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Originally published In Press as doi:10.1074/jbc.M205761200 on August 22, 2002

J. Biol. Chem., Vol. 277, Issue 46, 44093-44099, November 15, 2002
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Guinea Pig Phospholipase B, Identification of the Catalytic Serine and the Proregion Involved in Its Processing and Enzymatic Activity*

Michel NauzeDagger, Lauriane GoninDagger, Brigitte Chaminade, Christine Perès, Françoise Hullin-Matsuda, Bertrand Perret, Hugues Chap, and Ama Gassama-Diagne§

From the Institut Fédératif de Recherche en Immunologie Cellulaire et Moléculaire, INSERM Unité 563, Centre de Physiopathologie de Toulouse Purpan, Département "Lipoprotéines et Médiateurs Lipidiques," Hôpital Purpan, 31059 Toulouse Cedex, France

Received for publication, June 11, 2002, and in revised form, August 8, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Guinea pig phospholipase B (GPPLB) is a glycosylated ectoenzyme of intestinal brush border membrane. It displays a broad substrate specificity and is activated by trypsin cleavage. The primary sequence contains four tandem repeat domains (I to IV) and several serines in lipase consensus sequences. We used site-directed mutagenesis to demonstrate that only the serine 399 present in repeat II is responsible for the various enzymatic activities of GPPLB. Furthermore, we characterized for the first time the retinyl esterase activity of the enzyme. We also constructed and expressed in COS-7 cells, an NH2-terminal repeat I deletion mutant which was detected at a very low level by immunoblot. However, confocal microscopy study showed a strong intracellular accumulation with a weak membrane expression of the mutated protein, indicating a role of the NH2-terminal repeat I in the processing of GPPLB. Nevertheless, the Western blot-detected protein presented a glycosylation and trypsin sensitivity patterns similar to wild type PLB. The mutant is also fully active without trypsin treatment, in contrast to native enzyme. Thus, we propose a structural model for GPPLB, in which the repeat I constitutes a lid covering the active site and impairing enzymatic activity, its removal by trypsin leading to an active protein.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phospholipases B (PLB)1 are a heterogeneous group of enzymes that share the ability to hydrolyze both sn-1 and sn-2 acyl ester bonds of glycerophospholipids and then display both phospholipase A1 or phospholipase A2 and lysophospholipase activities. PLB activity has been reported in microorganisms such are Penicillium notatum and Saccharomyces cerevisae (1), in plants (2, 3), as well as in guinea pig (4), rabbit (5), and rat (6) brush border membranes. The mammalian enzymes also display a lipase activity (7). Despite this broad substrate specificity, we previously reported that guinea pig intestinal PLB (GPPLB) releases first in a calcium-independent manner the fatty acid in the sn-2 position of phospholipids (7). Accordingly, these PLB were considered as calcium-independent PLA2 (iPLA2) (8) and new ectomembrane phospholipases B (9).

It is generally accepted that digestion of lipids in the gastrointestinal tract requires hydrolysis reactions by the secretory enzymes originated from lingual, gastric, and pancreatic glands (10). However, our previous data indicated that phospholipid hydrolysis and absorption could be observed in vivo, in the absence of the secretory enzymes mentioned above, and could be due to intestinal PLB (11). Moreover, the bile salt-dependent retinyl esterase activity, intrinsically located in the brush border membranes, was suggested to be due to PLB (12), and a recent review proposed the involvement of intestinal PLB in digestion of dietary vitamin A (13). In addition, our previous data supported the view that PLB was expressed as a function of enterocyte differentiation, displaying the highest level in mature enterocytes of jejunum and ileum, which are the most implicated in lipid digestion (14). Taken together, these studies suggested that intestinal PLB could play a key role in hydrolysis of lipids and vitamin A from the diet in the lumen. PLB thus belongs to the group of brush border hydrolases such as lactase and sucrase isomaltase involved in terminal digestion of nutriments. A comparative study of PLB and sucrase isomaltase genes suggested a common structure for promoters and regulating transcription factors (15, 16).

