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• W2029006507 abstract "Enteropeptidase is a heterodimeric type II membrane protein of the brush border of duodenal enterocytes. In this location, enteropeptidase cleaves and activates trypsinogen, thereby initiating the activation of other intestinal digestive enzymes. Recombinant bovine enteropeptidase was sorted directly to the apical surface of polarized Madin-Darby canine kidney cells. Replacement of the cytoplasmic and signal anchor domains with a cleavable signal peptide (mutant proenteropeptidase lacking the amino-terminal signal anchor domain (dSA-BEK)) caused apical secretion. The additional amino-terminal deletion of a mucin-like domain (HL-BEK) resulted in secretion both apically and basolaterally. Further deletion of the noncatalytic heavy chain (L-BEK) resulted in apical secretion. Thus enteropeptidase appears to have at least three distinct sorting signals as follows: the light chain (L-BEK) directs apical sorting, addition of most of the heavy chain (HL-BEK) inhibits apical sorting, and addition of the mucin-like domain (dSA-BEK) restores apical sorting. Inhibition of N-linked glycosylation with tunicamycin or disruption of microtubules with colchicine caused L-BEK to be secreted equally into apical and basolateral compartments, whereas brefeldin A caused basolateral secretion of L-BEK. Full-length BEK was not found in detergent-resistant raft domains of Madin-Darby canine kidney cells or baby hamster kidney cells. These results suggest apical sorting of enteropeptidase depends on N-linked glycosylation of the serine protease domain and an amino-terminal segment that includes an O-glycosylated mucin-like domain and three potential N-glycosylation sites. In contrast to many apically targeted proteins, enteropeptidase does not form detergent-resistant associations with sphingolipid-cholesterol rafts. Enteropeptidase is a heterodimeric type II membrane protein of the brush border of duodenal enterocytes. In this location, enteropeptidase cleaves and activates trypsinogen, thereby initiating the activation of other intestinal digestive enzymes. Recombinant bovine enteropeptidase was sorted directly to the apical surface of polarized Madin-Darby canine kidney cells. Replacement of the cytoplasmic and signal anchor domains with a cleavable signal peptide (mutant proenteropeptidase lacking the amino-terminal signal anchor domain (dSA-BEK)) caused apical secretion. The additional amino-terminal deletion of a mucin-like domain (HL-BEK) resulted in secretion both apically and basolaterally. Further deletion of the noncatalytic heavy chain (L-BEK) resulted in apical secretion. Thus enteropeptidase appears to have at least three distinct sorting signals as follows: the light chain (L-BEK) directs apical sorting, addition of most of the heavy chain (HL-BEK) inhibits apical sorting, and addition of the mucin-like domain (dSA-BEK) restores apical sorting. Inhibition of N-linked glycosylation with tunicamycin or disruption of microtubules with colchicine caused L-BEK to be secreted equally into apical and basolateral compartments, whereas brefeldin A caused basolateral secretion of L-BEK. Full-length BEK was not found in detergent-resistant raft domains of Madin-Darby canine kidney cells or baby hamster kidney cells. These results suggest apical sorting of enteropeptidase depends on N-linked glycosylation of the serine protease domain and an amino-terminal segment that includes an O-glycosylated mucin-like domain and three potential N-glycosylation sites. In contrast to many apically targeted proteins, enteropeptidase does not form detergent-resistant associations with sphingolipid-cholesterol rafts. Enteropeptidase (enterokinase) is a protease of the duodenal brush border that cleaves and activates trypsinogen. The resultant trypsin then activates other pancreatic digestive zymogens within the lumen of the gut. Deficiency of enteropeptidase causes intestinal malabsorption (1Hadorn B. Tarlow M.J. Lloyd J.K. Wolff O.H. Lancet. 