©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Chaperone Function of a Grp 94-related Protein for Folding and Transport of the Pancreatic Bile Salt-dependent Lipase (*)

Nadine Bruneau (§) , Dominique Lombardo (¶)

From the (1) From INSERM Unité 260, Faculté de Médecine Timone, 27 boulevard Jean Moulin, 13385 Marseille Cedex 05, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In its fundamental attributes, the secretion pathway of the pancreatic bile salt-dependent lipase (BSDL) followed that described for all enzymes involved in regulated secretion. This route was inhibited by drugs that affect protein synthesis and intracellular transport. In the presence of monensin, BSDL was solely detected in microsome membrane fractions. The association of BSDL with intracellular membranes involved a protein complex, formed by at least two proteins of 94 and 56 kDa. In cells experiencing the metabolic stress due to azetidine-2-carboxylic acid, BSDL was additionally associated with a protein of 46 kDa. Affinity blotting showed that BSDL bound directly to the 94-kDa protein (p94). It was suggested that p94 could be a molecular chaperone, further identified as related to the 94-kDa glucose regulated protein (Grp 94). The membrane-associated BSDL (i.e. BSDL bound to the Grp 94-related p94) was O- and N-glycosylated and consequently appeared released from membranes in the trans-Golgi compartment. Therefore and for the first time, it is suggested that a multiprotein complex including the chaperone Grp 94-related p94 protein may play an essential role in the folding and transport of BSDL. One hypothesis is that the association of BSDL with membrane via the Grp 94-related p94 along its secretion pathway is required for its complete O-glycosylation, which occurs on the extended mucin-like structures present on the C-terminal part of the protein.


INTRODUCTION

Regulated cell secretion is the result of consecutive events comprising the synthesis and transport of proteins through intracellular compartments from the endoplasmic reticulum to zymogen granules. In response to the appropriate signal, zymogen granule membranes fuse with the luminal plasma membrane of the cell. This fusion allows the release of the soluble enzymes. During the secretory process, proteins are adequately folded and assembled and may undergo post-translational modification. Incompletely assembled, misfolded, or aggregated products are selectively retained in the endoplasmic reticulum (1) . In a similar way, transport to and from the Golgi apparatus occurs only when nascent proteins have been folded and assembled in the correct way (2) . A family of proteins known as molecular chaperones have been recognized participating in the correct folding of polypeptidic chains and in driving them into oligomeric structures, if required (3) . Molecular chaperones have been shown to be invaluable to cellular processes such as protein synthesis, transport, signal transduction, morphogenesis, immune and heat shock, or stress responses. Members of this family have been recently localized in distinct compartment along the secretory pathway of the pancreatic acinar cells (4) . Elements of the ``quality control'' mechanisms involved in recognizing, retaining, and degrading misfolded and misassembled proteins are therefore present in these cells (1) . The bile salt-dependent lipase (BSDL)() is an enzyme found in all examined pancreatic secretions (for a review, see Ref. 5). The size of this protein varies by species due to the consequence of the number of C-terminal tandem-repeated sequences (6, 7) , which are O-glycosylated (8) . The human BSDL possesses 16 of these tandem repeated sequences and represents the largest (100 kDa). The rat BSDL has only 4 of these repeats and has the smaller size (70 kDa). A site for N-linked glycosylation was located on Asn-187 (7) close to the Ser-194 of the active site (9) . This N-linked glycosylation was shown to be cotranslational and essential for the secretion and expression of a fully active enzyme by pancreatic cells (10) . A glyco-isoform of BSDL expressed by a neoplastic pancreas has been described (11, 12) . This glyco-isoform, referred to as fetoacinar pancreatic protein or FAP, is associated with oncogenesis and ontogenesis of the human pancreas (13) . Immunocytological experiments performed using J28, a monoclonal antibody that recognizes FAP, suggested that in tumor cells, this oncofetal form of BSDL was retained within the endoplasmic reticulum (14). Subcellular fractionation corroborated this localization and established that FAP might be associated with microsomal membranes (14). Defective processing of the N-linked glycosylation in the endoplasmic reticulum resulting from amino acid mutation was hypothesized to explain the retention of the protein (14) . However, we have recently shown that the maturation of the N-linked structure along the secretory pathway did not affect enzyme secretion (10). Variations were therefore observed for the N-linked glycosylation, which appeared of the biantennary complex type in the normal variant of the human enzyme (15) and of the high mannose type in the variant secreted by inflamed pancreas (12) . Moreover, in normal human pancreatic cells, BSDL was partly associated with membranes of microsomes. The association of BSDL with membranes was optimal between pH 5 and 6, which agrees with the pH of the endoplasmic reticulum vesicles. The BSDL can be released from microsome membranes by salt and trypsin treatment or at pH above 8.() Whereas in human, an in-depth study concerning the association of BSDL with microsome membranes was not possible due to the lack of a cell model, we turned to the rat AR 4-2J cell line. Our experiments showed that BSDL was retained without degradation within the cell upon tunicamycin or brefeldin A treatment of cells (10) . All these data suggest that the secretion or the folding of BSDL could be under the control of chaperones. In this paper we have further characterized microsomal proteins that bind to BSDL molecules synthesized in the presence of monensin, a drug that disrupts communication between trans-Golgi structures and secretory vesicules (16) . Results showed that unfolded, although glycosylated BSDL, formed a complex with proteins of 94 kDa (to which the enzyme is bound), 56 kDa, and 46 kDa. The 94-kDa protein is probably related to the 94-kDa glucose-regulated protein or Grp94.


EXPERIMENTAL PROCEDURES

Materials

Biotin-conjugated lectins, alkaline phosphatase-conjugated antibodies to biotin, alkaline phosphatase-conjugated antibodies anti-rabbit, goat anti-rat IgG, goat anti-mouse IgG, carbonyl cyanide chlorophenylhydrazone (CCCP), monensin, vinblastin sulfate, defatted serum albumin, Tween 20, nitro blue tetrazolium, 4-nitrophenyl hexanoate, soybean trypsin inhibitor, CNBr-activated Sepharose, biotinamidocaproate-N-hydroxysuccinimide ester, L-azetidine-2-carboxylic acid, and 5-bromo-4-chloro-3-indolyl phosphate were from Sigma. Nitrocellulose membranes were from Schleicher & Schüll (Dassel, Germany). -Glucosidase, endoglycosidase H (Endo H), 4-nitrophenyl--D-maltopentaoside, leupeptin, tunicamycin, and brefeldin A came from Boehringer (Mannheim, Germany). Polyvinylpyrolidone K40, phenylmethylsulfonyl fluoride, benzamidine, -phenyl propionate, heparin, and Coomassie Blue R 250 were from Fluka (Buchs, Switzerland). Cytochalasin B and cycloheximide were purchased from Serva (Heidelberg, Germany). [S]Methionine (TranS-label, >1000 Ci/mmol) was from ICN Biochemicals (Costa Mesa, CA).

Antibodies

Monoclonal antibody specific for Grp 94 (clone 9G10) was from StessGen Biotechnology Corp. (Victoria, Canada). Polyclonal antibodies raised using purified secretory rat BSDL and human -amylase were obtained in our laboratory. These antibodies did not react with the microsomes of the rat liver.() Antibodies were isolated by affinity chromatography on protein A-Sepharose as described previously (6) .

