Mass spectrometric evidence of covalently-bound tetrahydrolipstatin at the catalytic serine of Streptomyces rimosus lipase

We have recently detected that the lipase from Streptomyces rimosus belongs to a large but poorly characterised family of SGNH hydrolases having the αβα-fold. Our biochemical characterisation relates to the specific inhibition of an extracellular lipase from Streptomyces rimosus (SRL, 24.2 kDa, Q93MW7) by the preincubation method with tetrahydrolipstatin (THL). In high molar excess (THL/SRL = 590 at 25 °C, pH = 7.0) and after 2 h of incubation in an aqueous system, 56% of the enzyme inhibition was reached. Under the same conditions and in the presence of 50% (v/v) 2-propanol/water, 71% enzyme inhibition was obtained. Kinetic measurements are in agreement with pseudo-first-order kinetics. The nucleophilic attack of the catalytic serine residue 10 of SRL occurs via an opening of the β-lactone ring of tetrahydrolipstatin and formation of a covalent ester bond. The intact covalent complex of SRL-inhibitor was analysed by ESI and vacuum MALDI mass spectrometry and, furthermore, the exact covalent THL linkage was determined by vacuum MALDI high-energy collision-induced dissociation tandem mass spectrometry.     

Structural characterization of extracellular lipase from Streptomyces rimosus: assignment of disulfide bridge pattern by mass spectrometry

The cloning, sequencing and high-level expression of the gene encoding extracellular lipase from Streptomyces rimosus R6-554W have been recently described, and the primary structure of this gene product was deduced using a bioinformatic approach. In this study, capillary electrophoresis-on-the-chip and mass spectrometry were used to characterize native and overexpressed extracellular lipase protein from S. rimosus . The exact molecular mass of the wild-type and the overexpressed lipase, determined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, were in excellent agreement (Deltam=0.11 Da and Deltam=0.26 Da, respectively) with a value of 24165.76 Da calculated from the structure deduced from the nucleotide sequence, considering the mature enzyme with all six cysteines forming disulfide bridges. The primary structure derived from the nucleotide sequence was completely verified using a combination of tryptic digestion and formic acid cleavage of the protein, followed by peptide mass fingerprinting. Selected peptides were further investigated by MALDI low-energy collision-induced dissociation hybrid tandem mass spectrometry, allowing the unambiguous determination of their predicted amino acid sequence. No post-translational modifications of mature S. rimosus lipase were detected. Comparison of the peptide mass fingerprints from the reduced and non-reduced overexpressed enzyme unequivocally revealed three intramolecular disulfide bonds with the following linkages: C27-C52, C93-C101 and C151-C198.     

A novel streptomycete lipase: cloning,sequencing and high-level expression of the Streptomyces rimosus GDS(L)-lipase gene

An extracellular lipase from Streptomyces rimosus R6-554W has been recently purified and biochemically characterized. In this report the cloning, sequencing, and high-level expression of its gene is described. The cloned DNA contained an ORF of 804 bp encoding a 268-amino-acid polypeptide with 34 amino acid residues at the amino terminus of the sequence that were not found in the mature protein. The theoretical molecular mass (24.172 kDa) deduced from the amino acid sequence of the mature enzyme was experimentally confirmed. This lipase showed no overall amino acid sequence similarity to other lipases in the databases. However, two hypothetical proteins, i. e. putative hydrolases, derived from the genome sequencing data of Streptomyces coelicolor A3(2), showed 66% and 33% identity. In addition, a significant similarity to esterases from Streptomyces diastatochromogenes and Aspergillus terreus was found. Sequence analysis revealed that our novel S. rimosus lipase containing a GDS(L)-like consensus motif belongs to family II of lipolytic enzymes, previously unrecognized in Streptomyces. When the lipase gene was expressed in a S. rimosus lipase-deficient strain harboring the lipase gene on a high-copy-number vector, lipase activity was 22-fold higher than in the original strain.     

Identification of the active-site serine in human pancreatic lipase by chemical modification with tetrahydrolipstatin.

