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 from 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 allergenbound 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.
Twardosz
A
A
Institute of General and Experimental Pathology, AKH, University of Vienna, Austria.
Hayek
B
B
Seiberler
S
S
Vangelista
L
L
Elfman
L
L
Grönlund
H
H
Kraft
D
D
Valenta
R
R
D000485:Allergens / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000888:Antibodies, Anti-Idiotypic / Q000378:metabolism; D052179:Antigens, Plant; D001483:Base Sequence; D001937:Brassica; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D018744:DNA, Plant / Q000737:chemistry; D018985:Epitopes, B-Lymphocyte / Q000032:analysis; D004926:Escherichia coli; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006109:Poaceae; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D014197:Trees
D000485:Allergens; D000888:Antibodies, Anti-Idiotypic; D052179:Antigens, Plant; C108867:BETV4 protein, Betula pendula; D002135:Calcium-Binding Proteins; D018744:DNA, Plant; D018985:Epitopes, B-Lymphocyte; D010940:Plant Proteins; D011994:Recombinant Proteins; C026408:anti-IgE antibodies