Verger R

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Full name : Verger Robert

First name : Robert

Mail : CNRS University of Aix-Marseille Enzymologie Interfaciale et Physiologie de la Lipolyse UPR 9025, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20

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Country : France

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References (91)

Title : Development of a high-throughput assay for measuring lipase activity using natural triacylglycerols coated on microtiter plates - Serveau-Avesque_2013_Analyst_138_5230
Author(s) : Serveau-Avesque C , Verger R , Rodriguez JA , Abousalham A
Ref : Analyst , 138 :5230 , 2013
Abstract : We have designed a convenient, specific, sensitive and continuous lipase assay based on the use of natural triacylglycerols (TAGs) from the Aleurites fordii seed oil which contains alpha-eleostearic acid (9,11,13,cis,trans,trans-octadecatrienoic acid) and which was coated in the wells of microtiter plates. The coated TAG film cannot be desorbed by the various buffers used during the lipase assay. Upon lipase action, alpha-eleostearic acid is liberated and desorbed from the interface and then solubilized into the micellar phase. Consequently, the UV absorbance of the alpha-eleostearic acid is considerably enhanced due to the transformation from an adsorbed to a water soluble state. The lipase activity can be measured continuously by recording the variations with time of the UV absorption spectra. The rate of lipolysis was monitored by measuring the increase of OD at 272 nm, which was found to be linear with time and directly proportional to the amount of added lipase. This microtiter plate lipase assay, based on coated TAGs, presents various advantages as compared to the classical systems: (i) coated TAGs on the microtiter plates could be stored for a long-time at 4 degrees C, (ii) higher sensitivity in lipase detection, (iii) good reproducibility, and (iv) increase of signal to noise ratio due to high UV absorption after transfer of alpha-eleostearic acid from an adsorbed to a soluble state. Low concentrations, down to 1 pg mL(-1) of pure Thermomyces lanuginosus or human pancreatic lipase, could be detected under standard assay conditions. The detection sensitivity of this coated method is around 1000 times higher as compared to those obtained with the classical emulsified systems. This continuous high throughput lipase assay could be used to screen new lipases and/or lipase inhibitors present in various biological samples.
ESTHER : Serveau-Avesque_2013_Analyst_138_5230
PubMedSearch : Serveau-Avesque_2013_Analyst_138_5230
PubMedID: 23851449

Title : Lipases or esterases: does it really matter? Toward a new bio-physico-chemical classification - Ben Ali_2012_Methods.Mol.Biol_861_31
Author(s) : Ben Ali Y , Verger R , Abousalham A
Ref : Methods Mol Biol , 861 :31 , 2012
Abstract : Carboxylester hydrolases, commonly named esterases, consist of a large spectrum of enzymes defined by their ability to catalyze the hydrolysis of carboxylic ester bonds and are widely distributed among animals, plants, and microorganisms. Lipases are lipolytic enzymes which constitute a special class of carboxylic esterases capable of releasing long-chain fatty acids from natural water-insoluble carboxylic esters. However, up to now, several unsuccessful attempts aimed at differentiating "lipases" from "esterases" by using various criteria. These criteria were based on the first substrate used chronologically, primary sequence comparisons, some kinetic parameters, or some structural features.Lipids are biological compounds which, by definition, are insoluble in water. Taking into account this basic physico-chemical criterion, we primarily distinguish lipolytic esterases (L, acting on lipids) from nonlipolytic esterases (NL, not acting on lipids). In view of the biochemical data accumulated up to now, we proposed a new classification of esterases based on various criteria of physico-chemical, chemical, anatomical, or cellular nature. We believe that the present attempt matters scientifically for several reasons: (1) to help newcomers in the field, performing a few key experiments to figure out if a newly isolated esterase is lipolytic or not; (2) to clarify a debate between scientists in the field; and (3) to formulate questions which are relevant to the still unsolved problem of the structure-function relationships of esterases.
ESTHER : Ben Ali_2012_Methods.Mol.Biol_861_31
PubMedSearch : Ben Ali_2012_Methods.Mol.Biol_861_31
PubMedID: 22426710

Title : Phospholipase A2 purification and characterization: a case study - Karray_2012_Methods.Mol.Biol_861_283
Author(s) : Karray A , Gargouri Y , Verger R , Bezzine S
Ref : Methods Mol Biol , 861 :283 , 2012
Abstract : We compared here the purification procedures, the pH, the calcium, the bile salts, and the temperature dependencies as well as the catalytic activities on phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of two purified secreted PLA2 from chicken pancreatic (ChPLA2-IB) and chicken intestinal (ChPLA2-IIA) origins. Interestingly, ChPLA2-IB hydrolyzes efficiently both purified PC and PE, whereas ChPLA2-IIA hydrolyzes only PE and not PC, even after a long incubation period. These analytical results clearly indicate that the catalytic activity of ChPLA2-IIA, measured with the pH-stat and using egg yolk as substrate, is mainly due to the hydrolysis of the PE fraction present in egg yolk.
ESTHER : Karray_2012_Methods.Mol.Biol_861_283
PubMedSearch : Karray_2012_Methods.Mol.Biol_861_283
PubMedID: 22426725

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 : Purification, biochemical and kinetic properties of recombinant Staphylococcus aureus lipase - Horchani_2012_Methods.Mol.Biol_861_267
Author(s) : Horchani H , Fendri A , Louati H , Sayari A , Gargouri Y , Verger R
Ref : Methods Mol Biol , 861 :267 , 2012
Abstract : We have compared the purification procedures as well as the biochemical and kinetic properties of wild type (wt-SAL3), untagged recombinant (rec(-His)SAL3), and tagged recombinant (rec(+His)SAL3) purified forms of Staphylococcus aureus lipase (SAL3). We used the pH-stat method (with emulsified tributyrin and olive oil as substrates) and the monomolecular film technique (with the three dicaprin isomers spread in the form of monomolecular films at the air-water interface). The data obtained showed that the recombinant expression process as well as the presence of a his-tag at the N-terminus of recombinant SAL3 affects significantly many biochemical and catalytic properties. The effects of the heterologous expression process on the catalytic properties of the staphylococcal lipases are three times more deleterious than the presence of an N-terminal tag extension.
ESTHER : Horchani_2012_Methods.Mol.Biol_861_267
PubMedSearch : Horchani_2012_Methods.Mol.Biol_861_267
PubMedID: 22426724

Title : Staphylococcal lipases stereoselectively hydrolyse the sn-2 position of monomolecular films of diglyceride analogs. Application to sn-2 hydrolysis of triolein - Horchani_2010_J.Colloid.Interface.Sci_347_301
Author(s) : Horchani H , Ben Salem N , Chaari A , Sayari A , Gargouri Y , Verger R
Ref : J Colloid Interface Sci , 347 :301 , 2010
Abstract : Using the monomolecular film technique, a kinetic study on the stereoselectivity of nine staphylococcal lipase forms was carried out with three pairs of enantiomers from diglyceride analogs (didecanoyl-deoxyamino-O-methyl glycerol, DDG) containing a single hydrolysable decanoyl ester group and two lipase-resistant groups. Our results show that the kinetic profiles of the wild type, the recombinant untagged and the recombinant tagged forms of staphylococcal lipases are significantly different. As with most of the lipases investigated so far, these staphylococcal lipases showed higher catalytic rates with primary esters than with secondary esters. However, it is noteworthy that all these staphylococcal lipases were found to significantly hydrolyse the secondary ester group of diglyceride analogs, with a strong preference for the R configuration. This stereopreference, which was predicted on the basis of Kazlauskas' rule, was comparable to that of Candida rugosa and Pseudomonas glumae lipases. As was to be expected, all the staphylococcal lipases tested efficiently hydrolysed triolein at the sn-2 position. This hydrolytic activity was quantified by performing thin-layer chromatography to analyse the hydrolytic products of triolein. From the qualitative point of view, the sn-2 preferences observed with triolein and diglyceride analogs bearing a secondary ester function were in good agreement. Diglyceride analogs might therefore provide useful initial screening tools for use in future searches for strictly sn-2 specific lipases.
ESTHER : Horchani_2010_J.Colloid.Interface.Sci_347_301
PubMedSearch : Horchani_2010_J.Colloid.Interface.Sci_347_301
PubMedID: 20403605

Title : Heterologous expression and N-terminal His-tagging processes affect the catalytic properties of staphylococcal lipases: a monolayer study - Horchani_2010_J.Colloid.Interface.Sci_350_586
Author(s) : Horchani H , Sabrina L , Regine L , Sayari A , Gargouri Y , Verger R
Ref : J Colloid Interface Sci , 350 :586 , 2010
Abstract : The interfacial and kinetic properties of wild type, untagged recombinant and tagged recombinant forms of three staphylococcal lipases (SSL, SXL and SAL3) were compared using the monomolecular film technique. A kinetic study on the dependence of the stereoselectivity of these nine lipase forms on the surface pressure was performed using the three dicaprin isomers spread in the form of monomolecular films at the air-water interface. New parameters, termed Recombinant expression Effects on Catalysis (REC), N-Tag Effects on Catalysis (TEC), and N-Tag and Recombinant expression Effects on Catalysis (TREC), were introduced. The findings obtained showed that with all the lipases tested, the recombinant expression process and the N-terminal His-tag slightly affect the sn-1 preference for dicaprin enantiomers as well as the penetration capacity into monomolecular films of phosphatidylcholine but significantly decrease the catalytic rate of hydrolysis of three dicaprin isomers. This rate reduction is more pronounced at high surface pressures, i.e. at low interfacial energies. In conclusion, the effects of the heterologous expression process on the catalytic properties of the staphylococcal lipases are three times more deleterious than the presence of an N-terminal tag extension. In the case of the situation most commonly encountered in the literature, i.e. the heterologous expression of a tagged lipase, the rate of catalysis can be decreased by these processes by 42-83% on average in comparison with the values measured with the corresponding wild type form.
ESTHER : Horchani_2010_J.Colloid.Interface.Sci_350_586
PubMedSearch : Horchani_2010_J.Colloid.Interface.Sci_350_586
PubMedID: 20684959

Title : Action of Humicola lanuginosa lipase on mixed monomolecular films of tricaprylin and polyethylene glycol stearate - Ivanova_2008_Colloids.Surf.B.Biointerfaces_63_91
Author(s) : Ivanova T , Mircheva K , Dobreva G , Panaiotov I , Proust JE , Verger R
Ref : Colloids Surf B Biointerfaces , 63 :91 , 2008
Abstract : The hydrolysis catalyzed by Humicola lanuginosa lipase (HLL) of pure tricaprylin (TC) or stearate of polyethylene glycol 1500 (PEG-St) as well as their mixtures spread as monomolecular films were studied. The catalytic transformation of the two substrates TC or PEG-St into their respective reaction products was detected by measuring simultaneously the decrease in the film area and the surface potential using the "zero order" trough at constant surface pressure. A kinetic model describing the enzymatic hydrolysis was developed. The surface concentrations of the two substrates and their respective reaction products as well as the values of the global kinetic constants of hydrolysis were determined. The experimentally obtained global kinetic constants of the catalytic action of HLL against TC and PEG-St present in mixed monolayers of TC/PEG-St are approximately the same as in the case of pure monolayers. These obtained results give some indications that the activity of enzyme is not significantly affected by the different molecular environments in the mixed monolayers.
ESTHER : Ivanova_2008_Colloids.Surf.B.Biointerfaces_63_91
PubMedSearch : Ivanova_2008_Colloids.Surf.B.Biointerfaces_63_91
PubMedID: 18178069

