1275
Journal of Cell Science 108, 1275-1285 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
Characterization of mucins and proteoglycans synthesized by a mucinsecreting HT-29 cell subpopulation
G. Huet1,*, I. Kim2, C. de Bolos3, J. M. Lo-Guidice1, O. Moreau1, B. Hemon1, C. Richet1, P. Delannoy2,
F. X. Real3 and P. Degand1
1INSERM U377, Place de Verdun, 59045 Lille cedex, France
2UMR CNRS no. 111, UST Lille, 59655 Villeneuve d’Ascq cedex, France
3Institut Municipal d’Investigacio Mèdica, Universitat Autonoma de Barcelona,
Spain
*Author for correspondence
SUMMARY
HT-29 cells selected by adaptation to 10−5 M methotrexate
(HT-29 MTX) are a homogeneous cell population
producing high amounts of mucin. Intracellular mucins
and proteoglycans were isolated from these cells by ultracentrifugation of cell lysates on a cesium bromide gradient
and further separated by anion-exchange high perfomance
liquid chromatography. The major mucin fraction isolated
was characterized by a high hydroxy amino acid content
(40%), a Thr/Ser ratio of 1.52, a high sialic acid content,
and a low sulfate content. When the same procedure was
applied to undifferentiated HT-29 cells, a minor mucin
fraction was isolated which appeared less sialylated and
more sulfated. The major proteoglycan species identified in
HT-29 MTX cells showed less acidic behavior than the proteoglycan isolated from HT-29 cells. The effect of brefeldin
A and the sugar analog GalNAc-α-O-benzyl on the
synthesis and biochemical properties of mucins synthesized
by HT-29 MTX cells was examined. Brefeldin A induced
the synthesis of more-sulfated mucins. GalNAc-α-O-benzyl
treatment resulted in mucins with an increased content of
T antigen and a 13-fold lower sialic acid content. We show
that GalNAc-α-O-benzyl was metabolized by the cells to
Galβ1-3GalNAc-α-O-benzyl, which, in turn, was a potent
competitive inhibitor of the O-glycan α-2,3-sialyltransferase. These results illustrate the suitability of HT-29
MTX cells as a model to analyse mucin synthesis and sialylation.
INTRODUCTION
clonal cell line (cl.16-E) has been derived from HT-29 cultures
after treatment with sodium butyrate (Augeron and Laboisse,
1984) and a homogeneous mucin-secreting population has
been obtained by stepwise adaptation of HT-29 cells to 10−6
or 10−5 M methotrexate (MTX) (Lesuffleur et al., 1990). The
use of another antimetabolite, 5-fluorouracil (5-FU), has
resulted in the isolation of a population containing both absorptive and mucin-secreting cells (Lesuffleur et al., 1991). Antibodies raised against cl.16-E mucins stain goblet cells in both
normal colonic and normal gastric mucosa (Maoret et al.,
1989). Lesuffleur et al. (1990) have used rabbit antisera raised
against normal colonic mucosa and absorbed with stomach
tissue to identify mucins of ‘colonic’ specificity, and rabbit
antisera raised against normal gastric mucosa and absorbed
with colonic tissue to identify mucins of ‘gastric’ specificity.
The low proportion of mucin-secreting cells in parental HT-29
cultures expresses mucins of either gastric or colonic immunological specificity, whereas HT-29 MTX cells synthesize
mainly gastric-type mucins and HT-29 FU cells synthesize
colonic-type mucins (Lesuffleur et al., 1990, 1991). The characterization of these cell populations has been facilitated by the
isolation of cDNAs encoding human mucins (Gendler et al.,
1990; Gum et al., 1990, 1992; Porchet et al., 1991; Guyonnet-
Mucins and proteoglycans are high molecular mass glycoproteins whose biochemical functions depend on post-translational modifications. Alterations in these processes commonly
occur in transformed cells (Hardingham and Fosang, 1992;
Devine and McKenzie, 1992) and may contribute to the neoplastic phenotype. In human colorectal carcinomas, abnormalities in the oligosaccharide structures carried on mucins
(Kurosaka et al., 1983; Kim, 1992) and on proteoglycans
(Iozzo and Wight, 1982) have been described.
The analysis of the biosynthesis and composition of mucins
and proteoglycans, and the study of structure function relationships, would be greatly facilitated by the availability of cell
cultures with a wide range of phenotypic properties. The
parental HT-29 cell line fulfills this criterion. HT-29 cultures
are heterogeneous and in the postconfluent state consist of
>95% undifferentiated cells and a small proportion of differentiated mucin-secreting and absorptive cells (Pinto et al.,
1982; Augeron and Laboisse, 1984; Lesuffleur et al., 1990).
Differentiated populations of either absorptive or mucinsecreting phenotype can be obtained under various conditions
of metabolic stress. A stably differentiated mucin-secreting
Key words: mucin, proteoglycan, HT-29, aryl-N-acetyl-αgalactosaminide, brefeldin
1276 G. Huet and others
Duperat et al., 1994; Toribara et al., 1993; Bobek et al., 1993).
HT-29, HT-29 MTX and HT-29 FU cells show distinct
patterns of MUC1-MUC5 mucin gene expression and HT-29
MTX cells express mainly MUC1, MUC2, MUC3 and MUC5C
genes (Lesuffleur et al., 1993). However, less is known
regarding the main carbohydrate structures carried on the
products of these genes in HT-29 MTX cells (Dahiya et al.,
1992).
