From the Institut Fédératif de Recherche en Immunologie Cellulaire et Moléculaire, Université Paul Sabatier and Centre Hospitalo-Universitaire de Toulouse, Institut National de la Santé et de la Recherche Médicale, Unité 326, Phospholipides Membranaires, Signalisation Cellulaire et Lipoprotéines, Hôpital Purpan, F 31059 Toulouse Cedex, France and the § Laboratoire de Biochimie des Protéines, Sanofi Elf-Biorecherches, F 31676 Labège Innopole, France
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ABSTRACT |
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Guinea pig intestinal phospholipase B is a calcium-independent phospholipase hydrolyzing sequentially the acyl ester bonds at sn-2 and sn-1 positions of glycerophospholipids, promoting the formation of sn-glycero-3-phosphocholine from phosphatidylcholine. This 140-kDa glycoprotein from the brush border membrane of differentiated enterocytes contributes to lipid digestion as an ectoenzyme. The cDNA coding for guinea pig phospholipase B was revealed to be the homologue of AdRab-B, an mRNA appearing in rabbit upon intestine development. The sequence predicts a polypeptide of 1463 amino acids displaying four homologous repeats, two of them containing the lipase consensus sequence GXSXG. A 5-kilobase transcript was particularly abundant in mature ileal and jejunal enterocytes but was also detected in epididymis, where phospholipase B displayed a higher molecular mass (170 kDa versus 140 kDa in intestine), with no obvious evidence for enzyme activity. Trypsin treatment of phospholipase B immunoprecipitated from epididymal membranes reduced its size to 140 kDa, coinciding with the appearance of a significant phospholipase A2 activity. The same results were obtained in COS cells transfected with phospholipase B cDNA. Since sn-glycero-3-phosphocholine present at high concentrations in seminal plasma mainly stems from epididymis, this suggests a possible role of phospholipase B in male reproduction. This novel localization also unravels a mechanism of phospholipase B activation by limited proteolysis involving either trypsin in the intestinal lumen or a trypsin-like endopeptidase in the male reproductive tract.
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INTRODUCTION |
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PLB1 (EC 3.1.1.5) is defined as a hydrolase displaying both a PLA2 and a lysophospholipase activity, thus achieving the full deacylation of glycerophospholipids. Several PLB have been identified in various microorganisms (1-8) and in plants (9) as well as in the brush border membrane of mature enterocytes from three animal species including guinea pig (10-13), rat (14-17), and rabbit (17, 18). It was later found that intestinal PLB actually displays a broader substrate specificity, including diacylglycerol, monoacylglycerol (12), and retinyl esters (19). All of these data have led to the obvious conclusion that intestinal PLB participates in the digestion of various dietary lipids.
Despite its broad substrate specificity, PLB can be viewed as a PLA2 due to its ability to release first the fatty acid esterifying the sn-2 position of glycerophospholipids. Indeed, guinea pig intestinal PLB was recently considered as a subgroup of calcium-independent PLA2 (20). Moreover, a homologous sequence is shared by Penicillium notatum PLB and cytosolic PLA2, the major enzyme responsible for intracellular liberation of arachidonic acid (21). Our knowledge of PLA2 has considerably grown these last years with a number of enzymes being characterized at a molecular level (for a recent classification, see Ref. 22). This has allowed clarification of the role and the regulation of some of these enzymes, although there are still a number of uncertainties (23, 24).
The active site of guinea pig PLB is localized on the external face of the brush border membrane from which it can be solubilized either by detergents or by proteolytic cleavage (11), suggesting a structural arrangement similar to that of other brush border hydrolases such as sucrase-isomaltase, aminopeptidase N, or lactase-phlorizin hydrolase. All of these enzymes are stalked in the lipid bilayer by a hydrophobic segment connected to the larger, heavily glycosylated globular domain containing the catalytic site and protruding into the intestinal lumen (25-27). The mode of anchoring as well as other structural and functional features of these enzymes have been clearly determined by cloning and sequencing their cDNA (28-30). Thus, another interest of intestinal PLB is that, like other microvillar hydrolases, it might offer a very convenient tool to study general processes of cell biology such as protein glycosylation, differentiation of epithelial cells, or membrane traffic allowing proper addressing of proteins to apical membranes (25-27).
