Horse Pancreatic Lipase

Bourne, Yves; Martinez, C.; Kerfélec, Brigitte; Lagadic‐Gossmann, Dominique; Chapus, Catherine; Cambillau, Christian

doi:10.1006/jmbi.1994.1331

Cited by 103 publications

(67 citation statements)

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“…The complex contains the third threedimensional structure to be determined for a pancreatic lipase. As expected from the high degree of amino acid sequence homology, the porcine lipase structure is very similar to the corresponding human (17,18,22) and horse (19) enzymes. The porcine and human lipases are glycosylated at Asn…”

Section: Discussionsupporting

confidence: 58%

“…19; Protein Data Bank code HPL) as the search model. Rotation and translation searches, carried out using a 4 -12 Å resolution range, led to unique independent solutions for the two molecules of the asymmetric unit.…”

Section: X-ray Structure Determinationmentioning

confidence: 99%

“…Crystal structures of the human (17,18) and equine (19) lipases have shown that the polypeptide chain is divided into two domains bearing specific functions. The N-terminal domain contains the catalytic triad (Ser ) and is responsible for triglyceride hydrolysis.…”

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confidence: 99%

“…Because the pH of crystallization is very similar in the two cases, it is unlikely that the differences are due to variations in residue charges. Arg 386 also forms a salt bridge with Asp 390 in the uncomplexed equine enzyme (19). Lipase presents an intrinsic flexibility in the relative posi- Ϫ5 M) and various TGME or ␤OG concentrations.…”

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confidence: 99%

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Lipase Activation by Nonionic Detergents

Hermoso

Pignol

Kerfélec

et al. 1996

Journal of Biological Chemistry

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210

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The crystal structure of the ternary porcine lipasecolipase-tetra ethylene glycol monooctyl ether (TGME) complex has been determined at 2.8 Å resolution. The crystals belong to the cubic space group F23 with a ‫؍‬ 289.1 Å and display a strong pseudo-symmetry corresponding to a P23 lattice. Unexpectedly, the crystalline two-domain lipase is found in its open configuration. This indicates that in the presence of colipase, pure micelles of the nonionic detergent TGME are able to activate the enzyme; a process that includes the movement of an N-terminal domain loop (the flap). The effects of TGME and colipase have been confirmed by chemical modification of the active site serine residue using diisopropyl p-nitrophenylphosphate (E600). In addition, the presence of a TGME molecule tightly bound to the active site pocket shows that TGME acts as a substrate analog, thus possibly explaining the inhibitory effect of this nonionic detergent on emulsified substrate hydrolysis at submicellar concentrations. A comparison of the lipase-colipase interactions between our porcine complex and the human-porcine complex (van Tilbeurgh, H., Egloff, M.-P., Martinez, C., Rugani, N., Verger, R., and Cambillau, C. (1993) Nature 362, 814 -820) indicates that except for one salt bridge interaction, they are conserved. Analysis of the superimposed complexes shows a 5.4°rotation on the relative position of the N-terminal domains excepting the flap that moves in a concerted fashion with the C-terminal domain. This flexibility may be important for the binding of the complex to the water-lipid interface.Pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) plays a key role in dietary fat absorption in the intestine. It converts insoluble long chain triglycerides into more polar molecules that are able to cross the brush border membrane of enterocytes as mixed micelles with bile salts. Besides their role in triglyceride digestion through lipid emulsification and intestinal fat absorption, bile salts exert a strong inhibitory effect by preventing lipase adsorption through coating of the water-lipid interface (1). Lipase adsorption is a fundamental process because the enzyme catalyzes a heterogeneous reaction that involves an interfacial activation step. To counteract the inhibitory effect of bile salts, the pancreas secretes a small protein, colipase (molecular mass, 10 kDa), which anchors lipase to the bile salt-coated water-lipid interface in their presence (2, 3). Thus, lipolysis results from the combined effect of pancreatic lipase, colipase, and bile salts.Pancreatic lipase is a single polypeptide chain of 50 kDa. This protein has been characterized in several species, and the amino acid sequences of the enzyme from pig, man, horse, rabbit, rat, guinea pig, and coypu (4 -10) have been reported. In addition, related lipases (type 1 and 2) have been found in dog, rat man, guinea pig, and coypu (11-16) by screening pancreatic cDNA libraries. The localization and role of these related lipases is not clear.Crystal structures of the human ...

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Section: Discussionsupporting

confidence: 58%

Section: X-ray Structure Determinationmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Lipase Activation by Nonionic Detergents

Hermoso

Pignol

Kerfélec

et al. 1996

Journal of Biological Chemistry

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210

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“…Previously published structures of pancreatic lipases from human and horse were used as structural templates in conjunction with multiple sequence alignments of lipase sequences from several different species to generate three-dimensional models of human EL monomers. After model refinements using the MODELLER suite, the best scoring EL monomer models were aligned to the structure of the horse pancreatic lipase, which crystallized as a head-to-tail dimer (40). The EL dimer model (Fig.…”

Section: Comparison Of the Molecular Mass Of Uncleaved Wild-type El Amentioning

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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