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Proteolytic Cleavage of Ostrich and TurkeyPancreatic Lipases

Production of an Active N-Terminal Domain

Abir Ben Bacha,* Ahmed Fendri,* Youssef Gargouri, PhD,* Hafedh Mejdoub, PhD,*Þ and Nabil MiledAQ1 *

Abstract:AQ2 Pure ostrich pancreatic lipase (OPL) is a monomer and

has a molecular mass of approximately 45 kd. This enzyme

hydrolyzes short- and long-chain triacylglycerols (TGs) at compa-

rable rates. Ostrich pancreatic lipase is a serine enzyme because it is

inhibited by tetrahydralipstatin. In the absence of bile salts and

colipase, OPL failed to catalyze the hydrolysis of pure tributyrin and

did not efficiently hydrolyzed olive oil emulsion. When bile salts and

colipase were preincubated with the substrate, the OPL kinetic

behaviorAQ3 remained linear for more than 30 minutes. The enzyme

presented a penetration power into an egg phosphatidylcholine

monomolecular film that was comparable to that of human pancreatic

lipase and lower than that of turkey pancreatic lipase (TPL).

The effect of some proteolytic enzymes on OPL and TPL was

checked. Chymotrypsin, trypsin, and thermolysin were able to

hydrolyze OPL and TPL in different ways. In both cases, only N-

terminal fragments accumulated during the hydrolysis, whereas no

C-terminal fragment was obtained in either case. Tryptic cleavage of

OPL and TPL completely degraded the enzymes. Nevertheless,

chymotryptic attack generated 35- and 43-kd forms of TPL and OPL,

respectively. Interestingly, the OPL 43-kd form was inactive,

whereas the TPL 35-kd protein conserved its lipolytic activity.

Key Words: lipase, colipase, domains, interfacial denaturation,

penetration power

Abbreviations: HPL - human pancreatic lipase, PPL - porcine

pancreatic lipase, OPL - ostrich pancreatic lipase, TPL - turkey

pancreatic lipase, PVDF - polyvinylidene difluoride,

BSA - bovine serum albumin, NaDC - sodium deoxycholate,

NaTDC - sodium taurodeoxycholate, THL - tetrahydralipstatin,

E600 - diethyl-p-nitrophenyl phosphate, SDS-PAGE - sodium

dodecyl sulfate-polyacrylamide gel electrophoresis, CMC - critical

micellar concentration

(Pancreas 2007;00:00Y00)

L ipases (glycerol ester hydrolase, EC 3.1.1.3) are found inall living species of the animal kingdom, as well as in

plants and microorganisms such as yeast, bacteria, and fungi.Under physiological conditions, mammalian lipases hydro-lyze the ester bonds of acylglycerols. The digestion of dietarytriacylglycerol (TG) in mammals is initiated by preduodenallipases in the upper part of the digestive tract and then drivento completion by pancreatic lipases in the intestine.1Y3 It hasbeen clearly established that partial hydrolysis of dietary TGemulsions by a preduodenal lipase rapidly triggers thepancreatic lipase activity.3,4

Human pancreatic lipase (HPL) alone is inactive invitro on an emulsified triglyceride substrate in the presence ofsupramicellar concentrations of bile salts such as those foundin the small intestine. The inhibition by bile salts, however,can be reversed by the specific pancreatic lipase cofactorcolipase.5Y8 Colipase is a small amphiphilic pancreaticprotein whose function is to anchor lipase on the bilesaltYcoated lipid interface via the formation of a specific 1:1lipase-colipase complex.9,10

The 3-dimensional structure of classical HPL11 hasshown that the single polypeptide chain (449 amino acids) isfolded into 2 domains as postulated by Bousset-Risso et al12:a large N-terminal domain (residue 1-335), which shows atypical >A hydrolase fold,13 and a small C-terminal domain(residue 336-449), which is of A sandwich type.11

