Carriere F

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Full name : Carriere Frederic

First name : Frederic

Mail : CNRS, Aix-Marseille Universite, UMR 7282 Enzymologie Interfaciale et Physiologie de la Lipolyse, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20

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Country : France

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References (122)

Title : Interfacial adsorption and activity of pancreatic lipase-related protein 2 onto heterogeneous plant lipid model membrane - Kergomard_2023_Biochimie__
Author(s) : Kergomard J , Carriere F , Paboeuf G , Chonchon L , Barouh N , Vie V , Bourlieu C
Ref : Biochimie , : , 2023
Abstract : Pancreatic lipase related-protein 2 (PLRP2) exhibits remarkable galactolipase and phospholipase A1 activities, which depend greatly on the supramolecular organization of the substrates and the presence of surfactant molecules such as bile salts. The objective of the study was to understand the modulation of the adsorption mechanisms and enzymatic activity of Guinea pig PLRP2 (gPLRP2), by the physical environment of the enzyme and the physical state of its substrate. Langmuir monolayers were used to reproduce homogeneous and heterogeneous photosynthetic model membranes containing galactolipids (GL), and/or phospholipids (PL), and/or phytosterols (pS), presenting uncharged or charged interfaces. The same lipid mixtures were also used to form micrometric liposomes, and their gPLRP2 catalyzed digestion kinetics were investigated in presence or in absence of bile salts (NaTDC) during static in vitro, so called "bulk", digestion. The enzymatic activity of gPLRP2 onto the galactolipid-based monolayers was characterized with an optimum activity at 15 mN/m, in the absence of bile salts. gPLRP2 showed enhanced adsorption onto biomimetic model monolayer containing negatively charged lipids. However, the compositional complexity in the heterogeneous uncharged model systems induced a lag phase before the initiation of lipolysis. In bulk, no enzymatic activity could be demonstrated on GL-based liposomes in the absence of bile salts, probably due to the high lateral pressure of the lipid bilayers. In the presence of NaTDC (4 mM), however, gPLRP2 showed both high galactolipase and moderate phospholipase A1 activities on liposomes, probably due to a decrease in packing and lateral pressure upon NaTDC adsorption, and subsequent disruption of liposomes.
ESTHER : Kergomard_2023_Biochimie__
PubMedSearch : Kergomard_2023_Biochimie__
PubMedID: 37062468
Gene_locus related to this paper: human-PNLIPRP2

Title : The digestion of diacylglycerol isomers by gastric and pancreatic lipases and its impact on the metabolic pathways for TAG re-synthesis in enterocytes - Bakala-N'Goma_2022_Biochimie__
Author(s) : Bakala-N'Goma JC , Coudelo L , Vaysse C , Letisse M , Pierre V , Geloen A , Michalski MC , Lagarde M , Leao JD , Carriere F
Ref : Biochimie , : , 2022
Abstract : The specific activities of gastric and pancreatic lipases were measured using triacylglycerols (TAG) from rapeseed oil, purified 1,3-sn-DAG and 1,2(2,3)-sn-DAG produced from this oil, as well as a rapeseed oil enriched with 40% w/w DAG (DAGOIL). Gastric lipase was more active on 1,3-sn-DAG than on 1,2(2,3)-sn-DAG and TAG, whereas pancreatic lipase displayed a reverse selectivity with a higher activity on TAG than on DAG taken as initial substrates. However, in both cases, the highest activities were displayed on DAGOIL. These findings show that DAG mixed with TAG, such as in the course of digestion, is a better substrate for lipases than TAG. The same rapeseed oil acylglycerols were used to investigate intestinal fat absorption in rats with mesenteric lymph duct cannulation. The levels of TAG synthesized in the intestine and total fatty acid concentration in lymph were not different when the rats were fed identical amounts of rapeseed oil TAG, 1,2(2,3)-sn-DAG, 1,3-sn-DAG or DAGOIL. Since the lipolysis of 1,3-sn-DAG by digestive lipases leads to glycerol and not 2-sn-monoacylglycerol (2-sn-MAG) like TAG lipolysis, these results suggest that the re-synthesis of TAG in the enterocytes can entirely occur through the "glycerol-3-phosphate (G3P)" pathway, with the same efficiency as the 2-sn-MAG pathway predominantly involved in the intestinal fat absorption. These findings shed new light on the role played by DAG as intermediate lipolysis products. Depending on their structure, 1,2(2,3)-sn-DAG versus 1,3-sn-DAG, DAG may control the pathway (2-sn-MAG or G3P) by which TAG are re-synthesized in the enterocytes.
ESTHER : Bakala-N'Goma_2022_Biochimie__
PubMedSearch : Bakala-N'Goma_2022_Biochimie__
PubMedID: 35041857

Title : Cleaner degreasing of sheepskins by the Yarrowia lipolytica LIP2 lipase as a chemical-free alternative in the leather industry - Moujehed_2021_Colloids.Surf.B.Biointerfaces_211_112292
Author(s) : Moujehed E , Zarai Z , Khemir H , Miled N , Bchir MS , Gablin C , Bessueille F , Bonhomme A , Leonard D , Carriere F , Aloulou A
Ref : Colloids Surf B Biointerfaces , 211 :112292 , 2021
Abstract : Conventional degreasing of skins and hides in the leather industry requires high amounts of organic solvents and detergents that cause environmental issues. In this study, the LIP2 lipase from the yeast Yarrowia lipolytica (YLLIP2) was shown to be effective in degreasing sheepskins, thus reducing the amount of harmful chemicals. Using 6 mg of lipase/kg of raw skin, successful degreasing was achieved in only 15 min at pH 8 and 30 degreesC. ToF-SIMS mass spectra of chemically and enzymatically treated sheepskinsare consistent with a selective elimination process for the enzymatic treatment. Comparative SEM microscopy, ATR-FTIR spectroscopy and physicochemical analyses showed better properties of the enzymatically treated leather than those of the chemically treated leather. Effluent physicochemical parameters showed that the enzymatic treatment is a cleaner degreasing operation. Altogether, this work opens new horizons to use the YLLIP2 lipase as a more efficient alternative in the leather industry.
ESTHER : Moujehed_2021_Colloids.Surf.B.Biointerfaces_211_112292
PubMedSearch : Moujehed_2021_Colloids.Surf.B.Biointerfaces_211_112292
PubMedID: 34954514

Title : Quantitative monitoring of galactolipid hydrolysis by pancreatic lipase-related protein 2 using thin layer chromatography and thymol-sulfuric acid derivatization - Sahaka_2021_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_1173_122674
Author(s) : Sahaka M , Amara S , Lecomte J , Rodier JD , Lafont D , Villeneuve P , Gontero B , Carriere F
Ref : Journal of Chromatography B Analyt Technol Biomed Life Sciences , 1173 :122674 , 2021
Abstract : Galactolipids are the most abundant lipids on earth where they are mainly found in photosynthetic membranes of plant, algae, and cyanobacteria. Pancreatic lipase-related protein 2 (PLRP2) is an enzyme with galactolipase activity allowing mammals, especially herbivores, to digest this important source of fatty acids. We present a method for the quantitative analysis of galactolipids and galactosylated products resulting from their digestion by guinea pig PLRP2 (GPLRP2), using thin-layer-chromatography (TLC), thymol-sulfuric acid as derivatization reagent and scanning densitometry for detection. Thymol-sulfuric acid reagent has been used for the colorimetric detection of carbohydrates. It is shown here that the derivatization of galactosyl group from galactolipids by this reagent is not affected by the bound acyl glycerol, acyl chains length and number of galactose residues in the polar head. This allowed quantifying simultaneously the initial substrate and all galactosylated products generated upon the hydrolysis of monogalactosyl di-octanoylglycerol (C8-MGDG) by GPLRP2 using a single calibration with C8-MGDG as reference standard. The reaction products, monogalactosyl monooctanoyl glycerol (C8-MGMG) and monogalactosyl glycerol (MGG), were identified and quantified, MGG being recovered from the aqueous phase and analyzed by a separate TLC analysis. This method is therefore suitable to quantify the products resulting from the release of both fatty acids present in MGDG and thereby shows that PLRP2 can contribute to the complete digestion of galactolipids and further intestinal absorption of their fatty acids.
ESTHER : Sahaka_2021_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_1173_122674
PubMedSearch : Sahaka_2021_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_1173_122674
PubMedID: 33827017

Title : The 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester (DGGR) lipase assay in cats and dogs is not specific for pancreatic lipase - Lim_2020_Vet.Clin.Pathol_49_607
Author(s) : Lim SY , Xenoulis PG , Stavroulaki EM , Lidbury JA , Suchodolski JS , Carriere F , Steiner JM
Ref : Vet Clin Pathol , 49 :607 , 2020
Abstract : BACKGROUND: The measurement of pancreatic lipase is important for the diagnosis of feline and canine pancreatitis. Recent studies have claimed that lipase assays using the 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester (DGGR) as a substrate are more specific for measuring pancreatic lipase than traditional lipase assays. However, the analytical specificity of this assay for pancreatic lipase has not been demonstrated. OBJECTIVES: We aimed to determine whether hepatic and/or lipoprotein lipases can interfere with the DGGR-based assay results in cats and dogs. We, therefore, compared plasma lipase activities measured using DGGR-based and pancreatic lipase immunoreactivity (PLI) assays before and after administering heparin, known to cause the release of hepatic and lipoprotein lipases, in cats and dogs. METHODS: Heparin was administered in six cats and six dogs. Blood was collected at baseline and 10, 20, 30, 60, and 120 minutes after heparin administration. Lipase activity was measured using a DGGR-based assay, and PLI concentrations were measured using the Spec fPL and cPL assays for cats and dogs, respectively. RESULTS: Plasma lipase activities, as measured using the DGGR-based assay, increased significantly 10 minutes after heparin administration in both cats (P = .003) and dogs (P = .006) and returned to baseline by 120 minutes. In contrast, PLI concentrations showed no significant changes after heparin administration. CONCLUSIONS: DGGR is not only hydrolyzed by pancreatic lipase but also by hepatic lipase, lipoprotein lipase, or both, in cats and dogs. Since these extrapancreatic lipases are also naturally present in cats and dogs, they could contribute to the lack of analytical specificity for the DGGR-based assays.
ESTHER : Lim_2020_Vet.Clin.Pathol_49_607
PubMedSearch : Lim_2020_Vet.Clin.Pathol_49_607
PubMedID: 33111388

Title : Characterization of all the lipolytic activities in pancreatin and comparison with porcine and human pancreatic juices - Salhi_2020_Biochimie_169_106
Author(s) : Salhi A , Amara S , Mansuelle P , Puppo R , Lebrun R , Gontero B , Aloulou A , Carriere F
Ref : Biochimie , 169 :106 , 2020
Abstract : Porcine pancreatic extracts (PPE), also named pancreatin, are commonly used as a global source of pancreatic enzymes for enzyme replacement therapy in patients with exocrine pancreatic insufficiency. They are considered as a good substitute of human pancreatic enzymes and they have become a material of choice for in vitro models of digestion. Nevertheless, while the global PPE contents in lipase, protease and amylase activities are well characterized, little is known about individual enzymes. Here we characterized the lipase, phospholipase, cholesterol esterase and galactolipase activities of PPE and compared them with those of porcine (PPJ) and human (HPJ) pancreatic juices. The phospholipase to lipase activity ratio was similar in PPJ and HPJ, but was 4-fold lower in PPE. The galactolipase and cholesterol esterase activities were found at lower levels in PPJ compared to HPJ, and they were further reduced in PPE. The enzymes known to display these activities in HPJ, pancreatic lipase-related protein 2 (PLRP2) and carboxylester hydrolase/bile salt-stimulated lipase (CEH/BSSL), were identified in PPJ using gel filtration experiments, SDS-PAGE and LC-MS/MS analysis. The galactolipase and cholesterol esterase activities of PPE indicated that PLRP2 and CEH/BSSL are still present at low levels in this enzyme preparation, but they were not detected by mass spectrometry. Besides differences between porcine and human enzymes, the lower levels of phospholipase, galactolipase and cholesterol esterase activities in PPE are probably due to some proteolysis occurring during the production process. In conclusion, PPE do not provide a full substitution of the lipolytic enzymes present in HPJ.
ESTHER : Salhi_2020_Biochimie_169_106
PubMedSearch : Salhi_2020_Biochimie_169_106
PubMedID: 31288050

Title : The digestion of galactolipids and its ubiquitous function in Nature for the uptake of the essential alpha-linolenic acid - Sahaka_2020_Food.Funct_11_6710
Author(s) : Sahaka M , Amara S , Wattanakul J , Gedi MA , Aldai N , Parsiegla G , Lecomte J , Christeller JT , Gray D , Gontero B , Villeneuve P , Carriere F
Ref : Food Funct , 11 :6710-6744 , 2020
Abstract : Galactolipids, mainly monogalactosyl diglycerides and digalactosyl diglycerides are the main lipids found in the membranes of plants, algae and photosynthetic microorganisms like microalgae and cyanobacteria. As such, they are the main lipids present at the surface of earth. They may represent up to 80% of the fatty acid stocks, including a large proportion of polyunsaturated fatty acids mainly alpha-linolenic acid (ALA). Nevertheless, the interest in these lipids for nutrition and other applications remains overlooked, probably because they are dispersed in the biomass and are not as easy to extract as vegetable oils from oleaginous fruit and oil seeds. Another reason is that galactolipids only represent a small fraction of the acylglycerolipids present in modern human diet. In herbivores such as horses, fish and folivorous insects, galactolipids may however represent the main source of dietary fatty acids due to their dietary habits and digestion physiology. The development of galactolipase assays has led to the identification and characterization of the enzymes involved in the digestion of galactolipids in the gastrointestinal tract, as well as by microorganisms. Pancreatic lipase-related protein 2 (PLRP2) has been identified as an important factor of galactolipid digestion in humans, together with pancreatic carboxyl ester hydrolase (CEH). The levels of PLRP2 are particularly high in monogastric herbivores thus highlighting the peculiar role of PLRP2 in the digestion of plant lipids. Similarly, pancreatic lipase homologs are found to be expressed in the midgut of folivorous insects, in which a high galactolipase activity can be measured. In fish, however, CEH is the main galactolipase involved. This review discusses the origins and fatty acid composition of galactolipids and the physiological contribution of galactolipid digestion in various species. This overlooked aspect of lipid digestion ensures not only the intake of ALA from its main natural source, but also the main lipid source of energy for growth of some herbivorous species.
ESTHER : Sahaka_2020_Food.Funct_11_6710
PubMedSearch : Sahaka_2020_Food.Funct_11_6710
PubMedID: 32687132
Gene_locus related to this paper: helam-a0a2w1b5z2 , cavpo-2plrp

Title : Identification of a new natural gastric lipase inhibitor from star anise - Kamoun_2019_Food.Funct_10_469
Author(s) : Kamoun J , Rahier R , Sellami M , Koubaa I , Mansuelle P , Lebrun R , Berlioz-Barbier A , Fiore M , Alvarez K , Abousalham A , Carriere F , Aloulou A
Ref : Food Funct , 10 :469 , 2019
Abstract : The identification and isolation of bioactive compounds are of great interest in the drug delivery field, despite being a difficult task. We describe here an innovative strategy for the identification of a new gastric lipase inhibitor from star anise for the treatment of obesity. After plant screening assays for gastric lipase inhibition, star anise was selected and investigated by bioactivity guided fractionation. MALDI-TOF mass spectrometry and peptide mass fingerprinting allowed the detection of an inhibitor covalently bound to the catalytic serine of gastric lipase. A mass-directed screening approach using UPLC-HRMS and accurate mass determination searching identified the flavonoid myricitrin-5-methyl ether (M5ME) as a lipase inhibitor. The inhibitory activity was rationalized based on molecular docking, showing that M5ME is susceptible to nucleophilic attack by gastric lipase. Overall, our data suggest that M5ME may be considered as a potential candidate for future application as a gastric lipase inhibitor for the treatment of obesity.
ESTHER : Kamoun_2019_Food.Funct_10_469
PubMedSearch : Kamoun_2019_Food.Funct_10_469
PubMedID: 30632597
Gene_locus related to this paper: human-LIPF

Title : Inhibition of CpLIP2 Lipase Hydrolytic Activity by Four Flavonols (Galangin, Kaempferol, Quercetin, Myricetin) Compared to Orlistat and Their Binding Mechanisms Studied by Quenching of Fluorescence - Nasri_2019_Molecules_24_
Author(s) : Nasri R , Bidel LPR , Rugani N , Perrier V , Carriere F , Dubreucq E , Jay-Allemand C
Ref : Molecules , 24 : , 2019
Abstract : The inhibition of recombinant CpLIP2 lipase/acyltransferase from Candida parapsiolosis was considered a key model for novel antifungal drug discovery and a potential therapeutic target for candidiasis. Lipases have identified recently as potent virulence factors in C. parapsilosis and some other yeasts. The inhibition effects of orlistat and four flavonols (galangin, kaempferol, quercetin and myricetin) characterized by an increasing degree of hydroxylation in B-ring, were investigated using ethyl oleate hydrolysis as the model reaction. Orlistat and kaempferol (14 microM) strongly inhibited CpLIP2 catalytic activity within 1 min of pre-incubation, by 90% and 80%, respectively. The relative potency of flavonols as inhibitors was: kaempferol > quercetin > myricetin > galangin. The results suggested that orlistat bound to the catalytic site while kaempferol interacted with W294 on the protein lid. A static mechanism of interactions between flavonols and CpLIP2 lipase was confirmed by fluorescence quenching analyses, indicating that the interactions were mainly driven by hydrophobic bonds and electrostatic forces. From the Lehrer equation, fractions of tryptophan accessibility to the quencher were evaluated, and a relationship with the calculated number of binding sites was suggested.
ESTHER : Nasri_2019_Molecules_24_
PubMedSearch : Nasri_2019_Molecules_24_
PubMedID: 31398944
Gene_locus related to this paper: canpa-LIP2

Title : INFOGEST static in vitro simulation of gastrointestinal food digestion - Brodkorb_2019_Nat.Protoc_14_991
Author(s) : Brodkorb A , Egger L , Alminger M , Alvito P , Assuncao R , Ballance S , Bohn T , Bourlieu-Lacanal C , Boutrou R , Carriere F , Clemente A , Corredig M , Dupont D , Dufour C , Edwards C , Golding M , Karakaya S , Kirkhus B , Le Feunteun S , Lesmes U , Macierzanka A , Mackie AR , Martins C , Marze S , McClements DJ , Menard O , Minekus M , Portmann R , Santos CN , Souchon I , Singh RP , Vegarud GE , Wickham MSJ , Weitschies W , Recio I
Ref : Nat Protoc , 14 :991 , 2019
Abstract : Developing a mechanistic understanding of the impact of food structure and composition on human health has increasingly involved simulating digestion in the upper gastrointestinal tract. These simulations have used a wide range of different conditions that often have very little physiological relevance, and this impedes the meaningful comparison of results. The standardized protocol presented here is based on an international consensus developed by the COST INFOGEST network. The method is designed to be used with standard laboratory equipment and requires limited experience to encourage a wide range of researchers to adopt it. It is a static digestion method that uses constant ratios of meal to digestive fluids and a constant pH for each step of digestion. This makes the method simple to use but not suitable for simulating digestion kinetics. Using this method, food samples are subjected to sequential oral, gastric and intestinal digestion while parameters such as electrolytes, enzymes, bile, dilution, pH and time of digestion are based on available physiological data. This amended and improved digestion method (INFOGEST 2.0) avoids challenges associated with the original method, such as the inclusion of the oral phase and the use of gastric lipase. The method can be used to assess the endpoints resulting from digestion of foods by analyzing the digestion products (e.g., peptides/amino acids, fatty acids, simple sugars) and evaluating the release of micronutrients from the food matrix. The whole protocol can be completed in ~7 d, including ~5 d required for the determination of enzyme activities.
ESTHER : Brodkorb_2019_Nat.Protoc_14_991
PubMedSearch : Brodkorb_2019_Nat.Protoc_14_991
PubMedID: 30886367

Title : Galactolipase activity of Talaromyces thermophilus lipase on galactolipid micelles, monomolecular films and UV-absorbing surface-coated substrate - Belhaj_2018_Biochim.Biophys.Acta.Mol.Cell.Biol.Lipids_1863_1006
Author(s) : Belhaj I , Amara S , Parsiegla G , Sutto-Ortiz P , Sahaka M , Belghith H , Rousset A , Lafont D , Carriere F
Ref : Biochimica & Biophysica Acta Molecular & Cellular Biology Lipids , 1863 :1006 , 2018
Abstract : Talaromyces thermophilus lipase (TTL) was found to hydrolyze monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) substrates presented in various forms to the enzyme. Different assay techniques were used for each substrate: pHstat with dioctanoyl galactolipid-bile salt mixed micelles, barostat with dilauroyl galactolipid monomolecular films spread at the air-water interface, and UV absorption using a novel MGDG substrate containing alpha-eleostearic acid as chromophore and coated on microtiter plates. The kinetic properties of TTL were compared to those of the homologous lipase from Thermomyces lanuginosus (TLL), guinea pig pancreatic lipase-related protein 2 and Fusarium solani cutinase. TTL was found to be the most active galactolipase, with a higher activity on micelles than on monomolecular films or surface-coated MGDG. Nevertheless, the UV absorption assay with coated MGDG was highly sensitive and allowed measuring significant activities with about 10ng of enzymes, against 100ng to 10mug with the pHstat. TTL showed longer lag times than TLL for reaching steady state kinetics of hydrolysis with monomolecular films or surface-coated MGDG. These findings and 3D-modelling of TTL based on the known structure of TLL pointed out to two phenylalanine to leucine substitutions in TTL, that could be responsible for its slower adsorption at lipid-water interface. TTL was found to be more active on MGDG than on DGDG using both galactolipid-bile salt mixed micelles and galactolipid monomolecular films. These later experiments suggest that the second galactose on galactolipid polar head impairs the enzyme adsorption on its aggregated substrate.
ESTHER : Belhaj_2018_Biochim.Biophys.Acta.Mol.Cell.Biol.Lipids_1863_1006
PubMedSearch : Belhaj_2018_Biochim.Biophys.Acta.Mol.Cell.Biol.Lipids_1863_1006
PubMedID: 29859246
Gene_locus related to this paper: talth-f6lqk7

Title : IR spectroscopy analysis of pancreatic lipase-related protein 2 interaction with phospholipids: 1. Discriminative recognition of mixed micelles versus liposomes - Mateos-Diaz_2018_Chem.Phys.Lipids_211_52
Author(s) : Mateos-Diaz E , Bakala N'Goma JC , Byrne D , Robert S , Carriere F , Gaussier H
Ref : Chemistry & Physic of Lipids , 211 :52 , 2018
Abstract : Guinea pig pancreatic lipase-related protein 2 (GPLRP2) is an interesting model enzyme that can hydrolyze a large set of acylglycerols in vitro but displays however some selectivity depending on the supramolecular structure of substrate and the presence of surfactants like bile salts. We showed that GPLRP2 hydrolyzes 1,2-dipalmitoyl phosphatidylcholine (DPPC) present in mixed micelles with sodium taurodeoxycholate (NaTDC) but not in multilamellar (MLV) and large unilamellar (LUV) vesicles of DPPC. After characterization of these lipid aggregates by dynamic light scattering (DLS), the discriminative recognition of DPPC in DPPC/NaTDC micelles versus MLV and LUV by an inactive variant (S152G) of GPLRP2 to avoid the effect of substrate hydrolysis was investigated using Fourier transform infrared spectroscopy (FTIR). IR spectra were recorded after hydrogen/deuterium exchange, at pD 6 and various temperatures to study phase transitions. We analyzed the methylene asymmetric stretching (nu(CH2)as), the carbonyl stretching (nu(CO)) and the composite polar head-group vibration bands, first to characterized differences in DPPC micelles and vesicles, and second to estimate the degree of interaction of GPLRP2 S152G with phospholipid. Our results indicate that a significant interaction between GPLRP2 S152G and DPPC is only observed when NaTDC is added to the system to form micelles and this can be explained by the different organization of DPPC in mixed micelles compared to lamellar vesicles (higher hydration of polar head, higher mobility of alkyl chains) that favors GPLRP2 penetration into the phospholipid layer.
ESTHER : Mateos-Diaz_2018_Chem.Phys.Lipids_211_52
PubMedSearch : Mateos-Diaz_2018_Chem.Phys.Lipids_211_52
PubMedID: 28235448

Title : Probing Conformational Changes and Interfacial Recognition Site of Lipases With Surfactants and Inhibitors - Mateos-Diaz_2017_Methods.Enzymol_583_279
Author(s) : Mateos-Diaz E , Amara S , Roussel A , Longhi S , Cambillau C , Carriere F
Ref : Methods Enzymol , 583 :279 , 2017
Abstract : Structural studies on lipases by X-ray crystallography have revealed conformational changes occurring in the presence of surfactants/inhibitors and the pivotal role played by a molecular "lid" of variable size and structure depending on the enzyme. Besides controlling the access to the enzyme active site, the lid is involved in lipase activation, formation of the interfacial recognition site (IRS), and substrate docking within the active site. The combined use of surfactants and inhibitors has been critical for a better understanding of lipase structure-function relationships. An overview of crystal structures of lipases in complex with surfactants and inhibitors reveals common structural features and shows how surfactants monomers interact with the lid in its open conformation. The location of surfactants, inhibitors, and hydrophobic residues exposed upon lid opening provides insights into the IRS of lipases. The mechanism by which surfactants promote the lid opening can be further investigated in solution by site-directed spin labeling of lipase coupled to electron paramagnetic resonance spectroscopy. These experimental approaches are illustrated here by results obtained with mammalian digestive lipases, fungal lipases, and cutinases.
ESTHER : Mateos-Diaz_2017_Methods.Enzymol_583_279
PubMedSearch : Mateos-Diaz_2017_Methods.Enzymol_583_279
PubMedID: 28063495

Title : Constitutive expression of human gastric lipase in Pichia pastoris and site-directed mutagenesis of key lid-stabilizing residues - Sams_2017_Biochim.Biophys.Acta_1862_1025
Author(s) : Sams L , Amara S , Chakroun A , Coudre S , Paume J , Giallo J , Carriere F
Ref : Biochimica & Biophysica Acta , 1862 :1025 , 2017
Abstract : The cDNA encoding human gastric lipase (HGL) was integrated into the genome of Pichia pastoris using the pGAPZalpha A transfer vector. The HGL signal peptide was replaced by the yeast alpha-factor to achieve an efficient secretion. Active rHGL was produced by the transformed yeast but its levels and stability were dependent on the pH. The highest activity was obtained upon buffering the culture medium at pH5, a condition that allowed preserving enzyme activity over time. A large fraction (72+/-2%) of secreted rHGL remained however bound to the yeast cells, and was released by washing the cell pellet with an acid glycine-HCl buffer (pH2.2). This procedure allowed establishing a first step of purification that was completed by size exclusion chromatography. N-terminal sequencing and MALDI-ToF mass spectrometry revealed that rHGL was produced in its mature form, with a global mass of 50,837+/-32Da corresponding to a N-glycosylated form of HGL polypeptide (43,193Da). rHGL activity was characterized as a function of pH, various substrates and in the presence of bile salts and pepsin, and was found similar to native HGL, except for slight changes in pH optima. We then studied by site-directed mutagenesis the role of three key residues (K4, E225, R229) involved in salt bridges stabilizing the lid domain that controls the access to the active site and is part of the interfacial recognition site. Their substitution has an impact on the pH-dependent activity of rHGL and its relative activities on medium and long chain triglycerides.
ESTHER : Sams_2017_Biochim.Biophys.Acta_1862_1025
PubMedSearch : Sams_2017_Biochim.Biophys.Acta_1862_1025
PubMedID: 28694218
Gene_locus related to this paper: human-LIPF

