* Unité de Recherches sur la Biologie et la Physiopathologie des Cellules Mucipares, Institut National de la Sante et de la
Recherche Medicale (INSERM) U377, 59045 Lille Cedex, France; Unitat de Biologia Cellular i Molecular, Institut Municipal
d'Investigació Mèdica, Universitat Autónoma de Barcelona, E-08003, Barcelona, Spain; § Unité de Recherches sur la
Différenciation Cellulaire Intestinale, INSERM U178, 94807 Villejuif Cedex, France; and
Laboratoire de Chimie Biologique,
Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche (UMR)-111, Université des Sciences et
Technologies de Lille, 59655 Villeneuve d'Ascq Cedex, France
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Abstract |
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Exposure for 24 h of mucus-secreting HT-29
cells to the sugar analogue GalNAc--O-benzyl results
in inhibition of Gal
1-3GalNAc:
2,3-sialyltransferase, reduced mucin sialylation, and inhibition of their secretion (Huet, G., I. Kim, C. de Bolos, J.M. Loguidice, O. Moreau, B. Hémon, C. Richet, P. Delannoy, F.X. Real.,
and P. Degand. 1995. J. Cell Sci. 108:1275-1285). To determine the effects of prolonged inhibition of sialylation, differentiated HT-29 populations were grown under permanent exposure to GalNAc-
-O-benzyl. This
results in not only inhibition of mucus secretion, but
also in a dramatic swelling of the cells and the accumulation in intracytoplasmic vesicles of brush border-associated glycoproteins like dipeptidylpeptidase-IV, the
mucin-like glycoprotein MUC1, and carcinoembryonic
antigen which are no longer expressed at the apical
membrane. The block occurs beyond the cis-Golgi as
substantiated by endoglycosidase treatment and biosynthesis analysis. In contrast, the polarized expression
of the basolateral glycoprotein GP 120 is not modified. Underlying these effects we found that (a) like in mucins, NeuAc
2-3Gal-R is expressed in the terminal position of the oligosaccharide species associated with the
apical, but not the basolateral glycoproteins of the cells,
and (b) treatment with GalNAc-
-O-benzyl results in
an impairment of their sialylation. These effects are reversible upon removal of the drug. It is suggested that
2-3 sialylation is involved in apical targeting of brush
border membrane glycoproteins and mucus secretion in
HT-29 cells.
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Introduction |
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ENTEROCYTIC and mucus-secreting populations isolated from the human colon carcinoma cell line
HT-29 have proven extremely useful for the study
of intestinal cell differentiation (for review see Zweibaum
et al., 1991). The differentiated populations used in this
study were isolated from the mainly undifferentiated parental line (Fogh and Trempe, 1975
) by selection with increasing concentrations of methotrexate (MTX)1 (Lesuffleur et al., 1990
, 1991
). Cells selected with 10
6 and 10
5
M MTX form a homogeneous population of mucus-secreting cells (Lesuffleur et al., 1990
, 1991
), whereas selection
with 10
3 M MTX results in a population of enterocytic
phenotype (Lesuffleur et al., 1991
). Whatever their phenotype, these populations share common differentiation characteristics, namely (a) a postmitotic onset of the differentiation process and (b) a polarized organization of the cell
monolayer with the presence of an apical brush border endowed with several glycoproteins such as dipeptidylpeptidase-IV (DPP-IV), the carcinoembryonic antigen (CEA)
and the mucin-like glycoprotein MUC1. Mucus-secreting
cell populations selected by MTX have been extensively
analyzed as to the characteristics of their mucins: at the
mRNA level they mainly express MUC5AC (Lesuffleur et
al., 1993
, 1995
); at the protein level they secrete mucus of gastric immunoreactivity (Lesuffleur et al., 1990
) with a
main oligosaccharide species similar to the clone HT29-16E (Capon et al., 1992
), NeuAc
2-3Gal
1-3GalNAc-R
(sialyl T antigen) (Lesuffleur et al., 1993
; Huet et al.,
1995
).
Previous results have shown that short-term (24-h) exposure of postconfluent mucus-secreting HT-29 cells to
benzyl-2-acetamido-2-deoxy--D-galactopyranoside (GalNAc-
-O-benzyl), an inhibitor of mucin O-glycosylation
(Kuan et al., 1989
; Huang et al., 1992
; Byrd et al., 1995
), results in a decrease of mucus secretion, a lower sialic acid
content of newly synthesized mucins, and an increased
content of T antigen (Gal
1-3GalNAc-R) (Huet et al.,
1995
). Similar effects of GalNAc-
-O-benzyl have been
demonstrated in LS174-T colon cancer cells (Kuan et al.,
1989
) or KATO III gastric cells (Byrd et al., 1995
). In the case of HT-29 cells, the change in mucin glycosylation has
been shown to be a consequence of the metabolization
of GalNAc-
-O-benzyl into Gal
1-3GalNAc-
-O-benzyl
which, in turn, is a potent competitive inhibitor of the
Gal
1-3GalNAc
2,3-sialyltransferase (Huet et al., 1995
;
Delannoy et al., 1996
), the main sialyltransferase activity
expressed in HT-29 cells (Majuri et al., 1995
; Delannoy et al.,
1996
).
Because O-glycosylation may be part of the processing
of a wide variety of proteins and the biochemical changes
observed after 24 h of treatment may not fully reflect the
activity of GalNAc--O-benzyl, we set to analyze the effects of a permanent exposure to this drug on the proliferation and differentiation of mucus-secreting HT-29 cells.
