Bile Salt-stimulated Lipase Research Articles

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)

Selective Organic Precipitation/Extraction of Released N-Glycans Following Large-Scale Enzymatic Deglycosylation of Glycoproteins

A major difficulty with isolating enzymatically or chemically released oligosaccharides from large-scale glycoprotein deglycosylation reactions is the time-consuming chromatography, desalting, and concentration steps required to prepare a glycan fraction of manageable proportions. To overcome these time and preparative chromatography equipment requirements, we have developed a rapid organic solvent precipitation/extraction procedure that allows sequential isolation of endo-β-N-acetylglucosaminidase H (EC 3.2.1.96)-released high-mannose and hybrid, peptide-N4-(N-acetyl-β-glucosaminyl) Asn amidase (EC 3.5.1.52)-released complex, and β-eliminated O-linked glycans without the need for intermediate chromatography, desalting, or concentration steps. The method involves precipitation of protein and released glycans at −20°C in 80% acetone and extraction of the glycans from the pellet with 60% aqueous methanol after each deglycosylation step. Three pools of essentially salt- and detergent-free oligosaccharides (high-mannose/hybrid, complex, and O-linked) can be isolated in a high yield in 4 days with this protocol, which has been extensively tested using bovine RNase B, human bile salt-stimulated lipase expressed in Pichia pastoris, hen ovalbumin, bovine fetuin, bovine thyroglobulin, and several invertase preparations from wild-type and mutant yeast strains.

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.

Circulating antibodies against an exocrine pancreatic enzyme in type 1 diabetes.

In this article, we report the identification of a new autoantigen in type 1 diabetes originating from the exocrine pancreas. This antigen is a pancreatic enzyme termed bile salt-dependent lipase (BSDL). We show that antibodies present in the sera of newly diagnosed type 1 diabetic patients recognize BSDL and more specifically the COOH-terminal mucin-like region of the protein. Therefore, we engineered the COOH-terminal peptide of BSDL and demonstrated that autoreactivity was linked to specific glycosylation sites by at least two glycosyltransferases: the Core 2 beta(1-6)N-acetylglucosaminyltransferase and the alpha(1-3) fucosyltransferase FUT7. We next examined the prevalence of circulating anti-BSDL antibodies in type 1 diabetic patients and found 73.5% positivity (25 sera among 34 patients tested) at onset, whereas only 8.4% of normal individuals (7 of 83) were positive. Within a cohort of first-degree relatives of diabetic patients followed prospectively until development of diabetes, 6 of 19 (31.6%) were also positive. Interestingly, two prediabetic individuals were already positive for anti-BSDL antibodies (Abs), while islet cell cytoplasmic Abs and antibodies to GAD65, IA-2, and insulin were not detected. Anti-BSDL autoantibodies were weakly or not detected in patients suffering from pancreatitis or pancreatic adenocarcinoma or in patients with Graves' disease. Although autoreactivity to BSDL in prediabetic and newly diagnosed diabetic patients might reflect cross-reactivity, our results strongly suggest that in addition to pancreatic beta-cells, acinar cells may be also affected in type 1 diabetes.

The formation of the oncofetal J28 glycotope involves core-2 beta6-N-acetylglucosaminyltransferase and alpha3/4-fucosyltransferase activities.

