Glycosyltransferase Engineering to Dissect O-Mannose Glycosylation

Every single cell in our body displays on its surface a layer of sugars (glycans) that mediate interactions with pathogens, the immune system, and neighbouring cells. Glycans are commonly linked to proteins as their most abundant and complex modification. Thereby, glycans change the physical and biological properties of proteins and "fine-tune" their function in our body. Unsurprisingly, slight dysfunctions in the machinery that makes glycans can lead to severe disease. In contrast to other biomolecules, sugars are not directly encoded in the genome. Instead, they are synthesized by molecular machines called enzymes from simple building blocks. Hundreds of enzymes work together to manifest glycans in the right composition for a cell to function, usually as glycan "trees" with multiple building blocks being linked to each other. Among the manifold glycan types, so-called O-mannosyl (O-Man) glycans are perhaps the least understood type despite being of critical importance in physiology. O-Man glycans appear to be especially prevalent in the brain and other neuronal tissues, which suggests a special function in neurobiology. Dysfunctions in the biosynthesis of O-Man glycans are strongly linked to neuronal disorders such as abnormal brain development and muscular dystrophies. However, it is largely unclear how neuronal processes are conferred by these glycans because of a lack of suitable methods to investigate them. For instance, it is currently very challenging to understand how different parts of the same protein can carry O-Man glycans with different "trees" from different enzymatic processes. We know that three enzymes called POMGNT1, POMGNT2 and MGAT5B are important for decision-making of the right glycan "tree". But we do not know how these enzymes compete or collaborate with each other as well as with the hundreds of other enzymes in the cell. We also lack understanding on how individual, physiologically relevant glycan attachment sites are chosen by each enzyme. To understand how POMGNT1, POMGNT2 and MGAT5B function, we will develop chemical reporter tools that are specifically used by one of the enzymes. In our design, we will engineer each of the enzymes to transfer a sugar modified with a chemical tag to target proteins. The tag is amenable to bioorthogonal or "Click" chemistry, a concept that has been awarded the 2022 Nobel Prize in Chemistry. Using the bioorthogonal tag, we can adorn the sugars with a moiety that can be easily tracked or isolated, for instance a fluorescent molecule or a handle for glycoprotein enrichment. This tactic allows us to study which positions on which proteins are modified by which of the enzymes by employing cutting-edge instrumentation in mass spectrometry. Chemical tools are thus used to gain essential understanding in physiology. In an international collaboration, our work will establish a map of the fine details of O-Man glycans, with future applications in patient-relevant cells and organoids to probe which substrates these enzymes modify in a disease context. We have only in recent years gained the know-how needed to deliver on this project, underpinning the timeliness of our work. The project exactly matches the BBSRC scope to develop tools and technology underpinning biological research, and the scope to unravel some of the untapped potential of the glycosciences fits in the context of frontier bioscience to understand the rules of life. The postholders will be embedded in a diverse, supportive lab environment and experience outstanding multidisciplinary training opportunities.