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 : 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 : Gly311 residue triggers the enantioselectivity of Staphylococcus xylosus lipase: a monolayer study - Mosbah_2007_J.Colloid.Interface.Sci_310_196
Author(s) : Mosbah H , Sayari A , Verger R , Gargouri Y
Ref : J Colloid Interface Sci , 310 :196 , 2007
Abstract : Using emulsified triacylglycerols, we have shown recently [Mosbah et al., 2007, submitted for publication] that amino acid residue G311 of Staphylococcus xylosus lipase (SXL) is critically involved in substrate selectivity, pH and temperature dependency. Using the monomolecular film technique, we show in the present study that the four single mutants of this residue (G311L, G311W, G311D, and G311K), interact efficiently with egg-phosphatidyl choline (egg-PC) monomolecular films, comparably to the wild-type (G311). A critical surface pressure (pi(c)) of about 25 mN/m was obtained with the SXL wild-type (SXL-WT) and its mutants. These results support our conclusion that the G311 residue is not involved in the interfacial adsorption step of SXL. A kinetic study on the surface pressure dependency, stereoselectivity, and regioselectivity of SXL-WT and its G311 mutants was also performed using optically pure enantiomers of diacylglycerols (1,2-sn-dicaprin and 2,3-sn-dicaprin) and a prochiral isomer (1,3-sn-dicaprin) spread as monomolecular films at the air-water interface. Our results indicated that the mutation of one single residue at position 311 affects critically the catalytic activity, the stereo- and the regioselectivity of SXL. As previously observed with emulsified substrates [Mosbah et al., 2007, submitted for publication] we observed that an increase in the size of the 311 amino acid side chain residue was accompanied by a decrease of lipase activity measured on dicaprin monolayer. We also noticed that the substitution of G311 by a basic or acidic residue (G311K and G311D), induces a significant shift of the pH optimum from 8 to 9.5 or from 8 to 6.5, respectively.
ESTHER : Mosbah_2007_J.Colloid.Interface.Sci_310_196
PubMedSearch : Mosbah_2007_J.Colloid.Interface.Sci_310_196
PubMedID: 17335837

Title : Scorpion digestive lipase: kinetic study using monomolecular film technique - Zouari_2006_Colloids.Surf.B.Biointerfaces_49_8
Author(s) : Zouari N , Sayari A , Miled N , Verger R , Gargouri Y
Ref : Colloids Surf B Biointerfaces , 49 :8 , 2006
Abstract : Using the classical emulsified system and the monomolecular film technique, we compared the interfacial properties of the scorpion digestive lipase (SDL) with those of higher animals'. In the absence of bile slats, SDL does not hydrolyse efficiently pure tributyrin, as well as dicaprin films maintained at low surface pressure. The preincubation of bile salts with tributyrin seems to be a better substrate for SDL than the pure tributyrin. A kinetic study on the surface pressure dependency, stereospecificity and regioselectivity of SDL was performed using monomolecular films of either three dicaprin isomers or three pairs of didecanoyl-deoxyamino-O-methyl glycerol enantiomers (DDG) containing a single hydrolysable decanoyl ester bond. With all diacylglycerol isomers, SDL has a surface pressure threshold of about 15 m Nm(-1), below which enzymatic activity is undetectable. SDL seems to prefer vicinal ester groups of the diacylglycerol isomers, with preference for sn-1 position at both 15 and 23 m Nm(-1). Furthermore, the maximum SDL activity is measured with DDG having a primary ester bond (1,3DDG, SII). This shows that SDL has a preference for the sn-1 position of this diacylglycerol analogue. Moreover, this was in line with the fact that SDL is inactive on sn-2 position of both DDG isomers and a triacylglycerol. With diacylglycerol analogue isomers, SDL shows a preference for distal isomers contrary to what has been observed with diacylglycerol isomers. SDL interacts with egg-phosphatidyl choline (egg-PC) monomolecular films. The critical surface pressure value (13 m Nm(-1)) is comparable to those of pancreatic lipases.
ESTHER : Zouari_2006_Colloids.Surf.B.Biointerfaces_49_8
PubMedSearch : Zouari_2006_Colloids.Surf.B.Biointerfaces_49_8
PubMedID: 16580184

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 : Discrimination between closed and open forms of lipases using electrophoretic techniques - Miled_2005_Anal.Biochem_338_171
Author(s) : Miled N , Riviere M , Cavalier JF , Buono G , Berti L , Verger R
Ref : Analytical Biochemistry , 338 :171 , 2005
Abstract : The enhanced catalytic activity of lipases is often associated with structural changes. The three-dimensional (3D) structures showed that the covalently inhibited lipases exist under their open conformations, in contrast to their native closed forms. We studied the inhibition of various lipases--human and dog gastric lipases, human pancreatic lipase, and Humicola lanuginosa lipase--by the octyl-undecyl phosphonate inhibitor, and we measured the subsequent modifications of their respective electrophoretic mobility. Furthermore, the experimental values of the isoelectric points found for the native (closed) and inhibited (open) lipases are in agreement with theoretical calculations based on the electrostatic potential. We concluded that there is a significant difference in the isoelectric points between the closed (native) and open (inhibited) conformations of the four lipases investigated. Thus, analysis of the electrophoretic pattern is proposed as an easy experimental tool to differentiate between a closed and an open form of a given lipase.
ESTHER : Miled_2005_Anal.Biochem_338_171
PubMedSearch : Miled_2005_Anal.Biochem_338_171
PubMedID: 15745736

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 : N-terminal peptide of Rhizopus oryzae lipase is important for its catalytic properties - Sayari_2005_FEBS.Lett_579_976
Author(s) : Sayari A , Frikha F , Miled N , Mtibaa H , Ben Ali Y , Verger R , Gargouri Y
Ref : FEBS Letters , 579 :976 , 2005
Abstract : In a culture medium, the Rhizopus oryzae strain produces only one form of lipase, ROL32. When the concentrated culture medium was stored at 0 degrees C during several months or kept at 6 degrees C during a few days, we noticed the appearance of a second shorter form of ROL32 lacking its N-terminal 28 amino acid (ROL29). ROL29 was purified to homogeneity and its 21 N-terminal amino acid residues were found to be identical to the 29-49 sequence of ROL32. The cleavage of the N-terminal peptide reduced the specific activity of ROL29 by 50% using either triolein or tributyrin as substrates. In order to explain this decrease of the specific activity of ROL29, we measured its critical surface pressure of penetration into phosphatidyl choline from egg yolk films which was found to be 10 mN/m, in contrast to a value of 23 mN/m found in ROL32. A kinetic study on the surface pressure dependency, stereoselectivity and regioselectivity of ROL29 was performed using the three dicaprin isomers spread as monomolecular films at the air-water interface. Our results showed that in contrast to ROL32, ROL29 presented a preference for the distal ester groups of one diglyceride isomer (1,3-sn-dicaprin). Furthermore, ROL32 was markedly more stereoselective than ROL29 for the sn-3 position of the 2,3-sn-enantiomer of dicaprin. A structural explanation of the enhanced penetration capacity as well as the catalytic activity of ROL32 was proposed by molecular modeling. We concluded that the N-terminal peptide of ROL32 can play an important role in the specific activity, the regioselectivity, the stereoselectivity and the binding of the enzyme to its substrate.
ESTHER : Sayari_2005_FEBS.Lett_579_976
PubMedSearch : Sayari_2005_FEBS.Lett_579_976
PubMedID: 15710378
Gene_locus related to this paper: rhidl-lipas

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 : 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 : Inhibition of dog and human gastric lipases by enantiomeric phosphonate inhibitors: a structure-activity study - Miled_2003_Biochemistry_42_11587
Author(s) : Miled N , Roussel A , Bussetta C , Berti-Dupuis L , Riviere M , Buono G , Verger R , Cambillau C , Canaan S
Ref : Biochemistry , 42 :11587 , 2003
Abstract : The crystal structures of gastric lipases in the apo form [Roussel, A., et al. (1999) J. Biol. Chem. 274, 16995-17002] or in complex with the (R(P))-undecyl butyl phosphonate [C(11)Y(4)(+)] [Roussel, A., et al. (2002) J. Biol. Chem. 277, 2266-2274] have improved our understanding of the structure-activity relationships of acid lipases. In this report, we have performed a kinetic study with dog and human gastric lipases (DGL and HGL, respectively) using several phosphonate inhibitors by varying the absolute configuration of the phosphorus atom and the chain length of the alkyl/alkoxy substituents. Using the two previously determined structures and that of a new crystal structure obtained with the other (S(P))-phosphonate enantiomer [C(11)Y(4)(-)], we constructed models of phosphonate inhibitors fitting into the active site crevices of DGL and HGL. All inhibitors with a chain length of fewer than 12 carbon atoms were found to be completely buried in the catalytic crevice, whereas longer alkyl/alkoxy chains were found to point out of the cavity. The main stereospecific determinant explaining the stronger inhibition of the S(P) enantiomers is the presence of a hydrogen bond involving the catalytic histidine as found in the DGL-C(11)Y(4)(-) complex. On the basis of these results, we have built a model of the first tetrahedral intermediate corresponding to the tristearoyl-lipase complex. The triglyceride molecule completely fills the active site crevice of DGL, in contrast with what is observed with other lipases such as pancreatic lipases which have a shallower and narrower active site. For substrate hydrolysis, the supply of water molecules to the active site might be achieved through a lateral channel identified in the protein core.
ESTHER : Miled_2003_Biochemistry_42_11587
PubMedSearch : Miled_2003_Biochemistry_42_11587
PubMedID: 14529268

Title : Novel trifluoromethyl ketones as potent gastric lipase inhibitors - Kokotos_2003_Chembiochem_4_90
Author(s) : Kokotos G , Kotsovolou S , Verger R
Ref : Chembiochem , 4 :90 , 2003
Abstract : Novel inhibitors of human digestive lipases, lipophilic trifluoromethyl ketones, were developed. These analogues of the natural triacylglycerol substrates of lipases were designed to contain the carbonyl group of the trifluoromethyl ketone functionality in place of the carbonyl group of the scissile ester bond at the sn-1 position. The ester bond at the sn-3 position was replaced by an ether bond, while the secondary hydroxy group was either esterified or etherified. The inhibitors were prepared starting from solketal. The inhibition of human pancreatic and gastric lipases by the trifluoromethyl ketones was studied by the monolayer technique. 5,5,5-Trifluoro-1-(dodecyloxymethyl)-4-oxopentyl decanoate is the best synthetic inhibitor of human gastric lipase ever reported (inhibition constant alpha(50)=0.003).
ESTHER : Kokotos_2003_Chembiochem_4_90
PubMedSearch : Kokotos_2003_Chembiochem_4_90
PubMedID: 12512081

