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UBC Theses and Dissertations

Analysis of lecithin : cholesterol acyltransferase (LCAT) protein structure and its influence on binding to plasma lipoproteins Ayyobi, Amir Fardad


Lecithin: cholesterol acyltransferase (EC (LCAT) is synthesized by the liver in mammals and it is responsible for esterification of unesterified cholesterol (UC) in plasma. This plasma glycoprotein plays an important role in reverse cholesterol transport (RCT) and High density lipoprotein (HDL) metabolism. In this thesis, functional mutants of LCAT were utilized to study the factors influencing interaction of this protein with the plasma lipoproteins. McArdle 7777 cells were used to produce LCAT from a hepatic derived expression system. The expressed enzyme was uniformly glycosylated with biantennary oligosaccharide residues, which was significantly different from the glycosylation pattern observed in the plasma LCAT and the baby hamster kidney (BHK) expressed LCAT. Despite the differences in the glycosylation architecture, the new recombinant LCAT (rLCAT) displays similar kinetic properties to those of the plasma and BHK LCATs, suggesting that the differences in glycosylation architecture may not influence activity of the enzyme. Subsequently, interaction of rLCAT with lipoprotein substrates was studied to determine the binding characteristics of wild type (WT) and fish-eye disease (FED) plasma LCAT in vivo, and rLCAT in vitro. Endogenous LCAT was shown to remain bound to low-density lipoprotein (LDL) as well as HDL and that beta and alpha LCAT activity co-eluted with LDL and HDL particles, respectively. In vitro binding studies with whole plasma, total lipoproteins, and individual lipoproteins showed no differential association of rLCAT, either WT or FED (T123I), with HDL and LDL particles and that the majority of rLCAT did not bind to the plasma lipoproteins. BIAcore experiments using native plasma lipoproteins and proteoliposmes also showed little or no association of rLCAT with plasma lipoproteins, but a strong association with the synthetic HDL analogue particles. The final aspect of this thesis was concerned with the relationship of LCAT mutations with its structural and functional characteristics involving the study of two very unique mutations, which result in an increase in the LCAT activity. The kinetic data obtained from this study showed that the combination of the two mutations did not have an additive effect, but in fact resulted in reduction of LCAT activity. Although this effect may be due to lack of independence in mechanism of action or inability of the protein to tolerate sequence alterations, it suggests that the fourth oligosaccharide chain may influence LCAT function in more ways than just inhibiting substrate accessibility. In conclusion, although the glycosylation architecture of LCAT does not significantly alter the kinetic properties of the recombinant protein, it may influence binding/association of rLCAT to the plasma lipoproteins. In addition, the fourth oligosaccharide moiety of LCAT may play an important role (other than inhibitory) in LCAT activity.

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