Proteolytic processing of angiopoietin-like protein 4 by proprotein convertases modulates its inhibitory effects on lipoprotein lipase activity

Angiopoietin-like protein 4 (ANGPTL4) has been associated with a variety of diseases. It is known as an endogenous inhibitor of lipoprotein lipase (LPL), and it modulates lipid deposition and energy homeostasis. ANGPTL4 is cleaved by unidentified protease(s), and the biological importance of this cleavage event is not fully understood with respect to its inhibitory effect on LPL activity. Here, we show that ANGPTL4 appears on the cell surface as the full-length form, where it can be released by heparin treatment in culture and in vivo. ANGPTL4 protein is then proteolytically cleaved into several forms by proprotein convertases (PCs). Several PCs, including furin, PC5/6, paired basic amino acid-cleaving enzyme 4, and PC7, are able to cleave human ANGPTL4 at a consensus site. PC-specific inhibitors block the processing of ANGPTL4. Blockage of ANGPTL4 cleavage reduces its inhibitory effects on LPL activity and decreases its ability to raise plasma triglyceride levels. In summary, the cleavage of ANGPTL4 by these PCs modulates its inhibitory effect on LPL activity.     

Endothelial lipase: a new lipase on the block

Endothelial lipase (EL) is a newly described member of the triglyceride lipase gene family. It has a considerable molecular homology with lipoprotein lipase (LPL) (44%) and hepatic lipase (HL) (41%). Unlike LPL and HL, this enzyme is synthesized by endothelial cells and functions at the site where it is synthesized. Furthermore, its tissue distribution is different from that of LPL and HL. As a lipase, EL has primarily phospholipase A1 activity. Animals that overexpress EL showed reduced HDL cholesterol levels. Conversely, animals that are deficient in EL showed a marked elevation in HDL cholesterol levels, suggesting that it plays a physiologic role in HDL metabolism. Unlike LPL and HL, EL is located in the vascular endothelial cells and its expression is highly regulated by cytokines and physical forces, suggesting that it may play a role in the development of atherosclerosis. However, there is only a limited amount of information available about this enzyme. Some of our unpublished data in addition to previously published data support the possibility that the enzyme plays a role in the formation of atherosclerotic lesion.     

Hepatic proprotein convertases modulate HDL metabolism

The risk of atherosclerosis is inversely associated with plasma levels of high-density lipoprotein cholesterol (HDL-C). However, HDL metabolism is incompletely understood, and there are few effective approaches to modulate HDL-C levels. Here we show that inhibition in the liver of the classical proprotein convertases (PCs), but not the atypical PCs S1P and PCSK9, decreases plasma HDL-C levels. This metabolic effect of hepatic PCs is critically dependent on expression of endothelial lipase (EL), an enzyme that directly hydrolyzes HDL phospholipids and promotes its catabolism. Hepatic PCs reduce EL function through direct inactivating cleavage of EL as well as through activating cleavage of angiopoietin-like protein 3 (ANGPTL3), an endogenous inhibitor of EL. Thus, inhibition of hepatic PCs results in increased EL activity, leading to reduced HDL-C as well as impaired reverse cholesterol transport. The hepatic PC-ANGPTL3-EL-HDL pathway is therefore a novel mechanism controlling HDL metabolism and cholesterol homeostasis.     

Endothelial lipase mediates efficient lipolysis of triglyceride-rich lipoproteins

