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

项目概要 装饰细胞表面糖复合物的聚糖代表了丰富的分子语言 介导许多重要的生物功能。在细菌中，这些糖复合物至关重要 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。理解复杂的角色的一个重大挑战 细菌中的聚糖是单糖构建块和糖苷多样性的库 相对于真核生物，糖缀合物中反映的连接大大扩展。 因此，目前可用的聚糖结合蛋白，包括凝集素和单克隆 抗体根本不足以检测大多数细菌聚糖表位。鉴于 细菌聚糖在人类传染病中的重要性，实验的发展 试剂作为“聚糖读取器”，选择性地表征和监测病原体特异性聚糖 当前，决定因素至关重要。针对细菌聚糖的选择性聚糖读取器 表位有望成为鉴定细菌病原体和了解细菌病原体的有价值的试剂 糖缀合物在传染病中的生物学意义。  在这个探索性研究计划中，我们的目标是建立工程原理验证 基于蛋白质的聚糖阅读器，用于检测和分析病原体特异性聚糖和 糖复合物。该提案包括两个目标。 目标 1 将涉及制备化学定义的空肠弯曲菌聚糖表位，包括假胺 酸和N-乙酰基杆糖胺，它们是原核生物特异性碳水化合物。这些聚糖表位 将通过已建立的连接化学物质武装生物素，用于新型聚糖的定向进化 结合蛋白，基于 Sso7d 支架，使用酵母表面展示。 目标 2 将利用蛋白质工程开发进化的模块化聚糖结合剂的应用 方法，重点是研究与疾病相关的细菌聚糖的实用性。特别是， 分选酶介导的连接将用于修饰聚糖结合物的 C 和 N 末端 包括荧光团和生物素。这些“聚糖阅读器”对于以下应用很有价值： 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，叠氮化物修饰的聚糖阅读器将易于点击 与炔烃的基于化学的缀合反应能够实现多价显示等 利用贪欲效应。

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.