| Summary: | Lecithin: cholesterol acyltransferase (EC 2.3.1.43) (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. === Medicine, Faculty of === Pathology and Laboratory Medicine, Department of === Graduate
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