A chemical modification approach was used in this study to identify the active site serine residue of human pancreatic lipase. Purified human pancreatic lipase was covalently modified by incubation with [3H], [14C] tetrahydrolipstatin (THL), a potent inhibitor of pancreatic lipase. The radiolabeled lipase was digested with thermolysin, and the peptides were separated by HPLC. A single THL-peptide-adduct was obtained which was identical to that obtained earlier from porcine pancreatic lipase. This pentapeptide with the sequence VIGHS is covalently bound to a THL molecule via the side chain hydroxyl group of the serine unit corresponding to Ser-152 of the lipase. The selective cleavage of the THL-serine bond by mild acid treatment resulted in the formation of the delta-lactone Ro 40-4441 in high yield and clearly proves that THL is attached via an ester bond and with retention of stereochemistry at all chiral centers to the side chain hydroxyl group of Ser-152 of the lipase. The results obtained for human pancreatic lipase corroborate the inhibition mechanism of THL found on the porcine enzyme, and are in full agreement with the identification of the Ser-152 ... His-263 ... Asp-176 catalytic triad in the X-ray structure of human pancreatic lipase.     

The lipase inhibitor tetrahydrolipstatin binds covalently to the putative active site serine of pancreatic lipase

Tetrahydrolipstatin (THL) is a selective inhibitor of fat absorption. In animal models, it has anti-obesity and anti-hypercholesterolemic activity and is presently in clinical trials for these indications. THL binds covalently to pancreatic lipase. Complete inhibition of lipolytic activity is obtained concomitant with the incorporation of 1 mol of THL/mol of enzyme. Pancreatic lipase is the best studied lipase, but published results concerning its catalytic mechanism are still controversial. In order to learn more about the inhibitory mechanism of THL, a selective lipase inhibitor interacting at or near the catalytic site, and therefore, to obtain more information on the catalytic mechanism of lipase, we have determined the amino acid residue to which THL is bound. After proteolytic degradation of porcine pancreatic lipase inhibited with radioactively labeled THL, the labeled peptides were isolated and analyzed by quantitative amino acid analysis, N-terminal sequencing, and by mass spectrometry with fast atom bombardment ionization. The data clearly show that THL is bound as an ester to the serine 152 of the lipase.     

Characterization of a partially purified diacylglycerol lipase from bovine aorta

A partially-purified diacylglycerol (DG) lipase from bovine aorta has been characterized with respect to the effects of lipid metabolites and two lipase inhibitors, phenylboronic acid and tetrahydrolipstatin (THL). DG lipase activity was determined by the hydrolysis of the sn-1 position of 1-[1-⁴C]palmitoyl-2-oleoyl-sn-glycerol. The products of the lipase reaction, 2-monoacylglycerol (2-monoolein) and non-esterified fatty acids (oleate, arachidonate) produced a concentration-dependent (20–200 μM) inhibition of DG lipase activity. Oleoyl-CoA and dioleoylphosphatidic acid also inhibited aortic DG lipase activity, but lysophosphatidylcholine had little or no effect. The inhibition of aortic DG lipase by phenylboronic acid was competitive, with a K_i of approx. 4 mM. THL was a very potent inhibitor of aortic DG lipase; the concentration required for inhibition to 50% of control was 2–6 nM. THL was a very potent inhibitor of concentration of substrate in the assay was increased. Attempts to identify the aortic DG lipase by covalent-labelling with [¹⁴C]THL were unsuccessful. Immunoblotting experiments revealed that hormone-sensitive triacylglycerol lipase (HSL) could not be detected in bovine aorta.     

Mode of action of tetrahydrolipstatin: a derivative of the naturally occurring lipase inhibitor lipstatin