Molecular cloning of cDNA from GPPLB (17) indicated that it is homologous to rabbit AdRab-B (5) and rat phospholipase B/lipase (18). The sequence corresponded to a 170-180-kDa ectophospholipase with a short cytoplasmic carboxyl-terminal domain, stalked in the membrane by a hydrophobic segment connected to the larger, heavily glycosylated globular domain. The latter contains four tandem repeat domains and several lipase and phospholipase consensus sequences, i.e. the GXSXG, similar to a classic lipase motif found in alpha /beta -hydrolases (19) and the GDSL (20). It was shown that a unique serine was involved in rabbit PLB catalytic activity (21). However, in that study, PLB activity was analyzed toward water-soluble substrates. That could hamper the results, since our data indicated that the 170-kDa form of GPPLB was inactive against long chain phospholipids (17). In addition, recent studies indicated that only the second repeat of rat PLB contained the three essential residues involved in the catalytic triad responsible for all the PLB activities (22). Although both proteins are expected to display structures and properties very similar to GPPLB, analysis of the three species PLB sequences indicated that there were some differences concerning the number and the localization of the lipase consensus sequences (see Fig. 1A). Moreover, the third repeat of the guinea pig enzyme also contains all the residues of the catalytic triad.

In the present study, we used site-directed mutagenesis to clearly demonstrate that only the serine 399 residue is responsible for GPPLB activity. We thus demonstrated for the first time the retinyl esterase activity of GPPLB. Our precedent observation (17) indicated that native GPPLB needs limited proteolysis by trypsin to acquire its full enzymatic activity. This trypsin treatment induced an important reduction in the protein size and probably corresponds to removal of one repeat domain. In this study, we used a deletion mutant of PLB to demonstrate that this domain corresponds to the NH2-terminal repeat I, which constitutes a proregion involved in the regulation of PLB cellular localization and enzymatic activity.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- 1-Acyl-2-[14C]linoleoyl-sn-glycero-3-phosphocholine and 1-[3H]palmitoyl-sn-glycero-3-phosphocholine were from Amersham Biosciences Europe GmbH. Non-labeled 1-acyl-2-linoleoyl-sn-glycero3-phosphocholine, 1-palmitoyl-sn-glycero-3-phosphocholine, retinyl palmitate, and phospholipase C (from Bacillus cereus) were from Sigma.

Site-directed Mutagenesis-- The experiments were achieved by PCR using QuikChangeTM Site-Directed Mutagenesis Kit from Stratagene as indicated in the instruction manual. The mutagenesis reaction was performed using constructions containing small fragments (1.4-1.6 kbp) as template instead of full-length cDNA of PLB to decrease potential for random mutations during the thermal cycles. The cDNA corresponding to the first 1.5 kbp of PLB sequence, cloned in pGEMT-Easy vector (17), was used for the mutations of the serine residues 399 and 454, respectively, in the consensus GDSL and GXSXG (Fig. 1A). Two other constructions of the pBluescript SK vector containing 1.4- and 1.6-kpb fragments obtained by XhoI and XbaI digestions of PLB cDNA were used for the mutations of the serine residues 746 (GDSL) and 1155 (GXSXG), respectively (Fig. 1A). For each mutation, two complementary mutagenic oligonucleotides were designed, and the target serine in the sequence was replaced with an alanine by one nucleotide change. The resulting mutated plasmids were sequenced. For each of them, the insert was excised by digestion with restriction enzymes and ligated in pcDNA3 vector (Invitrogen) containing the full-length PLB cDNA (pcDNA3-PLB) deleted from the corresponding domain. Briefly, mutants for serines 399 and 454 were cut with a single EcoRI digestion, whereas mutants of serines 746 and 1155 were digested with EcoRV and XbaI or XbaI and ApaI, respectively, to obtain the different pcDNA3 constructions with full-length mutated PLB. They were named GDSL1, GXI, GDSL2, and GXII, respectively, for the serine 399, 454, 746, and 1155 mutants.

To obtain GDSL3, mutagenesis reaction was directly performed on pcDNA3-PLB by replacing serine 1102 with alanine without any subcloning and using QuikChangeTM XL Site-Directed Mutagenesis Kit from Stratagene as indicated in the instruction manual. The mutation was verified by sequencing.

