Development and application of glycan readers for the detection and analysis of bacterial glycoconjugates

项目摘要 修饰细胞表面糖偶联物的多糖代表了一种丰富的分子语言 这调节了无数重要的生物功能。在细菌中，这些糖偶联物是至关重要的 细菌群落之间以及致病菌和致病菌之间相互作用的决定因素 共生细菌和人类宿主细胞。理解Complex的作用的一个主要挑战 细菌中的葡聚糖是单糖池中的积木和多样性的糖苷 与真核生物相比，反映在糖共轭中的连接大大扩展。 因此，目前可用的糖链结合蛋白，包括凝集素和单抗 抗体，根本不足以检测大多数细菌多聚糖表位。鉴于 细菌多糖在人类感染性疾病中的重要性及其实验研究进展 作为“葡聚糖阅读器”的试剂，用于选择性地表征和监测病原体特异性的糖链 决定因素是当前最重要的。细菌多聚糖选择性读取器 表位有望成为识别细菌病原体和了解 糖结合物在传染病中的生物学意义。 在这个探索性的研究计划中，我们的目标是建立工程化的原则证明 基于蛋白质的糖链读取器，用于检测和分析病原体特异的糖链和 糖偶联物。这项提议包括两个目标。 目标1将涉及制备化学定义的空肠弯曲菌多聚糖表位，包括伪氨基 酸和N-乙酰杆菌胺，这是原核生物特有的碳水化合物。这些糖链表位 将通过已建立的连接物化学，用生物素武装新的糖链的定向进化 结合蛋白，基于Sso7d支架，使用酵母表面展示。 AIM 2将利用蛋白质工程开发进化的模块化多糖结合剂的应用 方法，重点是研究与疾病相关的细菌多糖的实用性。特别是， 将应用山梨酸酶介导的连接来修饰多糖结合体的C-端和N-端以 包括荧光团和生物素。这些“葡聚糖阅读器”将是有价值的应用，包括 多聚糖阵列可视化、实时和固定成像、荧光激活细胞分选(FACS)和 亲和层析。此外，叠氮修饰的葡聚糖阅读器将可以点击 基于化学的与炔烃的共轭反应，例如，使多价显示能够 利用贪婪效应。

Project Summary The glycans that decorate cell-surface glycoconjugates, represent a rich molecular language that mediates a myriad of important biological functions. In bacteria, these glycoconjugates are critical determinants in interactions amongst bacterial communities and between both pathogenic and symbiotic bacteria and human host cells. A major challenge in understanding the roles of complex glycans in bacteria is that the pool of monosaccharide building blocks and diversity of glycosidic linkages reflected in the glycoconjugates is greatly expanded relative to eukaryotic organisms. Therefore, the currently-available glycan-binding proteins, which include lectins and monoclonal antibodies, are simply inadequate for detecting a majority bacterial glycan epitopes. In light of the importance of bacterial glycans in human infectious disease, the development of experimental reagents, as “glycan readers”, to selectively characterize and monitor pathogen-specific glycan determinants is of utmost current importance. Selective glycan readers towards bacterial glycan epitopes promise to be valuable reagents for identifying bacterial pathogens and understanding the biological significance of glycoconjugates in infectious disease.  In this exploratory research program, we aim to establish proof-of-principle for engineered protein-based glycan readers for the detection and analysis of pathogen-specific glycans and glycoconjugates. This proposal includes two aims. Aim 1 will involve preparation of chemically-defined C. jejuni glycan epitopes, including pseudaminic acid and N-acetyl bacillosamine, which are prokaryote-specific carbohydrates. These glycan epitopes will be armed, via established linker chemistry, with biotin for directed evolution of novel glycan binding proteins, based on the Sso7d scaffold, using yeast surface display. Aim 2 will develop applications of the evolved modular glycan binders using protein engineering approaches, with a focus on utility for the study of disease-related bacterial glycans. In particular, sortase-mediated ligation will be applied for modifying the C- and N-termini of glycan binders to include fluorophores and biotin. These “glycan readers” will be valuable for applications including glycan array visualization, live and fixed imaging, fluorescence-activated cell sorting (FACS) and affinity chromatography. In addition, azide-modified glycan readers will be amenable to click chemistry-based conjugation reactions with alkynes enabling, for example, multivalent display to exploit avidity effects.