Takaharu Hayashi

Two-Dimensional IgE-Binding Spectrum of Japanese Cedar (Cryptomeria japonica) Pollen Allergens

Background: Japanese cedar (Cryptomeria japonica) pollen is one of the most prevalent sources of the allergens that elicit rhinitis and conjunctivitis. Only Cry j 1 and Cry j 2 have been well characterized as the major allergens of this pollen. Objectives: The aims of this study were to complete the repertoire of C. japonica pollen allergens, to investigate their variability with respect to IgE-reactive patterns and to identify the isoforms of Cry j 1 and Cry j 2 by proteome analysis. Methods: Proteins in C. japonica pollen separated on two-dimensional (2-D) polyacrylamide gel electrophoresis were immunodetected with IgE in sera of 40 subjects allergic to C. japonica pollen. Mass fingerprinting was used to elucidate the diversity of the major allergens. Results: 2-D immunolabeling with individual patients’ sera showed the distinguishable IgE-binding patterns inlaid with 4–87 spots from a total of 131 IgE-binding protein spots. At least 12 Cry j 1 (27.5–75% of IgE-binding frequency) and 3 Cry j 2 (32.5–40%) isoforms were localized. In total, 31 spots were found to be more reactive than the highest IgE-reactive isoform of Cry j 2. Conclusions: The proteomics approaches showed great interindividual variation of IgE-binding patterns to C. japonica proteins and contributed to the repertoire of numerous C. japonica allergens other than Cry j 1 and Cry j 2. Protein microsequencing demonstrated more complicated multiplicity in Cry j 1 than previously known and new isoforms in Cry j 2.

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.

Molecular cloning of a class IV chitinase allergen from Japanese cedar (Cryptomeria japonica) pollen and competitive inhibition of its immunoglobulin E‐binding capacity by latex C‐serum

Background Japanese cedar (Cryptomeria japonica) pollinosis is one of the most prevalent allergic diseases in Japan. Only three C. japonica allergens, Cry j 1, Cry j 2, and CJP‐6, have been characterized. The full IgE‐binding spectrum of C. japonica pollen allergens demonstrates that many allergens remain to be identified.

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. 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.

Decrease in the Allergenicity of Japanese Cedar Pollen Allergen by Treatment with Positive and Negative Cluster Ions

Background: Japanese cedar pollinosis is a severe allergic disease in Japan. The most effective means of decreasing allergic inflammation reactions is still avoidance of the aeroallergen. Recently, a novel air purification system using positively and negatively charged cluster ions was developed to create comfortable living environments. We aimed to assess the ability of existing technology to lower allergenicity of Japanese cedar pollen. Methods: A Japanese cedar pollen extract was nebulized from the top of a cylindrical container with 2 or 4 ion-generating devices. The extract in a mist was passed through the space filled with or without plasma cluster ions for 90 s, and the ion-treated or nontreated extract was then collected in a Petri dish at the bottom of the container. Results: The ion-exposed extract was significantly diminished in its reactivities to anti-Cry j 1 or anti-Cry j 2 antiserum and to human allergic sera IgE on ELISA. SDS-PAGE analysis revealed that ion exposure induced protein degradation in the pollen extract. Similarly, the ion treatment impaired about 80% of the binding to pooled sera IgE from patients allergic to Japanese cedar pollen on ELISA inhibition. Furthermore, intracutaneous and conjunctival reaction tests showed a remarkable diminution in the allergenicity of the ion-irradiated extract. Conclusion: Ion irradiation resulted in a remarkable decrease in in vitro and in vivo allergenicities of atomized Japanese cedar pollen extracts.

A New Lipid Transfer Protein Homolog Identified as an IgE-Binding Antigen from Japanese Cedar Pollen

Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal rhinitis and conjunctivitis in Japan, and an understanding of its full allergen repertoire is prerequisite for the development of future molecular diagnostics and immunotherapeutic strategies. Here we report the identification of a new C. japonica pollen IgE-binding antigen (CJP-8) homologous to lipid transfer proteins (LTPs), a class of plant cross-reactive allergens found in foods, latex, and pollen grains. The cjp-8 cDNA encodes a 165-amino acid polypeptide possessing the conserved eight cysteines characteristic of plant LTP family members. Escherichia coli-expressed recombinant CJP-8 (r-CJP-8) reacted with IgE antibody from Japanese cedar pollinosis patients at a 37.5% frequency (6/16).

