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Benzyl-N-acetyl-α-d-galactosaminide Induces a Storage Disease-like Phenotype by Perturbing the Endocytic Pathway

2003; Elsevier BV; Volume: 278; Issue: 14 Linguagem: Inglês

10.1074/jbc.m211909200

1083-351X

Fausto Ulloa, Francisco X. Real,

Glycosylation and Glycoproteins Research

The sugar analogO-benzyl-N-acetyl-α-d-galactosaminide (BG) is an inhibitor of glycan chain elongation and inhibits α2,3-sialylation in mucus-secreting HT-29 cells. Long-term exposure of these cells to BG is associated with the accumulation of apical glycoproteins in cytoplasmic vesicles. The mechanisms involved therein and the nature of the vesicles have not been elucidated. In these cells, a massive amount of BG metabolites is synthesized. Because sialic acid is mainly distributed apically in epithelial cells, it has been proposed that the BG-induced undersialylation of apical membrane glycoproteins is responsible for their intracellular accumulation due to a defect in anterograde traffic and that sialic acid may constitute an apical targeting signal. In this work, we demonstrate that the intracellular accumulation of membrane glycoproteins does not result mainly from defects in anterograde traffic. By contrast, in BG-treated cells, endocytosed membrane proteins were retained intracellularly for longer periods of time than in control cells and colocalized with accumulated MUC1 and ॆ1 integrin in Rab7/lysobisphosphatidic acid+ vesicles displaying features of late endosomes. The phenotype of BG-treated cells is reminiscent of that observed in lysosomal storage disorders. Sucrose induced a BG-like, lysosomal storage disease-like phenotype without affecting sialylation, indicating that undersialylation is not a requisite for the intracellular accumulation of membrane glycoproteins. Our findings strongly support the notion that the effects observed in BG-treated cells result from the accumulation of BG-derived metabolites and from defects in the endosomal pathway. We propose that abnormal subcellular distribution of membrane glycoproteins involved in cellular communication and/or signaling may also take place in lysosomal storage disorders and may contribute to their pathogenesis. The sugar analogO-benzyl-N-acetyl-α-d-galactosaminide (BG) is an inhibitor of glycan chain elongation and inhibits α2,3-sialylation in mucus-secreting HT-29 cells. Long-term exposure of these cells to BG is associated with the accumulation of apical glycoproteins in cytoplasmic vesicles. The mechanisms involved therein and the nature of the vesicles have not been elucidated. In these cells, a massive amount of BG metabolites is synthesized. Because sialic acid is mainly distributed apically in epithelial cells, it has been proposed that the BG-induced undersialylation of apical membrane glycoproteins is responsible for their intracellular accumulation due to a defect in anterograde traffic and that sialic acid may constitute an apical targeting signal. In this work, we demonstrate that the intracellular accumulation of membrane glycoproteins does not result mainly from defects in anterograde traffic. By contrast, in BG-treated cells, endocytosed membrane proteins were retained intracellularly for longer periods of time than in control cells and colocalized with accumulated MUC1 and ॆ1 integrin in Rab7/lysobisphosphatidic acid+ vesicles displaying features of late endosomes. The phenotype of BG-treated cells is reminiscent of that observed in lysosomal storage disorders. Sucrose induced a BG-like, lysosomal storage disease-like phenotype without affecting sialylation, indicating that undersialylation is not a requisite for the intracellular accumulation of membrane glycoproteins. Our findings strongly support the notion that the effects observed in BG-treated cells result from the accumulation of BG-derived metabolites and from defects in the endosomal pathway. We propose that abnormal subcellular distribution of membrane glycoproteins involved in cellular communication and/or signaling may also take place in lysosomal storage disorders and may contribute to their pathogenesis. benzyl-N-acetyl-α-d-galactosaminide