Triglyceride-rich lipoproteins (TRLs) are circulating reservoirs of fatty acids used as vital energy sources for peripheral tissues. Lipoprotein lipase (LPL) is a predominant enzyme mediating triglyceride (TG) lipolysis and TRL clearance to provide fatty acids to tissues in animals. Physiological and human genetic evidence support a primary role for LPL in hydrolyzing TRL TGs. We hypothesized that endothelial lipase (EL), another extracellular lipase that primarily hydrolyzes lipoprotein phospholipids may also contribute to TRL metabolism. To explore this, we studied the impact of genetic EL loss-of-function on TRL metabolism in humans and mice. Humans carrying a loss-of-function missense variant in LIPG, p.Asn396Ser (rs77960347), demonstrated elevated plasma TGs and elevated phospholipids in TRLs, among other lipoprotein classes. Mice with germline EL deficiency challenged with excess dietary TG through refeeding or a high-fat diet exhibited elevated TGs, delayed dietary TRL clearance, and impaired TRL TG lipolysis in vivo that was rescued by EL reconstitution in the liver. Lipidomic analyses of postprandial plasma from high-fat fed Lipg^-/- mice demonstrated accumulation of phospholipids and TGs harboring long-chain polyunsaturated fatty acids (PUFAs), known substrates for EL lipolysis. In vitro and in vivo, EL and LPL together promoted greater TG lipolysis than either extracellular lipase alone. Our data positions EL as a key collaborator of LPL to mediate efficient lipolysis of TRLs in humans and mice.     

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

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.     

Characterization of the lipolytic activity of endothelial lipase

Endothelial lipase (EL) is a new member of the triglyceride lipase gene family previously reported to have phospholipase activity. Using radiolabeled lipid substrates, we characterized the lipolytic activity of this enzyme in comparison to lipoprotein lipase (LPL) and hepatic lipase (HL) using conditioned medium from cells infected with recombinant adenoviruses encoding each of the enzymes. In the absence of serum, EL had clearly detectable triglyceride lipase activity. Both the triglyceride lipase and phospholipase activities of EL were inhibited in a dose-dependent fashion by the addition of serum. The ratio of triglyceride lipase to phospholipase activity of EL was 0.65, compared with ratios of 24.1 for HL and 139.9 for LPL, placing EL at the opposite end of the lipolytic spectrum from LPL. Neither lipase activity of EL was influenced by the addition of apolipoprotein C-II (apoC-II), indicating that EL, like HL, does not require apoC-II for activation. Like LPL but not HL, both lipase activities of EL were inhibited by 1 M NaCl. The relative ability of EL, versus HL and LPL, to hydrolyze lipids in isolated lipoprotein fractions was also examined using generation of FFAs as an end point. As expected, based on the relative triglyceride lipase activities of the three enzymes, the triglyceride-rich lipoproteins, chylomicrons, VLDL, and IDL, were efficiently hydrolyzed by LPL and HL. EL hydrolyzed HDL more efficiently than the other lipoprotein fractions, and LDL was a poor substrate for all of the enzymes.     

Design and synthesis of boronic acid inhibitors of endothelial lipase

Endothelial lipase (EL) and lipoprotein lipase (LPL) are homologous lipases that act on plasma lipoproteins. EL is predominantly a phospholipase and appears to be a key regulator of plasma HDL-C. LPL is mainly a triglyceride lipase regulating (V)LDL levels. The existing biological data indicate that inhibitors selective for EL over LPL should have anti-atherogenic activity, mainly through increasing plasma HDL-C levels. We report here the synthesis of alkyl, aryl, or acyl-substituted phenylboronic acids that inhibit EL. Many of the inhibitors evaluated proved to be nearly equally potent against both EL and LPL, but several exhibited moderate to good selectivity for EL.     

Angiopoietin-like Protein 3 Inhibits Lipoprotein Lipase Activity through Enhancing Its Cleavage by Proprotein Convertases