This paper describes a first step towards the biochemical and
structural characterization of the mucins and proteoglycans
synthesized by HT-29 MTX cells in comparison with those
produced by the parental cell population. To gain insight into
mucin biosynthesis in HT-29 MTX cells, we have used two
compounds that could potentially interfere with glycosylation
and alter the biochemical properties of mucins, brefeldin A
(BFA) and benzyl-N-acetyl-α-galactosaminide (GalNAcα-Obenzyl). BFA is a fungal antibiotic that disrupts protein
transport from the rough endoplasmic reticulum (RER) to the
Golgi apparatus, inducing a redistribution of Golgi proteins to
the RER (Lippincott-Schwartz et al., 1989). Aryl-N-acetyl-αgalactosaminides have been initially used as potential competitors of the glycosylation of GalNAc residues linked to the
core protein and have been shown to inhibit UDPGal:GalNAcβ1-3-galactosyltransferase in vitro (Kuan et al.,
1989). However, it was observed that GalNAcα-O-benzyl
treatment of HM7 colon cancer cells leads to an increased
expression of Tn antigen (GalNAc-Thr/Ser), but also of T
antigen (Galβl-3GalNAc), together with a decrease in sialylLex, sialyl-Lea and sulfomucin epitopes (Huang et al., 1992).
To explain the unexpected increase in T antigen activity, it has
been proposed that the in situ synthesis of Galβ1-3GalNAcαO-benzyl might lead to a more efficient inhibitor than
GalNAcα-O-benzyl itself; the benzyl disaccharide would
inhibit the elongation of T antigen by N-acetylglucosaminyltransferases, sialyltransferases and/or fucosyltransferase. Here,
we provide evidence that GalNAcα-O-benzyl induces a
marked increase in T antigen and a marked decrease in sialic
acid content of HT-29 MTX mucins. Furthermore, the
enzymatic mechanisms responsible for these changes in mucin
synthesis are partially elucidated.
in threonine-free Dulbecco’s minimum essential medium (Institut
Jacques Boy, Reims, France). After labeling, medium was collected
and cells were lysed in RIPA buffer (0.01 M Tris-HCl, pH 8.0, 0.01
M NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 0.5%
sodium deoxycholate, 1% phenylmethylsufonyl fluoride, 0.001 M
sodium ethylene diaminotetra-acetate).
In some experiments, the effect of GalNAcα-O-benzyl (Sigma, St
Louis, MO; 5 mM) or BFA (Sigma; 5 µg/ml) on mucin biosynthesis
was examined. Cells were cultured for 20 days as described above,
GalNAcα-O-benzyl or BFA were added for 24 hours, and cells were
processed.
Biochemical characterization
Ultracentrifugation
Twenty-one days after seeding, cells were rinsed twice with PBS,
scraped with a rubber policeman, and directly lysed by ultrasonication in PBS. Cell extracts were collected after centrifugation for 5
minutes at 1000 g at 4°C. After adding cesium bromide (0.42 g/ml),
cell lysates were ultracentrifuged (200,000 g for 72 hours) using a
Beckman 70.1 Ti rotor (Houdret et al., 1986). Fractions of 1 ml were
collected, weighed to determine density, and analysed for absorbance
at 280 nm, orcinol reactivity, and dimethylmethylene blue (DMB)
reactivity (De Jong et al., 1992).
HPLC chromatography
Anion-exchange high performance liquid chromatography (AEHPLC) was carried out using a TSK DEAE 5PW (7.5 mm × 75 mm)
Spherogel column (Beckman). The system was equilibrated with
0.005 M sodium/potassium phosphate buffer, pH 6.0, at a flow rate
of 0.8 ml/min. A total of 2 mg protein was injected. Elution was
carried out using a NaCl gradient (0 to 1 M) in the same buffer.
Eluates were collected in 0.8 ml fractions and analysed as described
above.
Compositional analyses
For amino acid analysis, samples were hydrolysed in 5.6 M HCl for
24 hours under a vacuum and processed using a 7300 Beckman amino
acid analyser (Palo Alto, CA) equipped with a high performance
sodium column (4 mm × 120 mm) (Beckman). Sugar analysis was
carried out by gas-liquid chromatography of trimethylsilyl derivatives
of methyl glycosides formed by methanolysis in 1.5 M HCl in
methanol at 80°C for 24 hours (Lamblin et al., 1984). To determine
sulfate content, samples were hydrolysed in 1 M HCl for 5 hours at
100°C and sulfate was determined by AE-HPLC (Lo-Guidice et al.,
1994). All compositional analyses were performed twice to examine
the reproducibility of the results.
MATERIALS AND METHODS
Cell culture
Parental HT-29 cells (referred to as HT-29) and HT-29 cells selected
by adaptation to 10−5 M methotrexate (MTX) were obtained from Dr
Thécla Lesuffleur (Unité INSERM U178, Villejuif, France). Cells
were grown in Dulbecco’s modified Eagle’s minimal essential
medium (Eurobio, Paris, France), supplemented with 10% inactivated
(30 minutes, 56°C) fetal bovine serum (Boehringer, Mannheim,
Germany). Cells were seeded at 1.5×106 cells in 75 cm2 flasks
(Corning Glassworks, Corning, NY) and cultured at 37°C in a 10%
CO2/90% air atmosphere. The medium was changed daily. Cultures
were studied in the late post-confluent period (21 days after seeding),
when all cells display a mucin-secreting phenotype (Lesuffleur et al.,
1990). Cell viability was determined by trypan blue dye exclusion.
