From the Unitat de Biologia Celular i Molecular, Institut Municipal d'Investigació Mèdica, Universitat Pompeu Fabra, Barcelona 08003, Spain
Received for publication, November 21, 2002, and in revised form, January 17, 2003
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ABSTRACT |
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The sugar analog
O-benzyl-N-acetyl- 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 (1, 2). 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 of
O-glycosylation are currently available (2).
Benzyl-N-acetyl- Our finding that BG treatment results in misprocessing of lysosomal
enzymes (14) is reminiscent of the effects of sucrose on fibroblasts
(15) and has led us to propose that BG could induce a reversible
lysosomal storage disease-like phenotype in HT-29 cells (14).
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 (16). A misrouting of lactosylceramide and possibly
other lipids appears to be characteristic of storage diseases, although
the precise mechanisms involved are unknown (17).
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 "BG phenotype" (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.
Cell Culture--
HT-29 colon cancer cells selected with
10 Antibodies and Lectins--
Rabbit polyclonal antiserum
detecting AAG was provided by Dr. A. Reuser (Erasmus University,
Rotterdam, The Netherlands) (19). Rabbit polyclonal antibody 1397 detecting Transmission Electron Microscopy--
Transmission electron
microscopy was performed on cells grown on plastic as previously
described (18). 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.
Fluorescence Microscopy--
For double immunofluorescence
staining, cells were grown on coverslips and fixed with 4%
paraformaldehyde for 10 min at room temperature, incubated with 50 mM NH4Cl for 30 min, and permeabilized with
0.5% Triton X-100 or 0.1% 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
0.05% 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 µg/ml) was added in
lectin buffer (50 mM Tris-HCl (pH 7.5), 15 mM
KCl, 5 mM MgCl2, and 0.1% saponin) for 1 h, followed by FITC-streptavidin (5 µg/ml). Fluorescence microscopy
analysis was performed using a Leica TCS-SP2 confocal unit.
To detect cholesterol, cells were grown on coverslips, fixed with 4%
paraformaldehyde for 10 min at room temperature, and stained with
filipin (Sigma) at 125 µg/ml in PBS for 30 min. Fluorescence analysis
was performed using conventional fluorescence microscopy.
Metabolic Labeling and Immunoprecipitation--
Cells were
maintained in methionine-free minimal essential medium containing 10%
dialyzed fetal bovine serum for 30 min, pulse-labeled for 1 h with
50 µCi/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 10% 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), 1% Triton X-100, 62.5 mM EDTA, 2 mM
Pefabloc, 1 µg/ml aprotinin, and 1 µg/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 0.2% 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, 0.1% SDS, 1% deoxycholate, and 1% Nonidet
P-40), three times with high salt buffer (10 mM Tris-HCl, 0.5 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, and
0.1% 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.
Quantitation of Protein Secretion--
Cells were metabolically
labeled as described above with 150 µCi/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 10% 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.
Endocytosis Assays--
Cells were incubated on ice with 0.5 mg/ml sulfosuccinimidyl
2-(biotinamido)ethyl-1,3'-dithiopropionate in PBS, 1 mM
MgCl2, 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 10% 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).
Lectin Blotting--
Cells were lysed in 50 mM
Tris-HCl (pH 8.0), 62.5 mM EDTA, and 1% 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
6% 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 (8).
Immunoblotting--
Cells were lysed in RIPA buffer supplemented
with protease inhibitors. Cell extracts (30 µg) were fractionated by
SDS-PAGE and transferred to a nitrocellulose membrane. Filters were
blocked with 5% 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
(8). 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).
-D-galactosaminide
(BG) is an inhibitor of glycan chain elongation and inhibits
2,3-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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-galactosaminide
(BG),1 as well as other
GalNAc analogs, was initially reported to inhibit
O-glycosylation, presumably as a result of the inhibition of
GalNAc-O-Ser/Thr elongation (3). 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 (4-10). 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) (8). In HT-29
glycoproteins, sialic acid is mainly linked in an
2,3-configuration,
and
2,3-sialyltransferase activity is profoundly inhibited upon
treatment with BG. This is accompanied by a marked decrease in the
sialylation of glycoproteins, detected with the
2,3-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) (4, 5, 8). 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 (5, 6). Based on these findings, on the apical
distribution of sialic acid in epithelial cells (11), and on the
proposed role of N- and O-glycans in apical targeting (12, 13), 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 (8, 9). We have shown that BG also perturbs the processing of
lysosomal enzymes such as acid
-glucosidase (AAG) and cathepsin D,
indicating broader effects of BG on intracellular traffic (14).
