Riviere M

References (13)

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 : 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 : Importance of the lid and cap domains for the catalytic activity of gastric lipases - Miled_2003_Comp.Biochem.Physiol.B.Biochem.Mol.Biol_136_131
Author(s) : Miled N , Bussetta C , de Caro A , Riviere M , Berti L , Canaan S
Ref : Comparative Biochemistry & Physiology B Biochem Mol Biol , 136 :131 , 2003
Abstract : Human gastric lipase (HGL) is an enzyme secreted by the stomach, which is stable and active despite the highly acidic environment. It has been clearly established that this enzyme is responsible for 30% of the fat digestion processes occurring in human. This globular protein belongs to the alpha/beta hydrolase fold family and its catalytic serine is deeply buried under a domain called the extrusion domain, which is composed of a 'cap' domain and a segment consisting of 58 residues, which can be defined as a lid. The exact roles played by the cap and the lid domains during the catalytic step have not yet been elucidated. We have recently solved the crystal structure of the open form of the dog gastric lipase in complex with a covalent inhibitor. The detergent molecule and the inhibitor were mimicking a triglyceride substrate that would interact with residues belonging to both the cap and the lid domains. In this study, we have investigated the role of the cap and the lid domains, using site-directed mutagenesis procedures. We have produced truncated mutants lacking the lid and the cap. After expressing these mutants and purifying them, their activity was found to have decreased drastically in comparison with the wild type HGL. The lid and the cap domains play an important role in the catalytic reaction mechanism. Based on these results and the structural data (open form of DGL), we have pointed out the cap and the lid residues involved in the binding with the lipidic substrate.
ESTHER : Miled_2003_Comp.Biochem.Physiol.B.Biochem.Mol.Biol_136_131
PubMedSearch : Miled_2003_Comp.Biochem.Physiol.B.Biochem.Mol.Biol_136_131
PubMedID: 12941646
Gene_locus related to this paper: human-LIPF

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

Title : The 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 : 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 : Site-directed removal of N-glycosylation sites in human gastric lipase - Wicker-Planquart_1999_Eur.J.Biochem_262_644
Author(s) : Wicker-Planquart C , Canaan S , Riviere M , Dupuis L
Ref : European Journal of Biochemistry , 262 :644 , 1999
Abstract : Human gastric lipase (HGL) is a highly glycosylated protein, as glycan chains account for about 15% of the molecular mass of the native HGL. Four potential N-glycosylation consensus sites (Asn15, 80, 252 and 308) can be identified from the HGL amino acid sequence. We studied the functional role of the individual N-linked oligosaccharide chains by removing one by one all the N-glycosylation sites, via Ala residue replacement by site-directed mutagenesis of Ser and Thr residues from the consensus sequences Asn-X-Ser/Thr. Mutagenized cDNA constructs were heterologously expressed in the baculovirus/insect cell system. Removal of oligosaccharides either at Asn15, 80 or 252 was found to have no significant influence on the enzymatic activity measured in vitro. However, the absence of glycosylation at Asn308, as well as a total deglycosylation, reduced the specific enzymatic activity of recombinant HGL (r-HGL), measured on short- and long-chain triglycerides, to about 50% of normal values. Furthermore, biosynthesis and secretion of r-HGL markedly dropped when all four potential glycosylation sites were mutated. The kinetics of the interfacial adsorption of r-HGL and the completely deglycosylated r-HGL (four-site mutant) were found to be identical when recording the changes with time of the surface pressure either at the air-water interface or in the presence of an egg phosphatidylcholine (PtdCho) monomolecular film spread at various initial surface pressures. This indicates that both recombinant HGLs are identical, as far as recognition of phospholipid film and adsorption on PtdCho are concerned. The N-glycosylation of HGL may contribute to the enzyme stability in the stomach, as under acidic conditions the degradation by pepsin of the unglycosylated r-HGL is increased.
ESTHER : Wicker-Planquart_1999_Eur.J.Biochem_262_644
PubMedSearch : Wicker-Planquart_1999_Eur.J.Biochem_262_644
PubMedID: 10411623

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 : 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 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 : Influence of various signal peptides on secretion of mammalian acidic lipases in baculovirus-insect cell system -
Author(s) : Dupuis L , Canaan S , Riviere M , Wicker-Planquart C
Ref : Methods Enzymol , 284 :261 , 1997
PubMedID: 9379938

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 : Cloning of a parathyroid hormone\/parathyroid hormone-related peptide receptor (PTHR) cDNA from a rat osteosarcoma (UMR 106) cell line: chromosomal assignment of the gene in the human, mouse, and rat genomes - Pausova_1994_Genomics_20_20
Author(s) : Pausova Z , Bourdon J , Clayton D , Mattei MG , Seldin MF , Janicic N , Riviere M , Szpirer J , Levan G , Szpirer C , Goltzman D , Hendy GN
Ref : Genomics , 20 :20 , 1994
Abstract : Complementary DNAs spanning the entire coding region of the rat parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) were isolated from a rat osteosarcoma (UMR 106) cell-line cDNA library. The longest of these clones (rPTHrec4) was used to chromosomally assign the PTHR gene in the human, rat, and mouse genomes. By somatic cell hybrid analysis, the gene was localized to human chromosome 3 and rat chromosome 8; by in situ hybridization, the gene was mapped to human chromosome 3p21.1-p22 and to mouse chromosome 9 band F; and by interspecific backcross analysis, the Pthr gene segregated with the transferrin (Trf) gene in chromosome 9 band F. Mouse chromosome 9 and rat chromosome 8 are known to be highly homologous and to also show synteny conservation with human chromosome 3. These three chromosomes share the transferrin gene (TF), the myosin light polypeptide 3 gene (MYL3), and the acylpeptide hydrolase gene (APEH). Our results add a fourth gene, the PTHR gene, to the synteny group conserved in these chromosomes.
ESTHER : Pausova_1994_Genomics_20_20
PubMedSearch : Pausova_1994_Genomics_20_20
PubMedID: 8020952