In an effort to understand both the enzymatic properties and the intracellular trafficking of guinea pig intestinal PLB in relation to enterocyte differentiation, we decided to clone the corresponding cDNA. The data reported below indicate that guinea pig PLB cDNA is actually the homologue of AdRab-B, an mRNA appearing in rabbit upon intestine development and enterocyte differentiation (18). However, an emerging observation of the present study was the detection of the mRNA of intestinal PLB in epididymis. GPC is the final product resulting from phosphatidylcholine breakdown by PLB. It is present at 1-3 mM concentrations in the seminal plasma of various animal species, and epididymis represents its major site of synthesis in the male reproductive tract, epididymal fluid containing as much as 100 mM GPC (31-38). Previous studies indicated that rat epididymis contains an androgen-dependent phospholipase A as well as a lysophospholipase activity able to produce GPC from phosphatidylcholine; however, no further molecular characterization of these enzymes has been achieved so far (39, 40). Together with the evidence that epididymal PLB is synthesized as a proenzyme requiring limited proteolytic cleavage to reveal full enzymatic activity, our data open interesting issues concerning the biological function as well as the regulation of this subgroup of calcium-independent PLA2.
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EXPERIMENTAL PROCEDURES |
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Isolation of Various Intestinal Cell Populations-- Intestinal cells were isolated and separated as a villus to crypt gradient according to Weiser (41).
Immunoprecipitation-- Intestinal brush border membrane vesicles were prepared according to Schmitz et al. (42). Proteins were solubilized by the addition of an equal volume of a twice concentrated immunoprecipitation buffer referred to as RIPA I buffer (43) and containing 40 mM Tris-HCl (pH 7.5), 300 mM NaCl, 4 mM EDTA, 1% (w/v) sodium deoxycholate, 2% (v/v) Triton X-100, and protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 5 µg/ml leupeptin). Samples were precleared by adding 25 µl of a 10% (v/v) suspension of protein A-Sepharose (Sigma), mixing for 30 min at 4 °C, and centrifuging for 2 min at 13,000 × g. Supernatants were then incubated overnight at 4 °C with 10 µl of polyclonal anti-guinea pig PLB obtained as described previously (13). Immunocomplexes were precipitated by the addition of 50 µl of the 10% suspension of protein A-Sepharose. After 4 h of incubation at 4 °C under gentle shaking, samples were then centrifuged at 13,000 × g for 2 min, and the pellet was washed twice in RIPA I buffer and once in RIPA II buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA), resuspended in Laemmli buffer (44) for SDS-PAGE or in phosphate-buffered saline containing 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 mM benzamidine for determination of PLA2 activity.
Sequencing of Guinea Pig PLB Tryptic Peptides-- Guinea pig PLB was immunoprecipitated as described above and analyzed on SDS-PAGE under reducing conditions. After Coomassie Blue staining of the gel, the band of interest was excised and digested in situ by porcine trypsin (45). The tryptic fragments were extracted from the gel and purified by reverse phase high pressure liquid chromatography according to Rosenfeld et al. (45). The peptides were sequenced on an Applied Biosystem sequencer (model 470 A), coupled to a phenylthiohydantoin-derivative analyzer model 120 A (Applied Biosystems, Les Ulis, France).
Polymerase Chain Reaction Amplification-- PCR was performed by standard techniques (46) with Taq DNA polymerase (Appligene Oncor) in a Crocodile III thermal cycler (Appligene Oncor). Amplified fragments were separated on 1% agarose gel and visualized by ethidium bromide staining. Oligonucleotides were synthesized in a PCR Mate DNA synthesizer (Applied Biosystems), except PLB5 and Ol2, which were obtained from Isoprim (Toulouse, France).