The feto-acinar pancreatic protein or FAPP, the oncofetal glycoisoform of bile salt-dependent lipase (BSDL), is characterized by the presence of the J28 glycotope recognized by mAbJ28. This fucosylated epitope is carried out by the O-linked glycans of the C-terminal mucin-like region of BSDL. This glycotope is expressed by human tumoral pancreatic tissues and by human pancreatic tumoral cell lines such as SOJ-6 and BxPC-3 cells. However, it is not expressed by the normal human pancreatic tissues and by MiaPaCa-2 and Panc-1 cells. Due to the presence of many putative sites for O-glycosylation on FAPP and BSDL, the structure of the J28 glycotope cannot be attained by classical physical methods. In the first part of the present study, we have determined which glycosyltransferases were differently expressed in pancreatic tumoral cell lines compared to normal tissues, focusing in part on fucosyltransferases (Fuc-T) and core-2 beta6-N-acetylglucosaminyltransferase (Core2GlcNAc-T). Our data suggested that alpha2-Fuc-T activity was decreased in the four cell lines tested (SOJ-6, BxPC-3, MiaPaCa-2, and Panc-1). The alpha(1-3) and alpha(1-4) fucosylations were decreased in tumor cells that do not express the J28 glycotope whereas alpha4-Fuc-T and Core2GlcNAc-T activities were significantly increased in SOJ-6 cells which best expressed the J28 glycotope. Therefore, we wished to gain information about glycosyltransferases involved in the building of this structure by transfecting the cDNA encoding the mucin-like region of BSDL in CHO-K1 also expressing Core2GlcNAc-T and/or FUT3 and/or FUT7 activities. These CHO-K1 cells have been previously transfected with the cDNA encoding Core2GlcNAc-T and/or FUT3 and/or FUT7. Data indicated that the C-terminal peptide of BSDL (Cter) produced by those cells did not carry out the J28 glycotope unless Core2GlcNAc-T activity is present. Further transfection with FUT3 cDNA, increased the antibody recognition. Nevertheless, transfection with FUT3 or FUT7 alone did not generate the formation of the J28 glycotope on the C-terminal peptide. Furthermore, the Cter peptide produced by CHO-K1 cells expressing Core2GlcNAc-T was more reactive to the mAbJ28 after in vitro fucosylation with the recombinant soluble form of FUT3. These data suggested that the J28 glycotope encompasses structures initiated by Core2GlcNAc-T and further fucosylated by alpha3/4-Fuc-T such as FUT3, likely on GlcNAc residues.

Carboxyl ester lipase activity in milk prevents fat-derived intestinal injury in neonatal mice.

Carboxyl ester lipase (bile salt-stimulated lipase) is a pancreatic enzyme capable of hydrolyzing esters of cholesterol and fat-soluble vitamins. It also efficiently digests triglycerides (TG) into free fatty acids and glycerol and is abundant in the milk of humans and several other species. We used the mouse as a model to test the hypothesis that milk-derived carboxyl ester lipase (CEL) digests milk TG and that without its activity milk lipids and their digestion intermediates can disrupt the intestinal epithelium of neonates. CEL protein and enzymatic activity were shown to be abundant in mouse milk. After 24-h administration of the CEL-specific inhibitor, WAY-121,751-5, the small intestines of treated and control neonates were analyzed histologically for signs of fat malabsorption and injury to their villus epithelium. In vehicle-fed controls, TG were digested and absorbed in the duodenum and jejunum, whereas, in inhibitor-fed littermates, large intracellular neutral lipid droplets accumulated in enterocytes of the ileum, resulting in damage to the villus epithelium. Similar results were observed in neonates nursed by CEL knockout females compared with heterozygous controls. The results suggest that lack of CEL activity causes incomplete digestion of milk fat and lipid accumulation by enterocytes in the ileum of neonatal mice.

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.

Digestion of ceramide by human milk bile salt-stimulated lipase.

