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Title: Enzymatic glycosylation of synthetic glycolipid substrates embedded in phospholipid vesicles
Author: Craven, Faye
ISNI:       0000 0005 0289 5036
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2018
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Cell Membranes are of great biological importance, as a platform for many intricately controlled vital processes including signal transduction and adhesion. Compartmentalisation of sphingolipids, proteins and cholesterol at the membrane interface are considered largely responsible for membrane functionality. A model system previously devised by the Webb group to mimic the glycocalyx and lipid raft formation in cell membranes was further investigated. The system was based on phospholipid vesicles embedded with synthetic glycolipids. Synthetic glycolipids were excluded from ordered phospholipid regions, allowing tuneable phase separation dependant on phospholipid composition. The model systems were used to study the effect of domain formation on various enzymatic reactions with glycolipid substrates. Experiments using PFEGlcNAc lipid embedded within phospholipid vesicles and galactosyl transferase enzyme (Î21,4-GalT1) were conducted. The enzyme was used to transfer galactose to the external lipid terminus. The Michaelis-Menten kinetics of the reaction were compared to prior results obtained by the Webb group. Results confirmed microdomain formation, significantly enhanced the rate of enzymatic transformation. To understand if this phenomenon was a unique feature of Î21,4-GalT1 or a generally observed principle, similar vesicular systems with microdomains were investigated with alternative enzymes. Perfluorinated glycolipids PFELac, PFEGal and PFEGlcNAc were synthesised and embedded within membranes of different phase states. Kinetic studies of enzymatic transformations with Trypanosoma cruzi Trans-sialidase and galactose oxidase were performed. A trisaccharide construct was built from consecutive enzymatic glycoaddition reactions on the membrane surface. The effect of lateral phase separation of substrates was studied by monitoring the formation rate of saccharide products, in membranes of different phase states.
Supervisor: Webb, Simon Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available