Construction of NH2-terminal Region-deleted Mutant (del PLB)-- The PLB cDNA fragment encoding amino acids 313-1463 was amplified by PCR using 5' sense primer incorporating a BamH1 restriction site, the Kozak and the signal sequences, nucleotides 1081-1104 of GPPLB coding sequence, and the antisense primer incorporating the nucleotides 4414-4428 fragment in-frame with a HindIII restriction site (Fig. 1B) to get a protein fused to the histidine and Myc tag of pcDNA3.1/myc-his C vector. The amplified fragment was first cloned in pGEMT-Easy vector (Promega), sequenced, digested by BamHI/HindIII and then subcloned in pcDNA3.1/myc-his C previously digested with the same enzymes to generate del PLB-Myc. This construction was used to obtain the non-tagged deleted form of PLB (del PLB). Briefly, del PLB-Myc was digested with BamHI and SacII, and the obtained fragment was inserted in pcDNA3 containing PLB full-length cDNA (PLB4) previously digested with the same restriction enzymes.

COS-7 Cell Culture and Transfection-- COS-7 cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 100 µg/ml penicillin/streptomycin (Invitrogen) in a humidified atmosphere containing 5% CO2. The cells were transfected with the different cDNA constructions obtained as described above, using SuperFect reagent (Qiagen) according to the manufacturer's protocol. Cells were rinsed twice and scraped in cold phosphate-buffered saline, sonicated, and used for protein and enzymatic activities determination. For immunoblotting, cells were directly collected in Laemmli buffer and boiled for 5 min.

Confocal Microscopy-- COS-7 cells were grown on sterile glass coverslips and transfected with cDNAs of del PLB and PLB4 for 48 h. Cells were then washed three times with phosphate-buffered saline, fixed for 15 min with 3.7% paraformaldehyde, permeabilized (or not) for 2 min with 0.2% Triton X-100, and saturated for 30 min with 0.2% gelatin. After 60 min of incubation with the anti-PLB antibody, immunostaining was performed with fluorescein isothiocyanate-labeled anti-rabbit IgG F(ab')2 antibody (Immunotech). The coverslips were examined after mounting using a Carl Zeiss, LSM 510, axiovert 100, confocal microscope with a 63× Plan-Apochromat objective (1.4 oil). An argon laser at 488 nm was used to detect fluorescein fluorochrome.

Trypsin Digestion of PLB-- Transfected COS cell homogenates were treated with trypsin (50 µg/ml) for the indicated time at 37 °C. The reaction was stopped by the addition of two volumes of soybean trypsin inhibitor (30 µg/ml) as described previously (17) for determination of enzymatic activities or by adding Laemmli buffer (23) for immunoblotting.

N-Glycosydase F and Endoglycosidase H Treatments-- Homogenates from COS cells, transfected with full-length PLB or NH2-terminal-deleted mutant (del PLB) were treated with N-glycosidase F and endoglycosidase H, as described by manufacturer (Biolabs, Ozyme).

Preparation of Fusion Protein and Polyclonal Antibodies-- The cDNA coding for full-length PLB was digested with XbaI and XhoI, and the obtained fragment (amino acid residues 353-532) corresponding to a part of the second repeat was inserted into pGEX-KG vector (a gift of Dr. J. E. Dixon, University of Michigan, Medical School). This construction was introduced into an Escherichia coli strain (XL1-blue), and production of the fusion protein was induced with isopropyl-1-thio-beta -D-galactopyranoside. The glutathione S-transferase fusion protein was purified by affinity chromatography on glutathione-agarose beads (Sigma) and used to produce a rabbit polyclonal antibody (Eurogentec, Seraing, Belgium).

Immunoblotting-- Protein concentration was determined according to Bradford (24), and samples were submitted to SDS-PAGE (23) and transferred onto nitrocellulose membranes according to standard protocol (25).

Assays of PLA2 and Lysophospholipase Activities-- These were achieved using 1-acyl-2-[14C]linoleoyl-sn-glycero-3-phosphocholine and 1-[3H]palmitoyl-sn-glycero-3-phosphocholine as substrates, respectively, as described previously (4).