Lipoprotein lipase (LPL)-mediated lipolysis of triglycerides is the first and rate-limiting step in chylomicron/very low density lipoprotein clearance at the luminal surface of the capillaries. Angiopoietin-like protein 3 (ANGPTL3) is shown to inhibit LPL activity and plays important roles in modulating lipoprotein metabolism in vivo. However, the mechanism by which it inhibits LPL activity remains poorly understood. Using cell-based analysis of the interaction between ANGPTL3, furin, proprotein convertase subtilisin/kexin type 5 (PCSK5), paired amino acid converting enzyme-4 (PACE4), and LPL, we demonstrated that the cleavage of LPL by proprotein convertases is an inactivation process, similar to that seen for endothelial lipase cleavage. At physiological concentrations and in the presence of cells, ANGPTL3 is a potent inhibitor of LPL. This action is due to the fact that ANGPTL3 can enhance LPL cleavage by endogenous furin and PACE4 but not by PCSK5. This effect is specific to LPL but not endothelial lipase. Both N- and C-terminal domains of LPL are required for ANGPTL3-enhanced cleavage, and the N-terminal domain of ANGPTL3 is sufficient to exert its effect on LPL cleavage. Moreover, ANGPTL3 enhances LPL cleavage in the presence of either heparan sulfate proteoglycans or glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1). By enhancing LPL cleavage, ANGPTL3 dissociates LPL from the cell surface, inhibiting both the catalytic and noncatalytic functions of LPL. Taken together, our data provide a molecular connection between ANGPTL3, LPL, and proprotein convertases, which may represent a rapid signal communication among different metabolically active tissues to maintain energy homeostasis. These novel findings provide a new paradigm of specific protease-substrate interaction and further improve our knowledge of LPL biology.     

Suppression of endothelial or lipoprotein lipase in THP-1 macrophages attenuates proinflammatory cytokine secretion

LPL and endothelial lipase (EL) are associated with macrophages in human atherosclerotic lesions, and overexpression of LPL in mouse macrophages is associated with a greater extent of atherosclerosis. To investigate potential mechanisms by which macrophage-derived lipase expression may mediate proatherogenic effects, we used lentivirus-mediated RNA interference to suppress the expression of either LPL or EL within THP-1 macrophages. After suppression of either LPL or EL, significant decreases in the concentration of interleukin-1beta, interleukin-6, monocyte chemoattractant protein-1, and tumor necrosis factor-alpha were observed. Incubation of THP-1 macrophages with either mildly or extensively oxidized LDL consistently decreased cytokine expression, which was additive to that contributed by lipase suppression. Decreased lipase expression was also associated with an altered lipid composition, with reduced percentages of cholesterol (unesterified and esterified), triglycerides, and lysophosphatidylcholine. Microarray data indicated a decreased expression of proinflammatory genes, growth factors, and antiapoptotic genes. By contrast, there was an increased expression of lipoprotein receptors (scavenger receptor 1, low density lipoprotein receptor, scavenger receptor class B type I, and CD36). Thus, we conclude that the suppression of either LPL or EL decreases proinflammatory cytokine expression and influences the lipid composition of THP-1 macrophages. These results provide further insight into the specific metabolic and potential pathological roles of LPL and EL in human macrophages.     

Comparative studies of vertebrate lipoprotein lipase: a key enzyme of very low density lipoprotein metabolism

Lipoprotein lipase (LIPL or LPL; E.C.3.1.1.34) serves a dual function as a triglyceride lipase of circulating chylomicrons and very-low-density lipoproteins (VLDL) and facilitates receptor-mediated lipoprotein uptake into heart, muscle and adipose tissue. Comparative LPL amino acid sequences and protein structures and LPL gene locations were examined using data from several vertebrate genome projects. Mammalian LPL genes usually contained 9 coding exons on the positive strand. Vertebrate LPL sequences shared 58-99% identity as compared with 33-49% sequence identities with other vascular triglyceride lipases, hepatic lipase (HL) and endothelial lipase (EL). Two human LPL N-glycosylation sites were conserved among seven predicted sites for the vertebrate LPL sequences examined. Sequence alignments, key amino acid residues and conserved predicted secondary and tertiary structures were also studied. A CpG island was identified within the 5'-untranslated region of the human LPL gene which may contribute to the higher than average (×4.5 times) level of expression reported. Phylogenetic analyses examined the relationships and potential evolutionary origins of vertebrate lipase genes, LPL, LIPG (encoding EL) and LIPC (encoding HL) which suggested that these have been derived from gene duplication events of an ancestral neutral lipase gene, prior to the appearance of fish during vertebrate evolution. Comparative divergence rates for these vertebrate sequences indicated that LPL is evolving more slowly (2-3 times) than for LIPC and LIPG genes and proteins.     

