Pancreatic Bile Salt-dependent Lipase Research Articles

Detection of bile salt-dependent lipase, a 110 kDa pancreatic protein, in urines of healthy subjects

Bile salt-dependent lipase (BSDL), a 110 kDa glycoprotein secreted by the pancreatic acinar cells, participates in the duodenal hydrolysis of dietary lipid esters. Recent in vitro and in vivo studies demonstrated that the BSDL reaches the blood via a transcytosis motion through enterocytes, suggesting that this enzyme may play a role in vascular biology. Once in the blood, BSDL should be eliminated. We address the hypothesis that BSDL may be filtered by the glomerulus and eliminated in urines. Immunological methods and proteomic were used to detect and to characterize BSDL in urine. The immunoreactive form of BSDL was detected in urines of 36 male subjects devoid of renal failure. Proteomic demonstrated that the immunoreactive protein is BSDL. Experiments using a monoclonal antibody to the oncofetal glycoform of pancreatic BSDL suggested that the protein is not expressed by renal cells but originates from the pancreas via circulation. We demonstrate that under normal physiological conditions, BSDL, a high-molecular weight blood glycoprotein, can be filtered by the renal glomerulus to be eliminated in urines.

In vitro polyphenol effects on activity, expression and secretion of pancreatic bile salt-dependent lipase

The relationship between cholesterol and atherosclerosis has gained wide credence and red wine polyphenols have been shown to have an anti-atherogenic activity. In the present in vitro studies, we have evaluated and compared the effects of resveratrol, an active compound of red wine, and of a whole red wine polyphenolic extract (RWE) on the pancreatic bile salt-dependent lipase (BSDL). BSDL is involved in the duodenal hydrolysis of lipid esters and in part of cholesteryl esters thus favoring the bioavailability of free cholesterol. Resveratrol and RWE decrease the human and rat enzyme activities. Resveratrol and RWE also impaired the secretion of BSDL by the rat pancreatic AR4-2J cells used as secreting model. This effect is reversed by the removal of resveratrol or RWE from the cell culture medium. Further, resveratrol (but not RWE) affects the transcription of the gene encoding BSDL and dramatically diminishes the quantity of the enzyme that is expressed and secreted by AR4-2J cells. Results suggest that the hypolipemic effects of red wine polyphenols could partly originate from the inhibition of BSDL activity and secretion in the duodenum. In vivo, these effects could decrease the hydrolysis of dietary lipid esters and likely the absorption of free cholesterol.

In Vitro Angiogenic Effects of Pancreatic Bile Salt-Dependent Lipase

Bile salt-dependent lipase (BSDL), a lipolytic enzyme secreted in the duodenum by pancreatic acinar cells, has been detected in the serum of all patients and in atheromatous plaque, suggesting its potential implication in vascular pathophysiology. In vitro pancreatic BSDL evokes human umbilical vein endothelial cell (HUVEC) proliferation and chemotactic migration. BSDL at mitogen concentration is capable to heal wounded HUVEC monolayer and to promote capillary network formation. HUVEC proliferation depends on the displacement of basic fibroblast growth factor and vascular endothelial growth factor from the extracellular matrix and the activation of extracellular signal-regulated kinases (ERK1/2), p38 mitogen-activated protein kinase, and focal adhesion kinase signaling pathways. For the first time to our knowledge, it is suggested that circulating BSDL could be involved in pathophysiological angiogenesis. We delineate the in vitro effects of pancreatic BSDL on endothelial cells, and we show that BSDL promotes proliferation, migration, capillary network formation, and wound-healing of HUVECs via the displacement of bFGF and VEGF from the ECM, suggesting that BSDL could be involved in angiogenesis.

A Role for Bile Salt-Dependent Lipase in Platelet Activation and in Thrombus Formation in Vivo.

