Microbial lipases: structures, function and industrial applications.
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Animals↗
Biomedical subjects
R Verger
Publications and source records attributed to R Verger.
Regulation of gastric and pancreatic lipase secretion by CCK and cholinergic mechanisms in humans.
Gastric lipase (HGL) contributes significantly to fat digestion. However, little is known about its neurohormonal regulation in humans. We studied the role of CCK and cholinergic mechanisms in the postprandial regulation of HGL and pancreatic lipase (HPL) secretion in six healthy subjects. Gastric emptying of a mixed meal and outputs of HGL, pepsin, acid, and HPL were determined with a double-indicator technique. Three experiments were performed in random order: intravenous infusion of 1) placebo, 2) low-dose atropine (5 micrograms.kg-.h-1), and 3) the CCK-A receptor antagonist loxiglumide (22 mumol.kg-.h-1). Atropine decreased postprandial outputs of HGL, pepsin, gastric acid, and HPL (P < 0.03) while slowing gastric emptying (P < 0.05). Loxiglumide markedly increased the secretion of HGL, pepsin, and acid while distinctly reducing HPL outputs and accelerating gastric emptying (P < 0.03). Plasma CCK and gastrin levels increased during loxiglumide infusion (P < 0.03). Atropine enhanced gastrin but not CCK release. Postprandial HGL, pepsin, and acid secretion are under positive cholinergic but negative CCK control, whereas HPL is stimulated by cholinergic and CCK mechanisms. We conclude that CCK and cholinergic mechanisms have an important role in the coordination of HGL and HPL secretion to optimize digestion of dietary lipids in humans.
Adult↗
A pancreatic lipase with a phospholipase A1 activity: crystal structure of a chimeric pancreatic lipase-related protein 2 from guinea pig.
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.
Amino Acid Sequence↗
Pancreatic lipase-related protein 2 but not classical pancreatic lipase hydrolyzes galactolipids.
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.
Animals↗
Regulation of lumen fat digestion: enzymic aspects.
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Amino Acid Sequence↗
Expression in insect cells and purification of a catalytically active recombinant human gastric lipase.
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.
Amino Acid Sequence↗
Purification and molecular characterization of lamb pregastric lipase.
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.
Amidohydrolases↗
Interfacial properties of the polyheme cytochrome c3 superfamily from Desulfovibrio.
In order to compare the interfacial behavior of the polyheme cytochromes c which belong to the cytochrome c3 superfamily, the monomolecular film technique was used to determine whether and how these metalloproteins interact with (phospho)lipids). Measurements of the variations of surface pressure and surface potential versus time have shown differences in their penetration capacity into phosphatidylcholine, dicaprin, and phosphatidylglycerol films. The Desulfovibrio vulgaris Hildenborough cytochrome with 16 hemes (Hmc) and Desulfovibrio desulfuricans Norway tetra- and octaheme cytochromes c3, which have been assumed to be soluble periplasmic molecules, may be considered as extrinsic membrane proteins, unlike the D. vulgaris Hildenborough cytochrome c3 (Mr 13 000). The interfacial properties are discussed in terms of the available three-dimensional structural data, the electrostatic potential calculation, and the results obtained by hydrophobic cluster analysis of the cytochrome sequences. The very different behavior of the two cytochromes c3 (Mr 13 000) enlightens the role of a particular surface loop in the interaction with a model membrane. A functional interpretation is proposed assuming that the D. vulgaris Hildenborough Hmc and both cytochromes c3 (Mr 13 000) and (Mr 26 000) from the Norway strain might provide the link between periplasmic hydrogen oxidation and cytoplasmic sulfate reduction.
Amino Acid Sequence↗
Interfacial binding of human gastric lipase to lipid monolayers, measured with an ELISA.
