Structural and Functional Characterization of Allergens

过敏原的结构和功能表征

基本信息

批准号：
8553806
负责人：
Robert E London
金额：
$ 24.47万
依托单位：
NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8553806
关键词：
Accident and Emergency department Accounting Advanced Glycosylation End Products Affinity Allergens Allergic Allergic Disease Allergy to peanuts Anaphylaxis Antibodies Asthma Atopic Dermatitis Autoantigens Binding Biological Process Breathing CD1 Antigens Cells Chemicals Chronic Complex Crystallization Cyclophilins Data Dendritic Cells Development Dictyoptera Encapsulated Epitope Mapping Epitopes Escherichia coli Fab Immunoglobulins Fc Receptor Felis catus Food Processing Future Gastrointestinal tract structure Goals Human Hypersensitivity IgE Immune Immune response Immune system Immunotherapeutic agent Immunotherapy Insect Proteins Knowledge Lead Ligands Lipids Lysine Malondialdehyde Maps Mass Spectrum Analysis Mediating Modification Molds Molecular N(6)-carboxymethyllysine Oleic Acids Oral Pathway interactions Patients Pattern Recognition Peanuts - dietary Phospholipids Plant Proteins Plants Pollen Property Protein Family Proteins Recombinants Recording of previous events Research Design Research Personnel Risk Risk Factors Roentgen Rays Role Serum Side Site Source Specificity Stearates Structure Suggestion System Tandem Repeat Sequences Techniques Testing Therapeutic Vinca Visit Western Blotting X-Ray Crystallography allergen Bla g 1 allergic response autoreactivity base cockroach allergen cross reactivity design electron density environmental allergen improved insight member novel protein folding receptor receptor binding receptor expression receptor for advanced glycation endproducts response structural biology success uptake