Bile Salt Dependent Lipase (BSDL) is an enzyme secreted by pancreatic acinar cells. BSDL, in the presence of primary bile salts, participates in the hydrolysis of dietary lipid esters in the duodenum lumen. This 105 kDa N and O-glycosylated protein has been detected in the plasma of normal subjects. Recent in vitro and in vivo studies demonstrated that pancreatic BSDL reaches the blood via transcytosis through enterocytes. Other studies showed that pancreatic human BSDL is captured by human umbilical vein endothelial cells and induces the proliferation of smooth muscle cells in vitro at BSDL concentrations found in blood, suggesting that this enzyme may play a role in hemostasis and thrombosis. However the specific role of circulating BSDL is unknown. The goal of this study was to determine the possible involvement of circulating BSDL in thrombus formation. We investigated the participation of circulating mouse BSDL in thrombus formation using widefield intravital microscopy in the cremaster muscle of living mice. Thrombi were formed following laser injury of the vessel wall of an arteriole in the cremaster muscle. Pancreatic mouse BSDL, a 74 kDa glycoprotein, was detected using several antibodies directed against either the whole human BSDL (pAbL64, pAbL32) or a peptide based on a sequence in the N-terminal domain of BSDL (Ser326-Thr350; pAbAntipeptide). Mouse and human BSDL share about 80% sequence homology, the main difference localized in the C-terminal domain, which is truncated to the mouse BSDL compared with the human enzyme. All the antibodies are able to specifically recognize the mouse pancreatic BSDL. Using antibodies pAbL64, pAbL32, or pAbAntipeptide we observed specific accumulation of circulating mouse BSDL into the growing thrombus. The circulating BSDL co-localized with platelets present in the thrombus. These results suggest that circulating BSDL is involved in thrombus formation in vivo. In order to determine if BSDL plays a role in platelet activation and aggregation, we performed in vitro studies on human washed platelets. BSDL increased both the amount of phosphatidylserine exposure on the surface of platelets and the activation of αIIbβ3 induced by thrombin. These results indicate that this enzyme can amplify the activation of platelets in vitro. While BSDL alone cannot induce the aggregation of platelets, this enzyme significantly increases the amount of platelet aggregation induced by SFLLRN peptide or thrombin. Altogether, these data suggeste that circulating BSDL participates in the thrombus formation after laser injury of the arterial wall and can amplify both the activation of platelets and the phosphatidylserine exposure, increasing the thrombotic response after vessel injury. This mechanism may be operative in the development of venous thromboembolic disease in pancreatic cancer.

Site-directed Mutagenesis of the Distal Basic Cluster of Pancreatic Bile Salt-dependent Lipase

Previous studies have postulated the presence of two bile salt-binding sites regulating the activity of the pancreatic bile salt-dependent lipase. One of these sites, located in an N-terminal basic cluster, has been identified as the specific bile salt-binding site. Interaction of primary bile salts with this proximal site induces the formation of a micellar binding site from a pre-existing nonspecific or pre-micellar bile salt-binding site. Here we have investigated the functional significance of another basic cluster comprised of amino acid residues Arg(423), Lys(429), Arg(454), Arg(458), and Lys(462), distal from the catalytic site. For this purpose these residues were mutagenized in Ile or Ala residues. The mutagenized enzyme lost activity on both soluble and emulsified substrates in the presence of bile salts. However, in the absence of bile salts, the mutagenized enzyme displayed the same activity on soluble substrate as the wild-type recombinant enzyme. Consequently, the distal basic cluster may represent the nonspecific (or pre-micellar) bile salt-binding site susceptible to accommodate primary and secondary bile salts. According to the literature, tyrosine residue(s) should participate in this site. Therefore, two tyrosine residues, Tyr(427) and Tyr(453), associated with the distal basic cluster were also mutagenized. Each tyrosine substitution to serine did not inhibit the enzyme activity on soluble substrate, independently of the presence of primary or secondary bile salts. However, the enzyme activity on cholesteryl oleate solubilized in primary bile salt micelles was decreased by mutations substantiating that these residues are part of the nonspecific bile salt-binding site.

