Human Hepatic Lipase Subunit Structure Determination

Hill, John S.; Davis, Richard C.; Yang, Dongliang; Wen, Jie; Philo, John S.; Poon, Pak H.; Phillips, Martin; Kempner, Ellis S.; Wong, Howard

doi:10.1074/jbc.271.37.22931

Cited by 45 publications

(37 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Another advantage to the radiation inactivation is that it is a method for determining the functional size of a protein without the need of prior purification. Uncleaved preparations of wild-type EL and EL-EL gave similar functional molecular mass estimates, consistent with the concept that EL is active as a homodimer and are in agreement with the data previously generated for LPL (23,46) and HL (25,43). Interestingly, cleavage of only one "side" of the homodimer results in an intermediate size form (Fig.…”

Section: Cellssupporting

confidence: 91%

“…Human HL was shown to have a molecular mass of 121 kDa (43), and similar studies of rat HL showed 113 kDa (26). Together, these data suggested that HL exists as a dimer.…”

Section: Discussionmentioning

confidence: 60%

“…There is some discrepancy in the literature with regard to the functional unit of HL. When recombinant human HL was subjected to radiation inactivation, the size of functional HL was calculated to be 109 kDa (unglycosylated), the size of a homodimer (43). An examination of rat HL from liver, adrenal gland, and ovaries showed that the liver enzyme had a functional molecular mass of 63 kDa (monomeric), but the enzymes from the adrenals and ovaries both showed func- tional molecular masses of 117 kDa (dimeric) (26).…”

Section: Cellsmentioning

confidence: 99%

See 2 more Smart Citations

Identification of the Active Form of Endothelial Lipase, a Homodimer in a Head-to-Tail Conformation

Griffon

Jin²,

Petty

et al. 2009

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Endothelial lipase (EL) is a member of a subfamily of lipases that act on triglycerides and phospholipids in plasma lipoproteins, which also includes lipoprotein lipase and hepatic lipase. EL has a tropism for high density lipoprotein, and its level of phospholipase activity is similar to its level of triglyceride lipase activity. Inhibition or loss-of-function of EL in mice results in an increase in high density lipoprotein cholesterol, making it a potential therapeutic target. Although hepatic lipase and lipoprotein lipase have been shown to function as homodimers, the active form of EL is not known. In these studies, the size and conformation of the active form of EL were determined. Immunoprecipitation experiments suggested oligomerization. Ultracentrifugation experiments showed that the active form of EL had a molecular weight higher than the molecular weight of a simple monomer but less than a dimer. A construct encoding a covalent head-to-tail homodimer of EL (EL-EL) was expressed and had similar lipolytic activity to EL. The functional molecular weights determined by radiation inactivation were similar for EL and the covalent homodimer EL-EL. We previously showed that EL could be cleaved by proprotein convertases, such as PC5, resulting in loss of activity. In cells overexpressing PC5, the covalent homodimeric EL-EL appeared to be more stable, with reduced cleavage and conserved lipolytic activity. A comparative model obtained using other lipase structures suggests a structure for the head-to-tail EL homodimer that is consistent with the experimental findings. These data confirm the hypothesis that EL is active as a homodimer in head-to-tail conformation.Three members of the triglyceride lipase family, lipoprotein lipase (LPL), 3 hepatic lipase (HL), and endothelial lipase (EL), contribute to lipoprotein catabolism in the plasma compartment. They are all secreted proteins that bind to heparan sulfate proteoglycans on the luminal side of endothelial cells where they interact with their lipoprotein substrates. They have different specificities for lipoproteins, and all hydrolyze triglycerides and phosphatidylcholine at the sn-1 position, albeit with widely differing efficiencies (1). The preferred lipoprotein substrates for LPL are the triglyceride-rich lipoproteins, chylomicrons, and very low density lipoproteins; the triglyceride lipase activity of LPL is more than 100-fold greater than its phospholipase activity. The primary lipoprotein substrates for HL are chylomicron remnants, intermediate density lipoproteins, and large triglyceridase-enriched HDL; its triglyceride lipase activity is about 20-fold higher than its phospholipase activity. EL is much more active on HDL, and its phospholipase activity is quite similar to its triglyceride lipase activity. These three lipolytic enzymes share a number of structural features. By analogy to the crystal structure of pancreatic lipase (2), another member of the triglyceride lipase family, each has a clearly defined N-terminal and C-terminal structural domain, ...

show abstract

Section: Cellssupporting

confidence: 91%

“…Human HL was shown to have a molecular mass of 121 kDa (43), and similar studies of rat HL showed 113 kDa (26). Together, these data suggested that HL exists as a dimer.…”

Section: Discussionmentioning

confidence: 60%

Section: Cellsmentioning

confidence: 99%

See 1 more Smart Citation

Identification of the Active Form of Endothelial Lipase, a Homodimer in a Head-to-Tail Conformation

Griffon

Jin²,

Petty

et al. 2009

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…In humans, plasma hHL activity can be increased by 1,000-fold upon infusion of heparin that disrupts hHL binding to heparan sulfate proteoglycans (HSPG) (18). Active hHL exists as a homodimer (19) and has a broad substrate specificity, catalyzing the hydrolysis of fatty acyl chains at the sn-1 position of phospholipids and of mono-, di-, and triacylglycerol that are associated with a variety of lipoproteins including HDL (20 -24). The dual phospholipase and triglyceride lipase activity of hHL is in contrast to LPL and EL that are predominantly triglyceride lipase and phospholipase, respectively (25).…”

mentioning

confidence: 99%

Severe Hypoalphalipoproteinemia in Mice Expressing Human Hepatic Lipase Deficient in Binding to Heparan Sulfate Proteoglycan

