Molecular cloning and immunochemical characterization of a new Japanese cedar pollen allergen homologous to aspartyl protease

Background:

Among allergenic pollens, Japanese cedar (Cryptomeria japonica) pollen represents the most important aeroallergens in Japan. It elicits rhinitis and conjunctivitis especially in younger generations. The two-dimensional IgE-binding spectrum of C. japonica pollen allergens demonstrated that many allergens remain to be identified. Here we present the molecular cloning and immunochemical characterization of a novel C. japonica pollen allergen belonging to aspartyl protease family.

Methods:

TOF-MS analysis of a high IgE-binding protein, termed CPA63, revealed its internal amino acid sequences. Based on these sequence information, cDNA-encoding CPA63 was cloned by RACE-PCR. The allergen was produced as a recombinant protein using baculovirus-insect cell culture system, and purified by double chromatographic technique using HisTrap and HiTrap Q columns. Putative mature recombinant CPA63 (r-CPA63) was produced upon autolysis by incubation in acetate buffer (pH 3.3), and used for ELISA experiment. Its proteolytic activity was tested using FITC-casein as a substrate at different pHs, and substrate specificity was evaluated by using series of protease inhibitors.

Results:

CPA63 cDNA encoded a 472 amino acid polypeptide with calculated molecular weight and isoelectric point of 51.1 kDa and 4.69, respectively. Homology search revealed that CPA63 polypeptide sequence showed about 40% identity with plant aspartyl protease/nucleoid DNA binding protein family members. ELISA demonstrated that purified r-CPA63 was recognized by pollinosis patient IgE at a frequency of 58% (18/31). The r-CPA63 also showed an aspartyl protease-like proteolytic activity, demonstrating its enzymatic maturation upon autolysis.

Conclusion:

CPA63 is the first plant aspartyl protease identified as an allergen. That might well open new investigations for other plant aspartyl protease allergens. The availability of CPA63 sequence and recombinant allergen production could be useful to develop future diagnostic technique and therapeutic approaches.