Title : Conformational changes and orientation of Humicola lanuginosa lipase on a solid hydrophobic surface: an in situ interface Fourier transform infrared-attenuated total reflection study - Noinville_2002_Biophys.J_82_2709
Author(s) : Noinville S , Revault M , Baron MH , Tiss A , Yapoudjian S , Ivanova M , Verger R
Ref : Biophysical Journal , 82 :2709 , 2002
Abstract : This study was done to better understand how lipases are activated at an interface. We investigated the conformational and solvation changes occurring during the adsorption of Humicola lanuginosa lipase (HLL) onto a hydrophobic surface using Fourier transform infrared-attenuated total reflection spectroscopy. The hydrophobic surfaces were obtained by coating silicon attenuated total reflection crystal with octadecyltrichlorosilane. Analysis of vibrational spectra was used to compare the conformation of HLL adsorbed at the aqueous-solid interface with its conformation in solution. X-ray crystallography has shown that HLL exists in two conformations, the closed and open forms. The conformational changes in HLL caused by adsorption onto the surface were compared with those occurring in three reference proteins, bovine serum albumin, lysozyme, and alpha-chymotrypsin. Adsorbed protein layers were prepared using proteins solutions of 0.005 to 0.5 mg/mL. The adsorptions of bovine serum albumin, lysozyme, and alpha-chymotrypsin to the hydrophobic support were accompanied by large unfoldings of ordered structures. In contrast, HLL underwent no secondary structure changes at first stage of adsorption, but there was a slight folding of beta-structures as the lipase monolayer became complete. Solvation studies using deuterated buffer showed an unusual hydrogen/deuterium exchange of the peptide CONH groups of the adsorbed HLL molecules. This exchange is consistent with the lipase being in the native open conformation at the water/hydrophobic interface.
ESTHER : Noinville_2002_Biophys.J_82_2709
PubMedSearch : Noinville_2002_Biophys.J_82_2709
PubMedID: 11964257

Title : Surface fluorescence resonance energy transfer studies on interfacial adsorption of Thermomyces (humicola) lanuginosa lipase, using monomolecular films of cis-parinaric acid - Yapoudjian_2002_Biopolymers_65_121
Author(s) : Yapoudjian S , Ivanova M , Douchet I , Zenatti A , Sentis M , Marine W , Svendsen A , Verger R
Ref : Biopolymers , 65 :121 , 2002
Abstract : The fluorescence resonance energy transfer (FRET) technique was adapted to study the process whereby lipase is adsorbed to monomolecular lipid films spread at the air-water interface. When cis-parinaric acid (cis-PnA) was spread over an aqueous subphase before the injection of sodium taurodeoxycholate (NaTDC) and Thermomyces lanuginosa lipase (TLL), no FRET was observed. Under these conditions, no adsorption of TLL was detected using an ELISA. In contrast, FRET occurred when cis-PnA was spread over an aqueous subphase containing NaTDC and TLL. The FRET signals observed were attributed to the interactions between the adsorbed TLL and the cis-PnA monomolecular films. Comparisons between the fluorescence emission spectra corresponding to the bulk phase and the aspirated film, in the presence and absence of TLL, showed that cis-PnA was undetectable in the bulk phase. We concluded that the FRET originated from the interface and not from the bulk phase. Using surface FRET, we estimated that the surface excess of the catalytically inactive mutant, TLL(S146A), was 1.6 higher than that present in the wild-type TLL. This finding is in agreement with independent measurements of the surface excess of TLL and TLL(S146A) on monomolecular films of cis-PnA.
ESTHER : Yapoudjian_2002_Biopolymers_65_121
PubMedSearch : Yapoudjian_2002_Biopolymers_65_121
PubMedID: 12209462

Title : Crystal structure of the open form of dog gastric lipase in complex with a phosphonate inhibitor - Roussel_2002_J.Biol.Chem_277_2266
Author(s) : Roussel A , Miled N , Berti-Dupuis L , Riviere M , Spinelli S , Berna P , Gruber V , Verger R , Cambillau C
Ref : Journal of Biological Chemistry , 277 :2266 , 2002
Abstract : Fat digestion in humans and some mammals such as dogs requires the successive intervention of two lipases: gastric lipase, which is stable and active despite the highly acidic stomach environment, followed by the classical pancreatic lipase secreted into the duodenum. We previously solved the structure of recombinant human gastric lipase (HGL) at 3.0-A resolution in its closed form; this was the first structure to be described within the mammalian acid lipase family. Here we report on the open structure of the recombinant dog gastric lipase (r-DGL) at 2.7-A resolution in complex with the undecyl-butyl (C11Y4) phosphonate inhibitor. HGL and r-DGL show 85.7% amino acid sequence identity, which makes it relevant to compare the forms from two different species. The open r-DGL structure confirms the previous description of the HGL catalytic triad (Ser(153), His(353), and Asp(324)) with the catalytic serine buried and an oxyanion hole (NH groups of Gln(154) and Leu(67)). In r-DGL, the binding of the C11Y4 phosphonate inhibitor induces part of the cap domain, the lid, to roll over the enzyme surface and to expose a catalytic crevice measuring approximately 20 x 20 x 7 A(3). The C11Y4 phosphonate fits into this crevice, and a molecule of beta-octyl glucoside fills up the crevice. The C11Y4 phosphonate inhibitor and the detergent molecule suggest a possible binding mode for the natural substrates, the triglyceride molecules.
ESTHER : Roussel_2002_J.Biol.Chem_277_2266
PubMedSearch : Roussel_2002_J.Biol.Chem_277_2266
PubMedID: 11689574
Gene_locus related to this paper: canfa-1lipg

Title : Synthesis of lipophilic aldehydes and study of their inhibition effect on human digestive lipases - Kotsovolou_2002_Org.Lett_4_2625
Author(s) : Kotsovolou S , Verger R , Kokotos G
Ref : Org Lett , 4 :2625 , 2002
Abstract : [reaction: see text] Novel inhibitors of human digestive lipases, aldehyde dialkyl and alkyl-acyl glycerol analogues, were developed. The inhibitors were prepared starting from 3-(benzyloxy)-1,2-propanediol. The inhibition of human pancreatic and gastric lipases by the aldehyde derivatives was studied using the monolayer technique. (1R)-1-[(Dodecyloxy)methyl]-4-oxobutyl decanoate caused a 50% decrease in HPL and HGL activities at 0.100 and 0.053 molar fractions, respectively.
ESTHER : Kotsovolou_2002_Org.Lett_4_2625
PubMedSearch : Kotsovolou_2002_Org.Lett_4_2625
PubMedID: 12153194

Title : An ultraviolet spectrophotometric assay for measuring lipase activity using long-chain triacyglycerols from Aleurites fordii seeds - Pencreac'h_2002_Anal.Biochem_303_17
Author(s) : Pencreac'h G , Graille J , Pina M , Verger R
Ref : Analytical Biochemistry , 303 :17 , 2002
Abstract : In this study, we designed a specific, continuous, and sensitive UV spectrophotometric lipase assay using natural triacylglycerols (TAGs) from the Aleurites fordii seed oil (tung oil). alpha-Eleostearic acid (9,11,13-cis, trans,trans-octadecatrienoic acid) is the main fatty acid component (it accounts for up to 70%) of the TAGs from tung oil. The conjugated triene present in alpha-eleostearic acid constitutes an intrinsic chromophore, which confers strong UV absorption properties on both the free fatty acid and the TAGs from tung oil. The lipase assay is based on the difference between the apparent molar extinction coefficients of the two types of alpha-eleostearic acid present, that which is esterified into TAGs and that which is released into the reaction medium. This difference is responsible for the variations in the UV absorption spectrum of the reaction medium occurring upon enzymatic TAGs hydrolysis. Using the purified lipase from Thermomyces lanuginosa (TLL) and the detergent sodium taurodeoxycholate (NaTDC, 4 mM), it was established that the most suitable method of measuring lipolysis consisted of monitoring the decrease in the OD at 292 nm, which was linear with time and proportional to the amount of lipase added. In order to be able to estimate the specific activity of TLL, we determined an apparent molar extinction coefficient of alpha-eleostearic acid (epsilon = 13,900 M(-1) cm(-1)) under the assay conditions. Amounts of pure TLL as small as 1 ng can be easily detected in the presence of 4 mM NaTDC. Interestingly, the NaTDC concentration can be decreased as far as 0.05 mM. In comparison with other well-known methods of lipase assay, the detection limit of this new method is 100-fold lower than with the pH-stat method and similar to that of a fluorescent assay recently developed at our laboratory.
ESTHER : Pencreac'h_2002_Anal.Biochem_303_17
PubMedSearch : Pencreac'h_2002_Anal.Biochem_303_17
PubMedID: 11906146

Title : Binding of Thermomyces (Humicola) lanuginosa lipase to the mixed micelles of cis-parinaric acid\/NaTDC - Yapoudjian_2002_Eur.J.Biochem_269_1613
Author(s) : Yapoudjian S , Ivanova MG , Brzozowski AM , Patkar SA , Vind J , Svendsen A , Verger R
Ref : European Journal of Biochemistry , 269 :1613 , 2002
Abstract : The binding of Thermomyces lanuginosa lipase and its mutants [TLL(S146A), TLL(W89L), TLL(W117F, W221H, W260H)] to the mixed micelles of cis-parinaric acid/sodium taurodeoxycholate at pH 5.0 led to the quenching of the intrinsic tryptophan fluorescence emission (300-380 nm) and to a simultaneous increase in the cis-parinaric acid fluorescence emission (380-500 nm). These findings were used to characterize the Thermomyces lanuginosa lipase/cis-parinaric acid interactions occurring in the presence of sodium taurodeoxycholate. The fluorescence resonance energy transfer and Stern-Volmer quenching constant values obtained were correlated with the accessibility of the tryptophan residues to the cis-parinaric acid and with the lid opening ability of Thermomyces lanuginosa lipase (and its mutants). TLL(S146A) was found to have the highest fluorescence resonance energy transfer. In addition, a TLL(S146A)/oleic acid complex was crystallised and its three-dimensional structure was solved. Surprisingly, two possible binding modes (sn-1 and antisn1) were found to exist between oleic acid and the catalytic cleft of the open conformation of TLL(S146A). Both binding modes involved an interaction with tryptophan 89 of the lipase lid, in agreement with fluorescence resonance energy transfer experiments. As a consequence, we concluded that TLL(S146A) mutant is not an appropriate substitute for the wild-type Thermomyces lanuginosa lipase for mimicking the interaction between the wild-type enzyme and lipids.
ESTHER : Yapoudjian_2002_Eur.J.Biochem_269_1613
PubMedSearch : Yapoudjian_2002_Eur.J.Biochem_269_1613
PubMedID: 11895431
Gene_locus related to this paper: humla-1lipa

Title : Synthetic routes and lipase-inhibiting activity of long-chain alpha-keto amides - Chiou_2001_Lipids_36_535
Author(s) : Chiou A , Verger R , Kokotos G
Ref : Lipids , 36 :535 , 2001
Abstract : Synthetic routes to primary and N-alkyl alpha-keto amides are presented in this paper. Primary alpha-keto amides may be prepared by using an aldehyde as starting material. Commercially available alpha-keto acids may be coupled in high yield with primary amines by the mixed carbonic anhydride method affording N-alkyl alpha-keto amides. Alternatively, N-alkyl alpha-keto amides may be prepared by coupling long-chain alpha-hydroxy acids with amino components, followed by oxidation with pyridinium dichromate or NaOCl in the presence of 4-acetamido-2,2,6,6-tetramethyl-1-piperidinyloxy free radical. The alpha-keto amide derivatives prepared according to these procedures were tested for their ability to form stable monomolecular films at the air/water interface. The inhibition of porcine pancreatic lipase by the alpha-keto amides, spread as mixed films with 1,2-dicaprin, was studied with the monolayer technique. Among the compounds tested in this study, methyl 2-[(2-ketododecanoyl)amino]hexadecanoate was shown to be the most potent inhibitor, causing a 50% decrease in lipase activity at a 0.09 molar fraction.
ESTHER : Chiou_2001_Lipids_36_535
PubMedSearch : Chiou_2001_Lipids_36_535
PubMedID: 11432468