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 defect
versus 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 (14).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
6 M 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 (18). 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) (15). 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 10% fetal bovine serum (Invitrogen) at 37 °C in a 5% CO2
atmosphere. 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.
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) (20, 21) 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) (22). mAb TS2/16 detecting
1 integrin was a
gift of Dr. F. Sánchez-Madrid (Hospital de la Princesa,
Universidad Autónoma de Madrid, Madrid, Spain) (23). mAb LICRLon
M8 detecting MUC1 was provided by Dr. D. Swallow (University
College, London, UK) (24). mAb 6D9 detecting the MAL proteolipid
was a gift of Dr. M. A. Alonso (Centro de Biología
Molecular-Consejo Superior de Investigaciones Científicas,
Madrid) (25, 26). 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) (27). 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
2,3-Neu5Ac-R and
Gal
1-3GalNAc-R, respectively, were purchased from Roche Molecular
Biochemicals (Mannheim, Germany). Biotin-conjugated MAL was obtained
from Vector Labs, Inc. (Burlingame, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
BG does not inhibit global protein secretion
in HT-29 M6 and IMIM-PC-1 cells. A, HT-29 M6 cells were
cultured in complete medium on Transwells for 15 days and subsequently
cultured in complete medium supplemented or not with 2 mM
BG for an additional 7 days. Cells were labeled with
[35S]Met/Cys for 1 h; apical and basolateral media
were collected; and cells were lysed. Proteins present in the culture
medium and cell lysates were precipitated in trichloroacetic acid, and
radioactivity was determined using a scintillation counter.
B, IMIM-PC-1 cells cultured on plastic were processed as
described for A, except that they were cultured on plastic,
as they are not polarized and do not form impermeable monolayers.
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.2 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 2,3-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.
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Endocytosed Membrane Glycoproteins Accumulate in Cytoplasmic
Vesicles in BG-treated Cells--
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 and
B). 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.
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Endocytosed Membrane Glycoproteins Accumulate in Vesicles
Containing MUC1 and 1 Integrin--
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
1
integrin, 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 (28, 29), strongly suggesting that the
cytoplasmic accumulation of these proteins is secondary to defective
recycling.
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Cytoplasmic Vesicles Exhibit Markers Typical of Late
Endosomes--
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)
(30), early endosomes (EEA1 and Rab5) (31, 32), and late endosomes
(LBPA and Rab7) (20, 34) was performed. Results are shown for
1 integrin colocalization experiments, and similar
findings were obtained with anti-MUC1 antibodies. Cells were cultured
for 8 days in the presence or absence of BG, fixed, and permeabilized.
Each of these markers displayed the expected distribution in control
cells (Fig. 5A, upper
panels). Immunofluorescence microscopy analysis of BG-treated cells showed no colocalization of MUC1 or
1 integrin
with either Golgi or early endosome markers (Fig. 5). The MAL
proteolipid, a proteolipid that is present in apical carrier vesicles,
did not colocalize with intracellularly accumulated MUC1 or
1 integrin, and MAL proteolipid expression levels were
very low in both control and BG-treated cells (data not shown). By
contrast, considerable co-labeling of late endosomal markers and MUC1
or
1 integrin was demonstrated, although
1 integrin+/Rab7
and
1 integrin
/Rab7+ vesicles were
also present in treated cells, emphasizing the heterogeneous nature of
the cytoplasmic vesicles (Fig. 5A). The transferrin
receptor, a basolateral protein subject to extensive recycling, also
colocalized with MUC1 or
1 integrin in some vesicles (Fig. 5A). Furthermore, BG-treated cells showed a marked
increase in the total amount of Rab7+ and LBPA+
vesicles and a slight increase in the amount of Rab5+
vesicles. Western blot analysis confirmed increased levels of Rab7 in
BG-treated cells (Fig. 5B). Because late endosomes are involved in the regulation of cholesterol transport (34), we also
examined cholesterol distribution in cells using filipin (35).
