ARTICLE |
Correspondence to: Moïse Bendayan, Département de Pathologie et Biologie Cellulaire, Faculté de Médecine, Univ. de Montréal, CP 6128, Succ Centre Ville, Montréal, Quebec, Canada H3C 3J7.
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Summary |
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In previous studies, we have shown that the bile salt-dependent lipase (BSDL) associates with the Grp94 molecular chaperone, an association that appears to play essential roles in the folding of BSDL. More recently, combined biochemical and immunocytochemical investigations were carried out to show that the transport of BSDL occurs via an association with the Grp94 all along the pancreatic secretory route (ERGolgigranules). The Grp94BSDL complex is secreted with the pancreatic juice into the acinar lumen and reaches the duodenal lumen, where it is internalized by enterocytes. The dissociation of the complex could take place within the endosomal compartment because BSDL continues further on its way to the basolateral membrane of the enterocyte. To localize the affinity binding sites of pancreatic BSDL in pancreatic and duodenal tissues, we have used an affinitygold ultrastructural technique. BSDL coupled to gold particles appears to interact with specific sites in tissue sections. This was confirmed by another indirect morphological approach using biotin-labeled BSDL and streptavidingold complexes on tissue sections. We have shown that BSDL associates with sites in the pancreatic secretory pathway compartments and in the microvilli, the endosomal compartment, and the basolateral membrane of enterocytes. By biochemical approaches, biotin-labeled BSDL displayed affinities with proteins of 180190 kD in both pancreatic and duodenal tissues. We have also shown that the Grp94BSDL complexes, which are insensitive to denaturing conditions, are present in pancreatic homogenate but not in duodenal lysate. Thus, BSDL is able to bind protein complexes formed by either BSDLGrp94 or Grp94 dimers. (J Histochem Cytochem 48:267276, 2000)
Key Words: bile salt-dependent lipase, Grp94, pancreas, intestine, affinitygold technique
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Introduction |
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THE BILE SALT-DEPENDENT LIPASE (BSDL, EC.3.1.1.13), also called cholesterol esterase or carboxyl ester lipase, is a lipolytic enzyme, that is biosynthesized and secreted by the pancreas. BSDL, in concert with other pancreatic lipolytic enzymes and gastric lipase, may act to complete digestion of dietary lipids. On activation by primary bile salts in the duodenal lumen, this enzyme catalyzes the hydrolysis of cholesterol esters into free cholesterol and fatty acids (
In previous studies on the pancreas-derived AR4-2J cell line, we have shown that the secretion of BSDL involves a multiprotein complex including the Grp94 chaperone (
High-resolution cytochemical labeling techniques are available to cell biologists for subcellular localization of most types of biological macromolecules, from antigenic sites to carbohydrate residues, nucleic acids, and other substrate molecules, through their interaction with antibodies, lectins, and/or specific enzymes (
Application of a proteingold complex for labeling tissue sections consists of a direct one-step postembedding technique. The protein coupled to gold particles interacts with its specific binding site exposed at the surface of the tissue section. Colloidal gold is the electron-dense marker of choice, allowing detection at high resolution (
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Materials and Methods |
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Antibodies
Affinity-purified rabbit polyclonal antibodies against rat pancreatic BSDL (
Preparation of the BSDLGold Complex
Colloidal gold particles (10-nm) were prepared using a protocol described by
BSDL Biotinylation
Human BSDL was isolated from pancreatic juice as already described (
Tissue Processing
Rat pancreatic and duodenal tissues were fixed by immersion with 1% (v/v) glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4, for 2 hr at 4C. The tissue samples were washed in the phosphate buffer, dehydrated by a series of graded ethanol solutions, and embedded in Unicryl or Lowicryl (British Biocell; Cardiff, UK) at -30C as described previously (
Cytochemistry
Labeling Protocol.
The rat pancreatic and duodenal tissue sections were incubated by floating them on drops of PBS, pH 5.6, and were transferred successively to 1% ovalbumin solution for 30 min and to BSDLgold complex for 30 min at room temperature (RT). Sections were then thoroughly washed with PBS and distilled water, dried, and stained with uranyl acetate. The grids were examined with a Philips 410 electron microscope. A similar protocol was carried out with biotin-labeled BSDL. Tissue sections were incubated on drops of PBS and transferred successively to 1% ovalbumin solution for 30 min and to biotin-labeled BSDL (10 µg/µl) for 30 min at RT. Sections were then washed with PBS. Free binding sites were blocked again by an incubation with the ovalbumin solution for 15 min and sections were finally incubated with the streptavidingold complex (Sigma; St Louis, MO) diluted 1:15 with PBS for 30 min. Thin sections were then extensively washed with PBS and distilled water and dried. They were stained with uranyl acetate and examined.