Title : Studying Gastric Lipase Adsorption Onto Phospholipid Monolayers by Surface Tensiometry, Ellipsometry, and Atomic Force Microscopy - Benarouche_2017_Methods.Enzymol_583_255
Author(s) : Benarouche A , Sams L , Bourlieu C , Vie V , Point V , Cavalier JF , Carriere F
Ref : Methods Enzymol , 583 :255 , 2017
Abstract : The access to kinetic parameters of lipolytic enzyme adsorption onto lipids is essential for a better understanding of the overall catalytic process carried out by these interfacial enzymes. Gastric lipase, for instance, shows an apparent optimum activity on triglycerides (TAG) at acidic pH, which is controlled by its pH-dependent adsorption at lipid-water interfaces. Since gastric lipase acts on TAG droplets covered by phospholipids, but does not hydrolyze these lipids, phospholipid monolayers spread at the air-water interfaces can be used as biomimetic interfaces to study lipase adsorption and penetration through the phospholipid layer, independently from the catalytic activity. The adsorption of recombinant dog gastric lipase (rDGL) onto 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) monolayers can be monitored by surface tensiometry at various enzyme concentrations, pHs, and surface pressures (Pi). These experimental data and the use of Langmuir adsorption isotherm and Verger-de Haas' lipase kinetics models further allow estimating various parameters including the adsorption equilibrium constant (KAds), the interfacial concentration [Formula: see text] , the molar fraction [Formula: see text] (PhiE*(%), mol%), and the molecular area [Formula: see text] of rDGL adsorbed onto the DLPC monolayer under various conditions. Additional insight into rDGL adsorption/insertion on phospholipid monolayers can be obtained by combining ellipsometry, Langmuir-Blodgett film transfer, and atomic force microscopy. When using multicomponent phospholipid monolayers with phase separation, these techniques allow to visualizing how rDGL preferentially partitions toward liquid expanded phase and at phase boundaries, gets adsorbed at various levels of insertion and impacts on the lateral organization of lipids.
ESTHER : Benarouche_2017_Methods.Enzymol_583_255
PubMedSearch : Benarouche_2017_Methods.Enzymol_583_255
PubMedID: 28063494

Title : Lysosomal Lipases PLRP2 and LPLA2 Process Mycobacterial Multi-acylated Lipids and Generate T Cell Stimulatory Antigens - Gilleron_2016_Cell.Chem.Biol_23_1147
Author(s) : Gilleron M , Lepore M , Layre E , Cala-De Paepe D , Mebarek N , Shayman JA , Canaan S , Mori L , Carriere F , Puzo G , De Libero G
Ref : Cell Chemical Biology , 23 :1147 , 2016
Abstract : Complex antigens require processing within antigen-presenting cells (APCs) to form T cell stimulatory complexes with CD1 antigen-presenting molecules. It remains unknown whether lipids with multi-acylated moieties also necessitate digestion by lipases to become capable of binding CD1 molecules and stimulate T cells. Here, we show that the mycobacterial tetra-acylated glycolipid antigens phosphatidyl-myo-inositol mannosides (PIM) are digested to di-acylated forms by pancreatic lipase-related protein 2 (PLRP2) and lysosomal phospholipase A2 (LPLA2) within APCs. Recombinant PLRP2 and LPLA2 removed the sn1- and sn2-bound fatty acids from the PIM glycerol moiety, as revealed by mass spectrometry and nuclear magnetic resonance studies. PLRP2 or LPLA2 gene silencing in APCs abolished PIM presentation to T cells, thus revealing an essential role of both lipases in vivo. These findings show that endosomal lipases participate in lipid antigen presentation by processing lipid antigens and have a role in T cell immunity against mycobacteria.
ESTHER : Gilleron_2016_Cell.Chem.Biol_23_1147
PubMedSearch : Gilleron_2016_Cell.Chem.Biol_23_1147
PubMedID: 27662254

Title : New lipase assay using Pomegranate oil coating in microtiter plates - Ulker_2016_Biochimie_120_110
Author(s) : Ulker S , Placidi C , Point V , Gadenne B , Serveau-Avesque C , Canaan S , Carriere F , Cavalier JF
Ref : Biochimie , 120 :110 , 2016
Abstract : Lipases play various roles in fat digestion, lipoprotein metabolism, and in the mobilization of fat stored in lipid bodies in animals, plants and microorganisms. In association with these physiological functions, there is an important field of research for discovering lipase inhibitors and developing new treatments of diseases such as obesity, atherosclerosis, diabetes and tuberculosis. In this context, the development of convenient, specific and sensitive analytical methods for the detection and assay of lipases and/or lipase inhibitors is of major importance. It is shown here that purified triacylglycerols (TAGs) from Punica granatum (Pomegranate) seed oil coated on microtiter plates can be used for the continuous assay of lipase activity by recording the variations with time of the UV absorption spectra at 275 nm. UV absorption is due the release of punicic acid (9Z,11E,13Z-octadeca-9,11,13-trienoic acid), a conjugated triene contained in Pomegranate oil. This new microtiter plate assay allows to accurately measure the activity of a wider range of lipases compared to the similar assay previously developed with Tung oil containing alpha-eleostearic acid (9Z,11E,13E-octadeca-9,11,13-trienoic acid), including the LipY lipase from Mycobacterium tuberculosis. Although punicic acid is a diastereoisomer of alpha-eleostearic acid, the Delta(13)cis double bound found in punicic acid gives a different structure to the acyl chain that probably favours the interaction of Pomegranate TAGs with the lipase active site. The microplate lipase assay using Pomegranate TAGs shows high sensitivity, reproducibility and remarkable relevance for the high-speed screening of lipases and/or lipase inhibitors directly from raw culture media without any purification step.
ESTHER : Ulker_2016_Biochimie_120_110
PubMedSearch : Ulker_2016_Biochimie_120_110
PubMedID: 26343557

Title : Slowing down fat digestion and absorption by an oxadiazolone inhibitor targeting selectively gastric lipolysis - Point_2016_Eur.J.Med.Chem_123_834
Author(s) : Point V , Benarouche A , Zarrillo J , Guy A , Magnez R , Fonseca L , Raux B , Leclaire J , Buono G , Fotiadu F , Durand T , Carriere F , Vaysse C , Couedelo L , Cavalier JF
Ref : Eur Journal of Medicinal Chemistry , 123 :834 , 2016
Abstract : Based on a previous study and in silico molecular docking experiments, we have designed and synthesized a new series of ten 5-Alkoxy-N-3-(3-PhenoxyPhenyl)-1,3,4-Oxadiazol-2(3H)-one derivatives (RmPPOX). These molecules were further evaluated as selective and potent inhibitors of mammalian digestive lipases: purified dog gastric lipase (DGL) and guinea pig pancreatic lipase related protein 2 (GPLRP2), as well as porcine (PPL) and human (HPL) pancreatic lipases contained in porcine pancreatic extracts (PPE) and human pancreatic juices (HPJ), respectively. These compounds were found to strongly discriminate classical pancreatic lipases (poorly inhibited) from gastric lipase (fully inhibited). Among them, the 5-(2-(Benzyloxy)ethoxy)-3-(3-PhenoxyPhenyl)-1,3,4-Oxadiazol-2(3H)-one (BemPPOX) was identified as the most potent inhibitor of DGL, even more active than the FDA-approved drug Orlistat. BemPPOX and Orlistat were further compared in vitro in the course of test meal digestion, and in vivo with a mesenteric lymph duct cannulated rat model to evaluate their respective impacts on fat absorption. While Orlistat inhibited both gastric and duodenal lipolysis and drastically reduced fat absorption in rats, BemPPOX showed a specific action on gastric lipolysis that slowed down the overall lipolysis process and led to a subsequent reduction of around 55% of the intestinal absorption of fatty acids compared to controls. All these data promote BemPPOX as a potent candidate to efficiently regulate the gastrointestinal lipolysis, and to investigate its link with satiety mechanisms and therefore develop new strategies to "fight against obesity".
ESTHER : Point_2016_Eur.J.Med.Chem_123_834
PubMedSearch : Point_2016_Eur.J.Med.Chem_123_834
PubMedID: 27543878
Gene_locus related to this paper: canfa-1lipg , human-LIPF

Title : Effect of preduodenal lipase inhibition in suckling rats on dietary octanoic acid (C8:0) gastric absorption and plasma octanoylated ghrelin concentration - Lemarie_2016_Biochim.Biophys.Acta_1861_1111
Author(s) : Lemarie F , Cavalier JF , Garcia C , Boissel F , Point V , Catheline D , Legrand P , Carriere F , Rioux V
Ref : Biochimica & Biophysica Acta , 1861 :1111 , 2016
Abstract : Part of medium chain fatty acids (MCFAs) coming from dietary triglycerides (TGs) can be directly absorbed through the gastric mucosa after the action of preduodenal lipase (lingual lipase in the rat). MCFA gastric absorption, particularly that of octanoic acid (C8:0), may have a physiological importance in the octanoylation of ghrelin, the orexigenic gastric peptide acting as an endogenous ligand of the hypothalamic growth hormone secretagogue receptor 1a (GHSR-1a). However, the amount of C8:0 absorbed in the stomach and its metabolic fate still haven't been clearly characterized. The purpose of the present study was to further characterize and quantify the importance of preduodenal lipase activity on the release and gastric absorption of dietary C8:0 and on the subsequent ghrelin octanoylation in the stomach mucosa. Fifteen days old rats received fat emulsions containing triolein or [1,1,1-(13)C]-Tri-C8:0 and a specific inhibitor of preduodenal lipase, 5-(2-(benzyloxy)ethoxy)-3-(3-phenoxyphenyl)-1,3,4-oxadiazol-2(3H)-one or BemPPOX. The fate of the (13)C-C8:0 was followed in rat tissues after 30 and 120min of digestion and octanoylated ghrelin was measured in the plasma. This work (1) demonstrates that part of C8:0 coming from Tri-C8:0 is directly absorbed at the gastric level, (2) allows the estimation of C8:0 gastric absorption level (1.3% of the (13)C-C8:0 in sn-3 position after 30min of digestion), as well as (3) the contribution of rat lingual lipase to total lipolysis and to duodenal absorption of dietary FAs (at least 30%), (4) shows no short-term effect of dietary Tri-C8:0 consumption and subsequent increase of C8:0 gastric tissue content on plasma octanoylated ghrelin concentration.
ESTHER : Lemarie_2016_Biochim.Biophys.Acta_1861_1111
PubMedSearch : Lemarie_2016_Biochim.Biophys.Acta_1861_1111
PubMedID: 27317984

Title : Efficient heterologous expression of Fusarium solani lipase, FSL2, in Pichia pastoris, functional characterization of the recombinant enzyme and molecular modeling - Jallouli_2016_Int.J.Biol.Macromol_94_61
Author(s) : Jallouli R , Parsiegla G , Carriere F , Gargouri Y , Bezzine S
Ref : Int J Biol Macromol , 94 :61 , 2016
Abstract : The gene coding for a lipase of Fusarium solani, designated as FSL2, shows an open reading frame of 906bp encoding a 301-amino acid polypeptide with a molecular mass of 30kDa. Based on sequence similarity with other fungal lipases, FSL2 contains a catalytic triad, consisting of Ser144, Asp198, and His256. FSL2 cDNA was subcloned into the pGAPZalphaA vector containing the Saccharomyces cerevisiae alpha-factor signal sequence and this construct was used to transform Pichia pastoris and achieve a high-level extracellular production of a FSL2 lipase. Maximum lipase activity was observed after 48h. The optimum activity of the purified recombinant enzyme was measured at pH 8.0-9.0 and 37 degrees C. FSL2 is remarkably stable at alkaline pH values up to 12 and at temperatures below 40 degrees C. It has high catalytic efficiency towards triglycerides with short to long chain fatty acids but with a marked preference for medium and long chain fatty acids. FSL2 activity is decreased at sodium taurodeoxycholate concentrations above the Critical Micelle Concentration (CMC) of this anionic detergent. However, lipase activity is enhanced by Ca2+ and inhibited by EDTA or Cu2+ and partially by Mg2+ or K+. In silico docking of medium chain triglycerides, monogalctolipids (MGDG), digalactolipids (DGDG) and long chain phospholipids in the active site of FSL2 reveals structural solutions.
ESTHER : Jallouli_2016_Int.J.Biol.Macromol_94_61
PubMedSearch : Jallouli_2016_Int.J.Biol.Macromol_94_61
PubMedID: 27620466
Gene_locus related to this paper: fushe-LIPASE

Title : Adsorption of gastric lipase onto multicomponent model lipid monolayers with phase separation - Bourlieu_2016_Colloids.Surf.B.Biointerfaces_143_97
Author(s) : Bourlieu C , Paboeuf G , Chever S , Pezennec S , Cavalier JF , Guyomarc'h F , Deglaire A , Bouhallab S , Dupont D , Carriere F , Vie V
Ref : Colloids Surf B Biointerfaces , 143 :97 , 2016
Abstract : The enzymatic lipolysis of complex natural lipoproteic assemblies such as milk fat globules is central in neonatal digestion. This process first requires the rapid adsorption of a lipolytic enzyme, gastric lipase, onto the membrane enveloping the triglyceride substrate before the onset of catalytic activity. The interactions governing lipase adsorption onto this complex lipid/water interface are not fully elucidated. This study was designed to unravel the interactions of recombinant dog gastric lipase (rDGL) with model monolayers presenting liquid-liquid phase coexistence and mimicking the outer leaflet of the milk fat globule membrane. Combining biophysical tools (ellipsometry, tensiometry and atomic force microscopy), it was evidenced that rDGL partitions toward liquid expanded phase and at phase boundaries. rDGL gets adsorbed at several levels of insertion suggesting molecular cooperation that may favor insertion and strongly impacts on the lipid phase lateral organization. The addition of phosphatidylserine, negatively charged, reinforced adsorption; hence besides hydrophobic interactions and as further investigated through surface potential modeling, rDGL adsorption is favored by electrostatic interactions.
ESTHER : Bourlieu_2016_Colloids.Surf.B.Biointerfaces_143_97
PubMedSearch : Bourlieu_2016_Colloids.Surf.B.Biointerfaces_143_97
PubMedID: 27011347

Title : The galactolipase activity of Fusarium solani (phospho)lipase - Jallouli_2015_Biochim.Biophys.Acta_1851_282
Author(s) : Jallouli R , Othman H , Amara S , Parsiegla G , Carriere F , Srairi-Abid N , Gargouri Y , Bezzine S
Ref : Biochimica & Biophysica Acta , 1851 :282 , 2015
Abstract : The purified (phospho)lipase of Fusarium solani (FSL), was known to be active on both triglycerides and phospholipids. This study aimed at assessing the potential of this enzyme in hydrolyzing galactolipids. FSL was found to hydrolyze at high rates of synthetic medium chains monogalactosyldiacylglycerol (4658+/-146U/mg on DiC8-MGDG) and digalactosyldiacylglycerol (3785+/-83U/mg on DiC8-DGDG) and natural long chain monogalactosyldiacylglycerol extracted from leek leaves (991+/-85U/mg). It is the microbial enzyme with the highest activity on galactolipids identified so far with a level of activity comparable to that of pancreatic lipase-related protein 2. FSL maximum activity on galactolipids was measured at pH8. The analysis of the hydrolysis product of natural MGDG from leek showed that FSL hydrolyzes preferentially the ester bond at the sn-1 position of galactolipids. To investigate the structure-activity relationships of FSL, a 3D model of this enzyme was built. In silico docking of medium chains MGDG and DGDG and phospholipid in the active site of FSL reveals structural solutions which are in concordance with in vitro tests.
ESTHER : Jallouli_2015_Biochim.Biophys.Acta_1851_282
PubMedSearch : Jallouli_2015_Biochim.Biophys.Acta_1851_282
PubMedID: 25529980
Gene_locus related to this paper: nech7-c7yuz1

Title : Biochemical characterization of Yarrowia lipolytica LIP8, a secreted lipase with a cleavable C-terminal region - Kamoun_2015_Biochim.Biophys.Acta_1851_129
Author(s) : Kamoun J , Schue M , Messaoud W , Baignol J , Point V , Mateos-Diaz E , Mansuelle P , Gargouri Y , Parsiegla G , Cavalier JF , Carriere F , Aloulou A
Ref : Biochimica & Biophysica Acta , 1851 :129 , 2015
Abstract : Yarrowia lipolytica is a lipolytic yeast possessing 16 paralog genes coding for lipases. Little information on these lipases has been obtained and only the major secreted lipase, namely YLLIP2, had been biochemically and structurally characterized. Another secreted lipase, YLLIP8, was isolated from Y. lipolytica culture medium and compared with the recombinant enzyme produced in Pichia pastoris. N-terminal sequencing showed that YLLIP8 is produced in its active form after the cleavage of a signal peptide. Mass spectrometry analysis revealed that YLLIP8 recovered from culture medium lacks a C-terminal part of 33 amino acids which are present in the coding sequence. A 3D model of YLLIP8 built from the X-ray structure of the homologous YLLIP2 lipase shows that these truncated amino acids in YLLIP8 belong to an additional C-terminal region predicted to be mainly helical. Western blot analysis shows that YLLIP8 C-tail is rapidly cleaved upon enzyme secretion since both cell-bound and culture supernatant lipases lack this extension. Mature recombinant YLLIP8 displays a true lipase activity on short-, medium- and long-chain triacylglycerols (TAG), with an optimum activity at alkaline pH on medium chain TAG. It has no apparent regioselectivity in TAG hydrolysis, thus generating glycerol and FFAs as final lipolysis products. YLLIP8 properties are distinct from those of the 1,3-regioselective YLLIP2, acting optimally at acidic pH. These lipases are tailored for complementary roles in fatty acid uptake by Y. lipolytica.
ESTHER : Kamoun_2015_Biochim.Biophys.Acta_1851_129
PubMedSearch : Kamoun_2015_Biochim.Biophys.Acta_1851_129
PubMedID: 25449652
Gene_locus related to this paper: yarli-LIP8

Title : Comparative genomics analysis of Lactobacillus species associated with weight gain or weight protection - Drissi_2014_Nutr.Diabetes_4_e109
Author(s) : Drissi F , Merhej V , Angelakis E , El Kaoutari A , Carriere F , Henrissat B , Raoult D
Ref : Nutr Diabetes , 4 :e109 , 2014
Abstract : BACKGROUND: Some Lactobacillus species are associated with obesity and weight gain while others are associated with weight loss. Lactobacillus spp. and bifidobacteria represent a major bacterial population of the small intestine where lipids and simple carbohydrates are absorbed, particularly in the duodenum and jejunum. The objective of this study was to identify Lactobacillus spp. proteins involved in carbohydrate and lipid metabolism associated with weight modifications.
METHODS: We examined a total of 13 complete genomes belonging to seven different Lactobacillus spp. previously associated with weight gain or weight protection. We combined the data obtained from the Rapid Annotation using Subsystem Technology, Batch CD-Search and Gene Ontology to classify gene function in each genome.
RESULTS: We observed major differences between the two groups of genomes. Weight gain-associated Lactobacillus spp. appear to lack enzymes involved in the catabolism of fructose, defense against oxidative stress and the synthesis of dextrin, L-rhamnose and acetate. Weight protection-associated Lactobacillus spp. encoded a significant gene amount of glucose permease. Regarding lipid metabolism, thiolases were only encoded in the genome of weight gain-associated Lactobacillus spp. In addition, we identified 18 different types of bacteriocins in the studied genomes, and weight gain-associated Lactobacillus spp. encoded more bacteriocins than weight protection-associated Lactobacillus spp.
CONCLUSIONS: The results of this study revealed that weight protection-associated Lactobacillus spp. have developed defense mechanisms for enhanced glycolysis and defense against oxidative stress. Weight gain-associated Lactobacillus spp. possess a limited ability to breakdown fructose or glucose and might reduce ileal brake effects.
ESTHER : Drissi_2014_Nutr.Diabetes_4_e109
PubMedSearch : Drissi_2014_Nutr.Diabetes_4_e109
PubMedID: 24567124

Title : A Cutinase from Trichoderma reesei with a Lid-Covered Active Site and Kinetic Properties of True Lipases - Roussel_2014_J.Mol.Biol_426_3757
Author(s) : Roussel A , Amara S , Nyyssola A , Mateos-Diaz E , Blangy S , Kontkanen H , Westerholm-Parvinen A , Carriere F , Cambillau C
Ref : Journal of Molecular Biology , 426 :3757 , 2014
Abstract : Cutinases belong to the alpha/beta-hydrolase fold family of enzymes and degrade cutin and various esters, including triglycerides, phospholipids and galactolipids. Cutinases are able to degrade aggregated and soluble substrates because, in contrast with true lipases, they do not have a lid covering their catalytic machinery. We report here the structure of a cutinase from the fungus Trichoderma reesei (Tr) in native and inhibitor-bound conformations, along with its enzymatic characterization. A rare characteristic of Tr cutinase is its optimal activity at acidic pH. Furthermore, Tr cutinase, in contrast with classical cutinases, possesses a lid covering its active site and requires the presence of detergents for activity. In addition to the presence of the lid, the core of the Tr enzyme is very similar to other cutinase cores, with a central five-stranded beta-sheet covered by helices on either side. The catalytic residues form a catalytic triad involving Ser164, His229 and Asp216 that is covered by the two N-terminal helices, which form the lid. This lid opens in the presence of surfactants, such as beta-octylglucoside, and uncovers the catalytic crevice, allowing a C11Y4 phosphonate inhibitor to bind to the catalytic serine. Taken together, these results reveal Tr cutinase to be a member of a new group of lipolytic enzymes resembling cutinases but with kinetic and structural features of true lipases and a heightened specificity for long-chain triglycerides.
ESTHER : Roussel_2014_J.Mol.Biol_426_3757
PubMedSearch : Roussel_2014_J.Mol.Biol_426_3757
PubMedID: 25219509
Gene_locus related to this paper: hypjq-g0rh85

Title : An interfacial and comparative in vitro study of gastrointestinal lipases and Yarrowia lipolytica LIP2 lipase, a candidate for enzyme replacement therapy - Benarouche_2014_Biochimie_102_145
Author(s) : Benarouche A , Point V , Carriere F , Cavalier JF
Ref : Biochimie , 102 :145 , 2014
Abstract : Lipolytic activities of Yarrowia lipolytica LIP2 lipase (YLLIP2), human pancreatic (HPL) and dog gastric (DGL) lipases were first compared using lecithin-stabilized triacylglycerol (TAG) emulsions (Intralipid) at various pH and bile salt concentrations. Like DGL, YLLIP2 was able to hydrolyze TAG droplets covered by a lecithin monolayer, while HPL was not directly active on that substrate. These results were in good agreement with the respective kinetics of adsorption on phosphatidylcholine (PC) monomolecular films of the same three lipases, YLLIP2 being the most tensioactive lipase. YLLIP2 adsorption onto a PC monolayer spread at the air/water interface was influenced by pH-dependent changes in the enzyme/lipid interfacial association constant (KAds) which was optimum at pH 6.0 on long-chain egg PC monolayer, and at pH 5.0 on medium chain dilauroylphosphatidylcholine film. Using substrate monolayers (1,2-dicaprin, trioctanoin), YLLIP2 displayed the highest lipolytic activities on both substrates in the 25-35 mN m(-1) surface pressure range. YLLIP2 was active in a large pH range and displayed a pH-dependent activity profile combining DGL and HPL features at pH values found in the stomach (pH 3-5) and in the intestine (pH 6-7), respectively. The apparent maximum activity of YLLIP2 was observed at acidic pH 4-6 and was therefore well correlated with an efficient interfacial binding at these pH levels, whatever the type of interfaces (Intralipid emulsions, substrate or PC monolayers). All these findings support the use of YLLIP2 in enzyme replacement therapy for the treatment of pancreatic exocrine insufficiency, a pathological situation in which an acidification of intestinal contents occurs.
ESTHER : Benarouche_2014_Biochimie_102_145
PubMedSearch : Benarouche_2014_Biochimie_102_145
PubMedID: 24650780

Title : Using the reversible inhibition of gastric lipase by Orlistat for investigating simultaneously lipase adsorption and substrate hydrolysis at the lipid-water interface - Benarouche_2014_Biochimie_101_221
Author(s) : Benarouche A , Point V , Carriere F , Cavalier JF
Ref : Biochimie , 101 :221 , 2014
Abstract : The lipolysis reaction carried out by lipases at the water-lipid interface is a complex process including enzyme conformational changes, adsorption/desorption equilibrium and substrate hydrolysis. Mixed monomolecular films of the lipase inhibitor Orlistat and 1,2-dicaprin were used here to investigate the adsorption of dog gastric lipase (DGL) followed by the hydrolysis of 1,2-dicaprin. The combined study of these two essential catalysis steps was made possible thanks to the highest affinity of DGL for Orlistat than 1,2-dicaprin and the fact that the inhibition of DGL by Orlistat is reversible. Upon DGL binding to mixed 1,2-dicaprin/Orlistat monolayers, an increase in surface pressure reflecting lipase adsorption was first recorded. Limited amounts of Orlistat allowed to maintain DGL inactive on 1,2-dicaprin during a period of time that was sufficient to determine DGL adsorption and desorption rate constants. A decrease in surface pressure reflecting 1,2-dicaprin hydrolysis and product desorption was observed after the slow hydrolysis of the covalent DGL-Orlistat complex was complete. The rate of 1,2-dicaprin hydrolysis was recorded using the surface barostat technique. Based on a kinetic model describing the inhibition by Orlistat and the activity of DGL on a mixed 1,2-dicaprin/Orlistat monolayer spread at the air-water interface combined with surface pressure measurements, it was possible to monitor DGL adsorption at the lipid-water interface and substrate hydrolysis in the course of a single experiment. This allowed to assess the kcat/KM* ratio for DGL acting on 1,2-dicaprin monolayer, after showing that mixed monolayers containing a low fraction of Orlistat were similar to pure 1,2-dicaprin monolayers.
ESTHER : Benarouche_2014_Biochimie_101_221
PubMedSearch : Benarouche_2014_Biochimie_101_221
PubMedID: 24508576

Title : Supported inhibitor for fishing lipases in complex biological media and mass spectrometry identification - Delorme_2014_Biochimie_107 Pt A_124
Author(s) : Delorme V , Raux B , Puppo R , Leclaire J , Cavalier JF , Marc S , Kamarajugadda PK , Buono G , Fotiadu F , Canaan S , Carriere F
Ref : Biochimie , 107 Pt A :124 , 2014
Abstract : A synthetic phosphonate inhibitor designed for lipase inhibition but displaying a broader range of activity was covalently immobilized on a solid support to generate a function-directed tool targeting serine hydrolases. To achieve this goal, straightforward and reliable analytical techniques were developed, allowing the monitoring of the solid support's chemical functionalization, enzyme capture processes and physisorption artifacts. This grafted inhibitor was tested on pure lipases and serine proteases from various origins, and assayed for the selective capture of lipases from several complex biological extracts. The direct identification of captured enzymes by mass spectrometry brought the proof of concept on the efficiency of this supported covalent inhibitor. The features and limitations of this "enzyme-fishing" proteomic tool provide new insight on solid-liquid inhibition process.
ESTHER : Delorme_2014_Biochimie_107 Pt A_124
PubMedSearch : Delorme_2014_Biochimie_107 Pt A_124
PubMedID: 25064360

Title : Enantioselective inhibition of microbial lipolytic enzymes by nonracemic monocyclic enolphosphonate analogues of cyclophostin - Point_2013_J.Med.Chem_56_4393
Author(s) : Point V , Malla RK , Carriere F , Canaan S , Spilling CD , Cavalier JF
Ref : Journal of Medicinal Chemistry , 56 :4393 , 2013
Abstract : Four nonracemic enolphosphonate analogues of Cyclophostin were obtained by asymmetric synthesis, and their absolute configurations at both phosphorus and C-5 carbon chiral centers were unambiguously assigned. The influence of chirality was studied by testing the inhibitory effects of these four stereoisomers toward the lipolytic activity of three microbial lipases: Fusarium solani cutinase, Rv0183, and LipY from Mycobacterium tuberculosis . Cutinase was highly diastereoselective for the (Sp) configuration using (Sc) inhibitors, whereas no obvious stereopreference at phosphorus was observed with (Rc) compounds. Conversely, Rv0183 exhibited strong enantioselective discrimination for (Sp) configuration regardless of the chirality at the asymmetric carbon atom. Lastly, LipY discriminated only the unusual diastereoisomeric configuration (Rc, Rp) leading to the most potent inhibitor. This work, which provides a fundamental premise for the understanding of the stereoselective relationships between nonracemic enolphosphonates and their inhibitory activity, also opens new prospects on the design and synthesis of highly specific enantioselective antimicrobial agents.
ESTHER : Point_2013_J.Med.Chem_56_4393
PubMedSearch : Point_2013_J.Med.Chem_56_4393
PubMedID: 23651298