项目摘要

During the past year, this project has succeeded in determining the structures of two important allergens. The structure of the cockroach allergen Bla g 1 revealed a previously uncharacterized protein fold that binds to lipid ligands, which is a common property of many allergens. The structure of the allergen Cat r 1 is the first example of a cyclophilin allergen derived from plants. Additionally, this project is beginning to move beyond the determination of single allergen structures and to investigate the interactions of allergens with the molecules in the immune system. This approach is currently proceeding with two molecular systems related to peanut allergy. First, we are looking at the chemical modification of peanut allergens due to roasting. Modifications such as those that result from roasting are commonly recognized by the receptor for advanced glycation end products (RAGE). These studies are the first to demonstrate that peanut allergens with modifications bind specifically to RAGE. Second, we are beginning an attempt to determine co-structures of antibodies bound to the major peanut allergen Ara h 2. Further accomplishments of each project are summarized below. Sensitization to cockroach allergens is a major risk factor for asthma. The cockroach allergen Bla g 1 and other homologous insect proteins are characterized by a tandem repeat of two sequences with about 25% identity, but the fold of the protein and the exact biological function are unknown. The structure of Bla g 1 was determined by X-ray crystallography. The Bla g 1 tandem repeat forms a novel fold with 6 helices that encapsulate a large and nearly spherical hydrophobic cavity of 3,750 A3. The X-ray data could identify some electron density in the cavity that might be long acyl-chain lipids. Using NMR and mass spectrometry, different lipid ligands were found associated with Bla g 1 depending on the source of the protein. Recombinant Bla g 1 from E. coli or P. pastoris bound phospholipids PE, PG, PS, and PC with various affinities, and cockroach frass was found to not contain phospholipids, but instead simple stearate and oleic acids. Further studies are needed to test whether or not these lipid ligands may contribute to the allergenicity of Bla g 1. Various phospholipids are known to bias the immune response towards a Th0/Th2 via presentation by dendritic cells on CD1 molecules, contributing to allergic disease. Cyclophilin allergens are considered pan-allergens due to their high crossreactivity; i.e. patients sensitized to just one source are usually highly allergic to the allergens from all other sources. This crossreactivity can include autoreactivity where the immune system mistakenly reacts against self-antigens. Indeed, some patients with chronic allergic disease, either asthma or atopic dermatitis, have demonstrated both humoral and cell-mediated autoreactivity. The reason for the crossreactivity is the high sequence identity between members of this protein family. In this period we determined the structure of the allergen Cat r 1, derived from the rosy periwinkle using NMR techniques. This is the first structure of a cyclophilin protein from plants. Using the structure, we have been able to better understand the important residues that likely account for the cross reactivity between plant and mould allergens, and potential residues involved in autoreactivity with human cyclophilins. This knowledge will help in the rationale design of immunotherapeutics in that researchers may now design hypo-allergens that also avoid encouraging autoreactivity. The protein Ara h 2 is the most potent peanut allergen recognized by >90% of peanut allergic patients. The natural allergen and the recombinant construct used to determine the structure showed different patterns of recognition by patient sera. Based on these comparisons a major site of interaction (an epitope) for about 50% of patients was identified. This success has encouraged us to further map the patient epitopes using a panel of antibodies with various specificities for Ara h 2 and the homologous Ara h 6 allergen. Currently we have selected and produced the Fab fragment of several antibodies and are currently performing crystallization trials with complexes of Ara h 2. It is our goal to further identify conformational epitopes on peanut allergens in order to better understand the patient response to peanut and to determine whether specific eptiope recognition correlates with any aspect of peanut allergic disease, e.g. risk of anaphylaxis, emergency room visits, or response to oral therapy. To do this we are collaborating Wesley Burks (UNC) in order obtain sera with well-documented patient histories. It is anticipated that further epitope mapping will provide information for the rational design of Ara h 2 hypoallergens for more effective and safer immunotherapies. It has been suggested that one of the reasons for the hypersensitivity to peanut allergens is due to food processing. Indeed, patient IgE more commonly recognizes roasted peanut proteins than raw. Roasting commonly leads to modifications of lysine side chains and formation of advanced glycation end products (AGEs). Recent immunonlogical data suggests that dendrictic cells preferentially uptake AGE modified protein and consequently upregulate expression of the receptor for AGE (RAGE). In our studies, AGE modifications were found on Ara h 1 and Ara h 3 that included carboxymethyllysine, carboxyethyllysine, malondialdehyde, and dihyropyridine, in both raw and roasted peanut extract. Very few modifications were found on Ara h 2. Based on mass spectrometric analysis and Western blotting with allergen specific antibodies, RAGE was demonstrated to selectively pull down Ara h 1, Ara h 3, and AHY-3 from peanut extract. No Ara h 2 binding to RAGE was detected by Western analysis. Recombinant Ara h 1 with no AGE modifications did not bind RAGE, however after AGE modification Ara h 1 bound to RAGE. If the suggestion that sensitization to peanut allergens occurs in dendritic cells recognizing AGE modified peanut proteins is correct, these cells are likely interacting with modified Ara h 1, and Ara h 3, and not Ara h 2. With a better understanding of the immune pathways that lead to sensitization, we will hopefully be able to identify novel future therapies.