Controls for Specificity. The specificity of the labelings was assessed through competition experiments. Incubations of tissue sections were carried out with a solution of free BSDL (1 µg/µl) for 1 hr at RT before incubation with the BSDLgold complex. Sections were also incubated with the streptavidingold complex omitting the biotinylated BSDL step. In addition, labelings were carried out in the presence of 1% albumin or 1% gelatin solution before incubation with gold- or biotin-labeled BSDL. In this case, the preincubation or the presence of another protein does not prevent labeling.
Quantitative Evaluations.
The labeling intensities were obtained as described previously (
Polyacrylamide Gel Electrophoresis and Western Blotting
Gel electrophoreses (SDS-PAGE) were performed on slab gels of polyacrylamide (7.5%) and sodium dodecyl sulfate (1.5%) under reducing conditions according to
Two-dimensional Gel Electrophoresis
Electrophoresis of pancreatic juice proteins was performed as described by
Ligand Affinity Blotting
Proteins that bind BSDL were detected by ligand affinity blotting on membranes obtained after SDS-PAGE and electrotransfer. Replicas were first blocked overnight with 0.1 M sodium phosphate buffer, pH 6.0 (3% bovine serum albumin, 50 mM NaCl) and incubated with biotin-labeled BSDL (5 µg/ml, 2 hr, 4C). After exhaustive washing (six times) with 0.1 M phosphate buffer (0.3% bovine serum albumin, 0.05% Tween-20), membranes were incubated (1 hr, RT) in 0.1 M sodium phosphate buffer, pH 7.4 (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-indoyl phosphate and 0.5 mM nitroblue tetrazolium in 0.1 M Tris-HCl, 0.1 M NaCl, 1 mM MgCl2 buffer, pH 9.5.
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Results |
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To morphologically determine the affinity binding sites of BSDL in rat pancreatic and duodenal tissues, we have applied a cytochemical approach for the ultrastructural localization of macromolecules. This technique is based on the affinity properties existing between purified proteins and putative binding sites on tissue sections. For this purpose, BSDL was coupled with colloidal gold particles and tissue sections were incubated with the BSDLgold complex to reveal BSDL binding sites.
In pancreatic acinar cells, binding sites for BSDLgold complexes were detected in the rough endoplasmic reticulum (RER), the Golgi apparatus, the zymogen granules, and the flocculent material present in the pancreatic acinar and duct lumen (Figure 1). In control experiments in which the tissue sections were incubated with unlabeled BSDL before incubation with the BSDLgold complexes, no interaction between BSDLgold and tissue binding sites was detected. This illustrates the specificity of the recognition of BSDLgold complexes for binding sites in the different compartments of the secretory pathway of the pancreatic acinar cell.
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In intestinal cells, labeling with BSDLgold complexes was restricted to the microvilli, the endosomal compartment, and the basolateral membrane of the enterocytes (Figure 1), suggesting that BSDL may have affinity binding sites in these compartments. Again, in this case, the preincubation of tissue sections with unlabeled BSDL abolished the reactivity of the BSDLgold complexes. These data indicate that BSDLgold complexes are interacting with sites on the enterocyte apical plasma membrane and associated with subcellular compartments of the transcytotic pathway.
To confirm these results, we applied a different cytochemical approach, making use of the streptavidinbiotin system. For this indirect procedure, BSDL was coupled to biotin and used for incubation of tissue sections. Affinity binding sites were detected by the streptavidingold complex. As shown in Figure 2, BSDLbiotin complexes were revealed over RER, Golgi, and secretory granules of pancreatic acinar cells (Figure 2A) and over microvilli, endosomal compartment, and basolateral membranes of the enterocyte (Figure 2B). Control experiments in which the tissue sections were incubated only with the streptavidingold complex (Figure 2C) showed no reactivity.
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Quantitative evaluations were performed and are shown in Table 1 and Table 2. They support the subjective observations showing that BSDL interacts with specific affinity binding sites in both tissues.
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We next attempted to determine whether BSDL could bind directly to one or several isolated proteins from pancreatic and duodenal tissues. For this purpose, the biotin-labeled BSDL was used in ligand affinity blotting experiments to detect proteins capable of binding BSDL. Rat pancreatic and duodenal tissues were lysed in 10 mM Hepes buffer containing 0.2 M NaCl, 1.5% Triton X-100, 2 mM CaCl2, 2 mM MgCl2, and 2 mM benzamidine. After centrifugation (2000 x g, 10 min) proteins were separated on SDS-PAGE and electrotransferred onto nitrocellulose membranes. When replicas were incubated with biotin-labeled BSDL, a doublet was detected by anti-biotin antibodies conjugated to alkaline phosphatase (Figure 3A). These bands display an electrophoretic migration corresponding to approximately 180190 kD in both pancreatic and duodenal tissues. In control experiments in which biotin-labeled BSDL was omitted (Figure 3B), no band at this Mr was detected. These data suggest that biotin-labeled BSDL binds to proteins that have a migration corresponding to 180190 kD in pancreatic and intestinal tissues.