Title : Effects of the propeptide of group X secreted phospholipase A(2) on substrate specificity and interfacial activity on phospholipid monolayers - Point_2013_Biochimie_95_51
Author(s) : Point V , Benarouche A , Jemel I , Parsiegla G , Lambeau G , Carriere F , Cavalier JF
Ref : Biochimie , 95 :51 , 2013
Abstract : Group X secreted phospholipase A(2) (GX sPLA(2)) plays important physiological roles in the gastrointestinal tract, in immune and sperm cells and is involved in several types of inflammatory diseases. It is secreted either as a mature enzyme or as a mixture of proenzyme (with a basic 11 amino acid propeptide) and mature enzyme. The role of the propeptide in the repression of sPLA(2) activity has been studied extensively using liposomes and micelles as model interfaces. These substrates are however not always suitable for detecting some fine tuning of lipolytic enzymes. In the present study, the monolayer technique is used to compare PLA(2) activity of recombinant mouse GX sPLA(2) (mGX) and its pro-form (PromGX) on monomolecular films of dilauroyl-phosphatidyl-ethanolamine (DLPE), -choline (DLPC) and -glycerol (DLPG). The PLA(2) activity and substrate specificity of mGX (PE approximately PG > PC) were found to be surface pressure-dependent. mGX displayed a high activity on DLPE and DLPG but not on DLPC monolayers up to surface pressures corresponding to the lateral pressure of biological membranes (30-35 mN/m). Overall, the propeptide impaired the enzyme activity, particularly on DLPE whatever the surface pressure. However some conditions could be found where the propeptide had little effects on the repression of PLA(2) activity. In particular, both PromGX and mGX had similar activities on DLPG at a surface pressure of 30 mN/m. These findings show that PromGX can be potentially active depending on the presentation of the substrate (i.e., lipid packing) and one cannot exclude such an activity in a physiological context. A structural model of PromGX was built to investigate how the propeptide controls the activity of GX sPLA(2). This model shows that the propeptide is located within the interfacial binding site (i-face) and could disrupt both the interfacial binding of the enzyme and the access to the active site by steric hindrance.
ESTHER : Point_2013_Biochimie_95_51
PubMedSearch : Point_2013_Biochimie_95_51
PubMedID: 22967966

Title : In vitro digestion of the self-emulsifying lipid excipient Labrasol() by gastrointestinal lipases and influence of its colloidal structure on lipolysis rate - Fernandez_2013_Pharm.Res_30_3077
Author(s) : Fernandez S , Jannin V , Chevrier S , Chavant Y , Demarne F , Carriere F
Ref : Pharm Res , 30 :3077 , 2013
Abstract : PURPOSE: Labrasol((a)) is a self-emulsifying excipient used to improve the oral bioavailability of poorly water-soluble drugs. It is a mixture of acylglycerols and PEG esters, these compounds being substrates for digestive lipases. The characterization of Labrasol((a)) gastrointestinal lipolysis is essential for understanding its mode of action. METHODS: Labrasol((a)) lipolysis was investigated using either individual enzymes (gastric lipase, pancreatic lipase-related protein 2, pancreatic carboxyl ester hydrolase) or a combination of enzymes under in vitro conditions mimicking first the gastric phase of lipolysis and second the duodenal one. Specific methods for quantifying lipolysis products were established in order to determine which compounds in Labrasol((a)) were preferentially hydrolyzed. RESULTS: Gastric lipase showed a preference for di- and triacylglycerols and the main acylglycerols remaining after gastric lipolysis were monoacylglycerols. PEG-8 diesters were also hydrolyzed to a large extent by gastric lipase. Most of the compounds initially present in Labrasol((a)) were found to be totally hydrolyzed after the duodenal phase of lipolysis. The rate of Labrasol((a)) hydrolysis by individual lipases was found to vary significantly with the dilution of the excipient in water and the resulting colloidal structures (translucent dispersion; opaque emulsion; transparent microemulsion), each lipase displaying a distinct pattern depending on the particle size. CONCLUSIONS: The lipases with distinct substrate specificities used in this study were found to be sensitive probes of phase transitions occurring upon Labrasol((a)) dilution. In addition to their use for developing in vitro digestion models, these enzymes are interesting tools for the characterization of self-emulsifying lipid-based formulations.
ESTHER : Fernandez_2013_Pharm.Res_30_3077
PubMedSearch : Fernandez_2013_Pharm.Res_30_3077
PubMedID: 23636839
Gene_locus related to this paper: human-PNLIPRP2

Title : Biochemical and structural characterization of non-glycosylated Yarrowia lipolytica LIP2 lipase - Aloulou_2013_Eur.J.Lipid.Sci.Tech_115_429
Author(s) : Aloulou A , Benarouche A , Puccinelli D , Spinelli S , Cavalier JF , Cambillau C , Carriere F
Ref : Eur.J.Lipid.Sci.Tech , 115 :429 , 2013
Abstract : The LIP2 lipase from the yeast Yarrowia lipolytica (YLLIP2) is assumed to be a good drug candidate for enzyme replacement therapy in patients with pancreatic exocrine insufficiency. Understanding and improving its biochemical properties are essential for its oral administration. YLLIP2 is a highly glycosylated protein, with glycan chains accounting for about 13% of the molecular mass of the native protein. Two potential N-glycosylation sites (N113IS and N134NT) can be identified from YLLIP2 amino acid sequence. YLLIP2 mutants with single (N113Q or N134Q) or combined (N113Q/N134Q) substitutions of these glycosylation sites were expressed in the yeast Pichia pastoris, purified and characterized. Lipase specific activity and adsorption at the lipidwater interface were found to be decreased in the absence of N-glycosylation. It was thus shown that the glycosylated enzyme had a better ability to bind and penetrate a DLPC monolayer than the non-glycosylated N113Q/N134Q mutant. Comparison of wild-type glycosylated and non-glycosylated YLLIP2 shows that the N-glycosylation clearly contributes to the high stability of YLLIP2 in the presence of pepsin in vitro, and to a lower extent in the presence of chymotrypsin. The X-ray structure of the YLLIP2 N113Q/N134Q double mutant was obtained at 2.6 angstrom resolution and was found to be identical to that of wild-type YLLIP2, with the lid in a closed conformation. Glycosylation is therefore not essential for a proper folding of YLLIP2. Practical applications: The LIP2 lipase from the yeast Yarrowia lipolytica is one of the most active lipases identified so far. Among the various applications envisioned for this enzyme, it seems particularly well adapted for enzyme replacement therapy in patients with pancreatic exocrine insufficiency. It is active and stable at low pH values, resistant to bile salts, and its glycosylation allows a high resistance to pepsin. All these properties are important for developing the oral administration of digestive enzymes used as drugs.
ESTHER : Aloulou_2013_Eur.J.Lipid.Sci.Tech_115_429
PubMedSearch : Aloulou_2013_Eur.J.Lipid.Sci.Tech_115_429
PubMedID:
Gene_locus related to this paper: yarli-lip2

Title : Partial deletion of beta9 loop in pancreatic lipase-related protein 2 reduces enzyme activity with a larger effect on long acyl chain substrates - Dridi_2013_Biochim.Biophys.Acta_1831_1293
Author(s) : Dridi K , Amara S , Bezzine S , Rodriguez JA , Carriere F , Gaussier H
Ref : Biochimica & Biophysica Acta , 1831 :1293 , 2013
Abstract : Structural studies on pancreatic lipase have revealed a complex architecture of surface loops surrounding the enzyme active site and potentially involved in interactions with lipids. Two of them, the lid and beta loop, expose a large hydrophobic surface and are considered as acyl chain binding sites based on their interaction with an alkyl phosphonate inhibitor. While the role of the lid in substrate recognition and selectivity has been extensively studied, the implication of beta9 loop in acyl chain stabilization remained hypothetical. The characterization of an enzyme with a natural deletion of the lid, guinea pig pancreatic lipase-related protein 2 (GPLRP2), suggests however an essential contribution of the beta9 loop in the stabilization of the acyl enzyme intermediate formed during the lipolysis reaction. A GPLRP2 mutant with a seven-residue deletion of beta9 loop (GPLRP2-deltabeta9) was produced and its enzyme activity was measured using various substrates (triglycerides, monoglycerides, galactolipids, phospholipids, vinyl esters) with short, medium and long acyl chains. Whatever the substrate tested, GPLRP2-deltabeta9 activity is drastically reduced compared to that of wild-type GPLRP2 and this effect is more pronounced as the length of substrate acyl chain increases. Changes in relative substrate selectivity and stereoselectivity remained however weak. The deletion within beta9 loop has also a negative effect on the rate of enzyme inhibition by alkyl phosphonates. All these findings indicate that the reduced enzyme turnover observed with GPLRP2-deltabeta9 results from a weaker stabilization of the acyl enzyme intermediate due to a loss of hydrophobic interactions.
ESTHER : Dridi_2013_Biochim.Biophys.Acta_1831_1293
PubMedSearch : Dridi_2013_Biochim.Biophys.Acta_1831_1293
PubMedID: 24046870
Gene_locus related to this paper: human-PNLIPRP2

Title : New insights into the pH-dependent interfacial adsorption of dog gastric lipase using the monolayer technique - Benarouche_2013_Colloids.Surf.B.Biointerfaces_111_306
Author(s) : Benarouche A , Point V , Parsiegla G , Carriere F , Cavalier JF
Ref : Colloids Surf B Biointerfaces , 111 :306 , 2013
Abstract : The access to kinetic parameters of lipolytic enzyme adsorption onto lipids is essential for a better understanding of interfacial enzymology and lipase-lipid interactions. The interfacial adsorption of dog gastric lipase (DGL) was monitored as a function of pH and surface pressure (Pi), independently from the catalytic activity, using non-hydrolysable 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) monomolecular films. The acid-stable DGL, which initiates fat digestion in the stomach, was then selected because its adsorption kinetics onto hydrophobic solid surfaces were already studied. This gastric lipase was therefore used as a model enzyme to validate both experimental and theoretical approaches. Results show that the adsorption process of DGL at the lipid/water interface depends on a pH-dependent adsorption equilibrium coefficient which is optimum at pH 5.0 (K(Ads) = 1.7 +/- 0.05 x 10(8)M(-1)). KAds values further allowed an indirect estimation of the molar fraction (PhiE*(%), mol%) as well as the molecular area (AE*) of DGL adsorbed onto DLPC monolayer. Based on these data, a model for DGL adsorption onto DLPC monolayer at pH 5.0 is proposed for a surface pressure range of 15-25 mNm(-1).
ESTHER : Benarouche_2013_Colloids.Surf.B.Biointerfaces_111_306
PubMedSearch : Benarouche_2013_Colloids.Surf.B.Biointerfaces_111_306
PubMedID: 23838197

Title : The galactolipase activity of some microbial lipases and pancreatic enzymes - Amara_2013_Eur.J.Lipid.Sci.Technol_115_442
Author(s) : Amara S , Lafont D , Parsiegla G , Point V , Chabannes A , Rousset A , Carriere F
Ref : Eur J Lipid Sci Technol , 115 :442 , 2013
Abstract : Several well known microbial lipases were screened for their ability to hydrolyze synthetic medium chain monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). Fusarium solani cutinase and Thermomyces lanuginosus lipase (TLL) were found to hydrolyze MGDG at high rates (984 +/- 62 and 450 +/-41 U/mg, respectively). These activities remained however lower than those measured with pancreatic lipase-related protein 2 (PLRP2) on the same substrate. As previously observed with PLRP2, galactolipid-bile salt mixed micelles were found to be the best substrate form for microbial enzymes. The galactolipid to bile salt molar ratios for measuring maximum galactolipase activities were found to be similar to those previously established with PLRP2, suggesting that bile salts have mainly an effect on the substrate and not on the enzyme itself. The galactolipase activity of cutinase and TLL, as well as human and guinea pig PLRP2s were also measured using galactolipid monomolecular films. Enzymes having a lid (TLL and human PLRP2) were found to act at higher surface pressures than those with no lid (cutinase and guinea pig PLRP2). In silico docking of medium chain MGDG and DGDG in the active site of guinea pig PLRP2 and TLL reveals some structural analogies between these enzymes
ESTHER : Amara_2013_Eur.J.Lipid.Sci.Technol_115_442
PubMedSearch : Amara_2013_Eur.J.Lipid.Sci.Technol_115_442
PubMedID:
Gene_locus related to this paper: bovin-balip , human-CEL

Title : Synthesis and kinetic evaluation of cyclophostin and cyclipostins phosphonate analogs as selective and potent inhibitors of microbial lipases - Point_2012_J.Med.Chem_55_10204
Author(s) : Point V , Malla RK , Diomande S , Martin BP , Delorme V , Carriere F , Canaan S , Rath NP , Spilling CD , Cavalier JF
Ref : Journal of Medicinal Chemistry , 55 :10204 , 2012
Abstract : A new series of customizable diastereomeric cis- and trans-monocyclic enol-phosphonate analogs to Cyclophostin and Cyclipostins were synthesized. Their potencies and mechanisms of inhibition toward six representative lipolytic enzymes belonging to distinct lipase families were examined. With mammalian gastric and pancreatic lipases no inhibition occurred with any of the compounds tested. Conversely, Fusarium solani Cutinase and lipases from Mycobacterium tuberculosis (Rv0183 and LipY) were all fully inactivated. The best inhibitors displayed a cis conformation (H and OMe) and exhibited higher inhibitory activities than the lipase inhibitor Orlistat toward the same enzymes. Our results have revealed that chemical group at the gamma-carbon of the phosphonate ring strongly impacts the inhibitory efficiency, leading to a significant improvement in selectivity toward a target lipase over another. The powerful and selective inhibition of microbial (fungal and mycobacterial) lipases suggests that these seven-membered monocyclic enol-phosphonates should provide useful leads for the development of novel and highly selective antimicrobial agents.
ESTHER : Point_2012_J.Med.Chem_55_10204
PubMedSearch : Point_2012_J.Med.Chem_55_10204
PubMedID: 23095026

Title : Inhibition of phospholipase A1, lipase and galactolipase activities of pancreatic lipase-related protein 2 by methyl arachidonyl fluorophosphonate (MAFP) - Amara_2012_Biochim.Biophys.Acta_1821_1379
Author(s) : Amara S , Delorme V , Record M , Carriere F
Ref : Biochimica & Biophysica Acta , 1821 :1379 , 2012
Abstract : Methyl arachidonyl fluorophosphonate (MAFP) is a known inhibitor of cytosolic phospholipase A2 and some other serine enzymes. MAFP was found here to be an irreversible inhibitor of human pancreatic lipase-related protein 2 (HPLRP2), an enzyme displaying lipase, phospholipase A1 and galactolipase activities. In the presence of MAFP, mass spectrometry analysis of HPLRP2 revealed a mass increase of 351Da, suggesting a covalent binding of MAFP to the active site serine residue. When HPLRP2 was pre-incubated with MAFP before measuring residual activity, a direct inhibition of HPLRP2 occurred, confirming that HPLRP2 has an active site freely accessible to solvent and differs from most lipases in solution. HPLRP2 activities on tributyrin (TC4), phosphatidylcholine (PC) and monogalactosyl dioctanoylglycerol (C8-MGDG) were equally inhibited under these conditions. Bile salts were not required to trigger the inhibition, but they significantly increased the rate of HPLRP2 inhibition, probably because of MAFP micellar solubilization. Since HPLRP2 is active on various substrates that self-organize differently in the presence of water, HPLRP2 inhibition by MAFP was tested in the presence of these substrates after adding MAFP in the course of the lipolysis reaction. In this case, the rates of inhibition of lipase, phospholipase A1 and galactolipase activities were not equivalent (triglycerides>PC>MGDG), suggesting different enzyme/inhibitor partitioning between the aqueous phase and lipid aggregates. The inhibition by MAFP of a well identified phospholipase A1 (HPLRP2), present in pancreatic juice and also in human monocytes, indicates that MAFP cannot be used for discriminating phospholipase A2 from A1 activities at the cellular level.
ESTHER : Amara_2012_Biochim.Biophys.Acta_1821_1379
PubMedSearch : Amara_2012_Biochim.Biophys.Acta_1821_1379
PubMedID: 22835523
Gene_locus related to this paper: human-PNLIPRP2

Title : Analysis of the discriminative inhibition of mammalian digestive lipases by 3-phenyl substituted 1,3,4-oxadiazol-2(3H)-ones. - Point_2012_Eur.J.Med.Chem_58_452
Author(s) : Point V , Pavan Kumar KV , Marc S , Delorme V , Parsiegla G , Amara S , Carriere F , Buono G , Fotiadu F , Canaan S , Leclaire J , Cavalier JF
Ref : Eur Journal of Medicinal Chemistry , 58 :452 , 2012
Abstract : We report here the reactivity and selectivity of three 5-Methoxy-N-3-Phenyl substituted-1,3,4-Oxadiazol-2(3H)-ones (MPOX, as well as meta and para-PhenoxyPhenyl derivatives, i.e.MmPPOX and MpPPOX) with respect to the inhibition of mammalian digestive lipases: dog gastric lipase (DGL), human (HPL) and porcine (PPL) pancreatic lipases, human (HPLRP2) and guinea pig (GPLRP2) pancreatic lipase-related proteins 2, human pancreatic carboxyl ester hydrolase (hCEH), and porcine pancreatic extracts (PPE). All three oxadiazolones displayed similar inhibitory activities on DGL, PLRP2s and hCEH than the FDA-approved anti-obesity drug Orlistat towards the same enzymes. These compounds appeared however to be discriminative of HPL (poorly inhibited) and PPL (fully inhibited). The inhibitory activities obtained experimentally in vitro were further rationalized using in silico molecular docking. In the case of DGL, we demonstrated that the phenoxy group plays a key role in specific molecular interactions within the lipase's active site. The absence of this group in the case of MPOX, as well as its connectivity to the neighbouring aromatic ring in the case of MmPPOX and MpPPOX, strongly impacts the inhibitory efficiency of these oxadiazolones and leads to a significant gain in selectivity towards the lipases tested. The powerful inhibition of PPL, DGL, PLRP2s, hCEH and to a lesser extend HPL, suggests that oxadiazolone derivatives could also provide useful leads for the development of novel and more discriminative inhibitors of digestive lipases. These inhibitors could be used for a better understanding of individual lipase function as well as for drug development aiming at the regulation of the whole gastrointestinal lipolysis process.
ESTHER : Point_2012_Eur.J.Med.Chem_58_452
PubMedSearch : Point_2012_Eur.J.Med.Chem_58_452
PubMedID: 23153815
Gene_locus related to this paper: canfa-1lipg , cavpo-2plrp , human-CEL , human-PNLIP , human-PNLIPRP2 , pig-1plip

Title : The molecular mechanism of human hormone-sensitive lipase inhibition by substituted 3-phenyl-5-alkoxy-1,3,4-oxadiazol-2-ones - Ben Ali_2012_Biochimie_94_137
Author(s) : Ben Ali Y , Verger R , Carriere F , Petry S , Muller G , Abousalham A
Ref : Biochimie , 94 :137 , 2012
Abstract : Hormone-sensitive lipase (HSL) plays an important role in the mobilization of free fatty acids (FFA) from adipocytes. The inhibition of HSL may offer a pharmacological approach to reduce FFA levels in plasma and diminish peripheral insulin resistance in type 2 diabetes. In this work, the inhibition of HSL by substituted 3-phenyl-5-alkoxy-1,3,4-oxadiazol-2-ones has been studied in vitro. 5-methoxy-3-(3-phenoxyphenyl)-1,3,4-oxadiazol-2(3H)-one (compound 7600) and 5-methoxy-3-(3-methyl-4-phenylacetamidophenyl)-1,3,4-oxadiazol-2(3H)-one (compound 9368) were selected as the most potent HSL inhibitors. HSL is inhibited after few minutes of incubation with compound 7600, at a molar excess of 20. This inhibition is reversed in the presence of an emulsion of lipid substrate. The reactivation phenomenon is hardly observed when incubating HSL with compound 9368. The molecular mechanism underlying the reversible inhibition of HSL by compound 7600 was investigated using high performance liquid chromatography and tandem mass spectrometry. The stoichiometry of the inhibition reaction revealed that specifically one molecule of inhibitor was bound per enzyme molecule. The inhibition by compound 7600 involves a nucleophilic attack by the hydroxy group of the catalytic Ser of the enzyme on the carbon atom of the carbonyl moiety of the oxadiazolone ring of the inhibitor, leading to the formation of covalent enzyme-inhibitor intermediate. This covalent intermediate is subsequently hydrolyzed, releasing an oxadiazolone decomposition product, carbon dioxide and the active HSL form. On the basis of this study, a kinetic model is proposed to describe the inhibition of HSL by compound 7600 in the aqueous phase as well as its partial reactivation at the lipid-water interface.
ESTHER : Ben Ali_2012_Biochimie_94_137
PubMedSearch : Ben Ali_2012_Biochimie_94_137
PubMedID: 22008857
Gene_locus related to this paper: human-LIPE

Title : MmPPOX inhibits Mycobacterium tuberculosis lipolytic enzymes belonging to the hormone-sensitive lipase family and alters mycobacterial growth - Delorme_2012_PLoS.One_7_e46493
Author(s) : Delorme V , Diomande SV , Dedieu L , Cavalier JF , Carriere F , Kremer L , Leclaire J , Fotiadu F , Canaan S
Ref : PLoS ONE , 7 :e46493 , 2012
Abstract : Lipid metabolism plays an important role during the lifetime of Mycobacterium tuberculosis, the causative agent of tuberculosis. Although M. tuberculosis possesses numerous lipolytic enzymes, very few have been characterized yet at a biochemical/pharmacological level. This study was devoted to the M. tuberculosis lipolytic enzymes belonging to the Hormone-Sensitive Lipase (HSL) family, which encompasses twelve serine hydrolases closely related to the human HSL. Among them, nine were expressed, purified and biochemically characterized using a broad range of substrates. In vitro enzymatic inhibition studies using the recombinant HSL proteins, combined with mass spectrometry analyses, revealed the potent inhibitory activity of an oxadiazolone compound, named MmPPOX. In addition, we provide evidence that MmPPOX alters mycobacterial growth. Overall, these findings suggest that the M. tuberculosis HSL family displays important metabolic functions, thus opening the way to further investigations linking the involvement of these enzymes in mycobacterial growth.
ESTHER : Delorme_2012_PLoS.One_7_e46493
PubMedSearch : Delorme_2012_PLoS.One_7_e46493
PubMedID: 23029536
Gene_locus related to this paper: myctu-Rv2970c

Title : Bis (monoacylglycero) phosphate interfacial properties and lipolysis by pancreatic lipase-related protein 2, an enzyme present in THP-1 human monocytes - Record_2011_Biochim.Biophys.Acta_1811_419
Author(s) : Record M , Amara S , Subra C , Jiang G , Prestwich GD , Ferrato F , Carriere F
Ref : Biochimica & Biophysica Acta , 1811 :419 , 2011
Abstract : The interfacial physical properties of bis(monoacylglycero)phosphate (BMP) and its derivatives with three oleoyl chains (hemi-BDP) and four oleoyl chains (bis(diacylglycero)phosphate, BDP) were investigated using Langmuir monomolecular films. The mean molecular area of BMP at the collapse surface pressure (45mN m(-1)) was similar to those measured with other phospholipids bearing two acyl chains (66 and 59.6A(2) molecule(-1) at pH 5.5 and 8.0, respectively). In Hemi-BDP and BDP, the mean molecular area increased by 26 and 35A(2) molecule(-1) per additional acyl chain at pH 5.5 and 8.0, respectively. When BMP was added to a phospholipid mixture mimicking late endosome membrane composition at pH 8.0, the mean phospholipid molecular area increased by 7% regardless of the surface pressure. In contrast, the variation in molecular area was surface pressure-dependent at pH 5.5, a pH value close to that of intra-endosomal content. BMP and hemi-BDP, but not BDP, were hydrolyzed by pancreatic lipase-related protein 2 (PLRP2), which exhibits phospholipase A(1) activity. At pH 5.5, the maximum activities of PLRP2 on BMP were recorded at high surface pressures (25-35mN/m). At pH 8.0, the PLRP2 activity vs. surface pressure showed a bell-shaped curve with maximum activities at 15mN/m for both BMP and hemi-BDP. This is a new activity for this enzyme which could degrade cellular BMP since both human PLRP2 (HPLRP2) and BMP were localized in human monocytic THP-1 cells. This is the first report on the cellular localization of HPLRP2 in human monocytes.
ESTHER : Record_2011_Biochim.Biophys.Acta_1811_419
PubMedSearch : Record_2011_Biochim.Biophys.Acta_1811_419
PubMedID: 21554982

Title : Identification of a putative triacylglycerol lipase from papaya latex by functional proteomics - Dhouib_2011_FEBS.J_278_97
Author(s) : Dhouib R , Laroche-Traineau J , Shaha R , Lapaillerie D , Solier E , Ruales J , Pina M , Villeneuve P , Carriere F , Bonneu M , Arondel V
Ref : Febs J , 278 :97 , 2011
Abstract : Latex from Caricaceae has been known since 1925 to contain strong lipase activity. However, attempts to purify and identify the enzyme were not successful, mainly because of the lack of solubility of the enzyme. Here, we describe the characterization of lipase activity of the latex of Vasconcellea heilbornii and the identification of a putative homologous lipase from Carica papaya. Triacylglycerol lipase activity was enriched 74-fold from crude latex of Vasconcellea heilbornii to a specific activity (SA) of 57 mumol.min(-1).mg(-1) on long-chain triacylglycerol (olive oil). The extract was also active on trioctanoin (SA = 655 mumol.min(-1).mg(-1) ), tributyrin (SA = 1107 mumol.min(-1).mg(-1) ) and phosphatidylcholine (SA = 923 mumol.min(-1).mg(-1) ). The optimum pH ranged from 8.0 to 9.0. The protein content of the insoluble fraction of latex was analyzed by electrophoresis followed by mass spectrometry, and 28 different proteins were identified. The protein fraction was incubated with the lipase inhibitor [(14) C]tetrahydrolipstatin, and a 45 kDa protein radiolabeled by the inhibitor was identified as being a putative lipase. A C. papaya cDNA encoding a 55 kDa protein was further cloned, and its deduced sequence had 83.7% similarity with peptides from the 45 kDa protein, with a coverage of 25.6%. The protein encoded by this cDNA had 35% sequence identity and 51% similarity to castor bean acid lipase, suggesting that it is the lipase responsible for the important lipolytic activities detected in papaya latex.
ESTHER : Dhouib_2011_FEBS.J_278_97
PubMedSearch : Dhouib_2011_FEBS.J_278_97
PubMedID: 21114629

Title : Galactolipase, phospholipase and triacylglycerol lipase activities in the midgut of six species of lepidopteran larvae feeding on different lipid diets - Christeller_2011_J.Insect.Physiol_57_1232
Author(s) : Christeller JT , Amara S , Carriere F
Ref : J Insect Physiol , 57 :1232 , 2011
Abstract : Galactolipase, phospholipase and triacylglycerol lipase activities were measured from the midgut of six species of lepidopteran larvae, two folivores, Epiphyas postvittana (Tortricidae) and Helicoverpa armigera (Noctuidae); two granivores, Plodia interpunctella (Pyralidae) and Ephestia kuehniella (Pyrallidae); a presumptive carnivore, Galleria mellonella (Pyralidae); and a keratinophage, Tineola bisselliella (Tineidae). Galactolipase has not been previously reported in insects. Galactolipase and phospholipase activities were high in the folivores and triacylglycerol lipase activity was low, matching the high galactolipid content of leaves. Conversely, galactolipase and phospholipase activities were low, but not absent, and triacylglycerol lipase activity high in the four other non-folivorous species, matching the high acylglycerol content of their diets. These data suggest the utility of reclassification, for evolutionary studies, of phytophagous lepidoptera into two feeding classes; folivore and granivore, the latter having similarity to the fungivore line of feeders in terms of its lipase activities and ability to retrieve essential polyunsaturated long chain fatty acids from their diets. All the digestive lipases have alkaline pH optima for activity, matching the pH of the lepidopteran midgut and their amino acid content show modifications likely to stabilize the proteins in that environment.
ESTHER : Christeller_2011_J.Insect.Physiol_57_1232
PubMedSearch : Christeller_2011_J.Insect.Physiol_57_1232
PubMedID: 21704634

Title : Effects of surfactants on lipase structure, activity, and inhibition - Delorme_2011_Pharm.Res_28_1831
Author(s) : Delorme V , Dhouib R , Canaan S , Fotiadu F , Carriere F , Cavalier JF
Ref : Pharm Res , 28 :1831 , 2011
Abstract : Lipase inhibitors are the main anti-obesity drugs prescribed these days, but the complexity of their mechanism of action is making it difficult to develop new molecules for this purpose. The efficacy of these drugs is known to depend closely on the physico-chemistry of the lipid-water interfaces involved and on the unconventional behavior of the lipases which are their target enzymes. The lipolysis reaction which occurs at an oil-water interface involves complex equilibria between adsorption-desorption processes, conformational changes and catalytic mechanisms. In this context, surfactants can induce significant changes in the partitioning of the enzyme and the inhibitor between the water phase and lipid-water interfaces. Surfactants can be found at the oil-water interface where they compete with lipases for adsorption, but also in solution in the form of micellar aggregates and monomers that may interact with hydrophobic parts of lipases in solution. These various interactions, combined with the emulsification and dispersion of insoluble substrates and inhibitors, can either promote or decrease the activity and the inhibition of lipases. Here, we review some examples of the various effects of surfactants on lipase structure, activity and inhibition, which show how complex the various equilibria involved in the lipolysis reaction tend to be.
ESTHER : Delorme_2011_Pharm.Res_28_1831
PubMedSearch : Delorme_2011_Pharm.Res_28_1831
PubMedID: 21234659