1969; i: 812-813Abstract Google Scholar, 2Haworth J.C. Gourley B. Hadorn B. Sumida C. J. Pediatr. 1971; 78: 481-490Abstract Full Text PDF PubMed Scopus (53) Google Scholar), and intrusion of enteropeptidase into the pancreas may contribute to hemorrhagic pancreatitis (3Hammond J.B. Mann N.S. Dig. Dis. 1977; 22: 182-188Crossref Scopus (10) Google Scholar, 4Grant D. Int. J. Pancreatol. 1986; 1: 167-183PubMed Google Scholar). Therefore, the localization of enteropeptidase is important to normal digestive physiology. Enteropeptidase consists of a disulfide-linked heterodimer with a heavy chain of 82–140 kDa and a light chain of 35–62 kDa. Both chains of mammalian enteropeptidases contain 30–50% carbohydrate, and this extensive glycosylation may contribute to the apparent variation in polypeptide masses (reviewed in Ref. 5Lu D. Sadler J.E. Barrett A.J. Rawlings N.D. Woessner Jr., J.F. Handbook of Proteolytic Enzymes. Academic Press Ltd., London1998: 50-54Google Scholar). Amino acid sequences deduced by cDNA cloning of bovine (6LaVallie E.R. Rehemtulla A. Racie L.A. DiBlasio E.A. Ferenz C. Grant K.L. Light A. McCoy J.M. J. Biol. Chem. 1993; 268: 23311-23317Abstract Full Text PDF PubMed Google Scholar, 7Kitamoto Y. Yuan X. Wu Q. McCourt D.W. Sadler J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7588-7592Crossref PubMed Scopus (148) Google Scholar), porcine (8Matsushima M. Ichinose M. Yahagi N. Kakei N. Tsukada S. Miki K. Kurokawa K. Tashiro K. Shiokawa K. Shinomiya K. Umeyama H. Inoue H. Takahashi T. Takahashi K. J. Biol. Chem. 1994; 269: 19976-19982Abstract Full Text PDF PubMed Google Scholar), human (9Kitamoto Y. Veile R.A. Donis-Keller H. Sadler J.E. Biochemistry. 1995; 34: 4562-4568Crossref PubMed Scopus (62) Google Scholar), mouse (10Yuan X. Zheng X.L. Lu D.S. Rubin D.C. Pung C.Y.M. Sadler J.E. Am. J. Physiol. 1998; 37: G342-G349Google Scholar), and rat enteropeptidase (11Yahagi N. Ichinose M. Matsushima M. Matsubara Y. Miki K. Kurokawa K. Fukamachi H. Tashiro K. Shiokawa K. Kageyama T. Takahashi T. Inoue H. Takahashi K. Biochem. Biophys. Res. Commun. 1996; 219: 806-812Crossref PubMed Scopus (35) Google Scholar) indicate that active two-chain enteropeptidase is derived from a single-chain precursor. The amino-terminal heavy chain contains domains that are homologous to sequences of O-glycosylated epithelial mucins, the low density lipoprotein receptor, complement components C1r and C1s, the macrophage scavenger receptor, and a recently described MAM motif. The carboxyl-terminal light chain is homologous to the trypsin-like serine proteases (reviewed in Ref. 5Lu D. Sadler J.E. Barrett A.J. Rawlings N.D. Woessner Jr., J.F. Handbook of Proteolytic Enzymes. Academic Press Ltd., London1998: 50-54Google Scholar). Studies of recombinant bovine enteropeptidase demonstrate that membrane association is mediated by a signal anchor sequence near the amino terminus (12Lu D. Yuan X. Zheng X. Sadler J.E. J. Biol. Chem. 1997; 272: 31293-31300Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The structures on enteropeptidase that direct it to apical membranes have not been characterized. Basolateral targeting generally is mediated by discrete amino acid sequence motifs in the cytoplasmic domains of transmembrane proteins, whereas the nature of apical targeting signals remains controversial. Apical targeting appears to depend on distributed features of protein ectodomains or transmembrane domains, and there may be several apical targeting mechanisms (13Keller P. Simons K. J. Cell Sci. 1997; 110: 3001-3009Crossref PubMed Google Scholar). For some proteins, apical targeting requires N-linked oligosaccharides (14Scheiffele P. Peranen J. Simons K. Nature. 