Structural characterization and IgE epitope analysis of arginine kinase from Scylla paramamosain.
Structural characterization and IgE epitope analysis of arginine kinase from Scylla paramamosain.
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DOI：
10.1016/j.molimm.2013.04.016
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发表时间：
2013-12
影响因子：
3.6
通讯作者：
Hai-Yan Mao;M. Cao;S. Maleki;Qiu-Feng Cai;W. Su;Yang Yang-Yang;Guangming Liu
中科院分区：
文献类型：
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作者：
Hai-Yan Mao;M. Cao;S. Maleki;Qiu-Feng Cai;W. Su;Yang Yang-Yang;Guangming Liu
精氨酸激酶（AK）是贝类的泛过敏原。然而，关于其IgE表位和结构特征的信息有限。本研究对拟穴青蟹（Scylla paramosain）AK进行了分离纯化和性质研究。纯化的AK是一种分子量为40 kDa的糖蛋白，它与其他贝类中存在的相关过敏原具有交叉反应性。S.克隆了paramamosainAK基因，编码357个氨基酸残基。通过生物信息学分析，预测了9个线性表位和7个构象表位。此外，在大肠杆菌BL 21（DE 3）中成功表达了完整的重组AK（rAK）和3个部分重组AK（rAK 1、rAK 2和rAK 3）。rAK 1、rAK 2和rAK蛋白与蟹过敏患者混合血清的IgE反应性较强，而rAK 3的IgE反应性较弱，说明AK的IgE表位主要分布在rAK 1和rAK 2区域。此外，使用合成的重叠肽，在rAK 1和rAK 2的区域中发现了三个实验线性表位（表位1：AA 127-141，表位2：AA 141-155和表位3：AA 211-225）。将实验线性表位定位到AK的蛋白质同源模型上。对9例蟹过敏患者血清的IgE结合试验中，只有3例血清与变性的线性AK发生反应，而8例血清与天然的折叠AK发生反应，表明S. paramamosainAK可能更占优势。
Arginine kinase (AK) is reported to be the pan-allergen of shellfish. However, there is limited information on its IgE epitopes and structural characteristics. In this study, AK fromScylla paramamosainwas purified and characterized. The purified AK is a glycoprotein with the molecular weight of 40 kDa and it demonstrates cross-reactivity with the related allergens present in other shellfish. The cDNA ofS. paramamosainAK was cloned, which encodes 357 amino acid residues. Nine linear epitopes and seven conformational epitopes were predicted following bioinformatics analysis. In addition, the entire recombinant AK (rAK) and three partial recombinant AKs (rAK1, rAK2, and rAK3) were successfully expressed inEscherichia coliBL21 (DE3). The proteins of rAK1, rAK2 and rAK have strong IgE reactivity with the pooled sera from crab allergic patients, while rAK3 has significantly weaker IgE reactivity, which indicates that the IgE epitopes of AK are mainly distributed in the regions of rAK1 and rAK2. Furthermore, three experimental linear epitopes (epitope 1: AA 127–141, epitope 2: AA 141–155, and epitope 3: AA 211–225) were discovered in the region of rAK1 and rAK2 using synthetized overlapping peptides. The experimental linear epitopes were mapped onto the protein homology model of AK. Meanwhile, in the IgE-binding assays of the sera from nine crab allergic patients, only three sera reacted with the denatured, linear AK as shown by Western-blotting, eight sera reacted with the native, folded AK by both dot-blotting and ELISA, which indicates that the conformational IgE epitopes ofS. paramamosainAK may be more predominant.