For metabolic labeling, cells were cultured as described above until
day 20 and were then labeled for 24 hours with [3H]glucosamine (1.48
MBq/ml) in low-glucose Dulbecco’s minimum essential H-16
medium (Gibco, Gaithesburg, MD), with [35S]sulfate (1.48 MBq/ml)
in Ham’s F-12 medium (Gibco), or with [3H]threonine (1.48 MBq/ml)
Electrophoresis and western blotting
SDS-PAGE
SDS-PAGE was performed with 2% to 5% gradient polyacrylamide
gels (Laemmli, 1970). For autoradiogaphy, gels were fixed in 40%
ethanol, 10% glycerol, 10% acetic acid (by vol.), soaked in Amplify
(Amersham, UK) for 20 minutes, dried on Whatman paper, and
exposed to Cronex 4 NIF film (Dupont).
Agarose gel electrophoresis
Mucin glycopeptides were obtained by Pronase digestion for 48 hours
at 37°C in 0.01 M calcium acetate buffer, pH 7, at an enzyme/substrate
ratio of 1/40 (w/w). Fresh enzyme was added after 24 hours of
digestion. Agarose gel electrophoresis was then performed in veronal
buffer at pH 8.2 and gels were stained with Schiff periodate (Marianne
et al., 1986).
Cellulose acetate electrophoresis
After immersion of cellulose acetate plates in 0.05 M barium acetate,
migration was carried out for 20 minutes. The plates were then stained
Biochemical characterization of HT-29 mucins 1277
in 0.02% DMB in 1% acetic acid for 10 minutes and destained in 10%
acetic acid (Wesslet, 1968).
Western blotting
After separation by 2-5% SDS-PAGE, proteins were transferred to a
nitrocellulose membrane as described by Vaessen et al. (1981).
Membranes were incubated with 10 µg/ml peroxidase-labeled lectins
(Sigma) in 10 mM Tris-glycine buffer containing 0.9% NaCl and 3%
bovine serum albumin at 37°C for 2 hours. After washing, membranes
were incubated with 4-chloro-1-naphthol (Sigma).
Immunocytochemistry
Indirect immunofluorescence was performed on cryostat sections of
cell layer rolls as reported by Lesuffleur et al. (1990), with minor
modifications. Sections were air-dried and fixed with acetone for 10
minutes. After washing with PBS, 5% normal horse serum was added.
Sections were incubated with FITC-labeled soybean (SBA), peanut
(PNA), and wheat germ (WGA) lectins for 60 minutes. After washing,
sections were mounted with glycerol. Alternatively, mouse monoclonal antibodies (mAb), that detect a large panel of mucin-associated
carbohydrate epitopes were used. mAb Cu-1, detecting Tn (Takahashi
et al., 1988), was obtained from Dr S. I. Hakomori (The Biomembrane Institute, Seattle, WA) and mAbs B72.3 and CC49, detecting
sialyl-Tn (Nuti et al., 1982), were obtained from Dr Kenneth O. Lloyd
(Sloan-Kettering Institute, New York, NY). To obtain better semiquantitative data, several lectin and antibody dilutions were used in
each experiment. Reactions were developed with goat anti-mouseTRlTC (Pierce, Rockford, IL) (5 µg/ml) and visualized using a Zeiss
Axioscop equipped with Plan-Neofluar lenses and scored as follows:
+++, when fluorescence signal was clearly seen at ×100 magnification; ++, when it was seen at ×200 magnification; +, when signal was
seen only at ×400 magnification. Percentage reactive cells was
expressed as an average of the whole section. Scoring was performed
independently by two investigators (C. de B. and F.X.R.) and all
experiments were repeated twice independently.
Sialyltransferase (ST) assays
Preparation of cell extracts
Cells were lysed at 0°C with 10 mM sodium cacodylate buffer, pH
6.5, containing 1% Triton X-100, 20% glycerol, 0.5 mM dithiotreitol,
and 5 mM MnCl2 (1 ml per 170 cm2 flask). After a 10 minute incubation under continuous stirring, cell homogenates were centrifuged
at 10,000 g for 15 minutes and the supernatants were used for
enzymatic assays. Protein concentration was determined according to
the method described by Peterson (1977) using bovine serum albumin
(BSA) as standard.
samples were centrifuged at 3,000 g for 5 minutes and supernatants
were directly processed for descending paper chromatography in ethyl
acetate/ pyridine/ water (10/4/3, by vol.) (Delannoy et al., 1993).
RESULTS
Comparative analysis of mucins and proteoglycans
from HT-29 MTX and HT-29 cells
The secretion of mucins and proteoglycans by HT-29 MTX
and HT-29 cells was first studied by metabolic labeling with
[3H]glucosamine and with [35S]sulfate followed by analysis of
culture medium by SDS-PAGE. After [3H]glucosamine
labeling (Fig. 1A), a broad band was observed in the case of
HT-29 MTX cells whereas HT-29 cells yielded a diffuse
smear. This result is relevant to the high rate of mucins
produced by HT-29 MTX cells and to the diffuse high
molecular mass band described by Dahiya et al. (1992). SDSPAGE analysis of culture media from [35S]sulfate-labeled HT29 MTX and HT-29 cells showed diffuse smears with a
mobility similar to that of [3H]glucosamine-labeled cells (Fig.
