IgE-binding capacity. By IgE-inhibition experiments it was demonstrated that recombinant Bet v 4 shared IgE-epitopes with natural Bet v 4 and a homologous timothy grass pollen allergen. Recombinant Bet v 4 may therefore be considered as a relevant crossreactive plant allergen, which may be used for diagnosis and treatment of patients suffering from multivalent plant allergies. (C) 1997 Academic Press

Calcium-binding allergens: From plants to man

Calcium-binding proteins contain a variable number of motifs, termed EF-hands, which consist of two perpendicularly placed alpha-helics and an inter-helical loop forming a single calcium-binding site. Due to their ability to bind and transport calcium as well as to interact with a variety of ligands in a calcium-dependent manner, they fulfill important biological functions in eukaryotic cells. After parvalbumin, a three EF-hand fish allergen, calcium-binding allergens were discovered in pollens of trees. grasses and weeds and, recently, as autoallergens in man. Although only a small percentage of atopic individuals displays IgE reactivity to calcium-binding allergens, these allergens may be important because of their ability to cross-sensitize allergic individuals. Confrontation and stability++ as well as IgE recognition of calcium-binding allergens greatly depend on the presence of protein-bound calcium ions. It is thus likely that hypoallergenic derivatives of calcium-binding allergens can be engineered by recombinant DNA technology for immunotherapy++ of sensitized patients

Calcium-binding allergens: From plants to man

Calcium-binding proteins contain a variable number of motifs, termed EF-hands, which consist of two perpendicularly placed alpha-helics and an inter-helical loop forming a single calcium-binding site. Due to their ability to bind and transport calcium as well as to interact with a variety of ligands in a calcium-dependent manner, they fulfill important biological functions in eukaryotic cells. After parvalbumin, a three EF-hand fish allergen, calcium-binding allergens were discovered in pollens of trees. grasses and weeds and, recently, as autoallergens in man. Although only a small percentage of atopic individuals displays IgE reactivity to calcium-binding allergens, these allergens may be important because of their ability to cross-sensitize allergic individuals. Confrontation and stability++ as well as IgE recognition of calcium-binding allergens greatly depend on the presence of protein-bound calcium ions. It is thus likely that hypoallergenic derivatives of calcium-binding allergens can be engineered by recombinant DNA technology for immunotherapy++ of sensitized patients