Title : Watching intracellular lipolysis in mycobacteria using time lapse fluorescence microscopy - Dhouib_2011_Biochim.Biophys.Acta_1811_234
Author(s) : Dhouib R , Ducret A , Hubert P , Carriere F , Dukan S , Canaan S
Ref : Biochimica & Biophysica Acta , 1811 :234 , 2011
Abstract : The fact that Mycobacterium tuberculosis mobilizes lipid bodies (LB) located in the cytosol during infection process has been proposed for decades. However, the mechanisms and dynamics of mobilization of these lipid droplets within mycobacteria are still not completely characterized. Evidence in favour of this characterization was obtained here using a combined fluorescent microscopy and computational image processing approach. The decrease in lipid storage levels observed under nutrient depletion conditions was correlated with a significant increase in the size of the bacteria. LB fragmentation/condensation cycles were monitored in real time. The exact contribution of lipases in this process was confirmed using the lipase inhibitor tetrahydrolipstatin, which was found to prevent LB degradation and to limit the bacterial cell growth. The method presented here provides a powerful tool for monitoring in vivo lipolysis in mycobacteria and for obtaining new insights on the growth of cells and their entry into the dormant or reactivation phase. It should be particularly useful for studying the effects of chemical inhibitors and activators on cells as well as investigating other metabolic pathways.
ESTHER : Dhouib_2011_Biochim.Biophys.Acta_1811_234
PubMedSearch : Dhouib_2011_Biochim.Biophys.Acta_1811_234
PubMedID: 21238605

Title : Probing structural transitions in both structured and disordered proteins using site-directed spin-labeling EPR spectroscopy - Longhi_2011_J.Pept.Sci_17_315
Author(s) : Longhi S , Belle V , Fournel A , Guigliarelli B , Carriere F
Ref : J Pept Sci , 17 :315 , 2011
Abstract : EPR spectroscopy is a technique that specifically detects unpaired electrons. EPR-sensitive reporter groups (spin labels or spin probes) can be introduced into biological systems via site-directed spin-labeling (SDSL). The basic strategy of SDSL involves the introduction of a paramagnetic group at a selected protein site. This is usually accomplished by cysteine-substitution mutagenesis, followed by covalent modification of the unique sulfydryl group with a selective reagent bearing a nitroxide radical. In this review we briefly describe the theoretical principles of this well-established approach and illustrate how we successfully applied it to investigate structural transitions in both human pancreatic lipase (HPL), a protein with a well-defined alpha/beta hydrolase fold, and the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (N(TAIL) ) upon addition of ligands and/or protein partners. In both cases, SDSL EPR spectroscopy allowed us to document protein conformational changes at the residue level. The studies herein summarized show that this approach is not only particularly well-suited to study IDPs that inherently escape atomistic description by X-ray crystallography but also provides dynamic information on structural transitions occurring within well-characterized structured proteins for which X-ray crystallography can only provide snapshots of the initial and final stages.
ESTHER : Longhi_2011_J.Pept.Sci_17_315
PubMedSearch : Longhi_2011_J.Pept.Sci_17_315
PubMedID: 21351321

Title : Carica papaya lipase: a naturally immobilized enzyme with interesting biochemical properties - Abdelkafi_2011_Plant.Foods.Hum.Nutr_66_34
Author(s) : Abdelkafi S , Barouh N , Fouquet B , Fendri I , Pina M , Scheirlinckx F , Villeneuve P , Carriere F
Ref : Plant Foods Hum Nutr , 66 :34 , 2011
Abstract : Triacylglycerol (TAG) lipases have been thoroughly characterized in mammals and microorganisms, whereas very little is known about plant TAG lipases. The lipolytic activity occurring in all the laticies is known to be associated with sedimentable particles, and all attempts to solubilize the lipolytic activity of Carica papaya latex have been unsuccessful so far. However, some of the biochemical properties of the lipase from Carica papaya latex (CPL) were determined from the insoluble fraction of the latex. The activity was optimum at a temperature of 37 degrees C and a pH of 9.0, and the specific activities of CPL were found to be 2,000 +/- 185 and 256 +/- 8 U/g when tributyrin and olive oil were used as substrates, respectively. CPL was found to be active in the absence of any detergent, whereas many lipases require detergent to prevent the occurrence of interfacial denaturation. CPL was inactive in the presence of micellar concentrations of Triton X-100, sodium dodecyl sulfate (SDS) and tetradecyl trimethylammonium bromide (TTAB), and still showed high levels of activity in the presence of sodium taurodeoxycholate (NaTDC) and the zwitterionic Chaps detergent. The effects of various proteases on the lipolytic activity of CPL were studied, and CPL was found to be resistant to treatment with various enzymes, except in the presence of trypsin. All these properties suggest that CPL may be a good candidate for various biotechnological applications.
ESTHER : Abdelkafi_2011_Plant.Foods.Hum.Nutr_66_34
PubMedSearch : Abdelkafi_2011_Plant.Foods.Hum.Nutr_66_34
PubMedID: 21267783

Title : In vitro stereoselective hydrolysis of diacylglycerols by hormone-sensitive lipase - Rodriguez_2010_Biochim.Biophys.Acta_1801_77
Author(s) : Rodriguez JA , Ben Ali Y , Abdelkafi S , Mendoza LD , Leclaire J , Fotiadu F , Buono G , Carriere F , Abousalham A
Ref : Biochimica & Biophysica Acta , 1801 :77 , 2010
Abstract : Hormone-sensitive lipase (HSL) contributes importantly to the mobilization of fatty acids in adipocytes and shows a substrate preference for the diacylglycerols (DAGs) originating from triacylglycerols. To determine whether HSL shows any stereopreference during the hydrolysis of diacylglycerols, racemic 1,2(2,3)-sn-diolein was used as a substrate and the enantiomeric excess (ee%) of residual 1,2-sn-diolein over 2,3-sn-diolein was measured as a function of DAG hydrolysis. Enantiomeric DAGs were separated by performing chiral-stationary-phase HPLC after direct derivatization from lipolysis product extracts. The fact that the ee% of 1,2-sn-diolein over 2,3-sn-diolein increased with the level of hydrolysis indicated that HSL has a preference for 2,3-sn-diolein as a substrate and therefore a stereopreference for the sn-3 position of dioleoylglycerol. The ee% of 1,2-sn-diolein reached a maximum value of 36% at 42% hydrolysis. Among the various mammalian lipases tested so far, HSL is the only lipolytic carboxylester hydrolase found to have a pronounced stereospecificity for the sn-3 position of dioleoylglycerol.
ESTHER : Rodriguez_2010_Biochim.Biophys.Acta_1801_77
PubMedSearch : Rodriguez_2010_Biochim.Biophys.Acta_1801_77
PubMedID: 19800417

Title : Evidence for the cytotoxic effects of Mycobacterium tuberculosis phospholipase C towards macrophages - Bakala_2010_Biochim.Biophys.Acta_1801_1305
Author(s) : Bakala N'goma J C , Schue M , Carriere F , Geerlof A , Canaan S
Ref : Biochimica & Biophysica Acta , 1801 :1305 , 2010
Abstract : Phospholipase Cs (PLCs) contribute importantly to the virulence and pathogenicity of several bacteria. It has been reported in previous studies that mutations in the four predicted plc genes of Mycobacterium tuberculosis inhibit the growth of these bacteria during the late phase of infection in mice. These enzymes have not yet been fully characterised, mainly because they are not easy to produce in large quantities. With a view to elucidating the role of all Mycobacterium tuberculosis phospholipase Cs (PLC-A, PLC-B, PLC-C and PLC-D), a large amount of active, soluble recombinant PLCs, were expressed and purified using Mycobacterium smegmatis as expression system. These enzymes showed different pH activity profiles. PLC-C was found to be the most active of the four recombinant PLCs under acidic conditions. All the enzymes tested induced cytotoxic effects on mouse macrophage RAW 264.7 cell lines, via direct or indirect enzymatic hydrolysis of cell membrane phospholipids. These results open new prospects for characterising biochemical and structural features of Mycobacterium tuberculosis PLCs, which might lead to the identification of novel anti-tuberculosis drug targets. All mycobacterial phospholipase Cs can now be studied in order to determine their role in the virulence and pathogenicity of bacteria of this kind.
ESTHER : Bakala_2010_Biochim.Biophys.Acta_1801_1305
PubMedSearch : Bakala_2010_Biochim.Biophys.Acta_1801_1305
PubMedID: 20736081

Title : Lipolysis of natural long chain and synthetic medium chain galactolipids by pancreatic lipase-related protein 2 - Amara_2010_Biochim.Biophys.Acta_1801_508
Author(s) : Amara S , Barouh N , Lecomte J , Lafont D , Robert S , Villeneuve P , de Caro A , Carriere F
Ref : Biochimica & Biophysica Acta , 1801 :508 , 2010
Abstract : Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the most abundant lipids in nature, mainly as important components of plant leaves and chloroplast membranes. Pancreatic lipase-related protein 2 (PLRP2) was previously found to express galactolipase activity, and it is assumed to be the main enzyme involved in the digestion of these common vegetable lipids in the gastrointestinal tract. Most of the previous in vitro studies were however performed with medium chain synthetic galactolipids as substrates. It was shown here that recombinant guinea pig (Cavia porcellus) as well as human PLRP2 hydrolyzed at high rates natural DGDG and MGDG extracted from spinach leaves. Their specific activities were estimated by combining the pH-stat technique, thin layer chromatography coupled to scanning densitometry and gas chromatography. The optimum assay conditions for hydrolysis of these natural long chain galactolipids were investigated and the optimum bile salt to substrate ratio was found to be different from that established with synthetic medium chains MGDG and DGDG. Nevertheless the length of acyl chains and the nature of the galactosyl polar head of the galactolipid did not have major effects on the specific activities of PLRP2, which were found to be very high on both medium chain [1786+/-100 to 5420+/-85U/mg] and long chain [1756+/-208 to 4167+/-167U/mg] galactolipids. Fatty acid composition analysis of natural MGDG, DGDG and their lipolysis products revealed that PLRP2 only hydrolyzed one ester bond at the sn-1 position of galactolipids. PLRP2 might be used to produce lipid and free fatty acid fractions enriched in either 16:3 n-3 or 18:3 n-3 fatty acids, both found at high levels in galactolipids.
ESTHER : Amara_2010_Biochim.Biophys.Acta_1801_508
PubMedSearch : Amara_2010_Biochim.Biophys.Acta_1801_508
PubMedID: 20083229
Gene_locus related to this paper: human-PNLIPRP2

Title : Amplitude of pancreatic lipase lid opening in solution and identification of spin label conformational subensembles by combining continuous wave and pulsed EPR spectroscopy and molecular dynamics - Ranaldi_2010_Biochemistry_49_2140
Author(s) : Ranaldi S , Belle V , Woudstra M , Bourgeas R , Guigliarelli B , Roche P , Vezin H , Carriere F , Fournel A
Ref : Biochemistry , 49 :2140 , 2010
Abstract : The opening of the lid that controls the access to the active site of human pancreatic lipase (HPL) was measured from the magnetic interaction between two spin labels grafted on this enzyme. One spin label was introduced at a rigid position in HPL where an accessible cysteine residue (C181) naturally occurs. A second spin label was covalently bound to the mobile lid after introducing a cysteine residue at position 249 by site-directed mutagenesis. Double electron-electron resonance (DEER) experiments allowed the estimation of a distance of 19 +/- 2 A between the spin labels when bilabeled HPL was alone in a frozen solution, i.e., with the lid in the closed conformation. A magnetic interaction was however detected by continuous wave EPR experiments, suggesting that a fraction of bilabeled HPL contained spin labels separated by a shorter distance. These results could be interpreted by the presence of two conformational subensembles for the spin label lateral chain at position 249 when the lid was closed. The existence of these conformational subensembles was revealed by molecular dynamics experiments and confirmed by the simulation of the EPR spectrum. When the lid opening was induced by the addition of bile salts and colipase, a larger distance of 43 +/- 2 A between the two spin labels was estimated from DEER experiments. The distances measured between the spin labels grafted at positions 181 and 249 were in good agreement with those estimated from the known X-ray structures of HPL in the closed and open conformations, but for the first time, the amplitude of the lid opening was measured in solution or in a frozen solution in the presence of amphiphiles.
ESTHER : Ranaldi_2010_Biochemistry_49_2140
PubMedSearch : Ranaldi_2010_Biochemistry_49_2140
PubMedID: 20136147

Title : Two cutinase-like proteins secreted by Mycobacterium tuberculosis show very different lipolytic activities reflecting their physiological function - Schue_2010_FASEB.J_24_1893
Author(s) : Schue M , Maurin D , Dhouib R , Bakala N'Goma JC , Delorme V , Lambeau G , Carriere F , Canaan S
Ref : FASEB Journal , 24 :1893 , 2010
Abstract : Cutinases are extracellular enzymes that are able to degrade cutin, a polyester protecting plant leaves and many kinds of lipids. Although cutinases are mainly found in phytopathogenic fungi or bacteria, 7 genes related to the cutinase family have been predicted in the genome of Mycobacterium tuberculosis. These genes may encode proteins that are involved in the complex lipid metabolism of the bacterium. Here, we report on the biochemical characterization of two secreted proteins of M. tuberculosis, Rv1984c and Rv3452, belonging to the cutinase family. Although their amino acid sequence shows 50% identity with that of the well-characterized cutinase from Fusarium solani pisi, and a high level of homology has been found to exist between these two enzymes, they show distinct substrate specificities. Rv1984c preferentially hydrolyzes medium-chain carboxylic esters and monoacylglycerols, whereas Rv3452 behaves like a phospholipase A(2), and it is able to induce macrophage lysis. The tetrahydrolipstatin inhibitor, a specific lipase inhibitor, abolishes the activity of both enzymes. Site-directed mutagenesis was performed to identify the catalytic triad of Rv1984c. Structural models for Rv1984c and Rv3452 were built, based on the crystal structure of F. solani cutinase, with a view to investigating the contribution of specific residues to the substrate specificity. Our findings open new prospects for investigating the physiological roles of cutinase-like proteins in the lipid metabolism and virulence of M. tuberculosis.
ESTHER : Schue_2010_FASEB.J_24_1893
PubMedSearch : Schue_2010_FASEB.J_24_1893
PubMedID: 20103719
Gene_locus related to this paper: myctu-cutas1 , myctu-RV3452

Title : A monoacylglycerol lipase from Mycobacterium smegmatis Involved in bacterial cell interaction - Dhouib_2010_J.Bacteriol_192_4776
Author(s) : Dhouib R , Laval F , Carriere F , Daffe M , Canaan S
Ref : Journal of Bacteriology , 192 :4776 , 2010
Abstract : MSMEG_0220 from Mycobacterium smegmatis, the ortholog of the Rv0183 gene from M. tuberculosis, recently identified and characterized as encoding a monoacylglycerol lipase, was cloned and expressed in Escherichia coli. The recombinant protein (rMSMEG_0220), which exhibits 68% amino acid sequence identity with Rv0183, showed the same substrate specificity and similar patterns of pH-dependent activity and stability as the M. tuberculosis enzyme. rMSMEG_0220 was found to hydrolyze long-chain monoacylglycerol with a specific activity of 143 +/- 6 U mg(-1). Like Rv0183 in M. tuberculosis, MSMEG_0220 was found to be located in the cell wall. To assess the in vivo role of the homologous proteins, an MSMEG_0220 disrupted mutant of M. smegmatis (MsDelta0220) was produced. An intriguing change in the colony morphology and in the cell interaction, which were partly restored in the complemented mutant containing either an active (ComMsDelta0220) or an inactive (ComMsDelta0220S111A) enzyme, was observed. Growth studies performed in media supplemented with monoolein showed that the ability of both MsDelta0220 and ComMsDelta0220S111A to grow in the presence of this lipid was impaired. Moreover, studies of the antimicrobial susceptibility of the MsDelta0220 strain showed that this mutant is more sensitive to rifampin and more resistant to isoniazid than the wild-type strain, pointing to a critical structural role of this enzyme in mycobacterial physiology, in addition to its function in the hydrolysis of exogenous lipids.
ESTHER : Dhouib_2010_J.Bacteriol_192_4776
PubMedSearch : Dhouib_2010_J.Bacteriol_192_4776
PubMedID: 20601476
Gene_locus related to this paper: mycs2-a0qnz7

Title : Continuous measurement of galactolipid hydrolysis by pancreatic lipolytic enzymes using the pH-stat technique and a medium chain monogalactosyl diglyceride as substrate - Amara_2009_Biochim.Biophys.Acta_1791_983
Author(s) : Amara S , Lafont D , Fiorentino B , Boullanger P , Carriere F , de Caro A
Ref : Biochimica & Biophysica Acta , 1791 :983 , 2009
Abstract : Galactolipids are the main lipids from plants and galactolipases play a major role in their metabolism. These enzymes were however poorly studied so far and only few assays have been developed. A specific and continuous galactolipase assay using synthetic medium chain monogalactosyl diacylglycerol (MGDG) as substrate was developed using the pH-stat technique and recombinant human (rHPLRP2) and guinea pig (rGPLRP2) pancreatic lipase-related protein 2 as model enzymes. PLRP2s are the main enzymes involved in the digestion of galactolipids in the gastrointestinal tract. Monogalactosyl di-octanoylglycerol was mixed with bile salt solutions by sonication to form a micellar substrate before launching the assay. The nature of the bile salt and the bile salt to MGDG ratio were found to significantly affect the rate of MGDG hydrolysis by rHPLRP2 and rGPLRP2. The maximum galactolipase activity of both enzymes was recorded with sodium deoxycholate (NaDC) and at a NaDC to MGDG ratio of 1.33 and at basic pH values (8.0-9.0). The maximum rates of hydrolysis were obtained using a MGDG concentration of 10(-2) M and calcium chloride was found to be not necessary to obtain the maximum of activity. Under these conditions, the maximum turnovers of rGPLRP2 and rHPLRP2 on mixed NaDC/MGDG micelles were found to be 8000+/-500 and 2800+/-60 micromol/min/mg (U/mg), respectively. These activities are in the same order of magnitude as the activities on triglycerides of lipases and they are the highest specific activities ever reported for galactolipases. For the sake of comparison, the hydrolysis of mixed bile salt/MGDG micelles was also tested using other pancreatic lipolytic enzymes and only native and recombinant human carboxyl ester hydrolase were found to display significant but lower activities (240+/-17 and 432+/-62 U/mg, respectively) on MGDG.
ESTHER : Amara_2009_Biochim.Biophys.Acta_1791_983
PubMedSearch : Amara_2009_Biochim.Biophys.Acta_1791_983
PubMedID: 19447192
Gene_locus related to this paper: cavpo-2plrp , human-PNLIPRP2

Title : Enhanced susceptibility to pancreatitis in severe hypertriglyceridaemic lipoprotein lipase-deficient mice and agonist-like function of pancreatic lipase in pancreatic cells - Wang_2009_Gut_58_422
Author(s) : Wang Y , Sternfeld L , Yang F , Rodriguez JA , Ross C , Hayden MR , Carriere F , Liu G , Hofer W , Schulz I
Ref : Gut , 58 :422 , 2009
Abstract : BACKGROUND AND AIMS: Recurrent pancreatitis is a common complication of severe hypertriglyceridaemia in patients with various gene mutations in lipoprotein lipase (LPL) or apolipoprotein CII. However, the exact pathogenetic mechanism has not yet been defined. METHODS: Susceptibility to pancreatitis in LPL-deficient mice was compared with that of wild-type mice after intraperitoneal injections of caerulein by determination of amylase release and pancreatic pathological scores. The effect of chylomicrons and fatty acids on enzyme release, Ca(2+) signalling and cell injury in isolated pancreatic acinar cells from wild-type and LPL-deficient mice was investigated. RESULTS: Caerulein induced higher levels of serum amylase and more severe inflammation in the pancreas of LPL-deficient mice than in wild-type mice. Addition of free fatty acids or chylomicrons to isolated pancreatic acinar cells led to the release of amylase and caused cell injury at higher concentrations. The effect of chylomicrons was partially blocked by orlistat, an inhibitor of pancreatic lipase. These results suggest that increased concentrations of free fatty acids from chylomicron hydrolysis by pancreatic lipase can induce acinar cell injury. Surprisingly, pancreatic lipase, whether in its active or inactive state could act like an agonist by inducing amylase secretion without cell injury. It caused an increase in cGMP levels and conversion of cell-damaging sustained elevations of [Ca(2+)] to normal Ca(2+) oscillations. CONCLUSIONS: LPL-deficient mice with severe hypertriglyceridaemia display enhanced susceptibility to acute pancreatitis. High levels of chylomicrons and free fatty acids result in pancreatic cell injury. Pancreatic lipase has a dual effect: generating free fatty acids from chylomicrons and preventing Ca(2+) overload in pancreatic acinar cells.
ESTHER : Wang_2009_Gut_58_422
PubMedSearch : Wang_2009_Gut_58_422
PubMedID: 18936103

Title : First evidence for the salt-dependent folding and activity of an esterase from the halophilic archaea Haloarcula marismortui - Muller-Santos_2009_Biochim.Biophys.Acta_1791_719
Author(s) : Muller-Santos M , de Souza EM , de Oliveira Pedrosa F , Mitchell DA , Longhi S , Carriere F , Canaan S , Krieger N
Ref : Biochimica & Biophysica Acta , 1791 :719 , 2009
Abstract : A gene encoding an esterase from Haloarcula marismortui, a halophilic archaea from the Dead Sea, was cloned, expressed in Escherichia coli, and the recombinant protein (Hm EST) was biochemically characterized. The enzymatic activity of Hm EST was shown to exhibit salt dependence through salt-dependent folding. Hm EST exhibits a preference for short chain fatty acids and monoesters. It is inhibited by phenylmethylsulfonyl fluoride, diethyl-p-nitrophenyl phosphate, and 5-methoxy-3-(4-phenoxyphenyl)-3H-[1,3,4]oxadiazol-2-one, confirming the conclusion from sequence alignments that Hm EST is a serine carboxylesterase belonging to the hormone-sensitive lipase family. The activity of Hm EST is optimum in the presence of 3 M KCl and no activity was detected in the absence of salts. Far-UV circular dichroism showed that Hm EST is totally unfolded in salt-free medium and secondary structure appears in the presence of 0.25-0.5 M KCl. After salt depletion, the protein was able to recover 60% of its initial activity when 2 M KCl was added. A 3D model of Hm EST was built and its surface properties were analyzed, pointing to an enrichment in acidic residues paralleled by a depletion in basic residues. This peculiar charge repartition at the protein surface supports a better stability of the protein in a high salt environment.
ESTHER : Muller-Santos_2009_Biochim.Biophys.Acta_1791_719
PubMedSearch : Muller-Santos_2009_Biochim.Biophys.Acta_1791_719
PubMedID: 19303051

Title : The role of free fatty acids, pancreatic lipase and Ca+ signalling in injury of isolated acinar cells and pancreatitis model in lipoprotein lipase-deficient mice - Yang_2009_Acta.Physiol.(Oxf)_195_13
Author(s) : Yang F , Wang Y , Sternfeld L , Rodriguez JA , Ross C , Hayden MR , Carriere F , Liu G , Schulz I
Ref : Acta Physiol (Oxf) , 195 :13 , 2009
Abstract : AIM AND METHODS: Recurrent pancreatitis is a common complication of severe hypertriglyceridaemia (HTG) often seen in patients carrying various gene mutations in lipoprotein lipase (LPL). This study investigates a possible pathogenic mechanism of cell damage in isolated mouse pancreatic acinar cells and of pancreatitis in LPL-deficient and in wild type mice. RESULTS: Addition of free fatty acids (FFA) or of chylomicrons to isolated pancreatic acinar cells caused stimulation of amylase release, and at higher concentrations it also caused cell damage. This effect was decreased in the presence of the lipase inhibitor orlistat. Surprisingly, pancreatic lipase whether in its active or inactive state could act like an agonist by inducing amylase secretion, increasing cellular cGMP levels and converting cell damaging sustained elevations of [Ca(2+)](cyt) to normal Ca(2+) oscillations. Caerulein increases the levels of serum amylase and caused more severe inflammation in the pancreas of LPL-deficient mice than in wild type mice. CONCLUSION: We conclude that high concentrations of FFA as present in the plasma of LPL-deficient mice and in patients with HTG lead to pancreatic cell damage and are high risk factors for the development of acute pancreatitis. In addition to its enzymatic effect which leads to the generation of cell-damaging FFA from triglycerides, pancreatic lipase also prevents Ca(2+) overload in pancreatic acinar cells and, therefore, counteracts cell injury.
ESTHER : Yang_2009_Acta.Physiol.(Oxf)_195_13
PubMedSearch : Yang_2009_Acta.Physiol.(Oxf)_195_13
PubMedID: 18983441

Title : Validation of lipolysis product extraction from aqueous\/biological samples, separation and quantification by thin-layer chromatography with flame ionization detection analysis using O-cholesteryl ethylene glycol as a new internal standard - Cavalier_2009_J.Chromatogr.A_1216_6543
Author(s) : Cavalier JF , Lafont D , Boullanger P , Houisse D , Giallo J , Ballester JM , Carriere F
Ref : Journal of Chromatography A , 1216 :6543 , 2009
Abstract : A general and easily accessible method for the extraction followed by the simultaneous separation and quantitative determination of triacylglycerols, diacylglycerols, monoacylglycerols and free fatty acids has been improved and optimized based on existing protocols using liquid-phase extraction and thin-layer chromatography coupled to flame ionization detection (TLC/FID Iatroscan). After lipid extraction in the presence of a suitable new synthetic internal standard, namely CholE1, a single elution step using n-heptane/diethyl ether/formic acid (55:45:1, v/v/v) was applied. This method was validated in line with international bioanalytical method validation guidelines using two different matrix systems: purified water and human gastro-intestinal fluid. Overall, the assay was found to have high levels of precision with coefficients of variation ranging from 1.48% to 11.0% and accuracy ranging from -13.3% to +5.79% RE. The confidence limits of the lipid mean recovery rates varied between 89.9% and 104%. This method is therefore highly suitable for quantifying the lipolysis products generated in vitro during the hydrolysis of various fats and oils by digestive lipases, as well as those collected from the gastro-intestinal tract in the course of human clinical studies on lipid digestion.
ESTHER : Cavalier_2009_J.Chromatogr.A_1216_6543
PubMedSearch : Cavalier_2009_J.Chromatogr.A_1216_6543
PubMedID: 19671473

Title : Lid opening and unfolding in human pancreatic lipase at low pH revealed by site-directed spin labeling EPR and FTIR spectroscopy - Ranaldi_2009_Biochemistry_48_630
Author(s) : Ranaldi S , Belle V , Woudstra M , Rodriguez J , Guigliarelli B , Sturgis J , Carriere F , Fournel A
Ref : Biochemistry , 48 :630 , 2009
Abstract : The structural changes induced in human pancreatic lipase (HPL) by lowering the pH were investigated using a combined approach involving the use of site-directed spin labeling coupled to electron paramagnetic resonance (SDSL-EPR) and Fourier transform infrared (ATR-FTIR) spectroscopy. The secondary structure of HPL observed with ATR-FTIR spectroscopy was found to be stable in the pH range of 3.0-6.5, where HPL remained active. Using a spin-label introduced into the lid of HPL at position 249, a reversible opening of the lid controlling the access to the active site was observed by EPR spectroscopy in the pH range of 3.0-5.0. In the same pH range, some structural changes were also found to occur outside the lid in a peptide stretch located near catalytic aspartate 176, using a spin-label introduced at position 181. Below pH 3.0, ATR-FTIR measurements indicated that HPL had lost most of its secondary structure. At these pH levels, the loss of enzyme activity was irreversible and the ability of HPL to bind to lipid emulsions was abolished. The EPR spectrum of the spin-label introduced at position 181, which was typical of a spin-label having a high mobility, confirmed the drastic structural change undergone by HPL in this particular region. The EPR spectrum of the spin-label at position 249 indicated, however, that the environment of this residue within the lid was not affected at pH 3.0 in comparison with that observed in the pH range of 3.0-5.0. This finding suggests that the disulfide bridge between the hinges of the lid kept the secondary structure of the lid intact, whereas the HPL was completely unfolded.
ESTHER : Ranaldi_2009_Biochemistry_48_630
PubMedSearch : Ranaldi_2009_Biochemistry_48_630
PubMedID: 19113953