Tetrahydrolipstatin is a specific lipase inhibitor derived from lipstatin, a lipid produced by Streptomyces toxytricini. In addition to pancreatic lipase, it is shown in the present study that tetrahydrolipstatin also inhibits human gastric lipase, carboxyl ester lipase (cholesterol esterase) of pancreatic origin and the closely related bile-salt-stimulated lipase of human milk. It does not inhibit the exocellular lipase from Rhizopus arrhizus or a lipase recently isolated from Staphylococcus aureus. In the presence of a water-insoluble substrate, such as tributyrin, the inhibition has the characteristics of an irreversible inactivation of the uncompetitive type, thus indicating that an enzyme.substrate.inhibitor complex is formed, which cannot undergo further reaction to yield the normal product. This reaction probably takes place at the aqueous/oil interface of the substrate. In aqueous solution, in the absence of substrate, the inhibition of carboxyl ester lipase by tetrahydrolipstatin has the characteristics of being reversible, and finally becomes of a temporary nature analogues to the trypsin-trypsin inhibitor system. It is suggested that an enzyme-inhibitor complex of an acyl-enzyme type is formed that is slowly hydrolysed, with water as the final acceptor, leaving an intact enzyme and an inactive form of the inhibitor. The enzyme thus consumes the inhibitor, which undergoes a chemical conversion, as indicated by a change in mobility in an appropriate thin-layer chromatographic system, indicating an increase in hydrophilicity. Evidence is presented that the reaction product is an acid and that the functional group of tetrahydrolipstatin is the beta-lactone reacting with the active site of the enzyme.     

The effects of the broad-specificity lipase inhibitor, tetrahydrolipstatin, on the growth, development and survival of the larvae of Epiphyas postvittana (Walker) (Tortricidae, Lepidoptera)

The effects of the lipase inhibitor, tetrahydrolipstatin (THL), on neonate Epiphyas postvittana (Walker) (Lepidoptera, Tortricidae) larvae were investigated by feeding on control artificial diets (with and without 2% ethanol) and diets containing 2% ethanol and one of three concentrations of THL (0.011%, 0.037% and 0.11%). Small but significant reductions in growth rate, percent pupation and time to pupation were observed for larvae feeding on 2% ethanol control diet compared with standard control diet, but larger reductions in all parameters occurred with increasing THL concentration. Third instar larvae fed 0.011% THL in the diet had 40% of the midgut lipase activity in the relevant control larvae and showed up-regulation of gene expression of the gastric lipase-like family but not the pancreatic lipase-like family of midgut lipases.     

A comparative kinetic study on human pancreatic and Thermomyces lanuginosa lipases: Inhibitory effects of tetrahydrolipstatin in the presence of lipid substrates

The inhibitory effects of tetrahydrolipstatin (THL) on the hydrolytic activity of human pancreatic lipase (HPL) and T. lanuginosa lipase (TLL) on various lipidic substrates ‘poisoned’ with THL as previously described was studied, using either the pH-stat, monomolecular film or oil drop technique.Prior to adding lipase (method C), an ethanolic solution of THL was injected in a tributyrin (TC4) or a purified soybean oil (PSO) emulsion prepared in a pH-stat vessel. Under these conditions, THL was found to be a potent HPL inhibitor. After being dissolved in the pure triglyceride phase (method D), THL also strongly inhibited HPL. However, with TC4 as substrate TLL was efficiently inhibited by THL only when method C was used and not method D. The very different inhibitory effects on HPL and TLL recorded with method D and PSO as substrate were confirmed using the monomolecular film and oil drop techniques.With a monomolecular film of dicaprin (di-C10) as substrate, 1 molecule of THL embedded in 400 000 molecules of di-C10 sufficed to reduce the HPL activity to half of its initial value.HPL was therefore efficiently inhibited by THL with all the methods and substrates tested here. Paradoxically, TLL was inhibited by THL molecules transiently present in the aqueous phase and not by the THL molecules present at the triglyceride/water interface. It should therefore be stressed that the inhibitory effects of THL on each lipase depend strongly on the method and the substrate used.     

Inhibition of lipases from Chromobacterium viscosum and Rhizopus oryzae by tetrahydrolipstatin

Tetrahydrolipstatin is known as an inhibitor for pancreatic lipase but not for microbial lipases. In this paper we demonstrate that in the presence of water-insoluble substrates like tributyrin or olive oil, tetrahydrolipstatin inhibits the lipases of Chromobacterium viscosum and Rhizopus oryzae, although with different potency. In contrast to porcine pancreatic lipase, which forms an irreversible and covalent enzyme-inhibitor complex with tetrahydrolipstatin, the inhibition of the microbial lipases is reversible as the inhibitor can be removed from the enzyme-inhibitor complex by solvent extraction. Moreover, after inhibition of Chromobacterium viscosum lipase tetrahydrolipstatin remains chemically unchanged.     

