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

描述(由申请人提供)：已知过敏原对呼吸道炎症和哮喘的诱导有直接影响。现在人们开始了解其中涉及的免疫学机制。最近的证据表明，一些过敏原可能在引发免疫反应方面特别有效，因为相同的分子(过敏原)具有与天然受体结合的能力，并充当传统的B和T细胞抗原。因此，尘螨的Derp2在募集细菌脂多糖(LPS)刺激Toll样受体4(TLR4)方面的行为与哺乳动物蛋白MD-2相似。根据这一假说，Derp2和一种来源的内毒素(或类似的脂基化合物)的存在可以启动固有系统的激活，最终导致TH细胞和IgE过敏反应的刺激以及相关的肺部疾病，如哮喘。我们建议使用一个称为酵母显示的高通量蛋白质表达和分析系统来研究MD-2、DERP2和其他相关过敏原与内毒素结合的分子细节。这项技术还将用于蛋白质工程，努力开发MD-2的显性负抑制药，可能作为此类不良反应的调节剂。其具体目的是：1)鉴定在内毒素和TLR4结合中重要的MD-2和DERP2残基，2)分析一组MD-2变应原同源物与内毒素和TLR4的结合，以及3)工程可溶性MD-2类似物，它们在内毒素募集中起到主要的DERP2负抑制作用。 公共卫生相关性：对于大多数哮喘患者来说，很明显，过敏可以诱发炎症级联反应。最近的证据表明，一些过敏原可能在引发免疫反应方面特别有效，因为相同的分子(过敏原)具有与天然受体结合的能力，并充当传统的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，并开发可以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.