Title : In vitro comparative study of three pancreatic enzyme preparations: dissolution profiles, active enzyme release and acid stability - Aloulou_2008_Aliment.Pharmacol.Ther_27_283
Author(s) : Aloulou A , Puccinelli D , Sarles J , Laugier R , Leblond Y , Carriere F
Ref : Aliment Pharmacol Ther , 27 :283 , 2008
Abstract : BACKGROUND: Various pancreatic enzyme preparations are used for the treatment of pancreatic insufficiency but their bioequivalence is often unknown. AIM: To determine in vitro the pH-dependent release and acid resistance of enzymes from three commercially available pancreatin capsules, two containing enteric-coated (Creon 25000; Eurobiol 25000) and one uncoated (Eurobiol 12500) microspheres.
METHODS: Dissolution experiments were performed at pH values ranging from 4.0 to 5.8. Lipase, chymotrypsin and amylase activities were measured in the solution as a function of time.
RESULTS: Eurobiol 25000 started to release its enzymes significantly at pH 5.0 (t(1/2) = 71 min), whereas the enzymes from Creon 25000 were only released at higher pH value (5.4; t(1/2) = 49.2 min). Unlike chymotrypsin, lipase and amylase were highly sensitive to acidic conditions at the lowest pH values tested. Both enzymes were also found to be sensitive to proteolytic inactivation at the highest pH values tested. Overall, Eurobiol 25000 released higher amounts of active amylase and lipase than Creon 25000 at the pH values usually found in duodenal contents. The uncoated Eurobiol 12500 preparation was, however, the only one that could immediately release rather high levels of active chymotrypsin and lipase at low pH (4.5). CONCLUSION: These findings suggest that pH-sensitive enteric-coated pancreatin products containing similar amounts of enzymes might not be bioequivalent depending on the pH of duodenal contents.
ESTHER : Aloulou_2008_Aliment.Pharmacol.Ther_27_283
PubMedSearch : Aloulou_2008_Aliment.Pharmacol.Ther_27_283
PubMedID: 17973644

Title : An analytical method for determining relative specificities for sequential reactions catalyzed by the same enzyme: application to the hydrolysis of triacylglycerols by lipases - Mitchell_2008_J.Biotechnol_133_343
Author(s) : Mitchell DA , Rodriguez JA , Carriere F , Baratti J , Krieger N
Ref : J Biotechnol , 133 :343 , 2008
Abstract : We propose a model for the sequential hydrolysis of ester bonds of triacylglycerols by lipases and use it as the basis for an analytical method for determining the relative specificity of the lipase for the various substrates with which it can react, when the substrates occur simultaneously in a single reaction system. We then apply the method to our own data and literature data involving the hydrolysis of triacylglycerols by lipases. Our model is able to fit well to most of the reaction profiles, enabling the estimation of relative specificities. We discuss the limitations and potential applications of our method.
ESTHER : Mitchell_2008_J.Biotechnol_133_343
PubMedSearch : Mitchell_2008_J.Biotechnol_133_343
PubMedID: 18068847

Title : Gastric lipase: an extremophilic interfacial enzyme with medical applications -
Author(s) : Aloulou A , Carriere F
Ref : Cell Mol Life Sciences , 65 :851 , 2008
PubMedID: 18213443

Title : Structure of human pancreatic lipase-related protein 2 with the lid in an open conformation - Eydoux_2008_Biochemistry_47_9553
Author(s) : Eydoux C , Spinelli S , Davis TL , Walker JR , Seitova A , Dhe-Paganon S , de Caro A , Cambillau C , Carriere F
Ref : Biochemistry , 47 :9553 , 2008
Abstract : Access to the active site of pancreatic lipase (PL) is controlled by a surface loop, the lid, which normally undergoes conformational changes only upon addition of lipids or amphiphiles. Structures of PL with their lids in the open and functional conformation have required cocrystallization with amphiphiles. Here we report two crystal structures of wild-type and unglycosylated human pancreatic lipase-related protein 2 (HPLRP2) with the lid in an open conformation in the absence of amphiphiles. These structures solved independently are strikingly similar, with some residues of the lid being poorly defined in the electron-density map. The open conformation of the lid is however different from that previously observed in classical liganded PL, suggesting different kinetic properties for HPLRP2. Here we show that the HPLRP2 is directly inhibited by E600, does not present interfacial activation, and acts preferentially on substrates forming monomers or small aggregates (micelles) dispersed in solution like monoglycerides, phospholipids and galactolipids, whereas classical PL displays reverse properties and a high specificity for unsoluble substrates like triglycerides and diglycerides forming oil-in-water interfaces. These biochemical properties imply that the lid of HPLRP2 is likely to spontaneously adopt in solution the open conformation observed in the crystal structure. This open conformation generates a large cavity capable of accommodating the digalactose polar head of galactolipids, similar to that previously observed in the active site of the guinea pig PLRP2, but absent from the classical PL. Most of the structural and kinetic properties of HPLRP2 were found to be different from those of rat PLRP2, the structure of which was previously obtained with the lid in a closed conformation. Our findings illustrate the essential role of the lid in determining the substrate specificity and the mechanism of action of lipases.
ESTHER : Eydoux_2008_Biochemistry_47_9553
PubMedSearch : Eydoux_2008_Biochemistry_47_9553
PubMedID: 18702514
Gene_locus related to this paper: human-PNLIPRP2

Title : Lipolytic enzymes in Mycobacterium tuberculosis - Cotes_2008_Appl.Microbiol.Biotechnol_78_741
Author(s) : Cotes K , Bakala N'goma J C , Dhouib R , Douchet I , Maurin D , Carriere F , Canaan S
Ref : Applied Microbiology & Biotechnology , 78 :741 , 2008
Abstract : Mycobacterium tuberculosis is a bacterial pathogen that can persist for decades in an infected patient without causing a disease. In vivo, the tubercle bacillus present in the lungs store triacylglycerols in inclusion bodies. The same process can be observed in vitro when the bacteria infect adipose tissues. Indeed, before entering in the dormant state, bacteria accumulate lipids originating from the host cell membrane degradation and from de novo synthesis. During the reactivation phase, these lipids are hydrolysed and the infection process occurs. The degradation of both extra and intracellular lipids can be directly related to the presence of lipolytic enzymes in mycobacteria, which have been ignored during a long period particularly due to the difficulties to obtain a high expression level of these enzymes in M. tuberculosis. The completion of the M. tuberculosis genome offered new opportunity to this kind of study. The aim of this review is to focus on the recent results obtained in the field of mycobacterium lipolytic enzymes and although no experimental proof has been shown in vivo, it is tempting to speculate that these enzymes could be involved in the virulence and pathogenicity processes.
ESTHER : Cotes_2008_Appl.Microbiol.Biotechnol_78_741
PubMedSearch : Cotes_2008_Appl.Microbiol.Biotechnol_78_741
PubMedID: 18309478

Title : Lipolysis of the semi-solid self-emulsifying excipient Gelucire 44\/14 by digestive lipases - Fernandez_2008_Biochim.Biophys.Acta_1781_367
Author(s) : Fernandez S , Rodier JD , Ritter N , Mahler B , Demarne F , Carriere F , Jannin V
Ref : Biochimica & Biophysica Acta , 1781 :367 , 2008
Abstract : Gelucire 44/14 is a semi-solid self-emulsifying excipient used for the oral delivery of poorly water-soluble drugs. It is composed of C8-C18 acylglycerols and PEG-32 esters, all of which are potential substrates for digestive lipases. Here we studied the lipolysis of Gelucire 44/14 by porcine pancreatic extracts, human pancreatic juice and several purified digestive lipases. Human pancreatic lipase (HPL), the main lipase involved in the digestion of triacylglycerols, did not show any significant activity on Gelucire 44/14 or on either of its individual compounds, C8-C18 acylglycerols and PEG-32 esters. Other pancreatic lipases such as human pancreatic lipase-related protein 2 (HPLRP2) showed low activity on Gelucire 44/14 although the highest activity of HPLRP2 was that observed on the C8-C18 acylglycerol fraction, which accounts for 20% (w/w) of Gelucire 44/14. In addition, HPLRP2 showed low activities on the PEG-32 esters, whether these were tested individually or mixed together. Carboxyl ester hydrolase (CEH) showed high activity on Gelucire 44/14, and the highest activities of CEH were those recorded on the total PEG-32 ester fraction and on each individual PEG-32 ester, except for PEG-32 monostearate. The highest activity of all the enzymes tested was that of dog gastric lipase (DGL) on Gelucire 44/14, although DGL showed low activity on the PEG-32 ester fraction and on each individual PEG-32 ester. We compared the lipolysis of Gelucire 44/14 with that of Labrasol, another self-emulsifying excipient, which is liquid at room temperature. Human pancreatic juice showed similar rates of activity on both Gelucire 44/14 and Labrasol. This finding means that these excipients are hydrolyzed in vivo during pancreatic digestion, mainly by CEH in the case of Gelucire 44/14 and by both HPLRP2 and CEH in that of Labrasol, whereas HPL showed very low activities on each of these two excipients. This is the first time the effects of PEG and acyl chain length on the lipolytic activity of digestive lipases on PEG esters have been investigated.
ESTHER : Fernandez_2008_Biochim.Biophys.Acta_1781_367
PubMedSearch : Fernandez_2008_Biochim.Biophys.Acta_1781_367
PubMedID: 18571509

Title : Novel chromatographic resolution of chiral diacylglycerols and analysis of the stereoselective hydrolysis of triacylglycerols by lipases - Rodriguez_2008_Anal.Biochem_375_196
Author(s) : Rodriguez JA , Mendoza LD , Pezzotti F , Vanthuyne N , Leclaire J , Verger R , Buono G , Carriere F , Fotiadu F
Ref : Analytical Biochemistry , 375 :196 , 2008
Abstract : In the present study, we propose a general and accessible method for the resolution of enantiomeric 1,2-sn- and 2,3-sn-diacylglycerols based on derivatization by isocyanates, which can be easily used routinely by biochemists to evaluate the stereopreferences of lipases in a time course of triacylglycerol (TAG) hydrolysis. Diacylglycerol (DAG) enantiomers were transformed into carbamates using achiral and commercially available reagents. Excellent separation and resolution factors were obtained for diacylglycerols present in lipolysis reaction mixtures. This analytical method was then applied to investigate the stereoselectivity of three model lipases (porcine pancreatic lipase, PPL; lipase from Rhizomucor miehei, MML; and recombinant dog gastric lipase, rDGL) in the time course of hydrolysis of prochiral triolein as a substrate. From the measurements of the diglyceride enantiomeric excess it was confirmed that PPL was not stereospecific (position sn-1 vs sn-3 of triolein), whereas MML and rDGL preferentially hydrolyzed the ester bond at position sn-1 and sn-3, respectively. The enantiomeric excess of DAGs was not constant with time, decreasing with the course of hydrolysis. This was due to the fact that DAGs can be products of the stereospecific hydrolysis of TAGs and substrates for stereospecific hydrolysis into monoacylglycerols.
ESTHER : Rodriguez_2008_Anal.Biochem_375_196
PubMedSearch : Rodriguez_2008_Anal.Biochem_375_196
PubMedID: 18162167

Title : Characterization of typo-, regio-, and stereo-selectivities of babaco latex lipase in aqueous and organic media - Cambon_2008_Biotechnol.Lett_30_769
Author(s) : Cambon E , Rodriguez JA , Pina M , Arondel V , Carriere F , Turon F , Ruales J , Villeneuve P
Ref : Biotechnol Lett , 30 :769 , 2008
Abstract : The unripe fruit of babaco (Vasconcellea x heilbornii; syn. Carica pentagona) contains a latex, similar to that in Carica papaya, which exhibits lipolytic activity. Herein, the regioselectivity, stereoselectivity and typoselectivity in both hydrolysis and acyltransfer reactions of babaco latex lipases were studied and compared to those of Carica papaya latex. In hydrolysis, both biocatalysts are 1,3-regioselective with ratios for 1,2-2,3-diacylglycerols/1,3-diacylglycerol of 6.5 and 21 for babaco and papaya, respectively. In contrast, papaya latex had a slight sn-3 stereopreference. Babaco latex displayed a higher activity on triacylglycerols with short chain and unsaturated fatty acids.
ESTHER : Cambon_2008_Biotechnol.Lett_30_769
PubMedSearch : Cambon_2008_Biotechnol.Lett_30_769
PubMedID: 18038267

Title : Development of an indirect method for measuring porcine pancreatic lipase in human duodenal fluid - Tuvignon_2008_Anal.Biochem_383_289
Author(s) : Tuvignon N , Abousalham A , Tocques F , De Caro J , de Caro A , Laugier R , Carriere F
Ref : Analytical Biochemistry , 383 :289 , 2008
Abstract : Patients with exocrine pancreatic insufficiency are usually treated with porcine pancreatic enzymes but the bioavailability of these enzymes in the gut remains a matter of discussion. In order to determine the duodenal availability of porcine pancreatic lipase (PPL) present in pancreatic extracts (PE) taken orally, we developed a method for quantifying PPL in samples containing both PPL and human pancreatic lipase (HPL). Total pancreatic lipase activity measurements using the pH-stat technique and tributyrin as substrate were combined with an HPL-specific ELISA. Based on the known specific activity of the purified HPL, its activity was deduced from the ELISA measurements, and the PPL activity was obtained by subtracting the HPL activity from the total pancreatic lipase activity. This assay was established and validated using various samples containing pure PPL and recombinant HPL or PE, mixed or not with human duodenal juice. Samples collected in vivo from patients treated with PE were also tested. It was found that PPL did not affect the HPL ELISA, and the indirect PPL assay gave a measurement accuracy of 6.6% with the samples containing pure PPL and 10% with those containing PE. This assay was also used successfully to discriminate between PPL and the endogenous HPL present in the duodenal contents of patients with severe pancreatic insufficiency treated with PE. This method might provide a useful means of assessing the availability of PEs at their site of action, in the absence of a PPL-specific ELISA.
ESTHER : Tuvignon_2008_Anal.Biochem_383_289
PubMedSearch : Tuvignon_2008_Anal.Biochem_383_289
PubMedID: 18814836

Title : An analytical method for determining relative specificities for sequential reactions catalyzed by the same enzyme: general formulation - Mitchell_2008_Biochim.Biophys.Acta_1784_705
Author(s) : Mitchell DA , Carriere F , Krieger N
Ref : Biochimica & Biophysica Acta , 1784 :705 , 2008
Abstract : We present a general formulation of a model that can be used to analyze reaction profiles in systems in which a single enzyme catalyzes several sequential reactions with the same molecular backbone. The analysis of these so-called "repeated-attack systems" allows estimation of the specificities that the enzyme has for the various intermediate substrates that appear in the reaction mixture, relative to the specificity that it has for the initial substrate. Our analytical method has the important advantage that it is not affected by competitive or uncompetitive inhibition, nor by denaturation of the enzyme during the reaction. We carry out case studies in three different systems, the lipase-catalyzed alcoholysis of triacylglycerols, the phytase-catalyzed removal of phosphate groups from phytic acid and the beta-amylase-catalyzed removal of maltose units from maltoheptaose. Our model fits well to all reaction profiles in which the phenomenon of processivity does not occur. It can therefore be used as a general tool for characterizing the relative specificities of "repeated-attack enzymes".
ESTHER : Mitchell_2008_Biochim.Biophys.Acta_1784_705
PubMedSearch : Mitchell_2008_Biochim.Biophys.Acta_1784_705
PubMedID: 18302946

Title : Occurrence of pancreatic lipase-related protein-2 in various species and its relationship with herbivore diet - De Caro_2008_Comp.Biochem.Physiol.B.Biochem.Mol.Biol_150_1
Author(s) : De Caro J , Eydoux C , Cherif S , Lebrun R , Gargouri Y , Carriere F , de Caro A
Ref : Comparative Biochemistry & Physiology B Biochem Mol Biol , 150 :1 , 2008
Abstract : The occurrence of classical pancreatic lipase (PL) and pancreatic lipase-related proteins 1 and 2 (PLRP1s and 2) in the pancreas of ten mammalian species (humans, pig, rat, guinea pig, coypu, rabbit, horse, ox, goat and sheep) and two bird species (ostrich and turkey) was investigated. The lipases were purified from delipidated pancreas and identified based on the results of Western blotting analysis with anti-human PLRP2 serum, the catalytic properties and N-terminal microsequencing data. PLRP2s were detected in the pancreas of monogastric herbivorous animals (guinea pig, coypu, rabbit and horse) but not in that of ruminant herbivorous animals (ox, goat and sheep). The pancreas of carnivorous animals (dogs and cats) does not have any detectable PLRP2, but contains high levels of PL and PLRP1. By contrast, the pancreas of omnivorous animals (humans and rats) contains PL, PLRP1 and PLRP2, with the exception of porcine pancreas, where no PLRP2 was detected. In the case of bird (ostrich and turkey) pancreases, only classical PL was detected. The substrate specificity of PLRP2s was investigated using phospholipid micelles and synthetic monomolecular galactolipid films. Like human PLRP2, rabbit and horse PLRP2s are galactolipases. In polygastric herbivorous animals (ruminants), however, galactolipids are hydrolyzed via microbial enzymatic processes (involving galactolipases). The absence of galactolipids in carnivorous animals' diet may explain why no PLRP2s were detected here in the pancreas of these species.
ESTHER : De Caro_2008_Comp.Biochem.Physiol.B.Biochem.Mol.Biol_150_1
PubMedSearch : De Caro_2008_Comp.Biochem.Physiol.B.Biochem.Mol.Biol_150_1
PubMedID: 18328758

Title : A comparative study on two fungal lipases from Thermomyces lanuginosus and Yarrowia lipolytica shows the combined effects of detergents and pH on lipase adsorption and activity - Aloulou_2007_Biochim.Biophys.Acta_1771_1446
Author(s) : Aloulou A , Puccinelli D , de Caro A , Leblond Y , Carriere F
Ref : Biochimica & Biophysica Acta , 1771 :1446 , 2007
Abstract : The effects of various detergents and pH on the interfacial binding and activity of two fungal lipases from Yarrowia lipolytica (YLLIP2) and Thermomyces lanuginosus (TLL) were investigated using trioctanoin emulsions as well as monomolecular films spread at the air-water interface. Contrary to TLL, YLLIP2 was found to be more sensitive than TLL to interfacial denaturation but it was protected by detergent monomers and lowering the temperature. At pH 7.0, both the interfacial binding and the activities on trioctanoin of YLLIP2 and TLL were inhibited by sodium taurodeoxycholate (NaTDC). At pH 6.0, however, YLLIP2 remained active on trioctanoin in the presence of NaTDC, whereas TLL did not. YLLIP2 activity on trioctanoin was associated with strong interfacial binding of the enzyme to trioctanoin emulsion, whereas TLL was mostly detected in the water phase. The combined effects of bile salts and pH on lipase activity were therefore enzyme-dependent. YLLIP2 binds more strongly than TLL at oil-water interfaces at low pH when detergents are present. These findings are particularly important for lipase applications, in particular for enzyme replacement therapy in patients with pancreatic enzyme insufficiency since high detergent concentrations and highly variable pH values can be encountered in the GI tract.
ESTHER : Aloulou_2007_Biochim.Biophys.Acta_1771_1446
PubMedSearch : Aloulou_2007_Biochim.Biophys.Acta_1771_1446
PubMedID: 18022403
Gene_locus related to this paper: humla-1lipa

Title : Probing the opening of the pancreatic lipase lid using site-directed spin labeling and EPR spectroscopy - Belle_2007_Biochemistry_46_2205
Author(s) : Belle V , Fournel A , Woudstra M , Ranaldi S , Prieri F , Thome V , Currault J , Verger R , Guigliarelli B , Carriere F
Ref : Biochemistry , 46 :2205 , 2007
Abstract : Access to the active site of human pancreatic lipase (HPL) is controlled by a surface loop (the lid) that undergoes a conformational change in the presence of amphiphiles and lipid substrate. The question of how and when the lid opens still remains to be elucidated, however. A paramagnetic probe was covalently bound to the lid via the D249C mutation, and electron paramagnetic resonance (EPR) spectroscopy was used to monitor the conformational change in solution. Two EPR spectral components, corresponding to distinct mobilities of the probe, were attributed to the closed and open conformations of the HPL lid, based on experiments performed with the E600 inhibitor. The open conformation of the lid was observed in solution at supramicellar bile salt concentrations. Colipase alone did not induce lid opening but increased the relative proportions of the open conformation in the presence of bile salts. The opening of the lid was found to be a reversible process. Using various colipase to lipase molar ratios, a correlation between the proportion of the open conformation and the catalytic activity of HPL was observed.
ESTHER : Belle_2007_Biochemistry_46_2205
PubMedSearch : Belle_2007_Biochemistry_46_2205
PubMedID: 17269661

Title : Characterization of an exported monoglyceride lipase from Mycobacterium tuberculosis possibly involved in the metabolism of host cell membrane lipids - Cotes_2007_Biochem.J_408_417
Author(s) : Cotes K , Dhouib R , Douchet I , Chahinian H , de Caro A , Carriere F , Canaan S
Ref : Biochemical Journal , 408 :417 , 2007
Abstract : The Rv0183 gene of the Mycobacterium tuberculosis H37Rv strain, which has been implicated as a lysophospholipase, was cloned and expressed in Escherichia coli. The purified Rv0183 protein did not show any activity when lysophospholipid substrates were used, but preferentially hydrolysed monoacylglycerol substrates with a specific activity of 290 units x mg(-1) at 37 degrees C. Rv0183 hydrolyses both long chain di- and triacylglycerols, as determined using the monomolecular film technique, although the turnover was lower than with MAG (monoacyl-glycerol). The enzyme shows an optimum activity at pH values ranging from 7.5 to 9.0 using mono-olein as substrate and is inactivated by serine esterase inhibitors such as E600, PMSF and tetrahydrolipstatin. The catalytic triad is composed of Ser110, Asp226 and His256 residues, as confirmed by the results of site-directed mutagenesis. Rv0183 shows 35% sequence identity with the human and mouse monoglyceride lipases and well below 15% with the other bacterial lipases characterized so far. Homologues of Rv0183 can be identified in other mycobacterial genomes such as Mycobacterium bovis, Mycobacterium smegmatis, and even Mycobacterium leprae, which is known to contain a low number of genes involved in the replication process within the host cells. The results of immunolocalization studies performed with polyclonal antibodies raised against the purified recombinant Rv0183 suggested that the enzyme was present only in the cell wall and culture medium of M. tuberculosis. Our results identify Rv0183 as the first exported lipolytic enzyme to be characterized in M. tuberculosis and suggest that Rv0183 may be involved in the degradation of the host cell lipids.
ESTHER : Cotes_2007_Biochem.J_408_417
PubMedSearch : Cotes_2007_Biochem.J_408_417
PubMedID: 17784850
Gene_locus related to this paper: myctu-rv0183

Title : Comparative study on digestive lipase activities on the self emulsifying excipient Labrasol, medium chain glycerides and PEG esters - Fernandez_2007_Biochim.Biophys.Acta_1771_633
Author(s) : Fernandez S , Jannin V , Rodier JD , Ritter N , Mahler B , Carriere F
Ref : Biochimica & Biophysica Acta , 1771 :633 , 2007
Abstract : Labrasol is a lipid-based self-emulsifying excipient used in the preparation of lipophilic drugs intended for oral delivery. It is mainly composed of PEG esters and glycerides with medium acyl chains, which are potential substrates for digestive lipases. The hydrolysis of Labrasol by porcine pancreatic extracts, human pancreatic juice and several purified digestive lipases was investigated in the present study. Classical human pancreatic lipase (HPL) and porcine pancreatic lipase, which are the main lipases involved in the digestion of dietary triglycerides, showed very low levels of activity on the entire Labrasol excipient as well as on separated fractions of glycerides and PEG esters. On the other hand, gastric lipase, pancreatic lipase-related protein 2 (PLRP2) and carboxyl ester hydrolase (CEH) showed high specific activities on Labrasol. These lipases were found to hydrolyze the main components of Labrasol (PEG esters and monoglycerides) used as individual substrates, whereas these esters were found to be poor substrates for HPL. The lipolytic activity of pancreatic extracts and human pancreatic juice on Labrasol(R) is therefore mainly due to the combined action of CEH and PLRP2. These two pancreatic enzymes, together with gastric lipase, are probably the main enzymes involved in the in vivo lipolysis of Labrasol taken orally.
ESTHER : Fernandez_2007_Biochim.Biophys.Acta_1771_633
PubMedSearch : Fernandez_2007_Biochim.Biophys.Acta_1771_633
PubMedID: 17418634

Title : Further biochemical characterization of human pancreatic lipase-related protein 2 expressed in yeast cells - Eydoux_2007_J.Lipid.Res_48_1539
Author(s) : Eydoux C , De Caro J , Ferrato F , Boullanger P , Lafont D , Laugier R , Carriere F , de Caro A
Ref : J Lipid Res , 48 :1539 , 2007
Abstract : Recombinant human pancreatic lipase-related protein 2 (rHPLRP2) was produced in the protease A-deficient yeast Pichia pastoris. A major protein with a molecular mass of 50 kDa was purified from the culture medium using SP-Sepharose and Mono Q chromatography. The protein was found to be highly sensitive to the proteolytic cleavage of a peptide bond in the lid domain. The proteolytic cleavage process occurring in the lid affected both the lipase and phospholipase activities of rHPLRP2. The substrate specificity of the nonproteolyzed rHPLRP2 was investigated using pH-stat and monomolecular film techniques and various substrates (glycerides, phospholipids, and galactolipids). All of the enzyme activities were maximum at alkaline pH values and decreased in the pH 5-7 range corresponding to the physiological conditions occurring in the duodenum. rHPLRP2 was found to act preferentially on substrates forming small aggregates in solution (monoglycerides, egg phosphatidylcholine, and galactolipids) rather than on emulsified substrates such as triolein and diolein. The activity of rHPLRP2 on monogalactosyldiglyceride and digalactosyldiglyceride monomolecular films was determined and compared with that of guinea pig pancreatic lipase-related protein 2, which shows a large deletion in the lid domain. The presence of a full-length lid domain in rHPLRP2 makes it possible for enzyme activity to occur at higher surface pressures. The finding that the inhibition of nonproteolyzed rHPLRP2 by tetrahydrolipstatin and diethyl-p-nitrophenyl phosphate does not involve any bile salt requirements suggests that the rHPLRP2 lid adopts an open conformation in aqueous media.
ESTHER : Eydoux_2007_J.Lipid.Res_48_1539
PubMedSearch : Eydoux_2007_J.Lipid.Res_48_1539
PubMedID: 17401110
Gene_locus related to this paper: human-PNLIPRP2

Title : Purification and biochemical characterization of the LIP2 lipase from Yarrowia lipolytica - Aloulou_2007_Biochim.Biophys.Acta_1771_228
Author(s) : Aloulou A , Rodriguez JA , Puccinelli D , Mouz N , Leclaire J , Leblond Y , Carriere F
Ref : Biochimica & Biophysica Acta , 1771 :228 , 2007
Abstract : The LIP2 lipase from the yeast Yarrowia lipolytica (YLLIP2) was obtained from two genetically modified strains with multi-copies of the lip2 gene and further purified using gel filtration and cation exchange chromatography. Four YLLIP2 isoforms were identified and subjected to N-terminal amino-acid sequencing and mass spectrometry analysis. These isoforms differed in their glycosylation patterns and their molecular masses ranged from 36,874 to 38,481 Da, whereas the polypeptide mass was 33,385 Da. YLLIP2 substrate specificity was investigated using short (tributyrin), medium (trioctanoin) and long (olive oil) chain triglyceride substrates at various pH and bile salt concentrations, and compared with those of human gastric and pancreatic lipases. YLLIP2 was not inhibited by bile salts at micellar concentrations with any of the substrates tested, and maximum specific activities were found to be 10,760+/-115 U/mg on tributyrin, 16,920+/-480 U/mg on trioctanoin and 12,260+/-700 U/mg on olive oil at pH 6.0. YLLIP2 was found to be fairly stable and still active on long chain triglycerides (1590+/-430 U/mg) at pH 4.0, in the presence of bile salts. It is therefore a good candidate for use in enzyme replacement therapy as a means of treating pancreatic exocrine insufficiency.
ESTHER : Aloulou_2007_Biochim.Biophys.Acta_1771_228
PubMedSearch : Aloulou_2007_Biochim.Biophys.Acta_1771_228
PubMedID: 17270492