We found that, in addition to an inhibition of mucus secretion, apical glycoproteins such as the transmembrane glycoprotein MUC1 (Gendler et al., 1987
), as well as the
N-glycosylated proteins DPP-IV (Semenza, 1989
; Misumi et
al., 1992
) and CEA (Fukushima et al., 1995
) considerably
accumulate within the cytoplasm and are no longer expressed at the apical membrane, this occurring without
modification of the polarization of the cells. Based on this
observation we further found that, like in mucins,
NeuAc
2-3 is the main sialylated determinant associated
with DPP-IV, MUC1, and CEA in differentiated HT-29
cells, whether of mucus-secreting or enterocytic phenotype, and that treatment with GalNAc-
-O-benzyl results
in a decreased sialylation of these proteins. These effects of GalNAc-
-O-benzyl are rapidly reversible upon removal of the drug. These results raise the question of
whether, in differentiated HT-29 cells,
2-3 sialylation is
required for mucus secretion as well as for the correct targeting of apical glycoproteins.
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Materials and Methods |
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Cell Culture
HT-29-differentiated subpopulations were derived from the original cell
line (Fogh and Trempe, 1975) that was obtained from the late J. Fogh
(Sloan-Kettering Memorial Cancer Center, Rye, NY). The cells adapted
to MTX 10
6, 10
5, and 10
3 M (Lesuffleur et al., 1990
, 1991
) were used
after several weekly passages of reversion to drug-free medium (10-40
passages) and are referred to as HT29-RevMTX10
6, RevMTX10
5 (mucus-secreting cells), and RevMTX10
3 (enterocytic cells). Cells were
grown in DME (Life Technologies, Inc., Cergy-Pontoise, France) supplemented with 10% inactivated FBS for 30 min at 56°C (Boehringer Mannheim Biochemicals, Mannheim, Germany). GalNAc-
-O-benzyl (Sigma
Chemical Co., St. Louis, MO) was solubilized in DME. All experiments
and maintenance of the cells were done in 25- or 75-cm2 T flasks (Corning
Glass Works, Corning, NY), and in 24-well cell culture clusters (Costar,
Cambridge, MA) at 37°C in a 10% CO2 / 90% air atmosphere. Cells were
seeded at 2 × 104 cells/cm2. The same conditions were applied to cells
grown on tissue culture-treated Transwell polycarbonate membranes with
a 24.5-mm diam and a 0.4-µm pore size (Costar). For maintenance purposes, cells were passaged weekly, using 0.025% trypsin in 0.53 mM
EDTA in PBS Ca2+Mg2+-free (PBS
). The medium was changed daily in
all culture conditions. For growth curves, cells grown in 24-well culture
clusters were detached with trypsin and counted with a hemocytometer.
Cell volume was determined in a hematocrit. Control Caco-2 cells were
cultured as previously reported (Pinto et al., 1983
) and analyzed between passages 70 and 80.
Antibodies and Lectins
Mouse mAbs HBB 3/775/42 (Hauri et al., 1985) and G1/136 (Eilers et al.,
1989
) specific for human DPP-IV and a 120-kD basolateral glycoprotein
respectively, were a gift of H.P. Hauri (Biocenter of the University of
Basel, Basel, Switzerland). Rat mAb 4H3 against human DPP-IV (Gorvel
et al., 1991
) was obtained from S. Maroux (CNRS Unité de Recherche
Associee 1820, Faculté des Sciences de Saint Jerôme, Marseille, France).
Mouse mAb 517 (Le Bivic et al., 1988
) against CEA was obtained from A. Le Bivic (Faculté des Sciences de Luminy, Marseille, France). Mouse
mAb BC-2 (Xing et al., 1989
), which recognizes a sequence in the tandem
repeat of the MUC1 gene product was obtained from I. McKenzie (Austin Cancer Research Institute, Heidelberg, Victoria, Australia). Mouse mAb
B72.3 against sialyl-Tn (Nuti et al., 1982
) was obtained from K.O. Lloyd
(Memorial Sloan-Kettering Cancer Center, New York). Mouse mAb
TS2/16 against the integrin
1 subunit (Arroyo et al., 1992
) was obtained
from F. Sanchez-Madrid (Universidad Autónoma de Madrid, Madrid,
Spain). Rabbit polyclonal Abs against porcine villin (Robine et al., 1985
)
and the tight junction protein ZO-1 (Willot et al., 1992
) were obtained
from D. Louvard (Institut Curie, Paris, France) and J.M. Anderson (Yale
University, New Haven, CT), respectively. For the detection of gastric
mucins we used the same rabbit polyclonal Ab L56/C as previously used for cloning the L31 mucin cDNA encoding the 3' end of MUC5AC
(Lesuffleur et al., 1995
). Fluorescein-conjugated Maackia amurensis agglutinin (MAA) (Wang and Cummings, 1988
), Sambucus nigra agglutinin
(SNA) (Shibuya et al., 1987
), and Peanut (Arachis hypogaea) agglutinin (PNA) (Lotan et al., 1975
), which recognize the oligosaccharide species
NeuAc
2-3Gal-R, NeuAc
2-6Gal, and Gal
1-3GalNAc-R, respectively, were from Vector Labs Inc. (Burlingame, CA).
Immunofluorescence and Histochemical Staining
Indirect immunofluorescence was performed on cryostat sections of cell
layer rolls as reported (Lesuffleur et al., 1990). Briefly, late (day 21) cultures of cells grown in 25-cm2 T flasks were rinsed with Ca2+Mg2+-free
PBS, the T flask was cut up with a soldering iron, and then the cell layer
gently was scraped with a rubber policeman and poured in a bath of liquid
nitrogen. The resulting frozen cell pellet was either stored in liquid nitrogen for further analysis, or immediately processed for cryostat sections.