1995; 378: 96-98Crossref PubMed Scopus (417) Google Scholar, 15Gut A. Kappeler F. Hyka N. Balda M.S. Hauri H.P. Matter K. EMBO J. 1998; 17: 1919-1929Crossref PubMed Scopus (175) Google Scholar), or juxtamembrane segments with clusteredO-linked oligosaccharides (16Yeaman C. Le Gall A.H. Baldwin A.N. Monlauzeur L. Le Bivic A. Rodriguez-Boulan E. J. Cell Biol. 1997; 139: 929-940Crossref PubMed Scopus (247) Google Scholar), or interactions of transmembrane domains (17Scheiffele P. Roth M.G. Simons K. EMBO J. 1997; 16: 5501-5508Crossref PubMed Scopus (570) Google Scholar) or glycosylphosphatidylinositol anchors (18Brown D.A. Crise B. Rose J.K. Science. 1989; 245: 1499-1501Crossref PubMed Scopus (304) Google Scholar,19Lisanti M.P. Caras I.W. Davitz M.A. Rodriguez-Boulan E. J. Cell Biol. 1989; 109: 2145-2156Crossref PubMed Scopus (375) Google Scholar) with the lipid bilayer. Apical sorting determinants may function, in part, by promoting association with sphingolipid-cholesterol rafts that deliver proteins to the apical cell surface (20Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8116) Google Scholar). However, some apical proteins appear not to associate with rafts and lack any of the currently recognized apical sorting signals (e.g. Ref. 21Alonso M.A. Fan L. Alarcon B. J. Biol. Chem. 1997; 272: 30748-30752Crossref PubMed Scopus (35) Google Scholar), and proteins may be sorted differently in different cell types (13Keller P. Simons K. J. Cell Sci. 1997; 110: 3001-3009Crossref PubMed Google Scholar). Therefore, the mechanism of apical protein targeting remains poorly understood. We have employed Madin-Darby canine kidney (MDCK) 1The abbreviations used are: MDCK, Madin-Darby canine kidney; BEK, recombinant bovine proenteropeptidase; dSA-BEK, mutant proenteropeptidase lacking the amino-terminal signal anchor domain; dSAdL-BEK, mutant proenteropeptidase lacking the signal anchor, mucin-like, and light chain domains; HL-BEK, mutant proenteropeptidase lacking the amino-terminal signal anchor and mucin-like domains; L-BEK, mutant proenteropeptidase containing the light chain and 17 carboxyl-terminal residues of the heavy chain; NHS-SS-biotin, sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; MOPS, 4-morpholinepropanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; BHK, baby hamster kidney; PNGase, peptide N-glycosidase; endo H, endoglycosidase H; MES, 4-morpholineethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; BSA, bovine serum albumin. cells, a well characterized system for the study of protein sorting (13Keller P. Simons K. J. Cell Sci. 1997; 110: 3001-3009Crossref PubMed Google Scholar, 22Matter K. Mellman I. Curr. Opin. Cell Biol. 1994; 6: 545-554Crossref PubMed Scopus (393) Google Scholar), to investigate the targeting of enteropeptidase. The results indicate that signals involved in apical delivery reside in the catalytic domain and in an amino-terminal segment that includes the mucin-like domain. Apical delivery of this type II transmembrane protein requires intact post-Golgi transport vesicles and depends on N-linked glycosylation. Unlike many other apically targeted proteins, delivery of enteropeptidase appears not to involve detergent-resistant association with sphingolipid-cholesterol rafts. Plasmid pBEK, containing the full-length cDNA sequence of bovine enteropeptidase, was assembled in vector pBluescript II KS+ (Stratagene) from cDNA clones isolated previously (7Kitamoto Y. Yuan X. Wu Q. McCourt