Exhaustive Allergenome Analysis of Japanese Cedar Pollen Allergen Molecules

日本雪松花粉症现在是一种全国性的过敏性疾病，尽管我们对致病因素，即日本雪松花粉(JCP)变应原分子谱系的了解仍然有限。本研究的目的是通过蛋白质组学和分子免疫学相结合的方法，对JCP变应原进行详尽的鉴定和鉴定，我们称之为“等位基因分析”。(1)JCP变应原的二维免疫印迹图谱显示：1)二维图谱上共发现131个变应原斑点，主要变应原Cry j1和Cry j2以多个亚型分布。2)不同花粉症患者的免疫球蛋白E结合谱有很大差异，提示量身定制的分子诊断是有效免疫治疗的关键。(2)新的主要JCP变应原的鉴定和免疫化学特性研究本研究利用飞行时间质谱仪和克隆技术对上述新变应原进行了结构和免疫化学分析。我们首次鉴定了四类几丁质酶同源物(CJP4)是一种新的主要变应原，其与IgE结合的频率为100%，高于Cry j1(71%)。我们还克隆了与β-1，3-葡聚糖酶同源的变应原CPA39和天冬氨酸蛋白酶/核糖核酸结合蛋白家族的CPA63，并利用杆状病毒-昆虫细胞培养系统对重组变应原进行了功能性表达。有趣的是，我们发现CJP4和Cpa39与其他植物如乳胶或橄榄花粉表现出Ig E交叉反应，提示JCP几丁质酶和/或β-1，3-葡聚糖酶可能是参与口腔变态反应综合征发病机制的泛变应原。

Japanese cedar pollinosis is now a nation-wide allergic disease, although our knowledge about the causing factors, i.e. repertoire of Japanese cedar pollen (JCP) allergen molecules, is still limited. Objective of this study is exhaustive identification and characterization of JCP allergens via proteomics in conjunction with molecular immunological strategies, which we call "Allergenome Analysis".(1) Mapping of JCP allergensTwo-dimensional (2D) IgE immunoblotting revealed that; 1) total of 131 allergen spots were found on the 2D map, and major allergens Cry j 1 and Cry j 2 spots distributed as multiple isoforms. 2) IgE-binding profile of each pollinosis patients was quite different from each other, suggesting that tailor-made molecular diagnosis is crucial for effective immunotherapy. 3) 31 allergen spots showed higher IgE binding frequency than that of Cry j 2 (40%).(2) Identification and immunochemical characterization of new major JCP allergensWe next tried structural and immunochemical analyses of above new CJP allergens using TOF-MS analysis and cDNA cloning. We first identified class IV chitinase homologue (CJP4) as a novel major allergen with 100% IgE binding frequency, which is higher than that of Cry j 1 (71%). We also cloned two other allergens homologous to β-1,3-glucanase (CPA39) and aspartyl protease/nucleoid DNA binding protein family (CPA63), and functionally expressed those recombinant allergens using baculovirus-insect cell culture system. Intriguingly, we found that CJP4 and CPA39 showed IgE crossreactivity with other plant species such as latex or olive pollen, suggesting that JCP chitinase and/or β-1,3-glucanase may serve as pan-allergens involved in the pathogenesis of oral allergy syndrome.