Development of platforms for manipulation and interrogation of glycan interactions and assessment of their effects on cellular signaling
The glycocalyx consists of a dense layer of carbohydrates which coat the surface of virtually all living cells, and plays pivotal roles in development, disease progression, and cellular signaling. Although glycans are ubiquitous and central to biology, our understanding of them is still rapidly evolving due to their multivalent properties, heterogenous composition, and genetically non-templated nature – which complicate their study. This dissertation is dedicated to the conception, development, and application of tools to both measure and manipulate glycans in biology.
Glycosaminoglycans are linear, charged polysaccharides which harbor binding sites for both cytokines and their receptors, and thus are key regulators of cell signaling. The small molecule aminoquinoline, surfen, is a reversible antagonist of interactions between heparan sulfate glycosaminoglycans and growth factors. In Chapter 2, we show that it can be used as a molecular tool to drug the glycome. Surfen is a potent molecule that can reversibly inhibit differentiation of stem cells while promoting maintenance of pluripotency, and as such provides a powerful alternative to genetic methods to control stem cell fate.
While glycosaminoglycans are of paramount importance in development, the inherent structural heterogeneity makes elucidating structure function relationships difficult. In Chapter 3, we engineer glycan microenvironments by conjugating chemically modified heparan sulfate to the gelatin matrix surrounding stem cells, where it influences growth factor binding and downstream cell signaling. By carefully controlling the glycan microenvironment around stem cells, we enable assessment of the contributions of extracellular heparan sulfate to growth factor binding and cell signaling. In Chapter 4, we developed a new method to remodel the cellular glycocalyx using photocleavable glycopolymers to enable a photopatterning approach to engineer the glycocalyx with spatial resolution.
Human epithelial surfaces contain hydrophilic mucin proteins which are richly colonized by bacteria in the human microbiome. Altered bacteria-glycan interactions are associated with inflammatory disease, and new methods to assess interactions would be of great use. In Chapters 5 and 6, we develop a new platform for assessment of glycan interactions in whole cell bacteria. By implementing a DNA-barcoding system to encode the identity of the glycans in conjugation with a mucin mimetic platform, we assessed the glycan interactions of whole cell E. coli using next generation sequencing techniques to provide a rapid readout of binding analysis.
Understanding the intricacies of glycobiology will pave the way for technological breakthroughs in science and medicine – however scientists need tools to rapidly interrogate and control the glycocalyx. The work described in this dissertation addresses these needs by expanding the modern scientists’ toolkit to probe and manipulate the glycome.