In 1985, Bousset-Risso et al12 have demonstrated thatmild chymotryptic digestion of native porcine pancreaticlipase (PPL) preferentially cleaved the Phe335-Ala336 bond,which is located in the linking region, releasing the intact C-terminal domain. However, the N-terminal domain could notbe isolated. Furthermore, during the chymotryptic hydrolysis, aloss of enzymatic activity was observed.12 Later, Abousalhamet al14 have demonstrated that the isolated PPL and HPL C-terminal domains were completely inactive toward emulsifiedtributyrin but were able to bind colipase, whereas in the horseenzyme, the cleavage of the Leu410-Thr411 bond gave rise toa large N-terminal (45 kd) and a small C-terminal (4 kd)fragment. The horse pancreatic lipase N-terminal fragmentretained the lipase activity but failed to correctly bind colipase.

Ostrich and turkey pancreatic lipases (OPL and TPL,respectively) were purified, and some kinetic properties AQ5wereestablished using emulsified substrates or monomolecularfilm techniques.15Y17 Some biochemical properties of theseenzymes are similar to those of mammals. However, somedifferences were observed in the specificity of these enzymestoward various substrates. It seems, therefore, of interest tocheck some other catalytic and biochemical properties ofOPL and TPL to gain more insights into their action mode onTG. Thus, we performed limited proteolysis experiments on

ORIGINAL ARTICLE

Pancreas & Volume 00, Number 0, Month 2007 1

From the *Laboratoire de Biochimie et de Genie Enzymatique des Lipases,Ecole Nationale d’Ingenieurs de Sfax; and †Unite de Service Communpour la Recherche BSequenceur de Proteines,^ Faculte des Sciences deSfax, Sfax, Tunisia.

This work received financial support from the Direction Generale de laRecherche Scientifique et TechniqueAQ4 granted to the Laboratoire deBiochimie et de Genie Enzymatique des Lipases.

Reprints: Nabil Miled, Laboratoire de Biochimie et de Genie Enzymatiquedes Lipases, Ecole Nationale d’Ingenieurs de Sfax, route de Soukra, 3038Sfax, Tunisia (e-mail: nmiled@yahoo.com).

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OPL and TPL using trypsin and chymotrypsin. Profilesregarding proteolysis and activity are reported.

MATERIALS AND METHODS

MaterialsChymotrypsin, trypsin, thermolysin, tributyroylglyc-

erol (tributyrin, 99% purissAQ7 ), egg phosphatidylcholine(egg-PC), benzamidine, bovine serum albumin, sodiumdeoxycholate (NaDC), sodium taurodeoxycholate (NaTDC),triacetylglycerol (triacetin, 99% purissAQ7 ), and liprocil (80%medium-chain TGs and 15% linoleic fatty acid) were fromSigma-Aldrich-Fluka Chimie (St-Quentin-Fallavier, France);gum arabic was from Mayaud Baker Ltd (Dagenham, UK);and tetrahydralipstatin (THL) was a generous gift fromHoffmann-La-Roche Ltd (Basel, Switzerland). All otherchemicals and solvents were reagent grade or better andwere obtained from local suppliers.

ProteinsOstrich pancreatic lipase and TPL were purified at our

laboratory as described by Ben Bacha et al15 and Sayariet al,16 respectively. Ostrich colipase was purified as de-scribed by Ben Bacha et al.18 Protein concentration wasdetermined using bovine serum albumin (E1%

1cm = 6.7) asreference as described by Bradford et al.19

Determination of Lipase ActivityThe lipase activity was measured titrimetrically at pH

8.5 and 37-C with a pH-stat (Metrohom) under the standardAQ8assay conditions described previously, using tributyrin(0.25 mL) or liprocil (0.25 mL) in 30 mL of 2.5 mMTris-HCl, 1 mM CaCl2, pH 8.5,20 or olive oil emulsion21

as substrates.

