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R Verger

Publications and source records attributed to R Verger.

At least 55 records · Page 3Linked to original sources
PubMed1998

Structural basis for the substrate selectivity of pancreatic lipases and some related proteins.

The classical human pancreatic lipase (HPL), the guinea pig pancreatic lipase-related protein 2 (GPLRP2) and the phospholipase A1 from hornet venom (DolmI PLA1) illustrate three interesting steps in the molecular evolution of the pancreatic lipase gene family towards different substrate selectivities. Based on the known 3D structures of HPL and a GPLRP2 chimera, as well as the modeling of DolmI PLA1, we review here the structural features and the kinetic properties of these three enzymes for a better understanding of their structure-function relationships. HPL displays significant activity only on triglycerides, whereas GPLRP2 displays high phospholipase and galactolipase activities, together with a comparable lipase activity. GPLRP2 shows high structural homology with HPL with the exception of the lid domain which is made of five amino acid residues (mini-lid) instead of 23 in HPL. The lid domain deletion in GPLRP2 allows the free access to the active site and reduces the steric hindrance towards large substrates, such as galactolipids. The role of the lid domain in substrate selectivity has been investigated by site-directed mutagenesis and the substitution of HPL and GPLRP2 lid domains. The addition of a large-size lid domain in GPLRP2 increases the substrate selectivity for triglycerides by depressing the phospholipase activity. The phospholipase activity is, however, not induced in the case of the HPL mutant with GPLRP2 mini-lid. Therefore, the presence of a full-length lid domain is not the unique structural feature explaining the absence of phospholipase activity in HPL. The 3D structure of the GPLRP2 chimera and the model of DolmI PLA1 reveal a higher hydrophilic/lipophilic balance (HLB) of the surface loops (beta5 loop, beta9 loop, lid domain) surrounding the active site, as compared to the homologous loops in HPL. This observation provides a potential explanation for the ability of GPLRP2 and DolmI PLA1 to hydrolyze polar lipids, such as phospholipids. In conclusion, the beta5 loop, the beta9 loop, and the lid domain play an essential role in substrate selectivity towards triglycerides, phospholipids and galactolipids.

Amino Acid Sequence↗
PubMed1998

Pancreatic lipase-related protein 1 (PLRP1) is present in the pancreatic juice of several species.

Pancreatic lipase-related protein 1 (PLRP1) was purified from human, canine, porcine and rat pancreatic juices. The four PLRP1s were identified using microsequencing methods after performing gel filtration on Ultrogel AcA-54 followed by chromatography on Heparin-Sepharose cation-exchanger. Polyclonal antibodies specific to human PLRP1 (HPLRP1) were raised in the rabbit using a synthetic decapeptide from HPLRP1. The results of Western blotting analysis showed that these antibodies recognized native HPLRP1 and recombinant HPLRP1 produced by insect cells, and cross-reacted only with rat PLRP1 (RPLRP1). No significant lipolytic activity was observed with native canine PLRP1 and recombinant HPLRP1 on various glycerides, phospholipid and vitamin esters, or on cholesterol esters. It was established for the first time that this protein is secreted in variable amounts by the adult exocrine pancreas of several species.

Amino Acid Sequence↗
PubMed1998

Reactivation of the totally inactive pancreatic lipase RP1 by structure-predicted point mutations.

Both classical pancreatic lipase (DPL) and pancreatic lipase-related protein 1 (DPLRP1) have been found to be secreted by dog exocrine pancreas. These two proteins were purified to homogeneity from canine pancreatic juice and no significant catalytic activity was observed with dog PLRP1 on any of the substrates tested: di- and tri-glycerides, phospholipids, etc. DPLRP1 was crystallized and its structure solved by molecular replacement and refined at a resolution of 2.10 A. Its structure is similar to that of the classical PL structures in the absence of any inhibitors or micelles. The lid domain that controls the access to the active site was found to have a closed conformation. An amino-acid substitution (Ala 178 Val) in the DPLRP1 may result in a steric clash with one of the acyl chains observed in the structures of a C11 alkyl phosphonate inhibitor, a transition state analogue, bound to the classical PL. This substitution was suspected of being responsible for the absence of DPLRP1 activity. The presence of Val and Ala residues in positions 178 and 180, respectively, are characteristic of all the known PLRP1, whereas Ala and Pro residues are always present in the same positions in all the other members of the PL gene family. Introducing the double mutation Val 178 Ala and Ala 180 Pro into the human pancreatic RP1 (HPLRP1) gene yielded a well expressed and folded enzyme in insect cells. This enzyme is kinetically active on triglycerides. Our findings on DPLRP1 and HPLRP1 are therefore likely to apply to all the RP1 lipases.

