Unravelling the biological function of heparan sulphate domain structure by three-dimensional analysis

这项研究的目的是揭开围绕被称为糖胺聚糖的大聚合物分子的一些谜团，糖胺聚糖在多细胞动物从单细胞生物进化的过程中起着至关重要的作用。在哺乳动物中，糖胺聚糖填充细胞之间的空隙，将它们连接在一起，赋予器官、关节和皮肤力量，同时允许细胞生长和变化。考虑到它们在体内无处不在，它们在哺乳动物中有许多不同的功能就不足为奇了。然而，在糖胺聚糖家族中，不同的成员在化学结构上可以识别出细微的差异，这些化学差异是如何导致观察到的不同生物学的问题仍然存在。例如，透明质酸润滑关节并填充眼球，硫酸软骨素是关节衬里和大脑的重要组成部分，硫酸皮肤素赋予心脏瓣膜弹性，硫酸肝素（有时称为肝素）存在于动脉壁上，通常在手术中用作抗凝血剂。我们研究的总体目标，包括这里所描述的项目，是通过使用我们在过去10年中完善的技术提供详细的微观分子三维信息，调查迄今为止尚不清楚的糖胺聚糖结构和功能之间的关系。在糖胺聚糖中，硫酸肝素已被发现具有最复杂和多样化的化学修饰。事实上，它已经被发现拥有一个分子条形码，这个条形码沿着它的长度被印记，并被硫酸盐化学和其他修饰编码。据推测，细胞可以读取这种条形码，并将其作为改变自身行为的信号，这表明，如果我们能够理解这种分子条形码及其读取器的本质，就有可能获得令人兴奋的生物学新见解和医学治疗。我们的研究导致了一个假设，硫酸肝素条形码编码在局部链的灵活性，其中可变的刚性部分是由柔性铰链分开。我们的目标是通过使用分子显微镜（核磁共振）来测试这一假设，原则上，它可以确定硫酸肝素链沿其长度和灵活性的形状。然而，虽然目前有很好的蛋白质分子显微镜技术（如x射线晶体学），但硫酸肝素的灵活性意味着现有技术无法使用，这是硫酸肝素局部灵活性尚未得到详细研究的关键原因。幸运的是，我们最近开创了一套新的技术，可以解开糖胺聚糖的三维形状和灵活性。通过将这些技术进步应用于硫酸肝素，我们的目标是揭示其分子形状和灵活性。这将有助于揭示分子条形码的确切性质，从而解释它如何与体内的细胞和其他分子相互作用。所获得的知识将帮助我们发现新的生物学，例如了解细胞如何自我组装，治疗因糖胺聚糖功能不正常而导致的疾病，并使组织工程和再生医学取得进展。这些信息还可以推动临床迫切需要的新型化学模拟物的发展（肝素的年销售额总计数十亿美元）。

This study aims to unravel some of the mysteries surrounding the large polymeric molecules called glycosaminoglycans, which have played a crucial part in the evolution of multicellular animals from single-celled organisms. In the mammal, glycosaminoglycans fill the space between cells, bonding them together, conferring strength to organs, joints and skin, while allowing cells to grow and change. Considering their omnipresence in the body it is not surprising that they have many different functions in mammals. However, within the glycosaminoglycan family, distinct members can be identified with slight differences in their chemical structure and question that remains is how these chemical differences lead to the observed diverse biology. For example, hyaluronan lubricates joints and fills the eyeballs, chondroitin sulphate is an essential constituent of joint linings and brain, dermatan sulphate gives elasticity to heart valves and heparan sulphate (sometimes called heparin) is found on the walls of arteries and commonly used as an anticoagulant during surgery. The overall aim of our research, including the project described here, is to investigate the, as yet, poorly understood relationship between structure and function in glycosaminoglycans, by providing detailed microscopic molecular three-dimensional information using techniques that we have perfected over the last 10 years. Of the glycosaminoglycans, heparan sulphate has been found to have the most complicated and diverse set of chemical decorations. In fact, it has been found to possess a molecular barcode, which is imprinted along its length and is encoded by the sulphate chemistry and other modifications. This barcode is read by cells, it is hypothesised, which use it as a signal to change their behaviour, suggesting that exciting new biological insights and medical therapies are possible if we can understand the nature of this molecular barcode and its reader. Our investigations have led to a hypothesis that the heparan sulphate barcode is encoded in the local chain flexibility, in which variable rigid sections are separated by flexible hinges. We aim to test this hypothesis by using a molecular microscope (nuclear magnetic resonance), which can, in principle, determine the shape of the heparan sulphate chain along its length and also its flexibility. However, while there are currently good molecular microscopy techniques for proteins (such as x-ray crystallography), the flexibility of heparan sulphate means that established techniques cannot be used, a key reason that heparan sulphate local flexibility has not been investigated in detail. Fortunately, we have recently pioneered a novel set of techniques that can unravel the three-dimensional shape and flexibility of glycosaminoglycans. By applying these technological advances to heparan sulphate, we aim to uncover its molecular shape and flexibility. This will help unravel the exact nature of the molecular barcode and hence explain how it interacts with cells and other molecules in the body. The knowledge gained will help us to uncover new biology, such as understanding how cells assemble themselves, treat diseases that result from incorrect functioning of glycosaminoglycans, and allow progress in tissue engineering and regenerative medicine. Such information can also drive the development of novel chemical mimetics that are urgently needed in the clinic (annual revenues from sales of heparin total many billions of dollars).