The combinatorial extension method reveals a sphingolipid binding domain on pancreatic bile salt-dependent lipase: role in secretion.

Structure similarity searches using a combinatorial extension approach revealed that a protein fold structurally related to the sphingolipid binding domain (SBD) of HIV-1 gp120 (V3 loop) is present on pancreatic bile salt-dependent lipase (BSDL). A synthetic peptide derived from the predicted V3-like domain of BSDL interacted with reconstituted monolayers of sphingolipids such as GalCer and GlcCer. Using Chinese hamster ovary cells stably transfected with the cDNA encoding the rat BSDL (CHO-3B clone) or pancreatic SOJ-6 cells expressing the human BSDL as models, we showed that the enzyme cofractionates with caveolin-1. The secretion of BSDL by CHO-3B cells was inhibited by permeable drugs affecting rafts structure (D609, PDMP, and filipin). Data suggest that the functional interaction between the BSDL SBD and lipid rafts is physiologically relevant and could be essential for sensing the BSDL folding prior to secretion. A tentative model accounting for the phosphorylation-induced dissociation of BSDL from rafts is presented.

Lectin-like Ox-LDL receptor is expressed in human INT-407 intestinal cells: involvement in the transcytosis of pancreatic bile salt-dependent lipase.

We have recently shown that the pancreatic bile salt-dependent lipase (BSDL) can be taken up by intestinal cells and transported to the blood circulation. This mechanism likely involves (specific) receptor(s) able to bind BSDL and located at the apical intestinal cell membrane. In this study, using Int407 human intestinal cells cultured to form a tight epithelium, we attempted to characterize (the) BSDL receptor(s). We found that an apical 50-kDa protein was able to bind BSDL. Further, we have demonstrated that Int407 cells expressed the lectin-like oxidized-LDL receptor (LOX-1), the upregulation of which by oxidized-LDL potentiates the transcytosis of BSDL, whereas carrageenan and to a lesser extent polyinosinic acid and fucoidan decrease the enzyme transcytosis. The mAb JTX92, which blocks the LOX-1 receptor function, also impaired the BSDL transcytosis. To confirm these results, the cDNA encoding the human intestinal receptor LOX-1 has been cloned, inserted into vectors, and transfected into Int407 cells. Overexpression of LOX-1 by these cells leads to a substantial increase in the BSDL transcytosis. Globally, these data support the view that LOX-1 could be an intestinal receptor for BSDL, which is implicated in the transcytosis of this enzyme throughout Int407 cells.

Circulating bile salt–dependent lipase originates from the pancreas via intestinal transcytosis

Background & Aims: Bile salt–dependent lipase (BSDL) has been detected in human blood, where it is assumed to play a substantial role in atherosclerosis. The origin of this circulating enzyme is unknown. The aim of this study was to show that blood BSDL originates from pancreatic exocrine secretions via a transcytotic motion across the intestinal epithelium. Methods: Fluorescein isothiocyanate– or [ 125I]-labeled human pancreatic BSDL was instilled into the lumen of intestinal loops of the rat, and combined biochemical and immunocytochemical investigations were performed in intestinal cells and in the blood of these animals. Results: In vivo pancreatic BSDL is internalized by duodenal enterocytes. The pancreatic enzyme was associated with microvilli and present in endocytic vesicles and Golgi apparatus as well as along the basolateral membrane of enterocytes. It was also detected in intestinal interstitial spaces. Radiolabeled pancreatic BSDL internalized by the duodenal epithelium is the one further detected in circulation. The radiolabeled protein was immunoprecipitated from plasma and had a 100-kilodalton molecular mass compatible with native pancreatic BSDL. In blood, BSDL was mainly associated with low-density lipoproteins. Conclusions: These in vivo data show that BSDL, normally present in blood, originates from exocrine pancreatic secretion and support the pathophysiologic relevance of BSDL transcytosis through the intestinal mucosa cell lining. Based on this, the implication of circulating BSDL in atherosclerosis merits careful attention.