Production, purification and characterization of thermophilic lipase from Bacillus sp. THL027

A low-cost medium, MGRS, has been developed for growth and lipase production from Bacillus THL027 at 65 degrees C and pH 7.0. MGRS was composed of 2% (v/v) buffer solution (7.3% (w/v) Na(2)HPO(4), 3.2% (w/v) KH(2)PO(4), pH 7.2), 40 microg ml(-1) FeSO(4) and 40 microg ml(-1) MgSO(4), 0.1% (w/v) (NH(4))(2)SO(4) supplemented with 3% NaCl, 0.1% glucose, 1.0% rice bran oil and 0.5% (w/v) rice bran. The lipase was purified 2.6-fold to apparent homogeneity by ultrafiltration and gel filtration chromatography. Its molecular mass was 69 kDa. The purified enzyme was characterized for its general physical properties.     

Inhibition of human pancreatic lipase by tetrahydrolipstatin: Further kinetic studies showing its reversibility

Tetrahydrolipstatin (THL, Orlistat) is a potent inhibitor of gastrointestinal lipases. Using the pH-stat technique we report that, in the absence of substrate, THL (at a molar excess of 100) inhibits rapidly (after few minutes of incubation) human pancreatic lipase (HPL). Bile salts over their critical micellar concentration (CMC) were found to accelerate the inhibition process.At variance with the generally accepted model of a covalent and quasi-irreversible acyl-lipase complex, we showed here that the inhibition of HPL could be rapidly and partially reversed in the presence of an emulsion of short- or long-chain triacylglycerols, as indicated by a kinetic reactivation process. The presence of bile salts in the incubation medium, containing THL and HPL, was found to stabilise the covalent complex as reflected by a decrease in the reactivation rate. Paradoxically, the presence of bile salts in the lipase assay enhanced this reactivation process probably by forming mixed micelles between bile salts and THL, which accelerates the deacylation phenomenon.On the basis of this kinetic study, a general model is proposed to describe the inhibition of lipases by THL in the aqueous phase as well as its partial reactivation process at the lipid–water interface.     

Assay and inhibition of diacylglycerol lipase activity

A series of N-formyl-α-amino acid esters of β-lactone derivatives structurally related to tetrahydrolipstatin (THL) and O-3841 were synthesized that inhibit human and murine diacylglycerol lipase (DAGL) activities. New ether lipid reporter compounds were developed for an in vitro assay to efficiently screen inhibitors of 1,2-diacyl-sn-glycerol hydrolysis and related lipase activities using fluorescence resonance energy transfer (FRET). A standardized thin layer chromatography (TLC) radioassay of diacylglycerol lipase activity utilizing the labeled endogenous substrate [1″-(14)C]1-stearoyl-2-arachidonoyl-sn-glycerol with phosphorimaging detection was used to quantify inhibition by following formation of the initial product [1″-(14)C]2-arachidonoylglycerol and further hydrolysis under the assay conditions to [1-(14)C]arachidonic acid.     

Inhibition of extracellular lipase from Streptomyces rimosus with 3,4-dichloroisocoumarin

Kinetic characterization of lipase inhibition was performed by activity measurement and mass spectrometry (MS), for the first time with serine-protease inhibitor 3,4-dichloroisocoumarin (DCI). Inhibition of Streptomyces rimosus extracellular lipase (SrLip), a member of the SGNH superfamily, by means of DCI follows the mechanism of two-step irreversible inhibition. The dissociation constant of the noncovalent E?I complex and first-order rate constant for inactivation were determined by incubation (K_i* = 26.6?±?2.8 µM, k₂ = 12.2?±?0.6 min–1) or progress curve (K_i* = 6.5?±?1.5 µM, k₂ = 0.11?±?0.01 min–1) method. Half-times of reactivation for lipase inhibited with 10-fold molar excess of DCI were determined by activity measurement (t_1/2 = 11.3?±?0.2?h), matrix-assisted laser desorption/ionization (MALDI, t_1/2 = 13.5?±?0.4?h), and electro-spray ionization (ESI, t_1/2 = 12.2?±?0.5?h) MS. The active SrLip concentration was determined by incubating the enzyme with near equimolar concentrations of DCI, followed by activity and MS measurement.     