Two sandwich enzyme linked immunosorbent assays (ELISA) were developed for evaluating the surface excess at the lipid/water interface of the human gastric lipase (HGL) and two anti-HGL monoclonal antibodies (mAbs). These assays were adapted to the monomolecular film technique used previously for measuring lipase kinetics. HGL and the two anti-HGL mAbs (4-3 and 218-13) were biotinylated without any significant loss of their biological activities occurring. They were further detected by ELISA using either anti-HGL or anti-mouse IgG polyclonal antibodies as specific captors before being revealed using a streptavidin--peroxidase conjugate as tracer. The detection limit was 25 and 85 pg in the case of HGL and mAb, respectively. By combining the above sandwich ELISA technique with the monomolecular film technique, it was possible for the first time to measure the enzymatic activity of HGL on 1,2-didecanoyl-sn-glycerol (dicaprin) monolayers as well as to determine the corresponding interfacial excess of the enzyme. The HGL turnover number increased steadily with the lipid packing. The specific activities determined on dicaprin films spread at 35 mN.m-1 were found to be in the range of the values measured under optimal bulk assay conditions, using tributyrin emulsion as a substrate [i.e., 1000 mumol/(min.mg of enzyme)]. At a given lipase concentration in the water subphase, the interfacial binding of HGL to the nonhydrolyzable egg yolk phosphatidylcholine (egg PC) monolayers was found to be 10 times lower than that in the case of dicaprin monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)
Adsorption↗
Kinetic behaviour of pancreatic lipase in five species using emulsions and monomolecular films of synthetic glycerides.
In the absence of colipase and bile salts, using tributyrin emulsions or monomolecular films of dicaprin at low surface pressure, we observed that no significant lipase activity can be measured with Human Pancreatic Lipase (HuPL), Horse Pancreatic Lipase (HoPL) or Dog Pancreatic Lipase (DPL). Only Porcine Pancreatic Lipase (PPL) and recombinant Guinea Pig Pancreatic Lipase Related Protein of type 2 (r-GPL) hydrolyse pure tributyrin in the absence of any additive, as well as dicaprin films at low surface pressures. The former lipases may lack enzyme activity because of irreversible interfacial denaturation due to the high energy existing at the tributyrin/water interface and at the dicaprin film surface at low surface pressures. The enzyme denaturation cannot be reflected in the number of disulfide bridges, since all the pancreatic lipases tested here contain six disulfide bridges, but behaved very differently at interfaces. We propose to use the surface pressure threshold, as determined using the monomolecular technique, as a criterion for classifying lipases in terms of their sensitivity to interfacial denaturation.
Animals↗
Relationships between structures and kinetic properties of pancreatic lipases.
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Animals↗
The 2.46 A resolution structure of the pancreatic lipase-colipase complex inhibited by a C11 alkyl phosphonate.
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.
Binding Sites↗
Human pancreatic lipase. Importance of the hinge region between the two domains, as revealed by monoclonal antibodies.
Several monoclonal antibodies (mAbs) were prepared against human pancreatic lipase (HPL). Two enzyme-linked immunosorbent assay (ELISA) procedures were set up for screening hybridomas producing specific antibodies. Four mAbs (81-23, 146-40, 315-25, and 320-24) of the IgG1 isotype were found to react with HPL in both simple sandwich and double sandwich ELISAs, while mAb 248-31, of the IgG2b isotype, reacted only with HPL in a double sandwich ELISA. The results of Western blot analysis carried out with native and SDS-denatured HPLs indicated that mAb 248-31 recognized only native HPL, while all the other mAbs recognized both forms of HPL. Since mAb 248-31 did not recognize SDS-denatured HPL, it was not possible to localize its epitope. To carry out epitope mapping along the primary sequence of HPL, four fragments (14, 26, 30, and 36 kDa) resulting from a limited chymotryptic cleavage of HPL were characterized by Western blotting as well as N-terminal amino acid sequence analysis. Of the above five anti-HPL mAbs, four (81-23, 248-31, 315-25, and 320-24) were found to inhibit the lipolytic activity of HPL (in both the presence and absence of bile salts and colipase), while mAb 146-40 had no inhibitory effects. The epitope recognized by mAb 146-40 was found to be located in the N-terminal domain (Lys1-Phe335). Combined immunoinactivation and epitope mapping studies showed that three inhibitory mAbs (81-23, 315-25, and 320-24) recognize overlapping epitopes from the hinge region between the N- and C-terminal domains of HPL, belonging to the 26-kDa fragment. In the presence of lipids, a significant decrease has been observed in the bending angle between the N- and C-terminal domains of the HPL tertiary structure (van Tilbeurgh, H., Egloff, M. P., Martinez, C., Rugani, N., Verger, R. and Cambillau, C. (1993) Nature 362, 814-820). From the present immunochemical data, we further propose that locking the hinge movement with mAbs may induce lipase immunoinactivation.