M. amurensis lectin peanut agglutinin acid α-glucosidase infantile sialic acid storage disease monoclonal antibody lysobisphosphatidic acid early endosomal antigen-1 5-N-acetylneuraminic acid phosphate-buffered saline bovine serum albumin fluorescein isothiocyanate tetramethylrhodamine isothiocyanate radioimmune precipitation assay Glycosylation plays a fundamental role in various biological processes such as the folding, oligomerization, and biochemical maturation of proteins, and glycans play an important role in cell-cell and cell-matrix adhesion during development and tumor progression and in host-pathogen interactions, among others (1Varki A. Glycobiology. 1993; 3: 97-130Google Scholar, 2Varki A. Cummings R. Esko J. Freeze H. Hart G. Marth J. Essentials of Glycobiology. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1999Google Scholar). The use of sugar analogs has been fundamental for the study of glycan function, mainly in the study of N-glycans, as no specific inhibitors ofO-glycosylation are currently available (2Varki A. Cummings R. Esko J. Freeze H. Hart G. Marth J. Essentials of Glycobiology. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1999Google Scholar). Benzyl-N-acetyl-α-d-galactosaminide (BG),1 as well as other GalNAc analogs, was initially reported to inhibitO-glycosylation, presumably as a result of the inhibition of GalNAc-O-Ser/Thr elongation (3Kuan S.F. Byrd J.C. Basbaum C. Kim Y.S. J. Biol. Chem. 1989; 264: 19271-19277Google Scholar). More recent studies of cells treated with BG or other GalNAc analogs have shown that their overall effects are likely due to complex modifications of glycan biosynthesis (4DiIulio N.A. Bhavanandan V.P. Glycobiology. 1995; 5: 195-199Google Scholar, 5Huet G. Kim I. de Bolos C. Lo-Guidice J.M. Moreau O. Hemon B. Richet C. Delannoy P. Real F.X. Degand P. J. Cell Sci. 1995; 108: 1275-1285Google Scholar, 6Delannoy P. Kim I. Emery N. de Bolos C. Verbert A. Degand P. Huet G. Glycoconj. J. 1996; 13: 717-726Google Scholar, 7Byrd J.C. Dahiya R. Huang J. Kim Y.S. Eur. J. Cancer. 1995; 31: 1498-1505Google Scholar, 8Huet 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: 1311-1322Google Scholar, 9Hennebicq-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: 283-295Google Scholar, 10Gouyer 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: 1455-1471Google Scholar). A detailed study of mucus-secreting HT-29 colon cancer cells treated chronically with BG revealed dramatic and pleiotropic effects, including a reduction in cell proliferation, an increase in cell size, and the accumulation of electron-lucid cytoplasmic vesicles containing predominantly undersialylated apical glycoproteins with N- and O-glycans (i.e. dipeptidyl peptidase IV and MUC1) (8Huet 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: 1311-1322Google Scholar). In HT-29 glycoproteins, sialic acid is mainly linked in an α2,3-configuration, and α2,3-sialyltransferase activity is profoundly inhibited upon treatment with BG. This is accompanied by a marked decrease in the sialylation of glycoproteins, detected with the α2,3-sialic acid-specific Maackia amurensis lectin (MAL), and an increase in the levels of glycoprotein-associated Gal-GalNAc precursor epitope detected with peanut agglutinin (PNA; Arachis hypogaea) (4DiIulio N.A. Bhavanandan V.P. Glycobiology. 1995; 5: 195-199Google Scholar, 5Huet G. Kim I. de Bolos C. Lo-Guidice J.M. Moreau O. Hemon B. Richet C. Delannoy P. Real F.X. Degand P. J. Cell Sci. 1995; 108: 1275-1285Google Scholar, 8Huet 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: 1311-1322Google Scholar). In these cells, changes in sialylation are a consequence of the synthesis of BG-derived metabolites, such as benzyl-GalNAc-Gal, that inhibit sialyltransferases, thus leading to the accumulation of the precursor carbohydrate chains of apical glycoproteins (5Huet G. Kim I. de Bolos C. Lo-Guidice J.M. Moreau O. Hemon B. Richet C. Delannoy P. Real F.X. Degand P. J. Cell Sci. 1995; 108: 1275-1285Google Scholar, 6Delannoy P. Kim I. Emery N. de Bolos C. Verbert A. Degand P. Huet G. Glycoconj. J. 1996; 13: 717-726Google Scholar). Based on these findings, on the apical distribution of sialic acid in epithelial cells (11Ulloa F. Real F.X. J. Histochem. Cytochem. 