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

项目总结 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-多糖的相互作用。唾液中的多糖成分 已被证明在口腔宿主微生物的生物学和免疫学方面发挥着重要作用。 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葡聚糖可以 在人类口腔中起到第一道宿主防线的作用，而多糖的识别有助于两者的定植 和清除口腔微生物。蛋白质-多糖相互作用研究中的一项革命性创新是 葡聚糖微阵列，其允许鉴定与生物相关的葡聚糖结合的葡聚糖配体 用显微镜上打印的数百个糖链结构同时询问蛋白质(GBP) 滑行。结合和非结合多聚糖的结构比较揭示了GBP的多聚糖专一性。 这些信息被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 我们将产生一个由代表唾液糖类的多糖组成的葡聚糖微阵列，即整个 唾液中的葡聚糖。这将通过首先产生一个纯化的自然产生的N-和N-的文库来实现 通过一种创新的氧化工艺从10升汇集的人唾液中释放O-连接的葡聚糖 使用简单的家用漂白剂释放天然多糖(ORNG)。糖链文库将打印为 葡聚糖微阵列和口服链球菌葡聚糖结合粘附素的询问以鉴定其 相应的天然高亲和力葡聚糖配体。我们将使用我们最近建立的链球菌工具箱 富丝氨酸重复蛋白粘附素，每个粘附素都含有不同的唾液酸结合的Siglec样结构域 唾液酸亚型在其底层亚末端多聚糖的更广泛的背景下的特异性。对于某些人来说 这些凝集素，天然的葡聚糖配体没有使用目前可用的阵列进行鉴定，推测是由于 到目前为止，检测中没有相应的天然葡聚糖配体。因为这些是凝集素 由口腔微生物表达的，我们预计它们的天然多糖配体很可能是 存在于人类唾液中的糖类，这些微生物在其中茁壮成长的天然生物液体。这 第一阶段项目应该提供一个例子，说明如何将完整的糖蛋白用作 确定一种新的蛋白质-多糖相互作用。一旦定义了葡聚糖配体的结构细节， 这些信息将成为知识产权，将导致治疗干预的策略 口腔及口腔以外的微生物定植或病原体感染。此平台可帮助您了解 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 开发一种改进的人工唾液，更接近于自然人类的多糖景观 唾液作为人乳低聚糖的添加，为婴儿配方奶粉的改进奠定了基础。

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.