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.     

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)     

Impairment of bile salt-dependent lipase secretion in human pancreatic tumoral SOJ-6 cells

Bile salt-dependent lipase (BSDL) was detected in human SOJ-6 and rat AR4-2J pancreatic cells. Whereas AR4-2J cells actively secreted the enzyme, BSDL was retained within the Golgi compartment of SOJ-6 cells. Because Rab6 is involved in vesicle transport in the Golgi apparatus and the trans-Golgi network, we confirmed the presence of Rab6 in these cells. In rat AR4-2J cells, Rab6 as well as Rab1A/B and Rab2, partitioned between the cytosol and microsomes. In SOJ-6 cells Rab1A/B and Rab2 also partitioned between the cytosol and microsomes, but Rab6 was strictly associated with microsome membranes, suggesting a specific defect of Rab6 cycling in human SOJ-6 cells. The apparent defect of cycling in these cells is not due to the expression of a defective Rab6 since its correct sequence was confirmed. We further demonstrated that AR4-2J and SOJ-6 cells express the Rab-GDIbeta and Rab-GDIalpha isoforms, respectively. However, the sequence of Rab-GDIbeta, which may be the main form expressed by SOJ-6 cells, identified a few substitutions located in regions that are essential for Rab-GDI function. We conclude that the deficient secretion of BSDL by SOJ-6 cells could be due to the expression of defective Rab-GDIbeta. In spite of the alterations in Rab-GDIbeta, membrane proteins such as CD71 and NHE3 were correctly localized to the cell plasma membrane of SOJ-6 cells, suggesting that two functional distinct secretory pathway coexist in pancreatic cells.     

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.     

Site-directed mutagenesis of the basic N-terminal cluster of pancreatic bile salt-dependent lipase. Functional significance

Previous studies have postulated the presence of a heparin-binding site on the bile salt-dependent lipase (BSDL), whereas two bile salt-binding sites regulate the enzyme activity. One of these sites may overlap with the tentative heparin-binding site at the level of an N-terminal basic cluster consisting of positive residues Lys(32), Lys(56), Lys(61), Lys(62), and Arg(63). The present study uses specific site-directed mutagenesis to determine the functional significance of this basic cluster. Mutations in this sequence resulted in recombinant enzymes that were able to bind to immobilized and to cell-associated heparin before moving throughout intestinal cells. Recombinant BSDL was fully active on soluble substrate, but mutants were less active on micellar cholesteryl oleate in comparison with the wild-type enzyme. Activation studies by primary (sodium taurocholate) and by secondary (sodium taurodeoxycholate) bile salts revealed that the activation of BSDL by sodium taurocholate at concentrations below the critical micellar concentration, and not that evoked by micellar bile salts, was affected by substitutions, suggesting that this N-terminal basic cluster likely represents the specific bile salt-binding site of BSDL. Substitutions also affected the activation of the enzyme promoted by anionic phospholipids, extending the function of this site to that of a cationic regulatory site susceptible to accommodate anionic ligands.     

Impairment of bile salt-dependent lipase secretion in AR4-2J rat pancreatic cells induces its degradation by the proteasome

Bile salt-dependent lipase (BSDL, EC 3.1.1.13) is a lipolytic enzyme normally secreted by the pancreatic acinar cell. Co- and post-translational modifications, such as N- and O-linked glycosylation, regulate the secretion of this enzyme; therefore it was of first importance to determine the behaviour of BSDL under conditions that impaired its secretion. Using AR4-2J pancreatic cells as model, we showed, particularly when BSDL secretion is impaired, that proteasome inhibitors increased the amount of intracellular BSDL, suggesting that the proteasome is involved in the degradation of this protein. This was strengthened by the detection of ubiquitinated BSDL and of degradation product. Our results suggested that both ubiquitination and degradation of the enzyme occurred at the level of the cytosolic side of microsome membranes. ATP hydrolysis appears essential in ubiquitinated BSDL association with membranes and degradation. Furthermore, under normal secretory conditions, we have shown that a fraction of ubiquitinated BSDL is neither O-glycosylated nor N-glycosylated, suggesting that the N-glycosylation-deficient proteasome substrate does not reach the Golgi and could be degraded by the ER-associated degradation machinery. However, another fraction of ubiquitinated BSDL that is deficient in O-glycosylation, carries out endoglycosidase H-insensitive N-linked glycans, meaning that a second system, that detects abnormal BSDL molecules, could also operate at the level of the Golgi compartment. Consequently, it appears that impairment of BSDL secretion consecutive to secretion inhibition or to a deficient glycosylation leads to the proteasome-ubiquitin-dependent degradation of the protein. Therefore, this pathway is part of the quality control involved in BSDL secretion.     

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.     

