Relationships between IgE/IgG4 Epitopes, Structure and Function in Anisakis simplex Ani s 5, a Member of the SXP/RAL-2 Protein Family
Relationships between IgE/IgG4 Epitopes, Structure and Function in Anisakis simplex Ani s 5, a Member of the SXP/RAL-2 Protein Family
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DOI：
10.1371/journal.pntd.0002735
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发表时间：
2014-03-01
影响因子：
3.8
通讯作者：
Rodriguez-Perez, Rosa
中科院分区：
文献类型：
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作者：
Flor Garcia-Mayoral, Maria;Angel Trevino, Miguel;Rodriguez-Perez, Rosa
背景：异尖线虫病是一种重新出现的全球性疾病，由食用受L3异尖线虫幼虫污染的生鱼或轻度煮熟的鱼引起。这种人畜共患疾病的特征是严重的胃肠道和/或过敏症状，可能被误诊为阑尾炎，胃溃疡或其他食物过敏。异尖线虫过敏原Ani s 5是属于SXP/RAL-2家族的蛋白质;仅在线虫中检测到。以往的研究表明SXP/RAL-2蛋白是活性抗原，但其结构和功能尚不清楚。本研究的目的是阐明Ani s 5的三维结构及其主要的IgE和IgG（4）bindingregions.Methodology/Principal Findings：重组Ani s 5在溶液中的三级结构的解决核磁共振。使用SDS-PAGE通过条带移动测定Mg 2+结合，但不测定Ca 2+结合。用免疫芯片和SPOTs膜分析了9例异尖线虫过敏患者血清中IgE和IgG（4）抗原表位。Ani s 5的三级结构由六个α螺旋（H）组成，具有钙调蛋白样折叠。H3是组织结构的长的中心螺旋，其中H1和H2包装在其N-末端，H4和H5包装在其C-末端。H6的方向是不确定的。关于由IgE和IgG 4免疫球蛋白识别的表位，源自Ani s 5的相同的11种肽被IgE和IgG两者结合（4）。肽14（L40-K59），26（A76-A95）和35（I103-D122）被9份血清中的3份所识别。镁离子结合和钙调蛋白的结构相似性，提示SXP/RAL-2蛋白的一些推定功能。此外，Ani s 5的IgE/IgG（4）结合区被鉴定为位于其表面的片段。这些数据将有助于更好地理解免疫球蛋白和过敏原之间发生的相互作用，反过来，有助于设计新的诊断测试和免疫策略。
Background: Anisakiasis is a re-emerging global disease caused by consumption of raw or lightly cooked fish contaminated with L3 Anisakis larvae. This zoonotic disease is characterized by severe gastrointestinal and/or allergic symptoms which may misdiagnosed as appendicitis, gastric ulcer or other food allergies. The Anisakis allergen Ani s 5 is a protein belonging to the SXP/RAL-2 family; it is detected exclusively in nematodes. Previous studies showed that SXP/RAL-2 proteins are active antigens; however, their structure and function remain unknown. The aim of this study was to elucidate the three-dimensional structure of Ani s 5 and its main IgE and IgG(4) binding regions.Methodology/Principal Findings: The tertiary structure of recombinant Ani s 5 in solution was solved by nuclear magnetic resonance. Mg2+, but not Ca2+, binding was determined by band shift using SDS-PAGE. IgE and IgG(4) epitopes were elucidated by microarray immunoassay and SPOTs membranes using sera from nine Anisakis allergic patients. The tertiary structure of Ani s 5 is composed of six alpha helices (H), with a Calmodulin like fold. H3 is a long, central helix that organizes the structure, with H1 and H2 packing at its N-terminus and H4 and H5 packing at its C-terminus. The orientation of H6 is undefined. Regarding epitopes recognized by IgE and IgG4 immunoglobulins, the same eleven peptides derived from Ani s 5 were bound by both IgE and IgG(4). Peptides 14 (L40-K59), 26 (A76-A95) and 35 (I103-D122) were recognized by three out of nine sera.Conclusions/Significance: This is the first reported 3D structure of an Anisakis allergen. Magnesium ion binding and structural resemblance to Calmodulin, suggest some putative functions for SXP/RAL-2 proteins. Furthermore, the IgE/IgG(4) binding regions of Ani s 5 were identified as segments localized on its surface. These data will contribute towards a better understanding of the interactions that occur between immunoglobulins and allergens and, in turn, facilitate the design of novel diagnostic tests and immunotherapeutic strategies.