Development of a Treatment for Eosinophil-Mediated Allergic Inflammatory Diseases Utilizing a Neutralizing Agent Targeting Eosinophil Granule Major Basic Protein

摘要 嗜酸性粒细胞是一种外周血白细胞，含有丰富的细胞质颗粒，富含阳离子。 蛋白质毒素。其中，摩尔含量最丰富的是主要的碱性蛋白-1，eMBP1。EMBP1 杀死蠕虫、细菌和许多细胞，如呼吸道上皮，但也激活细胞，包括 嗜碱性粒细胞和肥大细胞。对人类疾病的研究表明，eMBP1存在于患者的分泌物中 嗜酸性粒细胞介导的疾病，包括哮喘、慢性鼻窦炎和胃肠道疾病，以及 它被沉积在受损的目标上。这些研究表明，嗜酸性粒细胞介导其对寄生虫的损害。 通过将其富含毒素的颗粒排放到微生物目标和组织中。EMBP1合成为 前体，Pro-eMBP1，由eMBP1和一个非常酸性的前体片段序列组成。发展中 嗜酸性粒细胞合成原eMBP1，该原片段在颗粒成熟过程中被去除。PRO-PRO的分析 在不同的模型中显示，它可以中和毒性的eMBP1效应，也可以中和嗜酸性粒细胞的毒性 阳离子蛋白(ECP)。该项目提出，中和eMBP1可以作为一种治疗方法，以减轻组织 嗜酸性粒细胞相关疾病的损害。它将开发一种用于治疗嗜酸性粒细胞介导的前体产品。 通过中和eMBP1和其他颗粒毒素引起的疾病。目前，还没有针对这些疾病的治疗方法 解决颗粒蛋白质的中和问题。 在这个项目中，原件将被表达，相关的多糖将被修饰以确定最佳的 结合和中和eMBP1的分子形式。这些测试将确定糖基化是否 Pro-Pich通过分析N-或O-多糖和eMBP1的结合情况，重要地改变了其与eMBP1的结合 糖胺多聚糖在S62修饰其活性。该项目将确定以下最重要的亲件区域 结合eMBP1并通过产生重叠的多肽和增加氨基酸的突变体来中和它 活性区域特有的酸序列。预制件面板结合和抑制性能的评价 EMBP1将在几个体外模型中进行评估。这个项目的目标是确定一种活跃的形式 对eMBP1和其他颗粒毒性蛋白具有最佳抑制作用的原件。此表将进一步 在未来的项目中进行研究和开发。

ABSTRACT The eosinophil is a peripheral blood leukocyte containing an abundance of cytoplasmic granules, rich in cationic protein toxins. Among these, the most abundant on a molar basis is the major basic protein-1, eMBP1. eMBP1 kills helminths, bacteria, and numerous cells, such as respiratory epithelium, but also activates cells, including basophils and mast cells. Studies of human diseases show that eMBP1 is present in secretions from patients with eosinophil-mediated diseases, including asthma, chronic rhinosinusitis, and gastrointestinal diseases, and it is deposited on damaged targets. These studies show that the eosinophil mediates its damage to parasites and tissues by discharging its toxin rich granules onto microbial targets and tissues. eMBP1 is synthesized as a precursor, pro-eMBP1, composed of eMBP1 and a remarkably acidic pro-piece sequence. Developing eosinophils synthesize pro-eMBP1, and the pro-piece is removed during granule maturation. Analyses of pro- piece in different models show that it can neutralize the toxic eMBP1 effect and also the toxicity of the eosinophil cationic protein (ECP). This project proposes that neutralization of eMBP1 can be a treatment to mitigate tissue damage in eosinophil-related diseases. It will develop a pro-piece product for treatment of eosinophil-mediated diseases by neutralization of eMBP1 and other granule toxins. Currently, no therapies for these diseases address the neutralization of granule proteins. In this project, the pro-piece will be expressed and associated glycans will be modified to identify the optimal form of the molecule for binding to and neutralizing eMBP1. These tests will determine whether glycosylation of pro-piece importantly alters its binding to eMBP1 by analyzing whether N- or O- glycans and the glycosaminoglycan at S62 modify its activities. The project will identify the most important pro-piece regions for binding eMBP1 and neutralizing it by creating overlapping peptides and creating mutants with increased amino acid sequences characteristic of active regions. Evaluation of the pro-piece panel for binding to and inhibiting eMBP1 will be evaluated here in several in vitro models. The goal of this project is to identify an active form of the pro-piece with optimal inhibition effects on eMBP1 and other granule toxic proteins. This form will be further investigated and developed in future projects.