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

Lead Research Organisation: University of Leeds
Department Name: Sch of Biomedical Sciences


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Technical Summary

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.

Planned Impact

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.
Description We have made progress towards determining the 3-dimensional structure of human pentraxin-3 (PTX3) - an octameric protein that plays a key role in the organization of hyaluronan-rich matrices essential for ovulation/fertilization in mammals and also formed during inflammation. We have determined the structure of an individual C-terminal domain from human PTX3 using X-ray crystallography (to 2.7Å resolution). This facilitated the octameric organisation of the 8 C-terminal domains of PTX3 to be determined from cryo-Electron Microscopy (cryoEM) to 2.7Å. This region forms the central body of PTX3 where cryoEM has also resolved part of the N-terminal regions of the protein (18 amino acids next to the central body to 2.7Å and a further 28 amino acids to 6.5Å). This has revealed that four PTX3 monomers come together on each side of the central body and extend in opposite directions, i.e., to form two long arms (each composed of a tetrameric assembly of coil-coiled domains). The missing N-terminal regions have been modelled, and the final model has been corroborated by Small-Angle X-ray Scattering of full-length PTX3 and mutant constructs where a region of the N-terminal domain was truncated and/or a tetrameric PTX3 was analysed. Biophysical studies indicate that four Heavy Chains (HCs) of Inter-alpha-Inhibitor bind to each of the tetrameric N-terminal regions of PTX3 (i.e., with one binding site per protomer). Given HCs become covalently attached to the polysaccharide hyaluronan (during ovulation and inflammation), our studies provide a model for how PTX3-HC interactions may contribute to crosslinking of hyaluronan chains and thus modulate extracellular matrix structure. In addition, heterotypic interactions between different HCs have been analysed using biophysical methods providing further novel insights into crosslinking mechanisms.
Exploitation Route The outcome described above has not yet been published (just presented at several meetings), but we anticipate it will be of value to others in understanding the function of PTX3 (and Heavy Chains) in physiological and disease processes, with the potential for biomedical applications.
Sectors Healthcare