1B). Although [3H]glucosamine is predominantly incorporated
into mucins and [35S]sulfate into glycosaminoglycans, it was
not possible to distinguish between mucins and proteoglycans
using SDS-PAGE.
Mucins and proteoglycans synthesized by HT-29 MTX and
HT-29 cells were isolated by ultracentrifugation through a
CsBr gradient. Fig. 2A shows the absorbance profiles obtained
after the addition of orcinol, to detect carbohydrates; or DMB,
to detect glycosaminoglycans. AE-HPLC was then used to
separate mucins and proteoglycans from selected fractions of
HT-29 MTX (1.42-1.51 g/ml) and HT-29 (1.39-1.53 g/ml)
cells and the elution profiles are shown in Fig. 2B. In the HT29 MTX fraction, orcinol reactivity eluted as a major peak with
a retention time of 24 minutes, whereas DMB reactivity eluted
later and showed a complex pattern of peaks at the 26-37
ST assay using asialofetuin as acceptor
Cell extracts (40 µl) were carried to a final volume of 120 µl with 0.1
M sodium cacodylate, pH 6.5, 1% Triton X-100, 0.1% BSA, 0.2 M
galactose (an inhibitor of β-galactosidase), 1 mM 2,3-dehydro-2deoxy-Neu5Ac (an inhibitor of sialidases), 52.9 µM CMP[14C]Neu5Ac (0.58 GBq/mmol, 3.67 kBq/120 µl), containing 480 µg
of asialofetuin (prepared by mild acid hydrolysis of fetuin) as
exogenous substrate, and incubated at 37°C for 1 hour. The reaction
was stopped by adding 1 ml of ice-cold phosphotungstic acid (5% in
2 M HCl). The precipitate was collected on glass fiber filters, washed
extensively with 5% trichloroacetic acid, distilled water and ethanol,
and processed for scintillation counting (Vandamme et al., 1992).
ST assay using Galβ1-3GalNAcα-O-benzyl as acceptor
Incubations were performed under the conditions described above
using 40 µl of cell extract or 1 mU of purified porcine liver Galβ13GalNAc α-2,3-ST (Boehringer, Mannheim, Germany) as enzyme
source and Galβ1-3GalNAcα-O-benzyl (Sigma) (1 mM final concentration). The reaction was stopped by adding 1 volume of ethanol;
Fig. 1. SDS-PAGE autoradiogram of culture medium from HT-29
and HT-29MTX cells labeled with [3H]glucosamine (A) or
[35S]sulfate (B). Lane 1, HT-29 cells; lane 2, HT-29 MTX cells.
Equal amounts of protein were loaded for each cell type.
Electrophoretic migration was compared with that of a 200 kDa
molecular mass marker.
1278 G. Huet and others
Fig. 2. (A) Ultracentrifugation profiles of lysates from HT-29 MTX and HT-29 cells. (B) HPLC profiles of ultracentrifugation fractions from
HT-29 MTX and HT-29 cells.
minute time periods. For HT-29 cells, a low orcinol reactivity
was detected showing three small peaks at retention times of
4, 25, and 31 minutes, whereas, a high DMB reactivity was
apparent at the 26-40 minute time points. Preparative HPLC
chromatography was carried out for both cell types. Three and
two fractions were collected from HT-29 MTX (top Fig. 2B,
fraction A, 21-25 minutes; B1, 26-29 minutes; and B2, 30-37
minutes) and HT-29 cells (bottom Fig. 2B, fraction A, 21-27
minutes; B, 28-37 minutes), respectively. These fractions were
analysed for amino acid (Table 1), carbohydrate, and sulfate
composition (Table 2).
HT-29 MTX fraction A showed a high carbohydrate/protein
content (6.8 g sugar/g protein), high hydroxy amino acid
content (40.05%) with a Thr/Ser ratio of 1.52, and a high
proline content (11.83%), all typical features of mucins. Carbohydrate analysis revealed mainly GalNAc, Gal, GlcNAc and
sialic acid. The sialic acid content was very high (1.3 molar
ratio to GalNAc) whereas the sulfate content was very low (0.1
molar ratio to GalNAc). HT-29 MTX fractions B1 and B2 also
showed very high hydroxy amino acid contents (34.49% and
31.51%, respectively) with lower Thr/Ser ratios (1.30 and 1.11,
respectively), and lower Pro contents (9.73% and 8.62%,
respectively). Other major amino acid changes consisted of a
decrease in Arg and an increase in Cys, Glu/Gln, Gly and Tyr.