Preparation and Hydrolysis of Diacylglycerol-- The diacylglycerol was obtained as described previously (7). In summary, 1-acyl 2-[14C]linoleoyl-sn-glycero-3-phosphocholine was converted to diacylglycerol by treatment with B. cereus phospholipase C. The obtained 1-acyl 2-[14C]linoleoyl glycerol was dispersed by sonication in 0.1 ml of 0.2 M Tris-HCl (pH 8.5) containing 0.5%(w/v) arabic gum and 12 mM sodium deoxycholate. The mixture was incubated for 30 min at 37 °C under shaking after addition of 5-15 µl of homogenate. At the end of the reaction, 1.95 ml of chloroform, methanol, heptan (12/14/10, v/v/v), and 0.63 ml of 0.1 M carbonate borate (pH 10.5) were added. The mixture was vortexed for 15 s and centrifuged at 3,000 rpm, and the radioactivity corresponding to free fatty acids was determined in the upper phase.

Retinyl Esterase Activity-- Retinyl palmitate was taken to dryness from ethanol and dispersed by sonication as described for phosphatidylcholine. The reaction was started by addition of transfected cell homogenates. Incubation was carried out at 37 °C for 15 min under shaking and terminated by addition of 1 volume of ethanol and hexane. The free retinol formed was then extracted, analyzed by high performance liquid chromatography, and quantified by comparison of their integrate peak areas to calibrate areas from pure retinyl acetate standard as described previously (26).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of the Catalytic Serine of Guinea Pig Phospholipase B-- As represented in Fig. 1, the guinea pig PLB cDNA (GPPLB) sequence displays three serines in the consensus sequence GDSL (GDSL1, GDSL2, and GDSL3, respectively, for serines 399, 746, and 1102) and two serines in the consensus sequence GXSXG (GXI and GXII for serine 454 and 1155, respectively). To determine the involvement of these different serines in the catalytic activity of PLB, they were individually mutated to alanine, and COS-7 cells were transiently transfected with the corresponding cDNAs. Analysis of cell homogenates by immunoblotting (Fig. 2) indicated that all mutated proteins were expressed in COS-7 cells as a 170-kDa polypeptide (lanes 3-7), which corresponds to the size of the wild type cDNA (PLB4, lane 2). Nevertheless, with identical transfection conditions (about 40% efficiency), a lower expression level was observed for GDSL1 and GDSL3 mutants, suggesting that these mutations could affect the proper folding of the protein. No expression was observed in cells transfected with the vector alone used as negative control (pcDNA3, lane 1).


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  		Fig. 1.   A, shematic representation of the structure of GPPLB. Putative lipase consensus sequences are presented under their respective repeats and compared with rat and rabbit PLB. The number of serine residue is indicated for the five serines of GPPLB, and for other species, only the catalytic serine is numbered and presented in bold characters. encircled N = N-glycosylation site, N-ter = NH2-terminal region, C-ter = COOH-terminal region. B, sequences of oligonucleotides used in PCR for the construction of the NH2-terminal-deleted mutant.


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  		Fig. 2.   Immunoblot analysis of GPPLB and the different serine mutants in COS-7 cells. Site-directed mutagenesis was used to introduce serine to alanine mutations in GPPLB cDNA. COS-7 cells were transfected for 48 h with an equal amount of the different constructs and directly scraped in Laemmli buffer. The same volume of each transfection was loaded on SDS-PAGE, and immunoblot was performed with an anti-PLB polyclonal antibody as described under "Experimental Procedures."

The different enzymatic activities of PLB were assayed with different mutated proteins expressed in COS-7 cells and activated by trypsin treatment (17) (Fig. 3). We first investigated for PLA2 activity, and as presented in Fig. 3A, serine mutants 454, 746, 1102, and 1155 (GXI, GDSL2, GDSL3 and GXII, respectively) retained the catalytic activity. The enzymatic activity expressed according to the protein expression level is similar to that obtained with the wild type enzyme (PLB4). Conversely, mutation of the serine 399 (GDSL1) completely abolished the enzymatic activity. No activity was also detected in cells transfected with the vector alone used as negative control. Identical results were obtained for determinations of lysophospholipase (Fig. 3B), diacylglycerol lipase (Fig. 3C), and retinyl esterase (Fig. 3D) activities. These data clearly indicated that only the serine 399 is necessary for the enzymatic activity of guinea pig PLB.


