A Human Salivary Glycome Discovery Platform for Interrogating Glycan Function in Oral Innate Immunity

用于询问口腔先天免疫中聚糖功能的人类唾液糖发现平台

• 批准号: 10484608
• 负责人: David Fletcher Smith
• 金额: $ 31.5万
• 依托单位: NATGLYCAN, LLC
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10484608
• 关键词: Address; Affinity; Artificial Saliva; Bacterial Adhesins; Binding; Binding Proteins; Biological; Biological Assay; Biological Process; Biology; Carbohydrates; Catalogs; Chemicals; Chemistry; Communities; Complex; Defense Mechanisms; Deglutition; Development; Distant; Drug Targeting; Environment; Epithelial; Event; Evolution; Fluorescent Probes; Food; Fostering; Gatekeeping; Glycoproteins; Glycoside Hydrolases; High Pressure Liquid Chromatography; Host Defense; Host Defense Mechanism; Household; Human; Human Milk; Immune; Immunoglobulin G; Immunologics; Indigenous; Individual; Infant formula; Infection; Intellectual Property; Investigation; Label; Lead; Lectin; Libraries; Ligands; Link; Liquid substance; Mannose; Mechanics; Mediating; Medical Research; Methods; Microbe; Microscope; Minerals; Mucinous; Natural Products; Nucleic Acids; Oligosaccharides; Oral; Oral cavity; Pathologic; Phase; Play; Polysaccharides; Preparation; Probability; Procedures; Process; Proteins; Proteome; Recombinants; Role; Route; Sales; Saliva; Salivary; Salivary Proteins; Serine; Shotguns; Sialic Acids; Slide; Source; Specificity; Streptococcus; Structure; Surface; Technology; Therapeutic; Tissues; Viral; Virus; base; commensal microbes; design; drug development; improved; innovation; insight; microbial; microbial colonization; microbial host; microbiota; microorganism; milligram; novel; oral innate immunity; oral microbiome; oral streptococci; oxidation; pathogen; pathogenic microbe; phase 1 study; pressure; prevent; salivary mucins; scale up; sialic acid binding Ig-like lectin; tooth surface

Project summary The oral cavity is the first critical interface between potentially harmful substances or pathogens in the host environment, and evolution of the adaptive and innate immune defense mechanisms to inactivate or eliminate pathogenic microbes has to a great extent used protein-glycan interaction. The glycan components of saliva have been shown to play important roles in biological and immunological aspects of oral host-microbe, microbe- host, and microbe-microbe interactions, and we have shown that glycans attached salivary glycoproteins can act as a first line of host defense in the human mouth and that glycan recognition contributes to both colonization and clearance of oral microbes. A revolutionary innovation in studies of protein-glycan interactions was the glycan microarray, which permitted the identification glycan ligands for biologically relevant glycan binding proteins (GBP) by their simultaneous interrogation with hundreds of glycan structures printed on a microscope slide. Comparisons of the structures of bound and non-bound glycans reveal the glycan specificity of GBP, and this information is used in further biological studies to understand the biological function of GBP. In this project we will generate a glycan microarray composed of glycans that represent the salivary glycome, i.e. the entirety of glycans in saliva. This will be accomplished by first producing a library of purified, naturally occurring N- and O-linked glycans released from 10 liters of pooled human saliva by an innovative process for the Oxidative Release of Natural Glycans (ORNG) using simple household bleach. The library of glycans will be printed as a glycan microarray and interrogated with oral streptococcal glycan-binding adhesins to identify their corresponding natural high-affinity glycan ligands. We will use our recently established toolbox of streptococcal serine-rich-repeat protein adhesins that each contain sialic-acid-binding Siglec-like domains of differing specificity for subtypes of sialic acids in the wider context of their underlying subterminal glycans. For some of these lectins, the natural glycan ligands were not identified using currently available arrays, presumably due to the absence of their corresponding natural glycan ligands presented in the assays to date. Since these are lectins expressed by oral microbes, we anticipate that there is a high probability that their natural glycan ligands will be present in the glycome of human saliva, the natural biological fluid in which these microorganisms thrive. This Phase I project should provide an example of how a complete glycome can be used as a discovery platform for identifying a novel protein-glycan interaction. Once the details of the structure of the glycan ligand are defined, the information will become intellectual property that will lead to strategies to therapeutically interfere with microbial colonization or pathogen infection in the mouth and beyond. This platform for understanding the interactions of oral microorganisms and extraoral systemic pathogens with the human may ultimately lead to the development of an improved artificial saliva, more closely mimicking the glycan landscape of natural human saliva, as addition of human milk oligosaccharides has laid the groundwork for improvement of infant formula.

项目摘要 口腔是宿主中潜在有害物质或病原体之间的第一个关键接口 环境，以及自适应和先天免疫防御机制灭活或消除的环境的演变 致病性微生物必须在很大程度上使用蛋白质 - 聚糖相互作用。唾液的聚糖成分 已显示在口服宿主微叶片，微生物的生物学和免疫学方面起着重要作用 宿主和微生物 - 微生物相互作用，我们已经表明唾液糖蛋白可以 充当人口中宿主防御的第一线，而聚糖识别有助于两种殖民化 和口服微生物的清除。蛋白质 - 聚糖相互作用研究中的革命性创新是 Glycan微阵列，该微阵列允许识别用于生物学相关的聚糖结合的聚糖配体 蛋白质（GBP）通过其同时询问，并在显微镜上印刷了数百种聚糖结构 滑动。比较结构和非结合聚糖的结构揭示了GBP的聚糖特异性，以及 该信息用于进一步的生物学研究，以了解GBP的生物学功能。在这个项目中 我们将生成一个由代表唾液甘氨酸的聚糖组成的聚糖微阵列，即整个 唾液中的聚糖。这将通过首先制作出纯化的，自然发生的n-和 通过氧化的创新过程从10升合并的人类唾液中释放出O连接的聚糖 使用简单的家庭漂白剂释放天然聚糖（ORNG）。聚糖库将被打印为 聚糖微阵列并用口服链球菌聚糖结合粘合剂进行讯问以识别其 相应的天然高亲和力配体。我们将使用我们最近建立的链球菌工具箱 富含丝氨酸的重复蛋白粘合剂，每种含有不同的唾液 - 酸性结合siglec样域 在其潜在的亚末端聚糖的更广泛背景下，唾液酸亚型的特异性。对于某些 这些凝集素，天然聚糖配体未使用当前可用阵列鉴定，大概是由于 迄今为止，在测定中介绍了它们相应的天然聚糖配体。因为这些是凝集素 通过口服微生物表达，我们预计它们的天然聚糖配体可能很高 存在于人类唾液的糖果中，唾液是这些微生物繁殖的天然生物学流体。这 第一阶段项目应提供一个示例，说明如何将完整的糖作为发现平台 识别一种新型的蛋白质 - 聚糖相互作用。一旦定义了聚糖配体结构的细节， 该信息将成为知识产权，将导致治疗策略干扰 微生物定植或口腔中的病原体感染。这个平台用于理解 口服微生物和口腔系统病原体与人的相互作用最终可能导致 改进的人造唾液的发展，更紧密地模仿天然人类的聚糖景观 唾液作为添加人乳寡糖为改善婴儿配方奶粉的基础奠定了基础。