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.

项目概要 口腔是宿主体内潜在有害物质或病原体之间的第一个关键界面 环境，以及适应性和先天免疫防御机制的进化，以灭活或消除 病原微生物在很大程度上利用了蛋白质-聚糖相互作用。唾液中的聚糖成分 已被证明在口腔宿主微生物的生物学和免疫学方面发挥重要作用，微生物 宿主和微生物之间的相互作用，我们已经证明，附着在唾液糖蛋白上的聚糖可以 作为人类口腔中宿主防御的第一道防线，聚糖识别有助于定植 和口腔微生物的清除。蛋白质-聚糖相互作用研究的革命性创新是 聚糖微阵列，可以识别生物学相关聚糖结合的聚糖配体 通过在显微镜上打印的数百个聚糖结构同时询问蛋白质（GBP） 滑动。结合和非结合聚糖结构的比较揭示了 GBP 的聚糖特异性，并且 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糖组的聚糖组成的聚糖微阵列，即整体 唾液中的聚糖。这将通过首先产生纯化的、天然存在的 N-和 通过创新的氧化工艺，从 10 升汇集的人类唾液中释放出 O-连接聚糖 使用简单的家用漂白剂释放天然聚糖 (ORNG)。聚糖库将被打印为 聚糖微阵列并用口腔链球菌聚糖结合粘附素进行询问以鉴定其 相应的天然高亲和力聚糖配体。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附素，每个粘附素都含有不同的唾液酸结合 Siglec 样结构域 唾液酸亚型在其潜在的亚末端聚糖的更广泛背景下的特异性。对于一些 使用目前可用的阵列无法识别这些凝集素，天然聚糖配体，大概是由于 迄今为止的测定中不存在相应的天然聚糖配体。因为这些是凝集素 由口腔微生物表达，我们预计它们的天然聚糖配体很可能是 存在于人类唾液的糖组中，唾液是这些微生物繁衍生息的天然生物液体。这 第一阶段项目应该提供一个例子，说明如何将完整的糖组用作发现平台 鉴定一种新的蛋白质-聚糖相互作用。一旦聚糖配体的结构细节被确定， 这些信息将成为知识产权，从而制定治疗干扰的策略 口腔内外微生物定植或病原体感染。这个平台让大家了解 口腔微生物和口外系统病原体与人类的相互作用最终可能导致 开发改进的人造唾液，更接近地模仿自然人类的聚糖景观 唾液中添加人乳低聚糖，为婴儿配方奶粉的改进奠定了基础。