Structure-Based Engineering of Allergens to Enhance Digestibility

基于结构的过敏原工程可提高消化率

基本信息

批准号：
8053850
负责人：
Vikas Nanda
金额：
$ 23.17万
依托单位：
UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-01 至 2013-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8053850
关键词：
Acids Affect Allergens Amino Acids Aspartic Acid Benchmarking Binding Biological Assay Biological Models Charge Chemicals Comparative Study Complement Computer software Computer-Aided Design Computing Methodologies Conflict (Psychology)Confounding Factors (Epidemiology)Development Digestion Electrostatics Engineering Ensure Epitopes Essential Amino Acids Food Food Hypersensitivity Food Processing Future Gastrointestinal tract structure Genetically Modified Food Glutamic Acid Goals Health Histidine Human IgE Immune Immune response In Vitro Inflammatory Response Intestines Investigation Juice Knowledge Laboratories Lead Link Liquid substance Mass Spectrum Analysis Measures Mediating Methods Modeling Modification Molecular Mutation Outcome Pathway interactions Peanuts - dietary Plants Predisposition Process Property Protein Engineering Proteins Protocols documentation Reaction Reagent Recombinant Proteins Recombinants Research Resolution Resources Respiratory System Respiratory tract structure Role Safety Seeds Series Simulate Stomach Structural Protein Structure System Technology Testing Variant World Health Organization base cross reactivity crosslink design food allergen innovation instrument mutant novel prevent protein degradation protein expression protein folding protein purification soy structural biology

项目摘要

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.

描述（由申请人提供）：食物过敏研究领域中的一个至关重要和未解决的问题是为什么某些蛋白会引起IgE介导的免疫反应，而其他蛋白质则被容忍。一个令人信服的假设是，非过敏原更易于消化，从而导致胃中足够的蛋白质降解和肠道以使其余的片段在免疫学上使其呈惰性。尽管努力将过敏原和非过敏原的蛋白水解稳定性进行对比，但尚未建立消化率和过敏性之间的明确联系。混淆变量，例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性或致敏途径（例如，消化道和呼吸道）使实验结果的解释变得复杂。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反应之间的关系。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ARA H 1作为模型系统，我们将在胃液中迅速蛋白水解的计算设计酸敏感性。这些突变体将为比较研究提供最佳试剂，该试剂将pH稳定性与消化率且最终与过敏反应有关。我们将通过三个目标来实现这一目标：（1）基于ARA H 1蛋白的生物物理表征的基准pH稳定性计算，（2）ARA H 1中的工程师突变，该突变在低酸pH下专门降低稳定性，同时将结构和功能保存在中性pH条件下，并在中性pH条件下进行pH稳定性以及（3）使用两个模型的pH稳定性，并使用两个模型进行了模型，并进行了更先进的simit simber insation simberic simberic dynydion DynydiC dynydion DynydiC Dixpiention dyromid dynydiention DynydiC Dixpiention。该项目将分子静电学中的尖端计算方法应用于食品过敏研究的重要问题。对食物蛋白消化率的分子基础的详细理解将有助于定义其在过敏性中的作用，并允许我们开发更准确的方案来预测新的或转基因食品蛋白的过敏性。此外，这里提出的技术可能会在提高现有食物的安全性（例如低过敏性花生的设计）方面找到未来的应用。为了实现短期和长期目标，我们在许多重组系统（包括植物的重组系统以及肠道的免疫学研究）中建立了一个具有专业知识，生物物理方法，蛋白质表达和纯化方面具有专业知识的合作者团队。该小组拥有必要的专业知识和资源，以确保这个雄心勃勃的重要项目将成功。通常，食物中的蛋白质被完全分解，提供必需的氨基酸。但是，某些食物（例如花生）的不完全消化可能会引起对肠中吸收的完整蛋白质的免疫反应，从而导致食物过敏的发展。我们将在基因上设计一种主要的花生过敏原，以使其在胃的强酸性汁中非常容易降解。这些蛋白质变异将有助于我们更好地理解消化作为对过敏原的防御线的作用，并有可能导致花生和其他食物的低过敏性变体的发展。