This method has the double advantage of visualizing a large part of the
cell layer and allowing the concomitant detection of apical, basolateral, and intracellular proteins on the same section. Double immunofluorescence was performed on sections postfixed with 3.7% paraformaldehyde in PBS
for 10 min at room temperature using secondary antibodies fluorescein-coupled sheep anti-mouse or anti-rabbit Ig (Institut Pasteur Production, Marne-la-Coquette, France) or rhodamine-coupled sheep antiglobulins (Boehringer Mannheim Biochemicals). Desialylation was
performed by incubation for 16 h at 37°C of paraformaldehyde-fixed cryostat sections with sialidase from Clostridium perfringens (Sigma Chemical Co.) (50 mU/ml in 50 mM citrate buffer, pH 6.0, 0.9% NaCl, 0.1% CaCl2).
For confocal microscopy analysis, cells grown on glass coverslips were
fixed with 4% paraformaldehyde for 10 min, incubated with 50 mM
NH4Cl for 30 min, and then permeabilized with 0.1% saponin in 1% BSA/
PBS for 30 min. To detect
1 integrin, mAb TS2/16 (0.5 µg/ml in 0.1% saponin in 1% BSA/PBS) was added for 1 h, followed by FITC-conjugated
goat anti-mouse; MAA (Vector Labs, Inc.) (20 µg/ml in 0.1% saponin in
Tris-HCl, pH 7.5, 15 mM KCl, 5 mM MgCl2) was added for 1 h, followed
by streptavidin-rhodamine (Pierce Chemical Co., Rockford, IL). Confocal
microscopy analysis was performed using a Leica instrument (model TCS
4D; St. Gallen., Switzerland). Histological staining with alcian blue, pH
2.5, and nuclear red was done on cryostat sections postfixed in absolute ethanol for 10 min at room temperature.
Transmission EM and Ultrastructural Immunochemistry
Classical transmission EM was performed as previously reported (Lesuffleur et al., 1990, 1991
) on cells grown in 25-cm2 plastic flasks. Samples embedded in Epon (Polysciences, Inc., Warington, PA) were reembedded to
make sections perpendicular to the bottom of the flask. Thin sections
were stained with toluidine blue. Ultrastructural immunochemistry was
performed as previously described (Hennebicq-Reig et al., 1996
). After
rinsing three times in PBS, cells cultured in 25-cm2 flasks were fixed in
phosphate buffer containing 4% paraformaldehyde and 0.05% glutaraldehyde. The cell layer was scraped with a rubber policeman, the cell pellet
was infiltrated with phosphate buffer containing 2.3 M sucrose and 20%
polyvinylpyrolidone, and then frozen in liquid nitrogen. Ultrathin cryosections were successively incubated with PBS containing 10% FBS, mouse
mAb HBB 3/775/42 (DPP-IV), rabbit anti-mouse Ig antibody, and 8-nm
gold-conjugated protein A. All antibodies and gold-conjugated protein A
were diluted in PBS containing 10% FBS. The grids were finally counterstained with methylcellulose uranyl acetate and observed using an electron microscope (model 902; Carl Zeiss, Inc., Thornwood, NY). The same procedure was applied for mAb 517 (CEA) and mAb BC-2 (MUC1).
Northern Blot Analysis
For detection of DPP-IV and villin mRNAs, total RNA was isolated from
the cells 16 h after medium change by lysis with guanidium isothiocyanate
and centrifugation through a CsCl gradient (Chirgwin et al., 1979). Samples of total RNA, denatured in 1 M glyoxal (Thomas, 1980
), were fractionated by electrophoresis through 1% agarose gels and then transferred
to nylon (model Hybond N; Amersham Corp., Amersham, UK) in the
presence of 20× SSC. Filters were incubated overnight at 42°C in prehybridization buffer containing 50% formamide, 5× SSC, 10× Denhardt's
solution, 50 mM sodium phosphate, pH 6.5, and 250 µg/ml sonicated and
denatured salmon sperm DNA. Filters were then hybridized with the 32P-labeled probe for 20 h at 42°C in prehybridization buffer containing 10%
dextran sulfate (Thomas, 1980
). Blots were washed twice with 2× SSC,
0.1% SDS at room temperature, once with 0.1× SSC, 0.1% SDS at 50°C,
and once, using the same solution, at 65°C for 15 min. Blots were then
processed for autoradiography. To normalize for RNA, filters were dehybridized and stained with methylene blue. Methylene blue staining was
preferred to hybridization with actin or glyceraldehyde 3-phosphate dehydrogenase since it was found that the levels of these transcripts differ in dividing and postconfluent cells. DPP-IV was detected with cDNA DPI-101 (Darmoul et al., 1990
) and villin with cDNA V19 (Pringault et al., 1986
),
obtained from D. Louvard. The probe for human ST3Gal I (Kitagawa and
Paulson, 1994
) was a 537-bp PCR-amplified fragment from HepG2 cells
cDNA corresponding to the coding region from nucleotide 361 to 898 (Recchi et al., 1998
).
Measurement of Enzyme Activities
DPP-IV activity was measured in the cell homogenates as previously reported (Lesuffleur et al., 1990), according to the method of Nagatsu et al.
(1976)
using 1.5 mM glycyl-L-proline-4-nitroanilide as substrate. Results are
expressed as mU/mg of protein. One unit is defined as the activity that hydrolyzes 1 mmol of substrate/min at 37°C. Proteins were measured with
the BCA protein assay reagent (Pierce Chemical Co.). Gal
l-3GalNAc
2,3-sialyltransferase activity was measured in cell homogenates prepared
by lysing the cells at 0°C with 10 mM sodium cacodylate buffer, pH 6.5, containing 1% Triton X-100 (Sigma Chemical Co.), 20% glycerol, 0.5 mM
dithiothreitol, and 5 mM MnCl2 (1 ml per 2.5 × 107 cells). After 10 min of
incubation under continuous stirring, cell homogenates were centrifuged
at 10,000 g for 15 min and the supernatants were used for enzymatic assay.
