Molecular Cloning and Immunochemical Characterization of a Novel Major Japanese Cedar Pollen Allergen Belonging to the Aspartic Protease Family

Abstract

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. Objectives: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.

Integrated Innate Mechanisms Involved in Airway Allergic Inflammation to the Serine Protease Subtilisin

“…Thus, we hypothesize DCs might not be the main cell type that senses subtilisin and triggers type 2 immunity, at least, not in a direct way. Because basophils were implicated in the initiation of type 2 responses to papain (14), we also determined the effect of subtilisin stimulation on bone marrow-derived basophils, generated as previously described (44). Using the 4get reporter system, we did not find significant IL-4 production by murine basophils stimulated with active subtilisin when compared with the positive control stimulated with papain (data not shown).…”

Long-term sublingual immunotherapy for Japanese cedar pollinosis and the levels of IL-17A and complement components 3a and 5a

“…Proteases from Dermatophagoides species (particularly Der p3 and Der f3) have the ability to cleave C3 and C5 into their active fragments in vitro and to promote the generation of C3a/C5a during allergen sensitization [19]. Proteases from Japanese cedar pollen have been identified, and they might activate a complementary reaction [20]. Anaphylatoxins are effective inflammatory mediators targeting a broad spectrum of immune and nonimmune cells.…”

Immunodominance in allergic T-cell reactivity to Japanese cedar in different geographic cohorts

“…Both allergens are highly dominant, eliciting IgE reactivity in > 90% of JC-allergic patients tested 11 . In addition to Cry j 1 and 2, a number of reports have been published on other JC-derived allergenic proteins, including Cry j 3, a theumatin-like protein 12, 13 , Cry j 4, a calcium-biding protein 14 , Cry j isoflavone reductase (IFR), which is related to the minor birch allergen Bet v 6 (62 % sequence identity) 15 , Cry j chitinase, a class IV chitinase 16, 17 , Cry j Asp, an aspartic protease 18 , Cry j LTP, a lipid transfer protein 19 and Cry j CPA9, a serine protease 20 . Despite this relative abundance of allergenic proteins in Japanese cedar, only Cry j 1 and 2 are considered major allergens and as a result have been the focus of more detailed studies.…”