Immunological and biological properties of Bet v 4, a novel birch pollen allergen with two EF-hand calcium-binding domains.
We have isolated a cDNA clone coding for a birch pollen allergen, Bet v 4. The deduced amino acid sequence of Bet v 4 contained two typical EF-hand calcium-binding domains. Sequence similarities of Bet v 4 to calmodulin are primarily confined to the calcium-binding domains. However, significant sequence similarities extending outside the Ca2+-binding sites were found with a recently described group of pollen-specific allergens of Brassica and Bermuda grass. Both EF-hand domains of Bet v 4 are able to bind Ca2+, as demonstrated by 45Ca2+ blot overlay of wild type and calcium-binding deficient mutants of Bet v 4. Among pollen-allergic patients, protein-bound Ca2+ was not an absolute requirement for IgE recognition of Bet v 4. However, disruption of the carboxyl-terminal Ca2+-binding domain indicated that most IgE antibodies from allergic patients are directed against this site. IgE inhibition experiments suggested that Bet v 4 represents a highly cross-reactive pollen allergen. Pre-absorption of allergic sera with Bet v 4 drastically reduced IgE binding to proteins of similar molecular weight in pollen extracts from distantly related plant species (e.g. timothy grass, mugwort, lily) but not in extracts from plant-derived foodstuff. To test for a possible biological role in pollen germination and tube growth, we introduced recombinant Bet v 4 protein into growing lily pollen tubes by iontophoresis. As a result, cytoplasmic streaming stopped in the vicinity of the electrode tip, and a slight depolarization of the membrane voltage was measured. These effects were not observed with Ca2+-binding deficient mutants of Bet v 4. Thus, Bet v 4 and homologous proteins represent a new class of pollen-specific Ca2+-binding allergens that may have a physiological role as inhibitors of cytoplasmic streaming in outgrowing pollen tubes.
Engel
E
E
Institut für Genetik und Allgemeine Biologie, Austria.
Richter
K
K
Obermeyer
G
G
Briza
P
P
Kungl
A J
AJ
Simon
B
B
Auer
M
M
Ebner
C
C
Rheinberger
H J
HJ
Breitenbach
M
M
Ferreira
F
F
D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D007073:Immunoglobulin E / Q000378:metabolism; D007478:Iontophoresis; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011058:Pollen / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D016415:Sequence Alignment
D000485:Allergens; D052179:Antigens, Plant; C108867:BETV4 protein, Betula pendula; D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D010940:Plant Proteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D002118:Calcium