Protein concentration was determined according to Peterson (1977)
using
BSA as standard. Cell homogenates (40 µg of protein) were brought to
a final volume of 120 µl with 0.1 M sodium cacodylate buffer, pH 6.5, 1%
Triton X-100, 0.1 M galactose (as inhibitor of
-galactosidase), 1 mM 2,3-dehydro-2-deoxy-Neu5Ac (as inhibitor of sialidases), 52.9 µM CMP-[14C]-
Neu5Ac (0.58 GBq/mmol; 3.68 kBq/120 µl) (Amersham Corp.), containing
1 mM of Gal
l-3GalNAc
-O-pNp (Sigma Chemical Co.) and incubated
for 1 h at 37°C. The reactions were stopped by adding 1 vol of ethanol.
Samples were centrifuged at 3,000 g for 5 min and then supernatants were
directly developed by descending paper chromatography with ethyl acetate/pyridine/water (10:4:3 by vol) (Delannoy et al., 1993
). Assays were
performed in duplicate. The rates of reactions were linear with time, at
least for 1 h. The incorporation of [14C]-Neu5Ac was determined by subtraction of the radioactivity measured in the absence of exogenous acceptors and results are expressed as average values in nmol of Neu5Ac transferred per milligram of protein and per hour.
Electrophoresis and Western Blotting
Cells were homogenized by sonication in Tris/Mannitol buffer. Immunoprecipitation of DPP-IV, CEA, and MUC1 was performed as in Hauri et al.
(1985), using mAbs 3/775/42, 517, and BC-2 previously coated on protein
A-Sepharose beads (Pharmacia Fine Chemicals, Uppsala, Sweden). SDS-PAGE was performed under reducing conditions on 4-20% gradient
polyacrylamide gels (Laemmli, 1970
) either with 50 µg of total cellular
protein per lane or with DPP-IV, CEA, or MUC1 immunoprecipitates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane (model BioTrace NT; Gelman Sciences Inc., Ann Arbor, MI) as described in Vaessen et al. (1981)
. The membranes were then treated for 2 h
with polyvinylpyrolidone K-30 (2% in TBS). Immunodetection of DPP-IV,
CEA, and MUC-1 was performed with mAbs 4H3, 517, and BC-2, respectively, using secondary antibodies peroxidase-coupled anti-rat or anti-mouse Ig accordingly (Biosys, Compiègne, France). For glycan detection,
membranes were incubated with digoxigenin-labeled lectins from Boehringer Mannheim Biochemicals at concentrations of 5 µg/ml in TBS for MAA and SNA, and 2 µg/ml in TBS for PNA-digoxigenin. Then, the nitrocellulose membranes were incubated for 1 h with alkaline phosphatase-labeled antidigoxigenin Fab fragments (1 µg/ml in TBS) (Boehringer
Mannheim Biochemicals). After washing, labeled glycoproteins were revealed by 4-nitro blue tetrazolium chloride 5-bromo-4-chloro-3-indolyl-phosphate staining. Desialylation was performed as for cryostat sections
before incubation with the digoxigenin-labeled lectins. Digestion with endoglycosidase H (Boehringer Mannheim Biochemicals) was carried out in
0.1 M phosphate buffer, pH 5.5, containing 0.02% SDS, 1% Triton X-100,
1%
-mercaptoethanol, 1 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, 10 µg/ml pepstatin, using 10 mU of endoglycosidase H overnight
at 37°C. Digestion with endoglycosidase F/N-glycosidase F (Boehringer
Mannheim Biochemicals) was carried out in 0.1 M phosphate buffer, pH
7.0, containing 0.05% SDS, 1% Triton X-100, 1%
-mercaptoethanol, 1 mM
phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, and 10 µg/ml pepstatin,
using 0.5 U of endoglycosidase F/N-glycosidase F overnight at 37°C. Controls
were incubated in the same buffers overnight at 37°C without glycosidase.
Metabolic Labeling and Immunoprecipitation of DPP-IV
Cells were cultured in six-well plates with or without the presence of 2 mM
GalNAc--O-benzyl until day 11. Subsequently, untreated cells were
pulse labeled for 15 min with 200 µCi/well of [35S]-methionine (Amersham Corp.) in 1 ml of methionine-free medium, and then chased for the
indicated periods of time with 1 ml 0.01 M methionine in regular medium.
The same protocol was applied to treated cells, except for the presence of
2 mM GalNAc-
-O-benzyl throughout the experiment. Cells were rinsed
in PBS and lysed in 1 ml of RIPA buffer (0.001 M Tris-HCl, pH 8.0, 0.01 M
NaCl, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, 1% phenylmethylsulfonylfluoride, 0.001 M sodium ethylene diamine tetraacetate). Aliquots of 50 µg of proteins from cell lysates were incubated with
mAb 4H3 overnight at 4°C. Immunocomplexes were collected on protein
G-Sepharose 4B (Sigma Chemical Co.), eluted in SDS sample buffer (0.2 M
Tris-HCl buffer, pH 6.8, containing 2% SDS and 30% glycerol) at 60°C
for 5 min, and then analyzed on 5-30% SDS-polyacrylamide gels. For autoradiography, gels were fixed in 40% ethanol, 10% glycerol, 10% acetic
acid (by vol), soaked in Amplify (Amersham Corp.) for 20 min, dried on
Whatman paper, and then exposed to Cronex 4 NIF film (Dupont, Les
Ulis, France).
