Chimeras of lipoprotein lipase and hepatic lipase: Localization of the apolipoprotein C-II activation site of lipoprotein lipase

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-II (apo C-II), 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-II. 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-II 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_LPL65–68, HL_LPL73–79) or together (HL_LPLD). Similarly, the LPL chimeras were created in which the candidate regions were replaced with the corresponding HL sequence (LPL_HL77–80, LPL_HL85–91 and LPL_HLD ). Using a synthetic triolein substrate, lipase activity of the purified enzymes was measured in the presence and absence of apo C-II. Addition of apo C-11 to HL_LPL65–68 and HL_LPL73–79 did not significantly alter their enzyme activity. However, the activity of HL _LPLD increased ∼5-fold in the presence of apo C-II whereas the activity of native LPL increased ∼11-fold. Addition of apo C-II to LPL_HL77–80 resulted in ∼10-fold activation while only ∼6-fold and ∼4-fold activation in enzyme activity was observed in LPL_HL85–91 and LPL_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-II.