There is a renewed interest in metabolism of sphingolipids because of their role in signal transduction. Sphingomyelin is the dominating phospholipid in human milk but its metabolism and possible function in the gastrointestinal tract of breast fed infants is unknown. We explored whether bile salt-stimulated milk lipase has a role in sphingolipid metabolism. In vitro assays of sphingomyelinase and ceramidase activities, using radiolabeled substrates, human milk samples and purified native and recombinant variants of bile salt-stimulated milk lipase with or without known activators or inhibitors. Human whey and purified lipase catalysed hydrolysis of palmitoyl-labeled ceramide with the highest rate around pH 8.5-9.0. 1 mg of lipase hydrolysed 0.7 micromol ceramide in one hour at pH 8.5 in presence of 4 mM bile salt. The activity of whey was inhibited by antibodies towards human bile salt-stimulated milk lipase, indicating that this lipase accounted for virtually all ceramidase activity in the milk. In contrast, bile salt-stimulated milk lipase showed no activity against sphingomyelin. However we give evidence of a separate, hitherto unknown, acid sphingomyelinase in human milk. Under the used in vitro conditions this sphingomyelinase could account for hydrolysis of half of milk sphingomyelin in one hour. Human milk bile salt-stimulated milk lipase hydrolyses ceramide and may thus have a role in sphingomyelin digestion, but only after initial hydrolysis to ceramide and phosphorylcholine. Part of the latter could be carried out in the stomach by the acid milk sphingomyelinase now described. We speculate that these two milk enzymes may be of importance for optimal use of human milk sphingolipids.

Molecular Cloning of the Oncofetal Isoform of the Human Pancreatic Bile Salt-dependent Lipase

Specific transcripts for bile salt-dependent lipase (BSDL), a 100-kDa glycoprotein secreted by the human pancreas, were immunodetected in BxPC-3 and SOJ-6 pancreatic tumoral cell lines. Sequencing of fragments, obtained by mRNA reverse transcription and amplification, confirmed the presence of BSDL transcripts in these cancer cells. The protein was detected in lysates of pancreatic tumoral cells, where it was mainly associated with membranes. Only a minute amount of the enzyme was detected in the culture media. Immunofluorescence studies demonstrated that in SOJ-6 cells, BSDL colocates with the p58 Golgi protein and suggested that the protein may be sequestrated within the Golgi compartment. These results demonstrated that BSDL is expressed in human pancreatic tumoral cells and cannot be secreted (or for the least very poorly). Subsequently, a cDNA covering the entire sequence of BSDL was obtained by reverse transcription-polymerase chain reaction. The sequence of this cDNA indicated that the N-terminal domain encoded by exons 1-10 was identical to that of BSDL expressed by the human normal pancreas. However, the sequence corresponding to exon 11, which should code for the 16 tandem-repeated identical mucin-like sequences of BSDL, was deleted by 330 base pairs (bp) and encoded only 6 of these repeated sequences. We conclude that this truncated variant of BSDL would be its oncofetal form, referred to as feto-acinar pancreatic protein. We then investigated whether the deletion of 330 bp affected the secretion of the protein. For this purpose, the cDNA corresponding to the mature form of the BSDL variant expressed in SOJ-6 cells was cloned into an expression/secretion vector and transfected into CHO-K1 cells. Results indicated that the variant of BSDL isolated from SOJ-6 cells was expressed and secreted by transfected cells. However, the level of BSDL secreted by these transfected CHO-K1 cells was significantly higher than that observed for SOJ-6 cells. Consequently, the retention of the oncofetal variant of BSDL observed in human pancreatic tumoral cells might not result from inherent properties of the protein.

Effects of ethanol on the expression and secretion of bile salt-dependent lipase by pancreatic AR4-2J cells

The mechanisms by which ethanol administration alters pancreatic function are unknown. We have evaluated the effects of chronic ethanol treatment on secretion of a digestive enzyme: the bile salt-dependent lipase (BSDL), by the rat pancreatic cell line AR4-2J (as a model). We report that ethanol (50–300 mM) in culture medium induced a rise, in secreted and intracellular BSDL, that was a function of the duration of treatment and of the ethanol concentration. This effect was not abolished by pyrazole, which suggests a direct effect of ethanol. We have further established that the increase of BSDL activity was due to an enhanced biosynthesis of the enzyme consecutive to a major steady-state level of mRNA encoding BSDL. Also, the subcellular localization showed a specific accumulation of BSDL in the cytosolic fraction of cells chronically treated with ethanol. Given the enzymatic properties of BSDL, all these data could have some physiological consequences regarding the digestive function, plasma lipid metabolism and intracellular cholesterol homeostasis.

