Importance of N-glycosylation at the Neuromuscular Junction

When someone asks you: "what do sugars do in our body?" What do you think of? Do you just think about them as a source of calories? Well sugars are much more than just a source of calories! They can form a great variety of intricate and complex structures called glycans, which play many important roles in our body. Some of the most important roles they play is in helping proteins assemble and function properly. Glycans are synthesised and transferred onto proteins in an enzymatic process called glycosylation, which is one of the most common and important post-translational processes that proteins undergo. However, protein glycosylation is poorly understood, with very little information available for most of the enzymes involved. We also don't know the biological roles of the vast majority of glycans. Glycosylation is essential to all complex life including plants, animals and fungi. This is exemplified by what happens when the glycosylation process malfunctions. Mutations in nearly all the genes involved in glycosylation can lead to developmental disorders, with a spectrum of symptoms and severity. In the most severe cases, patients have the multisystem disorder congenital disease of glycosylation (CDG), and can pass away within one year of birth, or even in utero. An intriguing group of patients have congenital myasthenic syndromes (CMS), with symptoms restricted to the abnormal development of neuromuscular junctions (NMJ), which are essential for communication between nerves and muscles, and have fatiguable muscle weakness. I aim to use the study of these cases to provide a window for understanding the more severe multisystem disorders. Around 1,000 patient families have been identified so far with mutations in glycosylation genes, with more found every year. There are very few treatment options for these patients, most of which only temporarily help alleviate symptoms, and are ineffective in the long term. This proposal aims to improve our understanding of glycosylation and the diseases associated with it, and has 3 main objectives: 1) Better understand the fundamental properties of the enzymes that are involved in the protein glycosylation pathway, and how mutations change these properties to bring about disease. 2) Identify the changes in the glycosylation of key NMJ glycoproteins in muscle cells obtained from CMS patients and healthy controls. 3) Test therapeutic methods to correct the disease associated changes observed in the protein and cellular models of disease created in 1) and 2). To achieve these objectives, a multidisciplined approach will be employed combining cutting edge techniques in structural biology, biochemistry, molecular biology, cellular biology and glycomics. This research program will exploit the data gathered by the specialist genetics centres from the UK and around the world, maximising the benefits of modern genetics and sequencing technology to address fundamental questions in protein glycosylation, and the mechanisms underlying CMS and CDG. A biochemical and cellular platform will be created to test novel therapeutic approaches to treating glycosylation-associated CMS. The knowledge gained from this proposal will directly benefit CMS and CDG patients as well as the clinicians and scientists trying to help them. It will also benefit scientists from a variety of other fields including neuroscience, glycobiology, structural biology, and enzymology. My previous work in this area has already helped to develop novel antibiotics against the bacteria that causes tuberculosis. Glycans and glycoproteins are commonly used in medicines, therefore, information on their biosynthetic mechanisms will also have great benefits for the pharmaceutical and biotechnology industries.