Molecular cloning and immunochemical characterization of a novel major Japanese cedar pollen allergen belonging to the aspartic protease family.
BACKGROUND
Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal pollinosis in Japan. Protease activity in the pollen grains may trigger pro-allergic responses but no such proteases have yet been identified as pollen allergens.


OBJECTIVE
We report the molecular cloning and immunochemical characterization of a novel C. japonica pollen allergen belonging to the aspartic protease family.


METHODS
We focused on the C. japonica pollen allergen spot No. 63 (CPA63, 47.5% IgE binding frequency) on our 2-dimensional IgE immunoblot map. The internal amino acid sequences were determined using time-of-flight mass spectrometry. Full-length cpa63 cDNA was cloned by rapid amplification of cDNA ends (RACE)-PCR. Recombinant CPA63 (r-CPA63) was expressed using the baculovirus-insect cell culture system and its IgE binding capacity was analyzed by enzyme-linked immunosorbent assay (ELISA). The proteolytic activity of r-CPA63 was also assessed using a putative mature enzyme produced upon autolysis.


RESULTS
cpa63 cDNA encoded a 472 amino acid polypeptide showing about 40% sequence identity to members of the plant atypical aspartic protease family. ELISA showed that r-CPA63 was recognized by IgE antibodies in the serum of 58% (18/31) of Japanese cedar pollinosis patients. We also demonstrated an aspartic protease-like enzyme activity of the putative mature r-CPA63.


CONCLUSIONS
We have identified the first plant aspartic protease allergen from Japanese cedar pollen. The availability of the CPA63 sequence and its recombinant allergen production system are useful not only for pharmaceutical applications but also for further examination of the role of protease activity in the pathogenesis of cedar pollinosis.
Department of Molecular Biotechnology, Hiroshima University, Higashi-Hiroshima, Japan.
Ahmed Ragaa Nour
AR
Ibrahim
Seiji
S
Kawamoto
Tsunehiro
T
Aki
Yayoi
Y
Shimada
Satoshi
S
Rikimaru
Nobukazu
N
Onishi
Elfadil Elfadl
EE
Babiker
Isao
I
Oiso
Kunihiko
K
Hashimoto
Takaharu
T
Hayashi
Kazuhisa
K
Ono
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D052179:Antigens, Plant / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D057055:Aspartic Acid Proteases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D055162:Biocatalysis / Q000187:drug effects; D015153:Blotting, Western; D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D032485:Cryptomeria / Q000235:genetics / Q000276:immunology*; D004792:Enzyme Precursors / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology; D008969:Molecular Sequence Data; D010802:Phylogeny; D011058:Pollen / Q000737:chemistry / Q000276:immunology*; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D006255:Rhinitis, Allergic, Seasonal / Q000276:immunology; D017386:Sequence Homology, Amino Acid
D000906:Antibodies; D052179:Antigens, Plant; D004792:Enzyme Precursors; D006454:Hemoglobins; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D057055:Aspartic Acid Proteases; C551281:cpa63 allergen, Cryptomeria japonica