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  		Fig. 3.   Enzymatic activities of COS-7 cells expressing the different serine mutants of GPPLB. Cells were transfected as described in the legend of Fig. 2, homogenized in phosphate buffer saline, treated with trypsin, and used for the determination of the different enzymatic activities as described under "Experimental Procedures." According to the broad substrate specificity of GPPLB, enzymatic activities were measured toward 1-acyl-2-[14C]linoleoyl-sn-glycero-3-phosphocholine (A), 1-[3H]palmitoyl-sn-glycero-3-phosphocholine (B), and 1-acyl-2-[14C]linoleoyl-glycerol (C) and retinyl palmitate (D). The specific activity was expressed as nmol/min/expressed protein level, evaluated by densitometry analysis of Western blot of Fig. 2. The values are the means (±S.E.) of three different determinations.

Expression of the NH2-terminal Repeat I-deleted Mutant of GPPLB in COS-7 Cells-- A plasmid containing a cDNA corresponding to the deleted NH2-terminal repeat I of GPPLB cDNA was constructed as described under "Experimental Procedures" and used for transfection of COS-7 cells. After 6, 12, 24, 36, 48, and 72 h, the expressed protein (del PLB) was analyzed by Western blot, quantified, and compared with the expression of full-length PLB (PLB4) detected in the same experimental conditions. Data revealed marked difference in the expression level of these two proteins as indicated in Fig. 4A. PLB4 (170 kDa) expression level sharply increased at 36 h and continued to increase up to 72 h. By contrast, del PLB was expressed as a faint band of 140 kDa corresponding to the size of both brush border membrane PLB (14) and the trypsin-cleaved form of the recombinant protein (17). The kinetic study indicated only a slight increase in del PLB expression. Densitometry analysis of the Western blot revealed about 5-fold increase of PLB4 expression compared with del PLB (Fig. 4B). This variation suggested that the NH2-terminal repeat I is necessary for proper expression of GPPLB.


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  		Fig. 4.   Kinetic study of wild type (PLB4) and NH2-terminal first repeat deleted mutant (del PLB) of GPPLB expressed in COS-7 cells. COS-7 cells were transfected with the cDNA of PLB4 and del PLB. A, after the indicated period, cells were scraped in Laemmli buffer, and the same volume of each transfection was loaded on SDS-PAGE, and immunoblot was performed with an anti-PLB polyclonal antibody, as described under "Experimental Procedures." Revelation was done with iodine (125I)-labeled anti-rabbit IgG. B, densitometric analysis of the immunoblot presented in A. Shaded columns, PLB; white columns, del PLB.

Study of Cellular Localization of del PLB and PLB4 by Confocal Microscopy-- As in the same tranfection conditions, del PLB was expressed at a very low level compared with PLB4, we sought to determine for a role of the repeat I in the processing of GPPLB. Thus, we analyzed the cellular localization of del PLB by confocal microscopy. This was achieved on transfected COS cells and using an anti PLB antibody. Concerning del PLB, cells permeabilized with Triton X-100 displayed an intense intracellular staining around the nucleus, which seems to correspond to aggregates of misfolded proteins in the ER (Fig. 5A). Despite this strong intracellular localization, a few number of transfected cells revealed faint staining at the cell surface of non-permeabilized cells (Fig. 5B). These data indicated that only a small proportion of del PLB was correctly folded and transported to the membrane and could correspond to the low level of protein detected by immunoblot. On the contrary, an important membrane localization (Fig. 5D) and a faint intracellular signal (Fig. 5C) were observed for wild type PLB as expected. Taken together, these observations led us to conclude that deletion of the repeat I strongly impaired the processing of GPPLB.