Proteins and Enzyme Assays

Proteins were routinely assayed with the bicinchoninic acid method (Micro-BCA kit from Pierce) using bovine serum albumin as standard. The activity on 4-nitrophenyl hexanoate was measured at 410 nm and pH 7.4 in a thermostated cell at 30 °C as described elsewhere (17) . The -amylase activity was determined at 410 nm on 4-nitrophenyl maltopentaoside in pH 7.4, 0.1 M sodium phosphate, 50 mM NaCl buffer in the presence of -glucosidase (>24 units/ml).

Human BSDL Immobilization and Biotin Labeling

Human BSDL was isolated from human pancreatic juice as described previously (18) . The pure enzyme was immobilized on CNBr-activated Sepharose (1 mg of protein/0.5 g of wet gel) in 0.1 M sodium borate buffer (0.25 M NaCl, 5 mM CaCl, pH 8.0). After an overnight incubation at 4 °C under agitation, the gel was allowed to settle and quenched with 10 gel volumes of 0.1 M ethanolamine (4 h, 4 °C). Before use, the gel with immobilized BSDL was extensively washed alternatively with basic (0.1 M sodium phosphate, pH 8.0) and acidic (0.1 M MES, pH 5.5) buffers.

For use in ligand blotting, human BSDL was biotin-labeled as follows; 1 mg of BSDL in 1 ml of 0.1 M sodium bicarbonate, pH 8.0, buffer was biotinylated with biotinamidocaproate-N-hydroxysuccinimide ester (5 µl of a 40 mg/ml solution in dimethyl sulfoxide) for 1 h at 4 °C. The BSDL solution was then dialyzed twice for 24 h against water and diluted to 0.5 mg/ml before use. Approximately 1 ng of biotin-labeled BSDL can be detected using anti-biotin antibodies conjugated to alkalin phosphatase as detection system.

Polyacrylamide Gel Electrophoresis and Western Blotting

Gel electrophoresis (SDS-PAGE) were performed on a slab gel of polyacrylamide (5 or 7.5%) and 0.1% sodium dodecyl sulfate under reducing conditions according to Laemmli (19) using a Bio-Rad Mini-Protean II apparatus. Non-reducing conditions were obtained by omitting dithiothreitol in the denaturation buffer. Proteins were electrophoretically transferred to a nitrocellulose membrane (20) in 0.2 M Tris/HCl, pH 9.2, buffer (10% methanol), at 150 mA for 3 h in a cold room. Completeness of transfer was verified by staining polyacrylamide gels with Coomassie Blue R250. Destaining was performed in ethanol/acetic acid/water (2/3/35 by volume). Membranes were then treated as described previously (8) using the polyclonal antiserums to rat pancreatic BSDL. The BSDL protein mass was determined by densitometric scannings of Western blotting as described by Abouakil et al.(10) , corrected for the volume fraction loaded on SDS-PAGE. Alternatively, gels were stained using the Bio-Rad silver stain kit following the protocol given by the supplier.

Lectins and Ligand Blotting

After electrotransfer to nitrocellulose membranes, replicas were blocked 2 h with 50 mM Tris/HCl, 150 mM NaCl, pH 8.0, buffer containing 3% bovine serum albumin by weight. Glycoproteins were detected by lectin blotting using biotin-conjugated lectins (10 µg/ml) as described by Mas et al.(8) .

Proteins that bind to BSDL were detected by ligand blotting on membranes obtained after SDS-PAGE and electrotransfer. Membranes were first blocked overnight with 0.1 M sodium phosphate, pH 6.0, buffer (3% bovine serum albumin, 50 mM NaCl) and incubated with biotin-labeled BSDL (5 µg/ml, 2 h, 4 °C). After exhaustive washing (six times) with 0.1 M phosphate buffer (0.3% bovine serum albumin, 0.05% Tween 20), membranes were incubated (1 h, ambient temperature) in 0.1 M sodium phosphate, pH 7.4, buffer (3% bovine serum albumin, 150 mM NaCl) with anti-biotin antibodies conjugated to alkaline phosphatase. Bands were visualized by incubating replicas with 0.5 mM 5-bromo-4-chloro-3-indolyl phosphate and 0.5 mM nitro blue tetrazolium in 0.1 M Tris/HCl, 0.1 M NaCl, 1 mM MgCl, pH 9.5, buffer.

Cell Culture

AR 4-2J cells, a rat pancreatic tumor cell line derived from a transplantable tumor of the acinar pancreas (CRL.1492, ATCC, Rockville, MD), kindly provided by Dr. F. Clemente (INSERM, Unité 151, Toulouse, France), were grown in Dulbecco's modified Eagle's medium (DMEM) as described previously (10) . In this cell line, most of the activity on 4-nitrophenyl hexanoate (>85%) can be ascribed to BSDL (10) .

Isolation of Microsomes

The AR 4-2J cells grown to confluence were washed with incomplete PBS buffer (10 mM phosphate buffer, pH 7.0, 0.15 M NaCl without Ca and Mg), harvested by scraping with a rubber policeman, and homogenized with a Polytron in a 5 mM Tris/HCl, pH 7.4, buffer (0.25 M sucrose, 0.1 mM EDTA, 2 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride). Isolation of microsomes was done by serial centrifugation as follows. First, cell debris and nuclei were pelleted by centrifugation at 600 g for 10 min. The supernatant was first centrifuged at 3000 g, then at 8000 g, and finally at 15,000 g for 10 min to precipitate zymogen granules, mitochondria, and lysosomes, respectively. The microsomes were then pelleted by centrifugation of the post-lysosomal supernatant at 100,000 g for 1 h. The pellet was then washed with the Tris/HCl/sucrose buffer and once again centrifuged under the same conditions.

Differential Solubilization and Temperature-induced Phase Separation in Triton X-114

Separation of membrane proteins from soluble proteins was done by differential solubilization and temperature-induced phase separation using Triton X-114 according to the three-phase method described by Hooper and Bashir (21) .

Inhibitor Treatment

The rat pancreatoma AR 4-2J cells (80% confluence) were used for inhibition experiments. For this purpose, the drug was added from a stock solution (monensin, 10 mM in ethanol; brefeldin A, 1 mg/ml in PBS; cytochalasin B, 5 mg/ml in PBS; vinblastin sulfate, 1 mg/ml in PBS; CCCP, 5 mM in ethanol and cycloheximide, 1 mg/ml in PBS) to dishes at the appropriate final concentration. At the required time of incubation (3 h), the cell-free medium was withdrawn and used for the analysis of BSDL and -amylase activities. When variable concentrations of monensin were used, the BSDL activity was determined in the cell-free medium and the cell membrane fraction. For this purpose, after withdrawal of the cell-free medium, the corresponding cell layer was washed with PBS, scraped with a rubber policeman and lysed by sonication in a 10 mM Tris/HCl, pH 7.4, buffer (0.1 M NaCl, 0.1% Triton X-100, 2 mM benzamidine, and 1 mM EDTA). The homogenate was then centrifuged for 1 h at 17,000 g, the supernatant was discarded, and the membrane pellet was washed twice with a 10 mM Tris/HCl, pH 7.4, buffer and resuspended in the lysis buffer. Tunicamycin treatment of cells was performed as described previously before protein labeling with [S]methionine (10) . Briefly, cells were starved for 2.5 h in the presence of tunicamycin (8 µg/ml) in DMEM without methionine. After washing twice with PBS, cells were pulsed for 1.5 h with [S]methionine (20 µCi/ml) and then chased for 30 min while still in the presence of tunicamycin. Cells were washed and lysed as above.

