Analysis and engineering of MD-2 and related proteins from common allergens

描述（由申请方提供）：已知过敏原对诱导气道炎症和哮喘有直接影响。目前，人们已开始了解所涉及的免疫学机制。最近的证据表明，一些过敏原可能在引发免疫应答方面特别有效，因为相同的分子（过敏原）具有与先天受体结合的能力并充当常规的B和T细胞抗原。因此，来自尘螨的Derp 2已显示在募集细菌脂多糖（LPS）以刺激Toll样受体4（TLR 4）方面表现得像哺乳动物蛋白MD-2。根据该假说，Derp 2和LPS（或类似的基于脂质的化合物）的来源的存在可以启动先天系统的激活，最终导致TH细胞的刺激和IgE过敏反应以及相关的肺部疾病如哮喘。我们建议通过使用称为酵母展示的高通量蛋白表达和分析系统来研究MD-2，Derp 2和其他相关过敏原与LPS结合的分子细节。该技术也将用于蛋白质工程，以开发可能作为此类不良反应调节剂的MD- 2显性负抑制剂。具体目标是：1）表征在LPS和TLR 4结合中重要的MD-2和Derp 2残基，2）通过一组MD-2的过敏原同源物分析LPS和TLR 4结合，和3）工程化可溶性MD-2类似物，其在LPS募集中充当Derp 2的显性负抑制剂。 公共卫生相关性：对于大多数哮喘患者来说，很明显过敏会诱发炎症级联反应。最近的证据表明，一些过敏原可能在引发免疫应答方面特别有效，因为相同的分子（过敏原）具有与先天受体结合的能力并充当常规的B和T细胞抗原。我们建议使用高通量蛋白质工程和分析来了解先天相互作用的分子基础，并开发可作为特异性抗炎药物的蛋白质。

DESCRIPTION (provided by applicant): Allergens are known to have a direct affect on the induction of airway inflammation and asthma. The immunological mechanisms involved are now beginning to be understood. Recent evidence suggests that some allergens may be particularly potent at eliciting an immune response because the same molecule (allergen) has the ability to bind to innate receptors and serve as a conventional B and T cell antigen. Thus, Derp2 from the dust mite has been shown to behave like the mammalian protein MD-2 in recruiting bacterial lipopolysaccharide (LPS) to stimulate toll-like receptor 4 (TLR4). According to this hypothesis, the presence of Derp2 and a source of LPS (or similar lipid-based compounds), could initiate activation of the innate system, ultimately leading to stimulation of TH cells and IgE allergic reactions and associated pulmonary disease such as asthma. We propose to study the molecular details of MD-2, Derp2, and other related allergens in their binding to LPS by using a high-throughput protein expression and analysis system called yeast display. This technology will also be used for protein engineering in an effort to develop dominant negative inhibitors of MD- 2 that might serve as modulators of such adverse reactions. The specific aims are to: 1) characterize the MD-2 and Derp2 residues important in LPS and TLR4 binding, 2) analyze LPS and TLR4 binding by a panel of allergen homologs of MD-2, and 3) engineer soluble MD-2 analogs that act as dominant negative inhibitors of Derp2 in LPS recruitment. PUBLIC HEALTH RELEVANCE: For most patients with asthma, it is clear that allergies can induce the inflammatory cascade. Recent evidence suggests that some allergens may be particularly potent at eliciting an immune response because the same molecule (allergen) has the ability to bind to innate receptors and serve as a conventional B and T cell antigen. We propose to use high-throughput protein engineering and analysis to understand the molecular basis of the innate interactions, and to develop proteins that could act as specific anti-inflammatory drugs.