Havenith Heide, Kern Karolin, Rautenberger Paul, Spiegel Holger, Szardenings Michael, Ueberham Elke, Lehmann Jörg, Buntru Matthias, Vogel Simon, Treudler Regina, Fischer Rainer, Schillberg Stefan
Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
Fraunhofer Institute for Cell Therapy and Immunology IZI, Leipzig, Germany.
Biotechnol J. 2017 Feb;12(2). doi: 10.1002/biot.201600441.
Detailed IgE-binding epitope analysis is a key requirement for the understanding and development of diagnostic and therapeutic agents to address food allergies. An IgE-specific linear peptide microarray with random phage peptide display for the high-resolution mapping of IgE-binding epitopes of the major soybean allergen Gly m 4, which is a homologue to the birch pollen allergen Bet v 1 is combined. Three epitopes are identified and mapped to a resolution of four key amino acids, allowing the rational design and the production of three Gly m 4 mutants with the aim to abolish or reduce the binding of epitope-specific IgE. In ELISA, the binding of the mutant allergens to polyclonal rabbit-anti Gly m 4 serum as well as IgE purified from Gly m 4-reactive soybean allergy patient sera is reduced by up to 63% compared to the wild-type allergen. Basophil stimulation experiments using RBL-SX38 cells loaded with patient IgE are showed a decreased stimulation from 25% for the wild-type Gly m 4 to 13% for one mutant. The presented approach demonstrates the feasibility of precise mapping of allergy-related IgE-binding epitopes, allowing the rational design of less allergenic mutants as potential therapeutic agents.
详细的IgE结合表位分析是理解和开发用于解决食物过敏的诊断和治疗药物的关键要求。结合了一种具有随机噬菌体肽展示的IgE特异性线性肽微阵列，用于对主要大豆过敏原Gly m 4的IgE结合表位进行高分辨率定位，Gly m 4是桦树花粉过敏原Bet v 1的同源物。鉴定出三个表位并将其定位到四个关键氨基酸的分辨率，从而能够合理设计和生产三种Gly m 4突变体，旨在消除或减少表位特异性IgE的结合。在ELISA中，与野生型过敏原相比，突变型过敏原与多克隆兔抗Gly m 4血清以及从Gly m 4反应性大豆过敏患者血清中纯化的IgE的结合减少了高达63%。使用负载患者IgE的RBL-SX38细胞进行的嗜碱性粒细胞刺激实验表明，刺激率从野生型Gly m 4的25%降至一种突变体的13%。所提出的方法证明了精确绘制与过敏相关的IgE结合表位的可行性，从而能够合理设计低过敏原性突变体作为潜在的治疗药物。
