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

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).

An elaborate network of macromolecules holds together vertebrate tissue that is not only superstructural but also regulates cellular function, and thus the constituent molecules have unique biotechnological and medical applications. In particular, the physical matrix around cells, which extends up to a micrometre from the cell surface, comprises a large proportion of soluble glycosaminoglycans (GAGs) and GAG-containing proteoglycans (PGs), such as heparan sulphate proteoglycans (HSPGs). Their high negative charge density maintains tissue hydration and modulates cellular function during development, injury and inflammation. Furthermore, the binding of HSPGs with proteins (e.g., thrombin and fibroblast growth factors) is implicated in cell adhesion, enzyme regulation and cytokine action. Heparan sulphate chains have a domain structure, with sulphated and epimerised S-domains (which interact with proteins) punctuated by non-sulphated NA-domains. Little is known about the local and global 3D organisation HS and HSPGs, which is quite surprising considering their biological, biotechnological and medical importance. To address this need, we have developed a set of methodologies that can unravel the solution 3D organisation of sugars, both at the local and global levels and have applied them successfully to HS NA-domains. Based on these results we hypothesised that the S-domains are rigid while the NA-domains are flexible hinges, which allows HS to support complex multivalent interactions with proteins. In this proposal we aim to apply these approaches to S-domains from HS, to determine their local 3D structure and flexibility, and hence to confirm this hypothesis by comparison with our previous results from NA-domains. Furthermore, we aim to use this new local structural to develop coarse-grained models of HS polymers in solution that can be used to predict their physical properties and understand the relationship between HS decoration and function.