Alanine↗
PubMed1998

Human pancreatic lipase: an exposed hydrophobic loop from the C-terminal domain may contribute to interfacial binding.

Epitope mapping was performed using four anti-HPL monoclonal antibodies (mAb's 81-23, 146-40, 315-25, and 320-24) directed against human pancreatic lipase (HPL). Three HPL mutants produced in insect cells were tested for this purpose: (i) N-HPL, which consists of only the N-terminal domain of HPL, (ii) HPL(-lid), in which a short loop consisting of 5 amino acid residues replaces the full-length 23-residue lid domain present in HPL, and (iii) N-GPLRP2/C-HPL chimera, a chimeric mutant consisting of the N-terminal domain of the guinea pig pancreatic lipase related protein 2 (GPLRP2) fused to the C-terminal domain of HPL. The C-terminal domain of HPL (C-HPL) was prepared in a pure form after performing chymotryptic digestion of HPL. The mAb 146-40 recognizes HPL, HPL(-lid), and N-HPL but not GPLRP2, N-GPLRP2/C-HPL chimera, or the C-HPL. The antibody mAb 146-40 therefore specifically recognizes the N-terminal domain of HPL, and the epitope recognized does not include the amphiphilic lid. On the other hand, mAb's 81-23, 315-25, and 320-24 react specifically to the C-terminal domain of HPL, since they recognize HPL, HPL(-lid), the N-GPLRP2/C-HPL chimera, and the C-HPL but not N-HPL or GPLRP2. It was further established that these three mAb's recognize the same conformational epitope, the structure of which is stabilized by the N-terminal domain in the presence of SDS at concentrations greater than its critical micellar concentration. This conformational epitope was found to be located in the vicinity of Met 397 and Arg 414. These two residues delineate a highly exposed peptide stretch extending from the HPL C-terminal domain, which includes a hydrophobic surface loop (beta5'). Kinetic studies on the HPL/mAb's complexes showed that the lipase activity was much lower in these complexes than in HPL. The results of the present study suggest for the first time that the beta5' loop from the C-terminal domain may be involved in the interaction of HPL with a lipid/water interface.

Amino Acid Sequence↗
PubMed1998

An enzymatically active truncated form (-55 N-terminal residues) of rabbit gastric lipase. Correlation between the enzymatic activity and disulfide bond oxydo-reduction state.

Rabbit gastric lipase (RGL) was subjected to proteolysis with trypsin and led to cleavage occurring at three defined sites (Lys-4, Arg-55 and Arg-229). The tryptic hydrolysate contained four fragments: Gly-230-Lys-379 (T1), Gly-56-Arg-229 (T2), Ser-5-Arg-55 (T3), as well as a 45 kDa molecular form consisting of peptides T1 and T2 linked by a disulfide bridge. The tryptic hydrolysate of RGL as well as the 55 N-terminal amino acid deleted forms conserved 30% of the initial enzymatic activity in a tributyrin assay. Two out of the three cysteine residues which are present in all the known gastric lipases were found to be involved in a disulfide bridge. Unlike HGL, RGL appears to have a heterogenous pattern of cysteine residues. The 30% enzymatic activity of RGL persisting after trypsin treatment may be attributable to the 45 kDa molecular form (with the Cys-227-Cys-236 or Cys-227-Cys-244 disulfide bridge). Trypsin-treated HGL, which was completely inactivated, showed that a single location of the disulfide bridge existed between cysteine residues 236 and 244. It can be concluded that the existence of one disulfide bridge is necessary to maintain the lipase activity of the 45 kDa form of RGL.

