Photocrosslinking characterization of heat-labile toxin glycoreceptors

描述（由申请人提供）：产肠毒素大肠杆菌（ETEC）在发展中国家流行，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菌株产生的毒力因子称为热不稳定毒素（LT），可通过有害地破坏肠道电解质平衡而导致这些疾病症状。LT的五聚体B亚基（LTB）通过作为细菌凝集素激活这一过程，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到宿主细胞表面。迄今为止，LTB用于执行该过程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涎神经节苷脂GM1a，但其靶向结合袋也可以容纳携带末端N-乙酰乳胺（Gal¿1-4GlcNAc¿1-3-R）表位的糖缀合物（尽管亲和力明显较弱）。此外，还存在第二个（外周）结合位点，与GM1a口袋不同，能够结合血型a和B抗原，主要是通过与这些末端三糖的直接接触。目前，这些非gm1a配体在LTB细胞表面结合/发病机制中的作用尚不清楚，这些聚糖结构偶联的蛋白质和/或脂质的身份仍未确定。这是因为碳水化合物与蛋白质的相互作用具有瞬时/低亲和力，因此很难用传统的生化方法来研究。该研究项目的长期目标是：(1)通过在聚糖结构内的特定类型的单糖上插入交联剂，开发可用于共价捕获细胞表面结合伙伴的方法；(2)使用该技术来回答正在进行的问题，即在宿主细胞感染期间，热不稳定毒素的糖蛋白和糖脂结合伙伴负责其内化。本提案的目的1是生物合成地将重氮嘧啶修饰的唾液酸残基（又名SiaDAz）整合到人肠上皮细胞系的GM1a和其他细胞表面糖缀合物中（最接近模拟LTB感染部位）。在光照射下，重氮嘧啶被激活形成

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.