Title : Oil-bodies as substrates for lipolytic enzymes - Beisson_2001_Biochim.Biophys.Acta_1531_47
Author(s) : Beisson F , Ferte N , Bruley S , Voultoury R , Verger R , Arondel V
Ref : Biochimica & Biophysica Acta , 1531 :47 , 2001
Abstract : Plant seeds store triacylglycerols (TAGs) in intracellular organelles called oil-bodies or oleosomes, which consist of oil droplets covered by a coat of phospholipids and proteins. During seed germination, the TAGs of oil-bodies hydrolysed by lipases sustain the growth of the seedlings. The mechanism whereby lipases gain access to their substrate in these organelles is largely unknown. One of the questions that arises is whether the protein/phospholipid coat of oil-bodies prevents the access of lipase to the oil core. We have investigated the susceptibility of almond oil-bodies to in vitro lipolysis by various purified lipases with a broad range of biochemical properties. We have found that all the enzymes assayed were capable of releasing on their own free fatty acids from the TAG of oil-bodies. Depending on the lipase, the specific activity measured on oil-bodies using the pH-stat technique was found to range from 18 to 38% of the specific activity measured on almond oil emulsified by gum arabic. Some of these lipases are known to have a dual lipase/phospholipase activity. However, no correlation was found to exist between the ability of a lipase to readily and efficiently hydrolyse the TAG content of oil-bodies and the presence of a phospholipase activity. Kinetic studies indicate that oil-bodies behave as a substrate as other proteolipid organelles such as milk fat globules. Finally we have shown that a purified water-soluble plant lipase on its own can easily hydrolyse oil-bodies in vitro. Our results suggest that the lipolysis of oil-bodies in seedlings might occur without any pre-hydrolysis of the protein coat.
ESTHER : Beisson_2001_Biochim.Biophys.Acta_1531_47
PubMedSearch : Beisson_2001_Biochim.Biophys.Acta_1531_47
PubMedID: 11278171

Title : Kinetic studies of Rhizopus oryzae lipase using monomolecular film technique - Ben_2001_Biochimie_83_463
Author(s) : Ben Salah A , Sayari A , Verger R , Gargouri Y
Ref : Biochimie , 83 :463 , 2001
Abstract : Rhizopus oryzae lipase (ROL) was found to be a true lipase. This enzyme presents the interfacial activation phenomenon. The N-terminal amino acid sequence of ROL was compared to those of rhizopus lipases. Purified ROL possesses the same N-terminal sequence as the mature Rhizopus niveus lipase (RNL). This sequence was found in the last 28 amino acids of the propeptide sequence derived from the cDNA of Rhizopus delemar lipase (RDL). Using the baro-stat method, we have measured the hydrolysis rate of dicaprin films by ROL as a function of surface pressure. Our results show that Rhizopus oryzae lipase is markedly stereoselective of the sn-3 position of the 2,3 enantiomer of dicaprin. Polyclonal antibodies (PAB) directed against ROL have been produced and purified by immunoaffinity. The effects of these PAB on the interfacial behavior of ROL were determined. The immunoblot analysis with polyclonal antibodies anti-ROL (PAB anti-ROL) and various lipases shows a cross-immunoreactivity between the lipase from the rhizopus family (Rhizopus delemar lipase and Rhizopus arrhizus lipase).
ESTHER : Ben_2001_Biochimie_83_463
PubMedSearch : Ben_2001_Biochimie_83_463
PubMedID: 11506890
Gene_locus related to this paper: rhidl-lipas

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 : A conformational transition between an open and closed form of human pancreatic lipase revealed by a monoclonal antibody - Miled_2000_Biochim.Biophys.Acta_1476_165
Author(s) : Miled N , de Caro A , De Caro J , Verger R
Ref : Biochimica & Biophysica Acta , 1476 :165 , 2000
Abstract : The interfacial activation of human pancreatic lipase (HPL) probably involves the motion of a lid covering the active site of the enzyme. Here we observed that the presence of either bile salts or a small proportion of water-miscible organic solvents (called activator compounds) considerably enhances the enzymatic activity of HPL on a monomeric solution of tripropionin. This finding suggests that the activator compounds may favor the opening of the lid. This hypothesis was checked by comparing the immunoreactivity of HPL and HPL with a mini-lid (HPL(-lid)) towards anti-HPL monoclonal antibodies (mAbs), in the presence and absence of the activator compounds. A single conformational mAb (248-31) fails to immunoprecipitate HPL in the presence of activator compounds and HPL covalently inhibited with diethyl p-nitrophenyl phosphate (DP.HPL). This loss of recognition of HPL by mAb 248-31 was probably due to the motion of the lid, since HPL(-lid) was always recognized in the presence or absence of activator compounds. Furthermore, two other mAbs (81-23 and 146-40) immunoprecipitated HPL similarly whether or not the activator compounds were present. MAb 248-31 therefore specifically recognizes HPL in the closed but not the open conformation.
ESTHER : Miled_2000_Biochim.Biophys.Acta_1476_165
PubMedSearch : Miled_2000_Biochim.Biophys.Acta_1476_165
PubMedID: 10669782

Title : A novel extracellular esterase from Bacillus subtilis and its conversion to a monoacylglycerol hydrolase - Eggert_2000_Eur.J.Biochem_267_6459
Author(s) : Eggert T , Pencreac'h G , Douchet I , Verger R , Jaeger KE
Ref : European Journal of Biochemistry , 267 :6459 , 2000
Abstract : A novel gene lipB, which encodes an extracellular lipolytic enzyme, was identified in the Bacillus subtilis genomic DNA sequence. We have cloned and overexpressed lipB in B. subtilis and Escherichia coli and have also purified the enzyme from a B. subtilis culture supernatant to electrophoretic homogeneity. Four different lipase assays were used to determine its catalytic activity: pH-stat, spectrophotometry, fluorimetry and the monomolecular film technique. LipB preferentially hydrolysed triacylglycerol-esters and p-nitrophenyl-esters of fatty acids with short chain lengths of <= 10 carbon atoms. Triolein, which is a typical substrate for true lipases, was not hydrolysed at all. These results led us to classify LipB as an esterase rather than a lipase. The catalytic triad of LipB consists of residues Ser78, Asp134, and His157 as demonstrated by amino-acid sequence alignments and site-directed mutagenesis. The nucleophile Ser78 is located in a lipase-specific consensus sequence, which is Ala-X-Ser-X-Gly for most Bacillus lipases. All other bacterial lipases contain a glycine residue instead of the alanine at position-2 with respect to the catalytic serine. We have investigated the role of this alanine residue by constructing LipB variant A76G, thereby restoring the lipase-specific consensus motif. When compared with LipB this variant showed a markedly reduced thermostability but an increased stability at pH 5-7. Determination of the specific activities of wild-type LipB and variant A76G using a monomolecular film of the substrate monoolein revealed an interesting result: the A76G substitution had converted the esterase LipB into a monoacylglycerol hydrolase.
ESTHER : Eggert_2000_Eur.J.Biochem_267_6459
PubMedSearch : Eggert_2000_Eur.J.Biochem_267_6459
PubMedID: 11029590
Gene_locus related to this paper: bacsu-LIPB

Title : Covalent inhibition of digestive lipases by chiral phosphonates - Cavalier_2000_Acc.Chem.Res_33_579
Author(s) : Cavalier JF , Buono G , Verger R
Ref : Acc Chem Res , 33 :579 , 2000
Abstract : Designing and synthesizing specific inhibitors is of fundamental value for understanding the molecular mechanisms involved in the interfacial adsorption step as well as the catalytic activity of lipases. In this Account, we will review and discuss results obtained mostly at our laboratory concerning the covalent inhibition of human gastric and human pancreatic lipases by chiral phosphonates. Rather than presenting an exhaustive list of compounds tested so far with lipases of animal and microbial origin, we selected recent experimental data illustrating well the specific problems encountered during the covalent inhibition of these digestive lipases.
ESTHER : Cavalier_2000_Acc.Chem.Res_33_579
PubMedSearch : Cavalier_2000_Acc.Chem.Res_33_579
PubMedID: 10995195

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 : Assaying Arabidopsis lipase activity - Beisson_2000_Biochem.Soc.Trans_28_773
Author(s) : Beisson F , Arondel V , Verger R
Ref : Biochemical Society Transactions , 28 :773 , 2000
Abstract : A low lipase activity from a crude extract of Arabidopsis seedlings was assayed using three sensitive methods (radiolabelled triacylglycerols, commercial resorufin ester and triacylglycerols containing the naturally fluorescent parinaric acid as substrates). The specific activity of the extract was found to be similar using the three methods. However, the plant lipase activity measured using the radioactivity and the fluorescence assays could be abolished by heating the extract, contrary to the apparent activity measured using the commercial colorimetric assay. Unlike the radioactivity assay, the fluorescence assay can be monitored continuously. The parinaric acid-based method is therefore the only one to provide a sensitive, specific and continuous assay.
ESTHER : Beisson_2000_Biochem.Soc.Trans_28_773
PubMedSearch : Beisson_2000_Biochem.Soc.Trans_28_773
PubMedID: 11171203

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 : Inhibition of human gastric and pancreatic lipases by chiral alkylphosphonates. A kinetic study with 1,2-didecanoyl-sn-glycerol monolayer - Cavalier_1999_Chem.Phys.Lipids_100_3
Author(s) : Cavalier JF , Ransac S , Verger R , Buono G
Ref : Chemistry & Physic of Lipids , 100 :3 , 1999
Abstract : Enantiomerically pure alkylphosphonate compounds RR'P(O)PNP (R = CnH2n + 1, R' = OY with Y = Cn'H2n' + 1 with n = n' or n not equal to n'; PNP = p-nitrophenoxy) noted (RY), mimicking the transition state occurring during the carboxyester hydrolysis were synthesized and investigated as potential inhibitors of human gastric lipase (HGL) and human pancreatic lipase (HPL). The inhibitory properties of each enantiomer have been tested with the monomolecular films technique in addition to an enyzme linked immunosorbent assay (ELISA) in order to estimate simultaneously the residual enzymatic activity as well as the interfacial lipase binding. With both lipases, no obvious correlation between the inhibitor molar fraction (alpha 50) leading to half inhibition, and the chain length, R or Y was observed. (R11Y16)s were the best inhibitor of HPL and (R10Y11)s were the best inhibitors of HGL. We observed a highly enantioselective discrimination, both with the pure enantiomeric alkylphosphonate inhibitors as well as a scalemic mixture. We also showed, for the first time, that this enantioselective recognition can occur either during the catalytic step or during the initial interfacial adsorption step of the lipases. These experimental results were analyzed with two kinetic models of covalent as well as pseudo-competitive inhibition of lipolytic enzymes by two enantiomeric inhibitors.
ESTHER : Cavalier_1999_Chem.Phys.Lipids_100_3
PubMedSearch : Cavalier_1999_Chem.Phys.Lipids_100_3
PubMedID: 10640192

