Structure-Based Engineering of Allergens to Enhance Digestibility

描述（由申请人提供）：食物过敏研究领域中一个关键且未回答的问题是，为什么某些蛋白质引起IgE介导的免疫应答，而其他蛋白质是耐受的。一个令人信服的假设是，非过敏原更容易消化，导致胃和肠中的蛋白质充分降解，使剩余的片段具有免疫惰性。尽管努力对比过敏原和非过敏原的蛋白水解稳定性，但消化率和过敏性之间的明确联系尚未建立。混杂变量，如与食物基质中其他成分的相互作用，与其他过敏原的交叉反应，或致敏途径（如消化道与呼吸道），使实验结果的解释复杂化。在这个项目中，我们开发了一个高度定义的系统，用于探索消化率和过敏性之间的关系。我们假设蛋白质的消化率取决于其在酸性（pH < 3.0）条件下的稳定性。使用主要的花生过敏原，阿糖胞苷h 1，作为一个模型系统，我们将计算设计酸敏感的变种，在胃液中迅速蛋白水解。这些突变体将提供最佳的试剂进行比较研究有关的pH值稳定性的消化性，并最终致敏性。我们将通过三个目标来实现这一目标：（1）Ara h 1蛋白的生物物理特性的基准pH稳定性计算，（2）Ara h 1中的工程突变，其特异性地降低在低酸性pH下的稳定性，同时在中性pH条件下保留结构和功能，以及（3）使用已建立的模拟胃液测定和先进的动态消化模型将pH稳定性与消化率相关联。本计画将分子静电学的尖端计算方法应用于食物过敏研究的重要议题。对食物蛋白质消化率的分子基础的详细了解将有助于确定其在过敏性中的作用，并使我们能够开发更准确的方案来预测新的或转基因食物蛋白质的过敏性。此外，本文提出的技术可能会在未来应用于提高现有食品的安全性，例如设计低过敏性花生。为了实现短期和长期目标，我们建立了一支强大的合作团队，他们在计算结构生物学，生物物理方法，包括植物在内的许多重组系统中的蛋白质表达和纯化以及肠道免疫学研究方面具有专业知识。该小组拥有必要的专业知识和资源，以确保这一雄心勃勃的重要项目取得成功。通常，食物中的蛋白质被完全分解，提供必需氨基酸。然而，一些食物（如花生）的不完全消化可能会引起对肠道吸收的完整蛋白质的免疫反应，导致食物过敏的发展。我们将对一种主要的花生过敏原进行基因工程改造，使其在强酸性胃液中极易降解。这些蛋白质变体将帮助我们更好地理解消化作为对抗过敏原的防线的作用，并可能导致花生和其他食物的低过敏性变体的发展。

DESCRIPTION (provided by applicant): A crucial and unanswered question in the field of food allergy research is why certain proteins elicit an IgE mediated immune response, while others are tolerated. One compelling hypothesis is that non-allergens are more digestible, resulting in sufficient protein degradation in the stomach and intestine to render the remaining fragments immunologically inert. Despite efforts to contrast the proteolytic stability of allergens and non-allergens, a clear link between digestibility and allergenicity has yet to be established. Confounding variables such as interactions with other components in the food matrix, cross-reactivity with other allergens, or the pathway of sensitization (e.g. alimentary canal versus respiratory tract) complicate the interpretation of experimental outcomes. In this project, we develop a highly defined system for exploring the relationship between digestibility and allergenicity. We hypothesize that the digestibility of a protein is dependent on its stability under acidic (pH < 3.0) conditions. Using the major peanut allergen, Ara h 1, as a model system, we will computationally design acid-sensitive variants that are rapidly proteolyzed in gastric fluid. These mutants will provide optimal reagents for comparative studies relating pH-stability to digestibility and eventually to allergenicity. We will accomplish this goal through three aims: (1) benchmark pH-stability calculations on biophysical characterization of the Ara h 1 protein, (2) engineer mutations in Ara h 1 that specifically reduce stability at low, acid pH while preserving structure and function under neutral pH conditions and (3) relate pH-stability to digestibility using both established simulated gastric fluid assays and advanced dynamic digestion models. This project applies cutting-edge computational methods in molecular electrostatics to important issues in food allergy research. A detailed understanding of the molecular basis for food protein digestibility will help define its role in allergenicity, and allow us to develop more accurate protocols for predicting the allergenicity of new or genetically modified food proteins. Furthermore, the technologies proposed here may find future applications in increasing the safety of existing foods, such as the design of hypoallergenic peanuts. In order to achieve both short and long term goals, we have established a strong team of collaborators with expertise in computational structural biology, biophysical methods, protein expression and purification in a number of recombinant systems including plants, and immunological studies of the gut. This group has the necessary expertise and resources to ensure this ambitious and important project will succeed. Normally, proteins in food are completely broken down providing essential amino acids. However, incomplete digestion of some foods, such as peanuts, may induce an immune reaction against intact proteins absorbed in the intestine, resulting in the development of food allergies. We will genetically engineer a major peanut allergen such that it is highly susceptible to degradation in the strongly acidic juices of the stomach. These protein variants will help us better understand the role of digestion as a line of defense against allergens, and potentially lead to the development of hypoallergenic variants of peanuts and other foods.