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（由适用提供）：肠毒素大肠杆菌（ETEC）对发展中国家是内在的，其中急性感染性腹泻是儿童死亡的第二大最常见原因，而工业化世界中每年数百万的旅行者受到影响。某些ETEC产生的病毒因子称为热标记毒素（LT）可以通过确定肠中电解质平衡来导致这些疾病症状。 LT的五聚体B亚基（LTB）通过充当细菌授课来激发此过程，从而通过蛋白质 - 碳水化合物与适当的受体相互作用将毒素定位在宿主细胞表面上。迄今为止，LTB使用的绑定伙伴数量的复杂性可能被低估了。 LTB的主要宿主受体被认为是单糖蛋白GM1A的宿主受体，但其靶向结合口袋还可以容纳携带末端N-乙酰乳乙胺胺（GAL¿1-4GlCNACTO）（1-4GlCNAC？1-3-R）的糖缀合物（均具有明显的弱点）。此外，还存在第二个（周围）结合位点，与能够结合血型A和B抗原的GM1A袋不同，主要是通过与这些末端三糖的直接接触。当前，这些非GM1A配体在LTB细胞表面结合/发病机理中起的作用尚不清楚，并且这些聚糖结构被缀合以保持不确定。这是因为众所周知，由于其短暂性/低亲和力性质而难以研究碳水化的蛋白质相互作用，因此很难研究，因此，该研究计划的长期目标是（1）开发可用于共价捕获细胞表面捕获的细胞表面结合伙伴的细胞表面粘合剂在特定类型中的跨性别结构的方法和2（2热法毒素的糖蛋白和糖脂结合伙伴在宿主细胞感染期间的内在化负责。该提案的目标1是将生物合成的重氮嘧啶修饰的唾液酸保留（又称Siadaz）中的GM1A和其他细胞表面糖缀合物，该细胞表面糖缀合物（最紧密地模拟了LTB感染的部位）。光辐射后，重氮嗪被激活形成 一种高反应性的交联链，形成与近分子的共价加合物，因此可用于捕获LTB的结合伴侣。这些现在稳定的复合物中的修饰蛋白/脂质受体将通过质谱鉴定，并通过验证了LTB内在化的能力。 AIM 2将使用类似的方法来识别功能性GlcNAC修饰的LTB受体（又称GLCNDAZ），例如具有N-乙酰乳酸胺结构的功能。这些实验将标志着在理解ETEC介导疾病涉及的相关蛋白质方面的重要成就，从而发现了新的治疗靶标以完成现有治疗方法。