Selectivity of inhibitors of endocannabinoid biosynthesis evaluated by activity-based protein profiling

The endocannabinoid 2-arachidonoylglycerol (2-AG) has been implicated as a key retrograde mediator in the nervous system based on pharmacological studies using inhibitors of the 2-AG biosynthetic enzymes diacyglycerol lipase alpha and beta (DAGL-alpha/beta). Here, we show by competitive activity-based protein profiling that the DAGL-alpha/beta inhibitors, tetrahydrolipstatin (THL) and RHC80267, block several brain serine hydrolases with potencies equal to or greater than their inhibitory activity against DAGL enzymes. Interestingly, a minimal overlap in target profiles was observed for THL and RHC80267, suggesting that pharmacological effects observed with both agents may be viewed as good initial evidence for DAGL-dependent events.     

Identification of a putative triacylglycerol lipase from papaya latex by functional proteomics

Latex from Caricaceae has been known since 1925 to contain strong lipase activity. However, attempts to purify and identify the enzyme were not successful, mainly because of the lack of solubility of the enzyme. Here, we describe the characterization of lipase activity of the latex of Vasconcellea heilbornii and the identification of a putative homologous lipase from Carica papaya. Triacylglycerol lipase activity was enriched 74-fold from crude latex of Vasconcellea heilbornii to a specific activity (SA) of 57 μmol·min(-1)·mg(-1) on long-chain triacylglycerol (olive oil). The extract was also active on trioctanoin (SA = 655 μmol·min(-1)·mg(-1) ), tributyrin (SA = 1107 μmol·min(-1)·mg(-1) ) and phosphatidylcholine (SA = 923 μmol·min(-1)·mg(-1) ). The optimum pH ranged from 8.0 to 9.0. The protein content of the insoluble fraction of latex was analyzed by electrophoresis followed by mass spectrometry, and 28 different proteins were identified. The protein fraction was incubated with the lipase inhibitor [(14) C]tetrahydrolipstatin, and a 45 kDa protein radiolabeled by the inhibitor was identified as being a putative lipase. A C. papaya cDNA encoding a 55 kDa protein was further cloned, and its deduced sequence had 83.7% similarity with peptides from the 45 kDa protein, with a coverage of 25.6%. The protein encoded by this cDNA had 35% sequence identity and 51% similarity to castor bean acid lipase, suggesting that it is the lipase responsible for the important lipolytic activities detected in papaya latex.     

Surface behaviour of bile salts and tetrahydrolipstatin at air/water and oil/water interfaces.

The surface behaviour of two bile salts, sodium deoxycholate (NaDC) and sodium taurodeoxycholate (NaTDC), as well as that of tetrahydrolipstatin (THL), a potent gastrointestinal lipase inhibitor, was studied at air/water and oil/water interfaces, using interfacial tensiometry methods. The surface behaviour of NaDC and NaTDC was comparable at both oil/water and air/water interfaces. A fairly compact interfacial monolayer of bile salts is formed well below the critical micellar concentration (CMC) and can help to explain the well-known effects of bile salts on the kinetic behaviour of pancreatic lipases. Using the Wilhelmy plate technique, the surface pressure-molecular area curves recorded with THL at the air/water interface showed a collapse point at a surface pressure of 24.5 mN.m(-1), corresponding to a molecular area of 70 A(2). Surprisingly, using the oil drop method, a limiting molecular area of 160 A(2) was found to exist at the oil/water interface. On the basis of the above data, space-filling models were proposed for bile salts and THL at air/water and oil/water interfaces.     

