Importance of N-glycosylation at the Neuromuscular Junction

当有人问你：“糖在我们体内起什么作用？“想什么呢？你只是把它们当作卡路里的来源吗？糖不仅仅是卡路里的来源！它们可以形成各种各样的错综复杂的结构，称为聚糖，在我们的身体中扮演着许多重要的角色。它们发挥的一些最重要的作用是帮助蛋白质组装和正常发挥功能。聚糖在称为糖基化的酶促过程中合成并转移到蛋白质上，这是蛋白质经历的最常见和最重要的翻译后过程之一。然而，对蛋白质糖基化的了解很少，对于大多数涉及的酶的信息很少。我们也不知道绝大多数聚糖的生物学作用。糖基化对所有复杂的生命包括植物、动物和真菌都是必不可少的。当糖基化过程发生故障时会发生什么就是例证。几乎所有参与糖基化的基因的突变都可能导致发育障碍，具有一系列症状和严重程度。在最严重的情况下，患者患有多系统疾病先天性糖基化疾病（CDG），并可能在出生后一年内死亡，甚至在子宫内死亡。一组有趣的患者患有先天性肌无力综合征（CMS），症状仅限于神经肌肉接头（NMJ）的异常发育，而神经肌肉接头对于神经和肌肉之间的交流至关重要，并且具有疲劳性肌肉无力。我的目的是利用这些病例的研究，为了解更严重的多系统疾病提供一个窗口。到目前为止，大约有1,000个患者家庭被发现糖基化基因突变，每年都会发现更多。这些患者的治疗选择非常少，其中大多数只能暂时帮助缓解症状，长期无效。本提案旨在提高我们对糖基化及其相关疾病的认识，主要有3个目标：1）更好地理解参与蛋白质糖基化途径的酶的基本性质，以及突变是如何改变这些特性从而导致疾病的。2)鉴定从CMS患者和健康对照获得的肌细胞中关键NMJ糖蛋白的糖基化的变化。3）测试治疗方法以纠正在1）和2）中创建的疾病的蛋白质和细胞模型中观察到的疾病相关变化。为了实现这些目标，将采用多学科方法，结合结构生物学、生物化学、分子生物学、细胞生物学和糖组学。该研究计划将利用来自英国和世界各地的专家遗传学中心收集的数据，最大限度地发挥现代遗传学和测序技术的优势，以解决蛋白质糖基化的基本问题，以及CMS和CDG的机制。将创建一个生化和细胞平台来测试治疗糖基化相关CMS的新治疗方法。从这项建议中获得的知识将直接使CMS和CDG患者以及试图帮助他们的临床医生和科学家受益。它还将使来自其他各种领域的科学家受益，包括神经科学，糖生物学，结构生物学和酶学。我以前在这一领域的工作已经帮助开发了针对结核病细菌的新型抗生素。聚糖和糖蛋白通常用于药物中，因此，关于其生物合成机制的信息也将对制药和生物技术行业产生巨大的好处。

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.