2001; 49: 501-509Google Scholar), and on the proposed role of N- and O-glycans in apical targeting (12Fiedler K. Parton R.G. Kellner R. Etzold T. Simons K. EMBO J. 1994; 13: 1729-1740Google Scholar, 13Rodriguez-Boulan E. Gonzalez A. Trends Cell Biol. 1999; 9: 291-294Google Scholar), it was suggested that the altered subcellular distribution of apical glycoproteins and the accumulation of cytoplasmic vesicles in BG-treated cells might result from glycoprotein undersialylation and that sialic acid might play a role in apical targeting (8Huet 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: 1311-1322Google Scholar, 9Hennebicq-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: 283-295Google Scholar). We have shown that BG also perturbs the processing of lysosomal enzymes such as acid α-glucosidase (AAG) and cathepsin D, indicating broader effects of BG on intracellular traffic (14Ulloa F. Francı́ C. Real F.X. J. Biol. Chem. 2000; 275: 18785-18793Google Scholar). However, the available evidence did not allow us to unravel several key issues related to the biochemical effects of this drug: 1) no definite causal relationship has been established between the effects on sialylation and the abnormal subcellular distribution of membrane glycoproteins, and 2) there are few data supporting the contribution of an anterograde (Golgi-to-plasma membrane) traffic defectversus endocytic pathway defects leading to the vesicular accumulation of membrane glycoproteins in cells treated with BG for prolonged periods of time. In this regard, the best evidence for the contribution of an anterograde traffic defect comes from the analysis of AAG maturation in BG-treated cells, which showed a blockade in a post-trans-Golgi network compartment (14Ulloa F. Francı́ C. Real F.X. J. Biol. Chem. 2000; 275: 18785-18793Google Scholar). Our finding that BG treatment results in misprocessing of lysosomal enzymes (14Ulloa F. Francı́ C. Real F.X. J. Biol. Chem. 2000; 275: 18785-18793Google Scholar) is reminiscent of the effects of sucrose on fibroblasts (15Schmidt J.A. Mach L. Paschke E. Glössl J. J. Biol. Chem. 1999; 274: 19063-19071Google Scholar) and has led us to propose that BG could induce a reversible lysosomal storage disease-like phenotype in HT-29 cells (14Ulloa F. Francı́ C. Real F.X. J. Biol. Chem. 2000; 275: 18785-18793Google Scholar). Lysosomal storage diseases are a diverse group of inherited conditions characterized by functional defects in endosomes/lysosomes leading to the accumulation of intracellular vesicles. They are caused by mutations in a wide variety of genes and are associated with the accumulation of cholesterol and/or sphingolipids in late endosomes/lysosomes (16Simons K. Gruenberg J. Trends Cell Biol. 2000; 10: 459-462Google Scholar). A misrouting of lactosylceramide and possibly other lipids appears to be characteristic of storage diseases, although the precise mechanisms involved are unknown (17Chen C.S. Patterson M.C. Wheatley C.L. O'Brien J.F. Pagano R.E. Lancet. 1999; 354: 901-905Google Scholar). The aims of this work have been 1) to analyze the contribution of abnormalities in endocytic traffic to the BG phenotype, 2) to determine whether a causal relationship exists between the undersialylation of membrane glycoproteins induced by BG and their intracellular accumulation, and 3) to provide additional support to the hypothesis that BG induces a lysosomal storage-like phenotype. Our findings indicate that in HT-29 cells, as well as in IMIM-PC-1 pancreatic cancer cells, the 舠BG phenotype舡 (i.e. dramatic accumulation of cytoplasmic vesicles containing membrane glycoproteins) 1) is due, to a large extent, to defects in the endocytic pathway; 2) does not depend on a defect in glycoprotein sialylation; 3) is associated with the accumulation of intracellular vesicles with variable phenotype, including vesicles containing late endosomal markers as well as endocytosed membrane proteins, as in storage diseases; and 4) is similar to that induced by prolonged treatment with sucrose, except that the latter does not perturb glycoprotein sialylation. Altogether, these results support the notion that defective sialylation is not required for the abnormal subcellular distribution of membrane glycoproteins induced by BG and that the latter may also take place in lysosomal storage diseases and contribute to their pathogenesis. HT-29 colon cancer cells selected with 10−6m methotrexate (here designated as HT-29 M6 cells) were obtained from Drs. Alain Zweibaum and Thécla Lesuffleur (INSERM, Paris, France). IMIM-PC-1 pancreatic cancer cells were established in our laboratory (18Vilá M.R. Lloreta J. Schussler M.H. Berrozpe G. Welt S. Real F.X. Lab. Invest. 