Shotgun Glycomics: Linking Glycan Structure and Function

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DESCRIPTION (provided by applicant): The diversity and size of the human glycome is unknown. Recent studies have revealed that the human genome encodes thousands of glycoproteins, and that glycosylation is likely the most common type of post-translational modification. But how many different glycans are there? What are their relative levels of expression? Does each glycan structure have a function, either in human biology or in disease processes, such as innate immunity and resistance to infection? How does glycan expression change in response to age and disease? It has been known for decades that different cells and tissues make different glycan structures, and recent advances in methods for analysis of glycan structures has led to modern glycomics, which is mainly devoted to identifying glycan structures. Unfortunately, while knowing chemical structures is important, in a sense it only confirms what we already know; different cells make different glycan structures. While knowledge of all glycan structures may solve a chemical puzzle, it does not provide a biological solution to glycan function. We propose a new glycomic approach to link glycan structure to function/recognition using our new developments in "glyco-chemistry" that allow us to fluorescently tag free glycans released from specific cell or tissue glycoconjugates. These novel, bifunctional, fluorescent tags allow us to detect sub-microgram quantities of glycans that are otherwise undetectable, and purify them by chromatographic methods to obtain tissue- and/or cell- specific tagged-glycan libraries (TGLs). The tagged glycans retain a functional group to allow covalent "printing" of these libraries as microarrays. Such microarrays can subsequently be interrogated with glycan binding proteins (GBPs), pathogens, and cells to identify functionally recognized and biologically significant glycans, which will then be structurally defined. This novel approach avoids the laborious process of defining all glycan structures in a glycome regardless of their functional or recognition, and allows us to focus structural analyses on potentially biological relevant glycans. Developing glycomic TGLs is analogous to the "Shotgun" approach to defining the human genome, but allows us to target our analyses to link glycan structure to function. Defining all of the glycan structures of the human glycome is analogous to sequencing the human genome. The complexity of the human glycome and the lack of automated glycan sequencing methods make a "brute-force" approach to defining the glycome impractical. Here we propose a paradigm- shifting strategy, which is a combination of "shotgun" and targeted approaches. We will assemble amino functionalized, fluorescent-tagged glycan libraries from specific cells or tissues, print them as glycan microarrays, identify potentially biologically relevant glycans that are recognized by glycan binding proteins, and structurally define the recognized glycans. The stable, libraries and glycan microarrays produced will remain as a tangible resource for future analyses of both glycan structure and function.