1-Benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside Blocks the Apical Biosynthetic Pathway in Polarized HT-29 Cells
2003; Elsevier BV; Volume: 278; Issue: 39 Linguagem: Inglês
10.1074/jbc.m305755200
ISSN1083-351X
AutoresDelphine Delacour, Valérie Gouyer, Emmanuelle Leteurtre, Tounsia Aït‐Slimane, Hervé Drobecq, Christelle Lenoir, Odile Moreau-Hannedouche, Germain Trugnan, Guillemette Huet,
Tópico(s)Infant Nutrition and Health
ResumoIn previous work we reported that long term treatment of polarized HT-29 cells by 1-benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside (GalNAcα-O-bn) induced undersialylation and intracellular distribution of apical glycoproteins such as dipeptidyl peptidase IV (DPP-IV), and we suggested therefore that sialylation could act as an apical targeting signal. In this work, the apical direct biosynthetic route was studied after transfection of polarized enterocyte-like HT-29 5M12 cloned cells with a murine cDNA coding for a soluble form of DPP-IV, which was secreted into the apical medium. A 24-h treatment of transfected cells by GalNAcα-O-bn markedly inhibited the apical secretion and the sialylation of this soluble murine DPP-IV, which became blocked inside the cell. A similar short GalNAcα-O-bn treatment also induced an intracellular distribution of both endogenous transmembrane DPP-IV and proteins involved in the regulation of the apical trafficking such as the apical t-SNARE syntaxin-3 and the raft-associated protein annexin XIIIb, whereas the basolateral t-SNARE syntaxin-4 kept its normal localization. These apical membrane proteins moved efficiently from trans-Golgi network to apical carrier vesicles but failed to be transported from carrier vesicles to the apical plasma membrane. Isolation of membrane microdomains showed that GalNAcα-O-bn induced the formation of abnormal lipid-rich microdomains in comparison to normal rafts, as shown by their lower buoyant density and their depletion in annexin XIIIb. In conclusion, GalNAcα-O-bn blocks the anterograde traffic to the apical surface of polarized HT-29 cells at the transport level or docking/fusion level of carrier vesicles. In previous work we reported that long term treatment of polarized HT-29 cells by 1-benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside (GalNAcα-O-bn) induced undersialylation and intracellular distribution of apical glycoproteins such as dipeptidyl peptidase IV (DPP-IV), and we suggested therefore that sialylation could act as an apical targeting signal. In this work, the apical direct biosynthetic route was studied after transfection of polarized enterocyte-like HT-29 5M12 cloned cells with a murine cDNA coding for a soluble form of DPP-IV, which was secreted into the apical medium. A 24-h treatment of transfected cells by GalNAcα-O-bn markedly inhibited the apical secretion and the sialylation of this soluble murine DPP-IV, which became blocked inside the cell. A similar short GalNAcα-O-bn treatment also induced an intracellular distribution of both endogenous transmembrane DPP-IV and proteins involved in the regulation of the apical trafficking such as the apical t-SNARE syntaxin-3 and the raft-associated protein annexin XIIIb, whereas the basolateral t-SNARE syntaxin-4 kept its normal localization. These apical membrane proteins moved efficiently from trans-Golgi network to apical carrier vesicles but failed to be transported from carrier vesicles to the apical plasma membrane. Isolation of membrane microdomains showed that GalNAcα-O-bn induced the formation of abnormal lipid-rich microdomains in comparison to normal rafts, as shown by their lower buoyant density and their depletion in annexin XIIIb. In conclusion, GalNAcα-O-bn blocks the anterograde traffic to the apical surface of polarized HT-29 cells at the transport level or docking/fusion level of carrier vesicles. In recent years, data of the literature have shown the role of glycosylation in the apical biosynthetic route in polarized epithelial cells. Association of proteins with glycosphingolipids has been proposed for the apical delivery (1Simons K. Van Meer G. Biochemistry. 