1995; 72: 395-404Google Scholar). The A13 clone was obtained by seeding IMIM-PC-1 cells at 100 cells/plate in 10-cm diameter dishes and isolating single cell-derived populations with cloning cylinders. Human infantile sialic acid storage disease (ISSD) skin fibroblasts (GM5520) and control skin fibroblasts from the unaffected parents (GM5521) were kindly provided by Dr. Josef Glössl (University of Agricultural Sciences, Vienna, Austria) (15Schmidt J.A. Mach L. Paschke E. Glössl J. J. Biol. Chem. 1999; 274: 19063-19071Google Scholar). HT-29 M6 and IMIM-PC-1 cells were seeded on plastic at 2 × 104 and 5 × 103 cells/cm2, respectively. All cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen, Glasgow, UK) supplemented with 107 fetal bovine serum (Invitrogen) at 37 °C in a 57 CO2atmosphere. Unless specified otherwise, when cells were cultured in the presence of BG (Sigma), this drug was added to the medium 24 h after seeding at a final concentration of 2 mm. Rabbit polyclonal antiserum detecting AAG was provided by Dr. A. Reuser (Erasmus University, Rotterdam, The Netherlands) (19Wisselaar H.A. Kroos M.A. Hermans M.M.P. van Beeumen J. Reuser A.J.J. J. Biol. Chem. 1993; 268: 2223-2231Google Scholar). Rabbit polyclonal antibody 1397 detecting ॆ1 integrin was purchased from Chemicon International, Inc. (Temecula, CA). Rabbit anti-Rab5 and anti-Rab7 polyclonal antibodies, used for immunoblotting and immunofluorescence, were kindly provided by Dr. P. Chavrier (Institut Curie, Paris) (20Chavrier P. Parton R.G. Hauri H.P. Simons K. Zerial M. Cell. 1990; 62: 317-329Google Scholar, 21Meresse S. Gorvel J.P. Chavrier P. J. Cell Sci. 1995; 108: 3349-3358Google Scholar) and by Dr. A. Le Bivic (Institut de Biologie du Development de Marseilles, Marseilles, France), respectively. Sheep antibodies detecting TGN46, the human ortholog of the trans-Golgi network marker designated TGN38, was obtained from Dr. S. Ponnambalam (University of Dundee, Dundee, United Kingdom). Mouse monoclonal antibody (mAb) 6C4 detecting lysobisphosphatidic acid (LBPA) was kindly provided by Dr. J. Gruenberg (University of Genève, Genève, Switzerland) (22Kobayashi T. Stang E. Fang K.S. de Moerloose P. Parton R.G. Gruenberg J. Nature. 1998; 392: 193-197Google Scholar). mAb TS2/16 detecting ॆ1 integrin was a gift of Dr. F. Sánchez-Madrid (Hospital de la Princesa, Universidad Autónoma de Madrid, Madrid, Spain) (23Arroyo A.G. Sánchez-Mateos P. Campanero M.R. Martin-Padura I. Dejana E. Sánchez-Madrid F. J. Cell Biol. 1992; 117: 659-670Google Scholar). mAb LICRLon M8 detecting MUC1 was provided by Dr. D. Swallow (University College, London, UK) (24McIlhinney R.A. Patel S. Gore M.E. Biochem. J. 1985; 227: 155-162Google Scholar). mAb 6D9 detecting the MAL proteolipid was a gift of Dr. M. A. Alonso (Centro de Biologı́a Molecular-Consejo Superior de Investigaciones Cientı́ficas, Madrid) (25Martı́n-Belmonte F. Kremer L. Albar J.P. Marazuela M. Alonso M.A. Endocrinology. 1998; 139: 2077-2084Google Scholar, 26Puertollano R. Martı́n-Belmonte F. Millán J. de Marco M.C. Albar J.P. Kremer L. Alonso M.A. J. Cell Biol. 1999; 145: 141-145Google Scholar). Affinity-purified rabbit polyclonal antibodies detecting GRASP65 were a gift of Dr. A. Mallabiabarrena (Universitat Pompeu Fabra, Barcelona, Spain) and Dr. V. Malhotra (University of California at San Diego, La Jolla, CA) (27Sutterlin C. Lin C.Y. Feng Y. Ferris D.K. Erikson R.L. Malhotra V. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9128-9132Google Scholar). mAbs detecting early endosomal antigen-1 (EEA1) and p115 were purchased from Transduction Laboratories (San Diego, CA). Digoxigenin-conjugated MAL and PNA, recognizing the oligosaccharide species α2,3-Neu5Ac-R and Galॆ1–3GalNAc-R, respectively, were purchased from Roche Molecular Biochemicals (Mannheim, Germany). Biotin-conjugated MAL was obtained from Vector Labs, Inc. (Burlingame, CA). Transmission electron microscopy was performed on cells grown on plastic as previously described (18Vilá M.R. Lloreta J. Schussler M.H. Berrozpe G. Welt S. Real F.X. Lab. Invest. 1995; 72: 395-404Google Scholar). Samples embedded in Epon (Polysciences Inc., Warington, PA) were re-embedded to make sections perpendicular to the bottom of the flask. Ultrathin sections were visualized using a Phillips CM100 electron microscope. For double immunofluorescence staining, cells were grown on coverslips and fixed with 47 paraformaldehyde for 10 min at room temperature, incubated with 50 mm NH4Cl for 30 min, and permeabilized with 0.57 Triton X-100 or 0.17 saponin in phosphate-buffered saline (PBS)/bovine serum albumin (BSA) for 30 min. In the case of reactions employing antibody 6C4, the permeabilization step was carried out using 0.057 saponin in PBS/BSA. Cells were then sequentially incubated with primary antibodies and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse Ig or TRITC-conjugated goat anti-rabbit Ig (Dako, Glostrup, Denmark); antibodies were added for 1 h, followed by three washes with PBS. Biotin-conjugated MAL (20 ॖg/ml) was added in lectin buffer (50 mm Tris-HCl (pH 7.5), 15 mmKCl, 5 mm MgCl2, and 0.17 saponin) for 1 h, followed by FITC-streptavidin (5 ॖg/ml). Fluorescence microscopy analysis was performed using a Leica TCS-SP2 confocal unit. To detect cholesterol, cells were grown on coverslips, fixed with 47 paraformaldehyde for 10 min at room temperature, and stained with filipin (Sigma) at 125 ॖg/ml in PBS for 30 min. Fluorescence analysis was performed using conventional fluorescence microscopy. Cells were maintained in methionine-free minimal essential medium containing 107 dialyzed fetal bovine serum for 30 min, pulse-labeled for 1 h with 50 ॖCi/ml [35S]Met/Cys (Tran35S-label, ICN Biomedicals, Costa Mesa, CA) in the same medium, and chased with Dulbecco's modified Eagle's medium supplemented with 107 fetal bovine serum and 2 mm unlabeled methionine for 24 h. In the case of cells treated with BG, the culture medium was supplemented with the drug at the concentration indicated. Cells were washed three times with PBS and lysed with 50 mm Tris-HCl (pH 8), 17 Triton X-100, 62.5 mm EDTA, 2 mmPefabloc, 1 ॖg/ml aprotinin, and 1 ॖg/ml leupeptin. Lysates were centrifuged at 10,000 × g for 30 min, and supernatants were precleared with preimmune rabbit antiserum for 2 h and protein A-Sepharose (Roche Molecular Biochemicals) for 30 min. Antibodies were added to the precleared supernatants for 3–16 h in the presence of 0.27 SDS. When using mAbs, rabbit anti-mouse Ig (Dako) was added for 2 h prior to the incubation with protein A-Sepharose. Immunoprecipitates were washed three times with radioimmune precipitation assay (RIPA) buffer (10 mm Tris-HCl, 150 mm NaCl, 0.17 SDS, 17 deoxycholate, and 17 Nonidet P-40), three times with high salt buffer (10 mm Tris-HCl, 0.5 mm NaCl, 1 mm EDTA, 0.57 Nonidet P-40, and 0.17 SDS), and twice with PBS. All immunoprecipitation steps were carried out at 4 °C. Immunoprecipitates were resuspended in sample buffer, resolved by SDS-PAGE, and developed by fluorography. Cells were metabolically labeled as described above with 150 ॖCi/ml [35S]Met/Cys for 1 h and chased for 24 h at 37 °C; the medium was collected, and cells were lysed with RIPA buffer. Proteins from the medium and cell extracts were precipitated in 107 trichloroacetic acid for 1 h on ice. Samples were centrifuged at 13,000 rpm for 15 min; pellets were resuspended in 1 m NaOH; and labeled material was quantified in a scintillation counter. The proportion of secreted material was calculated as total secreted trichloroacetic acid-precipitable cpm/total cell lysate-associated trichloroacetic acid-precipitable cpm. Cells were incubated on ice with 0.5 mg/ml sulfosuccinimidyl 2-(biotinamido)ethyl-1,3′-dithiopropionate in PBS, 1 