Fatty acid specificity of bile salt-dependent lipase: enzyme recognition and super-substrate effects

A putative fatty acid specificity of bile salt-dependent lipases (BSDLs) has been re-investigated. The strategy was to use two evolutionally distant, homologous BSDLs (from human and cod), and to investigate their hydrolysis of different fatty acid esters at different assay conditions affecting the physicochemical phase of the substrate. Depending on assay conditions, large variations were seen in the hydrolysis rate for esters of different fatty acids. The two enzymes displayed similar fatty acid specificity patterns, with small, but significant differences that were maintained at various assay conditions. Compared to the human enzyme, the cod enzyme showed a preference for hydrolysis of long-chain polyunsaturated fatty acyl esters (up to 22 carbons in length). On the other hand, the human enzyme hydrolysed esters of shorter chain saturated fatty acids at significantly higher rates compared to the cod enzyme. Changing physicochemical factors affecting the substrate phase induced large changes in fatty acid specificity that affected both enzymes in similar manners. It is concluded that though the aliphatic chains of the fatty acids may not be recognized by the enzymes, these chains indirectly affect the conformation or interfacial availability of the carboxyl ester bond in the substrate, and the enzymes show minor specificities for variations in these structures.     

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.     

Binding of bile salts to pancreatic colipase and lipase.

The binding of conjugated bile salts to pancreatic colipase and lipase has been studied by equilibrium dialysis and gel filtration. The results indicate that at physiological ionic strength and pH, conjugated bile salts bind as micelles to colipase: 12-15 moles/mole of colipase for the dihydroxy conjugates and 2-4 for the trihydroxy conjugates. No binding of bile salt takes place from monomeric solutions. Under the same experimental conditions, only 1-2 moles of conjugated dihydroxy bile salts bind to pancreatic lipase.     

Human bile salt-dependent lipase efficiency on medium-chain acyl-containing substrates: control by sodium taurocholate

Bile salt-dependent lipase was purified to homogeneity from lyophilized human milk and used to screen the influence of the acyl chain length (2-16 carbon atoms) on the kinetic constants k(cat) and K(m) of the hydrolysis of para-nitrophenyl (pnp) ester substrates in the presence or absence of sodium taurocholate (NaTC: 0.02-20 mM). The highest k(cat) value (～3,500 s(-1)) was obtained with pnpC(8) as substrate, whereas the lowest K(m) (<10 μM) was that recorded with pnpC(10). In the absence of NaTC, the maximal catalytic efficiency (k(cat)/K(m)) was obtained with pnpC(8), while in the presence of NaTC k(cat)/K(m) was maximal with pnpC(8), pnpC(10) or pnpC(12). The bile salt activated the enzyme in two successive saturation phases occurring at a micromolar and a millimolar concentration range, respectively. The present data emphasize the suitability of this enzyme for the hydrolysis of medium-chain acyl-containing substrates and throw additional light on how BSDL is activated by NaTC.     

Purification and characterization of bovine pancreatic bile salt-activated lipase

An enzyme with lipase and esterase activity was purified from bovine pancreas. Furthermore, a non-radioactive lipase assay was developed which is 100 times more sensitive than the conventional methods and allowed the characterization of the lipase activity of the enzyme. The lipase activity increased 42 times in the presence of 10 mM sodium taurocholate, which for the first time provides direct evidence that a bile salt-activated lipase (bp-BAL) was isolated from bovine pancreas. This conclusion is further supported by the fact that the N-terminal amino acid sequence of this lipase/esterase is 88% homologous to human milk BAL and human pancreatic BAL. Staining with various lectins showed that bp-BAL is a glycoprotein which contains fucose residues. Previously from bovine pancreas a lysophospholipase has been purified and a gene was cloned and sequenced encoding an enzyme with cholesterol esterase/lysophospholipase activity. Comparison of the N-terminal amino acid sequence of bp-BAL with the deduced amino acid sequence of the latter revealed that they are identical. Furthermore, the molecular weight of the purified bp-BAL of 63,000, as estimated by SDS-PAGE, is very similar to that of the purified lysophospholipase (65,000) and to the theoretical molecular weight of 65,147 of the cholesterol esterase/lysophospholipase. These data suggest that these three enzymes are one and the same.     

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.     

Importance of arginines 63 and 423 in modulating the bile salt-dependent and bile salt-independent hydrolytic activities of rat carboxyl ester lipase

Previous studies using chemical modification approach have shown the importance of arginine residues in bile salt activation of carboxyl ester lipase (CEL) activity. However, the x-ray crystal structure of CEL failed to show the involvement of arginine residues in CEL-bile salt interaction. The current study used a site-specific mutagenesis approach to determine the role of arginine residues 63 and 423 in bile salt-dependent and bile salt-independent hydrolytic activities of rat CEL. Mutations of Arg(63) to Ala(63) (R63A) and Arg(423) to Gly(423) (R423G) resulted in enzymes with increased bile salt-independent hydrolytic activity against lysophosphatidylcholine, having 6.5- and 2-fold higher k(cat) values, respectively, in comparison to wild type CEL. In contrast, the R63A and R423A mutant enzymes displayed 5- and 11-fold decreases in k(cat), in comparison with wild type CEL, for bile salt-dependent cholesteryl ester hydrolysis. Although taurocholate induced similar changes in circular dichroism spectra for wild type, R63A, and R423G proteins, this bile salt was less efficient in protecting the mutant enzymes against thermal inactivation in comparison with control CEL. Lipid binding studies revealed less R63A and R423G mutant CEL were bound to 1,2-diolein monolayer at saturation compared with wild type CEL. These results, along with computer modeling of the CEL protein, indicated that Arg(63) and Arg(423) are not involved directly with monomeric bile salt binding. However, these residues participate in micellar bile salt modulation of CEL enzymatic activity through intramolecular hydrogen bonding with the C-terminal domain. These residues are also important, probably through similar intramolecular hydrogen bond formation, in stabilizing the enzyme in solution and at the lipid-water interface.     