1988; 27: 6197-6202Crossref PubMed Scopus (1090) Google Scholar, 2Simons K. Wandinger-Ness A. Cell. 1990; 62: 207-210Abstract Full Text PDF PubMed Scopus (422) Google Scholar). Glycosylphosphatidylinositol (GPI) 1The abbreviations used are: GPI, glycosylphosphatidylinositol; TGN, trans-Golgi network; GalNAcα-O-bn, 1-benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside; t-SNARE, target-soluble N-ethylmaleimide-sensitive factor attachment protein receptor; DPP-IV, dipeptidyl peptidase IV; sDPP-IV, secreted dipeptidyl peptidase IV; mAb, monoclonal antibody; MDCK, Madin-Darby canine kidney; MAA, M. amurensis lectin; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.-anchored proteins and glycosphingolipids could be isolated from a Triton X-100-insoluble fraction (3Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2610) Google Scholar). The dynamic clustering of glycosphingolipid-enriched microdomains in the TGN would constitute functional rafts for apical delivery of proteins (4Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8117) Google Scholar). Thus, proteins of apical destination, such as GPI-anchored proteins, would be specifically recruited in these apical raft carriers. Besides, data obtained by mutation, deletion, or addition of glycosylation sites showed that O- or N-glycosylation could influence the targeting of glycoproteins toward the apical side of the cells (5Danielsen E.M. Biochemistry. 1995; 34: 1596-1605Crossref PubMed Scopus (129) Google Scholar, 6Scheiffele P. Peranen J. Simons K. Nature. 1995; 378: 96-98Crossref PubMed Scopus (417) Google Scholar, 7Mirre C. Monlauzeur L. Garcia M. Delgrossi M.H. Le Bivic A. Am. J. Physiol. 1996; 271: 887-894Crossref PubMed Google Scholar, 8Yeaman 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, 9Monlauzeur L. Breuza L. Le Bivic A. J. Biol. Chem. 1998; 273: 30263-30270Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 10Gut A. Kappeler F. Hyka N. Balda M.S. Hauri H.P. Matter K. EMBO J. 1998; 17: 1919-1929Crossref PubMed Scopus (175) Google Scholar, 11Alfalah M. Jacob R. Preuss U. Zimmer K.P. Naim H. Naim H.Y. Curr. Biol. 1999; 9: 593-596Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 12Benting J.H. Rietvelo A.G. Simons K. J. Cell Biol. 1999; 146: 313-320Crossref PubMed Scopus (210) Google Scholar, 13Jacob R. Alfalah M. Grünberg J. Obendorf M. Naim H.Y. J. Biol. Chem. 2000; 275: 6566-6572Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 14Alfalah M. Jacob R. Naim H.Y. J. Biol. Chem. 2001; 277: 10683-10690Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The putative role of N- and O-glycans was also shown through the use of drugs affecting their processing, i.e. tunicamycin, deoxymannojirimycin, swainsonine for N-glycans, and 1-benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside (GalNAc-α-O-bn) for O-glycans (11Alfalah M. Jacob R. Preuss U. Zimmer K.P. Naim H. Naim H.Y. Curr. Biol. 1999; 9: 593-596Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 14Alfalah M. Jacob R. Naim H.Y. J. Biol. Chem. 2001; 277: 10683-10690Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 15Urban J. Parczyk K. Leutz A. Kayne M. Kondor-Koch C. J. Cell Biol. 1987; 105: 2735-2743Crossref PubMed Scopus (146) Google Scholar, 16Kitagawa Y. Sano Y. Ueda M. Higashio K. Narita H. Okano M. Matsumoto S. Sasaki R. Exp. Cell Res. 1994; 213: 449-457Crossref