Measurement of Lipase Penetration Intothe Egg-PC Monolayer

The surface pressure increase that was due to the pen-etration of lipase into the egg-PC/water interface was measuredin a cylindrical trough drilled in a Teflon block (surface area,7 cm2; total volume, 5 mL of 10 mM Tris-HCl buffer,pH 8.0, 150 mM NaCl, 21 mM CaCl2, and 1 mM EDTA).22

Limited ProteolysisLimited proteolysis of OPL and TPL was performed in

conditions similar to those reported for PPL.12 Lipase (1 mg)was dissolved in 1 mL of 50 mM Tris-HCl buffer, pH 8.5,without benzamidine. The lipase solution was digested at4-C, 25-C, 30-C, and 37-C with the selected endopeptidase.The endopeptidase/lipase molar ratio varied from 0.01 to 0.1.Samples (50 KL) were withdrawn from the incubationmixture at various times to assess the residual activity andthe electrophoretic profile. The reaction was stopped byaddition of benzamidine (4 mM final concentration).

Analytical MethodsAnalytical polyacrylamide gel electrophoresis of pro-

teins in the presence of sodium dodecyl sulfate was performed

using the method of Laemmli.23 Samples for sequencing wereelectroblotted according to Bergman and Jornvall.24

Amino Acid SequencingThe N-terminal sequence was determined by automated

Edman degradation using an Applied Biosystems ProteinSequencer Procise 492 equipped with 140 C high-performanceliquid chromatography system.25

RESULTS AND DISCUSSION

General CharacteristicsThe maximal activity of OPL was measured at pH 8.5

and 37-C. The optimum pH value for OPL activity is similarto that of TPL.16 Like mammalian pancreatic lipases, OPLwas found to be stable between pH 7 and pH 9, and it loses itsactivity at pH values greater than 9 or lesser than 5. Moreover,OPL was not stable at high temperatures because it iscompletely denaturated after incubation at 60-C for 30minutes. Specific activities of 6000, 5700, 5300, and 5000U/mg were measured on tributyrin, trioctanoin, liprocil, andolive oil emulsion as substrates, respectively, for OPL atpH 8.5 and at 37-C in the presence of colipase and 4 mMNaDC. These results show that in contrast to mammalianpancreatic lipases, which are mostly active on short-chainTG,26 OPL hydrolyzes both short- and long-chain TG atcomparable rates. Meanwhile, TPL16 was found to hydro-lyze long-chain TG more efficiently than short-chain ones.Interestingly, the OPL maintained approximately 35% of itsactivity when incubated at pH 3 for 1 hour, in contrast toall mammalian pancreatic lipases, which become inactiveunder these conditions. These differences among mamma-lian lipases, OPL, and TPL are likely because of a variationof the residues involved in the selectivity of the substrate.

Kinetic Studies of the OPL Activity on Tributyrinand Olive Oil Emulsions

It has been established that some mammalian pancre-atic lipases may lack enzyme activity when tributyrin is usedas substrate in the absence of bile salts and colipase. The highenergy existing at the tributyrin/water interface was sug-gested to explain their irreversible denaturation.27 Likewise,OPL failed to catalyze the hydrolysis of pure tributyrin. Totrigger the OPL activity on tributyrin, NaTDC and colipasewere added before the enzyme injection ( F1Fig. 1A). Whenolive oil was used as substrate, the OPL activity was found todecrease rapidly, in the absence of bile salts and colipase(Fig. 1B). In their presence, the kinetic behavior remainedlinear for more than 30 minutes (Fig. 1B). Similar resultswere obtained with HPL,27 whereas TPL was able to effi-ciently hydrolyze TG at a high interfacial energy (puretributyrin) without any denaturation and tolerate the presenceof long-chain free fatty acid at an olive oilYwater interface.17

This difference between TPL and OPL might be explained bystructural variation of exposed residues between the 2 lipases.