Relationship between serum HDL-C levels and common genetic variants of the endothelial lipase gene in Japanese school-aged children

Endothelial lipase (EL) is a new member of the triglyceride lipase family, the genes of which play a central role in dietary fat absorption, energy homeostasis, and plasma lipoprotein metabolism. One physiologic role of EL is thought to be hydrolysis of high-density lipoprotein (HDL) phospholipid, although the precise function of endothelial lipase has yet to be fully clarified. Furthermore, genetic variation in EL has been suggested to influence serum HDL-C levels. In the present study, we detected two common single nucleotide polymorphisms in the EL gene associated with serum HDL cholesterol levels in healthy school-aged children. Our data support the hypothesis that variations in the EL gene are one of the genetic determinants of serum HDL-C levels.     

Endothelial lipase modulates monocyte adhesion to the vessel wall. A potential role in inflammation

Endothelial lipase (EL), a new member of the lipoprotein lipase gene family, plays a central role in high density lipoprotein metabolism. Previous studies indicated that EL is expressed in endothelial cells, macrophages, and smooth muscle cells in atherosclerotic lesions in human coronary arteries. However, the functional role of EL in the local vessel wall remains obscure. In this study, we evaluated the ability of EL to modulate monocyte adhesion to the endothelial cell surface. EL mRNA and protein levels were markedly increased in tissues of the mouse model of inflammation induced by lipopolysaccharide injection. Adhesion assays in vitro revealed that overexpression of EL in COS7 or Pro5 cells enhanced monocyte bindings to the EL-expression cells. Heparin or heparinase treatment inhibited EL-mediated increases of monocyte adhesion in a dose-dependent manner. Moreover, ex vivo adhesion assays revealed that the number of adherent monocytes on aortic strips was significantly increased in EL transgenic mice and decreased in EL knock-out mice as compared with wild-type mice. These results suggest that EL on the endothelial cell surface can promote monocyte adhesion to the vascular endothelium through the interaction with heparan sulfate proteoglycans. Thus, the up-regulation of EL by inflammatory stimuli may be involved in the progression of inflammation.     

Endothelial and lipoprotein lipases in human and mouse placenta

Placenta expresses various lipase activities. However, a detailed characterization of the involved genes and proteins is lacking. In this study, we compared the expression of endothelial lipase (EL) and LPL in human term placenta. When placental protein extracts were separated by heparin-Sepharose affinity chromatography, the EL protein eluted as a single peak without detectable phospholipid or triglyceride (TG) lipase activity. The major portion of LPL protein eluted slightly after EL. This peak also had no lipase activity and most likely contained monomeric LPL. Fractions eluting at a higher NaCl concentration contained small amounts of LPL protein (most likely dimeric LPL) and had substantial TG lipase activity. In situ hybridization studies showed EL mRNA expression in syncytiotrophoblasts and endothelial cells and LPL mRNA in syncytiotrophoblasts. In contrast, immunohistochemistry showed EL and LPL protein associated with both cell types. In mouse placentas, lack of LPL expression resulted in increased EL mRNA expression. These results suggest that the cellular expression of EL and LPL in human placenta is different. Nevertheless, the two lipases might have overlapping functions in the mouse placenta. Our data also suggest that the major portions of both proteins are stored in an inactive form in human term placenta.     

Lipase maturation factor 1 is required for endothelial lipase activity

Lipase maturation factor 1 (Lmf1) is an endoplasmic reticulum (ER) membrane protein involved in the posttranslational folding and/or assembly of lipoprotein lipase (LPL) and hepatic lipase (HL) into active enzymes. Mutations in Lmf1 are associated with diminished LPL and HL activities ("combined lipase deficiency") and result in severe hypertriglyceridemia in mice as well as in human subjects. Here, we investigate whether endothelial lipase (EL) also requires Lmf1 to attain enzymatic activity. We demonstrate that cells harboring a (cld) loss-of-function mutation in the Lmf1 gene are unable to generate active EL, but they regain this capacity after reconstitution with the Lmf1 wild type. Furthermore, we show that cellular EL copurifies with Lmf1, indicating their physical interaction in the ER. Finally, we determined that post-heparin phospholipase activity in a patient with the LMF1(W464X) mutation is reduced by more than 95% compared with that in controls. Thus, our study indicates that EL is critically dependent on Lmf1 for its maturation in the ER and demonstrates that Lmf1 is a required factor for all three vascular lipases, LPL, HL, and EL.     