Effects of arachidonic and docosahexaenoic acids on secretion and degradation of bile salt-dependent lipase in AR4-2J cells

In this study we demonstrated that two polyunsaturated fatty acids, arachidonic acid (AA, n–6) and docosahexaenoic acid (DHA, n–3), modulate the secretion of bile salt-dependent lipase (BSDL) by pancreatic AR4-2J cells. The effects of AA and DHA were also compared with that of the monounsaturated fatty acid, oleic acid (OA). Our results showed that the chronic treatment of cells with AA or DHA, that did not affect the biosynthesis rate of BSDL, similarly decreased the amount of secreted BSDL and perturbed the intracellular partitioning of the enzyme, whereas OA had no effect. Particularly, AA and DHA induced the retention of the enzyme in microsomes and lowered its content in the cell cytosol. We have further shown that AA treatment decreased the ubiquitination of the protein, and consequently diminished its export toward the cytosol, a result that might explain the retention of BSDL in microsomes and correlated with membrane phospholipids alteration. The retained protein was further degraded by a nonproteasomal pathway that likely involves ATP-dependent endoplasmic reticulum proteases. These findings concerning the regulation of the pancreatic BSDL secretion by two polyunsaturated acids, AA and DHA, might be of physiological importance in the plasmatic and cellular cholesterol homeostasis. —Le Petit-Thévenin, J., N. Bruneau, A. Nganga, D. Lombardo, and A. Vérine. Effects of arachidonic and docosahexaenoic acids on secretion and degradation of bile salt-dependent lipase in AR4-2J cells.

Phosphorylation of the Oncofetal Variant of the Human Bile Salt-dependent Lipase: IDENTIFICATION OF PHOSPHORYLATION SITE AND RELATION WITH SECRETION PROCESS

In this paper, we report, for the first time, the localization of the phosphorylation site of the fetoacinar pancreatic protein (FAPP), which is an oncofetal variant of the pancreatic bile salt-dependent lipase. Using Chinese hamster ovary (CHO) cells transfected with the cDNA encoding FAPP, we radiolabeled the enzyme with (32)P, and then the protein was purified by affinity chromatography on cholate-immobilized Sepharose column and submitted to a CNBr hydrolysis. Analysis of peptides by high pressure liquid chromatography, associated with the radioactivity profile, revealed that the phosphorylation site is located at threonine 340. Site-specific mutagenesis experiments, in which the threonine was replaced by an alanine residue, were used to invalidate the phosphorylation of FAPP and to study the influence of the modification on the activity and secretion of the enzyme. These studies showed that CHO cells, transfected with the mutated cDNA of FAPP, kept all of their ability to synthesize the protein, but the loss of the phosphorylation motif prevented the release of the protein in the extracellular compartment. However, the mutated enzyme, which was sequestrated in the transfected CHO cells, remains active on bile salt-dependent lipase substrates.

Development of bile salt‐dependent lipase in larval turbot

Pancreatic bile salt‐dependent lipase (BSDL) was present with 0·5 μg BSDL larva−1 from hatching in turbot larvae. The enzyme content increased during the yolk sac phase to 1·1 μg BSDL larva−1. This suggests that larval turbot are able to digest lipids from the start of exogenous feeding. The BSDL synthesis was stimulated first by food about 5 days after the onset of first feeding. The content per larva increased exponentially in fed larvae to 20 μg BSDL larva−1 on day 23 after hatching and decreased in starved larvae. In contrast, the specific content decreased during the first feeding phase, meaning that smaller larvae had a higher content of enzyme related to their biomass than did bigger larvae.