Interaction of OPL With Egg-PC MonolayersTo measure the critical surface pressure of OPL, we

injected a lipase sample under a monomolecular film of

AQ3

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egg-PC at an initial surface pressure (Pi) that ranged from 1 to30 mN/m. The maximal surface pressure increase ($Pmax)reached at equilibrium, 50 minutes after the injection of the

AQ10 lipase into the stirred aqueous suphase, was determined andplotted as function of the Pi. $Pmax decreased linearly withincreasing Pi (F2 Fig. 2). The critical surface pressure (Pc) forthe lipase was estimated by a linear extrapolation to zerosurface pressure increase of the experimental points. A Pc

value of 19 mN/m was obtained with OPL. If one takes the Pc

value as an indicator of the protein capacity to penetrate into amonomolecular film, it can be concluded that OPL interactswith lipidic films with the same efficiency as HPL (18 mN/m)but less than that of TPL (29 mN/m).17 These results can becorrelated with the higher efficiency of TPL to hydrolyzetributyrin despite its interfacial energy or olive oil even in thepresence of long-chain free fatty acids.

Effect of the Tetrahydrolipstatinon the OPL Activity

To check if OPL, as all known lipases, is a serineenzyme, we assessed the effect of THL on lipase activity.Incubation of OPL with THL (THL/OPL molar ratio, 200)

FIGURE 2. Interaction of OPL with egg-PC monolayers.Maximal increase in surface pressure reached at equilibrium50 minutes after injection of enzyme under egg-PCmonomolecular films spread at various initial surface pressures.Assays were carried out in a cylindrical Teflon trough (volume,5 mL; surface, 7 cm2). Final enzyme concentration was5 nM. Buffer: 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 21 mMCaCl2, and 1 mM EDTA. For the sake of comparison, bothdataAQ11 for HPL and TPL21 (carried out under the same conditions)were reported.

FIGURE 3. Inhibition kinetic behavior of OPL by THL with orwithout 4mMof DOC. Ostrich pancreatic lipase was incubatedat 37-C with THL (THL/OPL molar ratio, 200). Lipolytic activitywas measured at pH 8.5 and 37-C using olive oil emulsion.

AQ3

AQ9FIGURE 1. Kinetic behavior AQ3ofhydrolysis of tributyrin (A) or oliveoil (B) emulsions by OPL (14 U).Lipolytic activity was measuredat pH 8.5 and 37-C.

Pancreas & Volume 00, Number 0, Month 2007 Proteolytic Cleavage of 2 Bird Pancreatic Lipases

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was performed with or without bile salts (NaDC) at 37-C andpH 8.0. The residual lipase activity was measured as afunction of incubation time (F3 Fig. 3). After 2.5 hours ofincubation, in the absence of NaDC, the activity of OPL wasreduced by approximately 75% (Fig. 3). This inhibition wasprobably because of covalent binding of THL to the catalyticserine of OPL, as was described for PPL and HPL.28 Theaddition of 4 mM NaDC in the incubation mediumaccelerated the inactivation of OPL (Fig. 3), which is likelydue to the fact that THL formed mixed micelles with bilesalts.29 The inhibitory effect of the well-known serineesterase inhibitor diethyl-p-nitrophenyl phosphate (E600)on OPL activity was also checked. Preincubating E600 withpure OPL did not affect the catalytic activity of the OPLmeasured under the same experimental conditions (data notshown). Ostrich pancreatic lipase was inhibited by E600 onlyin the presence of bile salts at a concentration above theircritical micellar concentration (data not shown). At thisconcentration, bile salts formed mixed micelles with the E600inhibitor, which induced the opening of the lid covering thelipase active site. Similar results were obtained with PPL,HPL, and human gastric lipase.30Y32 These results showedclearly that OPL, like all known lipases from various origins,is a serine enzyme.

