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Title: Investigating glycosaminoglycan-protein interactions : the 'sugar chip'
Author: Robinson, David Edward
Awarding Body: The University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2009
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The aim of these studies is to investigate the production of plasma polymer modified surfaces for the non-covalent binding of glycosaminoglycans (GAGs). These surfaces are designed to aid the investigation of GAG-protein interactions, and enable the elucidation of the important biological processes they may be involved in. It is also hoped that the discoveries in this thesis will translate into the production of new technology for studying GAG-protein interactions for example a sugar plate array that can be utilised in probe GAGprotein interactions and gycomics in a high-throughput platform. In this work investigations have been carried out on binding of heparin, heparin sulphate, chondroitin sulphate, dermatan sulphate and the unsulphated hyaluronic acid. Allylamine can be plasma-polymerised (PpAA) on to surfaces to provide a positivelycharged surface to which sulphated glycosaminoglycans (GAGs) such as heparin can bind. A range of plasma polymer chemistries were investigated, for the elucidation of the requirements for efficient binding of biologically active GAGs, initially using heparin as the . . model ligand. The influence of heparin concentration, solution ionic strength and incubation time were all explored as variables in the immobilization of heparin to ppAA modified microtitre plates. Gradients of surface chemistry were produced by mixing of allylamine with a hydrocarbon monomer octadiene to elucidate the optimal surface chemistry for the binding of heparin. The amount of heparin bound to the plates was monitored by X-ray photoelectron spectroscopy (XPS), using the S2p from heparin on the surface and radio-labelled esS] heparin. The biological activity of the surface immobilised GAGs were investigated using a range of protein binding assays. These systems involved using GAGs to capture proteins on the allylamine modified surfaces in a modified sandwich ELISA approach. Readout systems such as antibody specific binding to the proteins retained on the surface was utilised with a colourimetric change being read as an absorbance readout. The results showed that there are differences in binding by the GAGs for the different binding proteins. OPG protein appeared to be dependent on sulphation levels with higher sulphated GAGs binding more protein. Link _ TSG-6 protein showed the opposite, with less sulphated GAGs binding better in general. While TIMP-3 showed no correlation with sulphation levels, as heparan sulphates containing different sulphation levels bound equally well. This suggests a specific molecular binding moiety for binding may be utilised by TIMP- 3, meaning the protein structure for GAG interactions may be more specific. However, more research to elucidate these specific moieties is required. These results clearly demonstrate that if scaled down to a micro array format, these surfaces could act as a platform for a high-throughput sugar chip for the investigation of GAG-protein interactions. These could have some advantages over current assay formats that are widely used so far as they are inexpensive to produce and can bind native GAGs with no modifications. This allows avoidance of expensive and time consuming synthesis procedures for GAG production containing modified regions. As well as the risks involved in modifying a GAG such as altering its affinities for its binding proteins.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available