Pulse-chase Protocol

AR 4-2J cells were grown in methionine-free DMEM to starve the cells of this amino acid. After 45-60 min of starvation, dishes were pulse-labeled with [S]methionine (40 µCi/ml). The pulse medium was removed after 5 min followed by two quick washes with the chase medium, and cells were chased in DMEM with 10% fetal calf serum for different time intervals. The chase was stopped by aspirating the medium and adding ice-cold PBS without CaCl and MgCl. Cells were then lysed in 10 mM Hepes, pH 7.4, buffer (200 mM NaCl, 2 mM CaCl, 2.5 mM MgCl, 1.5% Triton X-100, 10 µg/ml leupeptin, 2 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride, 2 mM soybean trypsin inhibitor, and 2 mM -phenyl propionate). The cell lysates were centrifuged at 2000 g for 15 min at 4 °C to pellet nuclei and cell debris. The supernatant was used for immunoprecipitation. When indicated, chase medium was also used for immunoprecipitation. Some cells were further starved and pulsed in the presence of 5 mM azetidine-2-carboxylic acid.

Immunoprecipitation

Radiolabeled cells were lysed in 10 mM Hepes, pH 7.4, buffer in the presence of salts, detergent, and protease inhibitors as described above. Lysates were then clarified by centrifugation (2000 g, 15 min, 4 °C). One ml of either lysate or isolated microsomes was incubated overnight at 4 °C with 25 µg of antibodies to rat BSDL. Protein A-Sepharose (10 mg) pre-adsorbed with non-radioactive proteins from either lysate or microsome was added to antibody-antigen complexes and incubated for 4 h at 4 °C under agitation. At the end of the incubation, the antigen-antibody-protein A complexes were recovered by centrifugation (10,000 g, 15 min, 4 °C). When required, a two-cycle immunoprecipitation procedure slightly modified from Doolittle et al.(22) was used. The final pellet was then washed twice with the washing buffer (10 mM Tris/HCl, pH 7.4, buffer, 25 mM EDTA, and 1% Triton X-100), twice with the washing buffer supplemented with 1 M NaCl, and twice again with 10 mM Tris/HCl, pH 7.4, 5 mM EDTA buffer. The pellet was then transferred into the SDS-PAGE Laemmli's sample buffer, warmed for 2 min at 95 °C, centrifuged, and electrophoresed on SDS-PAGE. Gels were stained with Coomassie Blue R 250 and destained as above, immersed (30-60 min) in Amplify (Amersham), and autoradiographed using Hyperfilm MP (Amersham).

Endoglycosidase H Treatment

After metabolic labeling with [S]methionine, cells were lysed, clarified, and immunoprecipitated with antibodies to rat BSDL. The pellet was dissolved in 100 µl of Tris/HCl buffer (0.1 M Tris/HCl, pH 6.8, 2% SDS, and 10% glycerol), boiled for 2 min and centrifuged (10,000 g, 5 min). The supernatant was 10-fold diluted in 50 mM sodium phosphate, pH 5.6, buffer, 3% Triton X-100. Half of the sample was then treated by Endo H (40 milliunits/ml) for 10 h at 37 °C. The remaining was incubated in the absence of Endo H and used as control. Both parts were immunoprecipitated with antibodies to rat BSDL and analyzed on SDS-PAGE.


RESULTS

Effect of Monensin on Bile Salt-dependent Lipase Secretion by AR 4-2J Cell Line

We have shown recently that the rat pancreatic cell line AR 4-2J is able to synthesize and secrete the 74-kDa N-glycosylated BSDL (10) . The effect of various drugs affecting protein synthesis or transport mechanisms were therefore examined. As listed in , drugs that affect the integrity of the cytoskeleton such as cytochalasin B or vinblastin sulfate caused an approximately 40% decrease of the BSDL and -amylase activities, both detected in the cell free medium. CCCP, which is an uncoupler of the oxidative phosphorylation, had the same effect as the protein synthesis inhibitor cycloheximide. The most effective inhibitors of the secretion were the carboxyl ionophore monensin and the fungal metabolite brefeldin A, which impairs the trans-Golgi to secretory vesicles traffic and the endoplasmic reticulum to Golgi traffic, respectively. These two agents inhibited the secretion of BSDL by 80-90%; consequently, it appeared that the secretion (and transport) of the glycosylated BSDL was sensitive to energy depletion or to sodium/potassium equilibration in cells, as is the transport of -amylase. All these data suggested that the secretion pathway of the BSDL paralleled that of the -amylase and was therefore similar to that of all other secretory pancreatic enzymes (23) .

According to the above results, the effects of monensin, which is used extensively as an inhibitor of the trans-Golgi apparatus function (16) , were further examined. We first studied the effect of monensin on the time-dependent secretion of BSDL by the AR 4-2J cells. The rate of BSDL secretion was linear with time, but a dose-dependent decrease of this rate was observed in the presence of increasing concentrations of monensin. The ionophore, at a final concentration of 1 10M, totally inhibited the BSDL and the -amylase secretion in less than 3 h. Therefore, AR 4-2J cells were incubated for the appropriate time in the presence of increasing concentrations of monensin. As depicted in Fig. 1, increasing concentrations of monensin from 10 to 10M inhibited the BSDL secretion; meanwhile, the esterolytic activity on 4-nitrophenyl hexanoate increased in the cell membrane fraction. As expected, monensin induced the retention of BSDL within the cell membrane compartment. We were interested in whether the retention of BSDL was specific to a cell compartment. Electron microscopy indicated that upon menensin treatment of AR 4-2J cells (10M, 3 h), colloidal gold particles following the formation of the BSDL-antibody complexes accumulated in vacuoles likely originating from the Golgi apparatus. As these vacuoles were dispersed throughout the cytoplasm, microsomes of AR 4-2J cells were isolated from control cells and from cells after monensin treatment. Microsomes were then subjected to the temperature-induced phase separation with Triton X-114. Each phase was analyzed by SDS-PAGE and Western blot using polyclonal antibodies to rat BSDL (Fig. 2). In the absence of monensin, as judged from activity and Western blot scanning, BSDL was mainly detected in the aqueous phase. Following the monensin treatment of AR 4-2J cells, nearly 80% of the total BSDL protein mass was detected in the fractions containing membrane proteins (Triton X-114-rich phase and insoluble-detergent phase; Fig. 2). As expected, independent of monensin treatment, 80-90% of the -amylase determined from either its activity or by quantitation of Western blot was found in the aqueous phase of pancreatic microsomes. Hence, the association of BSDL with membrane microsomes was favored by monensin.


Figure 1: Effect of monensin on the secretion of the BSDL of rat pancreatic AR 4-2 J cells. Confluent AR 4-2 J cells were grown for 3 h with increasing concentrations of monensin. The BSDL activity was then determined either in cell-free medium () or in cell membrane fraction obtained after cell lysis (). Control activity was taken as the activity in cell-free medium or in cell-membrane fraction of AR 4-2J cells grown in the absence of monensin. Each value represents means ± S.E. from at least three independent experiments.