Lipoprotein lipases from cow, guinea-pig and man. Structural characterization and identification of protease-sensitive internal regions

Lipoprotein lipases from human, bovine or guinea-pig milk were purified, judged for domain relationships by characterization of sites sensitive to proteases, and structurally compared. The subunit of human lipoprotein lipase migrated slightly slower than those of bovine or guinea-pig lipoprotein lipases on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Bovine lipoprotein lipase is known to be a dimer of two non-covalently linked subunits of equal size, and the lipases from all three sources now yielded homogeneous N-terminal amino acid sequences (followed for 15-27 residues). The results indicate that the two subunits are identical. Bovine lipoprotein lipase had two additional N-terminal residues, Asp-Arg, compared to the human and guinea-pig enzymes, and the next two positions revealed residue differences, but further on homologies were extensive between all three enzymes as far as presently traced. Exposure of bovine lipoprotein lipase to trypsin led to production of three fragments (T1, T2a, and T2b), suggesting cleavage at exposed segments delineating domain borders. Time studies gave no evidence for precursor-product relationships between the fragments, and prolonged digestion did not lead to further cleavage. Fragments T2a and T2b had the same N-terminal sequence as intact lipase. Fragment T1 revealed a new sequence, and represents the C-terminal half of the molecule. Plasmin caused a similar cleavage as trypsin, whereas thrombin, factor Xa, and tissue plasminogen activator did not cleave the enzyme. Chymotrypsin cleaved off a relatively small fragment from the C-terminal of the molecule, after which exposure to trypsin still resulted in cleavage at the same sites as in intact lipase. Tryptic cleavage of guinea-pig lipoprotein lipase yielded two fragments. One had a similar size as bovine fragment T2b; the other had a similar size as bovine fragment T1 and an N-terminal sequence homologous with that of T1. Thus, trypsin recognizes the same unique site in guinea-pig lipoprotein lipase as in the bovine enzyme. This confirms the conclusion that this segment is the border between two domains in the subunit. The binding site for heparin was retained after both tryptic and chymotryptic cleavages and was identified as localized in the C-terminal part of the molecule.     

Lipase maturation factor 1: a lipase chaperone involved in lipid metabolism

Mutations in lipase maturation factor 1 (LMF1) are associated with severe hypertriglyceridemia in mice and human subjects. The underlying cause is impaired lipid clearance due to lipase deficiency. LMF1 is a chaperone of the endoplasmic reticulum (ER) and it is critically required for the post-translational activation of three vascular lipases: lipoprotein lipase (LPL), hepatic lipase (HL) and endothelial lipase (EL). As LMF1 is only required for the maturation of homodimeric, but not monomeric, lipases, it is likely involved in the assembly of inactive lipase subunits into active enzymes and/or the stabilization of active dimers. Herein, we provide an overview of current understanding of LMF1 function and propose that it may play a regulatory role in lipase activation and lipid metabolism. Further studies will be required to test this hypothesis and elucidate the full spectrum of phenotypes in combined lipase deficiency. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.     

