Characterization of pancreatic lipase-related protein 2 isolated from human pancreatic juice

Biochimica et Biophysica Acta 1701 (2004) 89 – 99 www.bba-direct.com

Characterization of pancreatic lipase-related protein 2 isolated from human pancreatic juice Josiane De Caro *, Barbara Sias, Philippe Grandval, Francine Ferrato, Hubert Halimi, Fre´de´ric Carrie`re, Alain De Caro Laboratoire d’Enzymologie Interfaciale et de Physiologie de la Lipolyse, UPR 9025 CNRS-Institut de Biologie Structurale et Microbiologie, 31, Chemin Joseph-Aiguier, 13402 Marseille Cedex 20, France Received 16 March 2004; received in revised form 1 June 2004; accepted 16 June 2004

Abstract Human pancreatic lipase-related protein 2 (HPLRP2) was identified for the first time in pancreatic juice using specific anti-peptide antibodies and purified to homogeneity. Antibodies were raised in the rabbit using a synthetic peptide from the HPLRP2 protein sequence deduced from cDNA. Western blotting analysis showed that these antibodies did not react with classical human pancreatic lipase (HPL) or human pancreatic lipase-related protein 1 (HPLRP1) but cross-reacted with native rat PLRP2 (RPLRP2), as well as with recombinant rat and guinea-pig PLRP2 (GPLRP2). Immunoaffinity chromatography was performed on immobilized anti-recombinant HPLRP2 polyclonal antibodies to purify native HPLRP2 after conventional chromatographic steps including gel filtration and chromatrography on an anionexchanger. The substrate specificity of HPLRP2 was investigated using various triglycerides, phospholipids and galactolipids as substrates. The lipase activity on triglycerides was inhibited by bile salts and weakly restored by colipase. The phospholipase activity of HPLRP2 on phospholipid micelles was very low. A significant level of galactolipase activity was measured using monogalactosyldiglyceride monomolecular films. These data suggest that the main physiological function of HPLRP2 is the hydrolysis of galactolipids, which are the main lipids present in vegetable food. D 2004 Elsevier B.V. All rights reserved. Keywords: Pancreatic juice; Pancreatic lipase-related protein; Anti-peptide antibody; Polyclonal antibody; Galactolipid; (Man); Galactolipase; Digestion

1. Introduction Pancreatic lipase (PL) is the main enzyme involved in the digestion of dietary triglycerides (TGs) in the small intestine and its biochemical and structural features have been well documented [1,2]. Besides the classical PL, mRNAs encoding two PL related proteins 1 and 2 (PLRP1 and PLRP2) were first isolated in humans [3]. During the last decade, numerous PL-related proteins have been identified in other species by isolating mRNAs from pancreas [4 –7] and other tissues [8,9], or isolated using classical protein purification procedures [4,6,7]. Since then, some important questions have arisen as to their gene regulation processes, catalytic properties, secretory levels and physiological functions. The amino acid sequences of PLRPs show a high level of

* Corresponding author. Tel.: +33-491-164-488; fax: +33-491-715-857. E-mail address: [email protected] (J. De Caro). 1570-9639/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2004.06.005

identity (65 –70%) with classical PL: they have an identical catalytic triad and the same two-domain pattern of structural organization, which is superimposable on that of classical PL [2,10,11], but PLRPs have different functional properties from those of classical PL. PL and PLRPs are expressed mainly in the exocrine pancreas, and in adult pancreas, the secretory level of each lipase varies greatly from one species to another. High levels of PLRP1 have been measured in dog [10] and cat [12], whereas low levels have been recorded in human, porcine and rat [13]. PLRP1 displays no lipase activity, due to the presence of two amino acid substitutions (V178, A180) as compared to classical PL (A178, P180). Replacing these HPLRP1 residues by those present in classical HPL at the same position restored a high level of lipase activity [10]. The exact physiological function of PLRP1 has not yet been elucidated. Most of the biochemical properties of the PLRP2s secreted by the exocrine pancreas of rat, guinea pig, coypu,