Radioactive Probes--
The PLB1 probe (486 bp) was obtained by
PCR using intestinal guinea pig cDNA, obtained by random priming
reverse transcription on 2 µg of poly(A)+ RNA, and the
couple of oligonucleotides, cob5' and cob3', derived from the guinea
pig PLB amino acid sequences B and C, respectively. The PCR protocol
comprised 1 min at 94 °C, 1 min at 45 °C, and 1 min 20 s at
72 °C for 30 cycles. The PLB2 probe (460 bp) was obtained by PCR on
2a1 clone plasmid DNA with P205 and M10 oligonucleotides (1 min at
94 °C, 1 min at 52 °C, and 1 min 30 s at 72 °C for
30 cycles). The glyceraldehyde-3-phosphate dehydrogenase probe (350 bp)
was prepared by PCR on human lung cDNA with specific
oligonucleotides determined according to the sequence (47). The
30-cycle program consisted of 1 min at 94 °C, 1 min at 58 °C, and
1 min at 72 °C. These probes were labeled by random primer extension
using the Nonaprimer Kit (Appligene Oncor) and
[-32P]dCTP from Amersham Pharmacia Biotech.
RNA Isolation-- Total RNA were extracted from different tissues or from isolated intestinal cells by cesium chloride/guanidium thiocyanate method (46). Poly(A)+ RNAs were prepared using an oligo(dT)-cellulose column from Amersham Pharmacia Biotech.
cDNA Library Construction--
Guinea pig intestinal
cDNA library was constructed in ZAP II vector using 5 µg of
poly(A)+ RNA from differentiated villus enterocytes
according to the manufacturer instructions (ZAP cDNA synthesis kit,
Stratagene, La Jolla, CA). The
library was packaged into phage
particles using the Gigapack II kit (Stratagene). The library was
amplified once in XLI-Blue MRF' Escherichia coli
(Stratagene), eluted into SM phage buffer (0.1 M NaCl, 8 mM MgSO4, 50 mM Tris-HCl, 0.01%
gelatin, pH 7.5), and stored at 4 °C.
Library Screening-- The library was plated on XLI-Blue MRF' E. coli and replicated on positively charged nylon membranes (Hybond N+, Amersham Pharmacia Biotech). After denaturing treatment (1.5 M NaCl; 0.5 M NaOH), phage DNA was covalently immobilized on the membrane by exposure to UV light. Filters were prehybridized for at least 2 h at 42 °C in 5× SSC, 50% formamide, 5× Denhardt's solution, 0.5% SDS, 7% dextran sulfate, 0.1 mg/ml salmon sperm DNA. Hybridization was performed for 16 h at 42 °C in prehybridization solution by adding radiolabeled cDNA probe at about 106 cpm/ml. Hybridized blots were washed four times, the final wash was carried out at 55 °C in 0.2× SSC, 0.1% SDS and exposed for 48 h to x-ray film (Hyperfilm MP, Amersham Pharmacia Biotech) using an intensifying screen. Plaque-purified positive clones were purified in three rounds and transformed into Bluescript plasmid by in vivo excision using ExAssist helper phage and SOLR E. coli (Stratagene). Plasmid DNA was extracted from an overnight culture of bacteria with the Wizard Miniprep DNA purification system from Promega (Madison, WI).
DNA Sequencing and Analysis-- Sequencing was carried out either by the Sanger dideoxy chain termination method using [35S]dATP and T7 polymerase (T7 sequencing kit from Amersham Pharmacia Biotech) or by automated methods (Genome Express, Grenoble, France). Nucleotide and protein sequences were analyzed by using the PC-gene software package (IntelliGenetics, Mountain View, CA). Hydropathy analysis was performed in accordance with Kyte and Doolittle (48).
5'-Rapid Amplification of cDNA Ends (RACE) Protocol-- The 5'-RACE was performed using Marathon cDNA amplification kit from CLONTECH (Palo Alto, CA) according to the manufacturer's instructions. Briefly, 2 µg of poly(A)+ RNAs from jejunal/ileal mucosa were reverse transcribed using oligo(dT) primer and converted to double strand, and an adaptor sequence was ligated at both ends of the cDNA. The ligated cDNA was diluted (1:200), and 5 µl were used to perform PCR reactions using the Advantage Klen Taq polymerase mix from CLONTECH. Adaptor primers and two primers designed at the 5'-end of 4a clone were used in the PCR protocol that comprises 1 min at 94 °C, 2 min or 2 min 30 s at 60 °C, and 2 min at 68 °C for 30 cycles.