D.W. Sadler J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7588-7592Crossref PubMed Scopus (148) Google Scholar). The cDNA insert of pBEK was cloned into theSmaI site of plasmid pNUT (23Palmiter R.D. Behringer R.R. Quaife C.J. Maxwell F. Maxwell I.H. Brinster R.L. Cell. 1987; 50: 435-443Abstract Full Text PDF PubMed Scopus (353) Google Scholar) to produce expression plasmid pNUTBEK. Plasmids pNUTHL, pNUTL, pBlue-HL, and pBlue-L were described previously (12Lu D. Yuan X. Zheng X. Sadler J.E. J. Biol. Chem. 1997; 272: 31293-31300Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In pNUTHL and pBlue-HL, the sequence encoding amino acids 1–197 was replaced by a signal peptide cassette consisting of the cleavable signal peptide of prothrombin, a His6 tag, thrombin cleavage site, and T7 epitope tag. In plasmid pNUTL and pBlue-L, the same signal peptide cassette replaced the sequence encoding amino acids 1–783. Additional plasmids derived from pBEK were constructed using oligonucleotide site-directed mutagenesis as described previously (12Lu D. Yuan X. Zheng X. Sadler J.E. J. Biol. Chem. 1997; 272: 31293-31300Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In plasmid pdSA, the sequence encoding the signal-anchor domain (residues 1–49) was replaced by the prothrombin signal peptide cassette from plasmid pNUTHL. To add the epitope tag DYKDDDDK to the carboxyl terminus of full-length recombinant enteropeptidase, the DNA sequence 5′-gac tac aag gac gac gat gac aag tag-3′ was inserted before the stop codon of plasmid pBEK, generating the plasmid pBEKflag. Plasmid pdSALflag was derived from pdSA by inserting the same oligonucleotide sequence before the codon for residue 787, thereby deleting the light chain and appending the DYKDDDDK tag. The cDNA inserts of pBEK, pBEKflag, pdSA, pdSALflag pBlue-HL, and pBlue-L were excised from the vectors with NotI and ApaI or NotI andHindIII and cloned into vectors pcDNA3, pcDNA3.1, or pcDNAI (Invitrogen) to yield plasmids pcDNA3-BEK, pcDNA3-BEKflag, pcDNA3.1-dSA, pcDNA3.1-dSAdLflag, pcDNAI-HL, and pcDNAI-L, respectively. Baby hamster kidney (BHK) cells were grown in six-well plates and transfected with 5 μg of plasmid pNUT-BEK, pNUT-HL, and pNUT-L, and 30 μg of Lipofectin (Life Technologies, Inc.) in serum-free Dulbecco's modified Eagle's medium (DMEM). After 5 h fetal bovine serum was added to 10%. After an additional 18 h, cultures were split 1:10 for selection in 0.5 mg/ml methotrexate for 10 days. Madin-Darby canine kidney (MDCK-II) cells (ATCC) were transfected with 5 μg of plasmid pcDNA3-BEK, pcDNA3-BEKflag, pcDNA3.1-dSA, pcDNA3.1-dSAdLflag, pcDNAI-HL, or pcDNAI-L and PerFect lipids (pfx-2) (Invitrogen) according to the manufacturer's recommendations, and clones were selected with 0.5 mg/ml geneticin (G418) (Life Technologies, Inc.). The cDNA sequence encoding the light chain of bovine enteropeptidase (amino acids 784–1035) was cloned into the XhoI site of plasmid pET-28a(+) (Novagen) to yield plasmid pET-L. Epicurean coli BL21 (DE3) (Stratagene, La Jolla, CA) transformed with plasmid pET-L were grown in LB broth containing ampicillin, and protein expression was induced with 1 mmisopropyl-1-thio-β-d-galactopyranoside, and cells were lysed with 40 mm MOPS, pH 7.5, 100 mm NaCl, 1% Triton X-100, 6 m urea, 300 mm imidazole, 10 mm dithiothreitol, and 100 μg/ml soybean trypsin inhibitor. After centrifugation, the supernatant solution was dialyzed against binding buffer (20 mm MOPS, pH 7.9, 50 mm NaCl, 0.5% Triton X-100) containing decreasing concentrations of urea (4 m to zero). Recombinant enteropeptidase light chain was purified by chromatography on Ni2+-nitrilotriacetic acid-agarose (12Lu D. Yuan X. Zheng X. Sadler J.E. J. Biol. Chem. 1997; 272: 31293-31300Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Polyclonal antibodies to the purified light chain were prepared in rabbits by standard methods (24Harlow E. Lane D. Antibodies: A Laboratory Manual. 1. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 92-135Google Scholar). Specific immune IgG against denatured light chain (anti-L) was purified by sequential affinity chromatography on protein A-agarose and on enteropeptidase light chain immobilized on Affi-Gel 10 (Bio-Rad). Antibodies against native enteropeptidase light chain were prepared similarly. The cDNA insert encoding amino acids 785–1035 of pBlue-L was cloned into baculovirus expression vector pVL1392 (PharMingen, San Diego, CA). Plasmid pVL1392-L was co-transfected into Sf9 cells (Life Technologies, Inc.) with BaculoGold DNA (PharMingen, San Diego, CA) to generate recombinant virus. Enteropeptidase light chain was expressed by infection of High Five cells (Life Technologies, Inc.) and purified by affinity chromatography on soybean trypsin inhibitor-agarose (Sigma). 2D. Lu, X. Zheng, and J. E. Sadler, manuscript in preparation. Polyclonal antibody against the purified active L-BEK was raised in rabbits (TANA Laboratories, Houston, TX), and the immune serum was designated anti-Lv. Stably transfected BHK or MDCK II cell lines were maintained in DMEM containing 10% fetal bovine serum. Cells were lysed directly in the plate at room temperature with sodium phosphate, pH 7.4, 150 mm NaCl (PBS) containing 1% Triton X-100, 0.5 mm phenylmethylsulfonyl fluoride (PMSF), and 3 μm aprotinin (1 ml/107 cells). After centrifugation at 15,000 rpm for 15 min in a microcentrifuge, the supernatants were stored at −70 °C. For Western blotting, proteins were fractionated in 4–15% gradient polyacrylamide electrophoresis gels (Bio-Rad) and transferred by electroblotting onto supported nitrocellulose membranes (pore size 0.4 μm, Bio-Rad). Membranes were blocked with 3% non-fat milk in 20 mm Tris-HCl, pH 7.5, 150 mm NaCl (TBS) containing 0.05% Tween 20 (TBST) at room temperature for 30 min. The blocked membrane was incubated with 1.5 μg/ml affinity purified anti-L-IgG or anti-Lv-antiserum (1:4000) diluted in TBST containing 1.5% non-fat milk at 4 °C overnight. Membranes were washed four times with TBST, once with TBS, and incubated with peroxidase-conjugated affinity purified swine anti-rabbit IgG (Dako Corp., Carpinteria, CA) 0.12 μg/ml in TBST containing 1.5% milk at room temperature for 1–2 h. Bound second antibody was detected with the chemiluminescent ECL detection system (Amersham Pharmacia Biotech). To analyze theN-glycosylation of proenteropeptidase, 10 μl of BHK cell lysate or conditioned medium was incubated at 100 °C for 5 min in 30 μl total volume of 20 mm sodium phosphate, pH 7.5, 50 mm EDTA-Na, 0.4% SDS, 4% β-mercaptoethanol, and 0.02% sodium azide. Denatured and reduced proteins (30 μl) were then digested without (control) or with 0.5 units of peptide-N-glycosidase F (Oxford Glycosciences, Bedford, MA) or 1 milliunit of endoglycosidase H (Boehringer Mannheim) in a total volume of 30 μl at 37 °C for 16 h. Samples (10 μl) of these digested materials were analyzed by Western blotting. For digestion with neuraminidase and O-glycanase, BHK cells expressing BEK were pulse-labeled with 200 μCi/ml Tran35S-label (>1000 Ci/mmol, containing 75% [35S]methionine, 15% [35S]cysteine, ICN Pharmaceuticals) at 37 °C for 30 min and chased for 60 min with complete DMEM containing an excess of unlabeled methionine (15 μg/ml) and cysteine (50 μg/ml). Cell lysates were prepared as described above, pre-cleared for 60 min with protein A-Sepharose 4B (50 μl/ml sample), and incubated with anti-Lv (5–10 μl/ml lysate) and 5 mg/ml