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  		Fig. 5.   Surface and intracellular expression of PLB4 and del PLB. COS-7 cells were transfected with cDNA corresponding to PLB4 or del PLB, treated as described under "Experimental Procedures" with (A, C) and without (B, D) permeabilization by Triton X-100, and analyzed by confocal microscopy. Typical images are illustrated.

N-Glycosylation Study of del PLB-- Phospholipase B is a highly N-glycosylated protein. Several roles such as involvement in protein folding, polarized sorting, and optimal expression have been described so far for glycosylation (27). We then decided to analyze the N-glycosylation pattern of del PLB compared with PLB4. Transfected COS-7 cell homogenates were treated with endoglycosidase H, which cleaves mannose-rich N-linked oligosaccharides, and with N-glycanase, which cleaves the complex type. As represented in Fig. 6A, PLB4 and the NH2-terminal repeat I-deleted mutant (del PLB) were cleaved by endoglycosidase H. However the mutant is more sensitive to endoglycosidase H treatment than PLB4. Furthermore, N-glycanase cleaved the total protein for PLB4 as well as for del PLB (Fig. 6B). For the latter, the molecular mass shifted from 140 to 108 kDa as previously observed with brush border GPPLB (14), suggesting that the weakly expressed repeat I-deleted mutant is correctly glycosylated and could correspond to the membrane expressed form observed in Fig. 5B.


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  		Fig. 6.   Effects of N-glycosydase F and endoglycosydase H on PLB4 and del PLB. Cells were transfected with the cDNA corresponding to PLB4 and del PLB, homogenized in phosphate buffer saline, and 100 µg were treated (+) or not (-) with endoglycosydase H (A) or N-glycosydase F (B), analyzed by SDS-PAGE, and detected with anti-PLB antibody.

Effects of Trypsin Treatment on Molecular Weight, Enzymatic Activity of GPPLB, and Its NH2-terminal Repeat I-deleted Mutant-- Native GPPLB is a proenzyme that needs limited proteolysis by trypsin to acquire its full enzymatic activity. This trypsin treatment induced an important reduction in the protein size that could probably correspond to removal of one repeat domain (17). To check for this hypothesis, we first analyzed the effect of trypsin treatment on the molecular weight of PLB4 and del PLB to determine its major cleavage sites. COS-7 cells were transfected for 48 h with the two cDNAs. The homogenates were submitted to trypsin treatment for different times and immunoblotted (Fig. 7). Within 5 min of trypsin treatment, the 170-kDa band of PLB4 disappeared, whereas a major 140-kDa form appeared remaining at the same intensity until 30 min of digestion. A product of 97 kDa was then observed until 60 min. Here again, as observed in Fig. 4, a lower expression was obtained for the mutant expressed as a 140-kDa protein, corresponding to the first cleavage product of PLB4 and indicating that the NH2-terminal repeat I could be the proregion deleted by trypsin treatment. A faint band of 97-kDa protein appeared after 30 min of trypsin treatment, suggesting a proper folding and processing of del PLB despite its low expression level. The 140-kDa band was essentially present in the membrane fraction but could be observed in soluble fraction, whereas the 97-kDa protein only appeared in the soluble fraction (data not shown). These data indicated the presence of two major trypsin cleavage sites in GPPLB.


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  		Fig. 7.   Effect of trypsin treatment on the molecular weight of PLB4 and del PLB. COS-7 cells were transfected with cDNA corresponding to PLB4 or del PLB, homogenized in phosphate buffer saline, and equal aliquots were treated with trypsin for the indicated time, analyzed on SDS-PAGE, and detected with anti-PLB antibody.

Transfected COS-7 cell homogenates described above were submitted or not to trypsin treatment for 15 min and assayed for different enzymatic activities. As expected, the wild type PLB was enzymatically active toward phosphatidylcholine, lysophosphatidylcholine, and diacylglycerol only upon trypsin treatment (Fig. 8). However, del PLB was fully active on the different substrates without any trypsin addition (Fig. 8), indicating that the NH2-terminal repeat I could be the proregion inhibiting PLB enzymatic activity in the native form.