The relative content of neutral monosaccharides was similar in
Table 1. Amino acid composition of HT-29 MTX and
HT-29 HPLC fractions (%)
HT-29 MTX HPLC fraction
Cys
Asp
Thr
Ser
Glu
Pro
Gly
Ala
Val
Ile
Leu
Tyr
Phe
His
Lys
Arg
HT-29 HPLC fractions
A
B1
B2
A
B
0.73
4.80
24.18
15.87
7.17
11.83
6.62
7.12
5.18
2.40
3.11
0.08
2.20
1.55
2.59
4.57
1.96
6.76
19.53
14.96
10.88
9.73
8.77
7.26
4.44
2.21
3.13
1.24
1.86
1.55
2.96
2.75
2.47
6.90
16.61
14.90
11.32
8.62
11.14
7.26
4.46
2.37
3.66
1.47
2.12
1.57
2.35
2.76
0.51
10.17
8.98
7.85
11.23
6.22
10.15
10.48
6.21
3.72
6.48
2.45
3.36
1.69
5.14
5.37
0.97
8.55
5.77
9.72
11.77
4.45
31.69
7.77
4.32
2.22
4.23
2.46
1.52
4.58
fractions A, B1 and B2. In contrast, fractions B1 and B2 had
lower relative sialic acid contents (0.9 and 0.5, respectively)
and higher sulfate contents (1.2 and 0.6, respectively). These
results suggest that fractions B1 and B2 correspond to more-
Biochemical characterization of HT-29 mucins 1279
Table 2. Carbohydrate composition and sulfate content of
HT-29 MTX and HT-29 HPLC fractions
HT-29 MTX HPLC fractions
Mannose
Galactose
GalNAc
GlcNAc
Sialic acid
Sulfate
Sulfate*
HT-29 HPLC fractions
A
B1
B2
A
B
1.4
=1
0.6
1.3
0.1
0.1
0.1
1.5
=1
0.5
0.9
1.2
2.6
0.2
1.1
=1
0.5
0.5
0.6
1.2
0.5
1
=1
1.2
0.3
2.1
1.7
ND
ND
ND
+
ND
+
1.6
All results are expressed as molar ratios to GalNAc.
*The sulfate content was also expressed as molar ratio to GlcNAc.
ND, not detectable.
acidic mucins, mixed with proteoglycans, as predicted by the
elution profile obtained after HPLC chromatography.
HT-29 fraction A showed a relatively low
carbohydrate/protein level (1.9 g sugar/g protein), a hydroxy
amino acid content of 16.83% with a Thr/Ser ratio of 1.14, and
a 6.22% Pro content. Carbohydrate analysis of this fraction
revealed mainly mannose, Gal, GalNAc, GlcNAc and sialic
acid. The sialic acid content was low (0.3 molar ratio to
GalNAc) whereas the sulfate content was relatively higher (2.1
molar ratio to GalNAc). HT-29 fraction B had a hydroxy amino
acid content of 15.49%, a Thr/Ser ratio of 0.59 and a high Gly
content (31.69%). Carbohydrate analysis revealed only
GlcNAc. Sulfate was also found in this fraction in a 1.6 molar
ratio to GlcNAc. Therefore, the biochemical characteristics of
HT-29 fraction B are those typical of a proteoglycan.
The presence of mucins in fraction A from HT-29 MTX and
HT-29 cells was further examined by Schiff periodate staining
of Pronase digestion products fractionated by agarose gel electrophoresis: a fast migrating band which stained intensely was
observed for HT-29 MTX cells whereas a weakly stained band
was observed for HT-29 cells (Fig. 3).
The electrophoretic behavior of proteoglycans from HT-29
MTX and HT-29 cells was analysed using cellulose acetate
strips and coloration with DMB (Fig. 4). HT-29 MTX fraction
B2 contained a proteoglycan with a slower migration than the
heparan sulfate standard, whereas HT-29 fraction B contained
a proteoglycan which migrated between the heparan sulfate
and dermatan sulfate standards. Consequently, the main proteoglycan detected in HT-29 cells is more acidic than the HT29 MTX proteoglycan.
Effect of BFA and GalNAcα-O-benzyl on mucin
biosynthesis by HT-29 MTX cells
The effects of BFA and GalNAcα-O-benzyl upon the biosynthesis and secretion of mucins were first studied through continuous labeling with [3H]threonine. In the presence of BFA,
cell-associated and secreted radioactivity were 435.9% and
42.1% of those of control cultures, respectively. In the
presence of GalNAcα-O-benzyl, cell-associated and secreted
radioactivity were 428.9% and 49% of those of control
cultures, respectively. Fig. 5 shows the electrophoretic pattern
of cell lysates and culture media after metabolic labeling with
[3H]threonine. A major band was observed in each cell lysate,
the migration of which appeared slightly increased after
treatment with BFA and slightly decreased after treatment with
Fig. 3. Agarose gel electrophoresis of mucin glycopeptides and
staining with Schiff periodate. Fractions A from HT-29 MTX cells
(lane 1) and from HT-29 cells (lane 2) were digested with Pronase
for 48 hours at 37°C and mucin glycopeptides were loaded on
agarose gel slides.
Fig. 4. Proteoglycan analysis by cellulose acetate electrophoresis and
DMB staining. Fraction B from HT-29 cells (A) and fraction B2
from HT-29 MTX cells (B) were compared with several standard
glycosaminoglycans. (A) Lane 1, chondroitin sulfate C; lane 2, HT29 fraction B; lane 3, chondroitin sulfate A; lane 4, dermatan sulfate;
lane 5, heparan sulfate. (B) Lane 1, chondroitin sulfate A; lane 2,
HT-29 MTX fraction B2; lane 3, chondroitin sulfate C; line 4,
dermatan sulfate; lane 5, heparan sulfate.
GalNAcα-O-benzyl. SDS-PAGE analysis of culture media
from BFA-treated and GalNAcα-O-benzyl-treated cultures
revealed faint smears with mobility similar to that of the corresponding cell lysates. This finding was in contrast to the
discrete band observed in culture medium from control HT-29
MTX cells.
