Cell-Surface Glycan Editing Tools for Mapping Glycan Expression and Interactions in Cancer and Immunity

Abstract

Glycans are complex carbohydrate structures that decorate the surfaces of cells and influence every major facet of biology. Glycans play important modulatory roles by serving as ligands for glycan-binding proteins to trigger crucial signaling and functional events. Dysregulation of glycan-mediated signaling is implicated in disease states, including cancer, autoimmunity, and infection. Sialic acid monosaccharides are particularly central to these interactions through recognition by the family of Siglec immune receptors. While the importance of glycans is well-established, they have been understudied compared to other biomolecules (e.g. DNA and proteins) due to a lack of convenient strategies to analyze their functions. There is thus great interest in characterizing the biology and functional interactions of glycans. This thesis describes the development and expansion of cell-surface glycan editing tools to study the expression and interactions of glycans and glycoconjugates in cancer and immunity. We use glycosyltransferase-mediated installation of nucleotide-sugars to endow cells with defined glycan epitopes to study their interactions, and for selective exo-enzymatic labeling (SEEL) with sugar derivatives to label certain glycan classes or epitopes. We first expanded the enzymatic toolkit for cell-surface installation of the biologically important α2,8-disialyl motif. We then applied SEEL to explore glycosylation patterns in breast cancer and breast cancer stem-like cells, identifying preliminary trends. Next, we developed a SEEL approach for installing photo-crosslinkable probes onto cells for capturing glycan-protein interactions. Diazirine-linked sialic acid derivatives were installed on cells and their utility for photo-crosslinking glycan-protein interactions was explored with Siglecs-2, -3, -7, and -15, toward identification of their glycoprotein ligands. This work represents a useful toolkit for exploring glycan expression and interactions that is highly relevant to the field of glycosciences for the development of glyco-focused therapeutics.