Brown

Gauthier

Parks

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

Unlike human hepatic lipase (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. The reduced HSPG binding of mHL is attributable to the C-terminal amino acids. To determine the functions of HSPG binding of hHL in vivo, we created adenovirus vectors encoding hHL or a chimeric protein (designated hHLmt) in which the C-terminal HSPG-binding sequences were replaced with the corresponding mouse sequences. Injecting hHLmt-expressing virus into C57BL/6J mice (1.8 ؋ 10 10 virus particles/mouse) resulted in a 3-fold increase in pre-heparin HL activity, whereas infection with an identical dose of hHL virus did not change pre-heparin HL activity. In hHLmt-expressing mice, the concentration of total cholesterol and phospholipids was inversely related to the hHL activity in pre-heparin plasma in a dose-and time-dependent manner, and the decrease was mainly attributable to high density lipoproteins (HDL) cholesterol and HDL phospholipids. The expression of hHL exhibited no change in plasma total cholesterol or phospholipid levels as compared with control mice infected with luciferase or injected with saline. The reduced HDL lipids in the hHLmt-expressing mice were accompanied by markedly decreased plasma and hepatic apolipoprotein (apo) A-I. In primary hepatocytes isolated from hHLmt-expressing mice, the concentration of cell-associated and secreted apoA-I was decreased by 2-3-fold as compared with hepatocytes isolated from control mice, whereas the levels of apoB and apoE were unaltered. Infection of primary hepatocytes with hHLmt virus ex vivo also resulted in reduced apoA-I secretion but had no effect on cell-associated apoA-I. These results suggest that expression of HSPG binding-deficient hHL has a profound HDL-lowering effect.

show abstract

“…Considering the possibility that EL might be active as a homodimer in a head-to-tail conformation as for HL and LPL (13)(14)(15)(16)(17)(18), and to further investigate the reasons for an unexpected partial effect of the EL lid exchange in substrate specificity, we investigated the role of the C-domain on the substrate specificity determined by the lid. To date, only the threedimensional structure of human pancreatic lipase has been resolved by X-ray diffraction (19).…”

Section: Discussionmentioning

confidence: 99%

Substrate specificity of lipoprotein lipase and endothelial lipase: studies of lid chimeras

Griffon

Budreck

Long

et al. 2006

Journal of Lipid Research

View full text Add to dashboard Cite

The triglyceride (TG) lipase gene subfamily, consisting of LPL, HL, and endothelial lipase (EL), plays a central role in plasma lipoprotein metabolism. Compared with LPL and HL, EL is relatively more active as a phospholipase than as a TG lipase. The amino acid loop or ''lid'' covering the catalytic site has been implicated as the basis for the difference in substrate specificity between HL and LPL. To determine the role of the lid in the substrate specificity of EL, we studied EL in comparison with LPL by mutating specific residues of the EL lid and exchanging their lids. Mutation studies showed that amphipathic properties of the lid contribute to substrate specificity. Exchanging lids between LPL and EL only partially shifted the substrate specificity of the enzymes. Studies of a double chimera possessing both the lid and the C-terminal domain (C-domain) of EL in the LPL backbone showed that the role of the lid in determining substrate specificity does not depend on the nature of the C-domain of the lipase. Using a kinetic assay, we showed an additive effect of the EL lid on the apparent affinity for HDL 3 in the presence of the EL C-domain. Endothelial lipase (EL) is a member of the triglyceride (TG) lipase gene family (1) that is synthesized by endothelial cells and by other cell types such as macrophages and hepatocytes (1-3). EL has a distinct substrate specificity profile and is relatively more active as a phospholipase than as a TG lipase (4). Thus, the ratio of TG lipase to phospholipase activity of EL is markedly less than those of LPL and HL. Relative to phospholipase activity, LPL displays the highest TG lipase activity and EL the lowest TG lipase activity of the three enzymes. EL also has greater preference for HDL, and LPL has the greater preference for VLDL (4, 5). Thus, LPL, HL, and EL seem to have evolved with distinct substrate specificities to accommodate the full spectrum of circulating lipoproteins.The lid covering the active site has been implicated as a major source of substrate specificity for LPL and HL. Dugi et al. (6) studied the hypothesis that the surface lid covering the catalytic pocket may modulate access of the substrate to the active site of LPL. Characterization of a number of mutants with altered amphipathic properties of the LPL lid showed that the disruption of the lid decreased its ability to hydrolyze an emulsified lipid substrate without affecting the ability to hydrolyze a water-soluble substrate. They proposed that the interaction between the lipoprotein substrates and the lid may in part determine substrate specificity. Chimeric lipases were also generated by exchanging the lid region between LPL and HL (7). The lid of LPL conferred preferential TG hydrolysis, as opposed to augmenting phospholipase activity in the case of the lid of HL. Preferential in vivo hydrolysis of phospholipids (PLs) was demonstrated in HL-deficient mice injected with adenovirus-expressing lipases containing the HL lid (HL or LPL with the lid of HL) compared with lipases containing the ...

show abstract

Human Hepatic Lipase Subunit Structure Determination

Cited by 45 publications

References 48 publications

Identification of the Active Form of Endothelial Lipase, a Homodimer in a Head-to-Tail Conformation

Identification of the Active Form of Endothelial Lipase, a Homodimer in a Head-to-Tail Conformation

Severe Hypoalphalipoproteinemia in Mice Expressing Human Hepatic Lipase Deficient in Binding to Heparan Sulfate Proteoglycan

Substrate specificity of lipoprotein lipase and endothelial lipase: studies of lid chimeras

Contact Info

Product

Resources

About