Figure 2: Triton X-114 phase separation of microsomes from AR 4-2 J cells. Microsomes of AR 4-2 J cells were isolated by centrifugation and then subjected to the temperature-induced phase separation induced by Triton X-114. Cells were previously treated for 3 h with (+ monensin) or without (- monensin) 10M monensin. 7 µg of protein from the aqueous phase (lanea), the Triton X-114-rich phase (laneb), and the detergent-insoluble phase (lanec) were separated by SDS-PAGE and electrotransferred on nitrocellulose membranes. Immunodetection was performed using polyclonal antibodies to rat BSDL. The total BSDL protein mass was determined by densitometric scanning, taking into account the volume fraction of each phase loaded onto SDS-PAGE. The BSDL activity was measured on 4-nitrophenyl hexanoate. ND, not detected. mU stands for 10 units.



The rate of synthesis and secretion of this protein was very fast as suggested by pulse-chase experiments, immunoprecipitation, and quantitation of band intensities on SDS-PAGE. After starvation (45 min) and a 5-min pulse with [S]methionine, the maximal synthesis of BSDL occurred after only a 10 min chase, while the enzyme could be immunoprecipitated from the extracellular medium after a chase of approximately 20 min (Fig. 3). The fast rate of secretion of BSDL by AR 4-2J cells gives a good explanation to the fact that in cells which were not treated with monensin, the membrane-associated BSDL cannot be detected. Because storage granules are poorly represented in AR 4-2J cells, we suggest that the rate of secretion of BSDL, which is about 30 min, corresponds to that of its synthesis, maturation, and transport. Consequently, BSDL is transiently associated with intracellular membranes and its release occurs within (or consecutive to) a cellular compartment sensitive to monensin. Because monensin affects regions of the trans-Golgi apparatus primarily associated with the final stages of secretory vesicle maturation (16) , it is suggested that BSDL reached those regions in a membrane-associated state. For the purpose of this study, AR 4-2J cells were (unless otherwise noted) grown for 3 h in the presence of 10M monensin before use. They will be referred to as monensin-treated AR 4-2J cells.


Figure 3: Time-dependent secretion of BSDL. The AR4-2J cells were grown in the absence of methionine (45 min), washed, pulsed for 5 min with [S]methionine, and chased for the time indicated. BSDL was then immunoprecipitated from the cell-free medium using antibodies to rat BSDL and analyzed on SDS-PAGE.



Glycosylation of the Membrane-associated Form of Bile Salt-dependent Lipase

The BSDL detected in the lysates of isolated microsomes from rat pancreatic monensin-treated AR 4-2J cells had a molecular mass of approximately 74 kDa (Fig. 2), compatible with that of the N-glycosylated BSDL of AR 4-2J cells (10) . Among proteins present in the Triton X-114-rich phase (Fig. 4, laneT), only one protein reacted with antibodies to rat BSDL (Fig. 4, lanea). In spite of the presence of many proteins in this phase only a small number of them reacted with lectins. Among these proteins, the membrane-associated BSDL from microsomes of monensin-treated AR 4-2J cells reacted with concanavalin A (Fig. 4, laneb, Con A) and Galanthusnivalis agglutinin (Fig. 4, lanec, GNA). These lectins recognized biantennary hybrid and high mannose type N-linked glycans, respectively, demonstrating that the membrane-associated BSDL was N-glycosylated. Nevertheless, incorporation of [H]fucose in BSDL of AR 4-2J (10) suggested that the rat enzyme, as its human homologue (8) , could also be O-glycosylated as indicated by the presence of four putative sites for O-glycosylation (24) . The presence of O-linked glycan structures was detected by means of lectin blotting using the peanut agglutinin (PNA) on the secretory rat BSDL (Fig. 4, laned) but also on the membrane-associated BSDL (Fig. 4, lanee). Prior to incubation with PNA, replicas were treated by sialidase as described by Mas et al.(8) .


Figure 4: Glycosylation of the membrane-associated rat BSDL. Rat pancreatic juice proteins were separated by SDS-PAGE and electrotransferred onto nitrocellulose membrane. Rat BSDL was localized on the replicate by immunodetection using antibodies to rat BSDL (lanea, 3 µg of loaded proteins). Proteins of the Triton X-114-rich phase from microsomes of monensin-treated (3 h, 10M) AR 4-2 J cells were separated on SDS-PAGE and transferred onto nitrocellulose, N-linked sugars were detected by lectin blotting with concanavalin A (ConA) and G. nivalis agglutinin (GNA) (lanes b and c, 7 µg of loaded proteins), and O-linked sugars were detected by lectin blotting with the PNA (lanee, 7 µg of loaded proteins). The presence of O-linked sugars on secretory rat BSDL was detected by lectin blotting as above (laned, 7 µg of loaded proteins). Silver staining of proteins of the Triton X-114-rich phase from microsomes of monensin-treated cells after SDS-PAGE was also shown (laneT, 5 µg of loaded proteins).



All these data clearly indicated that N-linked and O-linked oligosaccharide structures were present on the membrane-associated BSDL. The reactivity of PNA with the membrane-associated BSDL demonstrated the presence of Gal-GalNAc-O-Thr/Ser structures. The addition of galactose residues to oligosaccharide structures was a late event occurring in the trans-Golgi compartment (25) . The high reactivity of BSDL with lectins can be explained, first by the presence of both N- and O-linked carbohydrate structures on BSDL (8), and second because this protein was not degraded upon impairment of secretory processes (10) . Nevertheless one can note that Con A also strongly reacted with proteins of approximately 90-100 kDa while PNA recognized a 69-kDa protein. Other reactive proteins appeared after longer development times. As shown in Fig. 3, BSDL appeared outside the cell within 20 min of chase; hence, it is suggested that at this time the enzyme had passed through the Golgi apparatus where its N-linked glycosylated structure matured. Even though this fact has already been suggested by lectin blotting (Fig. 4), the sensitivity of BSDL to Endo H treatment was further determined. For this purpose BSDL was immunoprecipitated from the radiolabeled cell lysate following a 20-min chase (see Fig. 8for conditions) and treated with Endo H. As unglycosylated BSDL was still reactive with its specific antibodies (10) , the reaction product was again immunoprecipitated and analyzed by SDS-PAGE. As shown on Fig. 5, the electrophoretic mobility of BSDL was not modified following Endo H treatment. In comparison, intracellular BSDL from tunicamycin-treated cells presented a higher mobility. This indicates that within 20 min, BSDL may reach the trans-Golgi compartment, where its N-linked sugar matured, while still associated with membranes.


Figure 8: Association of BSDL to intracellular proteins in azetidine-2-carboxylic acid treated AR 4-2J cells. AR 4-2J cells grown in the absence of monensin were starved with methionine for 45 min in the presence (+) or absence (-) of 5 mM azetidine-2-carboxylic acid (Azetidine). Cells were then pulsed for 5 min with [S]methionine in the presence or absence of 5 mM azetidine-2-carboxylic acid and chased for the required time with complete DMEM. Cells were then lysed, and clarified lysates were immunoprecipitated with specific polyclonal antibodies to rat BSDL before electrophoresis on SDS-PAGE and autoradiography.




