Analysis of the structure and allergenicity of recombinant pro- and mature Der p 1 and Der f 1: Major conformational IgE epitopes blocked by prodomains
Analysis of the structure and allergenicity of recombinant pro- and mature Der p 1 and Der f 1: Major conformational IgE epitopes blocked by prodomains
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DOI：
10.1016/j.jaci.2004.11.024
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发表时间：
2005-03-01
影响因子：
14.2
通讯作者：
Ogawa, H
中科院分区：
文献类型：
--
作者：
Takai, T;Kato, T;Ogawa, H
背景资料：主要的屋尘螨1组过敏原Der p 1和Der f 1属于木瓜蛋白酶样半胱氨酸蛋白酶家族，是最有效的室内过敏原。目的：研究重组Der p 1和Der f 1的前体和成熟体与天然Der p 1和Der f 1的致敏性，并获得IgE结合表位的构象信息。在酵母中表达的分泌型pro-Der p 1和pro-Der f 1及其无高糖基化的突变体转化为成熟形式。我们纯化的前体和成熟的形式，并分析其表观分子大小和二级结构的获得过滤和圆二色性分析和过敏性IgE结合，IgE结合抑制，嗜碱性细胞组胺释放的测定。结果：重组成熟蛋白的分子大小、二级结构和致敏性与天然蛋白相似。另一方面，它们的前体表现出不同的二级结构，在所有血清和志愿者测试的过敏性低于成熟形式。分子建模显示，prosegment锚定在prosegment结合环和基板结合裂缝的成熟portion.Conclusions的表面上：我们的研究表明，前结构域的Der p 1和Der f 1降低过敏原性和常见的主要构象IgE表位在广泛的患者人群中存在的2个区域内的prosegments阻止。重组Der p 1和Der f 1以及本研究的结果将为过敏原标准化和更安全有效的过敏原疫苗设计奠定基础。
Background: The major house dust mite group 1 allergens Der p 1 and Der f 1, which belong to the papain-like cysteine protease family, are the most potent of indoor allergens. However, little information is available on the location of IgE epitopes.Objective: We investigated the allergenicities of recombinant proforms and mature forms of Der p 1 and Der f 1 to compare them with natural Der p 1 and Der f 1 and to obtain information on the conformational IgE-binding epitopes.Methods: Secreted pro-Der p 1 and pro-Der f 1 and their mutants without hyperglycosylation expressed in yeast were converted to mature forms. We purified the proforms and mature forms and analyzed their apparent molecular sizes and secondary structures by means of get-filtration and circular dichroism analysis and their allergenicities by means of assays for IgE binding, IgE-binding inhibition, and basophil histamine release. The tertiary structure of pro-Der f 1 was predicted by molecular modeling.Results: The recombinant mature forms exhibited similar molecular sizes, secondary structures, and allergenicities as their natural types. On the other hand, their proforms exhibited different secondary structures and less allergenicities than the mature forms in all sera and volunteers tested. Molecular modeling revealed that the prosegment is anchored at the prosegment-binding loop and the substrate-binding cleft on the surface of the mature portion.Conclusions: Our studies indicate that the prodomains of Der p 1 and Der f 1 reduce allergenicity and that the major conformational IgE epitopes commonly found in a broad population of patients exist within the 2 regions blocked by the prosegments. Recombinant Der p 1 and Der f 1 and the findings in the present study will be the basis for allergen standardization and the design of safer and more effective allergen vaccines.