Discovery of novel cholera toxin receptors

新型霍乱毒素受体的发现

• 批准号: 8236891
• 负责人: Jennifer J Kohler
• 金额: $ 19.84万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-07 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8236891
• 关键词: Affect; Affinity; Affinity Chromatography; Bacteria; Binding; Biological Assay; Biological Models; Caveolins; Cell Line; Cell surface; Cells; Characteristics; Chloride Ion; Cholera; Cholera Toxin; Cholera Toxin Protomer B; Clathrin; Complex; Data; Development; Diarrhea; Disease Outbreaks; Disease Progression; Endocytosis; Epithelial Cells; Event; Fluorescence Microscopy; Fractionation; Gangliosides; Glycolipids; Glycoproteins; Goals; Health Services Accessibility; Human; Hydration status; Imagery; Immunofluorescence Microscopy; Infection; Intestines; Intoxication; Invaded; Knowledge; Light; Liquid substance; Mass Spectrum Analysis; Mediating; Membrane Microdomains; Methods; Oral; Pathway interactions; Play; Post-Translational Protein Processing; Process; Prophylactic treatment; Reporting; Role; Salts; Surface; Techniques; Toxic effect; Toxin; Vibrio cholerae; cell type; choleragen receptor; combat; glycosylation; holotoxins; inhibitor/antagonist; intravenous administration; novel; novel strategies; public health relevance; receptor; research study

DESCRIPTION (provided by applicant): Cholera toxin subunit B (CTxB) binds to molecules presented on the surface of host intestinal epithelial cells. After the initial binding event, the holotoxin invades the cells, hijacking the cellular machinery to cause the profuse diarrhea that characterizes cholera infection. A complete understanding of the mechanistic details of cholera toxicity is necessary to facilitate the development of novel strategies to combat cholera outbreaks. While the ganglioside GM1a is widely described as the single receptor recognized by CTxB, recent data from our lab and others implies the existence of additional cholera toxin binding partners, which may be glycoproteins. The broad goals of this proposal are to identify novel CTxB binding partners and discover whether they function in cholera intoxication. The goal of Aim 1 is to identify cell surface molecules in T84 cells that are recognized by CTxB, making use of a novel photocrosslinking technique and modern mass spectrometry methods, along with traditional affinity purification approaches. Additional CTxB binding partners could play an auxiliary role, assisting in formation of the canonical CTxB-GM1a complex, or they might function as alternative receptors, enabling cholera toxin to bind cells in the absence of GM1a. The goal of Aim 2 is to distinguish between these possibilities, by using shRNAs to transiently knockdown expression of these alternative binding partners and glycosylation inhibitors to interfere with their post- translational modification. The results of these experiments will allow us to determine if the novel receptor or receptors are required for CTxB binding to T84 cells. While alternative binding partners might facilitate CTxB binding, a more critical question is whether they function in internalization of the toxin. The results of the immunofluorescence microscopy experiments and chloride ion secretion assays, proposed in Aim 3, will reveal whether previously unidentified CtxB receptors facilitate the process of cholera intoxication. This project challenges the existing paradigm that GM1a is both necessary and sufficient for CTxB binding and internalization. Along with the impact on our understanding of cholera disease progression, the results of the proposed experiments will be important for understanding and distinguishing among different mechanisms of endocytosis. Furthermore, because CTxB is commonly used to identify and visualize lipid rafts, a complete knowledge of CTxB binding partners will be vital to defining the composition and characteristics of membrane microdomains. PUBLIC HEALTH RELEVANCE: The bacteria Vibrio cholerae produce a toxin that binds to and invades human intestinal cells, causing the profuse diarrhea that characterizes cholera infection. The cholera toxin has long been believed to interact with a molecule known as GM1a, but our data suggest that the toxin may also interact with an additional molecule or molecules. We propose to identify the additional molecules with which cholera toxin interacts and to determine whether these interactions are critical to disease progression.

描述（由申请人提供）：霍乱毒素亚基B（CTXB）与宿主肠上皮细胞表面上的分子结合。在初始结合事件发生后，Holotoxin侵入细胞，劫持细胞机械，从而导致表征霍乱感染的大量腹泻。对霍乱毒性的机械细节的完全理解对于促进与霍乱疫情打击的新型策略的发展是必要的。虽然神经节GM1A被广泛描述为CTXB识别的单个受体，但我们实验室的最新数据意味着存在其他霍乱毒素结合伴侣，这可能是糖蛋白。该提案的广泛目标是确定新颖的CTXB结合伙伴，并发现它们是否在霍乱中毒中起作用。 AIM 1的目的是鉴定CTXB识别的T84细胞中的细胞表面分子，利用一种新型的Photocroslinking技术和现代的质谱方法以及传统的亲和力纯化方法。其他CTXB结合伴侣可以发挥辅助作用，有助于形成规范的CTXB-GM1A复合物，或者它们可以用作替代受体，从而使霍乱毒素在没有GM1A的情况下结合细胞。 AIM 2的目的是通过使用shrnas瞬时敲低这些替代结合伴侣的表达和糖基化抑制剂来区分这些可能性，以干扰其转化后修饰。这些实验的结果将使我们能够确定CTXB与T84细胞结合的新受体或受体是否需要。虽然替代结合伴侣可能会促进CTXB结合，但更关键的问题是它们是否在毒素的内在化中起作用。 AIM 3中提出的免疫荧光显微镜实验和氯离子分泌测定的结果将揭示以前未识别的CTXB受体是否有助于霍乱中毒的过程。该项目挑战了GM1A对于CTXB的结合和内在化既需要且足够的现有范式。除了对我们对霍乱疾病进展的理解的影响之外，提出的实验的结果对于理解和区分不同内吞作用机理至关重要。此外，由于CTXB通常用于识别和可视化脂质筏，因此对CTXB结合伴侣的完整知识对于定义膜微区的组成和特征至关重要。 公共卫生相关性：细菌弧菌霍乱产生与人类肠细胞结合并侵入人类肠道细胞的毒素，从而导致霍乱的大量腹泻，以表征霍乱感染。长期以来，霍乱毒素一直被认为与称为GM1A的分子相互作用，但是我们的数据表明毒素也可能与其他分子或分子相互作用。我们建议确定霍乱毒素相互作用的其他分子，并确定这些相互作用对疾病进展至关重要。

项目成果

Photoaffinity probes for studying carbohydrate biology.

用于研究碳水化合物生物学的光亲和探针。

• DOI: 10.1080/07328303.2012.676118
• 发表时间: 2012
• 期刊: Journal of carbohydrate chemistry
• 影响因子: 1
• 作者: Yu,Seok-Ho;Wands,AmberlynM;Kohler,JenniferJ
• 通讯作者: Kohler,JenniferJ