Figure 5: Endoglycosidase treatment of BSDL. AR4-2J cells were radiolabeled with [S]methionine, chased for 20 min in the absence of the radioactive amino acid, and lysed. The lysate was clarified and BSDL immunoprecipitated. The precipitated material was either treated by Endo H (40 milliunits/ml) for 10 h (+) or incubated without the glycosidase (-) and again immunoprecipitated with antibodies to rat BSDL before SDS-PAGE. For comparison, cells were preincubated in the presence of 8 µg/ml tunicamycin (Tm) in the absence of tunicamycin and radiolabeled with [S]methionine (30 min) while still in the presence (+) or absence (-) of tunicamycin. Cells were then washed twice and lysed. Cell lysates were then immunoprecipitated with antibodies to rat BSDL and submitted to SDS-PAGE.



Activity of the Membrane-associated Form of the Bile Salt-dependent Lipase

As shown on Fig. 1, the impairment of cell transport by monensin favored the association of BSDL with membranes. However, an increase in membrane-associated BSDL activity was not directly correlated with a decrease in secreted activity. These data suggested that membrane-associated BSDL was, at least, poorly active. We, therefore, attempted to determine whether membrane-associated BSDL was enzymatically active. For this purpose microsomes of monensin-treated AR 4-2J cells were subjected to temperature-induced phase separation with Triton X-114. Enzyme activity present in each phase was determined and corrected for their respective volume (see Fig. 2). Approximately 90% of the activity on 4-nitrophenyl hexanoate was detected in the soluble phase, in spite of the fact that only a small amount of BSDL protein (<5%) was associated with this phase. In contrast, the largest amount of BSDL protein was found in the Triton X-114-rich phase, where a very weak enzymatic activity on 4-nitrophenyl hexanoate was detected. In microsomes isolated from control cells, BSDL activity was exclusively detected in the soluble phase, in which we also detected all the BSDL protein. Furthermore, we observed that Triton X-114 did not affect BSDL activity, indicating that membrane-associated BSDL was poorly active. This could be the result of a tertiary structure, different from that of the soluble protein which displays full activity. In all probability, the impairment of the trans-Golgi apparatus function by monensin (16) interrupted the folding process of BSDL. Consequently, only incompletely folded forms of BSDL might be associated with intracellular membranes. We therefore attempted to gain information about the folding state of the membrane-associated BSDL by pulse-chase experiments. AR 4-2J cells were pulsed for 1-5 min and then chased for 2 min. Cell lysates were immunoprecipitated and analyzed on SDS-PAGE under reducing and non-reducing conditions. The electrophoretic mobility of the membrane-associated BSDL was not modified on SDS-PAGE under reducing or non-reducing running conditions. This means that the two disulfide bonds present on BSDL formed loops that were too small (7) to influence protein mobility once reduced. This point was supported by the fact that the electrophoretic mobility of the soluble form of BSDL was also not influenced by the SDS-PAGE running conditions.

Identification of Proteins That Bind to BSDL

Since the association of BSDL with microsome membranes involved neither a glycosyl phosphatidylinositol tail (54) nor a Golgi or ER retention signal (26, 27) , but appeared sensitive to KBr treatment, we sought to determine whether BSDL was associated with microsome membranes by means of membrane proteins. For this purpose, monensin-treated AR 4-2J cells were grown in the presence of [S]methionine and lysed in 5 mM MES, pH 6.0, buffer containing 0.5% Nonidet P-40, 0.1 M NaCl, and 1 mM EDTA. The cell lysates were clarified by centrifugation at 2000 g for 10 min and then chromatographed on an affinity column made of BSDL immobilized on Sepharose gel to isolate proteins that bind to BSDL. After elution of unbound fraction with the lysis buffer and washing of the column with the same buffer at pH 5.0, bound proteins were eluted at pH 8.0 (0.1 M sodium phosphate, 0.15 M NaCl, and 0.5% Nonidet P-40). The eluted [S]methionine-labeled proteins were electrophoresed on SDS-PAGE and autoradiographed. As shown in Fig. 6 (lanea), two bands at approximately 90-120 kDa and 74 kDa were detected. A third band at 56 kDa, although less intense, was also present. Control experiments performed on a column of Sepharose without immobilized BSDL indicated that no protein was nonspecifically absorbed by the gel. When microsomes, isolated from monensin-treated AR 4-2J cells, were chromatographed on the immobilized BSDL column two signal bands were detected on SDS-PAGE (Fig. 6, laneb). They corresponded to proteins of approximately 94 kDa and 70-74 kDa. Diffuse bands were also detected at 56 kDa and around 40-50 kDa. Because antibodies to rat BSDL reacted with the 70-74-kDa band in Western blotting experiment (Fig. 6, lanec), other bands detected on the autoradiogram were thought to be representative of proteins that specifically bound to BSDL. Therefore, BSDL present in microsomes was associated with at least two proteins of 94 kDa (p94) and 56 kDa (p56), respectively. A third protein at <50 kDa may also be suspected to bind to BSDL. To determine whether these proteins were all together associated with membranes or to the lumen content of microsomes, the soluble phase obtained after temperature-induced phase separation with Triton X-114 was chromatographed on the immobilized BSDL column as described above. No radioactive material was eluted from this column. Nevertheless, fractions eluted at pH 8.0 were collected, pooled, and concentrated before electrophoresis on SDS-PAGE and autoradiography; as expected, no signal was detected on the film. To confirm this result, radioactive material present in the Triton X-114-rich phase obtained after temperature-induced phase separation with Triton X-114 was immunoprecipitated with antibodies to rat BSDL and electrophoresed on SDS-PAGE. As shown on Fig. 6(laned), p94 and p56 as well as BSDL were found in this phase-containing membrane proteins (21) . Taken together, these data suggested that the multimeric complex might be effectively associated with membranes of microsomes. The band intensities, assuming the same methionine/cysteine contents for each protein, suggested that p94 and BSDL associate stoichiometrically while 1 or 2 p56 molecules could be involved in a putative complex.


Figure 6: Identification of proteins that bind to BSDL. [S]Methionine-labeled AR 4-2 J cell lysate or microsomes from monensin-treated AR 4-2J cells were chromatographed on an immobilized BSDL column. The column was washed with 5 mM MES, pH 5.0, buffer with 0.5% Nonidet P-40, 0.1 M NaCl, and 1 mM EDTA to elute unbound material. Bound proteins were then eluted with 0.1 M sodium phosphate, pH 8.0, buffer (0.15 NaCl and 0.5% Nonidet P-40). Eluted proteins were concentrated and electrophoresed on SDS-PAGE before autoradiography. Figure shows an autoradiogram of the material bound to the immobilized BSDL column starting from AR 4-2J cell lysate (lanea) or from AR 4-2J cell microsomes (laneb). Material eluted from the immobilized BSDL column starting from microsomes was electrophoresed on SDS-PAGE, transferred to a nitrocellulose membrane, and probed with antibodies to rat BSDL (lanec). The Triton X-114-rich phase obtained after temperature-induced phase separation with Triton X-114 of [S]methionine-labeled microsomes was immunoprecipitated with antibodies to rat BSDL. The precipitated material was then electrophoresed on SDS-PAGE and autoradiographed (lane d).