PubMed Scopus (77) Google Scholar, 17Naim H.Y. Joberty G. Alfalah M. Jacob R. J. Biol. Chem. 1999; 274: 17961-17967Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 18Ait-Slimane T. Lenoir C. Sapin C. Maurice M. Trugnan G. Exp. Cell Res. 2000; 258: 184-194Crossref PubMed Scopus (36) Google Scholar). We previously observed that long term treatment of polarized goblet or enterocytic HT-29 cells by GalNAcα-O-bn led (i) to a dramatic inhibition in the secretion of mucins, which are highly O-glycosylated; (ii) to a decrease in the apical membrane expression of brush border markers such as the transmembrane glycoprotein dipeptidylpeptidase-IV (DPP-IV), the GPI-anchored carcinoembryonic antigen, and the mucin-like glycoprotein MUC1; and (iii) to the abnormal presence of these glycoproteins inside the cells (19Huet G. Hennebicq-Reig S. De Bolos C. Ulloa F. Lesuffleur T. Barbat A. Carriere V. Kim I. Real F.X. Delannoy P. Zweibaum A. J. Cell Biol. 1998; 141: 1311-1322Crossref PubMed Scopus (88) Google Scholar, 20Hennebicq-Reig S. Lesuffleur T. Capon C. De Bolos C. Kim I. Moreau O. Richet C. Hemon B. Recchi M.A. Maes E. Aubert J.P. Real F.X. Zweibaum A. Delannoy P. Degand P. Huet G. Biochem. J. 1998; 334: 283-295Crossref PubMed Scopus (52) Google Scholar). In contrast, basolateral glycoproteins such as gp120 and gp525 kept a normal localization under GalNAcα-O-bn treatment (19Huet G. Hennebicq-Reig S. De Bolos C. Ulloa F. Lesuffleur T. Barbat A. Carriere V. Kim I. Real F.X. Delannoy P. Zweibaum A. J. Cell Biol. 1998; 141: 1311-1322Crossref PubMed Scopus (88) Google Scholar, 21Gouyer V. Leteurtre E. Delmotte P. Steelant W.F.A. Krzewinski-Recchi M.A. Zanetta J.P. Lesuffleur T. Trugnan G. Delannoy P. Huet G. J. Cell Sci. 2001; 114: 1455-1471Crossref PubMed Google Scholar). These data led us to analyze more deeply the interferences of exogenous GalNAcα-O-bn with the intracellular processes of glycosylation in HT-29 cells. In a previous work, we showed that GalNAcα-O-bn was highly converted into the benzyldisaccharide Galβ1–3GalNAcα-O-bn, which acts as a potent competitive inhibitor of α2,3-sialyltransferase ST3Gal I and involved in the terminal elongation of O-linked glycans (22Delannoy P. Kim I. Emery N. De Bolos C. Verbert A. Degand P. Huet G. Glycoconjugate J. 1996; 13: 717-726Crossref PubMed Scopus (51) Google Scholar). Thereafter, we reported that GalNAcα-O-bn was extensively metabolized beyond Galβ1–3GalNAcα-O-bn, showing that the glycosylation of endogenous substrates by several glycosyltransferases could be inhibited, and in particular the sialylation of N-glycans by α2,3-sialyltransferase ST3Gal IV (21Gouyer V. Leteurtre E. Delmotte P. Steelant W.F.A. Krzewinski-Recchi M.A. Zanetta J.P. Lesuffleur T. Trugnan G. Delannoy P. Huet G. J. Cell Sci. 2001; 114: 1455-1471Crossref PubMed Google Scholar, 23Zanetta J.-P. Gouyer V. Maes E. Pons A. Hemon B. Zweibaum A. Delannoy P. Huet G. Glycobiology. 2000; 10: 565-575Crossref PubMed Scopus (33) Google Scholar). In this way, the sialylation of N- and/or O-glycans was found inhibited on the endogenous glycoproteins DPP-IV, MUC1, and GPI-anchored carcinoembryonic antigen, and we suggested that undersialylation of glycoproteins may induce a defect in the direct apical targeting of glycoproteins (19Huet G. Hennebicq-Reig S. De Bolos C. Ulloa F. Lesuffleur T. Barbat A. Carriere V. Kim I. Real F.X. Delannoy P. Zweibaum A. J. Cell Biol. 1998; 141: 1311-1322Crossref PubMed Scopus (88) Google Scholar, 20Hennebicq-Reig S. Lesuffleur T. Capon C. De Bolos C. Kim I. Moreau O. Richet C. Hemon B. Recchi M.A. Maes E. Aubert J.P. Real F.X. Zweibaum A. Delannoy P. Degand P. Huet G. Biochem. J. 1998; 334: 283-295Crossref PubMed Scopus (52) Google Scholar, 21Gouyer V. Leteurtre