Lafont D

References (9)

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

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

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

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

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

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

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

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

Title : 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