Title : Effect of nonionic surfactants on Rhizopus homothallicus lipase activity: a comparative kinetic study - Diaz_2007_Mol.Biotechnol_35_205
Author(s) : Diaz JC , Cordova J , Baratti J , Carriere F , Abousalham A
Ref : Mol Biotechnol , 35 :205 , 2007
Abstract : Based on amino-terminal sequencing and mass spectrometry data on the Rhizopus homothallicus lipase extracted using solid (SSF) and submerged state fermentation (SmF) methods, we previously established that the two enzymes were identical. Differences were observed, however, in terms of the specific activity of these lipases and their inhibition by diethyl p-nitrophenyl phosphate (E600). The specific activity of the SSF lipase (10,700 mumol/min/mg) was found to be 1.2-fold that of SmF lipase (8600 mumol/min/mg). These differences might be the result of residual Triton X-100 molecules interacting with the SSF lipase. To check this hypothesis, the SmF lipase was incubated with submicellar concentrations of Triton X-100. The specific activity of the lipase increased after this treatment, reaching similar values to those measured with the SSF lipase. Preincubating SSF and SmF lipases with E600 at a molar excess of 100 for 1 h resulted in 80% and 60% enzyme inhibition levels, respectively. When the SmF lipase was preincubated with Triton X-100 for 1 h at a concentration 100 times lower than the Triton X-100 critical micellar concentration, the inhibition of the lipase by E600 increased from 60% to 80%. These results suggest that residual detergent monomers interacting with the enzyme may affect the kinetic properties of the Rh. homothallicus lipase.
ESTHER : Diaz_2007_Mol.Biotechnol_35_205
PubMedSearch : Diaz_2007_Mol.Biotechnol_35_205
PubMedID: 17652784

Title : Assaying lipase activity from oil palm fruit (Elaeis guineensis Jacq.) mesocarp - Ngando_2006_Plant.Physiol.Biochem_44_611
Author(s) : Ngando Ebongue GF , Dhouib R , Carriere F , Amvam Zollo PH , Arondel V
Ref : Plant Physiol Biochem , 44 :611 , 2006
Abstract : The mesocarp of mature oil palm fruit undergoes intensive triglycerides hydrolysis upon abscission and bruising. This generates such a high amount of free fatty acids that the oil might become unfit for human consumption without appropriate refining. The lipase (EC 3.1.1.3) involved in the breakdown of the oil is not stable after homogenization of the tissue in aqueous buffers. In this study, we have devised a solvent-based procedure that allowed us to obtain fractions with stable lipase activity. Using these fractions, we have determined the optimal conditions for assaying mesocarp lipase activity. The activity was highest at a temperature of 35 degrees C and a pH of 9. The lipase was found to be strictly calcium dependent. The specific activity of the lipase measured in optimal conditions was found to be 33 mumol fatty acids released min(-1) mg(-1) protein using olive oil as substrate. The mesocarp contains about 190 U of lipase g(-1) fresh weight. This activity was found to be inhibited by the lipase inhibitor tetrahydrolipstatin (THL), suggesting that the lipase is a serine hydrolase.
ESTHER : Ngando_2006_Plant.Physiol.Biochem_44_611
PubMedSearch : Ngando_2006_Plant.Physiol.Biochem_44_611
PubMedID: 17064925

Title : Val-407 and Ile-408 in the beta5'-loop of pancreatic lipase mediate lipase-colipase interactions in the presence of bile salt micelles - Freie_2006_J.Biol.Chem_281_7793
Author(s) : Freie AB , Ferrato F , Carriere F , Lowe ME
Ref : Journal of Biological Chemistry , 281 :7793 , 2006
Abstract : In a previous study, we demonstrated that the beta5'-loop in the C-terminal domain of human pancreatic triglyceride lipase (hPTL) makes a major contribution in the function of hPTL (Chahinian et al. (2002) Biochemistry 41, 13725-13735). In the present study, we characterized the contribution of three residues in the beta5'-loop, Val-407, Ile-408, and Leu-412, to the function of hPTL. By substituting charged residues, aspartate or lysine, in these positions, we altered the hydrophilic to lipophilic ratio of the beta5'-loop. Each of the mutants was expressed, purified, and characterized for activity and binding with both monolayers and emulsions and for binding to colipase. Experiments with monolayers and with emulsions suggested that the interaction of hPTL with a phospholipid monolayer differs from the interaction of the hPTL-colipase complex with a dicaprin monolayer or a triglyceride emulsion (i.e. neutral lipids). Val-407, Ile-408, and Leu-412 make major contributions to interactions with monolayers, whereas only Val-407 and Ile-408 appear essential for activity on triglyceride emulsions in the presence of bile salt micelles. In solutions of taurodeoxycholate at micellar concentrations, a major effect of the beta5'-loop mutations is to change the interaction between hPTL and colipase. These observations support a major contribution of residues in the beta5'-loop in the function of hPTL and suggest that a third partner, bile salt micelles or the lipid interface or both, influence the binding of colipase and hPTL through interactions with the beta5'-loop.
ESTHER : Freie_2006_J.Biol.Chem_281_7793
PubMedSearch : Freie_2006_J.Biol.Chem_281_7793
PubMedID: 16431912
Gene_locus related to this paper: human-PNLIP

Title : Human pancreatic lipase-related protein 2: tissular localization along the digestive tract and quantification in pancreatic juice using a specific ELISA - Eydoux_2006_Biochim.Biophys.Acta_1760_1497
Author(s) : Eydoux C , Aloulou A , De Caro J , Grandval P , Laugier R , Carriere F , de Caro A
Ref : Biochimica & Biophysica Acta , 1760 :1497 , 2006
Abstract : Human pancreatic lipase-related protein 2 (HPLRP2) was previously found to be secreted by the exocrine pancreas. HPLRP2 shows a high level of activity on galactolipids, and might be involved in the digestion of these common vegetable lipids. Specific antibodies were raised in rabbits using a synthetic HPLRP2 peptide selected for its weak amino acid homology with the corresponding peptides of classical human pancreatic lipase (HPL) and human pancreatic lipase-related protein 1 (HPLRP1). ELISA and Western blotting data showed that these antibodies did not react with HPL or HPLRP1. Various tissues from the digestive tract were subjected to Western blotting analysis with the specific anti-peptide HPLRP2 antibody and the expression of HPLRP2 was detected in the pancreas and colon. An ELISA was developed for specifically measuring the HPLRP2 levels in pure pancreatic juice. This procedure was performed using the anti-peptide HPLRP2 antibody as the captor antibody and a biotinylated anti-HPLRP2 polyclonal antibody as the detector antibody. The lowest HPLRP2 quantification limit was found to be 50 microg/L and the reference range for the present assay was 50 microg-500 microg/L. HPL and HPLRP2 levels were measured using specific ELISAs in pancreatic juice from patients with and without pancreatic disorders. Patients with chronic calcifying pancreatitis (CCP) had significantly lower levels of both HPL and HPLRP2 than the controls subjects. The mean HPLRP2 to HPL ratio was estimated to be 28.30% (w/w) and 23.96% (w/w) in controls subjects and CCP patients, respectively, and the difference was not significant. The levels of HPL and HPLRP2 are therefore similarly reduced in both healthy patients and CCP patients.
ESTHER : Eydoux_2006_Biochim.Biophys.Acta_1760_1497
PubMedSearch : Eydoux_2006_Biochim.Biophys.Acta_1760_1497
PubMedID: 16887271
Gene_locus related to this paper: human-PNLIPRP2

Title : How gastric lipase, an interfacial enzyme with a Ser-His-Asp catalytic triad, acts optimally at acidic pH - Chahinian_2006_Biochemistry_45_993
Author(s) : Chahinian H , Snabe T , Attias C , Fojan P , Petersen SB , Carriere F
Ref : Biochemistry , 45 :993 , 2006
Abstract : Gastric lipase is active under acidic conditions and shows optimum activity on insoluble triglycerides at pH 4. The present results show that gastric lipase also acts in solution on vinyl butyrate, with an optimum activity above pH 7, which suggests that gastric lipase is able to hydrolyze ester bonds via the classical mechanism of serine hydrolases. These results support previous structural studies in which the catalytic triad of gastric lipase was reported to show no specific features. The optimum activity of gastric lipase shifted toward lower pH values, however, when the vinyl butyrate concentration was greater than the solubility limit. Experiments performed with long-chain triglycerides showed that gastric lipase binds optimally to the oil-water interface at low pH values. To study the effects of the pH on the adsorption step independently from substrate hydrolysis, gastric lipase adsorption on solid hydrophobic surfaces was monitored by total internal reflection fluorescence (TIRF), as well as using a quartz crystal microbalance. Both techniques showed a pH-dependent reversible gastric lipase adsorption process, which was optimum at pH 5 (Kd = 6.5 nM). Lipase adsorption and desorption constants (ka = 147,860 M(-1) s(-1) and kd = 139 x 10(-4) s(-1) at pH 6) were estimated from TIRF experiments. These results indicate that the optimum activity of gastric lipase at acidic pH is only "apparent" and results from the fact that lipase adsorption at lipid-water interfaces is the pH-dependent limiting step in the overall process of insoluble substrate hydrolysis. This specific kinetic feature of interfacial enzymology should be taken into account when studying any soluble enzyme acting on an insoluble substrate.
ESTHER : Chahinian_2006_Biochemistry_45_993
PubMedSearch : Chahinian_2006_Biochemistry_45_993
PubMedID: 16411775

Title : Constitutive expression of human pancreatic lipase-related protein 1 in Pichia pastoris - Aloulou_2006_Protein.Expr.Purif_47_415
Author(s) : Aloulou A , Grandval P , De Caro J , de Caro A , Carriere F
Ref : Protein Expr Purif , 47 :415 , 2006
Abstract : High-level constitutive expression of the human pancreatic lipase-related protein 1 (HPLRP1) was achieved using the methylotrophic yeast Pichia pastoris. The HPLRP1 cDNA, including its original leader sequence, was subcloned into the pGAPZB vector and further integrated into the genome of P. pastoris X-33 under the control of the glyceraldehyde 3-phosphate dehydrogenase (GAP) constitutive promoter. A major protein with a molecular mass of 50 kDa was found to be secreted into the culture medium and was identified using anti-HPLRP1 polyclonal antibodies as HPLRP1 recombinant protein. The level of expression reached 100-120 mg of HPLRP1 per liter of culture medium after 40 h, as attested by specific and quantitative enzyme-linked immunosorbent assay. A single cation-exchange chromatography sufficed to obtain a highly purified recombinant HPLRP1 after direct batch adsorption onto S-Sepharose of the HPLRP1 present in the culture medium, at pH 5.5. N-terminal sequencing and mass spectrometry analysis were carried out to monitor the production of the mature protein and to confirm that its signal peptide was properly processed.
ESTHER : Aloulou_2006_Protein.Expr.Purif_47_415
PubMedSearch : Aloulou_2006_Protein.Expr.Purif_47_415
PubMedID: 16481202

Title : Use of an inhibitor to identify members of the hormone-sensitive lipase family - Ben Ali_2006_Biochemistry_45_14183
Author(s) : Ben Ali Y , Chahinian H , Petry S , Muller G , Lebrun R , Verger R , Carriere F , Mandrich L , Rossi M , Manco G , Sarda L , Abousalham A
Ref : Biochemistry , 45 :14183 , 2006
Abstract : Hormone-sensitive lipase (HSL) contributes importantly to the mobilization of fatty acids from the triacylglycerols stored in adipocytes, which provide the main source of energy in mammals. On the basis of amino acid sequence alignments and three-dimensional structures, this enzyme was previously found to be a suitable template for defining a family of serine carboxylester hydrolases. In this study, the HSL family members are characterized rather on the basis of their inhibition by 5-methoxy-3-(4-phenoxyphenyl)-3H-[1,3,4]oxadiazol-2-one (compound 7600). This compound inhibits mammalian HSL as well as other HSL family members, such as EST2 from the thermophilic eubacterium Alicyclobacillus acidocaldarius and AFEST from the hyperthermophilic archaeon Archaeoglobus fulgidus. Various carboxylester hydrolases that are not members of the HSL family were found not to be inhibited by compound 7600 under the same experimental conditions. These include nonlipolytic hydrolases such as Torpedo californica acetylcholinesterase and pig liver esterase, as well as lipolytic hydrolases such as human pancreatic lipase, dog gastric lipase, Thermomyces lanuginosus lipase, and Bacillus subtilis LipA. When vinyl esters were used as substrates, the residual activity of HSL, AFEST, and EST2 decreased with an increase in compound 7600 concentration in the incubation mixture. The inhibitor concentration at which the enzyme activity decreased to 50% after incubation for 5 min was 70, 20, and 15 nM with HSL, AFEST, and EST2, respectively. Treating EST2 and AFEST with the inhibitor resulted in an increase in the molecular mass, as established by performing matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis. This increase in the molecular mass, which corresponds approximately to the molecular mass of the inhibitor, indicates that a covalent enzyme-inhibitor complex has been formed. Surface-enhanced laser desorption ionization time-of-flight mass spectrometry analysis of a trypsin digest of AFEST treated with the inhibitor or not treated showed the occurrence of an increase in the molecular masses of the "GESAGG"-containing peptide, which is compatible with the formation of a covalent complex with the inhibitor.
ESTHER : Ben Ali_2006_Biochemistry_45_14183
PubMedSearch : Ben Ali_2006_Biochemistry_45_14183
PubMedID: 17115713
Gene_locus related to this paper: human-LIPE

Title : Exploring the specific features of interfacial enzymology based on lipase studies - Aloulou_2006_Biochim.Biophys.Acta_1761_995
Author(s) : Aloulou A , Rodriguez JA , Fernandez S , van Oosterhout D , Puccinelli D , Carriere F
Ref : Biochimica & Biophysica Acta , 1761 :995 , 2006
Abstract : Many enzymes are active at interfaces in the living world (such as in the signaling processes at the surface of cell membranes, digestion of dietary lipids, starch and cellulose degradation, etc.), but fundamental enzymology remains largely focused on the interactions between enzymes and soluble substrates. The biochemical and kinetic characterization of lipolytic enzymes has opened up new paths of research in the field of interfacial enzymology. Lipases are water-soluble enzymes hydrolyzing insoluble triglyceride substrates, and studies on these enzymes have led to the development of specific interfacial kinetic models. Structure-function studies on lipases have thrown light on the interfacial recognition sites present in the molecular structure of these enzymes, the conformational changes occurring in the presence of lipids and amphiphiles, and the stability of the enzymes present at interfaces. The pH-dependent activity, substrate specificity and inhibition of these enzymes can all result from both "classical" interactions between a substrate or inhibitor and the active site, as well as from the adsorption of the enzymes at the surface of aggregated substrate particles such as oil drops, lipid bilayers or monomolecular lipid films. The adsorption step can provide an alternative target for improving substrate specificity and developing specific enzyme inhibitors. Several data obtained with gastric lipase, classical pancreatic lipase, pancreatic lipase-related protein 2 and phosphatidylserine-specific phospholipase A1 were chosen here to illustrate these specific features of interfacial enzymology.
ESTHER : Aloulou_2006_Biochim.Biophys.Acta_1761_995
PubMedSearch : Aloulou_2006_Biochim.Biophys.Acta_1761_995
PubMedID: 16931141

Title : Syntheses of an alpha-D-Gal-(1-->6)-beta-D-Gal diglyceride, as lipase substrate - Lafont_2006_Carbohydr.Res_341_695
Author(s) : Lafont D , Carriere F , Ferrato F , Boullanger P
Ref : Carbohydr Res , 341 :695 , 2006
Abstract : Two different routes were explored to afford 3-O-(6-O-alpha-D-galactopyranosyl-beta-D-galactopyranosyl)-1,2-di-O-dodecanoyl-sn -glycerol. In the first one, the key step was the glycosylation of the 3-O-(2,3,4-tri-O-benzyl-beta-D-galactopyranosyl)-1,2-O-isopropylidene-sn-glycerol acceptor with 2-pyridyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D-galactopyranoside as the donor. In the second one, the key step was the coupling of 2,3,4-tri-O-acetyl-6-O-(2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl)-D-galact opyranosyl trichloroacetimidate donor with 1,2-O-isopropylidene-sn-glycerol. Even though the number of steps was the same in both pathways, the first one afforded a better overall yield (12.4%) than the second one (6.5%). This eight-step synthesis allowed the preparation of the expected glycolipid, which was used as substrate for recombinant GPLRP2 galactolipase using the monomolecular film technique.
ESTHER : Lafont_2006_Carbohydr.Res_341_695
PubMedSearch : Lafont_2006_Carbohydr.Res_341_695
PubMedID: 16458274

Title : Cloning and seasonal secretion of the pancreatic lipase-related protein 2 present in goat seminal plasma - Sias_2005_Biochim.Biophys.Acta_1686_169
Author(s) : Sias B , Ferrato F , Pellicer-Rubio MT , Forgerit Y , Guillouet P , Leboeuf B , Carriere F
Ref : Biochimica & Biophysica Acta , 1686 :169 , 2005
Abstract : The storage of frozen semen for artificial insemination is usually performed in the presence of egg yolk or skimmed milk as protective agents. In goats, the use of skimmed milk extenders requires, however, that most of the seminal plasma is removed before dilution of spermatozoa because it is deleterious for their survival. It has been previously demonstrated that a lipase (BUSgp60) secreted by the accessory bulbourethral gland was responsible for the cellular death of goat spermatozoa, through the lipolysis of residual milk lipids and the release of toxic free fatty acids. This lipase was purified from the whole seminal plasma of goat and was found to display both lipase and phospholipase A activities, this latter activity representing the main phospholipase activity detected in goat seminal plasma. Based on its N-terminal amino acid sequence, identical to that of BUSgP60 purified from bulbourethral gland secretion, and the design of degenerated oligonucleotides, the lipase was cloned from total mRNA isolated from bulbourethral gland. DNA sequencing confirmed it was the goat pancreatic-lipase-related protein 2 (GoPLRP2). The physiological role of GoPLRP2 is still unknown but this enzyme might be associated with the reproductive activity of goats. A significant increase in lipase secretion was observed every year in August and the level of lipase activity in the semen remained high till December, i.e., during the breeding season. A parallel increase in the plasmatic levels of testosterone suggested that GoPLRP2 expression might be regulated by sexual hormones. The lipase activity level measured in goat seminal plasma, which could reach 1000 U/ml during the breeding season, was one of the highest lipase activity measured in natural sources, including gastric and pancreatic juices.
ESTHER : Sias_2005_Biochim.Biophys.Acta_1686_169
PubMedSearch : Sias_2005_Biochim.Biophys.Acta_1686_169
PubMedID: 15629686

Title : Continuous monitoring of cholesterol oleate hydrolysis by hormone-sensitive lipase and other cholesterol esterases - Ben Ali_2005_J.Lipid.Res_46_994
Author(s) : Ben Ali Y , Carriere F , Verger R , Petry S , Muller G , Abousalham A
Ref : J Lipid Res , 46 :994 , 2005
Abstract : Hormone-sensitive lipase (HSL) contributes importantly to the hydrolysis of cholesteryl ester in steroidogenic tissues, releasing the cholesterol required for adrenal steroidogenesis. HSL has broad substrate specificity, because it hydrolyzes triacylglycerols (TAGs), diacylglycerols, monoacylglycerols, and cholesteryl esters. In this study, we developed a specific cholesterol esterase assay using cholesterol oleate (CO) dispersed in phosphatidylcholine and gum arabic by sonication. To continuously monitor the hydrolysis of CO by HSL, we used the pH-stat technique. For the sake of comparison, the hydrolysis of CO dispersion was also tested using other cholesteryl ester-hydrolyzing enzymes. The specific activities measured on CO were found to be 18, 100, 27, and 3 micromol/min/mg for HSL, cholesterol esterase from Pseudomonas species, Candida rugosa lipase-3, and cholesterol esterase from bovine pancreas, respectively. The activity of HSL on CO is approximately 4- to 5-fold higher than on long-chain TAGs. In contrast, with all other enzymes tested, the rates of TAG hydrolysis were higher than those of CO hydrolysis. The relatively higher turnover of HSL on CO observed in vitro adds further molecular insight on the physiological importance of HSL in cholesteryl ester catabolism in vivo. Thus, HSL could be considered more as a cholesteryl ester hydrolase than as a TAG lipase.
ESTHER : Ben Ali_2005_J.Lipid.Res_46_994
PubMedSearch : Ben Ali_2005_J.Lipid.Res_46_994
PubMedID: 15716583
Gene_locus related to this paper: human-LIPE

Title : Sensitive assay for hormone-sensitive lipase using NBD-labeled monoacylglycerol to detect low activities in rat adipocytes - Petry_2005_J.Lipid.Res_46_603
Author(s) : Petry S , Ben Ali Y , Chahinian H , Jordan H , Kleine H , Muller G , Carriere F , Abousalham A
Ref : J Lipid Res , 46 :603 , 2005
Abstract : The recent finding that p-nitrobenzofurazan (NBD)-FA is incorporated into and released from the acylglycerols of isolated rat adipocytes in an insulin-sensitive manner [G. Muller, H. Jordan, C. Jung, H. Kleine, and S. Petry. 2003. Biochimie. 85: 1245-1246] suggests that NBD-FA-labeled acylglycerols are cleaved by rat adipocyte hormone-sensitive lipase (HSL) in vivo. In the present study, we developed a continuous, sensitive in vitro lipase assay using a monoacylglycerol (MAG) containing NBD (NBD-MAG). NBD-MAG was found to provide an efficient substrate for rat adipocyte and human recombinant HSL. Ultrasonic treatment applied in the presence of phospholipids leads to the incorporation of NBD-MAG into the phospholipid liposomes and to a concomitant change of its spectrophotometric properties. The enzymatic release of NBD-FA and its dissociation from the carrier liposomes is accompanied by the recovery of the original spectrophotometric characteristics. The rate of lipolysis was monitored by measuring the increase in optical density at 481 nm, which was found to be linear with time and linearly proportional to the amount of lipase added. To assess the specific activity of recombinant HSL, we determined the molar extinction coefficient of NBD-FA under the assay conditions. This convenient assay procedure based on NBD-MAG should facilitate the search for small molecule HSL inhibitors.
ESTHER : Petry_2005_J.Lipid.Res_46_603
PubMedSearch : Petry_2005_J.Lipid.Res_46_603
PubMedID: 15627655
Gene_locus related to this paper: human-LIPE

Title : Closed and open conformations of the lid domain induce different patterns of human pancreatic lipase antigenicity and immunogenicity - Halimi_2005_Biochim.Biophys.Acta_1753_247
Author(s) : Halimi H , De Caro J , Carriere F , de Caro A
Ref : Biochimica & Biophysica Acta , 1753 :247 , 2005
Abstract : Epitope mapping was performed on human pancreatic lipase (HPL) using the SPOTscan method. A set of 146 short (12 amino acid residues) synthetic overlapping peptides covering the entire amino acid sequence of HPL were used to systematically assess the immunoreactivity of antisera raised in rabbits against native HPL, HPL without a lid (HPL(-lid)) and HPL covalently inhibited by diethyl p-nitrophenyl phosphate (DP-HPL). In the latter form of HPL, the lid domain controlling the access to the active site was assumed to exist in the open conformation. All the anti-lipase sera were tested in a direct ELISA, anti-HPL serum showing the greatest antibody titer. Although from the structural point of view, the differences between the various forms of HPL were restricted to the lid domain, differences in the antigenic properties of HPL were observed with the SPOTscan method, and the anti-DP-HPL antibodies showed the strongest reactivity. Most of the peptide stretches recognized included amino acid residues which are accessible at the surface of the lipase, except for those located near the active site. Two small peptides (T173-P180, V199-A207) were identified in the vicinity of the active site, their antipeptide antibodies were produced and their reactivity towards the various forms of HPL was tested in a double sandwich ELISA. No reactivity was observed under these conditions. Two antipeptide antibodies directed against two other selected peptides, P208-V221 (belonging to the beta9 loop) and I245-F258 (belonging to the lid domain) were prepared and found to react much more strongly with DP-HPL than with HPL or HPL(-lid) in a double sandwich ELISA. These antibodies should provide useful tools for monitoring the conformational changes taking place during the opening of the HPL lid domain.
ESTHER : Halimi_2005_Biochim.Biophys.Acta_1753_247
PubMedSearch : Halimi_2005_Biochim.Biophys.Acta_1753_247
PubMedID: 16203189

Title : Does the pancreas really produce much more lipase than required for fat digestion? - Carriere_2005_JOP_6_206
Author(s) : Carriere F , Grandval P , Gregory PC , Renou C , Henniges F , Sander-Struckmeier S , Laugier R
Ref : JOP , 6 :206 , 2005
Abstract : Thirty years ago, it was reported that a linear relationship does not exist between the amounts of human pancreatic lipase secreted in chronic pancreatitis and the degree of steatorrhea, which was considered to appear only after more than 90% of the pancreatic secretory capacity had been lost. From these observations, it was generally thought that the lipolytic potential of the pancreas is much higher than required. In recent years, however, it has been noted that: 1) the level of inhibition of digestive lipases and gastrointestinal lipolysis by the lipase inhibitor orlistat were almost linearly correlated with the amount of excreted fat; 2) in minipigs with experimentally-induced pancreatic exocrine insufficiency, the amounts of enteric-coated pancreatic extracts needed for restoring fat digestion to normal levels were estimated to be much higher than those usually administered; 3) human pancreatic lipase specific activity on meal triglycerides is 3 orders of magnitude lower than the very high specific activity usually measured under experimental in vitro conditions which are far from physiological conditions; 4) in patients with reduced human pancreatic lipase secretion, gastric lipase plays a significant role in fat digestion. This last observation might explain the absence of a linear relationship between human pancreatic lipase secretion in chronic pancreatitis and steatorrhea. From the low specific activity displayed by human pancreatic lipase on meal triglycerides, one can better understand why more lipase than expected is needed, why fat digestion lasts for more than a few minutes and, finally, why there is not such an excess secretory capacity for lipase as had been previously thought.
ESTHER : Carriere_2005_JOP_6_206
PubMedSearch : Carriere_2005_JOP_6_206
PubMedID: 15883471

Title : Human pancreatic lipase-related protein 2 is a galactolipase - Sias_2004_Biochemistry_43_10138
Author(s) : Sias B , Ferrato F , Grandval P , Lafont D , Boullanger P , de Caro A , Leboeuf B , Verger R , Carriere F
Ref : Biochemistry , 43 :10138 , 2004
Abstract : Human pancreatic lipase-related protein 2 (HPLRP2) was found to be expressed in the pancreas, but its biochemical properties were not investigated in detail. A recombinant HPLRP2 was produced in insect cells and the yeast Pichia pastoris and purified by cation exchange chromatography. Its substrate specificity was investigated using pH-stat and monomolecular film techniques and various lipid substrates (triglycerides, diglycerides, phospholipids, and galactolipids). Lipase activity of HPLRP2 on trioctanoin was inhibited by bile salts and poorly restored by adding colipase. In vivo, HPLRP2 therefore seems unlikely to show any lipase activity on dietary fat. In human pancreatic lipase (HPL), residues R256, D257, Y267, and K268 are involved in the stabilization of the open conformation of the lid domain, which interacts with colipase. These residues are not conserved in HPLRP2. When the corresponding mutations (R256G, D257G, Y267F, and K268E) are introduced into HPL, the effects of colipase are drastically reduced in the presence of bile salts. This may explain why colipase has such weak effects on HPLRP2. HPLRP2 displayed a very low level of activity on phospholipid micelles and monomolecular films. Its activity on monogalactosyldiglyceride monomolecular film, which was much higher, was similar to the activity of guinea pig pancreatic lipase related-protein 2, which shows the highest galactolipase activity ever measured. The physiological role of HPLRP2 suggested by the present results is the digestion of galactolipids, the most abundant lipids occurring in plant cells, and therefore, in the vegetables that are part of the human diet.
ESTHER : Sias_2004_Biochemistry_43_10138
PubMedSearch : Sias_2004_Biochemistry_43_10138
PubMedID: 15287741
Gene_locus related to this paper: human-PNLIPRP2

