Photocrosslinking characterization of heat-labile toxin glycoreceptors

热不稳定毒素糖受体的光交联表征

• 批准号: 8457586
• 负责人: Amberlyn M Wands
• 金额: $ 5.22万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8457586
• 关键词: Achievement; Acute; Address; Affect; Affinity; Antigens; Area; Binding; Binding Sites; Biochemical; Blood Group Antigens; Blood typing procedure; Carbohydrates; Cause of Death; Cell Line; Cell surface; Cells; Ceramides; Cessation of life; Chemicals; Child; Childhood; Cholera Toxin; Complement; Complex; Crosslinker; Data; Developed Countries; Developing Countries; Development; Diarrhea; Diazomethane; Disease; Electrolyte Balance; Engineering; Enterotoxins; Epithelial Cells; Epitopes; Escherichia coli Infections; Event; Future; Galactose; Gangliosides; Glycobiology; Glycoconjugates; Glycolipids; Glycoproteins; Goals; Heating; Human; Infection; Intestines; Intoxication; Knowledge; Lead; Lectin; Left; Life; Ligands; Lipids; Mammalian Cell; Mass Spectrum Analysis; Mediating; Methods; Modification; Molecular Weight; Monosaccharides; N-acetyllactosamine; Nature; Outcome; Pathogenesis; Peripheral; Persons; Play; Polysaccharides; Process; Protein-Carbohydrate Interaction; Proteins; Research; Role; Sialic Acids; Site; Structure; Symptoms; T-Lymphocyte; Techniques; Technology; Therapeutic; Toxin; Travel; Traveler' s diarrhea; Trisaccharides; Vibrio cholerae; Virulence Factors; Visit; adduct; analog; blood group; crosslink; enterotoxigenic Escherichia coli; new therapeutic target; novel; pathogen; poly-N-acetyllactosamine; programs; public health relevance; receptor; research study; sialogangliosides; sugar

DESCRIPTION (provided by applicant): Enterotoxigenic Escherichia coli (ETEC) is endemic to developing countries, in which acute infectious diarrhea is the second most common cause of childhood death, and millions of traveler's per year from the industrialized world are affected. A virulence factor produced by some strains of ETEC called the heat labile toxin (LT) can lead to these disease symptoms by detrimentally upsetting the balance of electrolytes in the intestine. The pentameric B subunit (LTB) of LT instigates this process by acting as a bacterial lectin, thereby localizing the toxin to the host cell surface through protein-carbohydrate interactions with the proper receptor(s). To date, the complexity of the number of binding partners utilized by LTB to carry out this process is likely underestimated. The primary host receptor for LTB is believed to be that of the monosialoganglioside GM1a, but its targeted binding pocket can also accommodate glycoconjugates carrying terminal N- acetyllactosamine (Gal¿1-4GlcNAc¿1-3-R) epitopes (albeit with significantly weaker affinity). Furthermore, there also exists a second (periphery) binding site distinct from the GM1a pocket capable of binding the blood group A and B antigens, primarily through direct contacts with these terminal trisaccharides. Currently, the role that these non-GM1a ligands play in LTB cell-surface binding/pathogenesis is still unclear, and the identity of the proteins and/or lipids that these glycan structures are conjugated to remains undetermined. This is because carbohydrate-protein interactions are notoriously difficult to study using traditional biochemical methods due to their transient/low affinity nature Therefore, the long term goals of this research program are (1) to develop methods that can be used to covalently capture cell-surface binding partners by inserting a crosslinker at specific types of monosaccharides within glycan structures and (2) to use this technology to answer the ongoing problem of which glycoprotein and glycolipid binding partners of the heat- labile toxin are responsible for its internalization during host cell infection. Aim 1 of this proposal is to biosynthetically incorporate diazirine-modified sialic acid residues (a.k.a, SiaDAz) into GM1a and other cell- surface glycoconjugates of human intestinal epithelial cell lines (which most closely mimic the site of LTB infection). Upon photoirradiation, the diazirine is activated to form a highly reactive crosslinker that forms a covalent adduct with nearby molecules, and thus can be used to capture binding partners of LTB. The modified protein/lipid receptors within these now stable complexes will be identified by mass spectrometry, and their ability to initiate LTB internalization verified. An analogous approach will be used in Aim 2 to identify functional GlcNAc-modified LTB receptors (a.k.a., GlcNDAz), such as those possessing N- acetyllactosamine structures. These experiments will mark an important achievement in understanding the relevant proteins involved in ETEC mediated disease, and thus uncover novel therapeutic targets to complement existing treatments.

描述（由申请人提供）：产肠毒素大肠杆菌（ETEC）是发展中国家的地方病，其中急性感染性腹泻是儿童死亡的第二大常见原因，每年有数百万来自工业化国家的旅行者受到某些细菌的影响。称为不耐热毒素 (LT) 的 ETEC 菌株可通过破坏肠道电解质平衡而导致这些疾病症状。 LT 的亚基 (LTB) 通过充当细菌凝集素来引发这一过程，从而通过蛋白质-碳水化合物与适当受体的相互作用将毒素定位到宿主细胞表面。迄今为止，所使用的结合配偶体数量非常复杂。 LTB 进行这一过程的作用可能被低估了，LTB 的主要宿主受体被认为是单唾液酸神经节苷脂 GM1a，但其靶向结合袋也可以容纳携带末端 N- 的糖缀合物。乙酰基乳糖胺（Gal¿ 1-4GlcNAc¿ 1-3-R）表位（尽管亲和力明显较弱）此外，还存在与 GM1a 口袋不同的第二个（外围）结合位点，能够主要通过与这些末端直接接触来结合 A 型和 B 型抗原。目前，这些非 GM1a 配体在 LTB 细胞表面结合/发病机制中所起的作用仍不清楚，并且这些聚糖结构所缀合的蛋白质和/或脂质的身份仍然不清楚。这是因为碳水化合物-蛋白质相互作用由于其瞬时/低亲和力性质而很难使用传统的生化方法进行研究。因此，该研究计划的长期目标是（1）开发可用于共价捕获的方法。通过在聚糖结构内的特定类型的单糖上插入交联剂来形成细胞表面结合伴侣，并且（2）使用该技术来解决目前存在的问题，即热不稳定毒素的糖蛋白和糖脂结合伴侣负责该提案的目标 1 是将二氮丙啶修饰的唾液酸残基（又名 SiaDAz）通过生物合成方法整合到人肠上皮细胞系的 GM1a 和其他细胞表面糖缀合物中（最接近地模拟 LTB 的位点）。光照射后，二嗪被激活形成。 一种高反应性交联剂，与附近的分子形成共价加合物，因此可用于捕获这些现已稳定的复合物中的修饰蛋白/脂质受体，并通过质谱法鉴定它们启动 LTB 内化的能力。目标 2 中将使用类似的方法来鉴定功能性 GlcNAc 修饰的 LTB 受体（又名 GlcNDAz），例如那些具有 N- 的受体。这些实验将标志着了解 ETEC 介导疾病相关蛋白质的重要​​成就，从而揭示新的治疗靶点来补充现有的治疗方法。