Title : The cysteine residues of recombinant human gastric lipase - Canaan_1999_Biochem.Biophys.Res.Commun_257_851
Author(s) : Canaan S , Riviere M , Verger R , Dupuis L
Ref : Biochemical & Biophysical Research Communications , 257 :851 , 1999
Abstract : Recombinant human gastric lipase (rHGL) and three of its cysteine mutants (cysteine 227, 236, and 244 substitued for threonine or serine) were expressed in the baculovirus/insect cell system and purified to homogeneity by performing a two-step procedure. Substituting Ser for Cys 227 and Cys 236 resulted in mutant lipases with a significantly lower level of activity (30% and 22%, respectively) on a short chain triglyceride (tribuyrin) substrate, while the mutation at position 244 only slightly reduced the activity. Using 4, 4'-dithiopyridine (4-PDS) as a sulfhydryl reagent on the above mutants, it was possible to clearly identify the single sulfhydryl residue at position 244 and consequently, the disulfide bridge at position 227-236. No potential disulfide bridges were formed during the protein folding between cysteines 227-244 or between cysteines 236-244, as thought to occur in the case of rabbit gastric lipase (RGL). The present results are consistent with the recently determined 3D-structure of rHGL.
ESTHER : Canaan_1999_Biochem.Biophys.Res.Commun_257_851
PubMedSearch : Canaan_1999_Biochem.Biophys.Res.Commun_257_851
PubMedID: 10208872

Title : Use of naturally fluorescent triacylglycerols from Parinari glaberrimum to detect low lipase activities from Arabidopsis thaliana seedlings - Beisson_1999_J.Lipid.Res_40_2313
Author(s) : Beisson F , Ferte N , Nari J , Noat G , Arondel V , Verger R
Ref : J Lipid Res , 40 :2313 , 1999
Abstract : The aim of this study was to design a convenient, specific, sensitive, and continuous lipase activity assay using natural long-chain triacylglycerols (TAGs). Oil was extracted from Parinari glaberrimum seed kernels and the purified TAGs were used as a substrate for detecting low levels of lipase activities. The purified TAGs are naturally fluorescent because more than half of the fatty acids from Parinari oil are known to contain 9,11,13, 15-octadecatetraenoic acid (parinaric acid) in its esterified form. The presence of detergents (sodium taurodeoxycholate, CHAPS, Sulfobetaine SB12, Tween 20, Brij 35, Dobanol, n-dodecylglucoside) above their critical micellar concentration dramatically increases the fluorescence of the parinaric acid released by various lipases. This increase in the fluorescence intensity is linear with time and proportional to the amount of lipase added. This new method, performed under non-oxidative conditions, was applied successfully to detecting low lipase levels in crude protein extracts from plant seeds and could be scaled down to microtiterplate measurements. Quantities as low as 0.1 ng of pure pancreatic lipase could be detected under standard conditions (pH 8). Lipase activity can also be assayed in acidic media (pH 5) using human gastric lipase. This simple and continuous assay is compatible with a high sample throughput and might be applied to detecting true lipase activities in various biological samples.
ESTHER : Beisson_1999_J.Lipid.Res_40_2313
PubMedSearch : Beisson_1999_J.Lipid.Res_40_2313
PubMedID: 10588957

Title : Gastric lipase: crystal structure and activity - Canaan_1999_Biochim.Biophys.Acta_1441_197
Author(s) : Canaan S , Roussel A , Verger R , Cambillau C
Ref : Biochimica & Biophysica Acta , 1441 :197 , 1999
Abstract : Fat digestion in humans requires not only the classical pancreatic lipase but also gastric lipase, which is stable and active despite the highly acidic stomach environment. We have solved the structure of recombinant human gastric lipase at 3.0 A resolution, the first structure to be described within the mammalian acid lipase family. This globular enzyme (379 residues) consists of a core domain, belonging to the alpha/beta hydrolase fold family, and an extrusion domain. It possesses a classical catalytic triad (Ser 153, His 353, Asp 324) and an oxyanion hole (NH groups of Gln 154 and Leu 67). Four N-glycosylation sites were identified on the electron density maps. The catalytic serine is deeply buried under the extrusion domain, which is composed of a 'cap' domain and a segment consisting of 30 residues, which can be defined as a lid. Its displacement is necessary for the substrates to access the active site. A phosphonate inhibitor was positioned in the active site which clearly suggests the location of the hydrophobic substrate binding site.
ESTHER : Canaan_1999_Biochim.Biophys.Acta_1441_197
PubMedSearch : Canaan_1999_Biochim.Biophys.Acta_1441_197
PubMedID: 10570247
Gene_locus related to this paper: human-LIPF

Title : Crystal structure of human gastric lipase and model of lysosomal acid lipase, two lipolytic enzymes of medical interest - Roussel_1999_J.Biol.Chem_274_16995
Author(s) : Roussel A , Canaan S , Egloff MP , Riviere M , Dupuis L , Verger R , Cambillau C
Ref : Journal of Biological Chemistry , 274 :16995 , 1999
Abstract : Fat digestion in humans requires not only the classical pancreatic lipase but also gastric lipase, which is stable and active despite the highly acidic stomach environment. We report here the structure of recombinant human gastric lipase at 3.0-A resolution, the first structure to be described within the mammalian acid lipase family. This globular enzyme (379 residues) consists of a core domain belonging to the alpha/beta hydrolase-fold family and a "cap" domain, which is analogous to that present in serine carboxypeptidases. It possesses a classical catalytic triad (Ser-153, His-353, Asp-324) and an oxyanion hole (NH groups of Gln-154 and Leu-67). Four N-glycosylation sites were identified on the electron density maps. The catalytic serine is deeply buried under a segment consisting of 30 residues, which can be defined as a lid and belonging to the cap domain. The displacement of the lid is necessary for the substrates to have access to Ser-153. A phosphonate inhibitor was positioned in the active site that clearly suggests the location of the hydrophobic substrate binding site. The lysosomal acid lipase was modeled by homology, and possible explanations for some previously reported mutations leading to the cholesterol ester storage disease are given based on the present model.
ESTHER : Roussel_1999_J.Biol.Chem_274_16995
PubMedSearch : Roussel_1999_J.Biol.Chem_274_16995
PubMedID: 10358049
Gene_locus related to this paper: human-LIPF

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 : Modulation of the expression level of human acidic lipases by various signal peptides -
Author(s) : Canaan S , Dupuis L , Riviere M , Verger R , Wicker-Planquart C
Ref : Methods Mol Biol , 109 :203 , 1999
PubMedID: 9918025

Title : Monolayer techniques for studying lipase kinetics -
Author(s) : Ransac S , Ivanova M , Panaiotov I , Verger R
Ref : Methods Mol Biol , 109 :279 , 1999
PubMedID: 9918030

Title : Structure and activity of rat pancreatic lipase-related protein 2 - Roussel_1998_J.Biol.Chem_273_32121
Author(s) : Roussel A , Yang Y , Ferrato F , Verger R , Cambillau C , Lowe M
Ref : Journal of Biological Chemistry , 273 :32121 , 1998
Abstract : The pancreas expresses several members of the lipase gene family including pancreatic triglyceride lipase (PTL) and two homologous proteins, pancreatic lipase-related proteins 1 and 2 (PLRP1 and PLRP2). Despite their similar amino acid sequences, PTL, PLRP1, and PLRP2 differ in important kinetic properties. PLRP1 has no known activity. PTL and PLRP2 differ in substrate specificity, bile acid inhibition, colipase requirement, and interfacial activation. To begin understanding the structural explanations for these functional differences, we solved the crystal structure of rat (r)PLRP2 and further characterized its kinetic properties. The 1.8 A structure of rPLRP2, like the tertiary structure of human PTL, has a globular N-terminal domain and a beta-sandwich C-terminal domain. The lid domain occupied the closed position, suggesting that rPLRP2 should show interfacial activation. When we reexamined this issue with tripropionin as substrate, rPLRP2 exhibited interfacial activation. Because the active site topology of rPLRP2 resembled that of human PTL, we predicted and demonstrated that the lipase inhibitors E600 and tetrahydrolipstatin inhibit rPLRP2. Although PTL and rPLRP2 have similar active sites, rPLRP2 has a broader substrate specificity that we confirmed using a monolayer technique. With this assay, we showed for the first time that rPLRP2 prefers phosphatidylglycerol and ethanolamine over phosphatidylcholine. In summary, we confirmed and extended the observation that PLRP2 lipases have a broader substrate specificity than PTL, we demonstrated that PLRP2 lipases show interfacial activation, and we solved the first crystal structure of a PLRP2 lipase that contains a lid domain.
ESTHER : Roussel_1998_J.Biol.Chem_273_32121
PubMedSearch : Roussel_1998_J.Biol.Chem_273_32121
PubMedID: 9822688
Gene_locus related to this paper: ratno-4plip

Title : Lipases: Interfacial Enzymes with Attractive Applications - Schmid_1998_Angew.Chem.Int.Ed.Engl_37_1608
Author(s) : Schmid RD , Verger R
Ref : Angew Chem Int Ed Engl , 37 :1608 , 1998
Abstract : Unusually versatile substrate specificity is shown by lipases. Not only do they hydrolyze triacylglycerols-for example, in the stomach and intestine during digestion of dietary fat-and various synthetic esters and amides, but their high stability in organic solvents permits their use in transesterification reactions and ester synthesis as well. Reactions based on lipase catalysis usually proceed with high regio- and enantioselectivity. Thus, the Ca(2+) antagonist diltiazem (1) was obtained with lipase from Serratia marcescens. Over 30 lipases have been cloned in the last few years. Since the tertiary structure of 12 lipases is known, there are presently significant efforts to improve this class of enzymes by protein engineering techniques, in view of their use in detergents and other fields of industrial application.
ESTHER : Schmid_1998_Angew.Chem.Int.Ed.Engl_37_1608
PubMedSearch : Schmid_1998_Angew.Chem.Int.Ed.Engl_37_1608
PubMedID: 29711530

Title : Purification and interfacial behavior of recombinant human gastric lipase produced from insect cells in a bioreactor - Canaan_1998_Protein.Expr.Purif_14_23
Author(s) : Canaan S , Dupuis L , Riviere M , Faessel K , Romette JL , Verger R , Wicker-Planquart C
Ref : Protein Expr Purif , 14 :23 , 1998
Abstract : Recombinant human gastric lipase (rHGL) (EC 3.1.1.3) was produced on a large scale (5-13 mg/liter) from recombinant baculovirus-infected insect cells using a bioreactor apparatus. Here an improved procedure is described for purifying rHGL involving the use of cation exchange chromatography followed by immunoaffinity column methods, which gives a total yield of 62% and a purification factor of 464, using 10% isopropanol in all the purification buffers. The presence of isopropanol was necessary to preserve the stability of the enzyme during the chromatographic separation steps. The specific activity of rHGL on tributyroylglycerol (700 U/mg) was lower than that of native HGL (nHGL) (1080 U/mg). The rHGL interfacial adsorption kinetics were studied by recording the changes in the surface pressure with time in the presence or absence of an egg phosphatidycholine monomolecular film spread at the air/water interface at various initial surface pressures. The surface behavior of rHGL was similar to that of nHGL. It can be concluded that the lipid binding affinity of rHGL is identical to that of the native lipase and, consequently, that the presence of detergents and lipids in the insect cell culture media did not affect the interfacial behavior of the purified rHGL. It will be therefore possible to specifically study the binding step of HGL mutants to a lipid monolayer.
ESTHER : Canaan_1998_Protein.Expr.Purif_14_23
PubMedSearch : Canaan_1998_Protein.Expr.Purif_14_23
PubMedID: 9758747