Title : Might the kinetic behavior of hormone-sensitive lipase reflect the absence of the lid domain? - Ben Ali_2004_Biochemistry_43_9298
Author(s) : Ben Ali Y , Chahinian H , Petry S , Muller G , Carriere F , Verger R , Abousalham A
Ref : Biochemistry , 43 :9298 , 2004
Abstract : Hormone-sensitive lipase (HSL) is thought to contribute importantly to the mobilization of fatty acids from the triacylglycerols (TAGs) stored in adipocytes, providing the main source of energy in mammals. To investigate the HSL substrate specificity more closely, we systematically assessed the lipolytic activity of recombinant human HSL on solutions and emulsions of various vinyl esters and TAG substrates, using the pH-stat assay technique. Recombinant human HSL activity on solutions of partly soluble vinyl esters or TAG was found to range from 35 to 90% of the maximum activity measured with the same substrates in the emulsified state. The possible existence of a lipid-water interface due to the formation of small aggregates of vinyl esters or TAG in solution may account for the HSL activity observed below the solubility limit of the substrate. Recombinant human HSL also hydrolyzes insoluble medium- and long-chain acylglycerols such as trioctanoylglycerol, dioleoylglycerol, and olive oil, and can therefore be classified as a true lipase. Preincubation of the recombinant HSL with a serine esterase inhibitor such as diethyl p-nitrophenyl phosphate in 1:100 molar excess leads to complete HSL inhibition within 15 min. This result indicates that the catalytic serine of HSL is highly reactive and that it is readily accessible. Similar behavior was also observed with lipases with no lid domain covering their active site, or with a deletion in the lid domain. The 3-D structure of HSL, which still remains to be determined, may therefore lack the lid domain known to exist in various other lipases.
ESTHER : Ben Ali_2004_Biochemistry_43_9298
PubMedSearch : Ben Ali_2004_Biochemistry_43_9298
PubMedID: 15260473
Gene_locus related to this paper: human-LIPE

Title : Characterization of pancreatic lipase-related protein 2 isolated from human pancreatic juice - De Caro_2004_Biochim.Biophys.Acta_1701_89
Author(s) : De Caro J , Sias B , Grandval P , Ferrato F , Halimi H , Carriere F , de Caro A
Ref : Biochimica & Biophysica Acta , 1701 :89 , 2004
Abstract : Human pancreatic lipase-related protein 2 (HPLRP2) was identified for the first time in pancreatic juice using specific anti-peptide antibodies and purified to homogeneity. Antibodies were raised in the rabbit using a synthetic peptide from the HPLRP2 protein sequence deduced from cDNA. Western blotting analysis showed that these antibodies did not react with classical human pancreatic lipase (HPL) or human pancreatic lipase-related protein 1 (HPLRP1) but cross-reacted with native rat PLRP2 (RPLRP2), as well as with recombinant rat and guinea-pig PLRP2 (GPLRP2). Immunoaffinity chromatography was performed on immobilized anti-recombinant HPLRP2 polyclonal antibodies to purify native HPLRP2 after conventional chromatographic steps including gel filtration and chromatrography on an anion-exchanger. The substrate specificity of HPLRP2 was investigated using various triglycerides, phospholipids and galactolipids as substrates. The lipase activity on triglycerides was inhibited by bile salts and weakly restored by colipase. The phospholipase activity of HPLRP2 on phospholipid micelles was very low. A significant level of galactolipase activity was measured using monogalactosyldiglyceride monomolecular films. These data suggest that the main physiological function of HPLRP2 is the hydrolysis of galactolipids, which are the main lipids present in vegetable food.
ESTHER : De Caro_2004_Biochim.Biophys.Acta_1701_89
PubMedSearch : De Caro_2004_Biochim.Biophys.Acta_1701_89
PubMedID: 15450178
Gene_locus related to this paper: human-PNLIPRP2

Title : Rapid exchange of pancreatic lipase between triacylglycerol droplets - Haiker_2004_Biochim.Biophys.Acta_1682_72
Author(s) : Haiker H , Lengsfeld H , Hadvary P , Carriere F
Ref : Biochimica & Biophysica Acta , 1682 :72 , 2004
Abstract : Two types of experiments were performed to study the reversibility of interfacial adsorption of pancreatic lipase (PL) to fat droplets during lipolysis. Lipolysis was measured in olive oil/gum arabic emulsions containing radiolabeled triolein in the presence of bile salts and lecithin at rate-limiting concentrations of porcine PL (PPL) or human PL (HPL). The lipolysis rate in a labeled emulsion, i.e. release of [(14)C]oleic acid, was immediately reduced by around 50% upon dilution with an equal amount of an unlabeled emulsion. Further, lipolysis was rapidly and completely suppressed when a non-exchanging lipase inhibitor was present in the second emulsion. These results indicate hopping of lipase between emulsion droplets. Alternative explanations were excluded. Hopping of PL between triolein droplets stabilized with gum arabic at supramicellar bile salt concentrations was observed only in the presence, not in the absence, of lecithin. Displacement from a trioctanoin-water interface of active HPL by an inactive mutant (S152G) was studied in the presence of bile salts by measuring HPL distribution between the water phase and the oil-water interface. Colipase was limiting for HPL binding to the oil-water interface (colipase to lipase molar ratio: 0.5) and, thus, for lipolysis. Upon adding S152G, which has the same affinity for colipase, inactive and active HPL were found to compete for binding at the oil-water interface. When equal amounts of HPL and HPL S152G were used, the lipolysis rate dropped to half the maximum rate recorded with HPL alone, suggesting that half the active HPL was rapidly desorbed from the oil-water interface. Therefore, under various conditions, PL does not remain irreversibly adsorbed to the oil-water interface, but can exchange rapidly between oil droplets, via an equilibrium between soluble and lipid-bound PL.
ESTHER : Haiker_2004_Biochim.Biophys.Acta_1682_72
PubMedSearch : Haiker_2004_Biochim.Biophys.Acta_1682_72
PubMedID: 15158758

Title : Critical evaluation of a specific ELISA and two enzymatic assays of pancreatic lipases in human sera - Grandval_2004_Pancreatology_4_495
Author(s) : Grandval P , de Caro A , De Caro J , Sias B , Carriere F , Verger R , Laugier R
Ref : Pancreatology , 4 :495 , 2004
Abstract : BACKGROUND AND AIMS: Human pancreatic lipases (HPL) include the classical HPL, and two related proteins known as pancreatic lipase-related proteins 1 and 2 (HPLRP1 and 2). The aim of this study was to develop an ELISA for specifically quantifying the classical-HPL level in sera of patients with and without pancreatic disorders. METHODS: The specific activity of various human (including classical-HPL) and microbial lipases was measured using Lipa Vitros and potentiometric (pH-stat) assays. A double sandwich ELISA was also set up, using an anti-classical-HPL polyclonal antibody and a biotinylated monoclonal antibody (mAb 146-40) specific to the classical-HPL. Sera (n = 53) were collected from patients with and without pancreatic disorders. The lipase concentration was deduced from the measured lipolytic activity and compared with the corresponding classical-HPL concentration, measured with the ELISA. RESULTS: Both the purified HPLRP2 and 3 lipases of microbial origin were found to have a significant and unexpected lipolytic activity under the standard Lipa Vitros assay, whereas the ELISA test developed in the present study was found to be specific for the classical-HPL, due to the absence of cross-reactivity between mAb 146-40, HPLRP1 and HPLRP2. The efficiency of the ELISA was assessed in terms of its reproducibility and accuracy. The lower detection limit of classical-HPL was found to be 0.03 microg/l. A good correlation was found to exist between the lipase concentrations obtained in the ELISA, pH-stat and Lipa Vitros tests, in both the control and pathological groups. CONCLUSION: This is the first time a specific method of measuring classical-HPL in human serum has been proposed. Using this ELISA, we established with the 53 sera selected in the present study, that the Lipa Vitros assay as well as the pH-stat assay were mostly detecting classical pancreatic lipase. However, it is possible that other lipases such as HPLRP2 or lipases of microbial origin, present in some pathological sera, may well interfere with the Lipa Vitros assay.
ESTHER : Grandval_2004_Pancreatology_4_495
PubMedSearch : Grandval_2004_Pancreatology_4_495
PubMedID: 15316225

Title : The beta 5' loop of the pancreatic lipase C2-like domain plays a critical role in the lipase-lipid interactions - Chahinian_2002_Biochemistry_41_13725
Author(s) : Chahinian H , Bezzine S , Ferrato F , Ivanova MG , Perez B , Lowe ME , Carriere F
Ref : Biochemistry , 41 :13725 , 2002
Abstract : The structural similarities between the C-terminal domain of human pancreatic lipase (C-HPL) and C2 domains suggested a similar function, the interaction with lipids. The catalytic N-terminal domain (N-HPL) and C-HPL were produced as individual proteins, and their partitioning between the water phase and the triglyceride-water interface was assessed using trioctanoin emulsions (TC8). N-HPL did not bind efficiently to TC8 and was inactive. C-HPL did bind to TC8 and to a phospholipid monolayer with a critical surface pressure of penetration similar to that of HPL (15 mN m(-1)). These experiments, performed in the absence of colipase and bile salts, support an absolute requirement of C-HPL for interfacial binding of HPL. To refine our analysis, we determined the contribution to lipid interactions of a hydrophobic loop (beta 5') in C-HPL by investigating a HPL mutant in which beta 5' loop hydrophobicity was increased by introducing the homologous lipoprotein lipase (LPL) beta 5' loop. This mutant (HPL-beta 5'LPL) penetrated into phospholipid monolayers at higher surface pressures than HPL, and its level of binding to TC8 was higher than that of HPL in the presence of serum albumin (BSA), an inhibitory protein that competes with HPL for interfacial adsorption. The beta 5' loop of LPL is therefore tailored for an optimal interaction with the surface of triglyceride-rich lipoproteins (VLDL and chylomicrons) containing phospholipids and apoproteins. These observations support a major contribution of the beta 5' loop in the interaction of LPL and HPL with their respective substrates.
ESTHER : Chahinian_2002_Biochemistry_41_13725
PubMedSearch : Chahinian_2002_Biochemistry_41_13725
PubMedID: 12427035

Title : Inhibition of gastrointestinal lipolysis by Orlistat during digestion of test meals in healthy volunteers - Carriere_2001_Am.J.Physiol.Gastrointest.Liver.Physiol_281_G16
Author(s) : Carriere F , Renou C , Ransac S , Lopez V , De Caro J , Ferrato F , de Caro A , Fleury A , Sanwald-Ducray P , Lengsfeld H , Beglinger C , Hadvary P , Verger R , Laugier R
Ref : American Journal of Physiology Gastrointest Liver Physiol , 281 :G16 , 2001
Abstract : The inhibition of digestive lipases by the antiobesity drug Orlistat along with lipolysis levels and fecal fat excretion were measured in healthy humans. Orlistat was found to be a powerful gastric lipase inhibitor, achieving 46.6--91.4% enzyme inhibition and thus greatly reducing gastric lipolysis of solid and liquid meals (11--33% of respective controls). Gastric lipase inhibition by Orlistat was extremely fast (half-inhibition time < 1 min). Duodenal lipolysis was reduced significantly by Orlistat given with the solid meal (32.6--37.6% of controls) but was only slightly reduced by Orlistat given with the liquid meal (74.5--100% of controls). Human pancreatic lipase (HPL) inhibition was found to be high (51.2--82.6%), however, regardless of the meal. These paradoxical results were explained when in vitro lipolysis experiments were performed. The rates of HPL inhibition by Orlistat were found to be similar with both types of meals (half-inhibition time 5--6 min), but the preemulsified triglycerides of the liquid meal were rapidly hydrolyzed by HPL before the enzyme was significantly inhibited by Orlistat. With the solid meal, the rate of hydrolysis of the meal triglycerides by HPL was slower than the rate of HPL inhibition by Orlistat. As predicted from the previous results, the effects of Orlistat on fat excretion levels were found to be much greater with the solid (40.5--57.4% of ingested fat) than with the liquid (4.2--18.8%) test meal.
ESTHER : Carriere_2001_Am.J.Physiol.Gastrointest.Liver.Physiol_281_G16
PubMedSearch : Carriere_2001_Am.J.Physiol.Gastrointest.Liver.Physiol_281_G16
PubMedID: 11408251

Title : The specific activities of human digestive lipases measured from the in vivo and in vitro lipolysis of test meals - Carriere_2000_Gastroenterology_119_949
Author(s) : Carriere F , Renou C , Lopez V , De Caro J , Ferrato F , Lengsfeld H , de Caro A , Laugier R , Verger R
Ref : Gastroenterology , 119 :949 , 2000
Abstract : BACKGROUND & AIMS: The lipolytic potential of digestive lipases in vivo has always been deduced so far from their in vitro activities under nonphysiologic conditions. In the present study, the specific activities of human gastric lipase (HGL) and pancreatic lipase (HPL) were measured on dietary triglycerides (TGs) during test meal lipolysis. METHODS: Healthy human volunteers ingested a liquid or solid meal. The specific activities of HGL and HPL were estimated from the lipase and free fatty acid (FFA) outputs at the postpyloric and duodenal levels, respectively. Based on the in vivo data, lipolysis was also performed in vitro by mixing the meal either with gastric juice and subsequently with pancreatic juice and bile or with purified HGL and HPL. FFAs were measured by thin-layer chromatography, and the specific activities of HGL and HPL were expressed as micromoles of FFA per minute per milligram of lipase. RESULTS: In vitro, the specific activities on the liquid meal TGs were 32 (gastric juice) and 34 (pure lipase) micromol x min(-1) x mg(-1) with HGL and 47 (pancreatic juice) and 43 (pure lipase) micromol x min(-1). mg(-1) with HPL. The specific activities on the solid meal TGs were 33 (gastric juice) and 32 (pure lipase) micromol x min(-1) x mg(-1) with HGL and 12 (pancreatic juice) and 15 (pure lipase) micromol x min(-1) x mg(-1) with HPL. The in vivo values obtained were in the same range. The secretory lipase outputs were 21.6+/-14.5 mg HGL and 253.5+/-95.5 mg HPL with the liquid test meal and 15.2+/-5.1 mg HGL and 202.9+/-96.1 mg HPL with the solid test meal. CONCLUSIONS: The specific activities of HGL and HPL on meal TGs were much lower than those measured in vitro under optimized assay conditions (1300-8000). However, these low specific activities are enough for the meal TGs to be completely lipolysed, given the amounts of HGL and HPL secreted during a meal.
ESTHER : Carriere_2000_Gastroenterology_119_949
PubMedSearch : Carriere_2000_Gastroenterology_119_949
PubMedID: 11040182

Title : Digestive lipases: from three-dimensional structure to physiology - Miled_2000_Biochimie_82_973
Author(s) : Miled N , Canaan S , Dupuis L , Roussel A , Riviere M , Carriere F , de Caro A , Cambillau C , Verger R
Ref : Biochimie , 82 :973 , 2000
Abstract : Human gastric lipase (HGL) is a lipolytic enzyme that is secreted by the chief cells located in the fundic part of the stomach. HGL plays an important role in lipid digestion, since it promotes the subsequent hydrolytic action of pancreatic lipase in duodenal lumen. Physiological studies have shown that HGL is able of acting not only in the highly acid stomach environment but also in the duodenum in synergy with human pancreatic lipase (HPL). Recombinant HGL (r-HGL) was expressed in the baculovirus/insect cell system in the form of an active protein with a molecular mass of 45 kDa. The specific activities of r-HGL were found to be similar to that of the native enzyme when tested on various triacylglycerol (TG) substrates. The 3-D structure of r-HGL was the first solved within the mammalian acid lipase family. This globular enzyme (379 residues) shows a new feature, different from the other known lipases structures, which consists of a core domain having the alpha/beta hydrolase fold and a cap domain including a putative 'lid' of 30 residues covering the active site of the lipase (closed conformation). HPL is the major lipolytic enzyme involved in the digestion of dietary TG. HPL is a 50 kDa glycoprotein which is directly secreted as an active enzyme. HPL was the first mammalian lipase to be solved structurally, and it revealed the presence of two structural domains: a large N-terminal domain (residues 1-336) and a smaller C-terminal domain (residues 337-449). The large N-terminal domain belongs to the alpha/beta hydrolase fold and contains the active site. A surface loop called the lid domain (C237-C261) covers the active site in the closed conformation of the lipase. The 3-D structure of the lipase-procolipase complex illustrates how the procolipase might anchor the lipase at the interface in the presence of bile salts: procolipase binds to the C-terminal domain of HPL and exposes the hydrophobic tips of its fingers at the opposite site of its lipase-binding domain. These hydrophobic tips help to bring N-terminal domain into close conformation with the interface where the opening of the lid domain probably occurs. As a result of all these conformational changes, the open lid and the extremities of the procolipase form an impressive continuous hydrophobic plateau, extending over more than 50 A. This surface might able to interact strongly with a lipid-water interface. The biochemical, histochemical and clinical studies as well as the 3-D structures obtained will be a great help for a better understanding of the structure-function relationships of digestive lipases.
ESTHER : Miled_2000_Biochimie_82_973
PubMedSearch : Miled_2000_Biochimie_82_973
PubMedID: 11099794

Title : The C-terminal domain of pancreatic lipase: functional and structural analogies with c2 domains - Chahinian_2000_Curr.Protein.Pept.Sci_1_91
Author(s) : Chahinian H , Sias B , Carriere F
Ref : Curr Protein Pept Sci , 1 :91 , 2000
Abstract : The 3D structure of pancreatic lipase (PL) consists of two functional domains. The N-terminal domain belongs to the alpha/beta hydrolase fold and contains the active site, which involves a catalytic triad analogous to that present in serine proteases. The beta-sandwich C-terminal domain of PL plays an important part in the binding process between the lipase and colipase, the specific PL cofactor. Recent structure-function studies have suggested that the PL C-terminal domain may have an extra role apart from that of binding colipase. This domain contains an exposed hydrophobic loop (beta5') which was found to be located on the same side as the hydrophobic loops surrounding the active site, and it may be involved in the lipid binding process. Indirect evidence for this new function of the PL C-terminal domain has been provided by studies with monoclonal antibodies directed against the beta5' loop. The catalytic activity of the PL-antibody complexes on water insoluble substrates decreased drastically, whereas their esterase activity on a soluble substrate remained unchanged. During the last few years, a number of protein structures (15-lipoxygenase, alpha-toxin from Clostridium perfringens) have been determined that contain domains with close structural homologies with the beta-sandwich C-terminal domain of PL. Generally speaking, these domains show structural homologies with the C2 domains occurring in a wide range of proteins involved in signal transduction (e.g. phosphoinositide-specific phospholipase C, protein kinase C, cytosolic phospholipase A2), membrane traffic (e.g. synaptotagmin I, rabphilin) and membrane disruption (e.g. perforin). Here it is proposed to review the structure and function of the C2 domains, based on the recent 3D structures and improved sequence alignments.
ESTHER : Chahinian_2000_Curr.Protein.Pept.Sci_1_91
PubMedSearch : Chahinian_2000_Curr.Protein.Pept.Sci_1_91
PubMedID: 12369922

Title : Human pancreatic lipase: colipase dependence and interfacial binding of lid domain mutants - Bezzine_1999_Biochemistry_38_5499
Author(s) : Bezzine S , Ferrato F , Ivanova MG , Lopez V , Verger R , Carriere F
Ref : Biochemistry , 38 :5499 , 1999
Abstract : Five key amino acid residues from human pancreatic lipase (HPL) are mutated in some pancreatic lipase-related proteins 2 (PLRP2) that are not reactivated by colipase in the presence of bile salts. One of these residues (Y403) is involved in a direct interaction between the HPL C-terminal domain and colipase. The other four residues (R256, D257, Y267, and K268) are involved in the interactions stabilizing the open conformation of the lid domain, which also interacts with colipase. Here we produced and characterized three HPL mutants: HPL Y403N, an HPL four-site mutant (R256G, D257G, Y267F, and K268E), and an HPL five-site mutant (R256G, D257G, Y267F, K268E, and Y403N), in which the HPL amino acids were replaced by those present in human PLRP2. Colipase reactivated both the HPL Y403N mutant and HPL, and Y403 is therefore not essential for lipase-colipase interactions. Both the HPL four-site and five-site mutants showed low activity on trioctanoin, were inhibited by bile salts (sodium taurodeoxycholate, NaTDC) and were not reactivated by colipase. The interfacial binding of the HPL four-site mutant to a trioctanoin emulsion was suppressed in the presence of 4 mM NaTDC and was not restored by addition of colipase. Protein blotting/protein overlay immunoassay revealed that the HPL four-site mutant-colipase interactions are not abolished, and therefore, the absence of reactivation of the HPL four-site mutant is probably due to a lid domain conformation that prevents the interfacial binding of the lipase-colipase complex. The effects of colipase were also studied with HPL(-lid), an HPL mutant showing an 18-residue deletion within the lid domain, which therefore has only one colipase interaction site. HPL(-lid) showed a low activity on trioctanoin, was inhibited by bile salts, and recovered its lipase activity in the presence of colipase. Reactivation of HPL(-lid) by colipase was associated with a strong interfacial binding of the mutant to a trioctanoin emulsion. The lid domain is therefore not essential for either the interfacial binding of HPL or the lipase-colipase interactions.
ESTHER : Bezzine_1999_Biochemistry_38_5499
PubMedSearch : Bezzine_1999_Biochemistry_38_5499
PubMedID: 10220337

Title : Colipase: structure and interaction with pancreatic lipase - van Tilbeurgh_1999_Biochim.Biophys.Acta_1441_173
Author(s) : van Tilbeurgh H , Bezzine S , Cambillau C , Verger R , Carriere F
Ref : Biochimica & Biophysica Acta , 1441 :173 , 1999
Abstract : Colipase is a small protein cofactor needed by pancreatic lipase for the efficient dietary lipid hydrolysis. It binds to the C-terminal, non-catalytic domain of lipase, thereby stabilising an active conformation and considerably increasing the overall hydrophobic binding site. Structural studies of the complex and of colipase alone have clearly revealed the functionality of its architecture. Interestingly, a structural analogy has recently been discovered between colipase and a domain in a developmental protein (Dickkopf), based on sequence analogy and homology modeling. Whether this structural analogy implies a common function (lipid interaction) remains to be clarified. Structural analogies have also been recognised between the pancreatic lipase C-terminal domain, the N-terminal domains of lipoxygenases and the C-terminal domain of alpha-toxin. These non-catalytic domains in the latter enzymes are important for interaction with membranes. It has not been established if these domains are also involved in eventual protein cofactor binding as is the case for pancreatic lipase.
ESTHER : van Tilbeurgh_1999_Biochim.Biophys.Acta_1441_173
PubMedSearch : van Tilbeurgh_1999_Biochim.Biophys.Acta_1441_173
PubMedID: 10570245

Title : Pancreatic lipase-related protein 1 (PLRP1) is present in the pancreatic juice of several species - De Caro_1998_Biochim.Biophys.Acta_1387_331
Author(s) : De Caro J , Carriere F , Barboni P , Giller T , Verger R , de Caro A
Ref : Biochimica & Biophysica Acta , 1387 :331 , 1998
Abstract : Pancreatic lipase-related protein 1 (PLRP1) was purified from human, canine, porcine and rat pancreatic juices. The four PLRP1s were identified using microsequencing methods after performing gel filtration on Ultrogel AcA-54 followed by chromatography on Heparin-Sepharose cation-exchanger. Polyclonal antibodies specific to human PLRP1 (HPLRP1) were raised in the rabbit using a synthetic decapeptide from HPLRP1. The results of Western blotting analysis showed that these antibodies recognized native HPLRP1 and recombinant HPLRP1 produced by insect cells, and cross-reacted only with rat PLRP1 (RPLRP1). No significant lipolytic activity was observed with native canine PLRP1 and recombinant HPLRP1 on various glycerides, phospholipid and vitamin esters, or on cholesterol esters. It was established for the first time that this protein is secreted in variable amounts by the adult exocrine pancreas of several species.
ESTHER : De Caro_1998_Biochim.Biophys.Acta_1387_331
PubMedSearch : De Caro_1998_Biochim.Biophys.Acta_1387_331
PubMedID: 9748646
Gene_locus related to this paper: canfa-1plip , human-PNLIPRP1 , ratno-3plip , pig-b8xy18

Title : Reactivation of the totally inactive pancreatic lipase RP1 by structure-predicted point mutations - Roussel_1998_Proteins_32_523
Author(s) : Roussel A , De Caro J , Bezzine S , Gastinel L , de Caro A , Carriere F , Leydier S , Verger R , Cambillau C
Ref : Proteins , 32 :523 , 1998
Abstract : Both classical pancreatic lipase (DPL) and pancreatic lipase-related protein 1 (DPLRP1) have been found to be secreted by dog exocrine pancreas. These two proteins were purified to homogeneity from canine pancreatic juice and no significant catalytic activity was observed with dog PLRP1 on any of the substrates tested: di- and tri-glycerides, phospholipids, etc. DPLRP1 was crystallized and its structure solved by molecular replacement and refined at a resolution of 2.10 A. Its structure is similar to that of the classical PL structures in the absence of any inhibitors or micelles. The lid domain that controls the access to the active site was found to have a closed conformation. An amino-acid substitution (Ala 178 Val) in the DPLRP1 may result in a steric clash with one of the acyl chains observed in the structures of a C11 alkyl phosphonate inhibitor, a transition state analogue, bound to the classical PL. This substitution was suspected of being responsible for the absence of DPLRP1 activity. The presence of Val and Ala residues in positions 178 and 180, respectively, are characteristic of all the known PLRP1, whereas Ala and Pro residues are always present in the same positions in all the other members of the PL gene family. Introducing the double mutation Val 178 Ala and Ala 180 Pro into the human pancreatic RP1 (HPLRP1) gene yielded a well expressed and folded enzyme in insect cells. This enzyme is kinetically active on triglycerides. Our findings on DPLRP1 and HPLRP1 are therefore likely to apply to all the RP1 lipases.
ESTHER : Roussel_1998_Proteins_32_523
PubMedSearch : Roussel_1998_Proteins_32_523
PubMedID: 9726421
Gene_locus related to this paper: canfa-1plip

Title : Human pancreatic lipase: an exposed hydrophobic loop from the C-terminal domain may contribute to interfacial binding - Bezzine_1998_Biochemistry_37_11846
Author(s) : Bezzine S , Carriere F , De Caro J , Verger R , de Caro A
Ref : Biochemistry , 37 :11846 , 1998
Abstract : Epitope mapping was performed using four anti-HPL monoclonal antibodies (mAb's 81-23, 146-40, 315-25, and 320-24) directed against human pancreatic lipase (HPL). Three HPL mutants produced in insect cells were tested for this purpose: (i) N-HPL, which consists of only the N-terminal domain of HPL, (ii) HPL(-lid), in which a short loop consisting of 5 amino acid residues replaces the full-length 23-residue lid domain present in HPL, and (iii) N-GPLRP2/C-HPL chimera, a chimeric mutant consisting of the N-terminal domain of the guinea pig pancreatic lipase related protein 2 (GPLRP2) fused to the C-terminal domain of HPL. The C-terminal domain of HPL (C-HPL) was prepared in a pure form after performing chymotryptic digestion of HPL. The mAb 146-40 recognizes HPL, HPL(-lid), and N-HPL but not GPLRP2, N-GPLRP2/C-HPL chimera, or the C-HPL. The antibody mAb 146-40 therefore specifically recognizes the N-terminal domain of HPL, and the epitope recognized does not include the amphiphilic lid. On the other hand, mAb's 81-23, 315-25, and 320-24 react specifically to the C-terminal domain of HPL, since they recognize HPL, HPL(-lid), the N-GPLRP2/C-HPL chimera, and the C-HPL but not N-HPL or GPLRP2. It was further established that these three mAb's recognize the same conformational epitope, the structure of which is stabilized by the N-terminal domain in the presence of SDS at concentrations greater than its critical micellar concentration. This conformational epitope was found to be located in the vicinity of Met 397 and Arg 414. These two residues delineate a highly exposed peptide stretch extending from the HPL C-terminal domain, which includes a hydrophobic surface loop (beta5'). Kinetic studies on the HPL/mAb's complexes showed that the lipase activity was much lower in these complexes than in HPL. The results of the present study suggest for the first time that the beta5' loop from the C-terminal domain may be involved in the interaction of HPL with a lipid/water interface.
ESTHER : Bezzine_1998_Biochemistry_37_11846
PubMedSearch : Bezzine_1998_Biochemistry_37_11846
PubMedID: 9718307

