Smart Anti-Glycan Reagents to Generate the Human Glycome Atlas

Beth Israel Deaconess Medical Center, Boston MA

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Abstract

? DESCRIPTION (provided by applicant): The key unsolved problems in the glycosciences are the temporal and spatial expression of glycans, as well as the identification of glycans, the proteins to which they are attached, and their biological partners (glycan-binding proteins or GBPs) that recognize them. To define glycan expression new techniques are needed that are applicable to flow cytometry, immunohistochemistry, and small numbers of cells and small amounts of glycoconjugates. Here we propose to develop a suite of anti-glycan reagents whose glycan-binding specificities are well defined. Such reagents and novel approaches to create them will provide novel information about the temporal and spatial expression of glycans in cells and tissues, and provide accessible reagents for `non-glyco' experts. We will exploit new technologies to generate `smart anti-glycan reagents' (SAGRs) whose anti-glycan specificities are defined. These will be created from both murine single-chain variable fragments (scfv) and lamprey-derived antibodies (variable lymphocyte receptors or VLRs). (Aim 1) We will assemble a panel of 100 glycans with different glycan determinants, which include well-defined glycans generated by chemo/enzymatic methods, purified and available at milligram scale. These glycans will be derivatized to contain a primary amine for covalent coupling to solid surfaces for screening. (Aim 2a) We will screen scfv libraries for recognition of specific glycan determinants and characterize the scfv for their specificity of glycan recognition using glycan microarray technologies. (Aim 2b) We will immunize lampreys with cells expressing known and unknown glycan determinants, and select and screen for specificity as above. (Aim 3) We will use this suite of novel SAGRs to explore glycan expression in many different primary cells, immortalized cell lines, and human tissues. These highly specific and targeted reagents will allow researchers to begin mapping the Human Glycome Atlas, which will compile information about glycan expression.