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  		Fig. 8.   Effect of trypsin treatment on PLB4 and del PLB enzymatic activities. COS-7 cells were transfected with cDNA corresponding to PLB4 or del PLB, homogenized in phosphate buffer saline, and equal aliquots awere treated (+) or not (-) with trypsin for 30 min and used to determine the different enzymatic activities. Data are means ± S.E. of three determinations for phospholipase A2, lysophospholipase, and diacylglycerol lipase. The specific activity is expressed in nmol/min/expressed protein level, evaluated by densitometry analysis of the corresponding protein detected by Western blot.

Proposed Model of the GPPLB Structure-- The different data obtained in this study, those obtained from GPPLB purification (4) and molecular cloning (17), have revealed the main structural features and the proteolytic cleavage sites of GPPLB as depicted in Fig. 9. The GPPLB is synthesized as an inactive precursor of 170 kDa with a short cytoplasmic carboxyl-terminal domain, a hydrophobic stretch that spans once the lipid bilayer and anchors the protein in the membrane. The large, highly glycosylated ectodomain contained four homologous domains. The catalytic serine (serine 399) is localized in the repeat II hidden beneath the proregion constituted by repeat I. Removal of this proregion by trypsin cleavage exposes the active site leading to a mature membrane protein of 140 kDa. A longer proteolysis leads to a cleavage between domain III and IV and generated a 97-kDa soluble form.


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  		Fig. 9.   Schematic representation of the structure of GPPLB in brush border membrane. Numbered boxes correspond to the four repeats; the active serine (Ser399) is in the second repeat (gray box). Shaded circles = N-linked sugar.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we demonstrated by site-directed mutagenesis that only the serine 399 in a GDSL consensus sequence is necessary for all the enzymatic activities of GPPLB. These data confirmed our previous finding, which indicated a same inhibition pattern for the PLA2, lysophospholipase, and lipase activities of purified GPPLB by 5,5'-dithiobis-(2-nitrobenzoic acid), diisopropyl fluorophosphate, and N-ethylmaleimide, suggesting the involvement of both serine and cysteine residues in a unique catalytic site responsible of all the activities (7). Concerning the role of the cysteine residue, we can hypothesize that an intramolecular disulfide bond necessary for the enzymatic function is present. Nevertheless, the serine esterase property of GPPLB was clearly demonstrated in this study in agreement with data obtained for rabbit (21) and rat intestinal PLB (22). This latter study indicated that the catalytic serine is part of a catalytic triad associating aspartate and histidine residues in the second repeat. By contrast, GPPLB contained such triad residues in both repeats II and IV, whereas the histidine of the triad in repeat IV is in the consensus EDCLH instead of the conserved stretch PDCFH, thus lacking the proline. However, only mutation of serine 399 in the GDSL consensus of repeat II completely abolished the enzymatic function and markedly affected the protein expression level. The change of the serine 1102 in repeat IV did not affect the enzymatic activity. Nevertheless, a low expression level was observed for both serine 399 (GDSL1) and serine 1102 (GDSL3) mutants, suggesting that they might be important for maintaining the three-dimensional structure.

Earlier studies strongly suggested that intestinal PLB is probably the intestinal bile salt-dependent long chain retinyl ester hydrolase (12). In this study, we clearly demonstrated for the first time that GPPLB has a retinyl esterase activity, associated to the unique catalytic site involving serine 399. Our present data emphasized the broad substrate specificity and the important role of intestinal brush border GPPLB in digestion of dietary lipids and vitamin A.