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horse and humans were determined using the recombinant form of the enzyme. Numerous kinetics studies have been carried out on PLRP2s to determine the substrate specificity and the effects of colipase and bile salts and to compare the biochemical properties of PLRP2s with those of the classical PL. The PLRP2s from guinea-pig (GPLRP2) [4,14,15], coypu (CoPLRP2) [16] and rat (RPLRP2) [11,15,17] show lipolytic activity on a variety of substrates, including triglycerides, phospholipids (phospholipase A1 activity) and galactolipids. The guinea-pig and coypu pancreas secretes high levels of classical PL and PLRP2 [6,14], but no evidence for the presence of phospholipase A2 was found [4,18]. The lipase activity of these PLRP2s is inhibited by micellar concentrations of bile salts, but, unlike classical PL, is not restored by colipase, which suggests that the PLRP2s have not lipase activity in vivo but may act like a phospholipase [16]. By contrast, the lipase activity of RPLRP2 is not affected by bile salts but the addition of colipase significantly increases its activity [17]. Lastly, it has been reported that horse PLRP2 is inhibited by micellar concentrations of bile salts, not restored by colipase, and displays a very low level of phospholipase activity [7]. It has been established that GPLRP2 and RPLRP2 hydrolyze galactolipids [15], which are major lipids present in vegetable food. The data available clearly show that the molecular characteristics of each PLRP2 vary considerably from one species to another. Structural comparisons between the PLRP2 gene and the classical PL and PLRP1 genes have shown a different exon – intron organization. The PL and PLRP1 genes have 13 exons, whereas the PLRP2 gene has only 12 exons because it contains one large exon that corresponds to exons I and II in the other two genes [19 –21]. The 1.8-kb mRNA encoding HPLRP2 was isolated from the total mRNA of human pancreas and the results of Northern blotting analysis showed that the rate of expression of HPLRP2 is 24-fold lower than that of HPL [3]. The HPLRP2 mRNA encodes a 469-amino-acid polypeptide, including a 17-residue signal peptide, and the theoretical molecular mass of the mature polypeptide (452 residues) is 50,081 Da. The deduced amino acid sequence of HPLRP2 contains two potential N-glycosylation sites (N336 and N411). By contrast, HPL has one potential glycosylation site, whereas HPLRP1 has no potential glycosylation sites. The recombinant HPLRP2 expressed in COS cells showed a lipolytic activity using triolein as substrate that was marginally dependent on the presence of colipase [3]. Andersson et al. [22] have suggested that PLRP2 may play a physiological role in the digestion of dietary galactolipids, consisting mainly of monogalactosyldiacylglycerol (MGalDG) and digalactosyldiacylglycerol (DGalDG). These authors established that human pancreatic juice contains two enzymes which are able to efficiently hydrolyse DGalDG, releasing digalactosylmonoacylglycerol (DGalMG), fatty acids and water-soluble galactose-containing compounds [22]. After separating these two enzymes by performing gel filtration chromatography, the first one was

identified as carboxyl ester lipase (CEL; 100 kDa) and the second one was co-eluted with HPL (50 kDa). Since HPL shows no activity towards galactolipids [15], it was suggested that HPLRP2 might display the galactolipase activity present in human pancreatic juice, but the native form could not be identified. This hypothesis was supported by data subsequently obtained by the same authors, indicating that rGPLRP2 and rRPLRP2 efficiently hydrolyze galactolipids [15]. Up to now, no native HPLRP2 has ever been identified in pancreatic juice and purified. In the present study, a PLRP2 from human pancreatic juice was identified and purified, and it was established for the first time that this enzyme is present in the exocrine pancreatic secretion. An anti-peptide antibody specifically recognizing HPLRP2 was produced in order to identify the native enzyme and to monitor its chromatographic behaviour during the purification procedure. In addition, we tested the lipase and phospholipase activities of the purified enzyme on triglycerides and lecithin, respectively. The galactolipase activity was measured using a monogalactosyldiglyceride (MGalDG) substrate and the monomolecular film technique. Our results throw light on the physiological role of HPLRP2 in the digestive tract and suggest that its main function is that of a galactolipase.

2. Materials and methods 2.1. Pancreatic juice and lipase samples Human pancreatic juice was obtained from patients devoid of pancreatic disease by performing endoscopic retrograde catheterization on the main pancreatic duct, at the Sainte-Marguerite Hospital (Marseille), under the supervision of Professor R. Laugier. Samples were immediately lyophilized and stored at 20 jC before use. Recombinant HPLRP1 and HPLRP2 were expressed in insect cells using the baculovirus expression system and purified to homogeneity after a one-step cation-exchange chromatography procedure on a Mono S column (Pharmacia) [23]. Recombinant RPLRP2 (rRPLRP2) and recombinant GPLRP2 (rGPLRP2) were generous gifts from Dr. M. Lowe (Washington University, St. Louis, USA) and Dr. S. Patkar (Novozymes, Denmark), respectively. Porcine pancreatic colipase was purified from pancreatic tissue as previously described [24]. 2.2. HPLRP1 and HPLRP2 synthetic peptides Regions of the amino acid sequences of HPLRP1 and 2 were identified as potential specific antigens with the criteria of weak amino acid conservation when compared to each other and to the classical HPL, and high surface accessibility based on the known 3-D structure of HPL [2]. For HPLRP1, a peptide was designed with the sequence QMLDILLTEY corresponding to residues 132 – 141 [13].

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For HPLRP2, two peptides with QLITGTEPDTI (Fig. 1) and TAFLIQALSTQLG sequences corresponding to HPLRP2 residues 51– 61 and 130 –142, respectively, were selected. These peptides were synthesized using the solidphase method developed by Barany and Merrifield [25]. An additional cysteine residue was added at the amino termini of the peptides to allow their selective coupling to maleimide-activated ovalbumin. Peptide purification was performed with a Beckman HPLC apparatus and a Merck C8 reverse phase column (10  125 mm). Amino acid sequences of peptides were controlled using an Applied Biosystems sequencer (Model 476 A).