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Results |
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GalNAc--O-benzyl Treatment Results in
a Dose-dependent Decrease of Mucus Secretion and
Swelling of Mucus-secreting HT-29 Cells
To assay for a dose-dependent effect of GalNAc--O-benzyl, mucus-secreting HT-29 cells (RevMTX10
6) were
treated, from the day of seeding on, with different concentrations of the drug in the 0.1-2 mM range. Regardless of
the concentration used, no effect on cell viability was observed, as assessed by the absence of cells in suspension
and trypan blue exclusion. As shown in Fig. 1, it has no effect on the doubling time of the cells in the first days in
culture, but results in a dose-dependent lower cell density
in the stationary phase. At the highest concentration (2 mM)
the cells stop growing before reaching total confluence, with the cell layer occupying 75-80% of the surface of the
flask (Fig. 1). This effect is associated with a dramatic
swelling of the cells (Figs. 1 and 2), with their cytoplasm
appearing filled with a honeycomb-like accumulation of
vesicles of various sizes during transmission electron microscopy (see Fig. 4 c). Concomitant with these changes,
the mucus gel is totally absent from the cells treated with
the highest concentrations, even when the cells are maintained for a longer period (<30 d). The decrease in mucus content was further demonstrated by the analysis of sections of the cell layer (Fig. 2). At the highest concentration
(2 mM), there was a total absence of alcian blue-stained
material (Fig. 2 e). In addition, the dense immunofluorescent staining of apical mucus droplets observed in control
cells with antigastric mucus antibodies was no longer observed in cells treated with 2 mM, having been replaced by
a diffuse staining of the cytoplasm (Fig. 2 f).
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|
Treatment of Mucus-secreting HT-29
Cells with GalNAc--O-benzyl Leads to an
Intracytoplasmic Accumulation of Brush Border
Membrane-associated Glycoproteins
Because the most clear-cut effect of GalNAc--O-benzyl
was observed at 2 mM, all further experiments were done
at this concentration. In control postconfluent HT29-RevMTX10
6 cells, DPP-IV, MUC1, and CEA are exclusively associated with the apical brush border of the cells,
as previously reported (Lesuffleur et al., 1993
) and shown
in Fig. 3, g, i, and k. In contrast, in GalNAc-
-O-benzyl-
treated cells, DPP-IV, MUC1, and CEA are present in the
totality of the cytoplasm (Fig. 3, h, j, and l); this occurs without modification of the morphological polarity of the
cells, substantiated by the apical expression of villin and
ZO1 (Fig. 3, a-d). Interestingly, and in contrast to what
observed for apical glycoproteins, the treatment had no effect on the basolateral expression of GP120 (Fig. 3, e and
f). The cytoplasmic accumulation of apical glycoproteins
in treated cells was further confirmed using immunoelectron microscopy: unlike in control cells where they are restricted to the apical brush border, DPP-IV, MUC1, and
CEA are localized in the numerous vesicles that fill the cytoplasm (Fig. 4). Using DPP-IV as a marker of the effect,
we further found that this altered distribution is associated
with an increased level of expression of the enzyme at
both mRNA (Fig. 5) and protein level as substantiated by
higher enzyme activities and higher protein content, with a
lower apparent molecular mass, however, as shown by
Western blot (Fig. 5). The same results were obtained with
mucus-secreting HT29-RevMTX 10
5 cells and enterocytic HT29-RevMTX10
3 cells (data not shown). The effect of GalNAc-
-O-benzyl on apical glycoproteins is not
dependent on the support the cells are cultured on, as exemplified by the swelling of the cells and the intracytoplasmic accumulation of DPP-IV also observed in treated filter-grown cells (data not shown).
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|
The Effects of GalNAc--O-benzyl Are Reversible
Switching back the cells to drug-free medium after 20 d of treatment results in a rapid reversal of the phenotype described above: within 24 h the volume of the cells decreases, and in the following days apical glycoproteins redistribute to the apical surface (Fig. 6 e). At the same time, mucus secretion resumes, judged by the occurrence of a visible gel on the surface of the cell layer demonstrated by alcian blue staining (data not shown) and immunofluorescence reactivity to antimucus antibodies of cell layer sections (Fig. 6 f).
|
NeuAc2-3Gal
1-3GalNAc Is a Major Oligosaccharide
Species Associated with Mucins and Other
Glycoproteins from Differentiated HT-29 Cells
To characterize the oligosaccharide species associated with
mucins and other glycoproteins from HT-29-differentiated cells, we used the lectin MAA, which reacts with
NeuAc2-3Gal-R terminal sequence and PNA, which reacts
with the O-linked T antigen (Gal
1-3GalNAc
1-O-Ser/Thr). As shown in Fig. 7 via double immunofluorescence, mucus
from control HT29-RevMTX10
6 cells is reactive with MAA
(Fig. 7, top panel, a-c), whereas only a small proportion of
the mucus reacts with PNA (Fig. 7, top panel, d-f). After
treatment with sialidase from Clostridium perfringens, all
the mucus is reactive with PNA (data not shown), confirming the fact that a large proportion of the mucus expresses the NeuAc
2-3Gal
1-3GalNAc sequence. Because mucus
droplets are concentrated in the apical cytoplasm and do
not allow to distinguish the reactivity of brush border-
associated glycoproteins to lectins in mucus-secreting cells,
we used enterocytic HT29-RevMTX10
3 cells to further
characterize the reactivity of these glycoproteins to lectins.
As shown on cryostat sections from postconfluent cells (Fig. 7, middle panel), MAA shows a strong apical reactivity
(Fig. 7, middle panel, a), whereas PNA is unreactive (Fig. 7,
middle panel, e). After sialidase treatment, there is no
longer any reactivity to MAA (Fig. 7, middle panel, c),
whereas PNA shows a strong apical staining (Fig. 7, middle
panel, g), thus testifying the presence of a large amount of
sialyl-T antigen linked to brush border-associated glycoproteins. In contrast, no basolateral labeling was observed with MAA by confocal microscopy (Fig. 7, bottom panel),
thus indicating that sialyl-T antigen is not associated with
basolateral glycoproteins in differentiated HT-29 cells. The
association of NeuAc
2-3Gal
1-3GalNAc
1-O-Ser/Thr with
glycoproteins in differentiated HT-29 cells was confirmed by
Western blot analysis of cell homogenates from the different HT-29 cell populations (Fig. 8) which suggest, based on the
reactivity to MAA and PNA before and after treatment
with sialidase, that NeuAc
2-3Gal
1-3GalNAc
1-O-Ser/
Thr sequence is associated not only to mucins, but also to a
number of glycoproteins with a molecular mass in the 80-
400 kD range. Among the glycoproteins that react with MAA
are DPP-IV, MUC1, and CEA as shown by immunoblot
analysis with MAA of these immunoprecipitated proteins
(see Fig. 10). NeuAc
2-3 glycosylation of T antigen is further supported by the observation that ST3Gal I is expressed at all stages of the culture in mucus-secreting as
well as in enterocytic HT-29 cells, as substantiated by analysis of ST3Gal I mRNA level and enzyme activity (Fig. 8).
