Understanding hyaluronan crosslinking mechanisms in ovulation and inflammation: CryoEM structural and interaction analysis of HC-HA/PTX3 complexes

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The extracellular matrix is found around and between virtually every cell in our bodies and it is this substance that organises cells into organs and provides our tissues with their particular mechanical properties. For example, bone is hard, brain is soft, and skin is strong but elastic. One important matrix component is hyaluronic acid, or HA for short. HA is a large polymer comprised of thousands of sugar molecules arranged in a long chain (a 'polysaccharide'). HA is present in every tissue of mammals and is believed to have evolved more than 500 million years ago. It is HA that hydrates our tissues and makes them resistant to compressive forces. For example, HA provides cartilage with its resilience, cushioning our joints when we walk and run. HA also plays a critical role in human reproduction, forming a very soft, but elastic, jelly-like coating around the egg just prior to it being released from the ovary at ovulation; here this elastic jelly allows the egg to travel down the oviduct and is required for the capture of a healthy sperm. HA is also necessary for embryonic development, directing the movement of cells during the formation of new organs. Given that HA has such an important role in mammalian biology, it is not surprising that HA is often affected during disease; for example, too much HA is made during cancer and during inflammatory conditions. HA also contributes to fibrosis, a process that contributes to approximately 40% of all deaths, because tissues become stiffened and no longer function correctly.Although HA is known to play a wide range of roles in health and disease, how it does this is not well understood. Despite HA being large, it is a very simple molecule, making it intriguing to understand how HA can contribute to such diverse functions. Evidence strongly suggests that the explanation lies in the association of HA with proteins. However, it is not clear exactly how this occurs in molecular terms. We have suggested that HA associates with different types of protein in different tissue locations and that this allows the formation of a diverse range of HA/protein 'composites' with distinct mechanical and functional properties. In other words, HA is like a biological plastic that can be moulded into different shapes with different degrees of softness and flexibility depending on which proteins it is combined with. We have also hypothesised, that these various HA/protein composites encode distinct molecular signals within the extracellular matrix that can be decoded and interpreted by cells, telling them what to do.The aim of the proposed research is to determine at a molecular level how HA/protein composites are organised into a three-dimensional network and how this dictates their mechanical properties. This will use a relatively new method (cryo-electron microscopy) to determine the precise shape (at atomic resolution) of the proteins that link together the HA chains, since more traditional methods have failed to achieve this. Our studies will generate fundamental new insights that will begin to allow us to understand how HA mediates its diverse and important functions, providing novel molecular concepts that can be applied widely across mammalian biology. To achieve our goals we will focus on a particular subtype of HA/protein composite (known as "HC-HA/PTX3 complexes") with compositions and functions that are already partly established. For example, this subtype of HA/protein composite has an essential role in ovulation and fertilisation, is formed in our joint cavities during arthritis, and can either promote or prevent fibrosis depending on the context in which it is made. Thus, the detailed findings of our research will have the potential to facilitate numerous biomedical applications. These include the design of tailored HC-HA/PTX3 complexes for use in improved fertility treatments and novel regenerative medicine strategies for a wide range of inflammatory and fibrotic diseases.