As described for lactase, another brush border hydrolase with structural characteristics similar to PLB (28), folding of a large multidomain protein as PLB is expected to be a complex process that implicates the proregion. The role of prosequences in modulating folding of proteins is largely described in a wide range of proteins like zymogens (29), proteases (30, 31) growth factors (32), hormones (33), hydrolases (34, 35), and lipase (36). Their cleavage was associated in many cases with biological activation of the final mature form of the protein. In this study, we sought to determine the role of the NH2-terminal repeat I as a proregion involved in the regulation of intracellular processing and enzymatic activity of GPPLB. The repeat I-deleted mutant was poorly detected by Western blot, compared with the wild type PLB. However confocal microscopy study indicated a low proportion reaching the membrane and a strong intracellular accumulation of the mutant around the nucleus. This could correspond to the presence in the ER of aggregates of mutated protein as high molecular weight complexes that could not be detected in our SDS-PAGE conditions. In fact, these aggregates range in size from dimers to large granules of several million daltons (37). This microscopy study indicated an important role of the repeat I in the folding of GPPLB in the ER or for its transport from ER to the Golgi apparatus. In fact, the conformational status of newly synthesized polypeptides is monitored in the lumen of the ER by an efficient quality control mechanism. Proteins that fail to acquire a correct three-dimensional structure are retained in the ER and ultimately degraded (37, 38). Some proteins exit the ER but are blocked in a pre-Golgi compartment instead of further transport to their final destination (39, 40). Nevertheless, due to the presence of a complex glycosylated form of mutant, we can conclude that correct folding occurs in the absence of the proregion, but at a very much low rate compared with wild type protein.

GPPLB is synthesized as a proenzyme activated by trypsin. In this work, the kinetic study of trypsin treatment indicated the presence of different cleavage sites in GPPLB leading to the two main enzymatic active forms. The first is a 140-kDa membrane protein that corresponds to the molecular weight of the brush border membrane enzyme and to that of the deleted mutant, strongly suggesting that the proregion consists of the NH2-terminal repeat I. The second is a 97-kDa soluble form, as previously obtained after papain treatment of brush border membrane (4). Trypsin treatment of the NH2-terminal-deleted mutant generated the 97-kDa form and thus suggested a proper folding of the immunoblot detected protein. These data also suggested that in some pathophysiological conditions associated with an increase of protease secretion, PLB can be released and acts as a soluble enzyme. These observations can be taken into account to investigate physiological function of PLB. In this context, an activation of PLB was already observed in WBN/Kob rats with pancreatic insufficiency (41).

Furthermore, we demonstrated that the repeat I-deleted protein displays the same enzymatic activities as the wild type, without trypsin treatment. These results underlined an important function of the repeat I in enzymatic activity of GPPLB. Thus, we proposed a structural model, indicating that PLB is synthesized as a proenzyme in which the large NH2-terminal repeat I constitutes a lid covering the catalytic site and inhibiting the enzyme by blocking access of the substrate. Proteolytic treatment removes the lid to produce a mature active enzyme. In the gastrointestinal tract, this cleavage could be achieved by pancreatic trypsin secreted in post-prandial periods. We do not know yet what is happening to the cleaved proregion. We can rule out its covalent association with another repeat by an intramolecular disulfide bound as described for bovine pancreatic trypsin inhibitor (42), because no change was observed in the molecular weight of trypsin-treated GPPLB in the presence or not of reducing agent (data not shown). The presence of a lid covering the active site is described for lipoprotein lipase (43) and cPLA2 (44). However, this concerns a flexible and smaller structure that must move to allow substrate access to the active site.

In conclusion, this work supplied new data for understanding the structure and function relationship of GPPLB. However, this enzyme is also present in epididymis, and we suggested a role in sperm maturation (17). Further studies and knock-out animals will give some insights concerning the more general function of this ectoenzyme.

    ACKNOWLEDGEMENTS

We thank Dr. Brigitte Perriquet and Anne-Marie Campo for important collaboration in the determination of retinyl esterase activity and Dr. Sabina Müller for the great assistance with confocal microscopy. Liliane Vrancken is acknowledged for helping with the bibliography.

    FOOTNOTES

* This work was supported by the Conseil Régional of Midi-Pyrénées.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger These authors contributed equally to this work.

§ To whom correspondence should be addressed. Tel.: 33-5-61-77-94-00; Fax: 33-5-61-77-94-01; E-mail: Ama.Gassama@toulouse.inserm.fr.

Published, JBC Papers in Press, August 22, 2002, DOI 10.1074/jbc.M205761200

    ABBREVIATIONS

The abbreviations used are: PLB, phospholipase B; GPPLB, guinea pig phospholipase B; PLA2, phospholipase A2; ER, endoplasmic reticulum.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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