Biophysical characterisation of tsg-6-ligand interactions: using dpi to investigate the interactome of a multi-functional inflammatory mediator

TSG-6 is a multifunctional, inflammation-induced, protein that protects/stabilises tissues during inflammation & that also has a critical role in mammalian ovulation/fertilisation; e.g. it can protect cartilage & bone from breakdown & we have on-going investigations into whether it has utility for treatment of arthritis & osteoporosis. TSG-6 mediates its diverse functions via interactions with a wide-range of protein & polysaccharide ligands (eg we have discovered >10 new binding partners in the last 5 years, including 7 BMPs, fibronectin, RANKL & 2 thrombospondins) [1,2]. While a few of these interactions have been studied in detail (eg with the glycosaminoglycans hyaluronan & heparin), in most cases we have little biophysical data. The aim of this project is to undertake a systematic analysis of TSG-6-ligand interactions to obtain information on their kinetics & establish the hierarchy of the TSG-6 interactome, including the identification of multimolecular complexes that serve as key nodal points. This will provide important new insights into TSG-6 structure/function inter-relationships and its mechanism of action, which will complement our current research on TSG-6 in arthritis (ARC Programmes 10/04-10/14) & ovulation (MRC Project 10/08-9/11). The project will also aid the development of generic methodologies that can be utilised for the investigation of the interactome of other extracellular matrix molecules, including the identification/characterisation of multi-component complexes & how they are conformationally regulated. These studies will be facilitated by the use of dual polarisation interferometry (DPI), a technique that has been pioneered by our industrial partner Farfield; an Analight 4D has been funded through the renewal of the Welcome Trust Centre for Cell-Matrix Research of which AJD/TAJ are part. This instrument can be used to determine structural changes (>0.1 Å) of molecules attached to a surface in real time, providing information on affinities & kinetics of individual interactions, but it can also monitor the assembly of multi-molecular complexes & provide information on their structure/stoichiometry. While DPI can be used to obtain kinetic information from mass-dependent changes at the sensor surface, obtaining kinetic data from mass independent conformational changes is also possible & this is being developed by Farfield, ie utilising a new low volume reaction cell. The student will contribute to these developments and compare kinetic data obtained by DPI and surface plasmon resonance (SPR). In isolation the relative affinities/kinetics of the individual TSG-6-ligand interactions will not reveal the hierarchy of binding, particularly if any of the associations involve allosteric mechanisms. The hierarchy of the TSG-6 interactome will be investigated using ligand-fishing techniques (ie with immobilised TSG-6 protein and defined mixtures of proteins) combined with mass spectrometric analyses; the effect of pH & glycosaminoglycans will be explored (to mimic particular microenvironments, for example an inflammatory milieu) and this will provide important insights into TSG-6 regulation. These studies will also identify key complexes formed between TSG-6 & its ligands. Their formation & stoichiometry will be investigated by DPI in conjunction with other biophysical techniques including QCM-D (quartz-crystal microbalance with dissipation monitoring) and SPR. In particular, Farfield will help us improve the methodology for orientation of protein molecules on the sensor surface to allow the assembly of complexes in a biologically relevant context. 1. Mahoney et al. (2008) TSG-6 regulates bone remodeling through inhibition of osteoblastogenesis and osteoclast activation. J. Biol. Chem. 283, 25952-62 2. Kuznetsova et al. (2008) TSG-6 binds via its CUB_C domain to the cell-binding domain of fibronectin and increases fibronectin matrix assembly. Matrix Biology 27, 201-10