Functional analysis of the carboxyl terminus of human hepatic lipase

Abstract

Human hepatic lipase (hHL) plays an important role in the metabolism of several lipoproteins. Unlike hHL that is mainly cell surface anchored via binding to heparan sulfate proteoglycans (HSPG), mouse HL (mHL) has a low affinity to HSPG and thus is largely blood borne. Studies have suggested that the carboxyl terminus of HL mediates cell surface binding. Recombinant hHL, mHL, and chimeric proteins (hHLmt and mHLht, in which the C-terminal 70 amino acids of hHL were exchanged with the corresponding sequence from mHL) were generated in a cell culture model. The hHL, mHL, and hHLmt proteins were catalytically active using triglyceride and phospholipid substrates. In transfected cells, the majority of hHL bound to the cell surface with only 4% of total extracellular hHL released into heparin-free media, whereas under the same conditions, 61% of total extracellular mHL were released. Like mHL, hHLmt showed decreased cell surface binding with 68% of total extracellular hHLmt released. A truncated hHL mutant (hHL471) was also generated by deleting the C-terminal five residues (KRKIR). The hHL471 also retained hydrolytic activity, and showed decreased cell surface binding with 40% of total extracellular protein released into the heparin-free media. To determine the functional role of HSPG binding by hHL in vivo, hHL and hHLmt were expressed in mice. Low levels of adenoviral expression (up to 1.8 x 10 10 virus particles per mouse) of hHL and the chimera hHLmt in C57BL/6J mice resulted in a 4- to 6-fold increase in post-heparin HL activity above uninfected mice. The hHLmt displayed reduced HSPG binding; the activity of hHLmt in pre-heparin plasma was 3-fold higher than that of hHL. In contrast to mice expressing hHL which had plasma total cholesterol and phospholipid levels comparable control mice, mice expressing hHLmt showed decreased plasma total cholesterol and phospholipids in a time-dependent manner by at least half, and the decrease was mainly attributable to HDL cholesterol and HDL phospholipids, as determined by FPLC. The reduced HDL lipids in the hHLmt-expressing mice were accompanied by markedly decreased plasma and hepatic apoA-I. In primary hepatocytes isolated from hHLmt-expressing mice, the concentration of cell-associated and secreted apoA-I were decreased by 2- to 3-fold as compared to hepatocytes isolated from control mice, whereas the levels of apoB and apoE were unaltered. Surprisingly, primary hepatocytes expressing hHLmt ex vivo had reduced secreted apoA-I but exhibited unaltered levels of cell-associated apoA-I. These data show that the C-terminal 70 amino acids of hHL play an important role in HSPG binding, and that the impaired HSPG binding of hHL has a profound HDL-lowering effect (hypoalphalipoproteinemia) in vivo.