Diabetes mellitus is a major, life-threatening worldwide disease wherein pathological complications include increased risk of infection and cardiovascular disease. The hallmark of diabetic disease is elevated plasma glucose. Within the immune system and vasculature, host-pathogen and regulatory host-host interactions can operate through binding of microbial or host oligosaccharides by proteins of the C-type lectin family. A number of these proteins recognise oligosaccharides rich in mannose and fucose: monosaccharide units with similar chemical structures to glucose. This raises the possibility that the high glucose conditions in diabetes affect immunological protein-oligosaccharide interactions via competitive inhibition. Mannose binding lectin, soluble recombinant DC-SIGN and DC-SIGNR, were tested for binding to high-mannose and high-fucose ligands in the presence of high glucose concentrations, typical of diabetes, via surface plasmon resonance and affinity chromatography. Complement activation assays were conducted in the presence of high glucose. Analysis of DC-SIGN and DCSIGNR expression in a range of tissues and cells was studied via immunohistochemistry and selective functional proteomics. High glucose greatly inhibited mannose binding lectin protein (MBL), DC-SIGN and DCSIGNR binding to high-mannose glycoprotein ligands, and binding of DCSIGN to a fucosylated ligands (ABO blood group trisaccharides) was abrogated in high glucose. Complement activation via the lectin pathway was inhibited in high glucose and also in high concentrations of trehalose, a nonreducing sugar with glucoside stereochemistry. Immunohistochemistry revealed DC-SIGN staining in adipose, brain and renal tissue, consistent with expression on dendritic cells. Selective functional proteomic analyses in leucocytes and placenta revealed thrombospondin-1 and pappalysins 1 and 2 as candidate DC-SIGN ligands with implications in vascular disease and pregnancy complications. Furthermore the carbohydrate recognition domains of DC-SIGN and DC-SIGNR were prepared in 15N stable isotope medium and analysed via solution NMR spectroscopy, providing a gateway to understanding the molecular dynamics of these lectins. Additional analyses of novel glycopolymers highlighted the potential therapeutic properties of these compounds. This thesis shows that high glucose disrupts C-type lectinmediated protein-oligosaccharide interactions, providing new mechanistic perspectives for the susceptibility to infectious and inflammatory disease in diabetes. The mechanisms involved are noncovalent, competitive, selective and reversible; in contrast to the broadly indiscriminate, covalent, widespread and irreversible glycation of proteins. The thesis illuminates a set of valuable new tools and ligand networks that will benefit diabetes and immunological research.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:535621 |
| Date | January 2010 |
| Creators | Ilyas, Rebecca |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/35516/ |
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