Expression of a 70-kDa Immunoreactive Form of Bile Salt-Dependent Lipase by Human Pancreatic Tumoral Mia PaCa-2 Cells

This work describes the characterization of an immunoreactive form of bile salt-dependent lipase (BSDL) expressed by the human pancreatic tumoral Mia PaCa-2 cell line. This BSDL-related protein, which has an Mr of 70 kDa, is enzymatically active and poorly secreted. Furthermore, a protein with the same electrophoretic migration can also be immunoprecipitated with polyclonal antibodies specific for the human pancreatic BSDL after in vitro translation of RNA isolated from Mia PaCa-2 cells. These data indicated that this BSDL-related protein might be poorly, or not, glycosylated. Reverse transcription and amplification of RNA extracted from Mia PaCa-2 cells using primers able to specifically amplify the full-length mRNA of the human BSDL resulted in a detectable 1.8-kb cDNA product, shorter than that of BSDL (2.2 kb). The sequence of this transcript corresponds to the mRNA sequence that codes for the mature human pancreatic BSDL. However, a deletion of 330 bp is located within the 3′-domain of this cDNA. Therefore data allowed us to conclude that the 70-kDa BSDL-related protein is a 612 amino acid length protein and represents a truncated form of BSDL. The deletion of 110 amino acids occurs in the C-terminal region of the protein, which encompasses 6 tandemly repeated sequences instead of the 16 normally present in the sequence of BSDL. Because feto-acinar pancreatic protein (FAPP), which is the oncofetal counterpart of BSDL, is a C-terminally truncated isoform of BSDL, it is suggested that the 70-kDa BSDL-related protein expressed in MiaPaCa-2 cells could be representative of the protein moiety of FAPP.

The affinity binding sites of pancreatic bile salt-dependent lipase in pancreatic and intestinal tissues.

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 (ER-Golgi-granules). The Grp94-BSDL 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 affinity-gold 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 streptavidin-gold 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 180-190 kD in both pancreatic and duodenal tissues. We have also shown that the Grp94-BSDL 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 BSDL-Grp94 or Grp94 dimers. (J Histochem Cytochem 48:267-276, 2000)

Immunodetection and molecular cloning of a bile-salt-dependent lipase isoform in HepG2 cells

In this article, we report the nucleotide sequence of the cDNA encoding an isoform of bile-salt-dependent lipase (BSDL) expressed by human hepatoma cells. The BSDL is a 100-kDa glycoprotein normally expressed by the human pancreas. Using a polyclonal antibody raised against an internal peptide located between Ile(327) and Glu(350) of the human pancreatic BSDL, we have immunodetected an isoform of human pancreatic BSDL, with an apparent molecular mass of about 62 kDa. This isoform of BSDL was mainly associated with the cytosol of a human hepatoma cell line (HepG2), the remaining protein being found in the microsome fraction. In addition, esterolytic activity on p-nitrophenyl hexanoate measured in microsomes and cytosol appeared very low and was weakly stimulated by bile salts, such as taurocholate. In contrast to human pancreatic BSDL, which is secreted as a component of pancreatic juice, this isoform appeared to be retained in the HepG2 cells. Reverse transcription, followed by PCR and amplification, performed on RNA extracted from HepG2 cells using specific primers hybridizing to the sequence coding for the entire normal human pancreatic BSDL, allowed us to amplify a 1. 7-kb transcript that appeared to be 0.5 kb shorter than the transcript of the pancreatic enzyme (2.2 kb). From the sequence of the transcript thus obtained, a protein with a molecular mass of 62 kDa might be predicted, which is in good agreement with the size of the isoform of BSDL immunodetected in HepG2 cells. The N-terminal amino-acid sequence, deduced from the 1.7-kb transcript sequence, matched that of the pancreatic BSDL. However, the C-terminal domain appeared truncated, bearing only a single mucin-like sequence compared with sixteen for the human pancreatic BSDL. The actual intracellular function of this human BSDL hepatoma isoform remains to be elucidated.

Participation of GRP94-related protein in secretion of pancreatic bile salt-dependent lipase and in its internalization by the intestinal epithelium.