Animals↗
Glyceride synthesis catalyzed by cutinase using the monomolecular film technique.
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.
Carboxylic Ester Hydrolases↗
Crystallographic study of the structure of colipase and of the interaction with pancreatic lipase.
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.
Amino Acid Sequence↗
Lipase structures at the interface between chemistry and biochemistry.
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).
Amino Acid Sequence↗
Phospholipase D from soybean (Glycine max L.) suspension-cultured cells: purification, structural and enzymatic properties.
Phospholipase D (phosphatidylcholine phosphatidohydrolase EC 3.1.4.4) from soybean (Glycine max L.) suspension-cultured cell was purified around 1,200-fold to homogeneity by acetone precipitation, Macro-Prep High Q anion exchange, and octyl-Sepharose CL-4B affinity chromatography. The purified enzyme released 1,600 mumol of choline per min per mg of protein. The enzyme is monomeric with a molecular mass of 92 kDa, as estimated by SDS-PAGE. One of the most interesting characteristics of the purified soybean phospholipase D was the dependence of the pH optimum on the Ca2+ ion concentration in the assay. With 10 mM, 20 mM and 40 mM Ca2+ ions, the optima were at pH 7.5, 6 and 5.5, respectively. The specific adsorption of phospholipase D onto octyl-Sepharose gel suggests that the molecule becomes more hydrophobic in the presence of Ca2+ ions. The amino acid sequence of the first 18 N-terminal residues of soybean phospholipase D revealed a high degree of homology with those previously published for cabbage leaf and castor bean endosperm enzymes. Western blots of the soybean phospholipase D showed an immunoreactivity with antibodies raised against a synthetic peptide corresponding to the 15 N-terminal amino acid residues of phospholipase D from cabbage leaves.
Amino Acid Sequence↗
Tryptic cleavage of gastric lipases: location of the single disulfide bridge.
Human (HGL) and rabbit (RGL) gastric lipases were cleaved by trypsin and the resulting peptides were characterized. Exposure of HGL to trypsin led to the production of three identified fragments (H1, H2 and H3) resulting from cleavage sites at Lys-4 and Arg-229. Fragments H2 (Lys-4-Arg-229) and H3 (Glu-230-Lys-379) were derived from fragment H1 (Lys-4-Lys-379). The single disulfide bridge (Cys-236-Cys-244) of the molecule is localized in fragment H3. Out of the three cysteine residues conserved in all known gastric lipases, the free sulfhydryl group (Cys-227) was localized in fragment H2. Immunoblots, carried out with the tryptic fragments of HGL and anti-HGL mAbs, revealed that five inhibitory mAbs immunoreacted selectively with the N-terminal fragment H2, whereas two other non inhibitory mAbs immunoreacted exclusively with the C-terminal fragment H3. Trypsin also cleaved RGL at two sites (Arg-55 and Arg-229) leading to four identifiable fragments (R1, R2, R3 and R4). One cleavage site (Arg-229) was found to be identical in both RGL and HGL. We propose that this latter site is localized between the two domains of native gastric lipases.
Amino Acid Sequence↗