bovine serum albumin at 4 °C overnight. After addition of protein A-Sepharose 4B (50 μl), samples were incubated for 60 min at room temperature. The protein A-Sepharose beads were washed four times with 20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.1% Triton X-100, 0.1% SDS, and once with 20 mm Tris-HCl, 150 mm NaCl, pH 7.4. Antigen-antibody complexes were eluted by boiling at 100 °C for 10 min with 100 μl of 20 mmTris-HCl, pH 7.4, 0.5% SDS. Samples of eluate (20 μl) or 20 ng of purified HL-BEK (12Lu D. Yuan X. Zheng X. Sadler J.E. J. Biol. Chem. 1997; 272: 31293-31300Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) were digested without or with 0.5 units of PNGase F, 20 milliunits of neuraminidase (Boehringer Mannheim) or 20 milliunits of neuraminidase plus 2 milliunits of O-glycanase (Genzyme Diagnostic, Cambridge, MA) in 20 mm sodium cacodylate, pH 6.5, 10 mm calcium acetate, 0.2% SDS, and 2% Nonidet P-40 at 37 °C for 20 h. The products were separated electrophoretically by 4–20% gradient SDS-PAGE; radiolabeled BEK and unlabeled HL-BEK were detected by autoradiography and silver staining, respectively. BHK cell lines were washed three times with ice-cold PBS, incubated with 1.5 mg/ml NHS-SS-biotin (Pierce) at 4 °C for 30 min, and reaction was stopped with 50 mm glycine in PBS. Cells were lysed in 1 ml of PBS containing 1% Triton X-100, 0.5 mm PMSF, and 3 μm aprotinin. Surface biotin-labeled proteins were precipitated with streptavidin-agarose at room temperature for 2 h. After washing four times with PBS containing 0.1% Triton X-100 and 0.1% SDS, the biotin-streptavidin-agarose complexes were eluted from beads by heating at 100 °C for 5 min with 30 μl of Laemmli sample buffer (Bio-Rad) containing 1% β-mercaptoethanol. The cell lysate, flow-through, and eluate fractions from streptavidin-agarose were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting. Transfected BHK cell lines in six-well plates were washed with PBS and treated without (control) or with 5 μg/ml trypsin-l-1-tosylamido-2-phenylethyl chloromethyl ketone (Worthington) in PBS at room temperature for 10 min. Residual trypsin was inactivated by addition of a 2-fold excess of soybean trypsin inhibitor. Cells were pelleted, washed with PBS, and lysed with PBS containing 0.5% Triton X-100, 0.1% SDS, 100 μg/ml PMSF, 24 μg/ml aprotinin, 1.5 μg/ml soybean trypsin inhibitor. Cell lysate was centrifuged at 15,000 rpm in a microcentrifuge, and the supernatant was collected for SDS-PAGE and Western blotting. Stably transfected MDCK II cells were grown on clear Costar Transwell filters (pore size 0.4 μm) until a tight monolayer was formed as shown by the transepithelial resistance. The filters were washed with PBS and fixed at 4 °C for 15 min in PBS containing 1.5% paraformaldehyde (25Beau I. Misrahi M. Gross B. Vannier B. Loosfelt H. Hai M.T. Pichon C. Milgrom E. J. Biol. Chem. 1997; 272: 5241-5248Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) or ethanol:acetic acid (9:1). Cells were washed sequentially with PBS followed by PBS containing 0.5% Triton X-100, 1% BSA, and 1% goat serum and then incubated in PBS containing 20 μg/ml rabbit anti-Lv serum (1:100) or anti-caveolin IgG (1:500) (Transduction Laboratories, Lexington, KY), 1% BSA, and 1% goat serum at 4 °C overnight. Cells were washed three times with PBS containing 0.05% Tween 20 and once with PBS and then incubated with Cy3-labeled goat anti-rabbit antibody (15 μg/ml diluted with TBS containing 1.5% non-immune goat serum) (Jackson ImmunoResearch, West Grove, PA) at room temperature for 60 min. After washing, the filters were cut out and mounted on slides with glycerol mounting medium (Sigma) and examined with a confocal laser-scanning microscope (Zeiss Axioplan/Bio-Rad MRC1024). To detect membrane-bound enteropeptidase, stably transfected MDCK cells were grown on six-well Costar Transwell filters (pore size 0.4 μm) until a tight monolayer was formed as shown by the transepithelial resistance. Cells were pulse-labeled with 150–200 μCi/ml Tran35S-label at 37 °C for 30 min and chased for 30 min with complete DMEM containing 150 μg/ml unlabeled methionine. Either the apical or basolateral cell surface was incubated with 1.5 mg/ml sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (NHS-SS-biotin, Pierce) at 4 °C for 30 min, and the reaction was stopped with 50 mm glycine in PBS (10 ml per 100-mm dish), and cell lysates were prepared as described above. Enteropeptidase was immunoprecipitated with anti-Lv (10–30 μg/ml sample) and protein A-Sepharose 4B (50 μl). Antigen-antibody complexes were eluted by boiling at 100 °C for 10 min with 100 μl of 20 mmTris-HCl, pH 7.4, containing 0.5% SDS. The eluate was diluted to 500 μl with 20 mm Tris-HCl, pH 7.4, 150 mm NaCl, and biotin-labeled proteins were precipitated with streptavidin-agarose as described above. Proteins were eluted from streptavidin-agarose with Laemmli sample buffer containing 1% β-mercaptoethanol, separated on 4–15% gradient mini-ready gels (Bio-Rad), fixed with 40% methanol, 10% acetic acid, treated with Amplify fluorographic reagent (Amersham Pharmacia Biotech) for 30 min, dried, and exposed to XAR5 film (Eastman Kodak Co.) at −70 °C. Alternatively, to detect secreted forms of enteropeptidase, conditioned medium from either apical or basolateral surfaces was collected, immunoprecipitated with anti-Ly, and fractionated by SDS-PAGE. The enteropeptidase then was visualized by either autoradiography (if labeled with Tran35S-label) or Western blotting. MDCK II cells transfected with pcDNAI-L were grown on Costar Transwell filters until formation of a tight monolayer as determined by transepithelial resistance measurements. Cells were then treated without or with 2 μg/ml tunicamycin or 10 μg/ml brefeldin A in serum-free DMEM for 24 h. The cell viability and integrity of the monolayer were confirmed after treatment by measuring the transepithelial resistance. Conditioned media were collected from either apical or basolateral surfaces and concentrated 10-fold by ultrafiltration (Centriprep-10). Samples (20 μl) of concentrated media were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting as described above. The amount ofL-BEK secreted from either surface was quantitated with NIH Image version 1.61 (26Marzolo M.P. Bull P. Gonzalez A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1834-1839Crossref PubMed Scopus (63) Google Scholar). Detergent extractability of enteropeptidase was determined according to Brown and Rose (27Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2610) Google Scholar). Confluent cell monolayers in 10-cm dishes were labeled with 200 μCi/ml Tran35S-label in serum-free DMEM without methionine for 30 min at 37 °C and chased at 37 or 20 °C with DMEM, F-12 HEPES medium containing an excess of unlabeled methionine (15 μg/ml) and cysteine (50 μg/ml). The cells were lysed with 1 ml of extraction buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Triton X-100, 0.5 mm PMSF, and 3 μm aprotinin) for 10 min on ice. Lysates were centrifuged for 5 min in a microcentrifuge at 15,000 rpm at 4 °C. The supernatant (Triton X-100 soluble fraction) was removed. The pellet (Triton X-100 insoluble fraction) was dissolved in 100 μl of 50 mm Tris-HCl, pH 8.0, 5 mm EDTA, and 1% SDS; DNA was sheared by passage through a 22-gauge needle, and the solution was diluted by addition of 900 μl of extraction buffer. Enteropeptidase was immunoprecipitated from soluble