Molecular cloning and characterization of a birch pollen minor allergen, Bet v 5, belonging to a family of isoflavone reductase-related proteins.
BACKGROUND
Birch pollen is a major cause of pollinosis and is responsible for cross-reactive oral allergies to fruits, nuts, and vegetables. The major allergen, Bet v 1, has been extensively characterized, and 3 minor allergens, Bet v 2, Bet v 3, and Bet v 4, have been cloned and sequenced. Recently, another birch pollen protein with an apparent mass of 35 kd was described as a new IgE-binding protein in birch pollen with cross-reacting homologues in plant foods.


OBJECTIVE
The aim of this study was to determine the primary structure of the 35-kd birch pollen allergen and to investigate its immunologic properties.


METHODS
On the basis of a known complementary DNA fragment, a PCR strategy was applied to obtain the full-length nucleotide sequence of the coding region. The protein was expressed as His-Tag fusion protein in Escherichia coli and purified by Ni-chelate affinity chromatography. Nonfusion protein was obtained by cyanogen bromide treatment of the fusion protein. IgE-binding characteristics and potential allergenicity were investigated by immunoblot, immunoblot inhibition analysis, rat basophil leukemia-cell mediator release assay, and basophil histamine release and compared with those of natural (n) Bet v 5, recombinant (r)Bet v 1, and rBet v 2.


RESULTS
Recombinant Bet v 5 has a mass of 33 kd, an isoelectric point of 9.0, and sequence identity of 60% to 80% to isoflavone reductase homologue proteins from various plants. On immunoblots the recombinant Bet v 5 bound IgE from 9 (32%) of 28 sera from patients allergic to birch pollen with a CAP class of at least 3; Bet v 1 was detected by 89% of these patients. IgE immunoblot and inhibition experiments showed that nBet v 5 and rBet v 5 shared identical epitopes. A rabbit antiserum raised against pea isoflavone reductase and patients' IgE reacted with Bet v 5 and proteins of similar size in several vegetable foods, including exotic fruits. A similar reaction pattern was obtained with 2 Bet v 5-specific mAbs. Furthermore, Bet v 5 triggered a dose-dependent mediator release from rat basophil leukemia 2H3 cells passively sensitized with murine anti-birch pollen IgE and from basophils of a Bet v 5-reactive subject with birch pollen allergy. In contrast, no mediator release could be induced from basophils of a subject who was monosensitized to Bet v 1.


CONCLUSIONS
This 33-kd protein, designated as Bet v 5, is a new minor allergen in birch pollen and may be responsible for pollen-related oral allergy to specific foods in a minority of patients with birch pollen allergy. Amino acid sequence comparison and immunoreactivity to anti-isoflavone reductase serum indicate that Bet v 5 is related to isoflavone reductase, a protein family that is involved in plant defense reactions.
Karamloo
F
F
Department of Allergology, Paul-Ehrlich-Institut, Langen, Germany.
Schmitz
N
N
Scheurer
S
S
Foetisch
K
K
Hoffmann
A
A
Haustein
D
D
Vieths
S
S
D000485:Allergens / Q000145:classification / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D052179:Antigens, Plant; D001483:Base Sequence; D003001:Cloning, Molecular; D003429:Cross Reactions; D018076:DNA, Complementary; D005638:Fruit / Q000276:immunology; D015870:Gene Expression; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000145:classification / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010936:Plant Extracts / Q000276:immunology; D010940:Plant Proteins / Q000145:classification / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D011058:Pollen; D051381:Rats; D017422:Sequence Analysis, DNA; D014197:Trees
D000485:Allergens; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; C401981:Bet v 5 protein, Betula pendula; D018076:DNA, Complementary; D010936:Plant Extracts; D010940:Plant Proteins; D007073:Immunoglobulin E; D010088:Oxidoreductases; C071072:vestitone reductase; D044925:Oxidoreductases Acting on CH-CH Group Donors