Lipopolysaccharide binding of the mite allergen Der f 2
Lipopolysaccharide binding of the mite allergen Der f 2
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DOI：
10.1111/j.1365-2443.2009.01334.x
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发表时间：
2009-09-01
期刊：
影响因子：
2.1
通讯作者：
Hatanaka, Hideki
中科院分区：
文献类型：
--
作者：
Ichikawa, Saori;Takai, Toshiro;Hatanaka, Hideki
脂质结合特性和/或参与宿主防御经常在过敏原蛋白中发现，这意味着这些内在的生物学功能可能有助于过敏原的过敏原性。组2主要螨过敏原，Der f 2和Der p 2，显示与MD-2，Toll样受体（TLR）4信号传导复合物的脂多糖（LPS）结合组分的结构同源性。通过结构研究阐明2型螨变应原的配体结合特性并鉴定相互作用位点对于探索变应原性与生物学功能之间的关系具有重要意义。在这里，我们报告了一种配体捕捞方法，其中His标记的Der f 2与超声处理的稳定同位素标记的大肠杆菌作为潜在的配体源一起孵育，然后通过HisTrap柱和NMR分析分离Der f 2结合的材料。我们发现Der f 2以纳摩尔亲和力与LPS结合，并且使用荧光和凝胶过滤测定，LPS以1：1的摩尔比与Der f 2结合。我们通过NMR微扰研究绘制了Der f 2的LPS结合界面，这表明LPS结合Der f 2的两个大的β-片层之间，类似于其与MD-2的结合，MD-2是先天免疫受体TLR 4的LPS结合组分。
Lipid-binding properties and/or involvement with host defense are often found in allergen proteins, implying that these intrinsic biological functions likely contribute to the allergenicity of allergens. The group 2 major mite allergens, Der f 2 and Der p 2, show structural homology with MD-2, the lipopolysaccharide (LPS)-binding component of the Toll-like receptor (TLR) 4 signalling complex. Elucidation of the ligand-binding properties of group 2 mite allergens and identification of interaction sites by structural studies are important to explore the relationship between allergenicity and biological function. Here, we report a ligand-fishing approach in which His-tagged Der f 2 was incubated with sonicated stable isotope-labelled Escherichia coli as a potential ligand source, followed by isolation of Der f 2-bound material by a HisTrap column and NMR analysis. We found that Der f 2 binds to LPS with a nanomolar affinity and, using fluorescence and gel filtration assays that LPS binds to Der f 2 in a molar ratio of 1 : 1. We mapped the LPS-binding interface of Der f 2 by NMR perturbation studies, which suggested that LPS binds Der f 2 between the two large beta-sheets, similar to its binding to MD-2, the LPS-binding component of the innate immunity receptor TLR4.