Bile salt-stimulated lipase (BSSL) distribution in rat, mouse and transgenic mouse expressing human BSSL.

In some species, including man and mouse, bile salt-stimulated lipase (BSSL) in milk catalyzes the hydrolysis of triacylglycerides into glycerol and free fatty acids, a reaction that is of particular importance during suckling. The enzyme is also secreted by the pancreas (referred to as carboxyl-ester hydrolase, CEH). We wished to localize sources and storage sites for BSSL/CEH in rats, in wild-type mice, and in transgenic mice producing recombinant human BSSL in milk. Immunoreactivity against several BSSL fragments was strong in the pancreatic acinar cells and moderate in the absorptive cells of the small intestine and in salivary duct cells of the mice, as well as in rats. Sections from lactating mammary glands of mouse, but not rat, also showed immunoreactivity for BSSL; the signal was strongest in the transgenic mice. Radioactive riboprobes for BSSL mRNA hybridized on sections of rat and mouse pancreatic acinar cells, and mouse mammary glands (both wild-type and transgenic). Using RT-PCR, it was possible to amplify BSSL mRNA from wild-type mouse pancreas and mammary gland, from rat submandibular glands, and, in a few cases, from rat liver. In transgenic mice, the BSSL mRNA was highly expressed only in lactating mammary gland, but could be detected in a few other organs as well.

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.

Molecular cloning of the bile salt-dependent lipase of ferret lactating mammary gland: an overview of functional residues

Ferret lactating mammary gland bile salt-dependent lipase (BSDL, EC 3.1.1.-) has been cloned by RT-PCR. The open reading frame consists of 1869 nucleotides which encode 623 amino acids of the functional enzyme. When compared to other species, the greatest homology is observed between residues 1 and 484, with little or no homology at the C-terminal end where seven repeated segments of similar sequence are located. Ferret mammary gland BSDL retains residues involved in the active site and the tentative heparin binding site at similar positions in comparison to other milk or pancreatic BSDL. Other important items, such as binding peptide to chaperone molecular, phosphorylation site(s) or bile salt binding sites, were also tentatively located in well conserved regions of seven available BSDL sequences.

Bile salt-stimulated lipase and digestion of non-breast milk fat.

13 Carbon (13C)-lipid breath tests are an effective, noninvasive way of repeatedly measuring fat digestion. The purpose of this study was to assess the contribution of bile salt-stimulated lipase (BSSL) in human milk to the digestion of non-breast-milk fat in Gambian infants. Twelve Gambian infants (aged 3-8 months) were studied on 4 days. 13C-Trioctanoin (7.5 mg/kg, digested by BSSL preduodenal and pancreatic lipases) and 13C-cholesteryl octanoate (25 mg/kg, digested by BSSL and pancreatic lipases) were used as substrates. The percentage dose recovery (PDR) of 13C in breath during 5 hours was compared after ingestion of each substrate with fresh, expressed breast milk (FBM) or heated, expressed breast milk (HBM). Gas isotope ratio-mass spectrometry was used to measure 13C enrichment, and breast milk samples were analysed for esterase activity. Heating breast milk significantly decreased esterase activity (mean +/- SD values: FBM = 12.2 +/- 2.9 IU/ml; HBM = 0.5 +/- 0.3 IU/ml), and there was no difference in the volumes of milk ingested on each test day (approximately 50 ml). The PDR of 13C was comparable to that previously described in healthy English infants and was not increased by BSSL. The mean +/- SD PDR of 13C from trioctanoin was 36.3 +/- 8.4% for FBM and 34.6 +/- 6.3% for HBM (NS). From cholesteryl octanoate, the mean +/- SD PDR of 13C was 24.3 +/- 8.7% for FBM and 27.1 +/- 7.5% for HBM (NS). Bile salt-stimulated lipase may enhance fat digestion in younger or malnourished infants who have a greater degree of pancreatic enzyme deficiency. However, this study suggests that it does not increase the digestion of non-breast-milk fat in healthy, well-nourished infants aged 3 to 8 months from an underprivileged background, who typically ingest frequent small quantities of breast milk.

