Detection of bile salt-dependent lipase, a 110 kDa pancreatic protein, in urines of healthy subjects
2006; Elsevier BV; Volume: 69; Issue: 6 Linguagem: Inglês
10.1038/sj.ki.5000133
ISSN1523-1755
AutoresBlandine Comte, C. Franceschi, Marie‐Odile Sadoulet, Françoise Silvy, Daniel Lafitte, L Benkoël, Alain Nganga, Laurent Daniel, J.-P. Bernard, Dominique Lombardo, Eric Mas,
Tópico(s)Diabetes Management and Research
ResumoBile 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. 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. Bile salt-dependent lipase (BSDL, E.C.3.1.1.13) is an enzyme involved in the duodenal hydrolysis of cholesteryl esters.1.Shamir R. Johnson W.J. Zolfaghari R. et al.Role of bile salt-dependent cholesteryl ester hydrolase in the uptake of micellar cholesterol by intestinal cells.Biochemistry. 1995; 34: 6351-6358Crossref PubMed Scopus (56) Google Scholar, 2.Howles P.N. Carter C.P. Hui D.Y. Dietary free and esterified cholesterol absorption in cholesterol esterase (bile salt-stimulated lipase) gene-targeted mice.J Biol Chem. 1996; 271: 7196-7202Crossref PubMed Scopus (163) Google Scholar The enzyme is synthesized in the endoplasmic reticulum of pancreatic acinar cells, then follows the secretory pathway of these cells to be secreted as a component of the pancreatic juice.3.Lombardo D. Bile salt-dependent lipase: its pathophysiological implications.Biochim Biophys Acta. 2001; 1533: 1-28Crossref PubMed Scopus (69) Google Scholar Once in the duodenum, BSDL is activated by bile salts and participates in the hydrolysis of dietary lipid esters. However, a fraction of BSDL is internalized by enterocytes via the oxidized low-density lipoprotein lectin-like receptor 1 (Lox-1) scavenger receptor4.Bruneau N. Richard S. Silvy F. et al.Lectin-like Ox-LDL receptor is expressed in human INT-407 intestinal cells: involvement in the transcytosis of pancreatic bile salt-dependent lipase.Mol Biol Cell. 2003; 14: 2861-2875Crossref PubMed Scopus (20) Google Scholar and transported to the blood compartment,5.Bruneau N. Bendayan M. Gingas D. et al.Circulating bile salt-dependent lipase originates from the pancreas via intestinal transcytosis.Gastroenterology. 2003; 124: 470-480Abstract Full Text PDF PubMed Scopus (25) Google Scholar where it partly associates with apolipoprotein B-containing lipoproteins.6.Caillol N. Pasqualini E. Mas E. et al.Pancreatic bile salt-dependent lipase activity in serum of normolipidemic patients.Lipids. 1997; 32: 1147-1153Crossref PubMed Scopus (30) Google Scholar BSDL was detected in atherosclerotic lesions of hypercholesterolemic monkeys and in the human aortic endothelium.7.Shamir R. Johnson W.J. Morlock-Fitzpatrick K. et al.Pancreatic carboxyl ester lipase: a circulating enzyme that modifies normal and oxidized lipoproteins in vitro.J Clin Invest. 1996; 97: 1696-1704Crossref PubMed Scopus (71) Google Scholar, 8.Augé N. Rebaï O. Le Petit-Thévenin J. et al.Pancreatic bile salt-dependent lipase induces smooth muscle cells proliferation.Circulation. 2003; 108: 86-91Crossref PubMed Scopus (16) Google Scholar The role of BSDL in blood and in part in atherosclerosis is still speculative.7.Shamir R. Johnson W.J. Morlock-Fitzpatrick K. et al.Pancreatic carboxyl ester lipase: a circulating enzyme that modifies normal and oxidized lipoproteins in vitro.J Clin Invest. 1996; 97: 1696-1704Crossref PubMed Scopus (71) Google Scholar, 8.Augé N. Rebaï O. Le Petit-Thévenin J. et al.Pancreatic bile salt-dependent lipase induces smooth muscle cells proliferation.Circulation. 2003; 108: 86-91Crossref PubMed Scopus (16) Google Scholar, 9.Rebaï O. Le Petit-Thevenin J. Bruneau N. et al.Pancreatic bile salt-dependent lipase induces smooth muscle cells proliferation.Arteriosclerosis Thrombosis Vasc Biol. 2005; 25: 359-364Crossref PubMed Scopus (21) Google Scholar, 10.Brodt-Eppley J. White P. Jenkins S. Hui D.Y. Plasma cholesterol esterase level is a determinant for an atherogenic lipoprotein profile in normolipidemic human subjects.Biochim Biophys Acta. 1995; 1272: 69-72Crossref PubMed Scopus (75) Google Scholar, 11.Hui D.Y. Howles P.N. Carboxyl ester lipase: structure-function relationship and physiological role in lipoprotein metabolism and atherosclerosis.J Lipid Res. 2002; 43: 2017-2030Crossref PubMed Scopus (151) Google Scholar Following its entrance into the blood stream, BSDL should be eliminated. This enzyme is a 110 kDa protein with a radius of 53.4 Å;12.Guy O. Lombardo D. Brahms J.G. Structure and conformation of human pancreatic carboxyl-ester hydrolase.Eur J Biochem. 