Fig. 5C shows an increase in filipin labeling upon treatment of IMIM-PC-1 cells with BG. Similar results were obtained using HT-29
M6 cells. Altogether, these results indicate that a large proportion of
the cytoplasmic vesicles that accumulate in BG-treated cells share
molecular features with late endosomes.
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BG-treated Cells Display Ultrastructural and Biochemical Properties Reminiscent of Those of Sialic Acid Storage Diseases-- We have previously shown that HT-29 M6 cells treated with BG display an abnormal processing of lysosomal enzymes, viz. AAG and cathepsin D (14). An abnormal processing of cathepsin B has also been demonstrated in fibroblasts from patients with ISSD (15), a recessive syndrome caused by mutations in the lysosomal sialic acid transporter (36, 37) leading to an accumulation of sialic acid in lysosomes (38). Therefore, we have proposed that similar mechanisms may contribute to the abnormal processing of the lysosomal enzymes observed in BG-treated HT-29 M6 cells and in ISSD cells, as the former synthesize massive amounts of sialylated BG-derived metabolites.
To further analyze this similarity, we examined AAG
processing in fibroblasts from one patient with ISSD and from the
patient's unaffected parents. AAG is synthesized as a precursor with a
molecular mass of ~110 kDa, which is subsequently processed to
intermediate and mature forms, which are the major species detected
after 1 h of pulse and 24 h of chase (19). In normal
fibroblasts, only the precursor AAG form was detected after 1 h of
labeling; after 4 h of chase, only the intermediate AAG form was
detected; and after 24 h of chase, both the intermediate and
mature AAG forms were detected (Fig. 6)
(19). We have previously reported a delay in AAG processing in
untreated HT-29 M6 cells, so the three forms are detected after 24 h of chase (39). By contrast, in BG-treated HT-29 M6 cells labeled for
1 h and chased for 24 h, anti-AAG antibodies immunoprecipitated molecules with a mobility that was intermediate to
that of the precursor and intermediate forms of AAG
(AAGBG), the abnormal AAG form found in cells cultured in
the presence of BG (14). This molecular species has not been described
in normal cells. ISSD fibroblasts showed defective maturation kinetics of AAG and the transient accumulation of an abnormally processed AAG
species, designated AAGISSD, with a mobility similar to
that of AAGBG (Fig. 6). This species has not been reported
in fibroblasts from normal individuals and was not found in fibroblasts
from the unaffected parents (Fig. 6). Therefore, similar defects in lysosomal protein processing are present in BG-treated cells and in
ISSD cells.
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BG and Sucrose Induce the Accumulation of Membrane Glycoprotein-containing Cytoplasmic Vesicles-- The results described above and those from the literature suggest that high concentrations of sialic acid or sialic acid-containing metabolites in endosomal/lysosomal compartments are associated with an accumulation of cytoplasmic vesicles. In BG-treated HT-29 cells, BG-derived metabolites accumulate up to 1 mg of hexose/mg of protein (40). To examine whether the acquisition of this phenotype is exclusive to this compound and whether reduced sialylation is a prerequisite, the effects of sucrose were studied. In normal fibroblasts, sucrose (50-100 mM) induces a lysosomal storage-like phenotype characterized by the intracellular accumulation of electron-lucid vesicles, designated sucrosomes (41, 42), as well as defects in cathepsin B processing (15).
IMIM-PC-1 cells were cultured for 8 days in medium containing 50 or 100 mM sucrose, and the morphology and subcellular distribution of MUC1, 1 integrin, and sialic acid were analyzed. At
the phase-contrast level, sucrose-treated cells showed morphological
changes similar to those observed in BG-treated cells (Fig.
7A). In addition, confocal
fluorescence microscopy demonstrated the intracellular accumulation of
MUC1 and
1 integrin in vesicles (Fig. 7, B
and C), especially at 100 mM. Lectin
cytochemistry showed that, unlike BG, sucrose had no effects on
glycoprotein sialylation. Reactivity with MAL and PNA was unchanged
(Fig. 7D), and vesicles in which MUC1 accumulated showed
strong reactivity with MAL (Fig. 7E).