Amino Acid Sequence↗
PubMed1998

Purification and interfacial behavior of recombinant human gastric lipase produced from insect cells in a bioreactor.

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

Animals↗
PubMed1998

An inactive pancreatic lipase-related protein is activated into a triglyceride-lipase by mutagenesis based on the 3-D structure.

Both classical dog pancreatic lipase (DPL) and dog pancreatic lipase-related protein 1 (DPLRP1) have been found to be secreted by the exocrine pancreas. These two proteins were purified to homogeneity from canine pancreatic juice and no significant catalytic activity was observed with DPLRP1 on any of the substrates tested: di- and tri-glycerides; phospholipids (PC); etc. DPLRP1 was crystallized and its structure solved by molecular replacement and refined at a resolution of 2.10 A. Its structure is similar to that of the classical pancreatic lipase (PL) structures determined in the absence of any inhibitors or micelles. The lid domain that controls the access to the active site was found to have a closed conformation. An amino-acid substitution (Ala 178 Val) in the DPLRP1 was suspected of being responsible for the absence of enzymatic activity by inducing a steric clash with one of the acyl chain observed in the structures of chiral C11 alkyl phosphonate inhibitors, bound to the classical PL. The presence of Val and Ala residues in positions 178 and 180, respectively, are characteristic of the three known pancreatic lipase-related protein 1 (PLRP1), whereas Ala and Pro residues are always present at the same positions in all the other members of the PL gene family. Introducing the double mutation Val 178 Ala and Ala 180 Pro into the human pancreatic-related protein 1 (HPLRP1) gene yielded a well expressed and folded enzyme in insect cells. This enzyme is kinetically active on tributyrin (1800 U/mg) as well as trioctanoin (2250 U/mg) and its activity is low in the presence of taurodeoxycholate and stimulated in the presence of colipase. Our findings on DPLRP1 and HPLRP1 are therefore likely to apply to all the PLRP1 lipases.

Amino Acid Sequence↗
PubMed1997

Study of fatty acid specificity of sunflower phospholipase D using detergent/phospholipid micelles.

The fatty acid specificity of phospholipase D purified from germinating sunflower seeds was studied using mixed micelles with variable detergent/phospholipid ratios. The main advantage of this approach is that since the substrate is integrated in the detergent micelles, comparisons can be made between the kinetic constants of a wide range of phosphatidylcholine (PtdCho) compounds with various fatty acid contents. Phospholipase D is subject to interfacial activation as it is most active on water-insoluble substrates. It is not active on sphingomyelin and only slightly on lysophosphatidylcholine. By fitting the curves based on the experimental kinetic data, the interfacial dissociation constant of phospholipase D, the maximum hydrolysis rate Vm and the kinetic constant Km(B), were determined with the micellar substrate. The specificity of various substrates was examined by comparing the Vm/Km(B) values, and it was noted that sunflower phospholipase D is most active on medium-chain fatty PtdCho compounds. With long-chain natural phospholipids, the specificity of phospholipase D was slightly dependent on the level of fatty acid unsaturation. The pure enzyme was able to hydrolyse the sunflower phospholipids present in mixed detergent micelles but not the phospholipids integrated in the natural sunflower oil body structure. We concluded, however, that during the germination of sunflower seeds, phospholipase D might be involved in the degradation of oil bodies, since other factors present in crude seed extracts may make phospholipids accessible to the enzyme.

Calcium↗
PubMed1997

Effects of colipase and bile salts on the catalytic activity of human pancreatic lipase. A study using the oil drop tensiometer.

Using the oil drop technique, we studied the effects of colipase and bile salts on the rate of hydrolysis of soybean oil by human pancreatic lipase (HPL) as well as on the interfacial binding. Upon continuously recording the decrease in the interfacial tension with time, a 10-15-fold increase in the HPL activity was found to occur in the presence of colipase. The catalytic rate constants of hydrolysis measured at the oil drop surface were found to be of the same order of magnitude as those obtained with monomolecular films spread at the air-water interface. Biotin-labeled HPL (HPL*) was used to determine the amount of adsorbed enzyme using an ELISA test. Less than 1% of the total amount of injected HPL* molecules was found to have adsorbed to the oil-water interface, and no significant effects of colipase on HPL* binding were observed. No significant changes in the hydrolysis rates or the binding of HPL* were detected in the presence of bile salts at concentrations ranging from below their critical micellar concentration (CMC) up to 100 microM. At the oil-water interface, in the absence or presence of bile salts below their CMC, it can be concluded that the colipase is a true lipase cofactor, i.e, it increases the enzyme turnover (approximately 10-15-fold) and does not affect the interfacial lipase adsorption.