1981; 117: 457-460Crossref PubMed Scopus (35) Google Scholar consequently it cannot be theoretically cleared from the circulation by renal filtration.13.D'Amico G. Bazzi C. Pathophysiology of proteinuria.Kidney Int. 2003; 63: 809-825Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar Data suggested that BSDL participates in the hepatic selective uptake of high-density lipoproteins; therefore, one possibility is that blood BSDL is partly eliminated via liver captation.14.Camarota L.M. Chapman J.M. Hui D.Y. Howles P.N. Carboxyl ester lipase cofractionates with scavenger receptor BI in hepatocyte lipid rafts and enhances selective uptake and hydrolysis of cholesteryl esters from HDL3.J Biol Chem. 2004; 279: 27599-27606Crossref PubMed Scopus (27) Google Scholar The high-density lipoprotein particle undergoes a retroendocytosis process during which lipids are separated from the holoparticle for delivery to the bile, whereas the protein moiety is re-secreted in the circulation15.Silver D.L. Wang N. Xiao X. Tall A.R. High density lipoprotein (HDL) particle uptake mediated by scavenger receptor class B type 1 results in selective sorting of HDL cholesterol from protein and polarized cholesterol secretion.J Biol Chem. 2001; 276: 25287-25293Crossref PubMed Scopus (200) Google Scholar supporting the fact that BSDL may not be eliminated by this way. Because BSDL is a sialylated glycoprotein,3.Lombardo D. Bile salt-dependent lipase: its pathophysiological implications.Biochim Biophys Acta. 2001; 1533: 1-28Crossref PubMed Scopus (69) Google Scholar another possibility is the liver clearance of plasma asialoglycoproteins.16.Mortensen B. Huseby N.E. Clearance of circulating gamma-glutamyltransferase by the asialoglycoprotein receptor. Enzyme forms with different sialic acid content are eliminated at different clearance rates and without apparent desialylation.Clin Chim Acta. 1997; 258: 47-58Crossref PubMed Scopus (17) Google Scholar In this study, we have examined the possible involvement of the kidney in the clearance of blood BSDL. We demonstrated that BSDL is present in urines of healthy subjects and likely originates from the pancreas via circulation. Morning urines were analyzed for their reactivity with antibodies against pancreatic BSDL. Urines were centrifugated to eliminate cell debris and other insoluble elements, then 25 μl of each sample was analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto nitrocellulose membranes. Membranes were probed with the pAbL64 or with the mAb8H8. All urines were reactive to these antibodies and a protein can be immunodetected at 110 kDa. This mass corresponds to that of the human pancreatic BSDL3.Lombardo D. Bile salt-dependent lipase: its pathophysiological implications.Biochim Biophys Acta. 2001; 1533: 1-28Crossref PubMed Scopus (69) Google Scholar. Figure 1 displays results obtained for healthy subjects Nb 1–8 given as an example. Along with a band at 110 kDa, some bands with a higher migration can be detected with antibodies. This could be because of a polymorphism of the immunoreactive protein or degradation occurring during sample handling. However, freeze–thawing cycles or urine incubation (24 h) at 37°C followed by aliquot analyses on SDS-PAGE and immunoblotting do not correlate with the degradation of the pAbL64 immunoreactive protein (data not shown). This favors a polymorphism of this protein in urines, which could reflect that of the pancreatic BSDL.17.Nilsson J. Hellquist M. Bjursell G. The human carboxyl ester lipase-like (CELL) gene is ubiquitously expressed and contains a hypervariable region.Genomics. 1993; 17: 416-422Crossref PubMed Scopus (25) Google Scholar, 18.Bengtsson-Ellmark S.H. Nilsson J. Orho-Melander M. et al.Association between a polymorphism in the carboxyl ester lipase gene and serum cholesterol profile.Eur J Hum Genet. 