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amine group to the wells. Specific antibodies were detected with peroxidase-conjugated anti-rabbit IgG antibody (Sigma, Ref. A-6154, St. Louis, MO, USA, dilution 1:10,000). 2.4. Preparation of the anti-rHPLRP2 column 2.4.1. Production of anti-rHPLRP2 polyclonal antibodies Polyclonal antibodies directed against rHPLRP2 produced in insect cells (to be published) were raised in rabbits using 3 mg of purified antigen for each animal. Rabbits were injected subcutaneously every 2 weeks with 0.5 mg of antigen. The titres of the sera were assessed by performing a direct binding ELISA.

2.3. Anti-peptide antibodies Anti-peptide antibodies were prepared in rabbits (Bourgogne Fawn) by injecting them with 100 Ag of peptide coupled to ImjectR maleimide-activated ovalbumin from Pierce (Ref. 77126, Rockford, IL, USA) used as the carrier. Eight subcutaneous injections of 100 Ag of peptide mixed with complete Freund’s adjuvant were performed on each rabbit at 2-week intervals. Rabbits were bled by ear vein puncture before the beginning of the immunization schedule to collect samples of pre-immune sera. Blood samples were subsequently taken 1 week after each peptide injection. The reactivity of the sera was evaluated by performing a simple ELISA using a microtiter plate (CovAbtest, CovalAb, Lyon, France) containing the peptide covalently linked via its

2.4.2. Purification of antibodies directed against rHPLRP2 Specific anti-rHPLRP2 antibodies were purified by affinity chromatography using an immobilized rHPLRP2 column. For this purpose, 2.5 mg of rHPLRP2 was immobilized on 3 ml of swollen Affi-Gel 15 (Bio-Rad) equilibrated with 10 mM MES buffer (pH 5.5) containing 100 mM NaCl. The unreacted gel groups were blocked with a solution of 1 M ethanolamine (pH 8.0). The gel was then poured into a glass column (1.6  1.5 cm) and washed successively with 30 ml of 0.2 M HCl –glycine buffer (pH 2.3) and 60 ml of 25 mM Tris – HCl buffer (pH 7.3) containing 150 mM NaCl (TBS). Under these conditions, more than 90% of the initial amount of rHPLRP2 was covalently coupled to the gel.

Fig. 1. (A) Water-accessible surface areas along the 47 – 66 peptide stretch of the HPL 3-D structure (Brookhaven Data Bank accession number 1LPA) calculated using the DSSP option [43] from the Turbo Frodo software program and amino acid sequence comparisons on the same peptide stretch between the three human pancreatic lipases. The 51 – 61 stretch synthesis was chosen to obtain a specific anti-HPLRP2 serum. (B) Alignment of the HPLRP2 undecapeptide (51 – 61) with the corresponding peptides of RPLRP2 and GPLRP2.

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Antisera were loaded onto the immobilized-rHPLRP2 column equilibrated with TBS. After a 12-h incubation period at 4 jC under rotative agitation (18 rpm), the gel was washed successively with TBS, 25 mM Tris – HCl (pH 7.3) containing 0.5 M NaCl and 0.2% Triton X-100, and again with TBS until the eluate had no optical absorbance at 280 nm. Pure antibodies directed against rHPLRP2 were eluted with 0.2 M HCl – glycine buffer (pH 2.3). The fractions (1 ml) were immediately neutralized with a solution of 2 M Tris – HCl (pH 9.0), pooled, concentrated to about 1.5 mg/ml, dialyzed against 20 mM phosphate buffer (pH 7.3) containing 150 mM NaCl and stored at 20 jC. 2.4.3. Preparation of the anti-rHPLRP2 column Antibodies directed against rHPLRP2 (13 mg) were immobilized on 5 ml of swollen Affi-Gel 10 equilibrated with the same dialysis buffer as that used above. The same procedure was used as that described above for rHPLRP2 immobilization. Ninety percent of the starting antibodies were coupled to the gel, which was then poured into a glass column (1.6  2.5 cm). This column was used for the final native HPLRP2 purification step.

containing 6 M lithium chloride. The fractions (1 ml) were immediately diluted with an equivalent volume of TBS, then pooled and concentrated on an Amicon ultra-filtration cell. The remaining lithium chloride was eliminated by successively diluting the Amicon cell contents with TBS. The concentration of HPLRP2 was determined by performing quantitative amino acid analysis using a Beckman 6300 amino acid analyzer. 2.6. Lipase and phospholipase activity determination The lipase activity was measured potentiometrically at 37 jC using a pH-stat (716 DMS Titrino, Metrohm). The substrates used were vigorously stirred emulsions of either tributyrin (short-chain) or trioctanoin (medium-chain) or olive oil (long-chain). The reaction vessel contained 0.5 ml of tributyrin or trioctanoin or 5 ml of olive oil emulsified (10%, wt/wt) in gum-arabic (10%, wt/wt) and a solution of 1 mM Tris –HCl buffer, pH 7.5 containing 0.1 M NaCl, 5 mM CaCl2, giving a final volume of 15 ml. Olive oil assays were run at pH 8.0. Phospholipase activity was measured potentiometrically using lecithin from egg yolk as described by de Haas et al. [27]. Specific activities were expressed as