Construction of Full-length PLB cDNA-- The PCR-amplified cDNA obtained by 5'-RACE protocol was used for a second amplification using primers M10 and PLB5, and the 1.5-kbp fragment obtained was cloned in the pGEMT-Easy vector (Promega). The insert cDNA was excised from the vector by EcoRI digestion and ligated in the clone 4a digested by EcoRI (the digestion removed 500 bp at the 5'-end of the insert). The resulting construction contained the full-length cDNA in Bluescript vector. The right inserted clones were screened by PCR, and the result was confirmed by sequencing of the insert.
PLB Expression in COS Cells-- The full-length PLB cDNA was excised from Bluescript vector with BamHI and ApaI, subcloned into eukaryotic expression vector pcDNA3 (Invitrogen) to give pcDNA3-PLB with the expression under cytomegalovirus promotor. COS-7 cells in 60-mm dishes were transfected using SuperFect (Qiagen) according to the manufacturer's protocol. After a 48-h transfection, cells were scraped and sonicated in phosphate-buffered saline containing 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 10 mM benzamidine. This homogenate was used for immunoprecipitation, Western blotting, and PLA2 activity assays.
Northern Blotting-- Total or poly(A)+ RNA were run on 1% agarose gels in the presence of 0.37 M formaldehyde, transferred by capillarity (46) to positively charged nylon membrane (Biohylon Z+, Bioprobe system) and covalently immobilized on the membrane by exposure to UV light. Filters were hybridized as described for library screening with a final wash at 65 °C in 0.1× SSC, 0.1% SDS for PLB1 probe and in 0.2× SSC, 0.1% SDS for glyceraldehyde-3-phosphate dehydrogenase probe. Deblotting of the membranes was obtained by treatment in 0.1% SDS at 100 °C.
Epididymis Subcellular Fractionation-- Animals were killed by a blow to the head, and the epididymis was removed. It was rinsed with ice-cold isotonic NaCl and homogenized on ice in Tris-sucrose buffer (250 mM sucrose buffered with 10 mM Tris-HCl, pH 7) using a Potter-Elvejhem homogenizer. The subcellular fractionation was carried out according to Diagne et al. (10) by a sequential centrifugation procedure, and the different pellets were resuspended in the homogenization buffer.
Trypsin Digestion of PLB-- Transfected COS cell homogenate or epididymal 100,000 × g pellet were incubated for 30 min at 37 °C in the presence or in the absence of trypsin (50 µg/ml). The reaction was stopped by adding soybean trypsin inhibitor (30 µg/ml) in 5 mM potassium phosphate (pH 6.8) as described by Quaroni et al. (49). The sample was used for gel electrophoresis and determination of PLA2 activity.
Immunoblotting-- Proteins were subjected to SDS-PAGE on a 7% (w/v) polyacrylamide gel under reducing conditions according to Laemmli (44) and transferred to nitrocellulose membranes (Hybond C Extra; Amersham Pharmacia Biotech). Immunoreactive proteins were detected using a 1:500 dilution of the polyclonal anti-PLB antibody, second antibody (goat anti-rabbit, Promega, France) being conjugated to alkaline phosphatase. The substrate was 5-bromo-4-chloro-3-indolyl phosphate, and the chromogen was nitro blue tetrazolium (Promega, France).
Epididymal cDNA Synthesis-- About 2 µg of poly(A)+ RNA from guinea pig epididymis were converted into single-strand cDNA using oligo(dT) priming and Moloney murine leukemia virus reverse transcriptase (Superscript Preamplification System; Life Technologies, Inc., Cergy, France) in a final volume of 100 µl, from which 10-µl aliquots were used for each PCR amplification.
Assays of PLA2, Lysophospholipase, and Lipase Activities-- These were achieved using 1-acyl-2-[14C]linoleoyl-sn-glycero-3-phosphocholine, 1-acyl-sn-glycero-3-phospho-[3H]choline, or [3H]oleoyl-labeled diolein as described previously (11, 12).
