Molecular cloning and characterization of a new Japanese cedar pollen allergen homologous to plant isoflavone reductase family.
BACKGROUND
Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal pollinosis, and more than 10% of Japanese people suffer from this allergic disorder. However, only two major pollen allergens, Cry j 1 and Cry j 2, have been identified and exclusively characterized.


OBJECTIVE
The aim of this study was to explore and identify important Japanese cedar pollen allergens other than Cry j 1 or Cry j 2.


METHODS
C. japonica cDNA library was immunoscreened by rabbit antiserum raised against a partially purified cedar pollen allergen fraction. An isolated cDNA clone was inserted into a glutathione S-transferase (GST)-tagged Escherichia coli expression vector to obtain recombinant GST fusion protein. Non-fusion recombinant protein was purified by glutathione Sepharose affinity chromatography in conjunction with factor Xa cleavage of the GST moiety. IgE-binding ability of the recombinant protein was then evaluated by western blot analysis and enzyme-linked immunosorbent assay (ELISA).


RESULTS
The cDNA encodes 306 amino acids with significant sequence similarity to those of plant isoflavone reductase-like proteins, which include a recently identified birch pollen allergen Bet v 5. Western blot analysis demonstrated that recombinant protein was recognized by cedar pollinosis patient IgE. In contrast to Bet v 5 being reported as a minor allergen, the recombinant protein exhibited 76% IgE binding frequency (19/25) against pollinosis patients.


CONCLUSIONS
Here we identified the third member of Japanese cedar pollen allergen homologous to isoflavone reductase. Its high IgE-binding frequency implicates that the isoflavone reductase homologue might be an additional major pollen allergen in C. japonica.
Kawamoto
S
S
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima, Japan. skawa@ hiroshima-u.ac.jp
Fujimura
T
T
Nishida
M
M
Tanaka
T
T
Aki
T
T
Masubuchi
M
M
Hayashi
T
T
Suzuki
O
O
Shigeta
S
S
Ono
K
K
D000485:Allergens / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D032485:Cryptomeria / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D007073:Immunoglobulin E / Q000276:immunology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010088:Oxidoreductases / Q000235:genetics* / Q000276:immunology; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011058:Pollen / Q000276:immunology*; D011994:Recombinant Proteins / Q000302:isolation & purification
D000485:Allergens; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D010088:Oxidoreductases; C071072:vestitone reductase; D044925:Oxidoreductases Acting on CH-CH Group Donors