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 : An enzymatically active truncated form (-55 N-terminal residues) of rabbit gastric lipase. Correlation between the enzymatic activity and disulfide bond oxydo-reduction state - De Caro_1998_Biochim.Biophys.Acta_1386_39
Author(s) : De Caro J , Verger R , de Caro A
Ref : Biochimica & Biophysica Acta , 1386 :39 , 1998
Abstract : Rabbit gastric lipase (RGL) was subjected to proteolysis with trypsin and led to cleavage occurring at three defined sites (Lys-4, Arg-55 and Arg-229). The tryptic hydrolysate contained four fragments: Gly-230-Lys-379 (T1), Gly-56-Arg-229 (T2), Ser-5-Arg-55 (T3), as well as a 45 kDa molecular form consisting of peptides T1 and T2 linked by a disulfide bridge. The tryptic hydrolysate of RGL as well as the 55 N-terminal amino acid deleted forms conserved 30% of the initial enzymatic activity in a tributyrin assay. Two out of the three cysteine residues which are present in all the known gastric lipases were found to be involved in a disulfide bridge. Unlike HGL, RGL appears to have a heterogenous pattern of cysteine residues. The 30% enzymatic activity of RGL persisting after trypsin treatment may be attributable to the 45 kDa molecular form (with the Cys-227-Cys-236 or Cys-227-Cys-244 disulfide bridge). Trypsin-treated HGL, which was completely inactivated, showed that a single location of the disulfide bridge existed between cysteine residues 236 and 244. It can be concluded that the existence of one disulfide bridge is necessary to maintain the lipase activity of the 45 kDa form of RGL.
ESTHER : De Caro_1998_Biochim.Biophys.Acta_1386_39
PubMedSearch : De Caro_1998_Biochim.Biophys.Acta_1386_39
PubMedID: 9675239

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 : Purification and characterization of a porcine liver microsomal triacylglycerol hydrolase - Lehner_1997_Biochemistry_36_1861
Author(s) : Lehner R , Verger R
Ref : Biochemistry , 36 :1861 , 1997
Abstract : We have purified an enzyme from porcine liver microsomes which catalyzes hydrolysis of triacylglycerols. The enzyme was solubilized from the membranes by the zwitterionic detergent 3-[(3- cholamidopropyl)dimethylammonio]-l-propansulfonate (CHAPS) and was purified to apparent homogeneity by sequential chromatography on Q-Sepharose, hydroxyapatite, Affi-Gel heparin, and Mono-Q. The purified hydrolase migrated in SDS-polyacrylamide gel electrophoresis (PAGE) as a single polypeptide band of an apparent molecular mass of 60 kDa. The enzyme hydrolyzed long-, medium-, and short-chain triacylglycerols, as well as a chromogenic lipase substrate, 1,2-O-dilauryl-rac-glycero-3-glutaric acid resorufin ester. The highest specific activity was obtained with tributyroylglycerol (240 mumol.min-1.mg-1). The reaction rate was maximal at pH 8.5. Sulfhydryl-directed reagents, such as N-ethylmaleimide (NEM), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and dodecyldithio-5-(2-nitrobenzoic acid) (C12-TNB) had no effect on the hydrolase activity; however, the enzyme was sensitive to HgCl2. Serine reagents, such as diethyl-p-nitrophenyl phosphate (E600) and diisopropyl fluorophosphate (DFP), used in 100-fold molar excess completely inhibited the activity, suggesting that it is a serine esterase. These results suggest that the enzyme may participate in the intracellular neutral lipid metabolism since the enzyme is located in the endoplasmic reticulum, an organelle where de novo triacylglycerol synthesis and assembly of lipoproteins take place.
ESTHER : Lehner_1997_Biochemistry_36_1861
PubMedSearch : Lehner_1997_Biochemistry_36_1861
PubMedID: 9048571

Title : Large-scale purification and kinetic properties of recombinant hormone-sensitive lipase from baculovirus-insect cell systems -
Author(s) : Holm C , Contreras JA , Verger R , Schotz MC
Ref : Methods Enzymol , 284 :272 , 1997
PubMedID: 9379939

Title : Covalent inactivation of lipases -
Author(s) : Ransac S , Gargouri Y , Marguet F , Buono G , Beglinger C , Hildebrand P , Lengsfeld H , Hadvary P , Verger R
Ref : Methods Enzymol , 286 :190 , 1997
PubMedID: 9309652

Title : Oil-drop tensiometer: applications for studying the kinetics of lipase action -
Author(s) : Labourdenne S , Cagna A , Delorme B , Esposito G , Verger R , Riviere C
Ref : Methods Enzymol , 286 :306 , 1997
PubMedID: 9309656

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 : Expression in insect cells and purification of a catalytically active recombinant human gastric lipase - Wicker-Planquart_1996_Protein.Eng_9_1225
Author(s) : Wicker-Planquart C , Canaan S , Riviere M , Dupuis L , Verger R
Ref : Protein Engineering , 9 :1225 , 1996
Abstract : Human gastric lipase (HGL) cDNA was synthesized by RT-PCR amplification and cloned into the PVL 1392 baculovirus transfer vector. The recombinant transfer vector was cotransfected with a modified baculovirus DNA (Baculogold) which contains a lethal deletion. Cotransfection of baculovirus DNA with the recombinant transfer vector rescues the lethal deletion of this virus DNA and reconstitutes viable virus particles inside the transfected insect cells. BTI-TN-5B1-4 insect cells (also called High Five cells) were used to express recombinant HGL. The level of HGL secretion was approximately 32 mg/l of culture medium. The insect cells also accumulated HGL intracellularly, which indicated the existence of rate-limiting steps in the secretion of HGL. Therefore we investigated the effect of replacing the HGL signal peptide (SP) by other SP of secreted proteins. The honeybee melittin SP and the human pancreatic lipase (HPL) SP were tested. The fusion of HGL with HPL SP resulted in a 2-fold increase in the amount of lipase secreted from the insect cells. The recombinant active HGL was not processed at the expected cleavage site of the natural enzyme, however, but at residue +3. On the other hand, High Five cells transfected with the vector encoding HGL fused to the melittin SP did not secrete any detectable active HGL. Recombinant HGL was identified using the Western blot procedure with rabbit polyclonal antibodies. The protein migrated with an apparent molecular mass of 45 kDa under SDS-PAGE analysis (compared with 50 kDa in the case of natural HGL), indicating that the insect cells have only a limited capacity to glycosylate HGL. The maximum specific activities of the recombinant lipase were 434, 730 and 562 units/mg using long-chain (Intralipid), medium-chain (trioctanoylglycerol) and short-chain (tributyroylglycerol) triacylglycerols, respectively.
ESTHER : Wicker-Planquart_1996_Protein.Eng_9_1225
PubMedSearch : Wicker-Planquart_1996_Protein.Eng_9_1225
PubMedID: 9010937

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 : The 2.46 A resolution structure of the pancreatic lipase-colipase complex inhibited by a C11 alkyl phosphonate - Egloff_1995_Biochemistry_34_2751
Author(s) : Egloff MP , Marguet F , Buono G , Verger R , Cambillau C , van Tilbeurgh H
Ref : Biochemistry , 34 :2751 , 1995
Abstract : Pancreatic lipase belongs to the serine esterase family and can therefore be inhibited by classical serine reagents such as diisopropyl fluoride or E600. In an attempt to further characterize the active site and catalytic mechanism, we synthesized a C11 alkyl phosphonate compound. This compound is an effective inhibitor of pancreatic lipase. The crystal structure of the pancreatic lipase-colipase complex inhibited by this compound was determined at a resolution of 2.46 A and refined to a final R-factor of 18.3%. As was observed in the case of the structure of the ternary pancreatic lipase-colipase-phospholipid complex, the binding of the ligand induces rearrangements of two surface loops in comparison with the closed structure of the enzyme (van Tilbeurgh et al., 1993b). The inhibitor, which could be clearly observed in the active site, was covalently bound to the active site serine Ser152. A racemic mixture of the inhibitor was used in the crystallization, and there exists evidence that both enantiomers are bound at the active site. The C11 alkyl chain of the first enantiomer fits into a hydrophobic groove and is though to thus mimic the interaction between the leaving fatty acid of a triglyceride substrate and the protein. The alkyl chain of the second enantiomer also has an elongated conformation and interacts with hydrophobic patches on the surface of the open amphipathic lid. This may indicate the location of a second alkyl chain of a triglyceride substrate. Some of the detergent molecules, needed for the crystallization, were also observed in the crystal. Some of them were located at the entrance of the active site, bound to the hydrophobic part of the lid. On the basis of this crystallographic study, a hypothesis about the binding mode of real substrates and the organization of the active site is proposed.
ESTHER : Egloff_1995_Biochemistry_34_2751
PubMedSearch : Egloff_1995_Biochemistry_34_2751
PubMedID: 7893686
Gene_locus related to this paper: human-PNLIP

Title : Glyceride synthesis catalyzed by cutinase using the monomolecular film technique - Melo_1995_Biochemistry_34_1615
Author(s) : Melo EP , Ivanova MG , Aires-Barros MR , Cabral JM , Verger R
Ref : Biochemistry , 34 :1615 , 1995
Abstract : The monomolecular film technique previously used to study the kinetics of lipase hydrolysis was adapted to synthesizing oleoyl glycerides (monoolein, diolein, and triolein). The water subphase was replaced by glycerol, and a film of oleic acid was initially spread on the glycerol surface. In this system a recombinant cutinase from Fusarium solani was able to catalyze oleoyl glyceride synthesis. More than 50% of the oleic acid film was acylated after 7 min of reaction. The surface pressure applied to the monomolecular film acts as a physical selectivity factor since glyceride synthesis can be steered so as to produce either diolein or triolein.
ESTHER : Melo_1995_Biochemistry_34_1615
PubMedSearch : Melo_1995_Biochemistry_34_1615
PubMedID: 7849021

