Alain De Caro

Characterization of an exported monoglyceride lipase from Mycobacterium tuberculosis possibly involved in the metabolism of host cell membrane lipids

The Rv0183 gene of the Mycobacterium tuberculosis H37Rv strain, which has been implicated as a lysophospholipase, was cloned and expressed in Escherichia coli. The purified Rv0183 protein did not show any activity when lysophospholipid substrates were used, but preferentially hydrolysed monoacylglycerol substrates with a specific activity of 290 units x mg(-1) at 37 degrees C. Rv0183 hydrolyses both long chain di- and triacylglycerols, as determined using the monomolecular film technique, although the turnover was lower than with MAG (monoacyl-glycerol). The enzyme shows an optimum activity at pH values ranging from 7.5 to 9.0 using mono-olein as substrate and is inactivated by serine esterase inhibitors such as E600, PMSF and tetrahydrolipstatin. The catalytic triad is composed of Ser110, Asp226 and His256 residues, as confirmed by the results of site-directed mutagenesis. Rv0183 shows 35% sequence identity with the human and mouse monoglyceride lipases and well below 15% with the other bacterial lipases characterized so far. Homologues of Rv0183 can be identified in other mycobacterial genomes such as Mycobacterium bovis, Mycobacterium smegmatis, and even Mycobacterium leprae, which is known to contain a low number of genes involved in the replication process within the host cells. The results of immunolocalization studies performed with polyclonal antibodies raised against the purified recombinant Rv0183 suggested that the enzyme was present only in the cell wall and culture medium of M. tuberculosis. Our results identify Rv0183 as the first exported lipolytic enzyme to be characterized in M. tuberculosis and suggest that Rv0183 may be involved in the degradation of the host cell lipids.

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.

Further biochemical characterization of human pancreatic lipase-related protein 2 expressed in yeast cells

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.

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 Å. 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. Proteins 32:523–531, 1998.

Immunocytochemical localization of pancreatic stone protein in the human digestive tract

Recently, in our laboratory, a protein extracted from human pancreatic stones was characterized and purified and a specific antibody was obtained. This pancreatic stone protein (PSP) was shown to have an inhibitory effect on the CaCO, crystal growth in vitro. The cellular origin of such a protein and its repartition along the digestive tract were studied by immunolocalization (protein A-colloidal gold method) at the ultrastructural level. Surgical biopsies of pancreata from normal or chronic pancreatitis patients, needle liver biopsies, gastric mucosa, and jejunum and duodenum biopsies were minced and fixed in the Karnovsky medium or in buffered 4% paraformaldehyde. The specimens were washed in buffer, dehydrated through ethanol, and embedded in Epon 812. Ultrathin sections, collected on uncoated nickel grids, were submitted to the following reactives at room temperature: protein A 1 mg/ml, anti-PSP (1:2 to 1:100), and protein A-colloidal gold. The specificity of the localization was checked by substituting buffer or nonimmune rabbit serum to anti-PSP. The stone protein was markedly present in the zymogen granules and condensing vacuoles of the normal pancreatic acinar cells, the label was found in the acinar and ductal lumen. In chronic pancreatitis, the localization of PSP, when it occurred, was extremely weak in the acinar cells. No PSP was specifically characterized in hepatocytes, gastric mucosa, and enterocytes. However, a weak but specific reaction was found in the secretory granules of Paneth cells. These results in pancreas confirm the acinar secretory origin of the PSP and are in good agreement with its possible function in stabilizing pancreatic juice in vivo, which is normally supersaturated in calcium carbonate. Moreover, the presence in the Paneth cells of a protein that can react with anti-PSP is an interesting finding that must be studied more precisely.

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.

Continuous measurement of galactolipid hydrolysis by pancreatic lipolytic enzymes using the pH-stat technique and a medium chain monogalactosyl diglyceride as substrate

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.

The two human chymotrypsinogens: Purification and characterization

The two chymotrypsinogens present in human pancreatic juice have been purified and characterized. The zymogens are two immunologically and electrophoretically different proteins. Chymotrypsinogen A, the major chymotryptic component (90% of the total potential N-acetyl-L-tyrosine ethylester activity) is stable in acidic medium. By its molecular weight (approx. 24 000), specific activity (530) and amino acid composition, human chymotrypsinogen A resembles chymotrypsinogens A and B form bovine and porcine pancreas. Chymotrypsinogen B is a minor chymotryptic component (7% of the total potential N-acetyl-L-tyrosine ethylester activity) unstable in acidic medium with a molecular weight slightly higher (approx. 27 000) and a specific activity slightly lower (300) than chymotrypsinogen A. The last 3% of the total potential N-acetyl-L-tyrosine ethylester activity corresponds to a proelastase that we have partially characterized.

Lipolysis of natural long chain and synthetic medium chain galactolipids by pancreatic lipase-related protein 2

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.

Purification of human gastric lipase by immunoaffinity and quantification of this enzyme in the duodenal contents using a new ELISA procedure

Human gastric lipase (HGL) is the first lipolytic enzyme involved in the digestion of dietary lipids along the gastrointestinal tract. We describe an improved procedure for isolating the enzyme using immunoaffinity chromatography in combination with ion-exchange chromatography. The purified enzyme, showing a single band on SDS-PAGE, expressed a specific activity of 1000 U/mg using tributyrin as the substrate. We also describe a specific enzyme-linked immunosorbent assay (ELISA) procedure for measuring duodenal HGL levels. The ELISA was performed using an anti-HGL polyclonal antibody (pAb) as the captor antibody and a biotinylated monoclonal antibody (mAb) as the detector antibody. With the double sandwich ELISA technique, HGL in the range of 1-60 ng/ml was measured in less than 5 h. Identical HGL concentrations were obtained using the above ELISA procedure when compared to those based on the enzymatic activity using the potentiometric method (correlation coefficient: r = 0.95). No significant interference from other duodenal components was observed, as proved by the quantitative HGL determinations performed on intestinal samples.