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RESULTS |
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The cDNA Coding for Guinea Pig Intestinal PLB Is the Homologue
of AdRab-B--
Guinea pig PLB was immunoprecipitated from brush
border membranes with a specific polyclonal antibody and migrated on
SDS-PAGE under reducing conditions. The 140-kDa Coomassie Blue-stained band was excised, treated with trypsin, and partially sequenced as
described under "Experimental Procedures." Three amino acid sequences (A, VGAFFNQA; B, LVNLVDFMNP; C, LGDSLTA) were obtained, and
the B and C sequences were used to design two oligonucleotides (cob3'
and cob5', respectively). A 486-bp fragment (PLB1) was PCR-amplified
from intestinal guinea pig cDNA with these two primers and used for
screening a cDNA library prepared in -ZAP II vector. Eight
positive clones were isolated and analyzed. They carried 3-3.5-kbp
cDNA insertions, and they were all sequenced on a few bases at
their 3'- and 5'-terminal ends. Six of them displayed an identical 3'
terminus (initiated at the poly(A) tail), indicating that they were
subclones of the same cDNA sequence. The longest cDNA (3.46 kbp, 2a1 clone) was totally sequenced, but it did not contain the ATG
initiation codon. In order to isolate the full-lengh cDNA, a 460-bp
probe (PLB2) was PCR-amplified on the 2a1 5'-terminal end between the
two specific primers: P205 and M10 (Fig.
1A). This probe was used for a
second screening of the cDNA library and allowed the isolation of
the 4a clone containing a 3.679-kbp cDNA.
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Intestinal Expression of Guinea Pig PLB Gene Is Maximal in Jejunum/Ileon and Follows Enterocyte Differentiation-- Using the Weiser procedure, expression of guinea pig PLB mRNA was analyzed along the crypt/villus axis with the PLB1 probe. As shown by Northern blot (Fig. 2A), a single 5-kilobase transcript was detected in all fractions, and we observed an increase of this specific mRNA level from undifferentiated cells (fraction 9) to differentiated ones (fractions 1-3). The same distribution was previously observed for protein expression (13), suggesting a transcriptional regulation of intestinal phospholipase B gene expression. The filter was deblotted and hybridization with a glyceraldehyde-3-phosphate dehydrogenase probe demonstrated that the amount of RNA loaded onto each lane was very similar.
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Epididymis Appears as a Second Site of PLB Gene Expression-- PLB mRNA expression was also investigated in other guinea pig tissues, confirming the selective localization of PLB in the digestive tract. However, an additional site of PLB expression was observed in epididymis, as clearly illustrated in Fig. 2B. Northern blot data were confirmed by PCR analysis of single-stranded epididymal cDNA. Four couples of intestinal PLB-specific primers (Fig. 2C) located on the four repeated domains were used in amplification reactions with epididymal cDNA or 4a cDNA as a matrix. With each couple of primers, epididymal cDNA gave a specific PCR amplification product of the expected size (Fig. 2C), indicating that it contains the four repeated sequences. Moreover, the cob5'/cob3' 486-bp product was sequenced and revealed a total identity with intestinal PLB cDNA.
This led us to investigate PLB protein expression in epididymis by subcellular fractionation of epididymal homogenates. Using a polyclonal anti-PLB antibody, a single 170-kDa polypeptide was detected by Western blotting in the different fractions and was found to be enriched in 10,000 × g and 100,000 × g pellets (Fig. 3A). This protein was also immunoprecipitated from the 100,000 × g pellet (Fig. 3A, first lane). As illustrated in Fig. 3B, treatment of epididimis PLB with N-glycanase reduced its molecular mass from 170 to 146 kDa, suggesting that epididymal PLB is highly glycosylated, as previously shown for intestinal enzyme (13).
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PLB Is Synthesized as a Proenzyme Activated by Proteolytic Cleavage-- To verify the presence of a proenzyme, PLB was immunoprecipitated from the 100,000 × g epididymal pellet, submitted to trypsin digestion, characterized by Western blotting, and tested for PLA2 activity. As shown in Fig. 4A, trypsin treatment decreased the molecular mass of epididymal PLB from 170 to 140 kDa, which corresponds to the size observed in intestinal brush border membrane. Identical results were obtained upon trypsin treatment of whole membranes from the 100,000 × g pellet under conditions allowing a total conversion of the 170-kDa band into a 140-kDa polypeptide. Occasionally, a smaller fragment also appeared at 116 kDa (data not shown). Interestingly, this shift in mass was associated with the appearance of PLA2 activity (Fig. 4B), clearly demonstrating a proteolytic regulation of the enzyme.