Incidence of molecular forms of bile salt-stimulated lipase in preterm and term human milk.

Preterm and term human milk samples obtained at various times after delivery were analyzed for the presence of molecular forms of the human milk enzyme, bile salt-stimulated lipase (BSSL). Thirty-five percent of both the preterm and term milk samples contained two molecular forms of BSSL, of variable molecular mass. The remainder contained only one molecular species of either 115 kD (50%) or 120 kD (15%). The number of molecular forms present was not related to length of lactation, maternal age, gestation, or maternal blood group. The specific activity of BSSL purified from term milk was similar to that purified from preterm milk, and there was no difference in specific activity whether one or two molecular forms were present. This study demonstrates heterogeneity of both molecular mass and molecular forms. We conclude that preterm babies fed their own mother's milk are unlikely to be disadvantaged with respect to fat digestion as BSSL secreted in preterm milk appears to be very similar to that produced in term milk, although we cannot exclude other functional differences.

Naturally Occurring Variants of Human Milk Bile Salt-Stimulated Lipase

Analysis of milk samples from a number of lactating women revealed molecular variants of bile salt-stimulated lipase (BSSL) of both lower and higher molecular mass than that commonly occurring. In contrast to previous observations, we report on individuals having only a variant of lower mass, both one of lower and one of common mass, or both one of lower and one of higher mass of the lipase. From two individuals we purified the lower molecular mass BSSL variant and characterized it. The amount of lipase in the milk of these two individuals was considerably less than average (mean of 10 women with BSSL of the most common molecular mass). The BSSL variant of lower mass showed the same bile salt activation, pH dependency, temperature stability as those most commonly occurring. We could localize the difference in mass to the large O-glycosylated repeat sequence close to the C-terminus of the protein. With respect to all characteristics studied, the BSSL variant of higher mass was also similar to that most commonly ocurring. Again, the difference in mass could be localized to the repeat region of the protein. Hence, it appears as if the repeat region, normally carrying 16 repeats of 11 amino acids each, varies in size between individuals.

Pancreatic bile salt-dependent lipase activity in serum of normolipidemic patients.

Bile salt-dependent lipase (BSDL, E.C. 3.1.1.-) is a digestive enzyme secreted by the pancreatic acinar cell. Once in the duodenum, the enzyme, upon activation by primary bile salts, hydrolyzes dietary lipid esters such as cholesteryl esters and lipid-soluble vitamin esters. This enzyme is partially transferred from the duodenum or pancreas to the circulation where it has been postulated to exert a systemic action on atheroma-generating oxidized-low density lipoprotein (LDL). In the present study, sera from 40 healthy normolipidemic volunteers were used to investigate the possible linkage between circulating BSDL, lipids, and lipoproteins. We showed, firstly, that pancreatic-like BSDL activity can be detected in these serums. Secondly, BSDL activity increased significantly with the level of LDL-cholesterol and was also positively linked to the serum concentration of Apo B100 and Apo A-I. Thirdly, we also established that BSDL was associated with LDL, in part by a specific interaction with Apo B100, while no interaction was found with Apo A-I. No linkage with other recorded parameters (triglycerides, phospholipids, and high density lipoprotein-cholesterol) was detected. Because an increase in LDL-cholesterol represents an important risk factor for atheroma, the concomitant increase in BSDL, which can metabolize atherogenic LDL, suggests for the first time that this circulating enzyme may exert a positive effect against atherosclerosis.

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.