2.5. Purification of human PL and PLRP2 Human PL and PLRP2 were purified from lyophilized pancreatic juice (0.6 –1 g) suspended in 6– 10 ml of 10 mM Tris – HCl buffer (pH 7.5) containing 0.5 M NaCl, 2 mM benzamidine, 2 mM phenylmethylsulfonylfluoride and one protease inhibitor cocktail tablet (complete Mini, Roche). After centrifugation (5500  g for 30 min), the insoluble pellet was discarded and the clear supernatant was applied to an Ultrogel AcA-54 column (2.4  200 cm, flow rate 24 ml/h, fraction volume 3.9 ml) equilibrated and eluted with the same buffer. The fractions containing lipase activity were pooled and concentrated with a Diaflo cell fitted with an Amicon PM 30 membrane. The concentrated sample was dialyzed against 5 mM Tris –HCl buffer (pH 8.0) containing 5 mM NaCl, 1 mM benzamidine, and then loaded onto a Macro-Prep DEAE column (1.6  5 cm, flow rate 9 ml/h, fraction volume 1.5 ml) equilibrated with the same buffer. A stepwise elution procedure was then performed using buffers containing 0.050, 0.1, 0.2 and 0.5 M NaCl. Fractions corresponding to the classical PL, PLRP1 and PLRP2 peaks were identified by measuring the lipase activity and by performing Western blotting using the specific anti-classical HPL monoclonal antibody (mAb 146-40) [26], antiHPLRP1 and anti-HPLRP2 peptide sera, respectively. Three peaks with lipase activity were detected. Only the peak eluted with the buffer containing 0.2 M NaCl showed a positive immunoreactive band with anti-HPLRP2 peptide serum. The fractions under this peak were pooled, concentrated on an Amicon ultra-filtration cell and loaded onto a column containing anti-rHPLRP2 antibodies. The lipase was eluted with a 25 mM Tris – HCl buffer (pH 7.3)

Fig. 2. Immunoreactivity of anti-classical HPL mAb 146-40, anti-HPLRP1 and anti-HPLRP2 peptide sera with classical HPL, rHPLRP1 and rHPLRP2, respectively. (A) SDS-PAGE (12%) Coomassie blue staining patterns obtained on the purified lipases. Lane 1, molecular mass markers; lane 2, classical HPL (2 Ag); lane 3, rHPLRP1 (2 Ag); lane 4, rHPLRP2 (2.8 Ag). (B – D) Immunoblot analysis of the lipases shown in the upper panel, using anti-classical HPL mAb 146-40 (B), anti-HPLRP1 peptide serum (C), and anti-HPLRP2 peptide serum (D), respectively.

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Fig. 3. Immunoblot analysis of human pancreatic juice (lane 1), nHPLRP2 (lane 2), rHPLRP2 (lane 3), rat pancreatic juice (lane 4), rRPLRP2 (lane 5), rGPLRP2 (lane 6) using anti-HPLRP2 peptide sera.

international lipase units (1 U = 1 Amol of fatty acid released per minute) per milligram of enzyme. 2.7. Galactolipase activity measurements using the monomolecular film technique Galactolipase activity was measured using monomolecular films of monogalactosyldiglyceride (1,2-di-O-dodecanoyl-3-O-h-D-galactopyranosyl-sn-glycerol; MGalDG) as a substrate. MGalDG was prepared by chemical synthesis and was a generous gift of Pr. Paul Boullanger. The kinetics of monomolecular film hydrolysis was performed on a KSV2200 barostat (KSV, Helsinki) using a ‘‘zero order’’ Teflon trough [28]. The trough was equipped with a mobile Teflon barrier, so as to keep the surface pressure constant during enzymatic hydrolysis of the substrate film, the products of the lipolysis (monododecanoyl-galactopyranosyl-glycerol and dodecanoic acid) being soluble in water. Surface pressure was measured using a Wilhelmy plate (perimeter 3.94 cm) attached to an electromicrobalance connected to a microprocessor controlling the movements of the mobile