Interestingly, no reactivity was observed by immunoblotting cell homogenates with mAb B72.3 against sialyl-Tn
(data not shown) or SNA that recognizes the terminal oligosaccharide species NeuAc
2-6Gal (Fig. 9).
|
|
|
|
Treatment with GalNAc--O-benzyl Results in a
Decreased
2,3-Sialylation of Glycoproteins
Western blot analysis of the reactivity to lectins of cell homogenates from postconfluent HT29-RevMTX106 cells
treated with various concentrations of GalNAc-
-O-benzyl shows a dose-dependent decrease of MAA-reacting
glycoproteins associated with a dose-dependent increase
in PNA-reacting glycoproteins, with a maximum effect observed at a drug concentration of 2 mM (data not shown).
This was further confirmed by the observation, using immunofluorescence, that regardless of their phenotype, the
cytoplasm of cells treated with 2 mM GalNAc-
-O-benzyl
is heavily stained with PNA, in contrast to control cells
(data not shown). Western blot analysis of immunoprecipitated DPP-IV, MUC1, and CEA with MAA and PNA
shows that MAA reactivity of these glycoproteins is reduced in cells treated with GalNAc-
-O-benzyl. Concomitant to these changes, a reactivity to PNA was observed
for MUC1 and DPP-IV, but not for CEA (Fig. 10). The
changes in glycosylation are reversible upon removal of
the drug; Western blot analysis of the reactivity to lectins
of cell homogenates from cells reverted to drug-free medium shows a reappearance of MAA reactivity and the
concomitant disappearance of PNA reactivity of a number
of glycoproteins in the 80-400 kD range (data not shown).
These include MUC1, DPP-IV, and CEA as shown by
Western blot analysis with lectins of the immunoprecipitates of these glycoproteins (Fig. 10).
The GalNAc--O-benzyl-dependent Secretory Block
Occurs beyond the cis-Golgi Compartment of the Cells
To further characterize at which level the block induced by
GalNAc--O-benzyl occurs, immunoprecipitates of apical
glycoproteins from control and treated cells were analyzed
for their sensitivity to endoglycosidase H and endoglycosidase F treatment. As exemplified for DPP-IV and CEA
(Fig. 11 A), the same results were observed in treated
and control cells with both proteins being endoglycosidase H-resistant and endoglycosidase F-sensitive, suggesting
that the block occurs after the cis-Golgi. This was further
confirmed by analysis of the biosynthesis of DPP-IV that
shows the processing of the enzyme is similar in treated
and in control cells (Fig. 11 B).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present results suggest that in HT-29 cells, 2,3-sialylation plays a crucial role in intracellular transport of
brush border membrane-associated glycoproteins and in
mucus secretion. They rely on the exceptional conjunction
of a number of factors: (a) the availability of polarized
cells, isolated from the HT-29 cell line, expressing either
an enterocytic or a mucus-secreting differentiated phenotype; (b) the neoplastic nature of these cells which, as such,
show a modified pattern of protein glycosylation as compared with their normal counterpart with shorter oligosaccharide side chains (for review see Lesuffleur et al., 1994
);
(c) the fact that the main sialyltransferase activities expressed in HT-29 cells catalyze the transfer of sialic acid to
the 3-position of Gal in the Gal
1-3GalNAc disaccharide
sequence (Dall'Olio et al., 1993
; Majuri et al., 1995
; Delannoy et al., 1996
) in contrast to most colon cancers (Sata et al.,
1991
) or cell lines, including the enterocytic cell line Caco-2,
which mainly express the Gal
1-4GlcNAc
2,6-sialyltransferase, ST6Gal I as reported by others (Dall'Olio et al.,
1992
, 1996
), and confirmed here by the reactivity to SNA of Caco-2 glycoproteins; (d) the availability of an O-glycosylation inhibitor, GalNAc-
-O-benzyl (Kuan et al., 1989
;
Huang et al., 1992
; Byrd et al., 1995
; DiIulio and Bhavanandan, 1995
), which enters the cells and is metabolized
into a compound that acts as a competitive inhibitor of
Gal
1-3GalNAc
-2,3-sialyltransferases (Huet et al., 1995
;
Delannoy et al., 1996
); and (e) the fact that, in differentiated HT-29 cells, NeuAc
2-3Gal
1-3GalNAc-R is the main oligosaccharide species associated not only with mucins, as previously reported (Capon et al., 1992
; Lesuffleur
et al., 1993
; Huet et al., 1995
), but also, as shown here, with
a number of glycoproteins of the brush border concomitantly expressed in these cells. The association of
NeuAc
2-3Gal
1-3GalNAc-R to apical glycoproteins relies exclusively on their reactivity to MAA, and not to a
biochemical characterization that would require a huge amount of cells to be performed. However, the reliability
of MAA characterization is validated by a recent structural characterization of carbohydrate chains of the mucus
from HT29-RevMTX10
5 mucus-secreting cells (that can
be easily performed since large quantities of mucus can be
harvested daily) which has confirmed that NeuAc
2-3Gal
1-3GalNAc-R is the main oligosaccharide species associated with the mucus of these cells (Hennebicq-Reig,
S., T. Lesuffleur, C. Capon, C. de Bolos, I. Kim, O. Moreau, C. Richet, B. Hémon, M.A. Recchi, E. Maës, J.P.