X-ray sequence ambiguities of Sclerotium rolfsii lectin resolved by mass spectrometry

X-ray crystallography, although a powerful technique for determining the three-dimensional structure of proteins, poses inherent problems in assigning the primary structure in residues Asp/Asn and Glu/Gln since these cannot be distinguished decisively in the electron density maps. In our recently published X-ray crystal structure of the Sclerotium rolfsii lectin (SRL) at 1.1 A resolution, amino acid sequence was initially deduced from the electron density map and residues Asp/Asn and Glu/Gln were assigned by considering their hydrogen bonding potential within their structural neighborhood. Attempts to verify the sequence by Edman sequencing were not successful as the N terminus of the protein was blocked. Mass spectrometry was applied to verify and resolve the ambiguities in the SRL X-ray crystal structure deduced sequence. From the Matrix assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI TOF-MS) and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis of tryptic and chymotryptic peptides of SRL, we could confirm and correct the sequence at five locations with respect to Asp/Asn and Glu/Gln. Analysis data also confirmed the positions of Leu/Ile, Gln/Lys residues and the sequence covering 118 of the total 141 residues accounting to 83.68% of the earlier deduced sequence of SRL.     

Effects of tetrahydrolipstatin, a lipase inhibitor, on absorption of fat from the intestine of the rat

Tetrahydrolipstatin (THL) derived by hydrogenation from lipstatin, a lipase inhibitor produced by Streptomyces toxytricini, has been shown to inhibit in vitro the activity of all three lipases secreted to the gastro-intestinal tract; gastric lipase, pancreatic lipase and carboxylester lipase (cholesterol ester hydrolase). The effects of THL on intestinal absorption of fat (transport to the thoracic duct chyle) has now been investigated after intraduodenal infusion in a rat model. Absorption of label from oleic acid when administered with monoolein in micellar bile salt solution was not affected by THL in concentrations up to 10(-4) M calculated on the volume of the aqueous phase. Absorption of free cholesterol in micellar bile salt solution of the lipolytic products of triolein; oleic acid and monoolein, is not significantly affected at a concentration of THL of 10(-4) M. Absorption of cholesterol from cholesteryl oleate under the same conditions is almost completely inhibited. The results indicate that absorption of free cholesterol is not dependent on the activity of pancreatic cholesterol ester hydrolase. The absorption of emulsified triolein was not significantly affected by 10(-5) M THL but decreased to around 30% of the controls by a concentration 10-times higher. There was no significant decrease of cholesterol absorption when administered in emulsified triolein while absorption of cholesteryl oleate was reduced at both concentrations of THL and almost completely at 10(-4) M. Radioactivity from [2-14C]THL when administered emulsified in triolein was recovered in urine, bile and thoracic duct lymph to 10-14, 8-13 and 1-3%, respectively, largely independent on dose administered. Label from [1"-14C] THL was recovered in the same amounts in lymph but much less in bile and urine indicating that the amino acid moiety has been split off early in the absorption process.     

Visualization of 2-arachidonoylglycerol accumulation and cannabinoid CB1 receptor activity in rat brain cryosections by functional autoradiography

In neuronal signalling mediated by the endocannabinoid 2-arachidonoylglycerol, both synthetic and inactivating enzymes operate within close proximity to the G(i/o)-coupled pre-synaptic CB(1) receptors, thus allowing for rapid onset and transient duration of this lipid modulator. In rat brain, 2-arachidonoylglycerol is inactivated mainly via hydrolysis by serine hydrolase inhibitor-sensitive monoacylglycerol lipase activity. We show in this study that comprehensive pharmacological elimination of this activity in brain cryosections by methyl arachidonylfluorophosphonate or hexadecylsulphonyl fluoride results in endocannabinoid-mediated CB(1) receptor activity, which can be visualized by functional autoradiography. URB597, a specific inhibitor of anandamide hydrolysis proved ineffective. TLC indicated that the bioactivity resided in 2-arachidonoylglycerol-containing fraction and gas chromatography-mass spectroscopy detected elevated levels of monoacylglycerols, including 2-arachidonoylglycerol in this fraction. Although two diacylglycerol lipase inhibitors, tetrahydrolipstatin (THL) and RHC80267, blocked the bulk of 2-arachidonoylglycerol accumulation in methyl arachidonylfluorophosphonate-treated sections, only THL reversed the endocannabinoid-dependent CB(1) receptor activity. Further studies indicated that at the used concentrations, THL rather specifically antagonized the CB(1) receptor. These findings confirm that in brain sections there is preservation of enzymatic pathways regulating the production of endogenous receptor ligands. Furthermore, the presently described methodology may serve as an elegant and intuitive approach to identify novel membrane-derived lipid modulators operating in the CNS.