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barrier. The aqueous subphase was composed of 10 mM Tris – HCl, 100 mM NaCl, 21 mM CaCl2 and 1 mM EDTA, pH 8.0. The enzyme (0.1 Ag; 0.1 nM final concentration) was injected through the film. The surface area of the reaction compartment was 100 cm2 and the volume was 100 ml. The reservoir was 27.9-cm long and 14.8-cm wide. Enzyme activity was estimated from the surface of the trough covered by the mobile barrier and the known molecular area of the substrate molecule. The molecular area of the MGalDG substrate was determined as a function of the surface pressure by performing a compression isotherm (data not shown). Enzyme activity was expressed in moles of substrate hydrolyzed per minute per surface unit (cm2), and reported to the overall molarity of the enzyme initially injected into the aqueous subphase (mol min 1 cm 2 M 1). 2.8. Gel electrophoresis and Western blotting analysis PAGE electrophoresis was performed in the presence of SDS using Laemmli’s procedure [29]. Proteins were transferred to nitrocellulose membranes as described by Gershoni [30]. Membranes were incubated with antipeptide sera diluted 1/1000 times and then with alkaline phosphatase conjugated goat anti-rabbit IgG (dilution 1/ 2000). The specific immunoreactivity of the antibodies was revealed with the alkaline phosphatase substrate solution. 2.9. N-terminal sequence analysis and mass spectrometry To determine the N-terminal sequence of the HPLRP2, the protein was first separated by means of the SDS-PAGE procedure using a 7.5% acrylamide gel and then electroblotted onto polyvinylidene difluoride membrane (PVDF, Bio-Rad). The peptide bands were subjected to sequence analysis on an Applied sequencer (model 476 A). MALDI-

Fig. 4. Purification of HPL and HPLRP2 on Macro-Prep DEAE column. The fractions (a – g) were eluted by making a stepwise increase in the NaCl concentration. Absorbances were measured at 280 nm (full lines) and lipase activity was measured using tributyrin as the substrate (broken lines). Pooled fractions are indicated by horizontal bars.

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Fig. 5. Analysis of the protein fractions recovered from Macro-Prep DEAE (a – g) and immunoaffinity chromatography steps. (A) SDS-PAGE (7.5%) Coomassie staining patterns. (B) Western blotting using anti-HPLRP2 peptide serum. Only fraction f gave a positive immunoreactive band. (C) Coomassie staining of immunopurified HPLRP2.

TOF analysis of HPLRP2 was carried out on a Perspective Biosystems Voyager DE-RP spectrometer.

anti-peptide HPLRP1 antibodies. The specificity of the anti-peptide antibodies was also tested on the native HPLRP1 and HPLRP2 present in samples of pancreatic juice from 12 patients devoid of pancreatic disease. In all the samples tested, Western blot analysis yielded a single positive 50-kDa immunoreactive band with each of the anti-peptide antibodies (data not shown). Western blot analysis was also performed to check the presence of a PLRP2 in bovine, canine, porcine and rat pancreatic juice. No immunoreactive form of PLRP2 was detected with anti-HPLRP2 peptide antibodies in the bovine, canine or porcine pancreatic juices, whereas a doublet protein band with a molecular mass of around 50 kDa was detected with the rat pancreatic juice (Fig. 3, lane 4). An identical immunoreactive pattern was obtained with purified rRPLRP2 (Fig. 3, lane 5). Purified rGPLRP2 also gave a positive immunoreactive band with anti-HPLRP2 peptide antibodies (Fig. 3, lane 6).

3. Results 3.2. Purification of native HPLRP2 3.1. Specificity of the anti-HPLRP1 and anti-HPLRP2 peptide sera To discriminate between the classical pancreatic lipase and the pancreatic lipase related proteins during their purification, antibodies were raised against synthetic peptides specific to HPLRP1 and HPLRP2, and a monoclonal antibody (mAb 146-40) was used to specifically detect the classical HPL (Fig. 2B). It was checked that this mAb did not cross-react with recombinant HPLRP1 and HPLRP2. Anti-peptide antibodies were raised against a peptide including residues 132 to 141 of HPLRP1 and two peptides including HPLRP2 residues 51 to 61 and 130 to 142. These antibodies were first tested against purified rHPLRP1 and rHPLRP2. With each enzyme and its specific anti-peptide antobodies, Western blotting analysis yielded a positive reaction with a 50-kDa band (Fig. 2C and D). We observed no cross-immunoreactivity between rHPLRP1 and antipeptide HPLRP2 antibodies or between rHPLRP2 and

Since the molecular masses of classical HPL and those of the related proteins are fairly similar (around 50 kDa), gel filtration was performed with human pancreatic juice loaded onto Ultrogel AcA-54 and used as the first step in the co-purification of the three pancreatic lipases. The fractions displaying the classical PL activity were eluted using a buffer volume equivalent to 1.3-fold the column void volume, and were then subjected to anionic exchange chromatography on a Macro-Prep DEAE column (Fig. 4). Proteins were then eluted using a stepwise increase in NaCl concentration (0.05, 0.1, 0.2 and 0.5 M). Three fractions containing lipase activity were obtained. Fractions a and b showed a high lipase activity (8500 U mg 1 ) corresponding to classical HPL, as deduced from the Western blotting data, which showed a strong reactivity with antiHPL mAb 146-40 and no reactivity with anti-HPLRP1 or anti-HPLRP2 peptide sera. SDS-PAGE analysis yielded a doublet protein band (Fig. 5A, lanes a and b) cor-

Table 1 Activities of PLRP2 against various triglyceride substrates in bulk phase assays Specific activity (U mg

1

)

Tributyrin

Trioctanoin

Triolein

(a)

(b)

(a)

(b)

(a)

(b)

80 0

230 n.d.