Title : Purification and molecular characterization of lamb pregastric lipase - De Caro_1995_Biochim.Biophys.Acta_1252_321
Author(s) : De Caro J , Ferrato F , Verger R , de Caro A
Ref : Biochimica & Biophysica Acta , 1252 :321 , 1995
Abstract : Lamb pregastric lipase (LPGL) was purified from pharyngeal tissues. The purification procedure was based on an aqueous extract containing 0.7% Tween 80 which was chromatographed on DEAE-cellulose anion-exchanger and adsorbed on HA-Ultrogel followed by gel filtration on Ultrogel AcA-54. The final enzymatic preparation, where the overall activity recovery was 3%, showed a single protein band on SDS-PAGE with a molecular mass of 50 kDa. LPGL is a glycoprotein containing approx. 14% (w/w) of carbohydrate. Extensive deglycosylation using peptide N-glycosidase F yielded a protein with an apparent molecular mass of 43 kDa. An uncontrolled proteolysis of LPGL during the purification lead to a 45 kDa form which was previously observed in human lysosomal acid lipase (HLAL) and rabbit gastric lipase (RGL). The labile bond X54-Leu55 was identified. Isoelectric focusing of LPGL reveals a major band corresponding to an isoelectric point of 4.8. The pure enzyme displayed specific activities of 950 U mg-1, 300 U mg-1 and 30 U mg-1 at pH 6.0, using tributyroylglycerol, trioctanoylglycerol and trioleoylglycerol as substrates, respectively. Using Western blot analysis, a cross-immunoreactivity of LPGL was observed with purified anti-human gastric lipase polyclonal antibodies. Determination of the amino-acid sequence of 62 residues revealed a high degree of homology with other known preduodenal lipases.
ESTHER : De Caro_1995_Biochim.Biophys.Acta_1252_321
PubMedSearch : De Caro_1995_Biochim.Biophys.Acta_1252_321
PubMedID: 7578240

Title : Crystallographic study of the structure of colipase and of the interaction with pancreatic lipase - Egloff_1995_Protein.Sci_4_44
Author(s) : Egloff MP , Sarda L , Verger R , Cambillau C , van Tilbeurgh H
Ref : Protein Science , 4 :44 , 1995
Abstract : Colipase (Mr 10 kDa) confers catalytic activity to pancreatic lipase under physiological conditions (high bile salt concentrations). Previously determined 3-A-resolution X-ray structures of lipase-colipase complexes have shown that, in the absence of substrate, colipase binds to the noncatalytic C-terminal domain of pancreatic lipase (van Tilbeurgh H, Sarda L, Verger R, Cambillau C, 1992, Nature 359:159-162; van Tilbeurgh et al., 1993a, Nature 362:814-820). Upon lipid binding, conformational changes at the active site of pancreatic lipase bring a surface loop (the lid) in contact with colipase, creating a second binding site for this cofactor. Covalent inhibition of the pancreatic lipase by a phosphonate inhibitor yields better diffracting crystals of the lipase-colipase complex. From the 2.4-A-resolution structure of this complex, we give an accurate description of the colipase. It confirms the previous proposed disulfide connections (van Tilbeurgh H, Sarda L, Verger R, Cambillau C, 1992, Nature 359:159-162; van Tilbeurgh et al., 1993a, Nature 362:814-820) that were in disagreement with the biochemical assignment (Chaillan C, Kerfelec B, Foglizzo E, Chapus C, 1992, Biochem Biophys Res Commun 184:206-211). Colipase lacks well-defined secondary structure elements. This small protein seems to be stabilized mainly by an extended network of five disulfide bridges that runs throughout the flatly shaped molecule, reticulating its four finger-like loops. The colipase surface can be divided into a rather hydrophilic part, interacting with lipase, and a more hydrophobic part, formed by the tips of the fingers. The interaction between colipase and the C-terminal domain of lipase is stabilized by eight hydrogen bonds and about 80 van der Waals contacts. Upon opening of the lid, three more hydrogen bonds and about 28 van der Waals contacts are added, explaining the higher apparent affinity in the presence of a lipid/water interface. The tips of the fingers are very mobile and constitute the lipid interaction surface. Two detergent molecules that interact with colipase were observed in the crystal, covering part of the hydrophobic surface.
ESTHER : Egloff_1995_Protein.Sci_4_44
PubMedSearch : Egloff_1995_Protein.Sci_4_44
PubMedID: 7773176

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 : 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 : Cutinase, a lipolytic enzyme with a preformed oxyanion hole - Martinez_1994_Biochemistry_33_83
Author(s) : Martinez C , Nicolas A , van Tilbeurgh H , Egloff MP , Cudrey C , Verger R , Cambillau C
Ref : Biochemistry , 33 :83 , 1994
Abstract : Cutinases, a group of cutin degrading enzymes with molecular masses of around 22-25 kDa (Kolattukudy, 1984), are also able to efficiently hydrolyse triglycerides (De Geus et al., 1989; Lauwereys et al., 1991), but without exhibiting the interfacial activation phenomenom (Sarda et al., 1958). They belong to a class of proteins with a common structural framework, called the alpha/beta hydrolase fold (Martinez et al., 1992; Ollis et al., 1992). We describe herein the structure of cutinase covalently inhibited by diethyl-p-nitrophenyl phosphate (E600) and refined at 1.9-A resolution. Contrary to what has previously been reported with lipases (Brzozowski et al., 1991; Derewenda et al., 1992; Van Tilbeurgh et al., 1993), no significant structural rearrangement was observed here in cutinase upon the inhibitor binding. Moreover, the structure of the active site machinery, consisting of a catalytic triad (S120, H188, D175) and an oxyanion hole (Q121 and S42), was found to be identical to that of the native enzyme, whereas the oxyanion hole of Rhizomucor lipase (Brzozowski et al., 1991; Derewenda et al., 1992), like that of pancreatic lipase (van Tilbeurgh et al., 1993), is formed only upon enzyme-ligand complex formation. The fact that cutinase does not display interfacial activation cannot therefore only be due to the absence of a lid but might also be attributable to the presence of a preformed oxyanion hole.
ESTHER : Martinez_1994_Biochemistry_33_83
PubMedSearch : Martinez_1994_Biochemistry_33_83
PubMedID: 8286366
Gene_locus related to this paper: fusso-cutas , orysa-LPL1

Title : Interfacial activation of the lipase-procolipase complex by mixed micelles revealed by X-ray crystallography - van Tilbeurgh_1993_Nature_362_814
Author(s) : van Tilbeurgh H , Egloff MP , Martinez C , Rugani N , Verger R , Cambillau C
Ref : Nature , 362 :814 , 1993
Abstract : The three-dimensional structure of the lipase-procolipase complex, co-crystallized with mixed micelles of phosphatidylcholine and bile salt, has been determined at 3 A resolution by X-ray crystallography. The lid, a surface helix covering the catalytic triad of lipase, adopts a totally different conformation which allows phospholipid to bind to the enzyme's active site. The open lid is an essential component of the active site and interacts with procolipase. Together they form the lipid-water interface binding site. This reorganization of the lid structure provokes a second drastic conformational change in an active site loop, which in its turn creates the oxyanion hole (induced fit).
ESTHER : van Tilbeurgh_1993_Nature_362_814
PubMedSearch : van Tilbeurgh_1993_Nature_362_814
PubMedID: 8479519
Gene_locus related to this paper: human-PNLIP

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 : Structure of the pancreatic lipase-procolipase complex - van Tilbeurgh_1992_Nature_359_159
Author(s) : van Tilbeurgh H , Sarda L , Verger R , Cambillau C
Ref : Nature , 359 :159 , 1992
Abstract : Interfacial adsorption of pancreatic lipase is strongly dependent on the physical chemical properties of the lipid surface. These properties are affected by amphiphiles such as phospholipids and bile salts. In the presence of such amphiphiles, lipase binding to the interface requires a protein cofactor, colipase. We obtained crystals of the pancreatic lipase-procolipase complex and solved the structure at 3.04 A resolution. Here we describe the structure of procolipase, which essentially consists of three 'fingers' and is topologically comparable to snake toxins. The tips of the fingers contain most of the hydrophobic amino acids and presumably form the interfacial binding site. Lipase binding occurs at the opposite side to this site and involves polar interactions. Determination of the three-dimensional structure of pancreatic lipase has revealed the presence of two domains: an amino-terminal domain, at residues 1-336 containing the active site and a carboxy-terminal domain at residues 337-449 (ref. 6). Procolipase binds exclusively to the C-terminal domain of lipase. No conformational change in the lipase molecule is induced by the binding of procolipase.
ESTHER : van Tilbeurgh_1992_Nature_359_159
PubMedSearch : van Tilbeurgh_1992_Nature_359_159
PubMedID: 1522902
Gene_locus related to this paper: human-PNLIP

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

Title : Inactivation of gastric and pancreatic lipases by diethyl p-nitrophenyl phosphate - Moreau_1991_Biochemistry_30_1037
Author(s) : Moreau H , Moulin A , Gargouri Y , Noel JP , Verger R
Ref : Biochemistry , 30 :1037 , 1991
Abstract : Reacting gastric and pancreatic lipases with mixed diethyl p-nitrophenyl phosphate/bile salt micelles resulted in a stoichiometric inactivation of these enzymes as tested on emulsified tributyroylglycerol and trioleoylglycerol as substrates. Diethyl p-nitrophenyl phosphate treated gastric lipases were also inactive on water-soluble p-nitrophenyl acetate, whereas the modified pancreatic lipase was still able to hydrolyze this water-soluble substrate. The binding of diethyl p-nitrophenyl phosphate modified pancreatic and gastric lipases to tributyroylglycerol/water interface was comparable to that of native lipases. The essential free sulfhydryl group of gastric lipases underwent no chemical changes due to the reaction with micellar diethyl p-nitrophenyl phosphate. All in all, these results indicate that, in both gastric and pancreatic lipases, the essential serine residue which was stoichiometrically labeled by this organophosphorus reagent is involved in catalysis and not in lipid binding.
ESTHER : Moreau_1991_Biochemistry_30_1037
PubMedSearch : Moreau_1991_Biochemistry_30_1037
PubMedID: 1989675
Gene_locus related to this paper: human-LIPF , human-PNLIP

Title : Stereoselectivity of lipases. II. Stereoselective hydrolysis of triglycerides by gastric and pancreatic lipases - Rogalska_1990_J.Biol.Chem_265_20271
Author(s) : Rogalska E , Ransac S , Verger R
Ref : Journal of Biological Chemistry , 265 :20271 , 1990
Abstract : In the present study, porcine pancreatic lipase, rabbit gastric lipase, and human gastric lipase stereospecificity toward chemically alike, but sterically nonequivalent ester groups within one single triglyceride molecule was investigated. Lipolysis reactions were carried out on synthetic trioctanoin or triolein, which are homogenous, prochiral triglycerides, chosen as models for physiological lipase substrates. Diglyceride mixtures resulting from lipolysis were derivatized with optically active R-(+)-1-phenylethylisocyanate, to give diastereomeric carbamate mixtures, which were further separated by high performance liquid chromatography. Resolution of diastereomeric carbamates gave enantiomeric excess values, which reflect the lipases stereobias and clearly demonstrate the existence of a stereopreference by both gastric lipases for the sn-3 position. The stereoselectivity of human and rabbit gastric lipases, expressed as the enantiomeric excess percentage, was 54% and 70% for trioctanoin and 74% and 47% for triolein, respectively. The corresponding values with porcine pancreatic lipase were 3% in the case of trioctanoin and 8% in that of triolein. It is worth noting that rabbit gastric lipase, unlike human gastric lipase, became more stereoselective for the triglyceride with shorter acyl chains (trioctanoin). This is one of the most striking catalytic differences observed between these two gastric lipases.
ESTHER : Rogalska_1990_J.Biol.Chem_265_20271
PubMedSearch : Rogalska_1990_J.Biol.Chem_265_20271
PubMedID: 2243091

