Fibronectin (Fn) is an extracellular matrix (ECM) protein involved in embryonic development, wound healing and tumorigenesis. Structurally Fn is mainly composed of three repeated modules: FI, FII and FIII, together with an alternatively spliced type III connecting segment (IIICS). The IIICS has no sequence homology to these repeated modules and contains integrin, proteoglycan and zinc binding sites. These sites facilitate adherence and spreading of leukocytes, peripheral neurons and melanoma cells, which can lead to disease states such as inflammation, autoimmunity and cancer metastasis. Therefore, there is the potential to develop therapeutic agents based on the IIICS structure. In this study, nuclear magnetic resonance (NMR) spectroscopy has been used to investigate the structure and dynamics of both the IIICS and its adjacent FIII15 module, 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 FIII15 module showed that FIII15 forms a rare six-stranded FIII fold, homologous to a typical seven-stranded FIII fold, with a disordered N-terminal linker sequence. NMR relaxation data and chemical shift analysis showed that the IIICS is an intrinsically disordered region with no areas of well-defined secondary structure. A structure was also calculated for FIII15 within a construct containing the IIICS, which showed that contrary to a previous hypothesis, the IIICS does not contribute to the FIII15 structure. In addition, structural comparisons between IIICS splice variants suggested that alternative splicing confers no stable structural features to the IIICS. Furthermore, ligand binding studies showed that, under conditions tested, neither zinc nor the proteoglycan heparin, induced the formation of any secondary structure to this region. Zinc binding did, however, induce oligomerisation of a IIICS containing construct and appeared to enhance the binding of heparin to the IIICS. Data was obtained to suggest that FIII15 forms a transient interaction with an adjacent module, which is likely to be FIII14. It is hoped that the work presented will contribute to further studies into this important area of Fn and may aid in the future development of novel therapeutics.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:703038 |
| Date | January 2017 |
| Creators | Blumson, Eve Charlotte |
| Contributors | Humphries, Martin ; Waltho, Jonathan |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/the-structure-and-function-of-the-type-iii-connecting-segment-iiics-region-of-fibronectin(9422ac4b-b8c1-405d-b10d-efdaff0ad9a7).html |
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