Mucins from HT-29 MTX cells cultured for 24 hours in the
presence of BFA or GalNAcα-O-benzyl were isolated by ultracentrifugation and AE-HPLC. Analysis of the mucin fractions
(Table 3) showed that BFA treatment led to a slight decrease
in the relative content of Gal and sialic acid and to a 4-fold
increase in sulfate content. GalNAcα-O-benzyl treatment
induced a 13-fold decrease in the relative amount of sialic acid
in HT-29 MTX fraction A. The composition of B fractions and
the electrophoretic behavior of the HT-29 MTX proteoglycan
were unchanged (data not shown).
To further study the effect of BFA and GalNAcα-O-benzyl
on HT-29 MTX mucins, their reactivity with a panel of mAbs
and lectins was examined by immunofluorescence on frozen
sections of cell rolls (Fig. 6 and Table 4), or by western blotting
(Fig. 7) on purified mucin fractions. BFA treatment did not
lead to major changes in the expression of mucin-associated
1280 G. Huet and others
Fig. 5. SDS-PAGE autoradiogram of HT-29 MTX cell
lysates (A) and culture media (B) after labeling with
[3H]threonine. Lane 1, control HT-29 MTX cells; lane 2,
HT-29 MTX cells cultured in the presence of GalNAcαO-benzyl for 24 hours; lane 3, HT-29 MTX cells
cultured in the presence of BFA for 24 hours. Equal
amounts of protein/sample were loaded.
carbohydrate epitopes. GalNAcα-O-benzyl treatment resulted
in an increase in the expression of Tn and T antigens as
detected with SBA and mAb Cu-l (Tn antigen) or PNA (Fig.
6 and Table 4). No changes in the reactivity with WGA were
observed (not shown). Western blotting particularly showed a
great increase in the T antigen reactivity of mucins obtained
after GalNAcα-O-benzyl treatment (Fig. 7).
To determine the mechanism(s) by which GalNAcα-Obenzyl treatment induced such a decrease in sialic acid content
and the concomitant increase in T antigen in HT-29 MTX
mucins, ST assays were performed. First, ST activity was
measured using asialofetuin as an in vitro substrate: a 30%
Table 3. Carbohydrate composition and sulfate content of
HT-29 MTX HPLC fraction A obtained after treatment
with brefeldin A or GalNAcα-O-benzyl
Galactose
GalNAc
GlcNAc
Sialic acid
Sulfate
HT-29 MTX
fraction A of
control HT-29
MTX cells
HT-29 MTX
fraction A
obtained after
treatment with
brefeldin A
HT-29 MTX
fraction A
obtained after
treatment with
GalNAcα-O-benzyl
1.4
=1
0.6
1.3
0.1
1.1
=1
0.6
1.1
0.4
1.4
=1
0.5
0.1
0.2
All results are expressed as molar ratios to GalNAc.
decrease in the transfer of [14C]sialic acid was detected in
GalNAcα-O-benzyl-treated cells (Fig. 8A).
GalNAcα-O-benzyl cannot be used as a substrate by sialyltransferases (Van den Eijden and Joziasse, 1993). On the other
hand, Galβ1-3GalNAcα-O-benzyl is a specific substrate for
Galβ1-3GalNAcα-2,3-sialyltransferase (α-2,3-ST(O), EC
2.4.99.4) and would therefore be able to compete for sialylation of asialofetuin in an in vitro assay.
The ability of Galβ1-3GalNAcα-O-benzyl and GalNAcαO-benzyl to inhibit the ST activity using asialofetuin as a
substrate was examined. As shown in Fig. 8B, Galβ13GalNAcα-O-benzyl was an effective inhibitor at a concentration of 100 µM, whereas GalNAcα-O-benzyl was not
inhibitory.
These results fitted well the hypothesis proposed by Huang
et al. (1992), that GalNAcα-O-benzyl could be converted in
vivo into Galβ1-3GalNAcα-O-benzyl and compete for the
elongation of T antigen in HM 7 colon cancer cells.
We then investigated the in situ synthesis of Galβ13GalNAcα-O-benzyl in GalNAcα-O-benzyl-treated HT-29
MTX cells. When the in vitro α-2,3-ST(O) assay was
performed with homogenates from GalNAcα-O-benzyl-treated
cells in the absence of the exogenous acceptor Galβ13GalNAcα-O-benzyl, a sialylated product that was absent
from control homogenates was detected. This sialylated
product comigrated with NeuAcα2-3Galβl-3GalNAcα-Obenzyl obtained by incubation of the Galβ1-3GalNAcα-O-
Table 4. Immunofluorescence on cryostat sections of HT-29 MTX cells untreated, or treated with brefeldin A or
GalNAcα-O-benzyl
% of labeled cells
Lectins
Antibodies
SBA, 20 µg/ml
PNA, 20 µg/ml
Cu-1
B72.3
CC.49
Specificity
Control HT-29
MTX cells
(%)
HT-29 MTX cells
treated with
brefeldin A (%)
HT-29 MTX cells
treated with
GalNAcα-O-benzyl (%)
Tn
T
Tn
Sialyl-Tn
Sialyl-Tn
<10
40-50(+)*
70 (++)
70 (++)
80 (++)
−
30
90 (+++)
80 (+++)
60 (+++)
100
70
100 (+++)
30 (++)
60 (++)
*See Materials and Methods for explanation of scoring method.
