Chinese Hamster Ovary Cells Expressing a Cell Surface-anchored Form of Hepatic Lipase

Abstract

The enzyme hepatic lipase may play several roles in lipoprotein metabolism. Recent investigation has suggested a role for the enzyme in lipoprotein and/or lipoprotein lipid uptake. To study this, a simple isolated system that mimics the in vivo system would be desirable. The enzyme is secreted by the hepatic parenchymal cell but exists, and presumably exerts its effects, while bound to capillary endothelial cells in the liver, adrenal gland, and the ovary. We constructed a cDNA that encodes the expression of a chimeric protein composed of rat hepatic lipase and the signal sequence for the addition of the glycophosphatidylinositol (GPI) anchor from human decay-accelerating factor. When transfected into Chinese hamster ovary (CHO) cells this gave rise to a cell population that had immunoreactive hepatic lipase on the cell surface. Cloning of the transfected cells produced several cell lines that expressed the chimeric protein bound to the cell surface by a GPI anchor. This was documented by demonstrating incorporation of [3H]ethanolamine into anti-hepatic lipase immunoprecipitable material; in addition, hepatic lipase was released from the cells by phosphatidylinositol-specific phospholipase C but not by heparin. Phosphatidylinositol-phospholipase C treatment of cells expressing the anchored lipase released material that comigrated with hepatic lipase on SDS-polyacrylamide gel electrophoresis and was immunoreactive with antibody to the cross-reacting determinant of GPI anchors. Cell lysates containing the anchored protein contained salt-resistant lipase activity, a known feature of the secreted hepatic lipase; thus it appears that these cells have a surface-anchored hepatic lipase molecule. Although it was not possible to demonstrate lipolysis by the enzyme while it was on the cell surface for technical reasons, the protein produced by these cells was active when studied in cell membranes. The ability of the cells to take up lipoproteins was studied. The cells demonstrated an increased affinity for low density lipoprotein (LDL) receptor mediated uptake of LDL. They did not, however, demonstrate any enhanced binding or removal of chylomicron remnants. With respect to LDL and remnants, the cells expressing anchored lipase behaved similarly to CHO cell that expressed secreted hepatic lipase. The cells expressing anchored hepatic lipase had a marked increase in the uptake of high density lipoprotein and high density lipoprotein cholesteryl ester when compared to that seen with CHO cells secreting hepatic lipase. This increase occurred primarily via the selective pathway, and was not reduced by addition of anti-LDL receptor or anti-hepatic lipase antibodies or the receptor-associated protein. Together the results suggest that hepatic lipase, when bound to the cell surface by a GPI anchor, plays a role in enhancing lipoprotein uptake. For LDL this may involve the provision of a second foot for particle binding, thus enhancing affinity for the LDL receptor. For chylomicron remnants an additional molecule or molecules are ...

Endogenously Produced Endothelial Lipase Enhances Binding and Cellular Processing of Plasma Lipoproteins via Heparan Sulfate Proteoglycan-mediated Pathway

“…Both CHO cell lines were maintained in Ham's F-12 medium supplemented with 10% fetal bovine serum. These three cell lines were chosen because they are readily infected with recombinant adenoviral constructs and were successfully used in the past to characterize effects of LpL and HL on lipoprotein metabolism (16,37,44,45).…”

“…In contrast, LpL had relatively small, if any, effects on HDL binding and holoparticle uptake, but significantly increased selective uptake of cholesterol esters from HDL particles (19 -22). Like LpL, HL has been shown to enhance binding and/or uptake of chylomicrons, chylomicron remnants, VLDL, and LDL by different cell types in vitro (15,16,23,24) via a HSPG-dependent process (15,16,23). In addition, in several studies HL efficiently increased holoparticle cellular uptake of HDL as well as selective uptake of cholesterol esters (16,25,26).…”

“…Like LpL, HL has been shown to enhance binding and/or uptake of chylomicrons, chylomicron remnants, VLDL, and LDL by different cell types in vitro (15,16,23,24) via a HSPG-dependent process (15,16,23). In addition, in several studies HL efficiently increased holoparticle cellular uptake of HDL as well as selective uptake of cholesterol esters (16,25,26). These bridging effects of LpL and HL require HSPGs and heparin-binding domains of the lipases but do not depend on their catalytical activities.…”

The Ligand-binding Function of Hepatic Lipase Modulates the Development of Atherosclerosis in Transgenic Mice

“…In this respect, HL could serve as a ligand that facilitates these interactions or, alternatively, could function as a coreceptor. In vitro studies have demonstrated that HL enhances the binding and/or uptake of chylomicrons, chylomicron remnants, ␤-very low density protein (VLDL), and LDL (15)(16)(17)(18)(19)(20) as well as HDL cholesterol (HDL-C) (18, 20 -22) in a variety of cell types. Cell-surface receptors, including the LDL receptor (LDLR), low density lipoprotein receptor-related protein (LRP), and SR-BI, as well as cellsurface proteoglycans, have been implicated in this process (15-17, 20, 22).…”

Hepatic Lipase, Lipoprotein Metabolism, and Atherogenesis

“…In vitro studies have demonstrated that hepatic lipase enhances the binding or uptake of chylomicrons, chylomicron remnants, VLDL, LDL, [53][54][55][56][57][58] and HDL cholesterol (HDL-C) 56,59 -61 into a variety of cell types. Cell surface receptors, including the LDL receptor (LDLr), 55 LDLr-related protein (LRP), 53,60 and scavenger receptor B1 (SR-B1), 59,61 as well as cell surface proteoglycans, 54,60 have been implicated in these processes.…”