95 n.d.

2000 [4] 200 [17]

0 [4] 1330 [17]

n.d. 400 [17]

30* [44] 1240 [17]

Native or recombinant Horse PLRP2 [7]

600 760

300 1700 1000 2000 [4] 2700 [17] 3900 [11] 140

560 3000

Native or recombinant Guinea pig PLRP2 Recombinant rat PLRP2

540 3000 1000 2000 [4] 500 [17]

170 150

0

20 20

1 1

Native HPLRP2 Recombinant Coypu PLRP2 [16]

The specific activities were determined (a) in the absence of NaTDC and colipase, (b) in the presence of 4 mM NaTDC and excess colipase, *10 mM NaDOC instead of 4 mM NaTDC. n.d., not determined.

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responding solely to the heterogeneity of the carbohydrate moities [31]. In addition, the N-terminal amino acid sequences obtained on 17 residues were identical to that of the classical HPL (KEVCYERLGCFSDDSPW). A protein eluted under peak d was identified as the HPLRP1 by performing Western blotting with anti-HPLRP1 peptide antibodies. Protein fraction f displayed a low lipase activity. SDS-PAGE yielded one major band and two minor protein bands (Fig. 5A, lane f). After the Western blotting procedure, the minor band migrating at a higher apparent molecular mass than the classical HPL displayed a positive reactivity with anti-HPLRP2 peptide sera (Fig. 5B) and no reactivity with either anti-HPLRP1 peptide serum or antiHPL mAb 146-40. The major protein band observed with fraction f at around 45 kDa was identified by performing N-terminal amino acid sequencing on 12 residues (KEDFVGHQVLRI) and found to correspond to either procarboxypeptidase A1 or A3. The N-terminal sequences of A1 and A3 molecular forms of procarboxypeptidase were found to be identical over the first 17 residues, and it was therefore obviously not possible to distinguish between these two forms. The solution recovered from Macro-Prep DEAE was then subjected to immunoaffinity chromatography using a column of immobilized recombinant HPLRP2. The breakthrough fraction contained only the procarboxypeptidase A1-3. The HPLRP2 eluted with a high salt concentration amounted to 70% of the initial activity applied to the column. SDS-PAGE analysis of the purified HPLRP2 yielded a single protein band corresponding to an apparent molecular mass of 50 kDa (Fig. 5C). Mass spectrometry analysis gave an experimental mass of 50,996 Da, which was 915 Da greater than the mass calculated from the sequence of the protein (50,081 Da). This finding suggests that only one of the two potential N-glycosylation sites is occupied by a short glycan chain. The N-terminal sequence obtained was completely identical to the sequence deduced from the HPLRP2 cDNA (KEVCYGQLGCFSDEK, nonconserved residues in comparison to HPL are indicated by thick letters). 3.3. Effects of bile salts and colipase The lipase activity of native HPLRP2 was determined on short-, medium- and long-chain triglycerides. The effects of bile salts and colipase on lipase activity were determined in a range of NaTDC concentrations (from 0 to 4 mM) in the presence and absence of colipase, and compared with those of PLRP2 from other species (see Table 1). HPLRP2 was inhibited by increasing concentrations of bile salts in the absence of colipase (Fig. 6A – C). Using tributyrin as a substrate, the specific activity was found to decrease at bile salt concentrations ranging from 0.5 to 2, before reaching a plateau corresponding to 80% inhibition at concentrations greater than 2 mM NaTDC. With medium- and long-chain triglycerides, the inhibitory

Fig. 6. Effects of NaTDC and colipase on the activity of HPLRP2 on emulsified tributyrin (panel A), trioctanoin (panel B) and olive oil (panel C).

effects were apparent as soon as NaTDC monomers were present, increased up to the critical NaTDC micellar concentration (1– 2 mM) and then reached a plateau value corresponding to 99% inhibition in the case of trioctanoin and 92% in that of olive oil. Adding colipase in a fivefold

