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

项目摘要 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，以消除或消除 病原微生物在很大程度上利用蛋白质-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、微生物 宿主和微生物-微生物相互作用，我们已经表明，聚糖附着的唾液糖蛋白可以 作为人体口腔中宿主防御的第一道防线，聚糖识别有助于定植 和清除口腔微生物。蛋白质-聚糖相互作用研究中的一项革命性创新是 聚糖微阵列，其允许鉴定用于生物学相关聚糖结合的聚糖配体 蛋白质（GBP）通过与显微镜上打印的数百个聚糖结构同时进行询问 滑盖.结合和非结合聚糖结构的比较揭示了GBP的聚糖特异性， 该信息用于进一步的生物学研究以理解GBP的生物学功能。在这个项目中 我们将产生由代表唾液糖组的聚糖组成的聚糖微阵列， 唾液中的聚糖这将通过首先产生纯化的、天然存在的N-和N-半胱氨酸的文库来实现。 通过创新工艺从10升混合人唾液中释放出O-连接聚糖， 使用简单家用漂白剂释放天然聚糖（ORNG）。聚糖库将打印为 聚糖微阵列，并用口服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 富含丝氨酸重复序列的蛋白质粘附素，其各自含有不同结构域的唾液酸结合Siglec样结构域， 在其基础亚末端聚糖的更广泛背景下，对唾液酸亚型的特异性。对于一些 这些凝集素，天然的聚糖配体，没有使用目前可用的阵列鉴定，大概是由于 在迄今为止的测定中不存在它们相应的天然聚糖配体。因为这些是凝集素 我们预计，它们的天然聚糖配体很有可能被口腔微生物表达， 存在于人类唾液的糖组中，唾液是这些微生物茁壮成长的天然生物液体。这 第一阶段项目应提供一个完整的糖组如何作为发现平台的例子， 鉴定新的蛋白质-聚糖相互作用。一旦确定了聚糖配体的结构细节， 这些信息将成为知识产权，这将导致治疗性干扰的策略， 微生物定植或口腔内外的病原体感染。这个平台可以让你了解 口腔微生物和口外系统性病原体与人类的相互作用可能最终导致 开发一种改进的人工唾液，更接近地模仿自然人类的聚糖景观， 唾液作为人乳低聚糖的添加为婴儿配方食品的改进奠定了基础。

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.