In previous studies on the AR4-2J cell line, we have shown that secretion of bile salt-dependent lipase (BSDL) involves a multiprotein complex, including a protein of 94 kDa (p94) that is immunologically related to the chaperone Grp94, which seems to play essential roles in the folding process of BSDL. Combined biochemical and immunocytochemical investigations were carried out to study the secretion of BSDL by normal pancreatic cells and its transport to the small intestine where this enzyme is thought to exert its physiological function. Both BSDL and Grp94 antigenic sites were localized and found to be associated all along the pancreatic acinar cell secretory pathway. Grp94 and BSDL remain associated from leaving the pancreas until arriving at the intestinal lumen. In pancreatic juice, both proteins appear as a complex of high molecular mass (180 kDa) containing at least one each of p94 and BSDL molecules, interacting by hydrophobic forces. At the intestinal level, associated Grp94 and BSDL were detected on microvilli and in the endosomal compartment of enterocytes. The BSDL mRNA, however, was not expressed by the intestinal mucosa. The pancreatic Grp94-BSDL complex was internalized through the endosomal compartment of enterocytes. Finally, the two proteins dissociated in this compartment and BSDL, but not Grp94, was transferred to the basolateral membrane.

An intracellular role for pancreatic bile salt-dependent lipase: evidence for modification of lipid turnover in transfected CHO cells

Pancreatic bile salt-dependent lipase (BSDL) hydrolyzes cholesteryl esters, triglycerides and phospholipids. BSDL is also capable of transferring free fatty acid to cholesterol. BSDL has been detected in many cells including fetal and tumor cells, hepatocytes, macrophages and eosinophils and in tissues such as adrenal glands and testes. The enzyme may be secreted or located within subcellular compartments such as the endoplasmic reticulum or the cytosol. Although the role of the secreted enzyme is well documented, that of the intracellular form(s) is still hypothetical. In the present study, we addressed the effects of BSDL on cell lipid metabolism. For that purpose, the cDNA of rat BSDL was transfected into CHO K1 cells (CHO K1-BSDL clone) which were then loaded with [ 3H]oleic acid. The results demonstrate that the transfected BSDL is secreted; in spite of that, a large fraction of catalytically active BSDL is found in cell lysate. The lipid metabolism of transfected cells is affected and BSDL induces an enhanced incorporation of [ 3H]oleic acid in cholesteryl esters whereas fatty acid incorporation in phosphatidylcholine is decreased. These effects were particularly important in the cytosol of transfected cells where transfected BSDL preferentially locates. These data suggested that BSDL could be implicated in the cycle of the cellular homeostasis of cholesterol which is particularly affected in tumoral cells leading to cholesteryl ester storage within cytosolic lipid droplets.

Association of bile-salt-dependent lipase with membranes of human pancreatic microsomes is under the control of ATP and phosphorylation.

Bile-salt-dependent lipase (BSDL) is secreted by the pancreas into the duodenum, where it catalyses the hydrolysis of dietary lipid esters on activation by bile salts. The secretion pathway of BSDL is comparable with that of other digestive enzymes produced by pancreatic acinar cells. However, in contrast with these other enzymes, BSDL is partly associated with endoplasmic reticulum membranes as part of a folding complex, including a Grp94-related protein to which BSDL is transiently linked. The release of BSDL from membranes occurs once its glycosylation is completed [Bruneau and Lombardo (1995) J. Biol. Chem. 270, 13524-13533]. In the present study, investigations concerning the mechanism of association/dissociation of BSDL with membranes of microsomes were performed. For this purpose the role of ATP and that of the possible phosphorylation of BSDL were examined. For the first time, it is shown that human pancreatic BSDL is phosphorylated, probably at a serine residue, during its transport within the acinar cell. The phosphorylation of BSDL is provoked by calphostin C, an inhibitor of protein kinase C. In the presence of 1-(isoquinolinesulphonyl)2-methylpiperazine, a non-specific inhibitor of serine/threonine protein kinase A, C or G, or of calcium chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N', N'-tetra-acetic tetra(acetoxymethyl)ester, the phosphorylation of BSDL elicited by calphostin C is abolished. These data suggested that the phosphorylation of BSDL within human pancreatic microsomes is under the control of a cascade of protein kinases. We have also shown that the phosphorylation of BSDL appears to be involved in the release of the enzyme from microsome membranes. Nevertheless ATP, which modifies the conformation of BSDL, triggers this association, and an unhydrolysable ATP analogue was unable to promote it.