E. Delmotte P. Steelant W.F.A. Krzewinski-Recchi M.A. Zanetta J.P. Lesuffleur T. Trugnan G. Delannoy P. Huet G. J. Cell Sci. 2001; 114: 1455-1471Crossref PubMed Google Scholar). Later on, the effect of GalNAcα-O-bn was studied by others using other cell lines, and the induction of a shift from the apical membrane to the basolateral membrane was reported in the targeting of the brush border glycoproteins sucrase isomaltase and DPP-IV using MDCK and Caco-2 cell lines (11Alfalah M. Jacob R. Preuss U. Zimmer K.P. Naim H. Naim H.Y. Curr. Biol. 1999; 9: 593-596Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 18Ait-Slimane T. Lenoir C. Sapin C. Maurice M. Trugnan G. Exp. Cell Res. 2000; 258: 184-194Crossref PubMed Scopus (36) Google Scholar). However, the expression of peripheral glycan epitopes is a cell type-specific process that depends on the expression pattern of glycosyltransferases. Interestingly, we and others found that GalNAcα-O-bn treatment of different cell lines in culture did not result in the identical alterations, regarding glycosylation and intracellular trafficking (21Gouyer V. Leteurtre E. Delmotte P. Steelant W.F.A. Krzewinski-Recchi M.A. Zanetta J.P. Lesuffleur T. Trugnan G. Delannoy P. Huet G. J. Cell Sci. 2001; 114: 1455-1471Crossref PubMed Google Scholar, 23Zanetta J.-P. Gouyer V. Maes E. Pons A. Hemon B. Zweibaum A. Delannoy P. Huet G. Glycobiology. 2000; 10: 565-575Crossref PubMed Scopus (33) Google Scholar, 24Huang J. Byrd J.C. Yoon W.H. Kim Y.S. Oncol. Res. 1992; 4: 507-515PubMed Google Scholar, 25Byrd J.C. Dahiya R. Huang J. Kim Y.S. Eur. J. Cancer. 1995; 9: 1498-1505Abstract Full Text PDF Scopus (33) Google Scholar, 26Gouyer V. Leteurtre E. Zanetta J.P. Lesuffleur T. Delannoy P. Huet G. Front. Biosci. 2001; 6: D1235-D1244Crossref PubMed Google Scholar, 27Leteurtre E. Gouyer V. Delacour C. Hemon B. Pons A. Richet C. Zanetta J.P. Huet G. J. Histochem. Cytochem. 2003; 51: 349-361Crossref PubMed Scopus (10) Google Scholar), suggesting that the cell type specificity in the cellular responses to GalNAcα-O-bn was connected to the cell type specificity in the modification of the glycosylation pattern. GalNAcα-O-bn appeared to be an interesting tool to study polarized HT-29 cells, regarding the fact that, in this cell type specifically, (i) the terminal sialylation was identified as the target of inhibition, and (ii) the normal apical localization of brush border glycoproteins was disrupted with intracellular localization. Thus, we investigated the effect of this inhibitor of glycosylation on de novo apical trafficking in enterocyte-like HT-29 cells. Cell Culture—HT-29 clone 5M12 cells (cloned from a HT-29 cell subpopulation resistant to methotrexate (Ref. 28Lesuffleur T. Violette S. Vasile-Pandrea I. Dussaulx E. Barbat A. Muleris M. Zweibaum A. Int. J. Cancer. 1998; 76: 383-392Crossref PubMed Scopus (47) Google Scholar)) were cultured as previously described (19Huet G. Hennebicq-Reig S. De Bolos C. Ulloa F. Lesuffleur T. Barbat A. Carriere V. Kim I. Real F.X. Delannoy P. Zweibaum A. J. Cell Biol. 1998; 141: 1311-1322Crossref PubMed Scopus (88) Google Scholar). Cells were cultured in 25-cm2 T flasks (Corning Glass Works, Corning, NY) for cell maintenance and on glass coverslips or 24.5-mm tissue culture-treated Transwell polyester membrane filters (0.4-μm pore size) (Costar, Cambridge, MA) for confocal microscopy and on 6-well culture dishes or 24.5-mm tissue culture-treated Transwell polyester membrane filters for metabolic labeling. GalNAcα-O-bn was used at a concentration of 2 mm in Dulbecco's modified Eagle's medium with fetal bovine serum during a short-term treatment (from 0 to 44 h) starting from confluence. In all experiments, GalNAcα-O-bn had no effect on cell viability, as