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Title: Heparan sulfate-protein interactions : evidence for sulfate group-dependent selectivity
Author: Ahmed, Yassir Ahmed Mohmed Ali
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2010
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Abstract:
Heparan sulfates (HS) are a family of structurally diverse, sulfated polysaccharides located at the cell surface and in the extracellular matrix. They are distributed widely in virtually all metazoan organisms. The structural diversity occurs via variability in the positions of sulfation along the HS chain and in (l L-iduronic acid and D› glucuronic acid substitutions. There is now strong evidence that these molecules perform diverse functions in vivo through their ability to regulate the activity of different proteins. A wide variety of proteins have been found to be ligands for liS, including growth factors, cytokines, receptors, adhesion and matrix molecules, enzymes, coagulation factors and a variety of bacterial and viral coat proteins. Hence, there is a very significant need to determine the molecular basis of HS activity and HS-protein interactions, addressing important issues such as the degree of molecular selectivity. In this study, two model protein systems were selected to test the hypothesis that selective saccharide sequence and sulfation patterns regulate protein binding and activity. In the first model, heparin and chemically modified heparin saccharides were screened for Slit and Robo binding and activity, as signalling by Slit requires two receptors, Robo transmembrane proteins and HS. How liS controls Slit-Robo signalling is unclear. Competition and direct enzyme linked immunoassay (ELISA) data showed that heparin derivatives enhance the affinity of Slit-Robo binary and ternary complexes. Analytical gel filtration chromatography demonstrated that Slit associates with a soluble Robo fragment and heparin derivatives to form a ternary complex. Furthermore, retinal growth cone collapse triggered by Slit requires cell surface HS or exogenously added heparin derivatives. The data indicate a complex relationship between HS binding and Slit-Robo proteins, which support selectivity in regulation of signalling responses, but do not suggest requirement for strictly defined sequence. In the second model, HS saccharides were prepared by heparinase treatment of porcine mucosal HS and purified by size-exclusion and SAX-IIPLC chromatography. Purified or semi-purified complex sulfated HS 6-mer and 8-mer sequences were prepared. This library was screened for binding to FGF-l and FGF-2 using a competition ELISA, and for corresponding bioactivity (regulation of FGF-l and FGF-2 signalling using a BaF3 cell assay with defined FGF receptors). This screen identified fractions that bound and activated these ligands, and ones that did not. Selected structures were subjected to activity analysis after further separation using ion-pairing reverse phase (IPRP-HPLC). Structures with similar or identical size and sulfation content, but distinct sulfation sequences, differed widely in their bioactivity, though binding data alone did not necessarily predict activity. The results provided information that supports the view that a significant degree of specificity for activation of FGF biological activity is encoded in the complex sulfation sequences observed in lIS chains. However, is not simply related to sulfation pattern. Over~ll: the results from these two model protein systems suggest that the degree of selectiVIty or specificity of HS structures for regulation of biological activity is complex, and varies according to the proteins involved. Nevertheless, they provide the basis for lIS to act as an important modulator of protein function in a wide variety of biological systems.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.539728  DOI: Not available
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