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

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.

The polysaccharide hyaluronan (HA) is present in all mammalian tissues dictating their elasticity, hydration and permeability. In addition to its structural role, HA directs cellular behaviour via engagement with cell-surface receptors, allowing it to mediate diverse functions during development, reproduction and inflammatory processes. Evidence indicates that the way the HA biopolymer is differentially organised through its interaction with a repertoire of specific HA-binding proteins is key to the diversity of its biological functions. However, this is not well understood at a molecular level.

In some biological contexts (e.g. ovulation and inflammation) HA becomes covalently modified with heavy chains (HCs), derived from the inter-alpha-inhibitor family of proteoglycans, in which many HCs can be attached to an individual HA polymer. This leads to the dynamic crosslinking of HA chains, through the association of HC-HA complexes with pentraxin-3 (PTX3), an octameric protein that can bind multiple HCs. However, the molecular details of how HC-HA/PTX3 complexes are organised are lacking, limiting our understanding of the way in which they dictate the mechanical and functional properties of tissues and how this is dysregulated in disease.

Therefore, the aim of this study is to unravel the mechanisms of HA crosslinking in ovulation and inflammation by determining the high-resolution structures for the protein complexes that act as HA 'crosslinking nodes' in these systems. We will use cryo-electron microscopy (cryoEM) in combination with other quantitative biophysical techniques in order to elucidate the interactions involved at a molecular level. Detailed structural insights will enable the targeted modulation of HA cross-linking, to interfere with pathological matrix development and to generate HA matrices with tailored properties in vitro, e.g. for applications in regenerative medicine and human fertility treatments.

We anticipate that the results obtained from this study will be of significant benefit to academics, clinicians and industry-based scientists, engaged in research in the areas of reproduction, ageing, inflammation, innate immunity, regenerative medicine, tissue engineering, biomaterials and cancer. Our findings will relate to a molecular process that, for example, occurs during ovulation and wherever/whenever there is inflammation, thus having potential for broad scientific impact. We will disseminate our results through participation at relevant conferences and through publications in peer-reviewed, open access, journals as outlined in the 'Academic Beneficiaries' section. We are committed to the wider dissemination of our scientific findings to the public in order to communicate their importance and relevance for society, e.g. with regard to new healthcare innovations, and to inspire young people to consider careers in the biosciences. To facilitate this we will to take part in public engagement activities (targeted at schools, families and the general public) organised through the Astbury Centre (Leeds) and the Wellcome Centre for Cell-Matrix Research (WCCMR, Manchester), such as the Astbury Conversation and Wellcome to the Matrix; we will also provide work experience opportunities for school children in our laboratories. We will report breakthroughs in the local, national and international press via media relations officers at our institutions.

The results of this study will be of particular benefit to the pharmaceutical and the biomaterials sectors with the potential for clinical translation, commercialisation and economic impact. For example, given the role of HC-HA/PTX3 complexes in reproductive biology our data could inform new approaches for the treatment of infertility. In addition, a new class of 'native' HA hydrogels could result that would have broad applicability for cell biology and tissue engineering applications. We will identify any commercialisable research through liaison with the IP offices (at the Universities of Leeds and Manchester) and engage with industry, for example, via the contacts of the Astbury Centre Industry Advisory Board and companies associated with the WCCMR's translational portfolio.

This inter-disciplinary proposal will lead to the training of the PDRAs, particularly in structural biology (including cryoEM, a new and sought after technique) and biophysics, building skills and capacity in these important and widely applicable areas. The research project, and our excellent research environments, will also provide opportunities for the PDRAs and technician to develop transferable skills that will be of value in their career development. Our Universities provide a wide range of training courses for all categories of staff to promote professional development and recent workshops aimed at PDRAs have included: "Planning a Fellowship", "Grant Reviewing", "Academic CV Writing" and a "Careers Day". These, and other workshops, aim to develop a range of skills including career planning, networking, project management, team working, critical peer review, communication and self-awareness, and we will encourage staff and the investigators to attend as appropriate.