Identification and mutational analysis of the immunodominant IgE binding epitopes of the major peanut allergen Ara h 2
Identification and mutational analysis of the immunodominant IgE binding epitopes of the major peanut allergen Ara h 2
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DOI：
10.1006/abbi.1997.9998
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发表时间：
1997-06-15
影响因子：
3.9
通讯作者：
Bannon, GA
中科院分区：
文献类型：
--
作者：
Stanley, JS;King, N;Bannon, GA
一种主要的花生过敏原Ara h 2可被90%以上的花生过敏症患者的血清IgE识别。该过敏原的生化特征表明，它是一种类似于17.5 kDa的糖蛋白。利用纯化的Ara h 2的N-末端氨基酸序列数据，合成寡核苷酸引物并用于鉴定来自花生cDNA文库的克隆（741 bp）。该克隆能够编码一个17.5 kDa的蛋白质，与种子贮藏蛋白的conglutin家族具有同源性。主要的线性免疫球蛋白E（IgE）结合表位的过敏原进行了映射使用重叠肽合成的活化纤维素膜和汇集的血清IgE从15花生敏感的患者。10个IgE结合表位被确定，分布在整个长度的Ara h 2蛋白。63%的表位中的氨基酸是极性不带电或非极性残基。为了确定10个表位中的哪一个（如果有的话）被大多数花生超敏性患者识别，用来自10个不同患者的血清IgE单独探测每组10种肽。所有测试的患者血清识别多个表位。三个表位（aa 27 -36，aa 57 -66，和aa 65 -74）被所有测试的患者所识别。此外，这三个肽结合更多的IgE比所有其他表位组合，表明它们是免疫显性表位的Ara h 2蛋白。Ara h 2表位的突变分析表明，单个氨基酸的变化导致IgE结合的丧失。aa 57 -74区域中的两个表位包含似乎是IgE结合所必需的氨基酸序列DPYSP。这些结果可能允许设计改进的诊断和治疗花生过敏的方法。(C)北京：科学出版社.
A major peanut allergen, Ara h 2, is recognized by serum IgE from >90% of patients with peanut hypersensitivity. Biochemical characterization of this allergen indicates that it is a glycoprotein of similar to 17.5 kDa. Using N-terminal amino acid sequence data from purified Ara h 2, oligonucleotide primers were synthesized and used to identify a clone (741 bp) from a peanut cDNA library. This clone was capable of encoding a 17.5-kDa protein with homology to the conglutin family of seed storage proteins. The major linear immunoglobulin E (IgE)-binding epitopes of this allergen were mapped using overlapping peptides synthesized on an activated cellulose membrane and pooled serum IgE from 15 peanut-sensitive patients. Ten IgE-binding epitopes were identified, distributed throughout the length of the Ara h 2 protein. Sixty-three percent of the amino acids represented in the epitopes were either polar uncharged or apolar residues. In an effort to determine which, if any, of the 10 epitopes were recognized by the majority of patients with peanut hypersensitivity, each set of 10 peptides was probed individually with serum IgE from 10 different patients. All of the patient sera tested recognized multiple epitopes. Three epitopes (aa27-36, aa57-66, and aa65-74) were recognized by all patients tested. In addition, these three peptides bound more IgE than all the other epitopes combined, indicating that they are the immunodominant epitopes of the Ara h 2 protein. Mutational analysis of the Ara h 2 epitopes indicate that single amino acid changes result in loss of IgE binding. Two epitopes in region aa57-74 contained the amino acid sequence DPYSP that appears to be necessary for IgE binding. These results may allow for the design of improved diagnostic and therapeutic approaches to peanut hypersensitivity. (C) 1997 Academic Press.