Aubert, F.X. Real, A. Zweibaum, P. Delannoy, P. Degand, and G. Huet, manuscript submitted for publication).
Finally, it must be noted, as demonstrated by confocal microscopy, that no MAA reactivity could be detected on the
basolateral membrane of the cells.
Based on both the sugar specificity of MAA and PNA
lectins and the results of sialidase treatment, the present
data show that in addition to mucins (i.e., in HT29-RevMTX106 and 10
5), apical O-glycosylproteins such as
MUC1 express NeuAc
2-3Gal
1-3GalNAc-R terminal
sequences. This being established, four main observations can be drawn from the experiments performed with GalNAc-
-O-benzyl. First, alteration of
2,3-sialylation of
mucins and of apical O-glycosylproteins is accompanied
by their accumulation into intracytoplasmic vesicles. This
accumulation, which is most likely responsible for the dramatic swelling of the cells, is not restricted to O-glycosylproteins, but is also observed for N-glycosylproteins such
as DPP-IV and CEA. Regarding CEA, the absence of PNA
binding in treated cells may be explained by a weak expression of the Gal
1-3GalNAc sequence, not in contradiction with the fact that the binding of MAA is decreased.
Second, the block induced by GalNAc-
-O-benzyl occurs after the cis-Golgi as substantiated by endoglycosidase H
resistance of apical glycoproteins and normal processing
of DPP-IV. Third, these effects are reversible upon removal of the drug, resulting in a concomitant restoration
of
2,3-sialylation and resumption of the secretion of mucins and of the apical delivery of brush border-associated
glycoproteins. Fourth, these effects occur without any
modification in the morphological polarity of the cells, as shown by the normal polarized expression of villin and ZO1,
and without modification of distribution of the basolateral
glycoprotein GP120.
How can the fact be explained that GalNAc--O-benzyl, an O-glycosylation inhibitor (Kuan et al., 1989
; Huang
et al., 1992
; Byrd et al., 1995
) which, in HT-29, is metabolized into a compound, acts as a competitive inhibitor of
Gal
1-3GalNAc
2,3-sialyltransferases (Huet et al., 1995
;
Delannoy et al., 1996
) may also inhibit the sialylation of
N-glycosylproteins? Recent progress in the molecular cloning of sialyltransferases (for review see Harduin-Lepers
et al., 1995
; Tsuji, 1996
; Tsuji et al., 1996
) indicates that
three different enzymes (ST3Gal I, ST3Gal II, and ST3Gal
IV), encoded by different genes, located on separate chromosomes (Chang et al., 1995
) are able to transfer sialic
acid residues in the 3-position of Gal onto the disaccharidic Gal
1-3GalNAc-R sequence. The substrate specificity of ST3Gal I and ST3Gal II is strictly restricted to this
particular disaccharidic sequence (Kojima et al., 1994
) but ST3Gal IV can use both Gal
1-3GalNAc and Gal
1-4GlcNAc as acceptor substrates (Kitagawa and Paulson,
1994
). The competitive inhibition of ST3Gal IV by Gal
1-3GalNAc-
-O-benzyl could explain the decrease of
2,3-sialylation of N-glycosylproteins in HT-29 cells. On the other hand, the presence of a high concentration of the
competitive substrate (i.e., Gal
1-3GalNAc-
-O-benzyl)
may also decrease the concentration of CMP-NeuAc in
the Golgi lumen and therefore can compete with the other
sialyltransferases expressed in HT-29 cells via the donor
substrate.
Whatever the mechanism leading to the decrease of
2,3-sialylation, these results show that lack of terminal
NeuAc
2-3Gal-R glycosylation is associated with a blockade of apical targeting of brush border membrane-associated glycoproteins and mucus secretion in HT-29 cells.
They further suggest that
2,3-sialylation could play a role
in regulating the intracellular traffic of these proteins, and
in some way, support the view by Fiedler and Simons
(1994)
that "it may be that oligosaccharide side chains play
a more important role in biosynthetic traffic than hitherto
recognized". Even if the role of glycans in the intracellular
targeting of newly synthesized lysosomal enzymes via the
mannose-6-phosphate receptor pathways was clearly demonstrated in human colon adenocarcinoma cell lines (Braulke
et al., 1992
), a role for glycans in the sorting machinery of
membrane proteins in polarized cells has long been excluded on the basis of experiments using inhibitors of
N-glycosylation such as tunicamycin, which blocks the
transfer of Glc3Man9GlcNAc2 from dolichol to Asn, thus
resulting in the accumulation of unprocessed proteins in
the rough ER (Green et al., 1981
), or castanospermine, or
1-deoxymannojirimycin which block processing before
trimming of the oligomannose chains (Duronio et al.,
1988
). Very few recent observations suggest, however, a
possible role for terminal glycans in this regulation.
Growth hormone, which is nonglycosylated and secreted
from both sides of MDCK cell layers, is secreted from the
apical side when N-glycosylated (Scheiffele et al., 1995
). The sialoglycoprotein gp114 that is expressed on the apical
membrane of MDCK cells is misglycosylated and predominantly basolateral in the MDCK mutant MDCKII-RCAr
(Le Bivic et al., 1993
). The O-glycosylated stalk domain is
required for apical sorting of neurotrophin receptors in
polarized MDCK cells (Yeaman et al., 1997
). In addition
to these particularities that concern glycoproteins, it has
been shown that among the increasing number of vesicular
proteins presumably involved in protein sorting (Rothman, 1994
), one of them, VIP36 (Fiedler et al., 1994
), presents some homology with leguminous lectins (Fiedler and
Simons, 1994
) and binds GalNAc (Fiedler and Simons,
1996
). Therefore, it is conceivable that terminal NeuAc
2-3Gal glycan sequences as signals, and lectins as receptors
for these signals, could be involved in the sorting machinery of glycoproteins in polarized HT-29 cells. Because the
glycosylation of both lectin-like resident Golgi proteins and in transit glycoproteins may be affected by GalNAc-
-
O-benzyl, the precise level at which the sorting machinery
is affected needs to be elucidated.