2004; 12: 627-632Crossref PubMed Scopus (17) Google Scholar To demonstrate that the pAbL64 and mAb8H8 immunoreactive protein in urine is BSDL, 5 ml of concentrated urine was desalted on Sephadex G-25. The eluted fractions associated with an activity on 4-nitrophenyl caproate (4-NPC) were also reactive with the mAb8H8, showing that the immunoreactivity and the esterolytic activity are present together in these fractions. The eluted fractions were pooled, concentrated by lyophilization and dissolved in 1 ml Tris/HCl buffer (10 mM, pH 7.8) 400 mM NaCl. The concentrated pool was active on cholesteryl esters (12.1±1.5 pmol/min/ml). This material was then loaded on a Sephacryl HRS-200 column. Fractions of 1 ml were collected after the void volume (40 ml) of the column. The 4-NPC activity is still associated with the pAbL64 reactivity (Figure 2). Eluted fractions 78–90 containing both esterolytic activity and immunoreactivity were pooled. At this stage, the specific activity of the enzyme is increased by approximately 300% (Table 1). The pooled fractions (12 ml) were dialyzed against water at 4°C and lyophilized. An SDS-PAGE showed a main protein band at 110 kDa, which was reactive with pAbL64 (Figure 3). Same results were obtained with all urines examined up to now.Table 1Purification step of BSDL from healthy subjects urinesProteinsEsterolytic activityaActivities determined on 4-NPC.Specific activityaActivities determined on 4-NPC.Purification step(μg/ml)(10−3 U/ml)(U/mg)Concentrated urines28 × 1042581 × 10−3Desalted fraction (Sephadex G25)4703472 × 10−3Molecular sieving (Sephacryl HRS-200)3510290 × 10−3a Activities determined on 4-NPC. Open table in a new tab Figure 3SDS-PAGE and immunodetection analysis of the material loaded and eluted from molecular sieving on Sephacryl HRS-200. (a) SDS-PAGE of material loaded (lane 1) and eluted (lane 2) from the column. Proteins were detected by Coomassie blue staining. (b) Immunodetection using pAbL64 of the material loaded (lane 1) and eluted (lane 2) from the column. Arrowheads (left) indicate the position of calibrated molecular mass standard proteins and arrows (right) indicate the apparent molecular size of BSDL detected in urines.View Large Image Figure ViewerDownload (PPT) These data support the presence of an active immunoreactive form of BSDL in urines of healthy subjects. To ascertain that this protein is actually BSDL, the material eluted from the molecular sieving column was loaded on a large SDS-PAGE. One lane of this gel was electrotransferred onto a nitrocellulose membrane and probed with pAbL64 to detect the immunoreactive protein. Another lane was treated with Coomassie blue, and the area of this band corresponding to the immunoreactivity was cut into four slices (Figure 4). A peptide mass fingerprinting was performed on each slice. The matrix-assisted laser desorption ionization (MALDI) mass spectrum of the digest of slice 1 (Figure 5, upper panels) showed 12 peaks identified as tryptic peptides of BSDL. Slice 2 gave the same results, whereas not enough material was detected in slices 3 and 4. The coverage of the BSDL sequence (Figure 5, lower panel) is close to 20% and suggested that the pAbL64 and mAb8H8 immunoreactive protein in urine is actually BSDL. Taking into account that the O-glycosylated C-terminal domain of BSDL (framed sequence in the lower panel of Figure 5) cannot be digested by trypsin, the sequence coverage (underlined sequence in the lower panel of Figure 5) is satisfactory. The glycosylation of pancreatic BSDL differs from the pathophysiological state of the pancreas, and BSDL expressed by tumoral pancreas bears the oncofetal glycotope recognized by mAbJ28.19.Escribano M.J. Cordier J. Nap M. et al.Differentiation antigens in fetal human pancreas. Reexpression in cancer.Int. J. Cancer. 1986; 38: 155-160Crossref PubMed Scopus (31) Google Scholar Any other tissue in humans even those expressing BSDL, displayed this epitope.19.Escribano M.J. Cordier J. Nap M. et al.Differentiation antigens in fetal human pancreas. Reexpression in cancer.Int. J. Cancer. 