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.

性状（由申请方提供）：霍乱毒素亚单位B（CTx B）与宿主肠上皮细胞表面呈递的分子结合。在最初的结合事件之后，全毒素侵入细胞，劫持细胞机器，导致霍乱感染的特征性大量腹泻。全面了解霍乱毒性的机制细节是必要的，以促进新的战略，以打击霍乱疫情的发展。虽然神经节苷脂GM 1a被广泛描述为CTxB识别的单一受体，但我们实验室和其他实验室的最新数据表明存在其他霍乱毒素结合伴侣，可能是糖蛋白。该提案的主要目标是确定新的CTxB结合伴侣，并发现它们是否在霍乱中毒中发挥作用。目的1的目标是鉴定T84细胞中被CTxB识别的细胞表面分子，利用新颖的光交联技术和现代质谱方法，沿着传统的亲和纯化方法。另外的CTxB结合伴侣可以发挥辅助作用，帮助形成典型的CTxB-GM 1a复合物，或者它们可以作为替代受体，使霍乱毒素能够在缺乏GM 1a的情况下结合细胞。目的2的目标是通过使用shRNA瞬时敲低这些替代结合配偶体的表达和糖基化抑制剂干扰其翻译后修饰来区分这些可能性。这些实验的结果将使我们能够确定CTxB与T84细胞结合是否需要新的受体。虽然替代结合伴侣可能促进CTxB结合，但更关键的问题是它们是否在毒素的内化中起作用。目标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之前未鉴定的CtxB受体是否促进霍乱中毒过程。该项目挑战了现有的范式，即GM 1a对于CTxB结合和内化是必要的和足够的。沿着对我们理解霍乱疾病进展的影响，所提出的实验的结果对于理解和区分内吞作用的不同机制将是重要的。此外，由于CTxB通常用于识别和可视化脂筏，CTxB结合伙伴的完整知识对于定义膜微区的组成和特性至关重要。 公共卫生相关性：霍乱弧菌产生一种毒素，这种毒素能结合并侵入人体肠道细胞，引起大量腹泻，这是霍乱感染的特征。长期以来，人们一直认为霍乱毒素与一种称为GM 1a的分子相互作用，但我们的数据表明，这种毒素也可能与其他一种或多种分子相互作用。我们建议确定与霍乱毒素相互作用的其他分子，并确定这些相互作用是否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