Title : Structural basis for the substrate selectivity of pancreatic lipases and some related proteins - Carriere_1998_Biochim.Biophys.Acta_1376_417
Author(s) : Carriere F , Withers-Martinez C , van Tilbeurgh H , Roussel A , Cambillau C , Verger R
Ref : Biochimica & Biophysica Acta , 1376 :417 , 1998
Abstract : The classical human pancreatic lipase (HPL), the guinea pig pancreatic lipase-related protein 2 (GPLRP2) and the phospholipase A1 from hornet venom (DolmI PLA1) illustrate three interesting steps in the molecular evolution of the pancreatic lipase gene family towards different substrate selectivities. Based on the known 3D structures of HPL and a GPLRP2 chimera, as well as the modeling of DolmI PLA1, we review here the structural features and the kinetic properties of these three enzymes for a better understanding of their structure-function relationships. HPL displays significant activity only on triglycerides, whereas GPLRP2 displays high phospholipase and galactolipase activities, together with a comparable lipase activity. GPLRP2 shows high structural homology with HPL with the exception of the lid domain which is made of five amino acid residues (mini-lid) instead of 23 in HPL. The lid domain deletion in GPLRP2 allows the free access to the active site and reduces the steric hindrance towards large substrates, such as galactolipids. The role of the lid domain in substrate selectivity has been investigated by site-directed mutagenesis and the substitution of HPL and GPLRP2 lid domains. The addition of a large-size lid domain in GPLRP2 increases the substrate selectivity for triglycerides by depressing the phospholipase activity. The phospholipase activity is, however, not induced in the case of the HPL mutant with GPLRP2 mini-lid. Therefore, the presence of a full-length lid domain is not the unique structural feature explaining the absence of phospholipase activity in HPL. The 3D structure of the GPLRP2 chimera and the model of DolmI PLA1 reveal a higher hydrophilic/lipophilic balance (HLB) of the surface loops (beta5 loop, beta9 loop, lid domain) surrounding the active site, as compared to the homologous loops in HPL. This observation provides a potential explanation for the ability of GPLRP2 and DolmI PLA1 to hydrolyze polar lipids, such as phospholipids. In conclusion, the beta5 loop, the beta9 loop, and the lid domain play an essential role in substrate selectivity towards triglycerides, phospholipids and galactolipids.
ESTHER : Carriere_1998_Biochim.Biophys.Acta_1376_417
PubMedSearch : Carriere_1998_Biochim.Biophys.Acta_1376_417
PubMedID: 9805004

Title : An inactive pancreatic lipase-related protein is activated into a triglyceride-lipase by mutagenesis based on the 3-D structure - Bezzine_1998_Chem.Phys.Lipids_93_103
Author(s) : Bezzine S , Roussel A , De Caro J , Gastinel L , de Caro A , Carriere F , Leydier S , Verger R , Cambillau C
Ref : Chemistry & Physic of Lipids , 93 :103 , 1998
Abstract : Both classical dog pancreatic lipase (DPL) and dog pancreatic lipase-related protein 1 (DPLRP1) have been found to be secreted by the exocrine pancreas. These two proteins were purified to homogeneity from canine pancreatic juice and no significant catalytic activity was observed with DPLRP1 on any of the substrates tested: di- and tri-glycerides; phospholipids (PC); etc. DPLRP1 was crystallized and its structure solved by molecular replacement and refined at a resolution of 2.10 A. Its structure is similar to that of the classical pancreatic lipase (PL) structures determined in the absence of any inhibitors or micelles. The lid domain that controls the access to the active site was found to have a closed conformation. An amino-acid substitution (Ala 178 Val) in the DPLRP1 was suspected of being responsible for the absence of enzymatic activity by inducing a steric clash with one of the acyl chain observed in the structures of chiral C11 alkyl phosphonate inhibitors, bound to the classical PL. The presence of Val and Ala residues in positions 178 and 180, respectively, are characteristic of the three known pancreatic lipase-related protein 1 (PLRP1), whereas Ala and Pro residues are always present at the same positions in all the other members of the PL gene family. Introducing the double mutation Val 178 Ala and Ala 180 Pro into the human pancreatic-related protein 1 (HPLRP1) gene yielded a well expressed and folded enzyme in insect cells. This enzyme is kinetically active on tributyrin (1800 U/mg) as well as trioctanoin (2250 U/mg) and its activity is low in the presence of taurodeoxycholate and stimulated in the presence of colipase. Our findings on DPLRP1 and HPLRP1 are therefore likely to apply to all the PLRP1 lipases.
ESTHER : Bezzine_1998_Chem.Phys.Lipids_93_103
PubMedSearch : Bezzine_1998_Chem.Phys.Lipids_93_103
PubMedID: 9720253

Title : Pancreatic lipase structure-function relationships by domain exchange - Carriere_1997_Biochemistry_36_239
Author(s) : Carriere F , Thirstrup K , Hjorth S , Ferrato F , Nielsen PF , Withers-Martinez C , Cambillau C , Boel E , Thim L , Verger R
Ref : Biochemistry , 36 :239 , 1997
Abstract : We designed chimeric mutants by exchanging the lid domains of the classical human pancreatic lipase (HPL) and the guinea pig pancreatic lipase related protein 2 (GPLRP2). This latter enzyme possesses naturally a large deletion within the lid domain and is not activated by lipid/water interfaces. Furthermore, GPLRP2 exhibits phospholipase A1 and lipase activities in the same order of magnitude, whereas HPL has no significant phospholipase activity and displays a clear interfacial activation. An HPL mutant [HPL(-lid)] with GPLRP2 mini-lid domain does not display interfacial activation. Its specific activity toward triglycerides is, however, dramatically reduced. A GPLRP2 mutant [GPLRP2(+lid)] with HPL full-length lid domain is not interfacially activated, and its lid domain probably exists under a permanent open conformation. Therefore, the phenomenon of interfacial activation in HPL is not only due to the presence of a full-length lid domain but also to other structural elements which probably allow the existence of stabilized closed and open conformations of the lid. GPLRP2(+lid) phospholipase activity is significantly reduced as compared to GPLRP2, whereas its lipase activity remains at the same level. Therefore, the lid domain plays a major role in substrate selectivity and can be considered as part of the active site. However, the presence of a full-length lid domain is not sufficient to explain the absence of phospholipase activity in HPL since HPL(-lid) does not display any phospholipase activity. We also produced a chimeric GPLRP2 mutant in which the C-terminal domain was substituted by the HPL C-terminal domain. The colipase effects, i.e., anchoring and stabilization of the lipase at the interface, are clearly observed with the chimera, whereas GPLRP2 is insensitive to colipase. The kinetic characterization of this chimera reveals for the first time that the interfacial stability of pancreatic lipases depends on the structure of the C-terminal domain.
ESTHER : Carriere_1997_Biochemistry_36_239
PubMedSearch : Carriere_1997_Biochemistry_36_239
PubMedID: 8993339

Title : In vivo and in vitro studies on the stereoselective hydrolysis of tri- and diglycerides by gastric and pancreatic lipases - Carriere_1997_Bioorg.Med.Chem_5_429
Author(s) : Carriere F , Rogalska E , Cudrey C , Ferrato F , Laugier R , Verger R
Ref : Bioorganic & Medicinal Chemistry , 5 :429 , 1997
Abstract : The stereoselectivity of dog gastric and dog pancreatic lipases was investigated both in vitro, under simulated physiological conditions, and in vivo, during the digestion of a liquid test meal. In vitro it was observed that although both lipases had a stereopreference for the sn-3 position in triglycerides, it was about three times higher in the case of the gastric lipase. On the other hand, both lipases clearly showed a comparable enantioselectivity for the sn-1 position when a racemic diolein was used as the substrate. In the case of pancreatic lipase, the enantiomeric excess of 1,2-sn-diolein generated in vitro by the hydrolysis of triolein was found to decrease significantly, and even to be slightly reversed, at high rates of hydrolysis (above 50%) due to the further stereoselective hydrolysis of diglycerides into monoglycerides. This finding may explain the low enantiomeric excess of the diglycerides observed in vivo during the early phase of intraduodenal digestion when pancreatic lipase plays a predominant role and the rate of triolein hydrolysis is already high. On the other hand, a large enantiomeric excess of 1,2-sn-diolein generated from triolein was always the fingerprint of the gastric lipase in vitro even at high hydrolysis rates. This fingerprinting of gastric lipase was observed during both the intragastric phase and the late intestinal phase of lipolysis. This feature was therefore taken as an index to determine the respective roles of gastric and pancreatic lipases during in vivo lipolysis. To the best of our knowledge, this is the first time that stereoselectivity has been used as a tool to discriminate between the activities of two enzymes hydrolyzing the same substrate in vivo.
ESTHER : Carriere_1997_Bioorg.Med.Chem_5_429
PubMedSearch : Carriere_1997_Bioorg.Med.Chem_5_429
PubMedID: 9061207

Title : A pancreatic lipase with a phospholipase A1 activity: crystal structure of a chimeric pancreatic lipase-related protein 2 from guinea pig - Withers-Martinez_1996_Structure_4_1363
Author(s) : Withers-Martinez C , Carriere F , Verger R , Bourgeois D , Cambillau C
Ref : Structure , 4 :1363 , 1996
Abstract : BACKGROUND: The guinea pig pancreatic lipase-related protein 2 (GPLRP2) differs from classical pancreatic lipases in that it displays both lipase and phospholipase A1 activities; classical pancreatic lipases have no phospholipase activity. The sequence of GPLRP2 is 63 % identical to that of human pancreatic lipase (HPL), but the so-called lid domain, is much reduced in GPLRP2. A phospholipase A1 from hornet venom (Dolml PLA1) is very similar to HPL and GPLRP2 but is devoid of lipase activity; Dolml PLA1 also contains a reduced lid domain and lacks a region termed the beta9 loop, which is located in the vicinity of the HPL and GPLRP2 active sites. The structure determination of a chimera of GPLRP2 and HPL and domain building of Dolml PLA1 were undertaken to gain a better understanding of the structural parameters responsible for the differences in lipase versus phospholipase activity among these structurally related enzymes. RESULTS: The crystal structure of a chimeric mutant of GPLRP2, consisting of the catalytic domain of GPLRP2 and the C-terminal domain of HPL, has been solved and refined to 2.1 A resolution. This enzyme belongs to the alpha/beta hydrolase fold family and shows high structural homology with classical pancreatic lipases. The active site is closely related to those of serine esterases, except for an unusual geometry of the catalytic triad. Due to the reduced size of the lid domain, the catalytic serine is fully accessible to solvent. Part of the beta9 loop, which stabilizes the lid domain in the closed conformation of the classical HPL, is totally exposed to the solvent and is not visible in the electron-density map. CONCLUSIONS: The structures of the related enzymes, GPLRP2 and HPL and the model of Dolml PLA1, provide insights into the role played by the loops located above the active site in controlling substrate selectivity towards triglycerides or phospholipids. In GPLRP2, the lid domain is reduced in size compared to HPL, and hydrophilic residues are exposed to solvent. GPLRP2 is thus able to accommodate the polar head of phospholipids. The beta9 loop is still present in GPLRP2, making it possible for this enzyme to still accommodate triglycerides. In Dolml PLA1, the beta9 loop is absent, and this enzyme is unable to process triglycerides retaining only the phospholipase A1 activity.
ESTHER : Withers-Martinez_1996_Structure_4_1363
PubMedSearch : Withers-Martinez_1996_Structure_4_1363
PubMedID: 8939760
Gene_locus related to this paper: human-PNLIP

Title : Pancreatic lipase-related protein 2 but not classical pancreatic lipase hydrolyzes galactolipids - Andersson_1996_Biochim.Biophys.Acta_1302_236
Author(s) : Andersson L , Carriere F , Lowe ME , Nilsson A , Verger R
Ref : Biochimica & Biophysica Acta , 1302 :236 , 1996
Abstract : The pancreatic lipase family contains three subfamilies, the 'classical' lipases and the pancreatic lipase-related proteins 1 (PLRP1) and 2 (PLRP2). Galactolipids are present in membranes of leaves and vegetables and consist of digalactosyldiacylglycerol (DGalDG) monogalactosyldiacylglycerol (MGalDG) and sulfoquinovosyldiacylglycerol (SQDG). These lipids were incubated with PLRP2 from guinea-pig (GPLRP2) and rat (RPLRP2). In the presence of bile salts DGalDG was efficiently hydrolyzed by GPLRP2 and, although less efficiently, by RPLRP2 to digalactosylmonoacylglycerol (DGalMG), free fatty acids and water-soluble galactose-containing compounds. Also, MGalDG and SQDG were hydrolyzed by GPLRP2 and RPLRP2. These data suggest a possible role of PLRP2 in the digestion of dietary galactolipids.
ESTHER : Andersson_1996_Biochim.Biophys.Acta_1302_236
PubMedSearch : Andersson_1996_Biochim.Biophys.Acta_1302_236
PubMedID: 8765145

Title : Cloning and expression in insect cells of two pancreatic lipases and a procolipase from Myocastor coypus - Thirstrup_1995_Eur.J.Biochem_227_186
Author(s) : Thirstrup K , Carriere F , Hjorth SA , Rasmussen PB , Nielsen PF , Ladefoged C , Thim L , Boel E
Ref : European Journal of Biochemistry , 227 :186 , 1995
Abstract : The physiological role of pancreatic lipases has traditionally been assigned solely to triacylglyceride metabolism, while the digestion of phospholipids requires the presence of the pancreatic phospholipase A2, a 14-kDa enzyme unrelated to pancreatic lipases. However, in the guinea pig, it was observed that the pancreatic phospholipase A2 was absent and that a guinea pig pancreatic-lipase-related protein 2 (GPL-RP2) was responsible for phospholipase activity, in contrast to the situation observed in other mammalian species. As the guinea pig is a member of the hystricomorph rodents, it was of interest to investigate if other species within this evolutionary suborder display similar characteristics. The coypu (Myocastor coypus) also a member of the hystricomorph rodents, was chosen for further investigations. The cDNAs encoding two pancreatic lipases and a procolipase from the coypu were cloned, expressed and characterized. One lipase, CoPL-RP2, was identified as belonging to the RP2 subfamily, while the second, CoPL, was found to belong to the classical pancreatic lipase subfamily. Enzymic characterization and sequence data suggest a role for coypu colipase as a specific cofactor for CoPL, while this coypu colipase cannot be an important cofactor for CoPL-RP2 in vivo. Also, the new lipase cDNA sequences were used in a phylogentic analysis to reinvestigate the taxonomical position of the hystricomorph rodents (e.g. coypu and guinea pig) with respect to the myomorph rodents (e.g. rat and mouse).
ESTHER : Thirstrup_1995_Eur.J.Biochem_227_186
PubMedSearch : Thirstrup_1995_Eur.J.Biochem_227_186
PubMedID: 7851384
Gene_locus related to this paper: myoco-1plip , myoco-2plrp

Title : Lipase structures at the interface between chemistry and biochemistry - Carriere_1995_Exs_73_3
Author(s) : Carriere F , Verger R , Lookene A , Olivecrona G
Ref : Exs , 73 :3 , 1995
Abstract : In this chapter we review recent molecular knowledge on two structurally related mammalian triglyceride lipases which have evolved from a common ancestral gene. The common property of the lipase family members is that they interact with non-polar substances. Pancreatic lipase hydrolyzes triglycerides in the small intestine in the presence of many dietary components, other digestive enzymes and high concentrations of detergents (bile salts). Lipoprotein lipase acts at the vascular side of the blood vessels where it hydrolyses triglycerides and some phospholipids of the circulating plasma lipoproteins. A third member of the gene family, hepatic lipase, is found in the liver of mammals. Also, this lipase is involved in lipoprotein metabolism. The three lipases are distantly related to some non-catalytic yolk proteins from Drosophila (Persson et al., 1989; Kirchgessner et al., 1989; Hide et al., 1992) and to a phospholipase A1 from hornet venom (Soldatova et al., 1993).
ESTHER : Carriere_1995_Exs_73_3
PubMedSearch : Carriere_1995_Exs_73_3
PubMedID: 7579978

Title : Lipid binding and activating properties of porcine pancreatic colipase split at the Ile79-Thr80 bond - Rugani_1995_Biochim.Biophys.Acta_1247_185
Author(s) : Rugani N , Carriere F , Thim L , Borgstrom B , Sarda L
Ref : Biochimica & Biophysica Acta , 1247 :185 , 1995
Abstract : Porcine colipase, the protein cofactor of pancreatic lipase, was isolated from pancreas freshly collected on animals and from a side fraction from the production of insulin (Novo Nordisk A/S). Samples of purified colipase were analyzed for homogeneity by polyacrylamide gel electrophoresis, reverse-phase high-performance liquid chromatography (RPLC), quantitative N-terminal sequence determination and mass spectrometry. The activating properties of colipase preparations were assayed against tributyrin, triolein or the commercial Intralipid emulsion, in presence of bile salt. Two fractions of colipase with the same specific activity were purified from fresh pancreas. The major fraction (85%) contained one single protein corresponding to fragment 1-93 of the 95-residue form of colipase (procolipase) previously characterized in porcine pancreatic juice. The other fraction (15%) corresponded to fragment 1-91 of procolipase. Also, two fractions of colipase were purified from the side fraction supplied by Novo. These fractions consisted of the 95-residue proform of colipase and of fragment 1-93, respectively, both specifically cleaved at the Ile79-Thr80 peptide bond with partial removal of isoleucine at position 79 and serine at position 78. Procolipase split at the 79-80 bond retained full activity on tributyrin and triolein and on the Intralipid emulsion but the kinetics of hydrolysis of triacylglycerol substrates showed much longer lag periods than those observed with native procolipase. Also, all forms of procolipase split at the 79-80 bond showed one peak in RPLC but their retention time was markedly decreased as compared to that of native procolipase which indicated a weaker hydrophobic binding capacity. The value of the retention time was of the same order of magnitude as that of inactive reduced procolipase. Treatment of native procolipase by pancreatic endopeptidases showed that elastase is likely responsible for specific cleavage at the 79-80 bond of procolipase purified from the Novo extract. Limited proteolysis by trypsin of the proforms of colipase split at the 79-80 bond reduced the lag period. Results presented in this communication provide the first direct evidence showing that the finger-shaped peptide segment between half-cystine residues at positions 69 and 87 is involved in colipase-lipid interaction as previously hypothesized from the three-dimensional structure of the protein.
ESTHER : Rugani_1995_Biochim.Biophys.Acta_1247_185
PubMedSearch : Rugani_1995_Biochim.Biophys.Acta_1247_185
PubMedID: 7696307

Title : Cloning of the classical guinea pig pancreatic lipase and comparison with the lipase related protein 2 - Carriere_1994_FEBS.Lett_338_63
Author(s) : Carriere F , Thirstrup K , Hjorth S , Boel E
Ref : FEBS Letters , 338 :63 , 1994
Abstract : Starting from total pancreatic mRNAs, the classical guinea pig pancreatic lipase was cloned using rapid amplification of 3' and 5' cDNA ends. Internal oligonucleotide primers were designed from a partial cDNA clone including the region coding for the lid domain. Using this strategy, we did not amplify the cDNA corresponding to the pancreatic lipase related protein 2 in which the lid domain is deleted. Amino acid sequences of the classical guinea pig pancreatic lipase and the related protein 2 were compared based on the primary and tertiary structures of the classical human pancreatic lipase. Their distinct physiological roles are discussed in the light of functional amino acid differences.
ESTHER : Carriere_1994_FEBS.Lett_338_63
PubMedSearch : Carriere_1994_FEBS.Lett_338_63
PubMedID: 8307159
Gene_locus related to this paper: cavpo-1plip

Title : Evidence for a pancreatic lipase subfamily with new kinetic properties - Thirstrup_1994_Biochemistry_33_2748
Author(s) : Thirstrup K , Verger R , Carriere F
Ref : Biochemistry , 33 :2748 , 1994
Abstract : Several new members of the pancreatic lipase family have been reported recently, and amino acid sequence comparison reveals that this family can now be divided into three subgroups: (1) "classical" pancreatic lipases, (2) related proteins 1 (RP1), and (3) related proteins 2 (RP2) (Giller, T., et al. (1992) J. Biol. Chem. 267(23), 16509-16516). Whereas "classical" pancreatic lipases are well characterized with respect to kinetic properties, i.e., interfacial activation and dependence on colipase in the presence of bile salts, the two latter subfamilies have been poorly investigated so far. The kinetic behavior of a lipase from guinea pig pancreas differs, however, from that of "classical" lipases (Hjorth, A., et al. (1993) Biochemistry 32, 4702-4707). This enzyme is highly homologous to RP2 lipases with the exception of a deletion in the so-called lid domain that regulates access to the active center of pancreatic lipases. We have now characterized a novel lipase from coypu (Myocastor coypus) pancreas. This enzyme, also belonging to the RP2 subfamily, possesses a full-length lid domain, but its kinetic properties are very similar to those of the guinea pig enzyme: (1) a high phospholipase activity, (2) the absence of interfacial activation, and (3) the absence of a colipase effect at high bile salt concentrations. Since both guinea pig and coypu pancreas produce a classical pancreatic lipase and no measurable phospholipase A2 activity, it is suggested that RP2 enzymes act as real phospholipases under physiological conditions. In fact, all RP2 lipases from other species might share phospholipase activity and fulfill new biological functions.
ESTHER : Thirstrup_1994_Biochemistry_33_2748
PubMedSearch : Thirstrup_1994_Biochemistry_33_2748
PubMedID: 8130186
Gene_locus related to this paper: myoco-2plrp

Title : Structure-function relationships in naturally occurring mutants of pancreatic lipase - Carriere_1994_Protein.Eng_7_563
Author(s) : Carriere F , Thirstrup K , Boel E , Verger R , Thim L
Ref : Protein Engineering , 7 :563 , 1994
Abstract : From primary structure comparison, the pancreatic lipase family is now divided into three subgroups: classical pancreatic lipases, pancreatic lipase-related proteins 1 (RPI) and pancreatic lipase-related proteins 2 (RP2). Among the RP2 subfamily, the guinea-pig and coypu enzymes share kinetic properties which differ from those of classical pancreatic lipases. Both enzymes display a high phospholipase activity and are not interfacially activated using a short chain triglyceride as substrate. Their activity towards insoluble triglycerides is inhibited by micellar concentrations of bile salts and is not restored by addition of colipase. These atypical kinetic properties are discussed in the light of amino acid sequence comparison between RP2 and classical pancreatic lipases, based on the closed and open conformations of the 3-D structure of human pancreatic lipase.
ESTHER : Carriere_1994_Protein.Eng_7_563
PubMedSearch : Carriere_1994_Protein.Eng_7_563
PubMedID: 8029213

Title : A structural domain (the lid) found in pancreatic lipases is absent in the guinea pig (phospho)lipase - Hjorth_1993_Biochemistry_32_4702
Author(s) : Hjorth A , Carriere F , Cudrey C , Woldike H , Boel E , Lawson DM , Ferrato F , Cambillau C , Dodson GG , Thim L , et al.
Ref : Biochemistry , 32 :4702 , 1993
Abstract : Typically pancreatic lipases are characterized by the following properties: (1) they are activated by lipid/water interfaces (interfacial activation), (2) they are inhibited by bile salts but reactivated by colipase (a small activator protein), and (3) they do not hydrolyze significantly phospholipids. A cDNA clone encoding a guinea pig pancreatic (phospho)lipase (GPL) has been sequenced and expressed. The enzyme (recombinant as well as native) differs from other pancreatic lipases in that (1) it is not interfacially activated, (2) its activity is unaffected by the presence of bile salts and/or colipase using tributyrin as substrate, and (3) it exhibits equally phospholipase A1 and lipase activities. The amino acid sequence of GPL is highly homologous to that of other known pancreatic lipases, with the exception of a deletion in the so-called lid domain that regulates access to the active centers of other lipases. We propose that this deletion is directly responsible for the anomalous behavior of this enzyme. Thus GPL challenges the classical distinction between lipases, esterases, and phospholipases.
ESTHER : Hjorth_1993_Biochemistry_32_4702
PubMedSearch : Hjorth_1993_Biochemistry_32_4702
PubMedID: 8490016
Gene_locus related to this paper: cavpo-2plrp , ratno-1plip

Title : Gastric and pancreatic lipase levels during a test meal in dogs - Carriere_1993_Scand.J.Gastroenterol_28_443
Author(s) : Carriere F , Laugier R , Barrowman JA , Douchet I , Priymenko N , Verger R
Ref : Scand J Gastroenterol , 28 :443 , 1993
Abstract : The levels of gastric and pancreatic lipases in the duodenum and the jejunum were measured during the digestion of a test meal in dogs. Using both a specific enzymatic titration and an enzyme-linked immunosorbent assay, it is shown for the first time that gastric lipase remains active in the duodenal and jejunal contents. An experimental device was set up for measuring the secretions and the intestinal flows of lipases during the digestion of a liquid test meal. In a dog equipped with gastric and duodenal cannulae, the secretion of gastric lipase was stimulated by food ingestion, reaching 3.0 +/- 0.3 mg/h (three times the basal secretion rate) during the 1st h of digestion. The total secretory outputs of gastric and pancreatic lipases recorded over a 3-h period of digestion were 7.2 +/- 1.2 mg and 18.7 +/- 1.2 mg, respectively.
ESTHER : Carriere_1993_Scand.J.Gastroenterol_28_443
PubMedSearch : Carriere_1993_Scand.J.Gastroenterol_28_443
PubMedID: 8511506

Title : Dog gastric lipase: stimulation of its secretion in vivo and cytolocalization in mucous pit cells - Carriere_1992_Gastroenterology_102_1535
Author(s) : Carriere F , Raphel V , Moreau H , Bernadac A , Devaux MA , Grimaud R , Barrowman JA , Benicourt C , Junien JL , Laugier R , Verger R
Ref : Gastroenterology , 102 :1535 , 1992
Abstract : Dog gastric lipase (DGL) secretion is stimulated in vivo by urecholine, pentagastrin, histamine, 16,16-dimethyl prostaglandin E2, and secretin. Under fasting conditions, DGL is irreversibly inactivated by gastric acid below pH 1.5; consequently, DGL output can be underestimated. This problem has been resolved by buffering the acid or by using an antisecretory drug such as omeprazole during stimulation. There is a clear parallelism between the secretion of DGL and of gastric mucus. This observation led to the present investigation of the cellular localization of DGL using immunofluorescence techniques. Results showed that DGL is cytolocalized in mucous pit cells of gastric glands. Pepsinogen is found in chief cells. To the authors' knowledge, this is the first description of an enzyme (gastric lipase) secreted by mucous-type gastric cells. In contrast to other species, gastric lipase of the dog is located in cardiac, fundic, and antral mucosae.
ESTHER : Carriere_1992_Gastroenterology_102_1535
PubMedSearch : Carriere_1992_Gastroenterology_102_1535
PubMedID: 1568562

Title : Isoform purification of gastric lipases. Towards crystallization - Moreau_1992_J.Mol.Biol_225_147
Author(s) : Moreau H , Abergel C , Carriere F , Ferrato F , Fontecilla-Camps JC , Cambillau C , Verger R
Ref : Journal of Molecular Biology , 225 :147 , 1992
Abstract : Several isoforms of rabbit and human gastric lipases have been purified. These isoforms have the same apparent molecular weight (Mr approximately 50,000), but very different isoelectric points. Some of these isoforms were purified: pI 7.2 and 6.5 in the case of rabbit gastric lipase; and pI 7.4 and 7.2 in that of human gastric lipase. All the purified isoforms were found to have the same specific lipase activity (around 1200 units per mg of protein, measured on tributyrin as substrate). The isoforms of dog gastric lipase are more closely related, and could not be separated. Partial enzymatic deglycosylation of human gastric lipase reduced the apparent molecular weight from Mr approximately 50,000 to Mr approximately 43,000 and induced a change in the isoelectrofocusing pattern and the emergence of a new isoform (pI 7.3). It is concluded that the charge heterogeneity of gastric lipases is at least partly due to the glycan moiety of the molecule, which amounts to approximately 14% of the total molecular weight. Several crystallization trials on purified native preparations of rabbit and human gastric lipases were unsuccessful, whereas crystals were obtained from native dog gastric lipase and all the purified isoforms of rabbit and human gastric lipases, some of which were crystallographically characterized.
ESTHER : Moreau_1992_J.Mol.Biol_225_147
PubMedSearch : Moreau_1992_J.Mol.Biol_225_147
PubMedID: 1583687

Title : Purification and biochemical characterization of dog gastric lipase - Carriere_1991_Eur.J.Biochem_202_75
Author(s) : Carriere F , Moreau H , Raphel V , Laugier R , Benicourt C , Junien JL , Verger R
Ref : European Journal of Biochemistry , 202 :75 , 1991
Abstract : A lipase was found to be present in dog stomach which appeared to be more abundant in the fundic than in the pyloric mucosa. Dog gastric lipase was extracted by soaking the gastric tissue and further purified after cation exchange, anion exchange and gel-filtration using fast protein liquid chromatography. The amino-acid composition, N-terminal amino-acid sequence, substrate specificity, interfacial and kinetic behavior and inactivation by sulfhydryl reagents were determined and compared with those of human and rabbit gastric lipases. We report for the first time that a gastric lipase is 13 times more active on long-chain than on short-chain triacylglycerols at pH 4.0, reaching a maximal specific activity of 950 U/mg on Intralipide emulsion.
ESTHER : Carriere_1991_Eur.J.Biochem_202_75
PubMedSearch : Carriere_1991_Eur.J.Biochem_202_75
PubMedID: 1935982
Gene_locus related to this paper: canfa-1lipg