O-Glycosylation of C-terminal Tandem-repeated Sequences Regulates the Secretion of Rat Pancreatic Bile Salt-dependent Lipase

Amino acid sequences rich in Pro, Glu, Ser, and Thr (PEST) are common to rapidly degraded proteins (Rogers, S., Wells, R. & Rechsteiner, M. (1986) Science 234, 364-368). On pancreatic bile salt-dependent lipase (BSDL), PEST sequences are present in the C-terminal region of the enzyme to which is associated the O-glycosylation. We have postulated that the O-glycosylation of BSDL may contribute to mask PEST sequences and to trigger the secretion of this enzyme instead of its delivery into a degradative pathway (Bruneau, N., and Lombardo, D. (1995) J. Biol. Chem. 270, 13524-13523). To further examine the role of the O-linked glycosylation on BSDL metabolism, rat pancreatic BSDL cDNA was stably transfected into two Chinese hamster ovary (CHO) cell lines, the CHO K1 wild-type line and the O-glycosylation defective CHO ldlD line. In these latter cells, O-glycosylation can be reversibly modulated by culture conditions. Results indicate that the rate of BSDL synthesis by transfected CHO K1 or CHO ldlD cells reflects, independently of culture conditions, the amount of mRNA specific for BSDL present in these transfected cells. Nevertheless, the rate of secretion of the enzyme depends upon cell culture conditions and increases with the cell capability to O-glycosylate C-terminal tandem-repeated sequences. Immunoprecipitation experiments performed on cell lysates suggested that a rapid degradation of BSDL occurred particularly when transfected CHO ldlD cells were cultured under non-permissive conditions. We further showed that BSDL secreted by CHO ldlD cells grown under non-permissive conditions that normally prevent O-glycosylation incorporated galactose and was reactive with peanut agglutinin, which recognizes the core structure of O-linked glycans. We concluded that the BSDL expressed by CHO ldlD cells grown under non-permissive conditions was rapidly degraded but a fraction of the enzyme was allowed to O-glycosylate and consequently was secreted.

Association of Bile‐Salt‐Dependent Lipase with Membranes of Human Pancreatic Microsomes

Immunolocalization studies indicated that, in contrast to other enzyme markers of human pancreatic secretion, bile-salt-dependent lipase (BSDL) was partly but specifically associated with endoplasmic reticulum membranes. In microsomes, temperature-induced phase separation using Triton X-114 elucidated the partition of BSDL between the aqueous phase and the detergent-rich phase containing hydrophilic and membrane proteins, respectively. The size of the membrane-associated BSDL (approx. 100 kDa) is compatible with that of the fully processed enzyme. Fucosylated O- and N-linked oligosaccharide structures were detected by means of specific lectins. The membrane-associated BSDL might therefore be released from membranes between the trans-Golgi compartment (where terminal fucose residues were added) and the zymogen granules where BSDL was mainly found in the soluble fraction. Even though BSDL associated with membranes was enzymically active, it appeared less efficient than the soluble form. The association of BSDL with membranes was pH-dependent and optimal association occurred between pH 5-6. The membrane-associated BSDL was released by KBr which suggests that the association of BSDL with microsomal membranes involves ionic interactions. Lipid-protein interactions are probably not involved in this association as BSDL did not associate with liver microsome membranes. We attempted to characterize the putative ligand and showed that BSDL and a 94-kDa protein, immunologically related to a glucose-regulated protein of 94 kDa (Grp94), were co-immunoprecipitated by specific antibodies directed against each individual species. It is suggested that the biogenesis of the human pancreatic BSDL involves an association with intracellular membranes and that its folding may be assisted by molecular chaperones.

Chaperone Function of a Grp 94-related Protein for Folding and Transport of the Pancreatic Bile Salt-dependent Lipase

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.