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molar excess had significant effects at all the NaTDC concentrations tested with tributyrin. Under the same conditions, colipase was found to have only slight effects with either trioctanoin or olive oil. It can be concluded that colipase does not fully reactivate the lipase activity of HPLRP2 in the presence of bile salt, contrary to what is known to occur with classical HPL. 3.4. Phospholipase and galactolipase activities of HPLRP2 A very low phospholipase activity (3 U/mg) was measured on egg yolk phospholipids, predominantly phosphatidylcholine. This activity value is identical to that obtained with horse PLRP2 (2 –5 U/mg; Ref. [7]) but is much lower than that measured with GPLRP2 (570 U/mg; Ref. [4]), coypu PLRP2 (180 U/mg; Ref. [16]) and rat PLRP2 (340 U/ mg; Ref. [17]). Contrary to GPLRP2, all the previously cited forms of PLRP2 possess a full-length lid domain (23 amino acid residues as against five in the case of GPLRP2) controlling the access to the active site [2]. It has been reported that a GPLRP2( + lid) mutant in which the GPLRP2 mini-lid domain was replaced by the HPL fulllength lid domain showed a lower phospholipase activity than GPLRP2, whereas both enzymes showed a similar level of lipase activity [32]. The galactolipase activity of HPLRP2 was measured on a MGalDG monolayer as a function of the surface pressure. As can be seen from Fig. 7, HPLRP2 showed galactolipase activity and its maximum activity (0.55 mol cm 2 min 1 M 1 at 15 mN/m) was six times lower than that of rGPLRP2 (3.5 mol cm 2 min 1 M 1 at 10 – 15 mN/m). HPL did not hydrolyze MGalDG under the same conditions.

Fig. 7. Variations with surface pressure in the galactolipase activities of rGPLRP2 and nHPLRP2 on MGalDG monomolecular film.

4. Discussion We previously detected the presence of a PLRP1 in human pancreatic juice and found no evidence that any PLRP2 was present [13]. Using specific synthetic peptides based on the amino acid sequences of HPLRP1 and HPLRP2 has made it possible, however, to obtain specific antibodies against HPLRP1 and HPLRP2. The lack of cross-reactivity of the anti-PLRP2 peptide antibody with classical HPL and rHPLRP1 provided a means of discriminating between HPLRP2 from the closely migrating (SDS-PAGE) classical HPL and HPLRP1. Two anti-peptide antibodies directed against the peptide stretches 51– 61 and 130 – 142, respectively, of HPLRP2 display an identical specificity towards HPLRP2, thus supporting the choice of these peptide stretches as specific antigens for raising antibodies against HPLRP2. We noted that among the others PLRP2 tested here, the anti peptide antibody directed against the peptide stretch 51 – 61 of HPLRP2 cross-reacted more strongly with RPLRP2 than with GPLRP2. The amino acid sequences of human, rat and guinea-pig PLRP2 were compared (Fig. 1B) and stretch 53 – 59 showed a higher degree of identity between HPLRP2 and RPLRP2 than that observed between HPLRP2 and GPLRP2. Identical results were obtained using the anti-peptide antibody directed against stretch 130– 142 (data not shown). This immunological approach has enabled us to clearly identify for the first time a PLRP2 in the human pancreatic juice, which means that it is produced by the exocrine pancreas and secreted. The complete isolation of the HPLRP2 from pancreatic juice involves several problems due to the small amount of enzyme available in comparison with the classical HPL, as well as the presence of procarboypeptidase isoforms, which are co-eluted with the three forms of lipase on an Ultrogel AcA-54 column. These proteins have very similar molecular masses and isoelectric points. The elution profile of procarboxypeptidase isoforms on a DEAE Macro-Prep column is in good agreement with that described by Pascual et al. [33] on a TSK-DEAE HPLC column. The pI value of HPLRP2 (5.03) is similar to those of the procarboxypeptidase A isoforms (4.9, 5.1 and 4.9 for A1, A2 and A3, respectively), which explains why these enzymes are co-eluted when performing anion exchange chromatography. In order to quickly and efficiently separate HPLRP2 from the procarboxypeptidase forms, we developed an immunoaffinity chromatography procedure involving the use of purified pAb directed against rHPLRP2. Similar amounts of HPLRP2 were isolated from the human pancreatic juice obtained from four different donors with a normal physiological pancreatic profile. The protein level of expression of HPLRP2 is about 20-fold lower than that of classical HPL in the normal exocrine secretion and is similar to that previously estimated in the case of HPLRP1 (0.3 –0.5% of the total protein content of pure pancreatic juice). The HPLRP2 to HPLRP2 + HPL