1986; 38: 155-160Crossref PubMed Scopus (31) Google Scholar, 20.Verine A. Bruneau N. Valette A. et al.Immunodetection and molecular cloning of a bile-salt-dependent lipase isoform in HepG2 cells.Biochem J. 1999; 342: 179-187Crossref PubMed Google Scholar Therefore, urine from a patient afflicted with pancreatic cancer was analyzed on SDS-PAGE, transferred onto a nitrocellulose membrane and probed with pAbL64, mAb8H8 and mAbJ28. BSDL in this urine sample is reactive with pAbL64 and mAb8H8 (Figure 6a). When the membrane was probed with mAbJ28 (lane 3) or mAb8H8 (lane 2) instead of pAbL64 (lane 1), a reactivity associated with a protein around 110 kDa was detected. Therefore, BSDL present in urines could harbor the J28 epitope, suggesting its pancreatic origin. To support this particular point, BSDL has been immunoprecipitated from desalted urines of this patient with the pAbL64. Immunoprecipitated material was separated on SDS-PAGE and electrotransferred on a nitrocellulose membrane further probed with pAbL64, mAb8H8 or mAbJ28. The pAbL64 immunoprecipitated material was of course reactive with pAbL64 polyclonal antibodies (Figure 6b, lane 1) and also with monoclonal antibodies mAb8H8 (lane 2) or mAbJ28 (lane 3); therefore, BSDL in urines of a patient afflicted with a pancreatic adenocarcinoma harbors the pancreatic-specific J28 epitope. In the first instance, these results suggest that BSDL in urine is representative of the pancreatic glycoform. If BSDL in urine comes from the pancreas, the enzyme should transit via the blood and the kidney glomerulus. Therefore, we have analyzed human kidney proteins. When these proteins were separated on SDS-PAGE, transferred onto nitrocellulose membrane and finally probed with pAbL64, mAb8H8 or mAbJ28, no material migrating with the known size of the pancreatic protein3.Lombardo D. Bile salt-dependent lipase: its pathophysiological implications.Biochim Biophys Acta. 2001; 1533: 1-28Crossref PubMed Scopus (69) Google Scholar could be detected (Figure 6d) whereas in tumoral pancreatic homogenate, BSDL was reactive with these three antibodies (Figure 6c). These data along with those demonstrating that no mRNA transcript encoding BSDL can be detected in human adult kidney21.Roudani S. Miralles F. Margotat A. et al.Bile salt-dependent lipase transcripts in human fetal tissues.Biochim Biophys Acta. 1995; 1264: 141-150Crossref PubMed Scopus (24) Google Scholar support that BSDL in urine originates from the blood. We next attempted to localize BSDL in the human normal kidney using immunohistochemistry. For this purpose, serial sections of human kidney were incubated with pAbL64 and markers of distal convoluted tubules (mAb anti-EMA), proximal tubules (mAb anti-vimentin) and endothelial cells (mAb anti-CD34). The antibody–antigen complexes were further detected with alkaline phosphatase-conjugated secondary antibodies. Distal convoluted tubules of the renal cortex characterized by a strong and diffuse staining with apical polarity with mAb anti-EMA were also stained by pAbL64 (Figure 7a). Proximal tubules showed an expression of vimentin at the basolateral membrane of cells, whereas BSDL was not detected (Figure 7b). A diffuse staining pattern was found in capillary arteries irrigating the glomerulus with mAb anti-CD34 and pAbL64 (Figure 7c). No staining was found in control sections without primary antibodies. However, cells within this cortical area were unreactive with these polyclonal antibodies supporting the fact that BSDL of urines is not expressed by kidney cells. These data suggested that circulating BSDL is filtered by the kidney glomerulus to be eliminated in urines without reabsorption by the proximal convoluted tubules. Quantitation of BSDL using the enzyme-linked immunosorbent assay method previously described to quantitate BSDL in sera22.Lombardo D. Montalto G. Roudani S. et al.Is bile salt-dependent lipase concentration in serum of any help in pancreatic cancer diagnosis?.Pancreas. 