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Robert Verger - One of the best experts on this subject based on the ideXlab platform.
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kinetic properties of pancreatic and intestinal spla2 from chicken and mammals using the Monomolecular Film technique
Joint International Conference on Information Sciences, 2011Co-Authors: Aida Karray, Robert Verger, Youssef Gargouri, Zied Zarai, Sofiane BezzineAbstract:Abstract The interfacial kinetic and binding data for the pancreatic and intestinal sPLA2 from bird and mammals show that these enzymes have dramatically different ability to bind and hydrolyse phospholipids. The main conclusions from our experimental data indicate that phosphatidylcholine monolayers (PC), in contrast to phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), were resistant to the hydrolysis by human intestinal sPLA2. Conversely, chicken intestinal sPLA2 was found to be able to hydrolyse all the phospholipids tested, including PC. The experiments show also that the interfacial penetrating ability of chicken sPLA2 (from intestine and pancreas) was higher than their mammalian’s orthologs. This observation is confirmed by the activity of pancreatic chicken PLA2 measured on PC Film showing that the interfacial pressure window that permits sPLA2 activity was very large, between 5 and 20 dynes cm −1 , compared with the porcine pancreatic sPLA2-IB which was inactive at pressure above 15 dynes cm −1 . In trying to establish a structure–function relationship, we examined the surface electrostatic potentials of the various sPLA2 from chicken and mammals. We reported in this study that the binding, orientation and persistence of sPLA2 at the lipid–water interface is probably governed by the electrostatic and hydrophobic forces operative at this surface. These variations argue strongly that these enzymes are not isoforms and that they are expected to have functions other than the release of lipid mediators for the biosynthesis of the eicosanoids.
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staphylococcal lipases stereoselectively hydrolyse the sn 2 position of Monomolecular Films of diglyceride analogs application to sn 2 hydrolysis of triolein
Journal of Colloid and Interface Science, 2010Co-Authors: Habib Horchani, Youssef Gargouri, Adel Sayari, Nadia Ben Salem, Ali Chaari, Robert VergerAbstract: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.
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continuous measurement of the lipoxygenase catalyzed oxidation of unsaturated lipids using the Monomolecular Film technique
Pharmaceutical Research, 2006Co-Authors: Abdelkarim Abousalham, Robert VergerAbstract:This paper presents the first detailed kinetic investigation involving the continuous measurement of the soybean lipoxygenase 1 (LOX1)-catalyzed oxidation of unsaturated lipids using the Monomolecular Film technique at an argon/water interface. The presence of oxidation products in the monolayer is qualitatively detected, at a constant area, by an increase in the monolayer surface pressure. Alternatively, the rate of lipid oxidation can be measured, at a constant surface pressure, by a backward movement of the mobile barrier, due to the oxidation-dependent increase in the monolayer area. For instance, the LOX1-catalyzed oxidation of 1,2-di[cis-9,12-octadecadienoyl]-sn-glycero-3-phosphocholine (diC18:2PC) monolayer was found to be characterized by a time dependent increase in the monolayer area, at constant surface pressure. However, the increase in the monolayer area was thought to be caused first by the penetration of the enzyme into the interface, and secondly, by the formation of hydroperoxides at the interface, due to the LOX1-catalyzed oxidation of the diC18:2PC Film. The rate of the LOX1-catalyzed oxidation of diC18:2PC Film was measured by subtracting the increase in the area due to the LOX1-penetration into the non-oxidizable 1,2-di[cis-9-octadecenoyl]-sn-glycero-3-phosphocholine (diC18:1PC) Film from the increase in the area due to LOX penetration and oxidation of the diC18:2PC Film. At a constant optimum surface pressure of 1 mN m−1, similar initial rates of LOX1-catalyzed oxidation are observed with both linoleic acid methyl ester (C18:2) and diC18:2PC. It is worth noting that the surface density of C18:2 acyl chains is also similar in both Films. We observed that a phosphatidylcholine (PC) Film with two potentially oxidizable chains (e.g., diC18:2PC) is oxidized at a rate which is twice that obtained with a PC containing a single oxidizable chain (e.g., 1-hexadecanoyl-2-[cis-9,12-octadecadienoyl]-sn-glycero-3-phosphocholine). The enzymatic lipid oxidation seems to occur when the monolayer is in the expanded state. This expanded state may possibly result in vivo from the lipolysis of a biomembrane and consequently lipolysis and lipid oxidation are coupled at the membrane level.
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Scorpion digestive lipase: Kinetic study using Monomolecular Film technique
Colloids and Surfaces B: Biointerfaces, 2006Co-Authors: Nacim Zouari, Adel Sayari, Robert Verger, Nabil Miled, Youssef GargouriAbstract: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 mN m−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 mN m−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 mN m−1) is comparable to those of pancreatic lipases.
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human pancreatic lipase related protein 2 is a galactolipase
Biochemistry, 2004Co-Authors: Barbara Sias, Dominique Lafont, Robert Verger, Francine Ferrato, Philippe Grandval, Paul Boullanger, Alain De Caro, B Leboeuf, Frederic CarriereAbstract: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.
Alain De Caro - One of the best experts on this subject based on the ideXlab platform.