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We then wished to collect information on these proteins and to determine whether BSDL and Grp94 are present in these complexes of high molecular mass. For this purpose, we carried out Western blots on pancreatic and duodenal proteins after SDS-PAGE separation and electrotransfer. Replicas were then probed with antibodies specific for BSDL and Grp94, respectively. In pancreatic and duodenal tissues, BSDL was detected as one major band with an apparent migration of 74 kD (Figure 3C). A second band migrating at approximately 180 kD was also detected in the pancreatic lysate but not in the duodenal one. This latter result indicates that BSDL appears to be part of this high molecular weight complex in pancreatic tissue but not in duodenal tissue.
With regard to Grp94, proteins of both pancreatic and duodenal homogenates were separated by SDS-PAGE, electrotransferred onto nitrocellulose membrane, and finally developed with the monoclonal antibodies to Grp94. As shown in Figure 4D (Lane 1), four proteins reacted with the anti-Grp94, indicating that Grp94 is present in different forms in pancreatic and duodenal lysates. One of these is the monomeric form of Grp94, migrating at 94 kD. The other three display a close migration between 180200 kD. The intensity of labeling of these latter bands representing complexed Grp94, compared to that of the monomer, suggests that most of the Grp94 in pancreas and in intestinal cells forms heterodimers and/or homodimers.
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The band migrating at 180 kD correlates with the heterodimeric complex immunoprecipitated from the rat pancreatic juice containing Grp94 and BSDL (
When rat pancreatic juice proteins were segregated in the first dimension according to their pI and then in a second dimension according to their size, a unique 180-kD band migrating with a pI of 4.6 and probably representing the BSDLGrp94 complex was detected in 2D electrophoresis (Figure 4C). Note that BSDL and Grp94 have a similar pI (4.5 and 4.6, respectively).
All these data strongly suggest that (a) the protein detected in Figure 3C and Figure 3D and migrating at 180 kD is probably the heterodimer BSDLGrp94, (b) BSDL is able to bind protein complexes composed of either BSDLGrp94 or Grp94 dimers, (c) the 180190-kD proteins that bind biotinylated BSDL could represent putative specific binding sites for BSDL that are present in pancreatic tissue and duodenal tissue, (d) the affinity binding sites of BSDL in the pancreatic secretory pathway are probably composed of heterodimer BSDLGrp94, (e) the BSDLGrp94 complexes internalized by the enterocytes were probably not detected because of their low amounts, and (f) Grp94 dimers present in the enterocyte are able to bind biotinylated BSDL.
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Discussion |
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Several molecular chaperones have been shown to participate in the secretion of pancreatic enzymes (
Among these secreted pancreatic chaperones, Grp94 is a glycoprotein that belongs to the glucose-regulated family of proteins. This chaperone, like Grp78 (also called Bip), possesses the KDEL C-terminal sequence, which is a signal for ER retention. Grp94 forms stable homodimers and is part of an oligomeric complex together with Grp78, calreticulin, calnexin, and similar proteins, and participates in the correct folding and assembly of ER proteins and their exit from this organelle (
Like all digestive enzymes synthesized by the pancreas, BSDL follows the regulated secretory pathway (
By combining immunocytochemical and biochemical techniques, we have recently shown that the association between BSDL and Grp94, which forms a 180-kD complex, takes place in the RER, continues in the Golgi apparatus and the zymogen granules, and is secreted in the pancreatic juice (
Two molecular populations of BSDL appear to be secreted in the pancreatic juice. One is monomeric BSDL, whereas the second is associated with Grp94 (Figure 4; and
The mechanism by which BSDL interacts with membranes appears to be specific.
As mentioned above, in enterocytes BSDL binding sites are associated with microvilli, the endosomal compartment, and basolateral membranes. However, BSDLGrp94 complexes were detected up to the endosomal compartment, BSDL alone (not associated with Grp94) being present in basolateral interdigitations (
In conclusion, we have recently shown (
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Footnotes |
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1 Present address: BIP, CNRS 31 Chemin Joseph Aiguier, Marseille, France.
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Acknowledgments |
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Supported by the Medical Research Council of Canada and by the Institut National de la Santé et de la Recherche Medicale (France). Dr N. Bruneau is recipient of a fellowship from the Fondation pour la Recherche Medicale.
The technical assistance of D. Gingras, C Rondeau, and G. Mayer and the photographic work of J. Léveillé and C. Crotte are greatly appreciated. We thank Dr M. Desjardins for his kind collaboration.
Received for publication May 19, 1999; accepted October 11, 1999.
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