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 und...