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

用于细菌糖复合物检测和分析的聚糖读数器的开发和应用

• 批准号: 9295172
• 负责人: Barbara Imperiali
• 金额: $ 23.21万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-16 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9295172
• 关键词: Acids; Address; Affinity; Affinity Chromatography; Alkynes; Antibodies; Avidity; Azides; Bacteria; Bacterial Infections; Binding; Binding Proteins; Biochemical; Biological; Biological Process; Biology; Biomedical Engineering; Biotin; Calorimetry; Campylobacter jejuni; Carbohydrates; Cell surface; Cells; Characteristics; Chemicals; Chemistry; Communicable Diseases; Communication; Communities; Complex; Detection; Development; Directed Molecular Evolution; Disaccharides; Disease; Dissociation; Engineering; Epithelial Cells; Epitopes; Equilibrium; Flagella; Flagellin; Fluorescence-Activated Cell Sorting; Gastrointestinal tract structure; Glycoconjugates; Glycolipids; Glycoproteins; Glycosides; Health; Hexoses; Human; Image; Imagery; Interferometry; Label; Language; Lectin; Libraries; Ligation; Light; Link; Mediating; Medical; Medicine; Microbiology; Molecular; Monitor; Mono-S; Monoclonal Antibodies; Monosaccharides; Organism; Pathogenesis; Pathogenicity; Play; Polymers; Polysaccharides; Population; Preparation; Prokaryotic Cells; Protein Engineering; Proteins; Reaction; Reader; Reagent; Research; Role; Scaffolding Protein; Specificity; Structure; Surface; Systemic infection; Technology; Titrations; Universities; Validation; Variant; Yeasts; base; biophysical techniques; carbohydrate receptor; cell motility; design; experience; experimental study; fluorophore; glycosylation; human disease; interest; microorganism; mutant; novel; pathogen; periplasm; practical application; programs; scaffold; sortase; sugar

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.

项目摘要 装饰细胞表面糖缀合物的聚糖代表一种丰富的分子语言 这介导了无数重要的生物学功能。在细菌中，这些糖缀合物很关键 确定细菌群落之间以及致病和之间的相互作用 共生细菌和人类宿主细胞。理解复杂角色的主要挑战 细菌中的聚糖是单糖构建块和糖苷多样性的池 相对于真核生物，反映在糖缀合物中的连接大大扩展。 因此，当前可用的聚糖结合蛋白包括讲座和单克隆 抗体只是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 作为“聚糖读取器”的试剂，有选择地表征和监测病原体特异性聚糖 决定因素至关重要。选择性聚糖读者朝着细菌聚糖 表位有望成为识别细菌病原体并了解的有价值试剂 糖缀合物在传染病中的生物学意义。 在此探索性研究计划中，我们旨在为工程的原理验证 基于蛋白质的聚糖读取器，用于检测和分析病原体特异性聚糖和 糖缀合物。该建议包括两个目标。 AIM 1将涉及化学定义的J. Jejuni Glycan表位，包括假胺激素 酸和N-乙酰基芽孢胺，它们是原核特异性碳氢化物。这些聚糖表位 通过既定的接头化学，将与生物素一起武装，用于新型聚糖的定向演变 使用酵母表面显示器基于SSO7D支架的结合蛋白。 AIM 2将使用蛋白质工程开发进化的模块化聚糖粘合剂的应用 方法，重点是用于研究与疾病相关细菌的效用。尤其， 分子酶介导的连接将用于将聚糖粘合剂的C和N末端修改为 包括荧光团和生物素。这些“聚糖读者”对于包括 聚糖阵列可视化，实时和固定成像，荧光激活的细胞分选（FACS）和 亲和色谱。此外，叠氮化物改性的聚糖读取器将可以单击 基于化学的结合反应与炔烃相同，例如，多价显示到 利用自然效应。