Biochemical characterization of HT-29 mucins 1281
Fig. 6. Immunocytochemistry of cryostat sections of HT-29 MTX cell rolls using lectins and antibodies recognizing different glycan epitopes.
Control untreated cells (a,d,g,j); BFA-treated cells (b,e,h,k); GalNAcα-O-benzyl-treated cells (c,f,i,l). SBA(a,b,c); PNA (d,e,f); mAb Cu-1
(g,h,i); mAb B72.3 (j,k,l). Bar, 100 µm.
benzyl substrate with purified porcine liver α-2,3-ST(O) (Fig.
9A). Furthermore, when denatured extracts from GalNAcα-Obenzyl-treated HT-29 MTX cells were used as substrates and
HT-29 MTX control extracts or purified porcine liver α-2,3-
ST(O) were used as enzyme source, NeuAcα2-3Galβ13GalNAcα-O-benzyl was also detected (Fig. 9B and C). Taken
together, these results indicate that GalNAcα-O-benzyl is
converted to Galβ1-3GalNAcα-O-benzyl, which in turn
1282 G. Huet and others
kDa
kDa
Fig. 7. Immunoblot with peroxidase-labeled Helix pomatia lectin (A)
and PNA lectin (B). Lane 1, untreated HT-29 MTX cells; lane 2,
BFA-treated HT-29 MTX cells; lane 3, GalNAcα-O-benzyl-HT-29treated MTX cells.
inhibits the α-2,3-ST(O) activity, resulting in the observed
increase in T antigen and decrease in sialic acid content of
mucins from HT-29 MTX cells.
DISCUSSION
The availability of mucin-producing HT-29 MTX cells and the
parental HT-29 cells from which the former population was
isolated has allowed the analytical characterization of mucins
synthesized by these two cell types. To interpret the findings,
one must bear in mind that the HT-29 MTX line represents a
homogeneous population of cells producing mucins of gastric
type, whereas HT-29 cultures contain >95% undifferentiated
cells and a low proportion of cells producing mucins of gastric
as well as colonic immunological specificity.
HT-29 MTX mucins barely enter 2% to 5% gradient polyacrylamide gels, in accordance with their formation of a
secreted mucous layer (Lesuffleur et al., 1990). The major
mucin fraction isolated by AE-HPLC is characterized by a high
hydroxy amino acid content and a Thr/Ser of 1.52. Prior studies
have shown that MUC1, MUC2, MUC3 and MUC5AC are
expressed in HT-29 MTX at the mRNA level, and the amino
acid composition of the major mucin fraction isolated is in
agreement with the amino acid sequences of the tandem repeats
of these genes (Gendler et al., 1990; Gum et al., 1990, 1992;
Porchet et al., 1991; Guyonnet Duperat et al., 1994). The carbohydrate composition of this fraction revealed a high GalNAc
and sialic acid content, and a low GlcNAc and sulfate content.
These characteristics are similar to those described for the
cl.16-E mucin-secreting clone isolated from HT-29 cells after
treatment with sodium butyrate (Augeron and Laboisse, 1984).
The analysis of mucin-containing fractions from HT-29 cells
revealed a lower amount of mucins, related to the small proportion of mucin-producing cells in this population (less than
0.5%) (Augeron and Laboisse, 1984; Lesuffleur et al., 1990,
1991), as well as less glycosylated mucins. Furthermore, the
composition of HT-29 mucins was different from that of HT29 MTX mucins in having: (1) lower hydroxy amino acid and
Fig. 8. Effect of GalNAcα-O-benzyl and Galβ1-3GalNAcα-Obenzyl on the transfer of Neu5Ac on asialofetuin. (A) Homogenates
of HT-29 MTX cells cultured with (j) or without (h) GalNAcα-Obenzyl were incubated with asialofetuin and the transfer of Neu5Ac
was comparatively measured. Results are expressed as nmol of
[14C]Neu5Ac transferred/mg of protein (mean values of two separate
experiments). (B) Homogenates of HT-29 MTX cells were incubated
with asialofetuin in the presence of GalNAcα-O-benzyl (j) or
Galβ1-3GalNAcα-O-benzyl (h) at different concentrations. Results
are expressed as the percentage of remaining activity.
proline content; (2) higher GlcNAc/GalNAc ratio; (3) lower
sialic acid content; and (4) higher sulfate content. The distinct
compositional features of HT-29 MTX and HT-29 mucins may
reflect differences in expression of mucin genes (Lesuffleur et
al., 1993) or be related to the altered compartmentalization of
resident Golgi proteins in HT-29 MTX cells (Egea et al., 1993).
Besides, the HT-29 cell line likely contains mucin-secreting
cells of different specificity, as shown using rabbit antisera
detecting gastric or colonic mucins (Lesuffleur et al., 1990).
Current efforts aim at characterizing the structure of carbohydrate chains of HT-29 MTX and HT-29 mucins in an attempt
to analyse the relationship between oligosaccharide chains and
the apomucin carrying them.
