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UBC Theses and Dissertations

Chimeras of lipoprotein lipase and hepatic lipase : localization of the apolipoprotein C-II activation site of lipoprotein lipase McIlhargey, Trina Leann


Hepatic lipase (HL) and lipoprotein lipase (LPL) are members of the same lipase gene family, along with pancreatic lipase, the pancreatic lipase-related lipases, endothelial lipase, and phosphatidylserine-specific phospholipase A1. Through their ability to hydrolyze triglycerides and phospholipids in a variety of circulating plasma lipoproteins including chylomicrons, very low and intermediate density lipoproteins (VLDL) and high density lipoproteins, HL and LPL greatly influence lipid metabolism. Unlike HL , however, LPL requires a specific cofactor, apolipoprotein C-ll (apo C-ll), to hydrolyze triglycerides in chylomicrons and VLDL. The aim of the present study is to identify residues within LPL which enable it to be responsive in the presence of apo C-ll. A previous study has identified a segment in the N-terminal domain of LPL (residues 65-86) as having the ability to bind an apo C-ll peptide fragment. This segment was found to contain regions of amino acid sequence dissimilarity when compared to the homologous residues in HL. Using site-directed mutagenesis, two sets of chimeras were created in which the two regions of human LPL (LPL residues 65-68 and 73-79) were exchanged with the corresponding human HL sequence. The HL chimeras consisted of a HL backbone with the suspected LPL regions replacing the corresponding HL sequence either individually (HL[sub LPL65-68], HL[sub LPL73-79]) or together (HL[sub LPLD]) Similarly, the LPL chimeras were created in which the candidate regions were replaced with the corresponding HL sequence (LPL[sub HL77-80], LPL[sub HL85-91] and LPL[sub HLD]). Using a synthetic triolein substrate, lipase activity of the purified enzymes was measured in the presence and absence of apo C-ll. Addition of apo C-ll to HL[sub LPL65-68] and HL[sub LPL73-79] did not significantly alter their enzyme activity. However, the activity of HL[sub LPLD] increased ~5-fold in the presence of apo C-ll whereas the activity of native LPL increased ~11-fold. Addition of apo C-ll to LPL[sub HL77-80] resulted in ~10-fold activation while only ~6- fold and ~4-fold activation in enzyme activity was observed in LPL[sub HL85-91] and LPL[sub HLD], respectively. In summary, our results have identified 11 amino acid residues within the amino-terminal domain of LPL (residues 65-68 and 73-79) which appear to act cooperatively to enable substantial activation of human LPL by apo C-ll.

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