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Title: Structural and Functional Characterisation of the G1 Domain from Human Versican
Author: Fielder , Helen Louise
ISNI:       0000 0001 3464 1867
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2008
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Versican, a chondroitin sulfate proteoglycan, fomis large extracellular link proteinstabilised complexes with hyaluronan (HA), thought to confer structural properties such as elasticity of blood vessels. HA binding is mediated through the N-terminal 01 domain of versican (VOl), which is composed of an Ig domain and two contiguous Link modules. In addition to the Link modules, the Ig domain may be required for HA .binding, perhaps stabilising the overall fold of the 01 domain. There is currently no high-resolution structure available for VOl, or for Type C HA-binding domains (HABO), which are comprised ofa pair of Link modules. Previously, VO I from human versican was expressed in Drosophila S2 cells as two N-glycoforms (dVOI); it was shown to interact with HA and this was unaffected by deglycosylation (Seyfried et aI., 2005b). Initial crystallisation attempts were limited by low expression yields (-0.5 mg/litre) and perhaps glycosylation. Here, VOl has - therefore been expressed a) in E. coli (eVGl) and b) in an unglycosylated form in Drosophila S2 cells in the presence of tunicamycin (tVO I), to generate material more suitable for structural studies. eVG1 was refolded by rapid dilution and, after ion exchange chromatography, 7-9 mg/litre pure protein was obtained. eVGl and tVOl have similar HA-binding properties to dVGl, but intrinsic fluorescence analysis indicated that only eVO1 was correctly folded. Glycosylation may therefore be required for correct folding of VG1 in eukaryotic systems. eVG1 also interacts with the TSO-6 Link module (as previously demonstrated for dVG1 (Kuznetsova et aI., 2006)). These functional studies, in combination with information from 10 NMR, ESI-TOF-MS and dynamic light scattering, indicated that eVGI was suitable for further biophysical/functional studies. eVG1 was screened against -1500 conditions, but an eVGI crystal was not obtained. Lack of success with crystallography may be due to a non-functional fraction of the eVOI preparation. Biotinylated-eVOI was found to be a useful tool for histological staining of HA in tissue sections. eVGI is also being used to raise a monoclonal antibody against the GI domain of versican and to investigate whether eVG1 can promote elastogenesis in vascular smooth muscle cells. Previously it has been hypothesised that VGI interacts with HA every HAlO and that this interaction displays all-or-nothing co-operativity (Seyfried et aI., 2006). By SECMALLS analysis it was found that eVOI 'footprints' a length of polymeric HA which is approximately double the length that was previously thought i.e. HAI8-22. Competition experiments using HA22 oligomers indicate that the interaction is positively co-operative, although further experiments need to be performed to ascertain whether the interaction shows all-or-nothing co-operativity. In sununary, a method of producing high yields of VOI has been developed and this material has facilitated structural, functional, cell biology and histology studies. Further characterisation of HA-VOI complex formation is improving our understanding of extracellular matrix organisation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available