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Altogether, these findings indicate that BG and sucrose induce a
similar storage disease-like phenotype characterized by the intracellular accumulation of vesicles containing membrane
glycoproteins. The effects of BG, but not those of sucrose, are
associated with reduced sialylation, as determined by MAL reactivity of
cellular glycoproteins. These findings demonstrate that a blockade in
sialylation is not required for the accumulation of membrane
glycoproteins in cytoplasmic vesicles.
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DISCUSSION |
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BG has been extensively used as an inhibitor of O-glycosylation in a variety of cell types (4, 5-7, 10, 43-45). Because no other drugs that effectively inhibit this process have been identified, it is important to unravel the mechanisms through which BG acts. Recent work has shown that, in fact, the effects of BG are much more complex than initially thought. (i) Acute (24 h) as well as prolonged (several days) exposure to BG is mainly associated with the inhibition of sialylation rather than with a blockade in O-glycan synthesis; (ii) both N- and O-glycans are affected by the drug; and (iii) in some cells, BG is used as a substrate by glycosyltransferases, and BG-derived metabolites are synthesized in large amounts, leading to their accumulation in cells and to their secretion to the culture medium (40). On the basis of these findings, it has been proposed that the accumulation of cytoplasmic vesicles containing membrane glycoproteins is due to defective sialylation of these proteins, leading to a blockade in their membrane targeting, and that sialic acid might constitute a membrane targeting signal (8).
Anterograde Traffic Versus Endocytosis/Recycling--
In this
work, we provide extensive evidence against the hypothesis described
above. First, we have shown that global anterograde protein traffic was
not significantly diminished in two cellular models in which BG induced
a similar phenotype, HT-29 M6 and IMIM-PC-1 cells. It has previously
been shown that BG induces reduced mucin secretion in mucus-secreting
HT-29 cells, a finding that led to the proposal of a blockade in
anterograde traffic (5, 6, 9). Nevertheless, mucin secretion
requires extensive post-translational modifications as well as
appropriate maturation of mucus droplets, and it is likely that defects
in these processes associated with BG treatment account for the
observed findings (9). By contrast, the majority of membrane
glycoproteins do not share such extensive glycosylation and thus appear
not to be affected by BG in a similar fashion. To provide an
alternative explanation for their intracellular accumulation, we
examined the kinetics of endocytosis of membrane glycoproteins and the
presence of endocytosed material in the cytoplasmic vesicles. As shown
above, we found that, in BG-treated cells, membrane proteins were
endocytosed slowly and remained intracellular for longer periods of
time, possibly due to defective recycling to the plasma membrane,
leading to their intracellular accumulation. In addition, we found that
vesicles that accumulated MUC1 and 1 integrin membrane
glycoproteins also contained endocytosed plasma membrane glycoproteins.
Overall, these findings strongly support a predominant role of abnormal
endocytosis, rather than altered anterograde traffic, in the generation
of the phenotype induced by BG.
The phenotype of BG-treated cells might be explained by a defect in vesicular fusion events related to late endosomes. Proteins of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) family, Rab proteins (regulators of vesicular traffic), and membrane lipids are candidates to play a role. Dominant-negative Rab7 proteins are able to disrupt lysosome biogenesis (46), suggesting that the overexpression of Rab7 present in BG-treated cells might be involved in the accumulation of cytoplasmic vesicles. The increased levels of LBPA and cholesterol present in BG-treated cells might directly result in changes in the biophysical properties of membranes, including the formation and distribution of rafts; and a role for LBPA in the regulation of the fusogenic properties of late endosomes has been proposed (47). In addition, the accumulation of cholesterol in the degradative compartments of cells treated with U18666A leads to changes in the membrane-to-cytosol recycling of Rab7 (48).
