Decoding structure-function relationships of glycans

Abstract

Glycans are an important class of biological molecules that regulate a variety of physiological processes such as signal transduction, tissue development and microbial pathogenesis. However, due to the structural complexity of glycans and the unique intricacies of glycan-protein interactions, elucidating glycan structure-function relationships is challenging. Thus, uncovering the biological function of glycans requires an integrated approach, incorporating structural analysis of glycans, and glycan-proteins interactions with functional analysis. In this thesis, I develop new tools and implement integrated approaches to study glycans and glycan-binding proteins (GBPs). I apply these approaches to study glycans and GBPs in two areas: i) the role of hemagglutinin-glycan receptor specificity in human adaptation and pathogenesis of influenza and ii) the function of glycan regulation of cell-microenvironment interaction in cancer progression. Section 1: Influenza poses a significant public health threat and there is a constant looming threat of a pandemic. Pandemic viruses emerge when avian viruses acquire mutations that enable human adaptation, leading to infection of an antigenically naive host. Influenza Hemagglutinin (HA), and HA-glycan receptor interactions, play a central role in host tropism, transmissibility, and immune recognition. In section one, I develop and apply an integrated approach comprised of structural modeling, inter-amino acid network analysis, biochemical assays, and bioinformatics tools to study the hemagglutinin-glycan interaction and, in some cases, HA's antigenic properties. Using this approach, we i) identify the structural determinants required, and potential mutational paths, for H5N1 to quantitatively switch it's binding specificity to human glycans receptors, ii) identify the mutations that enable the 2013 outbreak H7N9 HA to improve binding to human glycan receptors in the upper respiratory tract, iii) uncover H3N2 strains that are currently circulating in birds and swine that possess features of a virus that could potentially re-emerge and cause a pandemic, and iv) characterize the glycan binding specificity of a novel 2011 Seal H3N8 HA. The approaches implemented here and the findings of these studies provide a framework for improved surveillance of influenza viruses circulating in non-human hosts that pose a pandemic threat. Section 2: Glycans are abundant on the cell surface, and at the cell-ECM interface where they mediate interactions between cells and their microenvironment. Despite this, the function of glycans in cancer progression remains largely understudied. Here, I develop an integrated approach to characterize the cell surface glycome, including N-linked, 0-linked glycans, and HSGAGs. This approach integrates glycogene expression data, analytical tools, and glycan binding protein reagents. I demonstrate that this platform enables rapid and efficient characterization of the N- and 0-linked glycome in a model cell system, representing metastatic versus non-metastatic cancer cells. Next, I apply this integrated approach to uncover new roles of glycans. I study the role that HSGAGs play in regulating cancer stem cell (CSC) activity in breast cancer. Here, we report that SULF1, an HSGAG modifying enzyme, is required for efficient tumor initiation, growth and metastasis of CSCs. Furthermore, we identify a putative mechanism by which SULF1 regulates interactions between CSCs and their microenvironment. The approaches implemented here and the finding of these studies Overall, this thesis provides important tools, approaches and insights to enable and improve the study of glycans and glycan binding proteins. Together the work here provides a framework for decoding structure-function relationship of glycans.