The activity and metabolism of recombinant lecithin:cholesterol acyltransferase in vivo
Creators: name: Shaw, Stan Terry
Publisher: name: University of British Columbia
Year: 2009

description: Lecithin:cholesterol acyltransferase (LCAT; phosphatidylcholine-sterol acyltransferase, EC 2.3.1.43) is the plasma enzyme responsible for the formation of the majority of cholesteryl esters in human plasma. Its role in regulating plasma lipoprotein metabolism has been recognized for many years. However, despite extensive research, our knowledge of the structure and function of this enzyme is incomplete. With this in mind, the purpose of this thesis was to utilize a molecular biology approach to better understand the structure of LCAT through analysis of pre-dicted amino acid sequences and to utilize our ability to produce active recombinant LCAT to study the metabolism of LCAT in vivo. Specifically, we set out to: - Clone rabbit LCAT cDNA. Constitutively express rLCAT in a stable cell line Produce radiolabeled, biologically active rLCAT Determine the metabolic turnover rate of rLCAT in vivo. In order to achieve these aims, we cloned and expressed full length rabbit rLCAT and near full-length porcine rLCATThe near full-length porcine cDNA was obtained from screening a porcine A.-GT 11 library. Together with previously published sequence data from other species, the information obtained from the cloned porcine LCAT cDNA enabled us to design a set of consensus primers which were used to amplify overlapping nucleotide sequences from reverse-transcribed rabbit liver RNA. The nucleotide sequence identity of the rabbit LCAT cDNA sequence was 93% when compared to human LCAT . All major predicted structural features found in human LCAT were found in the rabbit sequence. This includes the lipase consensus region, the predicted N-linked glycosylation sites and potential bridged Cys residues. The rabbit LCAT cDNA was cloned into a pNUT expression vector, stably transfected into BHK cells and the enzymatically active recombinant protein was purified from the culture media. Characterization of the recombinant rabbit LCAT demonstrated that it was activated by both human and rabbit apo A-l and the Km of unesterified cholesterol in HDL analog substrates was 72.8 nmol/mL. All methods for radioiodination of the rLCAT (e.g.,Bolton-Hunter, Chloramine-T and lodobeads) resulted in an enzymatically inactive protein. However, endogenous incorporation of [³H]-leucine into BHK cells produced radiolabeled rLCAT with unaltered esterification activity. When incubated with plasma, 44.3% of [³H]-rLCAT was bound to lipoprotein fractions separated by size-exclusion chromatography. In vivo metabolic studies of rabbits injected with [³H]-rLCAT showed two catabolic components; the largest, with a halflife of 2.1-5.8 hours, and a second with a half-life of 52.7-62.6 hours. Interestingly, rabbits injected with enzymatically inactive [¹²⁵l]-rLCAT demonstrated no significant difference between the two components when compared to the turnover of [³H]- rLCAT. Since enzyme activity is maintained despite the failure of the majority of added rLCAT to bind to HDL in vitro and in vivo, the evidence suggests that esterification activity by LCAT does not require the enzyme to be permanently asso-ciated with plasma lipoproteins. In conclusion, this thesis provided novel information regarding the molecular organization of rabbit and porcine LCAT and the properties (in vitro) and catabolism of the recombinant enzyme {in vivo) in the New Zealand rabbit. These studies extend the growing body of knowledge of the structure and function of LCAT. In addition, the unexpectedly high initial rate of turnover of the rLCAT in vivo provides valuable insights into the utility of rLCAT for enzyme replacement therapy.