1993; 8: 581-588Crossref PubMed Scopus (31) Google Scholar did not give accurate results in urines. Therefore, quantitation of BSDL in urine was performed by dot-blot quantitations corrected for the unspecific reactivity (using secondary antibodies alone) and reported to the immunoreactivity of a standard solution of BSDL. Under these conditions, we can estimate that BSDL concentration in urines of healthy subjects ranges between 1100 and 100 μg/l (290±370 μg/l, n=9). Compared to the concentration of BSDL in serum as determined by enzyme-linked immunosorbent assay, that is 1.5 μg/l, this result suggests that the protein is efficiently filtrated and concentrated in urines. Albumin was also determined by dot-blot dilution using anti-human serum albumin (Sigma St Louis, MO). In our urine samples, the serum concentration of albumin was within the range of normal values, that is 1–5 mg/l (2.5±1.2 mg/l, n=9). From the literature, blood albumin concentration is close to 35–50 g/l. Clearly, these data demonstrate that BSDL is concentrated in urine. This study provides evidences that the circulating BSDL, which is of pancreatic origin,5.Bruneau N. Bendayan M. Gingas D. et al.Circulating bile salt-dependent lipase originates from the pancreas via intestinal transcytosis.Gastroenterology. 2003; 124: 470-480Abstract Full Text PDF PubMed Scopus (25) Google Scholar is in part eliminated from the blood by renal filtration. BSDL is found in all urines examined up to now and it is surprisingly intact and active both on water-soluble 4-NPC and on micellar cholesteryl esters. The presence of BSDL in urine was also ascertained by Western blot using a specific monoclonal antibody to the pancreatic enzyme. The protein was further isolated by molecular sieving from urine of healthy subjects. Finally, mass spectrometry allows unambiguous characterization of the enzyme. Immunohistochemistry images suggested that BSDL is concentrated in capillaries of Bowman's capsule and in distal convoluted tubules. Northern blots and reverse transcriptase-polymerase chain reaction performed on renal nucleic acids21.Roudani S. Miralles F. Margotat A. et al.Bile salt-dependent lipase transcripts in human fetal tissues.Biochim Biophys Acta. 1995; 1264: 141-150Crossref PubMed Scopus (24) Google Scholar and immunodetection experiments support that BSDL could be filtrated by the glomerulus and likely not expressed by any cells of the kidney. A recent proteomic profiling of low-density urinary membrane fractions produced by renal epithelial cells23.Pisitkun T. Shen R.F. Knepper M.A. Identification and proteomic profiling of exosomes in human urine.Proc Natl Acad Sci USA. 2004; 101: 13368-13373Crossref PubMed Scopus (1437) Google Scholar identified more than 250 proteins except BSDL. This suggests that BSDL might not originate from renal epithelial cells. The first question is why had BSDL not been detected in urine before this present work? The main reason is likely that the detection method used here is immunological and that previous studies produced selective maps.24.Bueler M.R. Wiederkehr F. Vonderschmitt D.J. Electrophoretic, chromatographic and immunological studies of human urinary proteins.Electrophoresis. 1995; 16: 124-134Crossref PubMed Scopus (33) Google Scholar Also two-dimentional electrophoreses profiling of proteins in urine followed by mass spectrometry and sequence database searches generally concern proteins of a size smaller than that of albumin (i.e. 53 Å).12.Guy O. Lombardo D. Brahms J.G. Structure and conformation of human pancreatic carboxyl-ester hydrolase.Eur J Biochem. 1981; 117: 457-460Crossref PubMed Scopus (35) Google Scholar Molecular modeling indicates that the N-terminal domain of BSDL is a dipole with the positive potential culminating at the N-terminal basic cluster.35.Feaster S.R. Quinn D.M. Barnett B.L. Molecular modeling of the structures of human and rat pancreatic cholesterol esterases.Protein Sci. 1997; 6: 73-79Crossref PubMed Scopus (17) Google Scholar This cationic head, with the extended C-terminal domain protruding on the opposite side of the molecule may serve to guide the protein within the pore and allow the attraction of the protein by heparan sulfate of the basement membrane. This problate ellipsoid with a stron
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