assessed by the absence of cells in suspension and trypan blue exclusion. For the analysis of cell culture media by two-dimensional gel electrophoresis, cells were cultured for 24 h in serum-free medium before collection. Transfection of Soluble Mouse DPP-IV—To generate a secreted mouse DPP-IV (sDPP-IV), the full-length mouse DPP-IV cDNA (29Marguet D. Bernard A.M. Vivier I. Darmoul D. Naquet P. Pierres M. J. Biol. Chem. 1992; 267: 2200-2208Abstract Full Text PDF PubMed Google Scholar) was digested with PvuII restriction enzyme to remove the first 105 nucleotides and was subcloned downstream of a sequence coding for a cleavable peptide signal into the eukaryotic expression vector pTEJ8 that contains the G418 resistance gene (18Ait-Slimane T. Lenoir C. Sapin C. Maurice M. Trugnan G. Exp. Cell Res. 2000; 258: 184-194Crossref PubMed Scopus (36) Google Scholar). HT-29 5M12 cells were transfected using LipofectAMINE according to the instructions from the manufacturer (Invitrogen). Resistant colonies growing in the presence of 400 μg/ml G148 were isolated using cloning cylinders and screened for secreted DPP-IV activity in the medium. Clone 5M12 Cl2 was selected for further experiments. DPP-IV activity was measured by a kinetic method with the fluorogenic substrate H-Gly-Pro-AMC (Bachem) (21Gouyer V. Leteurtre E. Delmotte P. Steelant W.F.A. Krzewinski-Recchi M.A. Zanetta J.P. Lesuffleur T. Trugnan G. Delannoy P. Huet G. J. Cell Sci. 2001; 114: 1455-1471Crossref PubMed Google Scholar). Antibodies—Mouse mAbs 525 against gp525, HBB 3/775/42 against human DPP-IV (30Hauri H.P. Sterchi E.E. Bienz D. Fransen J.A. Marxer A. J. Cell Biol. 1985; 101: 838-851Crossref PubMed Scopus (376) Google Scholar) were a gift from Dr. A. Le Bivic (IBDM, Marseille, France) and Dr. H. P. Hauri (Biocenter of the University of Basel, Basel, Switzerland), respectively. Rat mAbs 1082 and 773 against mouse DPP-IV were obtained from Didier Marguet (Centre d'Immunologie INSERM-CNRS de Marseille Luminy, France). Rabbit antibodies against human syntaxin-3, human annexin XIIIb, and human Munc18-2 were obtained from Dr. A. Le Bivic, Dr. J. Gordon (Washington University School of Medicine, St. Louis, MO) and Dr. M. Kauppi (National Public Health Institute, Helsinki, Finland), respectively. Mouse mAbs against syntaxin-4 (S40220) and against flotillin-1 (clone 1) were purchased from BD Transduction Laboratories (Lexington, KY). Mouse mAb against human DPP-IV (M-A261) was purchased from BD Pharmingen (San Diego, CA). Two-dimensional Gel Electrophoresis and MALDI Time-of-flight (TOF) Mass Fingerprinting of Tryptic Peptides—HT-29 5M12 Cl2 cells culture media were concentrated and precipitated with trichloroacetic acid. Amounts used for two-dimensional electrophoresis were normalized to the same amount of cells. Individual 17-cm ReadyStrips IPG strips (Bio-Rad), pH 3–10 or 4–7, were rehydrated with 400 μl of urea buffer (7 m urea, 2 m thiourea, 4% (w/v) CHAPS, 0.4% (v/v) Triton X-100, 0.5% (v/v) Bio-Lytes, 0.28% (w/v) dithiothreitol, 0.2% (v/v) bromophenol blue) containing the sample. Isoelectric focusing was carried out using a Protean IEF Cell according to the instructions from the manufacturer (Bio-Rad). Prior to the second dimension, the IPG gel strips were equilibrated twice in 10 ml of equilibration solution (50 mm Tris-HCl buffer, pH 8.8, containing 6 m urea, 3.3% (v/v) glycerol, 1% (w/v) SDS) for 20 min, first in the presence of dithiothreitol (65 mm) and then in the presence of iodoacetamide (87 mm). Second dimension gels consisted in a 6–16% acrylamide gradient, cross-linked with piperazine diacrylamide. Gels were run using the Protean II xi Cell (Bio-Rad), at a constant current of 15 mA/gel overnight. The analytical two-dimensional gels were silver-stained and scanned (ImageMaster, Amersham Biosciences). The preparative two-dimensional gels were stained for 4 days in Coomassie Brilliant Blue G solution. Electrophoretically separated proteins were excised from the Coomassie Blue-stained gels. Gel pieces were reswollen in a trypsin solution containing 0.2 μg of sequence-grade modified porcine trypsin (Promega, Madison, WI) at 37 °C during 18 h. The protein fragments were extracted with 25 μl of 50% acetonitrile, 0.1% trifluoroacetic acid for 30 min. The extracts were pooled, dried, and desalted with micro ZipTip C18 (Millipore) before the MALDI analysis. The peptide mixtures from the tryptic digests were crystallized in a matrix consisting of hydroxycinnaminic acid (Aldrich) prepared in 50% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid. Spectra were acquired using a PerSeptive Biosystems (Framingham, MA) Voyager-DE STR instrument and were internally calibrated using trypsin-autodigested peptides. The MALDI spectra were explored for database searches using the software Data Explorer (Applied Biosystems). The monoisotopic peptide masses obtained for each protein digest was submitted to Profound software to identify the proteins (prowl.rockfeller.edu/cgi-bin/Profound). Confocal Microscopy—Confocal microscopy was carried out on HT-29 5M12 cells using a Leica instrument (model TCS-NT) according to Gouyer et al. (21Gouyer V. Leteurtre E. Delmotte P. Steelant W.F.A. Krzewinski-Recchi M.A. Zanetta J.P. Lesuffleur T. Trugnan G. Delannoy P. Huet G. J. Cell Sci. 2001; 114: 1455-1471Crossref PubMed Google Scholar). To detect syntaxin-3, Munc18-2, annexin XIIIb, human DPP-IV, and syntaxin-4, respectively, anti-syntaxin-3 (1/50), anti-Munc18-2 (1/200), anti-annexin XIIIb (1/1000), M-A261 (1/100), or S40220 (1/100) in PBS containing 1% bovine serum albumin/0.2% saponin, were added overnight. Secondary fluorescein isothiocyanate-conjugated anti-rabbit or anti-mouse antibody was used. Serial sections of 0.5 μm were performed in z projection. Three xy sections at the top, middle, and bottom of the cell layers, and one xz section, are shown for each experiment. Quantitative analyses were performed by measuring the ratio apical fluorescence/inside fluorescence from confocal images. Fluorescence was quantified on a predefined surface for each section using the quantification program from Leica. The ratio apical fluorescence/inside fluorescence was calculated for the selected sections shown in the illustrations. Vesicle Isolation from Perforated Cells—Isolation of TGN-derived vesicles was carried out according to the procedure described by Wandinger-Ness et al. (31Wandinger-Ness A. Bennett M.K. Antony C. Simons K. J. Cell Biol. 1990; 111: 987-1000Crossref PubMed Scopus (216) Google Scholar). Briefly, HT-29 5M12 cells were seeded on 12 100-mm filters, which were cultured up to day 10. Six filters were treated by 2 mm GalNAcα-O-bn for 18 h before the experiment. After the transfer of filters to 14-cm dishes, cells were perforated using a nitrocellulose acetate filter (HATF, 0.45-μm pore) and carrier vesicles were released by incubation in the presence of an ATP-regenerating system for 1 h at 37 °C. The carriers vesicles were then isolated from the incubation medium by flotation onto a 0.25 m sucrose cushion, pelleted, and purified on a discontinuous density gradient (1.5 m/1.2 m). The perforated cells were collected from the filters. Ultrastructural Immunochemistry—Immunolabeling was carried out on isolated carrier vesicles of HT-29 5M12 cells according to the procedure described by Scheiffele et al. (32Scheiffele P. Verkade P. Fra A.M. Virta H. Simons K. Ikonen E. J. Cell Biol. 1998; 140: 795-806Crossref PubMed Scopus (264) Google Scholar). Briefly, the vesicle preparation was laid on parlodion-coated nickel grids and fixed with 4% paraformaldehyde for 5 min. Single or double labeling was performed. The grids were incubated with a first primary antibody (polyclonal anti-syntaxin-3 (1/20) or polyclonal anti-annexin XIIIb (1/40)) for one night at 4 °C in PBG buffer (PBS containing 0.5% bovine serum albumin and 0.2% gelatin), then with anti-rabbit Ig-coupled 18-nm gold particles (1/20) for 1 h at room temperature in PBG buffer. Subsequently, after a second fixation, grids were incubated with the second primary antibody (monoclonal anti-human DPP-IV antibody (1/20)) for 5 h at room temperature and then with anti-mouse Ig-coupled 12-nm gold particles. The grids were counterstained with 0.3% uranyl acetate and 1.8% methylcellulose. Controls grids were processed without the second primary antibodies and showed the absence of detection of 12-nm gold particles. Metabolic Labeling and Immunoprecipitation of Secreted Mouse DPP-IV and Endogenous Human DPP-IV—For monitoring the secretion of transfected sDPP-IV, HT-29 5M12 Cl2 cells were cultured on filters; otherwise, HT-29 5M12 cells were cultured in 6-well plates. Cells were cultured in standard conditions until day 10. Subsequently, control cells were pulse-labeled for 30 min with 200 μCi/well of [35S]methionine (ICN, Irvine, CA) in 1 ml of methionine-free medium, and then chased for the indicated periods of time with 1 ml of 0.01 m methionine in regular medium. A similar protocol was applied in the presence of 2 mm GalNAcα-O-bn throughout the experiment. To follow the secretion of sDPP-IV, we collected the apical medium and we immunoprecipitated sDPP-IV with rat mAbs 1082 and 773. For analyzing the detergent extractability of cellular endogenous human DPP-IV, we used the procedure described by Alfalah et al. (14Alfalah M. Jacob R. Naim H.Y. J. Biol. Chem. 2001; 277: 10683-10690Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). HT-29 5M12 cells were rinsed in PBS, and cells were solubilized in 25 mm Tris-HCl, pH 8.0, 50 mm NaCl, containing 1% Triton X-100 and protease inhibitors for 2 h at 4 °C. The detergent extracts were centrifuged at 100,000 × g for 1 h at 4 °C, and the supernatant was immunoprecipitated with the anti-human DPP-IV mAb HBB 3/775/42. The pellets were then solubilized in the same buffer for 20 min at 37 °C and immunoprecipitated with the anti-human DPP-IV mAb HBB 3/775/42. Immunocomplexes were collected on protein G-Sepharose-4B (Sigma), eluted in SDS sample buffer (0.2 m Tris-HCl buffer, pH 6.8, containing 2% SDS, 5% β-mercaptoethanol and 30% glycerol) at 60 °C for 15 min, and then analyzed on a 7% SDS-polyacrylamide gels. For autoradiography, gels were fixed in 40% ethanol, 10% glycerol, 10% acetic acid, soaked in Amplify (Amersham Biosciences) for 20 min, dried on Whatman paper, and then exposed to Hyperfilm-βmax (Amersham Biosciences). Isolation of Raft Microdomains—Rafts were isolated from series of 12 75-cm2 culture flasks of HT-29 5M12 cells according to the procedure of Fiedler et al. (33Fiedler K. Kobayashi T. Kurzchalia T.V. Simons K. Biochemistry. 1993; 32: 6365-6373Crossref PubMed Scopus (225) Google Scholar) slightly modified. The cell pellet equilibrated in 10 mm HEPES buffer, pH 7.4, with 2 mm EGTA, 0.25 m sucrose, and protease inhibitors, was homogenized by 10 passages in a 1-ml tip, 15 passages in a 22-gauge needle, and 10 passages in a Dounce homogenizer. A total membrane fraction was obtained from a post-nuclear supernatant after ultracentrifugation (38,000 rpm, 20 h) in a SW41 swinging rotor at the interface (1.2 m; 0.8 m) of a discontinuous sucrose gradient, washed in TNE buffer (25 mm Tris-HCl, pH 7.5, with 150 mm NaCl and 5 mm EDTA), treated with a mixture of TNE/Triton X-100 at 1% for 30 min on ice, and separated by ultracentrifugation (39,000 rpm, 18 h) on a discontinuous sucrose gradient (0.15 m; 1.1 m; 1.2 m) in a SW41 rotor. Individual fractions of 1 ml were isolated all over the gradient and examined for their protein content. For Western blotting, half of the entire volume of each fraction was used. Proteins were precipitated with trichloroacetic acid, washed with acetone, and solubilized in SDS sample buffer. Western Blotting—Samples were separated on a 5–30% SDS-PAGE (34Laemmli U.K. Nature. 1970; 227: 680-681Crossref PubMed Scopus (207227) Google Scholar), and the detection of proteins was carried out by luminescence using the ECL Western blotting system (Amersham Biosciences, Aylesbury, UK). In this work, we studied the effect of GalNAcα-O-bn on the direct apical biosynthetic route using a cloned population of HT-29 cells of enterocytic phenotype (HT-29 5M12) and short exposure times to GalNAcα-O-bn. In addition to the study of endogenous transmembrane DPP-IV, which is endocytosed and recycled to the apical membrane, we studied a soluble form of murine DPP-IV, which was expected to be secreted in the medium under a polarized fashion after transfection in HT-29 5M12 cells. Indeed, after transfection of a soluble form of rat DPP-IV in MDCK cells, Weisz et al. (35Weisz O.A. Machamer C.E. Hubbard A.L. J. Biol. Chem. 1992; 267: 22282-22288Abstract Full Text PDF PubMed Google Scholar) reported that this soluble form was predominantly secreted into the apical medium, indicating that the lumenal domain of DPP-IV was likely to contain the apical sorting information. Inhibition in the Secretion of a Soluble Murine DPP-IV Form—HT-29 5M12 cells of enterocytic phenotype were stably transfected with an eukaryotic expression vector coding for a secreted mouse DPP-IV, and we selected the clone HT-29 5M12 Cl2 that secreted DPP-IV enzymatic activity in the medium. We first investigated the secreted DPP-IV forms in the apical and basolateral medium using two-dimensional electrophoresis and protein identification by mass spectrometry of tryptic digests (Fig. 1A). Cells were cultured on filters in standard medium up to day 10 and then for 24 h in serum-free medium with or without GalNAcα-O-bn. Apical and basolateral cell culture media were collected, concentrated, and precipitated with trichloroacetic acid. The amounts of cell culture media used for two-dimensional electrophoresis were normalized in reference to the same number of cells. In the apical medium of control cells, a train of 5 spots of high intensity was visualized between 80 and 90 kDa with a range of pI (isoelectric point) from 5.5 to 6 approximately. These spots were excised, digested with trypsin and analyzed for their peptidic map by MALDI-TOF. Results showed that these spots corresponded to murine DPP-IV (data not shown). The two-dimensional pattern of the basolateral medium showed that sDPP-IV was not significantly secreted into this medium. This shows that the recombinant sDPP-IV is predominantly secreted in the apical medium of HT-29 5M12 Cl2 cells and confirmed the existence of apical targeting signal(s) in the ectodomain of the molecule, and also indicated the involvement of a specific machinery for its transport toward the apical side. In the apical medium collected after GalNAcα-O-bn treatment, we visualized in the area of sDPP-IV only three spots (spots 1, 2, and 3) with lower intensity. Spots 4 and 5, corresponding to the most acidic forms, were not detected, and spot 3, which accounted for the major spot, showed a marked decrease in comparison to spots 1 and 2. sDPP-IV was not recovered in the basolateral medium of treated HT-29 5M12 cells, although a s
Referência(s)