Since the co-isolation of p94 and p56 with BSDL implies their physical association as a complex, we sought to confirm this by taking advantage of the affinity of BSDL for heparin (28, 29) . For this purpose, radioactive material eluted from the immobilized BSDL column was loaded onto a heparin-Sepharose column equilibrated in a 10 mM Tris/HCl, pH 7.4, buffer. As shown on Fig. 7A, two radioactive peaks were eluted with the Tris/HCl buffer containing 0.4 M NaCl and 1 NaCl, respectively. Most of the radioactive material (70%) applied to the column was eluted with 0.4 M NaCl. Nearly 80% of the activity on 4-nitrophenyl hexanoate of the starting material was also detected in this fraction. The remaining radioactive material and BSDL activity was associated with the material eluted with 1 M NaCl and in the washing buffer. Radioactive material eluted in each fraction was electrophoresed on SDS-PAGE and autoradiographed. As shown on Fig. 7B (laneb), material eluted at 0.4 M NaCl included p94, BSDL, and p56, while the material with the lower mass (<50 kDa) became almost undetectable. No signal was obtained after analysis of the material eluted at 1 M NaCl (Fig. 7B, lane c) or eluted in the washing fractions (Fig. 7B, lanea). These data suggested that in microsomes at least two proteins, p94 and p56, formed a membrane multimeric complex that associates with BSDL.


Figure 7: Analysis of proteins that bind to BSDL on heparin-Sepharose column. A, radioactive material eluted at basic pH from the immobilized BSDL column was chromatographed on a heparin-Sepharose column (1 ml) eluted with 10 mM Tris/HCl buffer, pH 7.4. Bound material was eluted with the same buffer containing 0.4 M NaCl and 1 M NaCl. Radioactivity () and activity on 4-nitrophenyl hexanoate () were determined in each eluted fraction. B, SDS-PAGE and autoradiography of fractions eluted from the heparin-Sepharose column. Lanea, unbound fraction; laneb, fraction eluted with 0.4 M NaCl; lanec, fraction eluted with 1 M NaCl.



Preventing Folding of BSDL Induced the Formation of a Multimeric Complex

We tested whether the formation of the membrane multimeric complex involving BSDL, p94, and p56 or possibly lower molecular mass proteins was related to the folding state of BSDL, as suspected from the activity deficiency of the membrane-associated enzyme. For this experiment, monensin treatment was omitted and AR 4-2J cells were grown in methionine-free DMEM in the presence of the proline analogue, azetidine-2-carboxylic acid, which prevents folding of nascent proteins (30) . Under these conditions, misfolding of BSDL should be drastic, as proline represents about 7% of the amino acid residues of the protein (40/592). In addition, at the level of the O-glycosylated C-terminal tandem repeated sequences, 1 amino acid residue out of 3 is a proline (24, 29) . After starvation (45 min) in methionine-free DMEM in the presence of azetidine-2-carboxylic acid (5 mM), cells were pulsed for 5 min with [S]methionine while still in the presence of azetidine-2-carboxylic acid and chased with DMEM in the absence of the proline analogue. Cell lysates were subjected to immunoprecipitation with antibodies to rat BSDL before analysis on SDS-PAGE. As shown on Fig. 8, in cells experiencing the metabolic stress of azetidine-2-carboxylic acid, proteins of 94, 56, and 46 kDa were co-precipitated with BSDL. As expected, neither BSDL activity nor BSDL immunoreactive protein could be detected in the cell-free medium of AR 4-2J cells incubated in the presence of the proline analogue. However, when cells were grown in the absence of the proline analogue, only traces of p94 and p46 were co-precipitated with BSDL, while the enzyme was normally secreted. Therefore, misfolded BSDL interacts with proteins p94, p56, and p46 involved in the multiprotein complex, which appears to function as a folding complex. It is likely that this folding complex formed within the ER and continued up to the Golgi apparatus.

Ligand Blotting of Proteins in AR 4-2J Cells

We next attempted to determine whether BSDL could bind directly to 94-, 56-, or 46-kDa proteins to form a well ordered complex. For this purpose the biotin-labeled BSDL was used in ligand blotting to detect proteins able to bind BSDL. The rat monensin-treated AR 4-2J cells were lysed as described previously. After centrifugation (2000 g, 15 min), proteins were separated on SDS-PAGE and electrotransferred onto a nitrocellulose membrane. The electrophoretic migration of the BSDL was first observed by Western blotting using the polyclonal antibodies to the rat enzyme (Fig. 9, lanea). The enzyme electrophoresed as a doublet at 70 and 74 kDa, probably representing unglycosylated and glycosylated BSDL, respectively. When nitrocellulose membranes were incubated with biotin-labeled BSDL, two bands were immunodetected by anti-biotin antibodies conjugated to alkaline phosphatase (Fig. 9, laneb). The first one at 70-74 kDa corresponded to the self-aggregation of BSDL molecules (32) , and the second one, at approximately 95 kDa correlated well with the previously detected p94 protein that binds to the immobilized BSDL. Similar data were obtained when isolated microsomes were analyzed (Fig. 9, lanee). These two bands were not detected in the control experiment in which biotin-labeled BSDL was omitted (Fig. 9, laned). A heparin-like binding site was detected within the sequence of BSDL (24, 29) . This site, probably involved in the binding of the enzyme to the intestinal brush-border (32) , may be essential to the association of BSDL with microsome membranes. To test this possibility, we performed a ligand blotting in the presence of saturating amount of heparin (2 mg/ml). As shown on Fig. 9(lanec), heparin did not hinder the self-association of BSDL and its binding to the 95-kDa protein. This last feature means that the interaction between the 95-kDa protein and BSDL does not involve the heparin-binding site present on the enzyme.


Figure 9: Western blotting and ligand blotting. Clarified lysates of monensin-treated AR 4-2J cell were electrophoresed on SDS-PAGE and electrotransferred to nitrocellulose membrane (40 µg of cell proteins). Lanea, the membrane was incubated with antibodies to rat BSDL and the immunodetection performed with anti-IgG antibodies labeled with alkalin phosphatase; lanes b-d, the membrane was incubated with biotin-labeled BSDL and the binding reaction revealed with anti-biotin antibodies labeled with alkaline phosphatase (laneb). Incubation was performed in the presence of heparin (2 mg/ml, lanec), or the biotin-labeled BSDL was omitted (laned). Lanee, proteins from AR 4-2 J cells microsomes (10 µg) were separated on SDS-PAGE before transfer and affinity blotting performed as in lane b.



