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Modelling Glycocluster Effects Using Artificial Lipid Rafts
[Thesis]. Manchester, UK: The University of Manchester; 2012.
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Abstract
The interaction of carbohydrates with carbohydrate-binding proteins is key to a multitude of important biological events, such as cell-cell interactions and signal transduction. Carbohydrates are also essential for energy storage and transfer. Binding to the three-dimensional display of carbohydrates at the cell surface (the glycocalyx) is known to play a role in many disease states, such as carbohydrate binding by viruses. Furthermore, changes in the distribution and type of oligosaccharides is known to occur at tumour cell surfaces. The importance of these natural events at the cell membrane surface provided the motivation for their study in a biomimetic environment. Inspired by previous work in the field of carbohydrate-lectin binding and work in the Webb group into mimicry of cellular processes using supramolecular chemistry, vesicular structures of synthetic glycolipids in natural phospholipids were created. Several synthetic glycolipids were synthesised and found to be capable of lateral phase separation in ordered-phase phospholipid bilayers, forming artificial lipid rafts in the bilayer. The glycolipid vesicle systems were used to study the effects of the lateral clustering of glycolipids on two different biochemical events at the membrane surface: Binding of mannosyl-lipids by concanavalin A (ConA) and the enzymatic galactosylation of N-acetylglucosamine (GlcNAc)-lipids by bovine β-(1,4)-galactosyltransferase (β4GalT1). Fluorescence quenching titrations revealed that clustering of mannosyl-lipids had little effect on the strength of ConA binding. However, HPLC measurements showed that lateral clustering of GlcNAc-lipids could enhance their enzymatic galactosylation by β4GalT1. The work presented in this thesis represents the formulation of these vesicle systems and their study with ConA and β4GalT1. Further investigation with other phase-separating glycolipid-lectin/enzyme pairs is necessary to establish whether the effects of clustering observed herein are exclusive to ConA and β4GalT1, or are general phenomena observed at the membrane surface.
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CD-ROM containing appendix attached to hard bound copy of theses.