Causal Role of Undersialylation in the BG Phenotype--
A
second tenet of the models proposed to account for the effects of BG
has been that sialic acid might act as an apical targeting signal and
that undersialylation is responsible for the accumulation of
glycoproteins in cytoplasmic vesicles (8). Our data also rule out this
hypothesis. First, sucrose treatment of IMIM-PC-1 cells did not affect
glycoprotein sialylation, yet caused morphological changes, including
the accumulation of MUC1 and 1 integrin in cytoplasmic
vesicles, in a fashion similar to that observed with BG. Second,
because IMIM-PC-1 cells display some heterogeneity in their response to
BG, we derived clones by limiting dilution and analyzed their
sensitivity to BG treatment. One of the clones analyzed, designated
A13, displays a polarized phenotype and contains glycoproteins that are
mainly sialylated in the
2,3-position, as do the parental IMIM-PC-1
population and HT-29 cells. In A13 cells, BG induced a profound
reduction of overall
2,3-sialylation and MUC1-associated
2,3-sialic acid, without concomitant effects on
2,6-sialylation,
and a marked accumulation of cytoplasmic vesicles. Yet, MUC1 did not
accumulate intracellularly and displayed an apical distribution, as in
control cells (Fig. 8). Therefore, although changes in sialylation may be associated with changes in
the targeting (43) and/or subcellular distribution (8, 10) of specific
membrane glycoproteins, sialic acid is not required for the appropriate
apical targeting of MUC1. Furthermore, global constitutive apical and
basolateral secretion proceeded unaltered in cells showing
profound undersialylation.
|
Our findings are in agreement with data from other laboratories. Chinese hamster ovary Lec2 mutants contain inactive CMP-Neu5Ac transporters and show defective glycoprotein sialylation (49), yet no changes in the distribution of membrane glycoproteins have been described. Similarly, ricin-resistant Madin-Darby canine kidney cells show reduced sialylation of membrane glycoproteins, yet lack a vesiculated cytoplasm (50), again suggesting that sialylation defects are not the primary cause of the misdistribution of membrane glycoproteins.
In view of these findings, how can the effects of BG on HT-29 M6 and
IMIM-PC-1 cells be explained? Delannoy et al. (6) initially
reported that BG is metabolized to benzyl-GalNAc-Gal, and Zanetta
et al. (40) have subsequently shown that HT-29 cells synthesize massive amounts of BG-derived metabolites. Preliminary data
indicate that IMIM-PC-1 cells, but not other cells upon which BG has
little effect, also produced large amounts of BG-derived metabolites.3 Therefore, such
metabolites are candidates to play a role in the generation of the BG
phenotype. If so, one could predict that the levels of metabolites and
the effects of BG would depend on at least three factors: BG
concentration used, duration of treatment, and catabolism/secretion of
the BG-derived metabolites. Although a quantitative assessment of these
variables is not yet available, several observations support this
notion in a series of experiments shown in Fig.
9, in which dipeptidyl peptidase IV and
AAG processing was studied. HT-29 M6 cells were pretreated for various
periods of time with 2 mM BG, pulse-labeled with
[35S]Met/Cys, chased for 24 h in medium containing 2 mM BG, and used in immunoprecipitation assays. A 30-min
preincubation with BG was sufficient to inhibit dipeptidyl peptidase IV
sialylation, identified by changes in electrophoretic mobility
previously characterized as due to lack of sialic acid (8, 14). Under
these conditions, AAG processing was unaffected (Fig. 9A).
By contrast, pretreatment of cells for 4-10 days with BG led to
predominant processing of AAG to the AAGBG aberrant
species. In a similar series of experiments, cells were pretreated for
24 h with increasing concentrations of BG (0.05-2
mM), pulse-labeled, and chased in BG-containing medium at
the appropriate concentration, and dipeptidyl peptidase IV and AAG
immunoprecipitates were analyzed by SDS-PAGE. In these experiments, 0.5 mM BG led to inhibition of dipeptidyl peptidase IV
sialylation, but had no effect on AAG processing; a partial effect on
the latter was observed only at 2 mM (Fig. 9B).
Overall, these experiments indicate that higher BG concentrations or
longer exposure times are required for BG to affect AAG processing than to perturb dipeptidyl peptidase IV sialylation and that the effects of
BG on the processing of various types of molecules take place through
different mechanisms. Our findings are in agreement with the results
reported by Ait-Slimane et al. (43) in Caco-2 cells, upon
which BG induced changes in glycoprotein sialylation, but did not lead
to the accumulation of intracellular vesicles or to the inhibition of
AAG processing, even upon long-term exposure to the
drug.4
|
Nature of the Intracellular Vesicles--
Our data indicate that
the vesicles accumulating in BG-treated cells share molecular features
with late endosomes: they receive internalized membrane material, and
MUC1 and 1 integrin colocalize in vesicles with the late
endosomal markers Rab7 and LBPA, but not with early endosomal markers
such as EEA1 and Rab5. The acidic nature of the vesicles that
accumulate in HT-29 M6 cells, determined using the pH-sensitive marker
Lysosensor Green DND-189 (Molecular Probes, Inc., Leiden, The
Netherlands) (data not shown), further supports their endosomal nature.