Glycosylation of endothelial lipase at asparagine-116 reduces activity and the hydrolysis of native lipoproteins in vitro and in vivo

We previously identified that four of five putative N-linked glycosylation sites of human endothelial lipase (EL) are utilized and suggested that the substitution of asparagine-116 (Asn-116) with alanine (Ala) (N116A) increased the hydrolytic activity of EL. The current study demonstrates that mutagenesis of either Asn-116 to threonine (Thr) or Thr-118 to Ala also disrupted the glycosylation of EL and enhanced catalytic activity toward synthetic substrates by 3-fold versus wild-type EL. Furthermore, we assessed the hydrolysis of native lipoprotein lipids by EL-N116A. EL-N116A exhibited a 5-fold increase in LDL hydrolysis and a 1.8-fold increase in HDL2 hydrolysis. Consistent with these observations, adenovirus-mediated expression of EL-N116A in mice significantly reduced the levels of both LDL and HDL cholesterol beyond the reductions observed by the expression of wild-type EL alone. Finally, we introduced Asn-116 of EL into the analogous positions within LPL and HL, resulting in N-linked glycosylation at this site. Glycosylation at this site suppressed the LPL hydrolysis of synthetic substrates, LDL, HDL2, and HDL3 but had little effect on HL activity. These data suggest that N-linked glycosylation at Asn-116 reduces the ability of EL to hydrolyze lipids in LDL and HDL2.     

Vertebrate endothelial lipase: comparative studies of an ancient gene and protein in vertebrate evolution

Endothelial lipase (gene: LIPG; enzyme: EL) is one of three members of the triglyceride lipase family that contributes to lipoprotein degradation within the circulation system and plays a major role in HDL metabolism in the body. In this study, in silico methods were used to predict the amino acid sequences, secondary and tertiary structures, and gene locations for LIPG genes and encoded proteins using data from several vertebrate genome projects. LIPG is located on human chromosome 18 and is distinct from other human ‘neutral lipase’ genes, hepatic lipase (gene: LIPC; enzyme: HL) and lipoprotein lipase (gene: LPL; enzyme: LPL) examined. Vertebrate LIPG genes usually contained 10 coding exons located on the positive strand for most primates, as well as for horse, bovine, opossum, platypus and frog genomes. The rat LIPG gene however contained only 9 coding exons apparently due to the presence of a ‘stop’ codon’ within exon 9. Vertebrate EL protein subunits shared 58–97% sequence identity as compared with 38–45% sequence identities with human HL and LPL. Four previously reported human EL N-glycosylation sites were predominantly conserved among the 10 potential N-glycosylation sites observed for the vertebrate EL sequences examined. Sequence alignments and identities for key EL amino acid residues were observed as well as conservation of predicted secondary and tertiary structures with those previously reported for horse pancreatic lipase (PL) (Bourne et al. 1994). Several potential sites for regulating LIPG gene expression were observed including CpG islands near the LIPG gene promoter and a predicted microRNA binding site near the 3’-untranslated region. Promoter regions containing functional polymorphisms that regulate HDL cholesterol in baboons were conserved among primates but not retained between primates and rodents. Phylogenetic analyses examined the relationships and potential evolutionary origins of the vertebrate LIPG gene subfamily with other neutral triglyceride lipase gene families, LIPC and LPL. It is apparent that the triglyceride lipase ancestral gene for the vertebrate LIPG gene predated the appearance of fish during vertebrate evolution >500 million years ago.     

GPIHBP1, an endothelial cell transporter for lipoprotein lipase

Interest in lipolysis and the metabolism of triglyceride-rich lipoproteins was recently reignited by the discovery of severe hypertriglyceridemia (chylomicronemia) in glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1)-deficient mice. GPIHBP1 is expressed exclusively in capillary endothelial cells and binds lipoprotein lipase (LPL) avidly. These findings prompted speculation that GPIHBP1 serves as a binding site for LPL in the capillary lumen, creating "a platform for lipolysis." More recent studies have identified a second and more intriguing role for GPIHBP1-picking up LPL in the subendothelial spaces and transporting it across endothelial cells to the capillary lumen. Here, we review the studies that revealed that GPIHBP1 is the LPL transporter and discuss which amino acid sequences are required for GPIHBP1-LPL interactions. We also discuss the human genetics of LPL transport, focusing on cases of chylomicronemia caused by GPIHBP1 mutations that abolish GPIHBP1's ability to bind LPL, and LPL mutations that prevent LPL binding to GPIHBP1.