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further biochemical characterization of human pancreatic lipase related protein 2 expressed in yeast cells
Journal of Lipid Research, 2007Co-Authors: Cecilia Eydoux, Dominique Lafont, Frederic Carriere, Francine Ferrato, Paul Boullanger, Josiane De Caro, R Laugier, Alain De CaroAbstract: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.
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human pancreatic lipase related protein 2 is a galactolipase
Biochemistry, 2004Co-Authors: Barbara Sias, Dominique Lafont, Robert Verger, Francine Ferrato, Philippe Grandval, Paul Boullanger, Alain De Caro, B Leboeuf, Frederic CarriereAbstract: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.
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A study on human gastric lipase and complexes with monoclonal antibodies using the Monomolecular Film technique
Colloids and Surfaces B: Biointerfaces, 1993Co-Authors: Margarita G. Ivanova, Alain De Caro, Mustapha Aoubala, Cécile Daniel, Joelle Hirn, Robert VergerAbstract:Abstract Five monoclonal antibodies (MAbs) directed against human gastric lipase (HGL) were used as probes to characterize and locate the functional sites (catalytic and lipid binding domains) on the surface of the enzyme. Taking advantage of the low surface activity of IgG molecules, the capacity of the MAbs to inhibit the penetration of native and sulfhydryl (SH)-modified HGL into lipid monolayers was investigated using the Monomolecular Film technique. A schematic representation of the topography of the epitopes recognized by the MAbs in relation to the catalytic and lipid binding sites of the enzyme is proposed. The region containing the free SH group essential for lipase activity is identified.
Frederic Carriere - One of the best experts on this subject based on the ideXlab platform.
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further biochemical characterization of human pancreatic lipase related protein 2 expressed in yeast cells
Journal of Lipid Research, 2007Co-Authors: Cecilia Eydoux, Dominique Lafont, Frederic Carriere, Francine Ferrato, Paul Boullanger, Josiane De Caro, R Laugier, Alain De CaroAbstract: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.
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syntheses of an α d gal 1 6 β d gal diglyceride as lipase substrate
Carbohydrate Research, 2006Co-Authors: Dominique Lafont, Frederic Carriere, Francine Ferrato, Paul BoullangerAbstract:Two different routes were explored to afford 3-O-(6-O-α-d-galactopyranosyl-β-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-β-d-galactopyranosyl)-1,2-O-isopropylidene-sn-glycerol acceptor with 2-pyridyl 2,3,4,6-tetra-O-benzyl-1-thio-β-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-α-d-galactopyranosyl)-d-galactopyranosyl 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.
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human pancreatic lipase related protein 2 is a galactolipase
Biochemistry, 2004Co-Authors: Barbara Sias, Dominique Lafont, Robert Verger, Francine Ferrato, Philippe Grandval, Paul Boullanger, Alain De Caro, B Leboeuf, Frederic CarriereAbstract: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.
Paul Boullanger - One of the best experts on this subject based on the ideXlab platform.
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further biochemical characterization of human pancreatic lipase related protein 2 expressed in yeast cells
Journal of Lipid Research, 2007Co-Authors: Cecilia Eydoux, Dominique Lafont, Frederic Carriere, Francine Ferrato, Paul Boullanger, Josiane De Caro, R Laugier, Alain De CaroAbstract: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.
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syntheses of an α d gal 1 6 β d gal diglyceride as lipase substrate
Carbohydrate Research, 2006Co-Authors: Dominique Lafont, Frederic Carriere, Francine Ferrato, Paul BoullangerAbstract:Two different routes were explored to afford 3-O-(6-O-α-d-galactopyranosyl-β-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-β-d-galactopyranosyl)-1,2-O-isopropylidene-sn-glycerol acceptor with 2-pyridyl 2,3,4,6-tetra-O-benzyl-1-thio-β-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-α-d-galactopyranosyl)-d-galactopyranosyl 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.
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human pancreatic lipase related protein 2 is a galactolipase
Biochemistry, 2004Co-Authors: Barbara Sias, Dominique Lafont, Robert Verger, Francine Ferrato, Philippe Grandval, Paul Boullanger, Alain De Caro, B Leboeuf, Frederic CarriereAbstract: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.
Dominique Lafont - One of the best experts on this subject based on the ideXlab platform.
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further biochemical characterization of human pancreatic lipase related protein 2 expressed in yeast cells
Journal of Lipid Research, 2007Co-Authors: Cecilia Eydoux, Dominique Lafont, Frederic Carriere, Francine Ferrato, Paul Boullanger, Josiane De Caro, R Laugier, Alain De CaroAbstract: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.
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syntheses of an α d gal 1 6 β d gal diglyceride as lipase substrate
Carbohydrate Research, 2006Co-Authors: Dominique Lafont, Frederic Carriere, Francine Ferrato, Paul BoullangerAbstract:Two different routes were explored to afford 3-O-(6-O-α-d-galactopyranosyl-β-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-β-d-galactopyranosyl)-1,2-O-isopropylidene-sn-glycerol acceptor with 2-pyridyl 2,3,4,6-tetra-O-benzyl-1-thio-β-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-α-d-galactopyranosyl)-d-galactopyranosyl 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.
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human pancreatic lipase related protein 2 is a galactolipase
Biochemistry, 2004Co-Authors: Barbara Sias, Dominique Lafont, Robert Verger, Francine Ferrato, Philippe Grandval, Paul Boullanger, Alain De Caro, B Leboeuf, Frederic CarriereAbstract: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.