Animals↗
PubMed1997

Purification and characterization of a porcine liver microsomal triacylglycerol hydrolase.

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

Amino Acid Sequence↗
PubMed1997

Pancreatic lipase structure-function relationships by domain exchange.

We designed chimeric mutants by exchanging the lid domains of the classical human pancreatic lipase (HPL) and the guinea pig pancreatic lipase related protein 2 (GPLRP2). This latter enzyme possesses naturally a large deletion within the lid domain and is not activated by lipid/water interfaces. Furthermore, GPLRP2 exhibits phospholipase A1 and lipase activities in the same order of magnitude, whereas HPL has no significant phospholipase activity and displays a clear interfacial activation. An HPL mutant [HPL(-lid)] with GPLRP2 mini-lid domain does not display interfacial activation. Its specific activity toward triglycerides is, however, dramatically reduced. A GPLRP2 mutant [GPLRP2(+lid)] with HPL full-length lid domain is not interfacially activated, and its lid domain probably exists under a permanent open conformation. Therefore, the phenomenon of interfacial activation in HPL is not only due to the presence of a full-length lid domain but also to other structural elements which probably allow the existence of stabilized closed and open conformations of the lid. GPLRP2(+lid) phospholipase activity is significantly reduced as compared to GPLRP2, whereas its lipase activity remains at the same level. Therefore, the lid domain plays a major role in substrate selectivity and can be considered as part of the active site. However, the presence of a full-length lid domain is not sufficient to explain the absence of phospholipase activity in HPL since HPL(-lid) does not display any phospholipase activity. We also produced a chimeric GPLRP2 mutant in which the C-terminal domain was substituted by the HPL C-terminal domain. The colipase effects, i.e., anchoring and stabilization of the lipase at the interface, are clearly observed with the chimera, whereas GPLRP2 is insensitive to colipase. The kinetic characterization of this chimera reveals for the first time that the interfacial stability of pancreatic lipases depends on the structure of the C-terminal domain.

Amino Acid Sequence↗
PubMed1997

In vivo and in vitro studies on the stereoselective hydrolysis of tri- and diglycerides by gastric and pancreatic lipases.

The stereoselectivity of dog gastric and dog pancreatic lipases was investigated both in vitro, under simulated physiological conditions, and in vivo, during the digestion of a liquid test meal. In vitro it was observed that although both lipases had a stereopreference for the sn-3 position in triglycerides, it was about three times higher in the case of the gastric lipase. On the other hand, both lipases clearly showed a comparable enantioselectivity for the sn-1 position when a racemic diolein was used as the substrate. In the case of pancreatic lipase, the enantiomeric excess of 1,2-sn-diolein generated in vitro by the hydrolysis of triolein was found to decrease significantly, and even to be slightly reversed, at high rates of hydrolysis (above 50%) due to the further stereoselective hydrolysis of diglycerides into monoglycerides. This finding may explain the low enantiomeric excess of the diglycerides observed in vivo during the early phase of intraduodenal digestion when pancreatic lipase plays a predominant role and the rate of triolein hydrolysis is already high. On the other hand, a large enantiomeric excess of 1,2-sn-diolein generated from triolein was always the fingerprint of the gastric lipase in vitro even at high hydrolysis rates. This fingerprinting of gastric lipase was observed during both the intragastric phase and the late intestinal phase of lipolysis. This feature was therefore taken as an index to determine the respective roles of gastric and pancreatic lipases during in vivo lipolysis. To the best of our knowledge, this is the first time that stereoselectivity has been used as a tool to discriminate between the activities of two enzymes hydrolyzing the same substrate in vivo.

Animals↗