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.

This Proposal has 3 aims:
Aim 1: study the structure and function of proteins involved in the N-glycosylation pathway, and how mutations found in patients affect their function and interactions.
Methods: use suspension adapted insect and mammalian cells for large scale protein production, LIC methods to produce DNA vectors, and high throughput purification and crystallisation methods to obtain protein crystals. X-ray diffraction data will be collected at Diamond Light Source. Enzyme kinetics will be assessed using colorimetric assays, rapidfire-QTOF, and TLC based assays using radioisotope labelled substrates. Protein-protein interactions will be assessed in vitro using co-affinity purification, SEC-MALS, and SPR, and in cells using super resolution microscopy.
Scientific opportunities: create protein structural and biochemical tools for studying the fundamental properties of these proteins, as well as the effect of disease genotypes.
Aim 2: examine how mutations identified in CMS patients lead to changes in the glycosylation of key glycoproteins involved in NMJ development and function.
Methods: immortalised muscles cells derived from CMS patients and controls will be differentiated into myotubes. Agrin will be added to stimulate AChR clustering. NMJ glycoproteins will be purified from these patient and control cells using cell fractionation, affinity purification, SEC, and IEC. Glycomics analysis of purified glycoproteins to determine the differences between the glycosylation of patient and control cells.
Scientific opportunities: create a tissue platform for studying glycosylation-associated CMS.
Aim3: Test methods of treatment.
Methods: Use protein and cell platforms developed in 1) and 2) to test different methods of altering the enzymatic flux of the N-glycosylation pathway, and see if they can rescue the effect of disease causing genotypes.
Medical opportunities: produce potential therapeutic methods for treating glycosylation-associated CMS.

The research detailed in this proposal will investigate the fundamental biology of proteins involved in the N-glycosylation pathway, and how mutations in these proteins can lead to changes in neuromuscular junction development and function, and disease. There are many potential beneficiaries to the research, who will be split and discussed in 3 categories:
1) Clinicians and patients
2) General public
3) Biotechnology and pharmaceutical industries
1) The primary aim of this proposal is to better understand the biological mechanisms underlying N-glycosylation-associated Congenital Myasthenic Syndromes (CMS) and Congenital Diseases of Glycosylation (CDG), and to create a biochemical and cellular platform to test novel treatments for these diseases. With advances in next-generation DNA sequencing, and genetic screening procedures helping to pick up cases earlier, this study will help clinicians provide a definitive diagnoses and a basis for prognosis advice, and more personalised treatment plans for patients. This information will also be disseminated to patient charities to help patient families better understand the illness and give them encouragement that progress is being made in research, and there may be hope for novel therapies in the future. In the long term, I hope work from this proposal will be the first step on a path to finding effective treatments for these patients.
2) Sugars and carbohydrates are fundamentally important to all life on earth, yet most of the general public only know that they make you fat, make your teeth rotten, and give you diabetes. There has been a lot of negative publicity over recent years about this class of biochemicals, and the general public need to know more about the many different roles carbohydrates play in our bodies, and just how important they are. Therefore, a variety of different public engagement activities, designed to suit different sectors of the public, will be undertaken to introduce them to some of the other important roles that sugars play in our bodies.
3) There is considerable economic potential to the scientific data generated from the research described in this proposal. One of the key objectives of this proposal is to create a biochemical and cellular platform to test novel therapeutic strategies to correct the molecular changes produced by CMS-associated mutations in genes involved in N-glycosylation. The pharmaceutical industry is increasingly interested in targeting rare metabolic diseases, and there are few therapies available for the majority of patients with disorders of N-glycosylation. During this proposal I will use this platform to test 1-2 different therapeutic strategies, which if effective could lead to interest from the commercial sector to develop them further.
Many small-molecule drugs such as antibiotics, antivirals and anticancer drugs contain glycans as part of their core structure. Most biotherapeutic products are glycoproteins, such as erythropoietin, cytokines, antibodies, glycosyltransferases, and glycosidases. Another key scientific objective of this proposal is to better understand the structure and biochemistry of enzymes in the N-glycosylation pathway. Work done to achieve this objective will reveal scientific information on the biosynthetic mechanisms of complex glycans and glycoproteins, which will be of great interest to the pharmaceutical and biotechnology industries.