Proteolytic Cleavage of OPL and TPL

Cleavage of OPL

Lipase was incubated with trypsin, thermolysin, orchymotrypsin at 30-C using a protease/lipase molar ratio of

FIGURE 4. A, Remaining activity of OPL incubated with trypsin, chymotrypsin, and thermolysin. Ostrich pancreatic lipase wasincubated with chymotrypsin, trypsin, or thermolysin at 30-C (20:1 wt/wt) as described in Materials and Methods. The remainingactivity of OPL cleaved by chymotrypsin (Ì), trypsin (h), and thermolysin (&) toward tributyrin emulsion in the presence of 0.1 mMNaDC and in the presence of an excess of colipase was measured at various incubation times. Samples were withdrawn from theincubation mixtures at various times and analyzed. B, Sodium dodecyl sulfateYgel electrophoresis analysis of the chymotrypticcleavage of OPL as a function of time. The gel was stained with Coomassie blue to reveal proteins. Lane 1, molecular mass markers;lane 2, OPL; lane 3, after incubation of OPL with chymotrypsin for 48 hours. C, Localization of the cleaved fragments resulting fromthe chymotryptic cleavage of OPL on the primary sequence. The arrow indicates the chymotryptic cleavage side in OPL.

TABLE 1. Sequences Comparison of Proteic FragmentsIsolated After Chymotryptic Proteolysis of OPL With HPL

For comparison, bold, underlined, and italics symbols of residues indicate identical,homologous, and different amino acids, respectively; asterisk, amino acid residues of thecatalytic triad; arrow, chymotryptic site of OPL.

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0.05. The OPL activity was reduced by 50% within 3, 6, and12 hours by trypsin, thermolysin, and chymotrypsin, respec-tively, using tributyrin as substrate in the presence of

noninhibitory concentration of bile salts (0.1 mM NaTDC)( F4Fig. 4A). When using trypsin and thermolysin, the 45-kdband corresponding to OPL disappeared concomitantly with

FIGURE 5. A, Remaining activity of TPL incubated with trypsin, chymotrypsin, and thermolysin. Turkey pancreatic lipase wasincubated with chymotrypsin, trypsin, or thermolysin at 30-C (20:1 wt/wt) as described in Materials and Methods. The remainingactivity of TPL cleaved by chymotrypsin (Ì), trypsin (h), and thermolysin (&) toward tributyrin in the presence of 0.1 mMNaDC was measured at various incubation times. Samples were withdrawn from the incubation mixtures at various times andanalyzed. B, Sodium dodecyl sulfateYgel electrophoresis analysis of the tryptic cleavage of TPL as a function of time. The gelwas stained with Coomassie blue to reveal proteins. Lane 1, molecular mass markers; lane 2, TPL; lane 3, after incubation ofTPL with chymotrypsin for 3 hours. C, Sodium dodecyl sulfateYgel electrophoresis analysis of the chymotryptic cleavage of TPL asa function of time. The gel was stained with Coomassie blue to reveal proteins. Lane 1, molecular mass markers; lane 2, TPL;lanes 3 and, after incubation of TPL with chymotrypsin for 30 and 120 minutes, respectively. D, Effect of NaTDC on the activityof native TPL (h) and incubated TPL with chymotrypsin for 2 hours (r) in the presence of 5-fold M excess of colipase. The activitywas measured on emulsified tributyrin at pH 8.2 and at 37-C. E, Schematic patterns of chymotryptic cleavage of TPL.

AQ12

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the appearance of many bands of comparable intensitieshaving molecular masses varying from 10 to 40 kd (data notshown). Notably, when using chymotrypsin, the OPL banddisappeared during the hydrolysis, whereas 4 majors bands(P1, P2, P3, and P4) of different sizes (~43, 25, 14, and 10 kd,respectively) accumulated (Fig. 4B). These fragments weretransferred on a polyvinylidene difluoride membrane, andtheir N-terminal amino acids were sequenced. The results,together with the corresponding sequences from the HPL, aregiven inT1 Table 1. The N-terminal sequencing showed that thelarger 43-kd fragment (P1) corresponds to an N-terminaltruncated form of OPL starting at residue S31. As determinedby the N-terminal sequencing of OPL,15 residue 30 is a Trp.The N-terminal truncated OPL form (43 kd) was thusgenerated upon cleavage by chymotrypsin of the W30-S31bond of OPL. The P4 band has the same N-terminal sequenceas the P1 peptide, whereas the P3 band corresponds to a smallfragment produced by cleavage at the position 114 of the OPLsequence. The P2 peptide started at residue E253. Based on itsmolecular mass (25 kd), this band corresponds to the OPL (45kd) lacking N-terminal 252 amino acids. Based on theirmolecular masses, it is very likely that P4 (10 kd), P3 (14 kd),and P2 (25 kd) peptides were the products of the cleavage ofP1 at positions 114 and 252 (Fig. 4C). These results suggestthat OPL possesses aromatic residues at positions 114 and 252corresponding to Y and W amino acids for HPL, respectively,and allowing the chymotryptic cleavage to occur.

