Calcium Triggers Folding of Lipoprotein Lipase into Active Dimers

Zhang, Liyan; Lóokene, Aivar; Wu, Gengshu; Olivecrona, Gunilla

doi:10.1074/jbc.m507252200

Cited by 58 publications

(63 citation statements)

References 50 publications

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“…First, assembly of LPL monomers into active dimers takes hours in vitro ( 39 ) but only minutes in the ER ( 40 ), suggesting that this process is likely to be aided by a chaperone(s), such as Lmf1. Likewise, lipase homodimers rapidly dissociate to misfolded monomers in vitro unless stabilized by binding to factors such as heparin ( 39 ). In contrast, the LPL dimer is highly stable in the ER lumen, suggesting that an ERspecifi c stabilizing factor prevents such dissociation in vivo ( 40,41 ).…”

Section: Discussionmentioning

confidence: 99%

“…Instead, the formation of aggregates likely refl ects the accumulation of monomers due to ineffi cient homodimer assembly and/or stabilization in the absence of Lmf1. Indeed, the LPL monomer has been demonstrated to be prone to aggregation, probably due to exposure of a hydrophobic surface area that is shielded in the assembled homodimer ( 39,42 ). Finally, phylogenetic Epitope-tagged Lmf1 and affi nity-tagged lipase proteins were coexpressed in HEK293 cells.…”

Section: Discussionmentioning

confidence: 99%

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Lipase maturation factor 1 is required for endothelial lipase activity

Ben-Zeev

Hosseini

Lai

et al. 2011

Journal of Lipid Research

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This article is available online at http://www.jlr.org hydrolyze two principal lipid substrates associated with lipoprotein particles, triglycerides (TGs), and phospholipids. The released fatty acids (FAs) resulting from lipase hydrolysis are taken up by subjacent tissue and used for energy storage (adipose), oxidation, and energy production (skeletal muscle and heart) and the synthesis of bioactive metabolites (variety of tissues).LPL is principally a TG lipase involved in the metabolism of TG-rich lipoproteins (chylomicrons and very-lowdensity lipoproteins) in adipose and muscle and heart tissues ( 1 ). Its defi ciency and overexpression have been linked to metabolic abnormalities such as hypertriglyceridemia, insulin resistance, and cardiomyopathy, indicating the critical role of this enzyme in TG metabolism ( 4-10 ). Unlike LPL, HL has comparable TG lipase and phospholipase activities and is involved in the hepatic metabolism of high-density lipoprotein (HDL) as well as apolipoprotein B (apoB)-containing lipoproteins (LpBs) ( 11,12 ). Overexpression of HL reduces plasma levels of HDL and LpBs, whereas HL defi ciency has the opposite effect ( 13-16 ). EL is predominantly a phospholipase affecting HDL metabolism, but it also shares a redundant role with HL in the metabolism of LpBs ( 11,17 ). Indeed, modulation of EL activity in mice leads to changes in plasma HDL levels similar to those of HL (18)(19)(20), refl ecting their related substrate specifi cities. Consistent with their multifaceted involvement in lipoprotein metabolism, LPL, HL, and EL are strongly associated with plasma lipid levels in the general population ( 21 ). Abstract 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 defi ciency") 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 copurifi es 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. The vascular lipase family is composed of three evolutionarily related enzymes, lipoprotein lipase (LPL), hepatic lipase (HL), and endothelial lipase (EL) ( 1-3 ). Localized to the luminal face of tissue capillaries, lipases

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Lipase maturation factor 1 is required for endothelial lipase activity

Ben-Zeev

Hosseini

Lai

et al. 2011

Journal of Lipid Research

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“…The radiation inactivation method was developed to determine the size of enzymes (45) and has been previously used to determine the functional molecular mass of both LPL (23,46) and HL (25,44). For bovine milk LPL, the combined data of radiation inactivation under a number of different conditions yielded a functional size of 72 kDa for the unglycosylated protein, which is close to that expected for a dimer, 77 kDa (23).…”

Section: Cellsmentioning

confidence: 99%

“…associated almost exclusively with the dimer fraction (24). Sucrose density gradient centrifugation was also used to determine that monomeric LPL refolded in the presence of calcium becomes dimeric (44). A complicating issue in determining the functional size of catalytically active EL relates to its susceptibility to proteolytic cleavage by proprotein convertases (19,20).…”

Section: Table 1 Triglyceride Lipase and Phospholipase Activities Of mentioning

confidence: 99%

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

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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, ...

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“…Biotinylation and Separation of Bovine LPL by Sucrose Density Gradient Centrifugation-100 g of bovine LPL was biotinylated with a Sulfo-NHS-Biotin kit (Pierce) and was then re-purified by sucrose density gradient ultracentrifugation to separate low density lipoprotein dimers and monomers as described elsewhere (23,37). Briefly, 5-20% sucrose density gradient was made up in a buffer containing 20 mM Tris-HCl (pH 7.4), 2 M NaCl, and 2 mg/ml BSA.…”

Section: Surface-enhanced Laser Desorption/ionization Time-of-flight mentioning

confidence: 99%

A Highly Conserved Motif within the NH2-terminal Coiled-coil Domain of Angiopoietin-like Protein 4 Confers Its Inhibitory Effects on Lipoprotein Lipase by Disrupting the Enzyme Dimerization

Yau

Wang

Lam

et al. 2009

Journal of Biological Chemistry

105

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Lipoprotein lipase (LPL) is a principal enzyme responsible for the clearance of chylomicrons and very low density lipoproteins from the bloodstream. Two members of the Angptl (angiopoietin-like protein) family, namely Angptl3 and Angptl4, have been shown to inhibit LPL activity in vitro and in vivo. Here, we further investigated the structural basis underlying the LPL inhibition by Angptl3 and Angptl4. By multiple sequence alignment analysis, we have identified a highly conserved 12-amino acid consensus motif that is present within the coiled-coil domain (CCD) of both Angptl3 and Angptl4, but not other members of the Angptl family. Substitution of the three polar amino acid residues (His 46 , Gln 50 , and Gln 53 ) within this motif with alanine abolishes the inhibitory effect of Angptl4 on LPL in vitro and also abrogates the ability of Angptl4 to elevate plasma triglyceride levels in mice. The CCD of Angptl4 interacts with LPL and converts the catalytically active dimers of LPL to its inactive monomers, whereas the mutant protein with the three polar amino acids being replaced by alanine loses such a property. Furthermore, a synthetic peptide consisting of the 12-amino acid consensus motif is sufficient to inhibit LPL activity, although the potency is much lower than the recombinant CCD of Angptl4. In summary, our data suggest that the 12-amino acid consensus motif within the CCD of Angptl4, especially the three polar residues within this motif, is responsible for its interaction with and inhibition of LPL by blocking the enzyme dimerization.

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Calcium Triggers Folding of Lipoprotein Lipase into Active Dimers

Cited by 58 publications

References 50 publications

Lipase maturation factor 1 is required for endothelial lipase activity

Lipase maturation factor 1 is required for endothelial lipase activity

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

A Highly Conserved Motif within the NH2-terminal Coiled-coil Domain of Angiopoietin-like Protein 4 Confers Its Inhibitory Effects on Lipoprotein Lipase by Disrupting the Enzyme Dimerization

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