Nevertheless, it should be noted that the vesicles that accumulate in
both HT-29 and IMIM-PC-1 cells display a considerable degree of
molecular heterogeneity, a feature that is also characteristic of late
endosomes (47). The working model proposed on the basis of the findings
reported above is summarized in Fig.
10.
|
The accumulation of intracellular vesicles observed in some cells upon exposure to 2 mM BG appears to be closely related to changes in lysosomal enzyme processing and to a storage disease phenotype. Here, we also provide indirect evidence supporting the notion that the accumulation of BG-derived metabolites observed in HT-29 and IMIM-PC-1 cells and the accumulation of sialic acid described in cells from patients with ISSD lead to a similar morphological and molecular phenotype, as determined by the processing of lysosomal enzymes and the increased levels of cholesterol in cells.
The similarities between the cellular and molecular phenotypes of ISSD
and BG-treated cells, together with the fact that a dramatic
accumulation of plasma membrane glycoproteins takes place in the
latter, provide support for a hypothesis that has recently been
proposed, but for which there is so far no evidence: changes in the
subcellular distribution of plasma proteins might take place in cells
from patients with lysosomal storage diseases. MUC1, 1
integrin, and CD44 are some of the membrane glycoproteins that have
been shown to display a subcellular misdistribution in BG-treated
cells. All these proteins participate in cellular communication
processes, including cell-cell and cell-matrix adhesion. It is
conceivable that altered plasma membrane expression of these as well as
other not yet studied membrane proteins as a result of the alterations
in traffic that are typical of lysosomal storage diseases might
secondarily contribute to the pathogenesis of these disorders. Simons
and Gruenberg (16, 33) have proposed that the amount of raft lipids
tolerated by raft endosomes is limited and that the accumulation of one
type of raft lipids, cholesterol or sphingolipids, would cause the
accumulation of the other type of lipid, eventually leading to a
traffic jam that contributes to the cellular and tissue phenotype
associated with these conditions. A better understanding of these
molecular events might provide valuable information for the treatment
of lysosomal storage disorders. In this regard, the reversible nature
of the effects of BG on cells might be of help in identifying
pharmacological strategies for the systemic therapy of these disorders.
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ACKNOWLEDGEMENTS |
---|
We thank the investigators mentioned under "Materials and Methods" for providing cells and reagents and G. Huet and P. Delannoy for valuable discussions and for sharing unpublished results. We are grateful to J. Lloreta, S. Castel, A. Mallabiabarrena, and X. Mayol for valuable contributions.
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FOOTNOTES |
---|
* This work was supported in part by Grant SAF97-0085 from the Comisión Interministerial de Ciència y Tecnología, Grants SGR-00245 and SGR-0410 from the Comissió Interdepartamental de Ciència i Tecnología (Generalitat de Catalunya), and a grant from the Mizutani Foudation for Glycoscience.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a predoctoral fellowship from the MUTIS Program.
§ To whom correspondence and reprint requests should be addressed. Tel.: 34-93-221-1009; Fax: 34-93-221-3237; E-mail: preal@imim.es.
Published, JBC Papers in Press, January 21, 2003, DOI 10.1074/jbc.M211909200
2 F. Ulloa and F. X. Real, unpublished data.
3 P. Delannoy, G. Huet, F. X. Real, F. Ulloa, and J. P. Zanetta, unpublished data.
4 C. Francí and F. X. Real, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
BG, benzyl-N-acetyl--D-galactosaminide;
MAL, M. amurensis lectin;
PNA, peanut agglutinin;
AAG, acid
-glucosidase;
ISSD, infantile sialic acid storage disease;
mAb, monoclonal antibody;
LBPA, lysobisphosphatidic acid;
EEA1, early
endosomal antigen-1;
Neu5Ac, 5-N-acetylneuraminic acid;
PBS, phosphate-buffered saline;
BSA, bovine serum albumin;
FITC, fluorescein
isothiocyanate;
TRITC, tetramethylrhodamine isothiocyanate;
RIPA, radioimmune precipitation assay.
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