Structural mechanism for recognition of host receptor by botulinum neurotoxin A

A型肉毒杆菌神经毒素识别宿主受体的结构机制

基本信息

批准号：
9238660
负责人：
Rongsheng Jin
金额：
$ 19.31万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-03-07 至 2019-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9238660
关键词：
Achievement Adverse effects Affinity Antibodies Avidity Bacteria Bacterial Toxins Binding Biological Biological Assay Bioterrorism Bontoxilysin Botox Botulinum Toxin Type A Botulism Cell surface Cells Clostridium botulinum Complex Cosmetics Crystallization Data Dermatologic Dermatology Disease Escherichia coli Evolution GTP-Binding Protein alpha Subunits, Gs Gangliosides Gastrointestinal Diseases Glycoproteins Goals HIV-1 Human In Vitro Lethal Dose 50 Link Mediating Medicine Membrane Proteins Modeling Molecular Motor Neurons Mutagenesis Nature Nerve Neurologic Neurons Ophthalmology Paper Paralysed Pathway interactions Pharmaceutical Preparations Polysaccharides Post-Translational Protein Processing Process Protein Isoforms Proteins Race Recycling Reporting Serotyping Site-Directed Mutagenesis Specificity Structure Synaptic Membranes Synaptic Vesicles Syndrome Toxic effect Toxin Vertebral column Vertebrates Work X-Ray Crystallography arm base botulinum toxin type B clinical application clinical efficacy design env Gene Products fighting glycosylation improved inhibitor/antagonist insight interest man novel novel therapeutic intervention pathogen prevent protein protein interaction public health relevance receptor receptor binding therapeutic development uptake urologic weapons

项目摘要

DESCRIPTION (provided by applicant): Structural mechanism for recognition of host receptor by botulinum neurotoxin An Abstract Botulinum neurotoxins (BoNTs), produced by the bacterium Clostridium botulinum, are the causative agents of neuroparalytic disease botulism. The extraordinary toxicity of BoNTs relies on the highly specific uptake of BoNTs by neuronal cells. A well-accepted dual-receptor model suggests that the cell surface binding and uptake process of BoNTs is mediated synergistically by specific protein receptors and gangliosides. BoNTs then exploit the synaptic vesicle recycling pathway entering the nerve terminus mediated by the protein receptors. However, it is largely unknown how various BoNTs develop serotype-specific mechanisms for protein receptor recognition, which is believed to account for the differences in BoNTs' biological activity. Our study is focused on BoNT/A serotype because it is a major concern for bioterrorism and is also the most commonly used medicine among the seven BoNT serotypes (BoNT/A-G). The goal is to understand the molecular mechanism by which BoNT/A specifically targets motoneurons through synaptic vesicle glycoprotein 2 that has three isoforms (SV2A, 2B, and 2C). Our preliminary studies show that BoNT/A and SV2C bind to each other through a relatively small protein-protein interface mostly involving backbone-backbone interactions, which is not sufficient to provide the high receptor binding affinity and specificity that BoNT/A needs. Remarkably, we found that BoNT/A takes advantage of SV2 glycosylation, a major form of post- translational modification of synaptic membrane proteins, to compensate for the "shortfall" on protein-mediated recognition. BoNT/A directly binds to an N-linked glycan of SV2, which is conserved in SV2A, 2B, and 2C and highly conserved across different vertebrates, to significantly enhance receptor binding affinity and specificity. This represents a new paradigm of intricate host-pathogen interactions. The specific aims are (1) to understand the structural basis for recognition of SV2C glycans by BoNT/A; and (2) to understand the affinity and specificity requirements for the BoNT/A-SV2 recognition. We will use an integrated approach that combines X-ray crystallography, site-directed mutagenesis, and binding assays. The achievement of our goal will guide the design of novel therapeutic approaches to prevent and treat botulism by preventing cell entry of BoNT/A, provide new insights into activity and side- effects of BoNT/A-based drugs, help improve their clinical efficacy, and suggest novel applications. Furthermore, this study will have impact on our basic biological understanding of the everlasting host-pathogen arms race, which may stimulate new ideas for therapeutic development.

描述（由申请人提供）：肉毒杆菌神经毒素识别宿主受体的结构机制摘要肉毒杆菌神经毒素（BoNT）由肉毒杆菌产生，是神经麻痹性疾病肉毒杆菌中毒的致病因子。神经元细胞对 BoNT 的特异性摄取。 BoNT 的细胞表面结合和摄取过程由特定的蛋白质受体和神经节苷脂协同介导，然后利用蛋白质受体介导的突触小泡回收途径进入神经末端。然而，目前尚不清楚各种 BoNT 如何形成血清型特异性机制。蛋白质受体识别，这被认为是 BoNT 生物活性差异的原因。我们的研究重点是 BoNT/A 血清型，因为它是一个主要问题。用于生物恐怖主义，也是七种 BoNT 血清型 (BoNT/A-G) 中最常用的药物。目标是了解 BoNT/A 通过具有三种亚型（SV2A、2B）的突触囊泡糖蛋白 2 特异性靶向运动神经元的分子机制。和 2C）我们的初步研究表明，BoNT/A 和 SV2C 通过相对较小的蛋白质-蛋白质界面相互结合，主要涉及骨架-骨架。值得注意的是，我们发现 BoNT/A 利用 SV2 糖基化（突触膜蛋白翻译后修饰的主要形式）来进行补偿。 BoNT/A 直接与 SV2 的 N 连接聚糖结合，该聚糖在 SV2A、2B 和 2C 中保守，并且在整个细胞中高度保守。不同的脊椎动物，显着增强受体结合亲和力和特异性，这代表了复杂的宿主-病原体相互作用的新范例，具体目标是（1）了解 BoNT/A 识别 SV2C 聚糖的结构基础；为了了解 BoNT/A-SV2 识别的亲和力和特异性要求，我们将使用结合 X 射线晶体学、定点诱变和结合测定的综合方法来指导我们目标的设计。通过阻止 BoNT/A 进入细胞来预防和治疗肉毒杆菌中毒的新治疗方法，为基于 BoNT/A 的药物的活性和副作用提供新的见解，有助于提高其临床疗效，并提出新的应用。影响我们对永恒的宿主-病原体军备竞赛的基本生物学理解，这可能会激发治疗开发的新思路。