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影响。由某些ETEC菌株产生的称为热不稳定毒素（LT）的毒力因子可通过破坏肠道内电解质平衡而导致这些疾病症状。LT的五聚体B亚基（LTB）通过充当细菌凝集素来引发该过程，从而通过与适当受体的蛋白质-碳水化合物相互作用将毒素定位于宿主细胞表面。到目前为止，LTB用来进行这一过程的结合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唾液酸神经节苷脂GM 1a的受体，但其靶向结合口袋也可以容纳携带末端N-乙酰乳糖胺（Gal <$1 -4GlcNAc <$1 -3-R）表位的糖缀合物（尽管亲和力明显较弱）。此外，还存在不同于GM 1a口袋的第二（外周）结合位点，其能够主要通过与这些末端三联体直接接触而结合血型A和B抗原。目前，这些非GM 1a配体在LTB细胞表面结合/发病机制中发挥的作用仍不清楚，这些聚糖结构缀合的蛋白质和/或脂质的身份仍不确定。这是因为碳水化合物-蛋白质相互作用由于其瞬时/低亲和力的性质而非常难以使用传统的生物化学方法进行研究。这项研究计划的长期目标是（1）开发通过在聚糖结构内的特定类型单糖上插入交联剂来共价捕获细胞表面结合配偶体的方法，和（2）利用这项技术来回答目前存在的问题，即不耐热毒素的糖蛋白和糖脂结合配偶体负责其在宿主细胞感染期间的内化。该提议的目的1是将二氮丙啶修饰的唾液酸残基（a.k.a，SiaDAz）生物合成地掺入人肠上皮细胞系的GM 1a和其他细胞表面糖缀合物（其最接近地模拟LTB感染的位点）中。在光照射时，二氮丙啶被活化以形成 一种高反应性交联剂，与附近分子形成共价加合物，因此可用于捕获LTB的结合配偶体。这些现在稳定的复合物中的修饰的蛋白质/脂质受体将通过质谱法鉴定，并且它们启动LTB内化的能力得到验证。在目的2中将使用类似的方法来鉴定功能性GlcNAc修饰的LTB受体（也称为，GlcNDAZ），如具有N-乙酰基乳糖胺结构的那些。这些实验将标志着在理解ETEC介导的疾病中涉及的相关蛋白质方面的重要成就，从而发现新的治疗靶点，以补充现有的治疗方法。