Adenovirus-mediated human pancreatic lipase gene transfer to rat bile: gene therapy of fat malabsorption

This study explored the potential of using the gene therapy approach, based on adenovirus-mediated expression of pancreatic lipase in the hepatobiliary tract, to increase lipid digestion in the intestinal lumen and promote lipid absorption through the gastrointestinal tract. Recombinant adenovirus containing the human pancreatic lipase cDNA (AdPL) was shown to transduce and mediate pancreatic lipase biosynthesis in rat IEC-6 epithelial cells in vitro. Retrograde infusion of recombinant adenovirus (3 x 10(8) plaque-forming units) containing the bacterial LacZ gene (AdLacZ) into the bile duct of rats resulted in positive X-gal reaction products in the periportal liver cells 7 days after AdLacZ infusion. A high level of human pancreatic lipase was detected in bile after retrograde bile duct infusion of rats with AdPL but not in the bile of animals infused with AdLacZ. Triglyceride hydrolytic activity in the bile of AdPL-infused rats was equivalent to that present in pancreatic juice. In contrast, serum obtained from these animals did not contain any detectable pancreatic lipase activity. These results suggest that ectopic expression of pancreatic enzymes in the hepatobiliary tract may be an alternative therapeutic strategy for treating fat malabsorption due to pancreatic insufficiency.     

Val-407 and Ile-408 in the beta5'-loop of pancreatic lipase mediate lipase-colipase interactions in the presence of bile salt micelles

In a previous study, we demonstrated that the beta5'-loop in the C-terminal domain of human pancreatic triglyceride lipase (hPTL) makes a major contribution in the function of hPTL (Chahinian et al. (2002) Biochemistry 41, 13725-13735). In the present study, we characterized the contribution of three residues in the beta5'-loop, Val-407, Ile-408, and Leu-412, to the function of hPTL. By substituting charged residues, aspartate or lysine, in these positions, we altered the hydrophilic to lipophilic ratio of the beta5'-loop. Each of the mutants was expressed, purified, and characterized for activity and binding with both monolayers and emulsions and for binding to colipase. Experiments with monolayers and with emulsions suggested that the interaction of hPTL with a phospholipid monolayer differs from the interaction of the hPTL-colipase complex with a dicaprin monolayer or a triglyceride emulsion (i.e. neutral lipids). Val-407, Ile-408, and Leu-412 make major contributions to interactions with monolayers, whereas only Val-407 and Ile-408 appear essential for activity on triglyceride emulsions in the presence of bile salt micelles. In solutions of taurodeoxycholate at micellar concentrations, a major effect of the beta5'-loop mutations is to change the interaction between hPTL and colipase. These observations support a major contribution of residues in the beta5'-loop in the function of hPTL and suggest that a third partner, bile salt micelles or the lipid interface or both, influence the binding of colipase and hPTL through interactions with the beta5'-loop.     

Production of recombinant human bile salt-stimulated lipase in Pichia pastoris.

Recombinant human bile salt-stimulated lipase (rhBSSL) was efficiently expressed under the control of the AOX1 gene promoter in Pichia pastoris. Human BSSL has 16 successively repeated sequences in the carboxy terminal region. The sequence consists of 11 amino acid residues. The coding sequence for the middle 11 of the 16 repeats was removed from hBSSL cDNA to facilitate efficient secretory expression. The clone used for fermentation was a transformant of GS115 (his4) integrated with four copies of the expression cassette containing the modified hBSSL cDNA. Unique fermentation conditions were required for efficient expressions of rhBSSL in the high cell-density fermentation. A sufficient glycerol feed at 30 degrees C and pH 4 under an adequate concentration of dissolved oxygen in the growth phase make the cells active over a long induction period of approximately 15 days. On methanol induction, the concentration of dissolved oxygen should be maintained very low in the presence of sorbitol and skimmed milk at 20 degrees C and pH 5.7. Under these conditions, 0.8-1 g of rhBSSL was secreted in 1 liter of the medium. By immunoelectron microscopy, rhBSSL-tagged gold particles were located in secretion microbodies after the beginning of methanol induction. The secreted rhBSSL was efficiently captured and purified by expanded bed adsorption chromatography.