Hydrogen/deuterium exchange memory NMR reveals structural epitopes involved in IgE cross-reactivity of allergenic lipid transfer proteins
Hydrogen/deuterium exchange memory NMR reveals structural epitopes involved in IgE cross-reactivity of allergenic lipid transfer proteins
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DOI：
10.1074/jbc.ra120.014243
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发表时间：
2020-12-18
影响因子：
4.8
通讯作者：
Gadermaier, Gabriele
中科院分区：
文献类型：
--
作者：
Di Muzio, Martina;Wildner, Sabrina;Gadermaier, Gabriele
抗体结合表位的鉴定对了解免疫机制至关重要。特别值得关注的是，在脂质转移蛋白（LTP）综合征中观察到的具有高交叉反应性的过敏原蛋白，其特征是严重的过敏反应。艾草花粉LTP Art v 3经常参与这种交叉反应，但迄今尚未确定抗体结合表位。为了揭示Art v 3的人类ige结合区，我们制作了3个小鼠高亲和力单抗，显示艾草花粉过敏患者的致敏表位覆盖率为70-90%。由于缺乏可靠的方法来确定在天然条件下紧密相互作用的完整抗体的结构表位，我们开发了一种直接的NMR方法，称为氢/氘交换记忆（HDXMEM）。它依赖于不可见的抗原-单抗复合物和自由的n- 15标记抗原之间的缓慢交换，其H-1-N-15相关性被检测到。由于记忆效应，测量了抗体结合过程中NH保护的变化。H/D交换率的差异和mAb对同源LTPs的反应性分析揭示了三个结构表位：α -螺旋2和4周围的两个部分交叉反应区域以及C端新的Art v - 3特异性表位。具有交换表位残基的蛋白变体证实了抗体结合位点，并显示出强烈降低的IgE反应性。利用新的HDXMEM进行核磁共振表位定位，可以鉴定出过敏性花粉LTP的第一个结构表位。这一知识可以改善LTP过敏患者的交叉反应性预测，并促进治疗方法的设计。
Identification of antibody-binding epitopes is crucial to understand immunological mechanisms. It is of particular interest for allergenic proteins with high cross-reactivity as observed in the lipid transfer protein (LTP) syndrome, which is characterized by severe allergic reactions. Art v 3, a pollen LTP from mugwort, is frequently involved in this cross-reactivity, but no antibody-binding epitopes have been determined so far. To reveal human IgE-binding regions of Art v 3, we produced three murine high-affinity mAbs, which showed 70-90% coverage of the allergenic epitopes from mugwort pollen-allergic patients. As reliable methods to determine structural epitopes with tightly interacting intact antibodies under native conditions are lacking, we developed a straightforward NMR approach termed hydrogen/deuterium exchange memory (HDXMEM). It relies on the slow exchange between the invisible antigen-mAb complex and the free N-15-labeled antigen whose H-1-N-15 correlations are detected. Due to a memory effect, changes of NH protection during antibody binding are measured. Differences in H/D exchange rates and analyses of mAb reactivity to homologous LTPs revealed three structural epitopes: two partially cross-reactive regions around alpha-helices 2 and 4 as well as a novel Art v 3-specific epitope at the C terminus. Protein variants with exchanged epitope residues confirmed the antibody-binding sites and revealed strongly reduced IgE reactivity. Using the novel HDXMEM for NMR epitope mapping allowed identification of the first structural epitopes of an allergenic pollen LTP. This knowledge enables improved cross-reactivity prediction for patients suffering from LTP allergy and facilitates design of therapeutics.