在过去的一年中，该项目成功地确定了两个重要的过敏原的结构。蟑螂过敏原bla g 1的结构揭示了一种先前未表征的蛋白质折叠，该蛋白折叠与脂质配体结合，这是许多过敏原的常见特性。过敏原CAT R 1的结构是源自植物的环磷脂过敏原的第一个例子。此外，该项目开始超越单个过敏原结构的确定，并研究过敏原与免疫系统中分子的相互作用。目前，这种方法正在使用与花生过敏有关的两个分子系统。首先，我们正在研究由于烘烤而对花生过敏原的化学修饰。诸如烘烤导致的修饰通常被高级糖基化终产物（RAGE）的受体认识到。这些研究是第一个证明具有修饰的花生过敏原特异性与愤怒结合的研究。其次，我们开始尝试确定与主要花生过敏原ARA H 2结合的抗体的共结构。每个项目的进一步成就总结了下面。对蟑螂过敏原的敏化是哮喘的主要危险因素。蟑螂过敏原BLA G 1和其他同源昆虫蛋白的特征是两个序列的串联重复，但蛋白质的折叠和确切的生物学功能尚不清楚。 BLA G 1的结构通过X射线晶体学确定。 BLA G 1串联重复形成一个新型折叠，并带有6个螺旋，该螺旋封装了3,750 A3的大且几乎球形的疏水腔。 X射线数据可以识别腔中可能是长酰基链脂质的某些电子密度。使用NMR和质谱法，发现不同的脂质配体与Bla G 1相关，具体取决于蛋白质的来源。来自大肠杆菌或P. p. pastoris结合的磷脂，PG，PS和PC的重组BLA G 1具有各种亲和力，并且发现蟑螂Frass不含磷脂，而是简单的硬脂酸和油酸。需要进一步的研究来测试这些脂质配体是否可能有助于Bla g 1的过敏性。已知各种磷脂可以通过树突细胞对CD1分子的呈现对TH0/TH2的免疫反应偏见，从而有助于过敏性疾病。环磷脂过敏原因其高反应性而被认为是泛过敏原。即，仅对一个来源敏感的患者通常对所有其他来源的过敏原过敏。这种交叉反应性可以包括自动反应性，其中免疫系统错误地反应自我抗原。实际上，一些患有慢性过敏性疾病的患者，无论是哮喘还是特应性皮炎，都表现出体液和细胞介导的自身反应性。交叉反应性的原因是该蛋白质家族成员之间的高序列身份。在此期间，我们确定了使用NMR技术源自玫瑰色的毛刺的过敏原cat r 1的结构。这是植物中环磷脂蛋白的第一个结构。使用该结构，我们已经能够更好地理解可能解释植物和霉菌过敏原之间交叉反应性的重要残基，以及与人环蛋白相关的潜在残基。这些知识将有助于免疫治疗剂的基本原理设计，因为研究人员现在可能会设计低过敏原，从而避免鼓励自动反应性。蛋白ARA H 2是> 90％的花生过敏患者识别的最有效的花生过敏原。用于确定结构的天然过敏原和重组构建体显示患者血清的识别模式不同。基于这些比较，确定了约50％的患者的相互作用（表位）的主要部位。这种成功鼓励我们使用一组具有各种特异性的抗体和同源ARA H 6过敏原的抗体进一步绘制患者表位。目前，我们已经选择并生产了几种抗体的Fab片段，目前正在进行具有ARA H 2的复合物进行结晶试验。我们的目标是进一步识别花生过敏原上的构象表位，以便更好地了解患者对花生的反应并确定特定的Eptiope识别是否与花生氏疾病的任何方面相关。过敏反应的风险，急诊室就诊或对口服治疗的反应。为此，我们正在协作韦斯利·伯克斯（Wesley Burks）（UNC），以便获得具有据可查的患者历史的血清。可以预计，进一步的表位映射将为ARA H 2低过敏原的合理设计提供信息，以提供更有效，更安全的免疫疗法。有人提出，对花生过敏原过敏的原因之一是由于食物加工。实际上，患者IgE比RAW更常见的是烤花生蛋白。烘烤通常会导致赖氨酸侧链的修饰和高级糖基化终产物（年龄）的形成。最近的免疫数据表明，树突细胞优先摄取年龄修饰的蛋白质，从而在年龄（RAGE）上上调受体的表达。在我们的研究中，在原始和烤花生提取物中发现了包括羧甲基乙醇胺，羧乙基透析，丙二醇和二氢吡啶的ARA H 1和ARA H 3的年龄修饰。基于质谱分析和使用过敏原特异性抗体的蛋白质印迹，在ARA H 2上发现了很少的修饰，愤怒被证明可以选择性地从花生提取物中拉下Ara H 1，Ara H 3和Ahy-3。通过西方分析未检测到与愤怒的Ara H 2结合。重组ARA H 1没有年龄修改没有结合愤怒，但是年龄修改后Ara H 1与愤怒结合。如果在识别年龄修饰的花生蛋白的树突状细胞中发生敏化对花生过敏原是正确的，那么这些细胞可能与修饰的ARA H 1和ARA H 3相互作用，而不是ARA H 3，而不是ARA H 2。在更好地了解导致敏感的免疫途径的情况下，我们有望能够识别出新的新型未来疗法。