Whether or not 2,3-sialylation is exclusively involved
in the apical sorting of glycoproteins in HT-29 cells cannot
be concluded from the present work since it was only focused on brush border membrane glycoproteins and mucins. From the results obtained with Western blot, it is
clear that the effects of GalNAc-
-O-benzyl are shared by
a number of other proteins that remain to be characterized as to their nature and localization. Nevertheless, the
present data show that a shift in the predominant glycosylation pattern of glycoproteins from NeuAc
2-3Gal
1-3GalNAc-R to Gal
1-3GalNAc-R results in what appears
as a glycoprotein traffic jam that dramatically alters their
normal delivery and also leads to cell hypertrophy.
Analysis of whether the present results uncover a more
general involvement of terminal glycans in the intracellular protein transport machinery needs to consider two
main evidences. First, there is the high polymorphism of
terminal glycans which differ from one individual to another. Second, and with regard to a putative role of animal
lectins as receptors for the glycan signal (Fiedler and Simons, 1994), there is the strict oligosaccharide specificity of lectins. This implies that if the results obtained with HT-29 cells rely on a general mechanism, each cellular system,
whether from human or animal origin, is unique and only
representative of the genetic background of the individual
it originates from. With regard to normal or malignant intestinal cells it must be noted that there are only very few
indications in which oligosaccharide species are associated
with apical glycoproteins. The only available data concern
the observation that, in the normal adult intestine, ABH
blood group antigens are the main terminal glycans associated with the intestinal brush border hydrolases in humans
(Triadou et al., 1983
; Green et al., 1988
) as well as in rabbits (Gorvel et al., 1982
). With regard to differentiated colon cancer cells, the results reported here are the first observation showing that in HT-29 cells, the main terminal
oligosaccharide species associated with apical glycoproteins is NeuAc
2-3Gal. Preliminary results obtained in the
laboratory indicate that, as in rat developing enterocytes
(Roth, 1993
), the apical membrane of intestinal enterocytes from 10-20 gestational wk fetuses is strongly reactive
with MAA; in contrast, epithelial cells in the adult human
small intestine lack MAA reactivity (our unpublished results), differing from the situation found in the adult colon
which has been shown to express an apical reactivity to
MAA (Sata et al., 1991
), but which is devoid of brush border-associated hydrolases. Therefore, the type of glycosylation demonstrated in HT-29-differentiated cells likely
represents one more feature of the fetal type of differentiation which is known to be associated with the malignant
phenotype (Zweibaum et al., 1991
).
With regard to the oligosaccharidic individual variability
it is clear that the results obtained with HT-29 cells cannot
be extrapolated as such to other cellular systems. For example, in Caco-2 cells, in which the main expressed sialyltransferase is ST6Gal I (Dall'Olio et al., 1992, 1996
) GalNAc-
-O-benzyl has no effect, even at much higher
concentrations (10 mM), on the morphology of the cells
and the apical polarity of brush border-associated glycoproteins (our unpublished results). This absence of effect is obviously consistent with the fact that in Caco-2 cells,
apical glycoproteins such as sucrase-isomaltase or DPP-IV
most likely express NeuAc
2-6, as suggested from the
SNA reactivity of Caco-2 glycoproteins and that GalNAc-
-O-benzyl has no effect on ST6Gal I.
Finally the present results draw attention to the necessity of characterizing, in each experimental system, which oligosaccharide species is associated with the apical glycoproteins. This is a prerequisite for any further analysis of the role of terminal oligosaccharides in the intracellular traffic of apical glycoproteins. It also implies that new strategies should be developed to specifically block the terminal glycosylation of apical glycoproteins. One challenge is to analyze whether, for example, ABH blood group antigens are involved in the intracellular traffic of intestinal brush border hydrolases in humans.
![]() |
Footnotes |
---|
Received for publication 17 June 1997 and in revised form 29 April 1998.
Address all correspondence to Alain Zweibaum, INSERM U178, 16 Avenue Paul-Vaillant-Couturier, 94807 Villejuif Cedex, France. Tel.: (33) 1-45-59-50-41. Fax: (33) 1-46-77-02-33. E-mail: zweibaum{at}infobiogen.frWe thank V. van Miegem (CNRS UMR III [Lille]), B. Hémon, and O. Moreau (both from INSERM U377 [Lille]) for excellent technical assistance. We are grateful to the Scientific and Medical Services of the University of Barcelona (Barcelona, Spain) for confocal microscopy analysis.
This work was supported in part by INSERM Consejo Superior de Investigaciones Científicas (CSIC) Cooperation Agreement, and grants from Fondo de Investigacion Sanitaria (94/1128), Generalitat de Catalunya (GRQ 93-01), Comisión Interministerial de Ciencia y Tecnología (CICYT) (SAF 97-0085), and Université Paris XI.
![]() |
Abbreviations used in this paper |
---|
CEA, carcinoembryonic antigen;
DPP-IV, dipeptidylpeptidase-IV;
GalNAc--O-benzyl, benzyl-2-acetamido-2-deoxy-
-D-galactopyranoside;
MAA, Maackia amurensis agglutinin;
MTX, methotrexate;
PNA, Arachis hypogaea agglutinin;
SNA, Sambucus nigra agglutinin. The abbreviations for sialyltransferases are
according to the new systematic nomenclature proposed by Tsuji et al.
(1996)
.
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