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protein ratio was found to be identical to the mRNA ratio (4%) reported by Giller et al. [3]. The specific activities of HPLRP2 on several TGs (short-, medium- or long-chain) were found to be much lower than those observed with classical HPL. In the presence of 4 mM bile salts and colipase, the specific activity of HPLRP2 on long-chain TGs was 31-fold lower than that of classical HPL (95 vs. 3000 U mg 1; Ref. [34]). The fact that lipase activity of HPLRP2 is inhibited by bile salts and not fully restored by colipase suggests that, in vivo, the dietary TGs are mostly hydrolyzed by classical HPL and only marginally by HPLRP2. In order to explain the weak effect of colipase on HPLRP2 activity, we have considered the following two possibilities: (1) the lid residues and the C-terminal domain residues involved in the interactions between classical HPL and colipase [35] are mostly conserved in the HPLRP2 sequence [36]; (2) the residues are involved in the stabilization of the open lid conformation, this form being able to interact with colipase. The interactions R256-Y267 and D257-K268 stabilizing the open lid conformation in HPL [35] are not conserved in HPLRP2 [36]. It seems likely that the lack of stabilization of the open lid conformation induces weaker HPLRP2– colipase interactions than those existing between HPL and colipase. HPLRP2 shows a much lower specific activity on egg yolk phospholipids (3 U mg 1) than the specific activity measured on the same substrate with phospholipase A2 (600 U mg 1; Ref. [37]) present in human pancreatic juice. It therefore seems likely that HPLRP2 may not play a crucial role in the digestion of dietary phospholipids. Unlike the classical PL and PLRP1, PLRP2 can be expressed not only in the pancreas, but also in various tissues and cell types depending on the species. For this reason, various physiological functions have been proposed for PLRP2. The patterns of mRNA expression of PL and PLRPs differ significantly during development. In rodents [38,39], the PLRP2 mRNA levels are high at birth and low in adults, whereas no PL mRNA expression is detectable in the fetal pancreas. These levels develop rapidly around the suckling to weaning transition period and persist in high levels into adulthood. A similar pattern of mRNA expression has been also described in the human genes encoding these lipases [40]. The temporal pattern of PLRP2 mRNA expression suggests that PLRP2 plays an essential role in dietary fat digestion in suckling mammals. This hypothesis was confirmed by the fact that PLRP2-deficient suckling mice had fat malabsorption deficits, whereas adult mice had normal fat absorption processes [39]. This hypothesis is also consistent with our own data, since in the absence of bile salts, as at birth, HPLRP2 displays a significant level of lipase activity (Fig. 6). Nevertheless, the acinar cell location of RPLRP2 suggests that this enzyme may have another function. Wishart et al. [5] have detected the presence of RPLRP2 (also named GP-3), attached to the inner surface of zymogen granule membranes: this finding suggests that the enzyme may possibly contribute to zymogen granule fusion

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with the acinar cell plasma membrane and exocytosis of the zymogen granule contents. Despite its membrane association, RPLRP2 was found to be present in pancreatic juice [41] and may therefore be involved in intestinal lipolysis. Murine PLRP2 was initially identified as an interleukin-4 inducible gene in cytotoxic T lymphocytes [8], which led to the hypothesis that PLRP2 may mediate cell lysis by hydrolyzing membrane lipids. The role of PLRP2 in immunological defence was further suggested by the decrease in T cell cytotoxicity observed in PLRP2-deficient mice [39]. Murine PLRP2 mRNA was also found to be expressed throughout the small intestine in both enterocytes and paneth cells [9]. If PLRP2 is synthesized in paneth cells, it may have an anti-microbial activity, but it seems unlikely that PLRP2 may have a digestive function in the distal small intestine. The results of the present study suggest that, in vivo, HPLRP2 can be classified as a galactolipase. Galactolipids are to be found in many parts of plants (leaves, fruits, roots), and humans consume 200 mg of these substances everyday on average. The digestion of dietary galactolipids is probably carried out by two lipolytic pancreatic enzymes, CEL and HPLRP2, but CEL displays a very low level of activity on galactolipids in vitro [15,22]. Up to now, little information has been available in the literature about the digestion and absorption of galactolipids and the selectivity of galactolipases for these substrates. The question arises as to why PLRP2 hydrolyzes galactolipids and classical HPL does not. In order to understand the structure – function relationships between these related lipases, Withers-Martinez et al. [42] have modeled a galactolipid molecule within the catalytic groove of GPLRP2. In GPLRP2, the lid domain is much smaller than in the classical HPL and the 3-D structure of GPLRP2 around the active site shows a higher hydrophilic/lipophilic balance in the surface loops (h5 loop, h9 loop, lid domain) surrounding the active site, as compared to the homologous loops in HPL [42]. GPLRP2 is therefore able to accommodate the polar head of the digalactosyldiglyceride. However, HPLRP2 and RPLRP2 possess a full-length lid domain and are also able to hydrolyze a substrate having a polar head such as MGalDG. This finding probably means that the lid domain does not play a crucial role in the substrate selectivity of PLRP2 towards galactolipids, contrary to what is observed with phospholipids [32]. Further biochemical and crystallographic studies are now required to explain the substrate specificity of PLRP2 towards galactolipids. It would be also interesting to study the PLRP2 levels present in herbivorous animals and the relationships between these PLRP2 levels and those resulting in humans from the vegetables they consume.

Acknowledgements We would like to thank Re´gine Lebrun for MALDI-TOF analysis and Jacques Bonicel for N-terminal sequence

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analysis. We are grateful to Dr. Dominique Lafont and Pr. Paul Boullanger (Universite´ de Lyon, Laboratoire de Chimie Organique II, Villeurbanne, France) for providing us with synthetic MGalDG. We acknowledge the help of Dr. Jessica Blanc in revising the English manuscript.

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