and insoluble fractions with anti-Lv (5 μl/ml) for analysis by SDS-PAGE and autoradiography as described above. Detergent-insoluble glycosphingolipid-enriched raft domains were prepared by a modification of the Triton X-100 procedure of Pike and Casey (28Pike L.J. Casey L. J. Biol. Chem. 1996; 271: 26453-26456Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Confluent cells in 100-mm dishes were rinsed twice with ice-cold PBS and scraped into 1 ml of lysis buffer containing 25 mm MES, pH 6.5, 150 mm NaCl, 1% Triton X-100, 10 mm benzamidine, 1 mmphenylmethylsulfonyl fluoride, and 0.3 mm aprotinin. Lysates were incubated for 20 min on ice with intermittent gentle agitation and then mixed with an equal volume of 80% sucrose in MBS (25 mm MES, pH 6.5, 150 mm NaCl). Six ml of 30% sucrose in MBS followed by 4 ml of 5% sucrose in MBS were layered on top of each sample. The gradients were centrifuged at 4 °C for 23 h at 175,000 × g (39,000 rpm, Beckman SW40 rotor). Fractions of 1.2 ml were collected, and the small insoluble pellet was resuspended in 600 μl of lysis buffer by homogenization (25,000 rpm, 1 min) with a Brinkmann homogenizer (Kinematica AG, Switzerland). Proteins in the fractions were precipitated with 10% trichloroacetic acid on ice for 30 min, and pellets were resuspended in 100 μl of 0.2 n NaOH. Samples (20 μl) of trichloroacetic acid-concentrated fractions were analyzed by SDS-PAGE and immunoblotting with either anti-Lv (1:4,000) or anti-caveolin IgG (1:10,000) (Transduction Laboratories, Lexington, KY). BHK cells expressing BEK were cultured in 150-mm dishes to 90% confluency in DMEM, 10% fetal bovine serum and washed twice with 10 ml of ice-cold PBS. Cells were incubated without (control) or with anti-Lv (preabsorbed with non-transfected BHK cell lysate coupled on Affi-Gel 10) at a dilution of 1:1000 in serum-free DMEM, 0.1% BSA at 12 °C for 60 min. After washing three times with 10 ml of ice-cold PBS, cells were further incubated with goat anti-rabbit IgG (1:1000) at 12 °C for 60 min. Cells were scraped into 25 mm MES, pH 6.5, 150 mm NaCl, 1% Triton X-100, 10 mm benzamidine, 1 mmphenylmethylsulfonyl fluoride, and 0.3 mm aprotinin. Sucrose gradient centrifugation and Western blotting were performed as described above. For immunofluorescence, cells grown on slide chambers were either fixed with acetic acid:ethanol (1:9) on ice for 10 min and incubated with anti-Lv (1:1000) and Cy3-conjugated anti-rabbit IgG (1:50), or first cross-linked with primary and secondary antibodies at 12 °C for 60 min before fixing with acetic acid ethanol (1:9) on ice for 10 min. After washing with PBS, cells were mounted with 30% glycerol in 20 mm Tris-HCl, pH 7.4, and photographed with a Zeiss Axiophot microscope. BHK cells expressed two major species of cell-associated full-length proenteropeptidase (BEK) with molecular masses of 180 and 150 kDa (Fig.1 A, lane 1). These apparent sizes are similar to those for secreted and intracellular forms, respectively, of proenteropeptidase lacking the transmembrane and mucin-like domains, HL-BEK (Fig. 1 A, lanes 4 and7), as reported previously (12Lu D. Yuan X. Zheng X. Sadler J.E. J. Biol. Chem. 1997; 272: 31293-31300Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Digestion of BEK (Fig.1 A, lane 3) or HL-BEK (Fig. 1 A, lanes 6 and9) with protein N-glycosidase F (PNGase) reduced the apparent mass of all species to ≈120 kDa, indicating that much of the difference between the ob" @default.
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• W2029006507 title "Apical Sorting of Bovine Enteropeptidase Does Not Involve Detergent-resistant Association with Sphingolipid-Cholesterol Rafts" @default.
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