mmMgCl2, and 1 mm CaCl2 for 15 min to label membrane proteins; subsequently, cold Dulbecco's modified Eagle's medium was added for 15 min as a blocking reagent. Cells were incubated at 37 °C for the indicated periods of time with Dulbecco's modified Eagle's medium supplemented with 107 prewarmed fetal bovine serum. Cells were placed on ice, and biotin bound to membrane proteins was stripped by incubating the cells twice in cold 60 mm glutathione in PBS for 20 min. Cells were lysed with RIPA buffer supplemented with protease inhibitors; lysates were centrifuged at 13,000 rpm for 30 min at 4 °C; and supernatants were fractionated by SDS-PAGE and transferred to a nitrocellulose membrane. Biotinylated proteins were detected with peroxidase-conjugated streptavidin (Zymed Laboratories Inc., South San Francisco, CA). The proportion of biotinylated proteins was determined by densitometric analysis using GenQuant software (Amersham Biosciences). Cells were lysed in 50 mmTris-HCl (pH 8.0), 62.5 mm EDTA, and 17 Triton X-100, supplemented with protease inhibitors, and lysates were centrifuged at 13,000 rpm for 30 min at 4 °C. Protein extracts were fractionated by 67 SDS-PAGE and transferred to a nitrocellulose membrane. MAL blotting was carried out with reagents from the digoxigenin glycan differentiation kit (Roche Molecular Biochemicals) according to the manufacturer's instructions. Lectin reactivity of MUC1 molecules immunoprecipitated from IMIM-PC-1 cell lysates was examined as described (8Huet 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: 1311-1322Google Scholar). Cells were lysed in RIPA buffer supplemented with protease inhibitors. Cell extracts (30 ॖg) were fractionated by SDS-PAGE and transferred to a nitrocellulose membrane. Filters were blocked with 57 skim milk for 30 min, incubated with primary antibodies for 1 h, washed, and incubated with peroxidase-conjugated goat anti-rabbit or anti-mouse Ig antibodies. Proteins were detected by the enhanced chemiluminescence system (Amersham Biosciences) according to the manufacturer's instructions. Treatment of HT-29 M6 cells with BG leads to the accumulation of undersialylated apical glycoproteins in cytoplasmic vesicles. The interpretation of these effects has relied upon the assumption that BG induces a blockade in the anterograde traffic, leading to the vesicular accumulation of apical glycoproteins en route to the plasma membrane (8Huet 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: 1311-1322Google Scholar). To obtain insight into the rate of overall anterograde protein traffic, HT-29 M6 cells were cultured on Transwells, and the impermeability of the cell layers was assessed using [14C]mannitol. BG was or was not added to the medium of impermeable cell cultures for 7 days; cells were metabolically labeled with [35S]Met/Cys; apical and basolateral media and labeled cells were collected; and total constitutive protein secretion and cell-associated proteins were quantified. The secreted/cell-associated label ratio was expressed as an index. There was no significant inhibition of total apical or basolateral protein secretion (Fig. 1A). To obtain additional evidence that BG has no significant effect on total protein secretion, we performed similar experiments using IMIM-PC-1 pancreatic cancer cells. This cell line was selected from a panel of lines in which the effects of prolonged BG treatment had been tested. 2F. Ulloa and F. X. Real, unpublished data. Upon treatment with BG, IMIM-PC-1 cells displayed a phenotype that is similar to that of BG-treated HT-29 M6 cells and is characterized by reduced cell proliferation, increased cell volume, reduced α2,3-glycoprotein sialylation, and accumulation of electron-lucid cytoplasmic vesicles (Fig. 2, A–D). Unlike HT-29 M6 cells, BG-treated IMIM-PC-1 cells displayed the intracellular accumulation of both apical (i.e. MUC1) and basolateral (i.e. ॆ1 integrin) glycoproteins (Fig.2E). As shown for HT-29 M6 cells, BG did not significantly decrease total protein secretion in IMIM-PC-1 cells (Fig.1B). Therefore, the BG-induced cytoplasmic accumulation of membrane glycoproteins is not primarily due to alterations in anterograde