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Title: Expression and structure-functional studies of human apolipoprotein CIII
Author: Liu, Haiqun
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2000
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Apolipoprotein (Apo) CIII plays a key role in triglyceride (TG)-rich lipoprotein metabolism and is a risk factor for coronary heart disease (CHD). The study involved in this thesis is the first in vitro structure-functional study using recombinant apoCIII proteins. The physicochemical properties of recombinant wild type and A23T, a naturally occurring mutation that is associated with apoCIII deficiency and lower plasma TG levels, as well as three site-directed mutants of apoCIII, designed by molecular modelling and implicated in lipid binding (L9T/T20L, F64A/W65A) or lipoprotein lipase (LPL) inhibition (K21A), were compared. Relative lipid binding efficiencies of each apoCIII variants to 1.2-dimyristoyl-sn-glycero-3- phosphatidylcholine (DMPC) were: L9T/T20L > WT > K21A > A23T > F64A/W65A with an inverse correlation with size of the discoidal complexes formed. Physicochemical analysis (Trp fluorescence, circular dichroism (CD) and GdnHC1 denaturation) suggested that the stability of the resulting apoCIII:DMPC complexes were dependent on their lipid binding properties. The displacement of apoE by apoCIII variants were tested by gel filtration of apoE:dipalmitoylphosphatidylcholine (DPPC) discoidal complexes mixed with the various apoCIII variants. All apoCIII proteins bound to the apoE:DPPC complexes and the capacity to displace apoE from the complex was dependent on their lipid binding affinity. All the recombinant apoCIII proteins inhibited LPL in the presence or in the absence of apoCII, with F64A/W65A displaying the most inhibition, suggesting that apoCIII inhibition of LPL is independent of lipid binding and therefore due to the protein:protein interaction with apoCII and/or LPL. Taken together, our data suggest that the hydrophobic residues F64 and W65 are crucial for the lipid binding properties of apoCIII and the redistribution of the N- terminal helix of apoCIII (L9T/T20L permutation) can enhance the lipid binding properties of the protein. Additionally, the reduced lipid binding capacity of the naturally occurring mutation A23T could lead to reduced plasma apoCIII and lower plasma TG levels in carriers.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry