MOLECULAR CHARACTERIZATION, EXPRESSION IN ESCHERICHIA-COLI, AND EPITOPE ANALYSIS OF A 2 EF-HAND CALCIUM-BINDING BIRCH POLLEN ALLERGEN, BET-V-4

Birch pollen belongs to the most potent elicitors of Type I allergic r eactions in early spring. Using serum IgE from a birch pollen allergic patient, two cDNA clones (clone 6 and clone 13) were isolated hom a b irch pollen expression cDNA library constructed in phage lambda gt11. Clone 6 encoded a 9.3 kD two EF-hand calcium-binding protein, designat ed Bet v 4, with significant end to end sequence homology to EF-hand c alcium-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 redu ction 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 s ignificantly to the proteins IgE-binding capacity. By IgE-inhibition e xperiments it was demonstrated that recombinant Bet v 4 shared IgE-epi topes with natural Bet v 4 and a homologous timothy grass pollen aller gen. Recombinant Bet v 4 may therefore be considered as a relevant cro ssreactive plant allergen, which may be used for diagnosis and treatme nt of patients suffering from multivalent plant allergies. (C) 1997 Ac ademic Press.