transport. To determine whether intracellular glycoprotein accumulation results from altered endocytosis/recycling of plasma membrane glycoproteins, IMIM-PC-1 cells were used because they are flatter and allow a better morphological resolution than HT-29 M6 cells and because they express lower levels of membrane-associated mucins that impair cell-surface biotinylation. IMIM-PC-1 membrane proteins were biotinylated at 4 °C and allowed to internalize at 37 °C for various periods of time. Biotin that remained bound to plasma membrane glycoproteins that had not been endocytosed was dissociated using glutathione, so only the internalized glycoproteins originating from the plasma membrane could bind streptavidin. Endocytosis of biotinylated membrane proteins was analyzed and quantified by Western blotting with peroxidase-conjugated streptavidin (Fig. 3A). In addition, the subcellular distribution of biotinylated glycoproteins was examined using FITC-streptavidin by confocal fluorescence microscopy (Fig. 3B). In control and BG-treated cells, labeled proteins were distributed mainly at the plasma membrane 10 min after shifting the temperature to 37 °C, and intracellular labeling increased progressively during the first 2 h. Subsequently, a decrease in cytoplasmic labeling was observed only in control cells. In BG-treated cells, intracellular labeling progressively increased during the first 30 min after the temperature shift, and an intracellular accumulation of labeled proteins was demonstrated for up to 24 h. Quantitative analysis showed that endocytosis occurred at a relatively slower rate in BG-treated cells during the first 2 h, whereas intracellular accumulation of biotinylated glycoproteins occurred to a greater extent than in control cells (Fig. 3, A andB). At 24 h, BG-treated cells showed a 5.5-fold higher accumulation of membrane proteins compared with control cells. These results indicate that the phenotype of BG-treated cells involves the intracellular accumulation of endocytosed membrane glycoproteins. To determine whether compartments in which MUC1 and ॆ1 integrin accumulate also contain endocytosed biotinylated membrane proteins, cells were cultured for 8 days in the presence of BG, surface-biotinylated at 4 °C, and incubated at 37 °C for various periods of time. As described above, in BG-treated IMIM-PC-1 cells, MUC1 and ॆ1 integrin colocalized and accumulated in cytoplasmic vesicles (Fig.4). A progressive increase in the amount of biotinylated membrane glycoproteins was observed in MUC1- and ॆ1 integrin-containing vesicles starting 30 min after initiation of internalization for up to 24 h (data not shown). At the 4-h time point, numerous vesicles showed double labeling of endocytosed membrane proteins with either MUC1 or ॆ1integrin, whereas others contained exclusively one type of protein marker (Fig. 4). These findings indicate that the cytoplasmic vesicles that accumulate in BG-treated cells indeed receive endocytosed membrane material. MUC1 and ॆ1 integrin have been shown to be recycled to the plasma membrane (28Litvinov S.V. Hilkens J. J. Biol. Chem. 1993; 268: 21364-21371Google Scholar, 29Regen C.M. Horwitz A.F. J. Cell Biol. 1992; 119: 1347-1359Google Scholar), strongly suggesting that the cytoplasmic accumulation of these proteins is secondary to defective recycling. To obtain insight into the nature of the cytoplasmic vesicles, double immunolabeling for MUC1 or ॆ1 integrin and markers corresponding to the Golgi complex (GRASP65 and TGN46) (30Prescott A.R. Lucocq J.M. James J. Lister J.M. Ponnambalam S. Eur. J. Cell Biol. 1997; 72: 238-246Google Scholar), early endosomes (EEA1 and Rab5) (31Stenmark H. Aasland R. Toh B.-H. D'Arrigo A. J. Biol. Chem. 1996; 271: 24048-24054Google Scholar, 32Gorvel J. Chavrier P. Zerial M. Gruenberg J. Cell. 1991; 64: 915-925Google Scholar), and late endosomes (LBPA and Rab7) (20Chavrier P. Parton R.G. Hauri H.P. Simons K. Zerial M. Cell. 1990; 62: 317-329Google Scholar, 34Kobayashi T. Beuchat M.H. Lindsay M. Frias S. Palmiter R.D. Sakuraba H. Parton R.G. Gruenberg J. Nat. Cell Biol. 1999; 1: 113-118Google Scholar) was performed. Results are shown for ॆ1 integrin colocalization exper