Characterization of the p94 as a Grp 94-related Protein

The presence of chaperone proteins along the secretion pathway of acinar pancreatic cells has been described recently (4) . Because of the size homology between the p94 and the 94-kDa glucose-regulated protein (Grp 94) and because the Grp 94 was frequently associated with proteins around 40-50 kDa (2) as found here, we investigated the possible participation of this former chaperone protein in the secretory process of BSDL. The Grp 94 chaperone was detected in monensin-treated AR 4-2J cell lysates by Western blot using a specific monoclonal antibody (clone 9G10). Since this chaperone protein may be involved in the multimeric complex and might assist the folding of BSDL, we first attempted to detect Grp 94 in the material isolated from the immobilized BSDL column. As shown above, when microsomes isolated from monensin-treated AR 4-2J cells were chromatographed on the immobilized BSDL column, a multimeric complex was isolated (see Fig. 6, laneb or Fig. 10A, lanea). In this complex, we demonstrated that the 70-74-kDa protein was the BSDL as this material reacted with antibodies to rat BSDL (see Fig. 6, lanec). We then, used monoclonal antibody to Grp 94 to determine whether this chaperone protein could be detected in the multimeric complex involving BSDL. As shown in Fig. 10A (laneb), the antibody specific for Grp 94 actually recognized the 94-kDa band. The antibody to Grp 94 was therefore used to immunoprecipitate the radioactive material eluted from the column of immobilized BSDL and concentrated by lyophilization. After SDS-PAGE and autoradiography, immunoprecipitated proteins resolved into proteins at 94 and 74 kDa (Fig. 10A, lanec). The two other bands detected at 56 and 46 kDa were also co-precipitated (Fig. 10A, lanea). Presumably, these two bands corresponded to those already detected (see Fig. 8 ). These results suggested that the Grp 94 was retained by the immobilized BSDL column and was actually involved in the multimeric complex that associates p56, p46, and BSDL. In another set of experiments, monensin-treated AR 4-2J cells were labeled with [S]methionine and lysed. The cell lysate was clarified by centrifugation and immunoprecipitated with antibody to Grp 94. Analysis of the immunoprecipitated material on SDS-PAGE indicated that essentially three proteins of 120, 70/74, and 58 kDa co-precipitated with the Grp 94 (Fig. 10B, lanea, asterisk), one of which was suspected to be BSDL (arrow). A second immunoprecipitation was therefore performed on this material after dissociation of complexes (22) using either monoclonal antibody specific for Grp 94 or polyclonal antibodies to rat BSDL. As shown on Fig. 10B, the first antibody immunoprecipitated two proteins of 95 and 87 kDa (laneb), while antibodies to rat BSDL precipitated only one protein, the size of which correlated with BSDL (lanec). The 87-kDa protein could be the unprocessed Grp 94 (33) . When the two-cycle immunoprecipitation procedure was performed as above, first with antibodies to rat BSDL and subsequently with either antibodies to rat BSDL or Grp 94, the same pattern was obtained. As shown on Fig. 10C, the first immunoprecipitation (lanea) indicates that BSDL was effectively precipitated (arrow) and that a protein at 94 kDa (asterisk) is co-precipitated along with proteins at 50/56 and 43 kDa. Subsequent precipitation with either antibodies to rat BSDL (laneb) or Grp 94 (lanec) detected the respective protein. Therefore, we can conclude that the p94 might be related to the Grp 94. The association between the Grp 94-related protein and BSDL was indicated first by the co-precipitation of the two proteins by specific antibodies directed against each individual species, and second by their co-isolation by chromatography on immobilized BSDL column (see Fig. 6, lanec and Fig. 10A, laneb).


Figure 10: Characterization of p94 as a Grp 94-related protein. A, detection of the Grp 94 chaperone protein in the multimeric complex that binds to the immobilized BSDL column. [S]Methionine-labeled proteins from microsomes of monensin-treated AR 4-2J cells were chromatographed on the immobilized BSDL column. Material eluted at basic pH was concentrated and submitted to SDS-PAGE (lanea) or electrotransferred to nitrocellulose membrane and probed with the monoclonal antibody to Grp 94 (laneb). The material eluted at basic pH was also concentrated and immunoprecipitated using the monoclonal antibody to Grp 94 (lanec). B and C, two-cycle immunoprecipitation. Two-cycle immunoprecipitation was performed on cell lysate of monensin-treated AR 4-2J cells after metabolic labeling with [S]methionine. The first cycle was performed using either monoclonal antibody to Grp 94 (B, lanea) or antibodies to rat BSDL (C, lane a), followed by a second immunoprecipitation with monoclonal antibody to Grp 94 (B, laneb; C, lanec) or polyclonal antibodies to rat BSDL (B, lanec; C, laneb).




DISCUSSION

The central dogma of the synthesis and secretion of pancreatic protein established by Palade and co-workers (23) stipulated that at the end of the translocation process, the signal peptide is cleaved by endogenous signal-peptidase, thus releasing proteins into the soluble cisternal content. Comparative examination of the intracellular transport of many secreted proteins showed that the transport from the endoplasmic reticulum to the Golgi apparatus is selective and can be the rate-limiting step in the secretion process (34, 35) . This selectivity may be associated to different rates of transport between the endoplasmic reticulum and Golgi apparatus for different secreted proteins within cells (36) . This led to the hypothesis that secretory proteins express specific transport signals within their own structure (34, 35). This transport may also be receptor-mediated (34, 35, 37) .

In its fundamental attributes, the secretory route of BSDL was similar to that of other pancreatic secretory proteins. As -amylase, the secretion of BSDL was sensitive to the impairment of the Golgi apparatus by monensin and brefeldin A. Cytoskeleton-disrupting agents (cytochalasin B and vinblastin sulfate) had a poor effect on the secretion (at least at the concentration used here). CCCP, a respiratory inhibitor, blocked the secretion of both -amylase and BSDL, probably by inhibiting the fusion of secretory vesicules with target membranes, an event that is energy-dependent (38) . As shown by temperature-induced phase separation with Triton X-114, the impairment of the secretion pathway by the ionophore monensin induced the association of BSDL to microsome membranes. This was in contrast to the behavior of -amylase which, even after cell treatment by monensin, remained associated with the soluble fraction of microsomes. The association of the former enzyme did not involve signal sequence (KDEL sequence or other) or anchoring by means of lipids (acylation or glycosyl phosphatidylinositol tail) but by a membrane multiprotein complex. This multiprotein complex implicated at least three proteins, p94, p56, and p46, of which p94 binds directly to BSDL. The association of BSDL with microsome membranes upon monensin treatment might therefore be due to a direct interaction of the enzyme with the 94-kDa protein. The membrane-associated BSDL was enzymatically inactive or poorly active in contrast to its soluble form. This provides evidence that the membrane-associated BSDL might not be correctly folded, either because monensin interrupted the secretory process and consequently stopped the folding process or because this drug acted directly on the folding process by affecting, for example, the maturation of the BSDL glycosylation (39) . In the absence of monensin, the multiprotein complex cannot be co-immunoprecipitated with BSDL which therefore appeared transiently associated to p94. In conditions of metabolic stress, such as protein synthesis in the presence of the proline analogue azetidine-2-carboxylic acid, the association appeared permanent. A model consistent with all these findings suggests that the multimeric complex, more precisely the association with p94, may facilitate the proper folding of BSDL.