Molecular Cloning and Immunochemical Characterization of a New Japanese Cedar Pollen Allergen Homologous to Plant Subtilisin-Like Serine Protease

Protease activities in allergen sources are thought to be involved in triggering allergic inflammation through the disruption of epithelial barrier or the induction of proinflammatory cytokines. Protease allergens may also work as type 2 helper T cell (TH2) adjuvants through the cleavage of cell surface receptors. Here, we report molecular cloning and immunochemical characterization of a new Japanese cedar (Cryptomeria japonica) pollen allergen (CPA9) homologous to serine protease, which is initially found as a high IgE-binding spot on our two-dimensional (2-D) IgE immunoblotting map. The cpa9 cDNA encoded a 757 amino acid polypeptide showing a significant sequence identity with plant subtilisin-like serine protease family members including melon major allergen Cuc m 1. We found that native CPA9 purified from C. japonica pollen showed a high IgE-binding frequency and IgE cross-reactivity with melon extract.

Der f 34, a Novel Major House Dust Mite Allergen Belonging to a Highly Conserved Rid/YjgF/YER057c/UK114 Family of Imine Deaminases

The high prevalence of house dust mite (HDM) allergy is a growing health problem worldwide, and the characterization of clinically important HDM allergens is a prerequisite for the development of diagnostic and therapeutic strategies. Here, we report a novel HDM allergen that belongs structurally to the highly conserved Rid/YjgF/YER057c/UK114 family (Rid family) with imine deaminase activity. Isolated HDM cDNA, named der f 34, encodes 128 amino acids homologous to Rid-like proteins. This new protein belongs to the Rid family and has seven conserved residues involved in enamine/imine deaminase activity. Indeed, we demonstrated that purified Der f 34 had imine deaminase activity that preferentially acted on leucine and methionine. Native Der f 34 showed a high IgE binding frequency as revealed by two-dimensional immunoblotting (62.5%) or ELISA (68%), which was comparable with those of a major HDM allergen Der f 2 (77.5 and 79%, respectively). We also found that Der f 34 showed cross-reactivity with another prominent indoor allergen source, Aspergillus fumigatus. This is the first report showing that the Rid family imine deaminase represents an additional important pan-allergen that is conserved across organisms.

Der f 35: an MD-2−like house dust mite allergen that cross-reacts with Der f 2 and Pso o 2

Dermatophagoides farinae is a source of airborne house dust mite (HDM) allergens. We elucidated IgE‐reactive allergens from D. farinae by two‐dimensional immunoblotting‐based allergenome analysis, and identified one new allergen, named Der f 35, that possesses IgE‐binding capacity comparable to that of Der f 2. The aim of this study was to clarify the allergenic capacity of new HDM allergen Der f 35.

Prominent IgE-binding and cytokine-inducing capacities of a newly cloned N-terminal region of Der f 14, an apolipophorin-like house dust mite allergen

We previously characterized a 177-kDa allergen, M-177, from Dermatophagoides farinae. Thereafter, a counterpart to M-177 for Euroglyphus maynei was cloned as Eur m 14, and its sequence revealed that two environmental allergens, Mag 1 and Mag 3, are digested fragments of M-177. The aims of this study were to clone the cDNA of Der f 14 corresponding to M-177 and to elucidate the allergenic capacities of the N-terminal fragment of Der f 14 (Der f 14-N). Recombinant allergens were produced as trigger-factor-fused proteins in Escherichia coli. Der f 14-N showed the highest IgE-binding frequency among Der f 14-derived fragments in patients allergic to house dust mite by enzyme-linked immunosorbent assay. Der f 14-N showed the highest capacity to induce cell proliferation in murine lymphocyte and human peripheral mononuclear cells among Der f 14-derived fragments. Der f 14-N induced IL-13, IFN-γ and IL-17 production more than Der f 1 and Der f 2 in mouse, and induced IL-5 and IFN-γ production at levels comparable to those of Der f 1 and Der f 2 in some patients. The high prevalence of IgE binding to the Der f 14-N indicates that it could be an important mite allergen.