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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

基本信息

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
项目类别：
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

项目摘要

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.

项目总结