Cleavage of TPL

The cleavage of TPL by trypsin, chymotrypsin, andthermolysin was checked under the same conditions asdescribed above. Tryptic cleavage of TPL generated 2 majorfragments: T1 (16 kd) and T2 (10 kd), with a decrease of lipaseactivity by half within 90 minutes (F5 Figs. 5A, B). These 2 smallpeptides having the same N-terminal sequence as the matureTPL thus correspond to C-terminal truncated forms lackingcatalytic residues, which explains their inactivity. Conversely,when incubated with chymotrypsin, the protein band corre-sponding to the native TPL disappeared within 2 hours,generating 3 major fragments (B1, B2, and B3) of differentsizes (~35, 14, and 10 kd, respectively; Fig. 5C). TheN-terminal sequence of the B1 peptide was the same as thenative TPL. This shows that this 35-kd fragment is thusissued from a C-terminal truncation of the TPL. Based on itsmolecular mass (35 kd), the C-terminal truncated TPL form(B1) corresponds with the N-terminal domain, which leads tothe complete degradation of the C-terminal domain uponchymotryptic cleavage.AQ13 In contrast to what has been foundfor TPL, HPL and PPL chymotryptic digestion degraded theN-terminal domain and generated an intact C-terminal one.12

Based on their N-terminal sequences (data not shown), B2 andB3 fragments appeared to result from internal cleavage ofTPL. Interestingly, chymotryptic cleavage was not accom-panied by any loss of lipolytic activity assayed on tributyrin.Furthermore, no C-terminal domain was detected afterproteolysis. The N-terminal TPL domain alone appeared tobe active, despite the absence of the C-terminal domain. Thisis in line with the fact that TPL, whose N-terminal domain hasa higher hydrophobic surface, displayed the highest interac-

tion capacity with a lipidic substrate among the otherpancreatic lipases.33 In the presence of bile salts, the truncatedform of the TPL failed to be reactivated by colipase (Fig. 5D)as was observed for the 45-kd C-terminal truncated form of thehorse pancreatic lipase.14 This is in agreement with theabsence of the C-terminal colipase binding domain. Interest-ingly, the N-terminal TPL fragment was fully active ontributyrin emulsion in the absence of colipase and in thepresence of low-concentration bile salts (0.1 mM NaTDC).Attempts to produce an active N-terminal pancreaticdomain by limited proteolysis failed except with the horsepancreatic lipase14 and the TPL (present study). The HPLN-terminal domain produced in baculovirus-insect cellsystem34 had a very low lipolytic activity, and the authorssuggested that the C-terminal domain is necessary for lipaseactivity. This was attributed to the key role played by theC-terminal domain through colipase binding and interactionof the A5¶ loop with an interface. The N-terminal domain ofTPL thus appears to be more resistant to chymotrypticcleavage and correctly folded even in the absence of theC-terminal domain. This domain is composed of a basic >A

hydrolase fold, which is present in small bacterial lipases (eg,Rhizomucor miehei lipase, Thermomyces lanuginosa lipase). AQ13

Ostrich, turkey, and mammalian pancreatic lipases sharea high amino acid sequence homology. Further investigationsare, however, needed to identify key residues involved insubstrate recognition responsible for the biochemical differ-ences between the 2 classes of lipases.

ACKNOWLEDGMENTThe authors thank M. Saadaoui (FSS) for his technical

assistance.

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Pancreas & Volume 00, Number 0, Month 2007 Proteolytic Cleavage of 2 Bird Pancreatic Lipases

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