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Properties of Recombinant PLB Expressed in COS Cells-- To follow PLB expression with the pcDNA3 construction and to confirm the data obtained with epididymal PLB, COS cells were transfected as described under "Experimental Procedures." Analysis of cell homogenates and immunoprecipitates by Western blotting (Fig. 4C) indicated that PLB was not expressed in pcDNA3-transfected COS cells (lanes 1-3), whereas a 170-kDa polypeptide was recognized by the anti-PLB antibody in COS cells transfected with pcDNA3-PLB (lanes 4-6). In immunoprecipitated fractions (lane 5), the 140-kDa proteolytic form was slightly detectable but increased significantly upon trypsin treatment (lane 6). Here again, trypsin digestion was concomitant with a dramatic increase in PLA2 activity (Fig. 4D), demonstrating that proteolytic cleavage was required for the protein to express a full enzymatic activity.
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DISCUSSION |
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This study was based on the molecular cloning of the cDNA coding for guinea pig intestinal PLB, which revealed its identity with AdRab-B previously isolated in rabbit (18). Although both proteins are expected to display very similar structures and properties, one small difference concerns the lipase consensus sequence GXSXG, which was identified in domains II and IV of guinea pig PLB, whereas an additional motif (GSSEG) is present in repeat IV of AdRab-B. In a very recent study, Wacker et al. (55) identified the active serine of the product of AdRab-B, which they now call "adult only" esterase/phospholipase A. Unexpectedly, serine 400 was the only residue labeled with [14C]diisopropyl fluorophosphate under conditions where enzyme activity toward both long chain fatty acyl esters and phosphatidylcholine was abolished. This active serine is located in a sequence (GDSLT) similar to lipase consensus sequence and is absolutely conserved in repeats II, III, and IV of both rabbit and guinea pig phospholipases B (see Fig. 1C). Further studies based on site-directed mutagenesis will be necessary to identify other residues of the catalytic triad and to explain why only one of the four repeated domains apparently bears the enzymatic activity. In addition, there might be some significant differences between guinea pig and rabbit PLB. For instance, the latter one was found to be insensitive to sulfhydryl blocking reagents (18), whereas the guinea pig enzyme is fully inhibited by N-ethyl maleimide (12). Interestingly, cytosolic PLA2 is inhibited by SH-blocking reagents; however, a serine but not a cysteine residue was identified in the catalytic triad, where histidine is replaced by an arginine residue (21, 56). This apparently small difference would deserve some attention in future studies dealing with the identification of PLB active site(s).
A first use of PLB cDNA was to follow the pattern of mRNA expression in guinea pig intestine, which was identified so far as the only site of PLB synthesis. These and previous data all support the view that the PLB gene is expressed as a function of enterocyte differentiation, probably at a transcriptional level, resulting in a maximal synthesis of PLB in cells that are the most implicated in lipid digestion, i.e. mature enterocytes of jejunum and ileon (10-18).
We would like to focus on the most crucial point of this study,
i.e. our finding that guinea pig intestinal PLB is also
strongly expressed in epididymis. This is actually not the first
example of a pancreatic or intestinal enzyme with an ectopic expression suggesting other roles than a simple digestive function. For instance, pancreatic (type I) PLA2 has been found in lung (57, 58), spleen (59), gastric mucosa (58, 60-61), pancreatic -cells (62),
and kidney (63). It is noteworthy that a membrane receptor specific for
secretory PLA2 was also found in some of these tissues (64-66). A pancreatic lipase was identified in mouse cytotoxic T
lymphocytes (67) and is now classified in a subgroup of pancreatic lipase-related proteins (68-73), whose first member was actually discovered in guinea pig pancreas (74). Finally, intestinal dipeptidyl
peptidase IV (also called CD26) is expressed in T-lymphocytes and
thymocytes, where it appears as an activation signal-transducing molecule as well as having its intrinsic enzymatic activity (75, 76). A
first issue of these various observations would be to investigate which
factors regulate a so tight but so different tissue expression of these
enzymes. This could be particularly interesting for guinea pig PLB,
insofar as its appearance in the male genital tract might be under the
influence of androgen secretion, as described for the PLB activity
previously observed (39, 40).