Title : Gastric lipases: biochemical and physiological studies -
Author(s) : Gargouri Y , Moreau H , Verger R
Ref : Biochimica & Biophysica Acta , 1006 :255 , 1989
PubMedID: 2688745

Title : Minireview on pancreatic lipase and colipase - Chapus_1988_Biochimie_70_1223
Author(s) : Chapus C , Rovery M , Sarda L , Verger R
Ref : Biochimie , 70 :1223 , 1988
Abstract : By hydrolyzing the dietary triacylglycerols, pancreatic lipase causes catalysis in heterogeneous medium. In vivo, lipase action cannot take place without colipase due to the presence of bile salts. The cofactor enables lipase anchoring to the water-lipid interface. The lipase-colipase system furnishes an excellent example of specific interactions (protein-protein and protein-lipid). The studies of lipase catalytic properties brought to light the importance of certain parameters related to the 'quality of the interface'. The structure-function relationship analyses revealed a certain number of functional amino acid residues in lipase and colipase involved either in the catalytic site of the enzyme or in the recognition sites (lipase-colipase and protein-interface). Comparisons of the sequences of lipases derived from different sources display interesting similarities in certain cases.
ESTHER : Chapus_1988_Biochimie_70_1223
PubMedSearch : Chapus_1988_Biochimie_70_1223
PubMedID: 3147715

Title : Purification, characterization and kinetic properties of the rabbit gastric lipase - Moreau_1988_Biochim.Biophys.Acta_960_286
Author(s) : Moreau H , Gargouri Y , Lecat D , Junien JL , Verger R
Ref : Biochimica & Biophysica Acta , 960 :286 , 1988
Abstract : Rabbit gastric lipase was purified from an acetonic powder of rabbit stomach fundus. 25 mg of pure rabbit gastric lipase (glycerol ester hydrolase, EC 3.1.1.3) was obtained from 30 rabbit stomachs after ammonium sulfate fractionation, Sephadex G-100 gel filtration and cation exchange (mono S column) using a fast protein liquid chromatography (FPLC) system. The pure enzyme obtained was resistant to acidic pH conditions, and had specific activities of 1200, 850 and 280 U/mg, using, respectively, short- (tributyroylglycerol (TC4)), medium- (trioctanoyl- to tridecanoylglycerol (TC8-TC10)) and long-chain (soybean oil) triacylglycerols. The amino-acid composition was determined, and the first 30 N-terminal amino-acid residues were sequenced. Interfacial denaturation and catalytic properties on triacylglycerol emulsions were studied. Rabbit gastric lipase turned out to be structurally and kinetically very similar to human gastric lipase.
ESTHER : Moreau_1988_Biochim.Biophys.Acta_960_286
PubMedSearch : Moreau_1988_Biochim.Biophys.Acta_960_286
PubMedID: 3382677

Title : Molecular cloning of a human gastric lipase and expression of the enzyme in yeast - Bodmer_1987_Biochim.Biophys.Acta_909_237
Author(s) : Bodmer MW , Angal S , Yarranton GT , Harris TJ , Lyons A , King DJ , Pieroni G , Riviere C , Verger R , Lowe PA
Ref : Biochimica & Biophysica Acta , 909 :237 , 1987
Abstract : The molecular cloning of a cDNA coding for human gastric lipase and its expression in yeast is described. A lipase present in human gastric aspirates was purified and its N-terminal amino-acid sequence was determined. This was found to be homologous with the N-terminal sequence of rat lingual lipase. A cDNA library was constructed from mRNA isolated from human stomach tissue and probed with cloned rat lingual lipase DNA. One clone, pGL17, consisting of approximately 1450 base-pairs, contained the entire coding sequence for a human gastric lipase. The amino-acid sequence from the isolated protein and the DNA sequence obtained from the cloned gene indicated that human gastric lipase consists of a 379 amino acid polypeptide with an unglycosylated Mr of 43,162. Human gastric lipase and rat lingual lipase amino-acid sequences were closely homologous but were unrelated to porcine pancreatic lipase apart from a 6 amino-acid sequence around the essential Ser-152 of porcine pancreatic lipase. A yeast expression plasmid containing the phosphoglycerate kinase promoter and terminator sequences together with the human gastric lipase gene was constructed. Yeast transformed with this vector synthesised the lipolytically active enzyme.
ESTHER : Bodmer_1987_Biochim.Biophys.Acta_909_237
PubMedSearch : Bodmer_1987_Biochim.Biophys.Acta_909_237
PubMedID: 3304425
Gene_locus related to this paper: human-LIPF

Title : Kinetic assay of human gastric lipase on short- and long-chain triacylglycerol emulsions - Gargouri_1986_Gastroenterology_91_919
Author(s) : Gargouri Y , Pieroni G , Riviere C , Sauniere JF , Lowe PA , Sarda L , Verger R
Ref : Gastroenterology , 91 :919 , 1986
Abstract : Under optimal conditions, assay for pure human gastric lipase was carried out with short- and long-chain triacylglycerol emulsions. Maximal specific activities of 1160 and 620 U/mg were obtained with tributyrin and soybean emulsion, respectively. We observed that with a tributyrin substrate, bovine serum albumin or bile salts must be added before the addition of the enzyme in order to prevent its irreversible interfacial denaturation. With long-chain triacylglycerols as substrate, a decrease with time in the rate of hydrolysis was associated with release of protonated long-chain fatty acids. The inhibitory effect of protonated fatty acids was also observed using tributyrin at pH 3.0. These observations support the conclusion that human gastric lipase shows no intrinsic specificity for short-chain triacylglycerols and that its apparent specificity is modulated by pH and presence of amphiphile in the incubation medium. Our conclusions support the view that, in the human, gastric lipolysis may play an important role in long-chain fat digestion.
ESTHER : Gargouri_1986_Gastroenterology_91_919
PubMedSearch : Gargouri_1986_Gastroenterology_91_919
PubMedID: 3743968

Title : Inhibition of lipases by proteins: a binding study using dicaprin monolayers - Gargouri_1986_Biochemistry_25_1733
Author(s) : Gargouri Y , Pieroni G , Riviere C , Sarda L , Verger R
Ref : Biochemistry , 25 :1733 , 1986
Abstract : We previously reported that the inhibition of pancreatic and Rhizopus delemar lipases by proteins is due to the protein associated with lipid and is not caused by direct protein-enzyme interaction in the aqueous phase [Gargouri, Y., Pieroni, G., Riviere, C., Sugihara, A., Sarda, L., & Verger, R. (1985) J. Biol. Chem. 260, 2268-2273]. In this study, using radiolabeled lipases, serum albumin, and beta-lactoglobulin A, we investigated their respective binding with respect to lipolysis of dicaprin monolayers. Lipase inhibition was found to be correlated with a lack of lipase binding to mixed protein-dicaprin films or to a desorption of lipase from the interface when inhibitory protein was added later. Since a large proportion of the lipid film remained potentially accessible to the enzyme in the presence of inhibitory protein, it was concluded that the observed decrease in lipase binding to the interface was due to a variation of the physiochemical properties of the lipid-water interface following binding of inhibitory protein. On the basis of the results presented here, it is proposed that mixed protein-glyceride films could be used to characterize the interaction of various lipases with lipid substrates and to classify these enzymes according to their penetration power.
ESTHER : Gargouri_1986_Biochemistry_25_1733
PubMedSearch : Gargouri_1986_Biochemistry_25_1733
PubMedID: 3707907

Title : Molecular cloning and nucleotide sequence of rat lingual lipase cDNA - Docherty_1985_Nucleic.Acids.Res_13_1891
Author(s) : Docherty AJ , Bodmer MW , Angal S , Verger R , Riviere C , Lowe PA , Lyons A , Emtage JS , Harris TJ
Ref : Nucleic Acids Research , 13 :1891 , 1985
Abstract : Purified rat lingual lipase (EC3113), a glycoprotein of approximate molecular weight 52,000, was used to generate polyclonal antibodies which were able to recognise the denatured and deglycosylated enzyme. These immunoglobulins were used to screen a cDNA library prepared from mRNA isolated from the serous glands of rat tongue cloned in E. coli expression vectors. An almost full length cDNA clone was isolated and the nucleotide and predicted amino acid sequence obtained. Comparison with the N-terminal amino acid sequence of the purified enzyme confirmed the identity of the cDNA and indicated that there was a hydrophobic signal sequence of 18 residues. The amino acid sequence of mature rat lingual lipase consists of 377 residues and shares little homology with porcine pancreatic lipase apart from a short region containing a serine residue at an analogous position to the ser 152 of the porcine enzyme.
ESTHER : Docherty_1985_Nucleic.Acids.Res_13_1891
PubMedSearch : Docherty_1985_Nucleic.Acids.Res_13_1891
PubMedID: 3839077
Gene_locus related to this paper: ratno-1lipg

Title : Action of lipoprotein lipase on mixed triacylglycerol\/phosphatidylcholine monolayers. Activation by apolipoprotein C-II -
Author(s) : Vainio P , Virtanen JA , Kinnunen PK , Gotto AM, Jr. , Sparrow JT , Pattus F , Bougis P , Verger R
Ref : Journal of Biological Chemistry , 258 :5477 , 1983
PubMedID: 6853529
Gene_locus related to this paper: human-LPL

Title : Action of lipoprotein lipase on phospholipid monolayers. Activation by apolipoprotein C-II -
Author(s) : Vainio P , Virtanen JA , Kinnunen PK , Voyta JC , Smith LC , Gotto AM, Jr. , Sparrow JT , Pattus F , Verger R
Ref : Biochemistry , 22 :2270 , 1983
PubMedID: 6860664
Gene_locus related to this paper: human-LPL

Title : Product activation of pancreatic lipase. Lipolytic enzymes as probes for lipid\/water interfaces - Wieloch_1982_J.Biol.Chem_257_11523
Author(s) : Wieloch T , Borgstrom B , Pieroni G , Pattus F , Verger R
Ref : Journal of Biological Chemistry , 257 :11523 , 1982
Abstract : During the action of pancreatic lipase and colipase on racemic 1,2-didodecanoylglycerol monolayers in the absence of bile salts, biphasic kinetics was observed under conditions of high lipid packing. Similar kinetics has earlier been reported using phospholipid-emulsified triolein droplets (Borgstrom, B. (1980) Gastroenterology 78, 954-962). These kinetics are characterized by a lag time tau d, dependent on products accumulation at the substrate/water interface. This lag time is differentiated from the previously described enzyme concentration independent lag time tau i in systems of low lipid packing (Verger, R., Mieras, M. C. E., and de Haas, G. H. (1973) J. Biol. Chem. 248, 4023-4034). Both tau i and tau d reflect a rate-limiting step due to the slow enzyme penetration into the substrate interface. The variation of tau d under different conditions (change in pH and concentration of Ca2+, enzyme, bovine serum albumin, and lipolytic products) lead us to propose a model for the product activation during lipolysis. We will discuss the use of the pancreatic lipase-colipase system to probe the lipid packing of emulsified triglyceride particles and lipoproteins using tau d as a reference value.
ESTHER : Wieloch_1982_J.Biol.Chem_257_11523
PubMedSearch : Wieloch_1982_J.Biol.Chem_257_11523
PubMedID: 7118893

Title : Enzyme kinetics of lipolysis -
Author(s) : Verger R
Ref : Methods Enzymol , 64 :340 , 1980
PubMedID: 7374455