Mapping Spatiotemporal Glycoprotein Interactions in Cells

When we think about proteins, the major players of cellular interactions, we tend to visualise simple structures based on a sequence of amino acids. Fundamentally, this is true, however, a single protein can have an enormous number of structural forms, each of which interacts with other biomolecules in different ways and fine-tunes how a protein functions or where it moves in the cell or even how long it lasts in the body. Protein structural diversity is derived from chemical modifications at specific amino acids and the most common and complex type is called glycosylation - an intricate, non-template driven process involving over 500 enzymes that work together to assemble complex sugars, termed glycans, to specific sites on a protein. Glycosylation is vitally important across biology, defects in enzymatic machinery for glycan biosynthesis is detrimental to normal cell survival. This is exemplified by the human disease Congenital Disorders of Glycosylation, where mild defects manifest in severe multisystem disfunction, developmental delay and premature death. Glycoproteins are prevalent in the biopharmaceutical industry as the safety and efficacy of many protein-based drugs, including monoclonal antibodies, are directly linked to their glycan structures. Glycosylation is also important for structure-based vaccines; this is because viral surface proteins are extensively covered under "glycan shields" that are derived from the host glycosylation enzymatic machinery and hence the sugars on a good vaccine will mimic the sugars on the virus. Although we know glycosylation is important for human health, disease and the design of drugs, we know surprisingly little of how glycans control protein functions through their interactions with other biomolecules. This is in large part due to their structural complexity and how glycans contribute to binding other proteins (e.g. a cell surface receptor) that drives function. A major obstacle in linking glycoprotein structure with function is a general lack of capable tools designed for studying glycoprotein interactions in complex systems, such as inside cells or tissues. This proposal will help solve this problem by establishing a new platform capable of mapping glycoprotein interactions with high levels of spatial and temporal precision. To do this we will exploit the complex glycoprotein biosynthetic pathway to introduce non-natural chemical “tags” that will allow us to trap and measure specific interactions using new chemical crosslinking molecules that we can characterise using mass spectrometry, the leading technology for studying glycoproteins. Importantly, the new methods and chemical reagents we will develop during this work will be widely applicable for tracking glycoprotein-specific interactions across life science research, and we anticipate that they will become widely used by ourselves and others in a wide range of research applications. This is because our approach has several benefits over the current advanced methods that are used to characterise interactomes. We will demonstrate the potential of our new method by characterising interactions between two viruses, Ebola and SARS-CoV-2 and their host cells. Understanding the dynamics of their interactions has the potential to uncover new biomolecules involved in viral entry and therefore drug targets.