We have recently demonstrated that the N-linked glycosylation of BSDL was essential for the correct folding and secretion of the enzyme, nevertheless the trimming and maturation of the N-linked glycosyl structure did not influence the enzyme secretion (10) . Therefore, possible alterations of the N-linked glycosylation processes by monensin that acted late in the processing continuum (39) cannot be advocated as a retention mechanism (10, 12) . The size of the membrane-associated BSDL correlated with that of the glycosylated protein. This was confirmed by lectin reactivity (8, 40) and by the lack of effect of the Endo H on the BSDL electrophoretic mobility, which agreed with the presence of matured N-linked glycans. The incorporation of fucose residues in BSDL (10) and the reactivity of the membrane-associated protein with the peanut agglutinin after sialidase action agreed with the presence of Gal-GalNAc-O-Thr/Ser O-linked structures, possibly fucosylated and sialylated. This suggested that the glycosylation of BSDL was processed while the protein is associated with membranes. Since terminal glycosyltransferases such as galactosyltransferases are localized at the trans-cisternae and trans-Golgi network (25, 41) , we believe that BSDL remains associated with membranes while transported from the endoplasmic reticulum to the trans-Golgi apparatus. The release of the enzyme might take place in a cell compartment posterior to the trans-Golgi or within it. This agrees with the effect of monensin, which is described as an inhibitor of trans-Golgi functions and blocked the normal formation of secretory vesicles (16) . Impairment of the secretory process in cancer pancreatic cells may, therefore, be responsible for the association of the oncofetal form of BSDL with microsome membranes (14) .

We further showed that p94 was probably related to Grp 94. Grp 94 is a resident chaperone of the endoplasmic reticulum (42) , which associates with unassembled immunoglobulin (43) , unassembled histocompatibility complex class II polypeptides (44) , and mutant of herpesvirus glycoproteins (45) . In all these cases, additional proteins were found, although their nature and role is still unknown (36, 43, 45) . Grp 94 is a glycoprotein that belongs to the Hsp 90 family implicated in the transport of important biological molecules within the cell (46) . Grp 94 has the KDEL C-terminal sequence for retention of proteins in the lumen of the endoplasmic reticulum (33) . Given the fact that chaperones are identified merely by their molecular weight on SDS-PAGE, it is possible that Grp 94-related p94 may be the endoplasmic resident Grp 94, or an apparently identical protein associated with the Golgi apparatus (2) . Interestingly, the size of both p56 and p46 correlated with those of -galactoside 1,2-fucosyltransferase and -galactoside 2,6-sialyltransferase, respectively, two transferases most probably involved in the terminal glycosylation of BSDL (15) . The protein disulfide isomerase (molecular mass 56 kDa), the expression of which is induced by the calcium ionophore A 23187 (47), may also be involved in the folding complex of BSDL. Unfortunately, precipitating antibodies to these proteins are not available to test this hypothesis.

A model compatible with all the data presented here could be as follows. The N-terminal sequence of the protein, where the two disulfide bridges of BSDL (7) are located, folds up either with the help of an undetected chaperone or spontaneously around the N-linked glycosylation as suggested by recent data: (i) the maturation of the N-linked structure did not affect the BSDL secretion; (ii) N-glycosylation of BSDL was essential for the expression of a fully active enzyme (10) . Additionally, the functionality of the heparin-binding site (see Fig. 7A) located between residues 61-66 (29, 32) argues for the ``nearly correct'' folding of the N-terminal sequence of the enzyme. This mechanism might be common to many secretory pancreatic enzymes such as -amylase. Therefore, the particular secretion pathway of BSDL, involving the association with the Grp 94-related p94, may be due to specific structural properties of this protein. Although multiple reasons may be advocated, the most probable one would lie in the fact that BSDL is the unique secretory pancreatic protein with mucin-like tandem-repeated sequences on which O-linked oligosaccharides are clustered. This cluster encoded by exon 11 (48) is located on the extended C-terminal domains rich in serine, threonine, and proline residues (7) . When this domain was deleted by recombinant DNA techniques, the truncated BSDL was normally expressed and displayed full activity (49, 50) . In contrast, transfection of BSDL cDNA with 88% of the exon 11 residues deleted from the sequence resulted in a protein that was not secreted (49) . These data suggested that a domain encoded by exon 11 of BSDL is required for intracellular processing and secretion of the normal enzyme (49) . This domain, between residues 490-534 (49) , may be responsible for the interaction between p94 and BSDL. We can therefore suggest that the association of BSDL with the Grp 94-related p94 protein is essential for the processing (i.e. glycosylation) of the C-terminal tandem-repeated sequences. Proteins that are rapidly degraded within eukaryotic cells contain regions rich in proline, glutamic acid, serine, and threonine (PEST region) associated with abundant doublets of basic amino acids (51). Many of these doublets are present within the sequence of BSDL, and the PEST regions coexist with the cluster of O-linked oligosaccharides (24, 52) . A survey of the literature demonstrated that these sequence features are absent in sequences of other enzymes of the pancreatic secretion. Of significant importance is that no intracellular degradation of BSDL occurred in pancreatic cells upon treatment with drugs that inhibits the enzyme secretion (10) . Hence, association with membranes by means of the Grp 94-related p94 chaperone protein and consequently glycosylation of PEST sequences may contribute to take the pancreatic BSDL away from a possible degradation route and to its secretion by acinar pancreatic cells. Once fully glycosylated, the enzyme is released from the Grp 94-related p94 either in or after the trans-Golgi compartment and aggregated in the trans-Golgi network (31) . The releasing mechanism is still unknown, but proteoglycans involved in the condensing process of secretory proteins may play a role (53) . Whenever the PEST sequences and the domain 490-534 are deleted or absent, BSDL enters the pathway shared by all pancreatic secreted proteins (23) .

Therefore and for the first time, it is suggested that a multimeric complex including the chaperone Grp 94-related p94 protein may play an essential role in the secretory process of BSDL, first by accompanying BSDL from its site of synthesis to its site of release, and second by keeping this protein in close proximity to membrane glycosyltransferases and presenting the right conformation of the peptide core of BSDL to these transferases. Finally, the association with p94 may prevent premature aggregation of BSDL molecules (31) .

  
Table: Effect of drugs on the secretion of BSDL and -amylase by AR 4-2J cells

AR 4-2J cells were incubated (3 h) in the presence of each inhibitor at the appropriate final concentration, then cell-free medium was withdrawn and used for the determination of bile salt-dependent lipase (BSDL) and -amylase activity. Values are means ± S.E. of at least six independent experiments.



FOOTNOTES

*
This work was supported by grants from the Association pour la Recherche sur le Cancer (ARC, Villejuif, France) and the Conseil Général des Bouches-du-Rhône (Marseille, France). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a fellowship from the ARC.

To whom correspondence should be addressed: INSERM U-260, Faculté de Médecine Timone, 27 Blvd. Jean Moulin, 13385 Marseille Cedex 05, France. Tel.: 33-91-83-44-02; Fax: 33-91-83-01-87.

Abbreviations used: BSDL, bile salt-dependent lipase (EC 3.1.1.-); Grp, glucose-regulated protein; Hsp, heat shock protein; Endo H, endoglycosidase H (EC 3.2.1.96).

N. Bruneau, P. Lechne, V. Sbarra, and D. Lombardo, unpublished results.

N. Bruneau and D. Lombardo, unpublished results.


ACKNOWLEDGEMENTS

We are indebted to V. Sbarra for invaluable technical assistance, to V. Mariottini for careful typing of the manuscript, and to B. Khalil and C. Crotte for the artwork. We acknowledge Dr. B. Lerique for autoradiogram quantitation and Dr. F. Clemente (INSERM Unité 151, Toulouse, France) for providing us with the AR 4-2J cell line. We thank Drs. P. Lechne de la Porte and S. Mathieu for performing microscopy experiments and for fruitful discussions. We are also indebted to Dr. Franck Haigler III for careful reading of the manuscript and for suggestions.


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