Fibronectin (Fn) is an extracellular matrix (ECM) protein involved in embryonicdevelopment, wound healing and tumorigenesis. Structurally Fn is mainly composedof three repeated modules: FI, FII and FIII, together with an alternatively splicedtype III connecting segment (IIICS). The IIICS has no sequence homology to theserepeated modules and contains integrin, proteoglycan and zinc binding sites. Thesesites facilitate adherence and spreading of leukocytes, peripheral neurons andmelanoma cells, which can lead to disease states such as inflammation,autoimmunity and cancer metastasis. Therefore, there is the potential to developtherapeutic agents based on the IIICS structure.In this study, nuclear magnetic resonance (NMR) spectroscopy has been used toinvestigate the structure and dynamics of both the IIICS and its adjacent FIII15module, two of the few Fn regions for which a structure has not been elucidated.An ensemble of solution state NMR structures calculated for the isolated FIII15module showed that FIII15 forms a rare six-stranded FIII fold, homologous to atypical seven-stranded FIII fold, with a disordered N-terminal linker sequence. NMRrelaxation data and chemical shift analysis showed that the IIICS is an intrinsicallydisordered region with no areas of well-defined secondary structure. A structurewas also calculated for FIII15 within a construct containing the IIICS, which showedthat contrary to a previous hypothesis, the IIICS does not contribute to the FIII15structure. In addition, structural comparisons between IIICS splice variantssuggested that alternative splicing confers no stable structural features to the IIICS.Furthermore, ligand binding studies showed that, under conditions tested, neitherzinc nor the proteoglycan heparin, induced the formation of any secondarystructure to this region. Zinc binding did, however, induce oligomerisation of a IIICScontaining construct and appeared to enhance the binding of heparin to the IIICS.Data was obtained to suggest that FIII15 forms a transient interaction with anadjacent module, which is likely to be FIII14.It is hoped that the work presented will contribute to further studies into thisimportant area of Fn and may aid in the future development of novel therapeutics.
|Date of Award||1 Aug 2017|
- The University of Manchester
|Supervisor||Martin Humphries (Supervisor) & Jonathan Waltho (Supervisor)|