Reengineering of Glycan Binding Specificity for Targeted Cellular Delivery.

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There are four major groups of biological molecules, nucleic acids (DNA and RNA), proteins, carbohydrates (sugars) and lipids (fats). The reengineering of these biological molecules is the focus of the field of synthetic biology; whereby proteins and nucleic acids are designed from scratch, or redesigned based on a natural blueprint, to perform functions not seen in nature. Proteins are the major workhorses of biological processes and over the last 20 years many advances have been made to reengineer proteins to perform new tasks. Proteins are responsible for many biological functions, for example: individually as enzymes; with each other to respond to environmental change; with nucleic acids to turn gene expression on or off; with sugars for communication between cells; or with fats to form transporters across biological membranes. Of these protein-based interactions, correspondingly little synthetic biology work has focused on the design of protein-sugar interactions, despite sugars being a major component of biological systems. Sugars are most commonly known as a foodstuff, such as sucrose (table sugar), or for maintaining structure, such as cellulose in trees. However, sugars are also present in great abundance on the outside of all our cells, forming a structure called the 'glycocalyx' (sugar-husk). The sugar molecules which form this husk are attached to the cell via proteins or lipids. Interaction with the glycocalyx allows for cellular adhesion and communication, controls cellular behaviour and defence, and forms a physical barrier against infection by microbes. There are many variations in the types of sugars which make up this glycocalyx and different cell types have a different sugary composition. In fact, the sugar composition of a cell can change during the phases of replication and when cells are diseased, such as in cancer. We call this sugar composition the 'glyco-code', and in this work, I will use this glyco-code to differentiate between cell types, including healthy from diseased.Often pathogens, microbial toxins and viruses, can use these sugars in the glycocalyx to trick the cell into absorbing them, including a group of toxins known as the AB5 toxin family, which includes cholera toxin from Vibrio cholera. This toxin attaches to a specific sugar which is found predominately in the glycocalyx of cells in the intestine. As a result, the toxin is absorbed, and the cell behaviour is changed, causing it to expel water into the intestine leading to the familiar cholera poisoning symptom, diarrhoea. These AB5 toxins are formed from two components the toxic A-subunit and the non-toxic B5-subunit. The cholera toxin B5-subunit (CTB), although non-toxic, is important as it binds to the sugars on the outside of the cell, and triggers absorption. This natural mechanism can be hijacked by redesigning CTB to bind to different sugar molecules; hence, reengineering this CTB molecule as a cell selective absorption tool would allow an attached cargo to be transported into the cell interior.In this fellowship I will use novel computational approaches combined with experimental selection and directed evolution to reengineer these B5-subunits to bind to a different sugar, which is only found on the surface of some cancer cells, including melanoma (skin cancer) and neuroblastoma (a childhood cancer of the nervous tissue). By reengineering CTB to bind to these sugars, it can be used to transport diagnostic and therapeutic molecules into specific cancer cells. The demonstration of this redesign process will allow future engineering of other protein-sugar interactions. Such as reengineering other B5-subunits against a range of glycolipids, to generate a selection of molecules which can specifically target a range of cells; such a synthetic biology toolkit will be of great importance in diagnostics and drug delivery.