In the latter case, it is tempting to suggest that epididymal PLB might fulfill a very critical role in sperm maturation. Spermatozoa are known to reside for about 12-14 days in epididymis, where they acquire a number of proteins essential for their motility and for oocyte fertilization (77-81). Moreover, during epididymal transit, spermatozoa loose up to 50% of their membrane phospholipids (82-85). This balances the production of up to 80% of GPC (a main component of seminal plasma probably essential for sperm protection and/or metabolism) by epididymis (31-38). However, spermatozoa might not represent the unique source of epididymal GPC, which could also be derived from phosphatidylcholine of blood lipoproteins (86).
The detection in epididymis of both mRNA and protein corresponding to intestinal PLB stimulated us to search for mechanism(s) able to trigger the corresponding enzyme activities. The fact that trypsin digestion activates this latent enzyme strongly argues in favor of the view that a similar mechanism might operate in vivo. Proteolytic maturation of intestinal PLB has been suggested to occur during intracellular traffic in a way similar to that of other brush border hydrolases such as lactase-phlorizin hydrolase (18). More recently, Wacker et al. (55) found that a trypsin cleavage site after Arg-363, which is strictly conserved between rabbit and guinea pig proteins (Arg-362 in guinea pig), is present at the junction between repeats I and II and might be responsible for enzyme processing in the intestinal lumen. Roughly, this would result in a 25% reduction of PLB molecular mass, which is identical to the 26% difference determined between 146 and 108 kDa (deglycosylated forms). However, proteolytic digestion of the glycosylated form resulted in a reduction of apparent molecular mass limited to 18% (170 versus 140 kDa). This could be explained by the fact that the N-terminal part of the molecule until Arg-362 contains only one of the 12 N-glycosylation sites present in the entire molecule.
Our observation that native PLB needs limited proteolysis to acquire its full enzymatic activity is reminiscent of pancreatic PLA2, which is produced as a zymogen activated upon removal of its N-terminal heptapeptide (87, 88). This has been the object of a number of functional and structural studies showing that activation of pro-PLA2 results in a conformational change allowing the enzyme to interact with the lipid-water interface by an "interface recognition site" (87, 88). In the case of PLB, trypsin treatment induces a more drastic reduction in the protein size, probably upon the removal of repeat I, as discussed above. Since a priori this domain does not display any enzymatic activity, it will be interesting in future studies to define whether it can act as an intrinsic inhibitor of PLB, impairing for instance its interaction with the lipid-water interface. This can be suggested by the fact that recombinant 170-kDa PLB displayed a significant esterase activity toward water-soluble substrates (18), whereas both recombinant and epididymal 170-kDa PLB were virtually inactive against phospholipids (this study).
The point of PLB adsorption at lipid-water interfaces would be particularly crucial to understand a possible interaction between epididymal PLB and sperm membrane. In our hands, guinea pig intestinal PLB is a poorly penetrating enzyme requiring biliary salts to achieve significant degradation of phospholipids. In a recent study, we provided evidence that secretory nonpancreatic PLA2 (type II PLA2), which is largely involved in inflammatory processes, is another poorly penetrating phospholipase unable to attack phospholipids in the membrane of intact cells (89). However, we also showed that type II PLA2 becomes active on membranes having lost their asymmetric transverse distribution of phospholipids. Our present finding will certainly stimulate further studies dealing with the characterization of sperm membrane organization in relation to its susceptibility to intestinal/epididymal PLB.
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FOOTNOTES |
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* 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF045454.
Supported by a fellowship from Ministère de la Recherche et
de l'Enseignement Supérieur, de la Recherche et de la
Technologie.
§ These authors contributed equally to this work.
To whom correspondence should be addressed: INSERM Unité
326, Hôpital Purpan, 31059 Toulouse Cedex, France. Tel.:
33-5-61-77-94-00; Fax: 33-5-61-77-94-01; E-mail:
chap{at}purpan.inserm.fr.
1 The abbreviations used are: PLB, phospholipase B; PLA2, phospholipase A2; GPC, sn-glycero-3-phosphocholine; RIPA, radioimmune precipitation buffer; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); kbp, kilobase pair(s); RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction.
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REFERENCES |
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