The study of high molecular mass glycoproteins from these
two cell populations has also shown differences in the structure
of proteoglycans, although this analysis is hampered by the
high amount of mucins synthesized by HT-29 MTX. Indeed, a
certain level of mixing of these two types of molecules is
almost inevitable. In any case, the proteoglycans of HT-29
c.p.m. (×10−3)
c.p.m. (×10−3)
Biochemical characterization of HT-29 mucins 1283
Fig. 9. Detection of Galβ1-3GalNAcα-O-benzyl in the homogenates
of HT-29 MTX cells treated with 5 mM of GalNAcα-O-benzyl.
(A) Homogenates of cells cultured with (j) or without (h)
GalNAcα-O-benzyl were incubated in standard conditions of α-2,3ST(O) assay without exogenous acceptor, and sialylated products
were detected as described in Materials and Methods. 1, CMPNeu5Ac; 2, Neu5Ac; 3, the arrowed bar indicates the position of
Neu5Acα2-3Galβ1-3GalNAcα-O-benzyl obtained by the sialylation
of Galβ1-3GalNAcα-O-benzyl with porcine liver α-2,3-ST(O).
(B,C) Homogenates of cells cultured with GalNAcα-O-benzyl were
denatured by heating and used as substrate source in α-2,3-ST(O)
assay. Homogenates from control cells (B) or porcine liver α-2,3ST(O) (C) were used as enzyme sources. Sialylated products were
detected as described in Materials and Methods.
MTX and HT-29 cells differ with regard to their electrophoretic behavior: the less-acidic nature of HT-29 MTX
proteoglycans might result from a larger amount of unsulfated
heparan chains. Such structures were shown to coexist with
sulfated heparan chains on the same protein core of the heparan
sulfate proteoglycan (HSPG) from the colon carcinoma cell
line WiDr (Iozzo, 1989), a cell line that has been reported by
the ATCC as possibly being the same as HT-29. The studies
of high Mr HSPG produced by different cell types have
suggested that a common precursor protein core could undergo
cell-specific processing, thus generating different forms of proteoglycans (Murdoch et al., 1992). The data from mucin and
proteoglycan analysis suggest that the degree of sulfatation of
these molecules may be modulated by the state of differentiation of HT-29 cells.
To examine mucin glycosylation in greater detail, we have
analysed the effect of BFA and GalNAcα-O-benzyl on the
structure of mucins synthesized by HT-29 MTX cells. BFA,
which blocks transport from the RER to the Golgi apparatus
and induces the redistribution of resident Golgi proteins to the
RER (Lippincott-Schwartz et al., 1989), induced an increase in
sulfate content and an increase in the electrophoretic mobility
of mucins in SDS-PAGE, events that are possibly related.
Indeed, the high carbohydrate content of mucins may prevent
effective binding of SDS to the protein core and the intrinsic
negative charge of mucin molecules could influence their
migration, as described for the MUC1 gene product (Hilkens
and Buijs, 1988). The mechanisms by which these changes
occur are not known, but our findings suggest that compartmentalization of sulfotransferases is affected by brefeldin A.
GalNAcα-O-benzyl induced an increased expression of T
antigen and a marked decrease in the sialylation of HT-29
MTX mucins. These changes were accompanied by a decrease
in the electrophoretic mobility of mucins by SDS-PAGE,
possibly due to the synthesis of less-acidic mucins. Indeed the
extensively sialylated form of MUC1 has a higher mobility in
SDS-PAGE than the incompletely sialylated premature form
(Hilkens and Buijs, 1988). GalNAcα-O-benzyl treatment of
the HM7 high-mucin variant of LS174T colon cancer cells also
led to an increase in T antigen reactivity. This result, now
reproduced in HT-29 MTX cells, was unexpected as
GalNAcα-O-benzyl behaves as an in vivo competitive
inhibitor of the galactosyltransferase responsible for the
synthesis of T-antigen (Huang et al., 1992). One of the
hypotheses proposed by these authors was the in situ generation of the disaccharide Galβ1-3GalNAcα-O-benzyl, which
would behave as an efficient inhibitor of the elongation of T
antigen. This hypothesis would be in agreement with the
decreased sialylation of HT-29 MTX mucins observed in our
study. Galβ1-3GalNAcα-O-benzyl is a potential substrate for
α-2,3-ST(O) but also for Galβ1-3GalNAc-R β-1,6-N-acetylglucosaminyltransferase (EC 2.4.1.102) and sulfotransferases.
Thus changes in the incorporation of GlcNAc and sulfate might
also be expected. Our findings indicate that incorporation of
GlcNAc and sulfate was unaffected, whereas sialic acid incorporation into mucin was almost completely inhibited. In fact,
using asialofetuin as substrate, we have demonstrated that the
ST activity in GalNAcα-O-benzyl-treated homogenates is
decreased and that Galβ1-3GalNAcα-O-benzyl is a powerful
competitive inhibitor of the ST activity of HT-29 MTX
homogenates. Among the different ST, only the Galβ13GalNAc α-2,3-ST can use Galβ1-3GalNAcα-O-benzyl as
substrate. These observations suggest that α-2,3-ST(O) is the
predominant ST expressed in HT-29 MTX cells, a finding that
would be in agreement with the fact that NeuAcα2-3Galβ13GalNAc-O is a major carbohydrate component of the mucins
synthesized by HT-29 MTX cells (unpublished observations)
as well as of the mucins from HT-29-derived cl. 16E (Capon
et al., 1992). The marked inhibition of sialic acid incorporation into mucins may be related to the striking inhibition of
mucin secretion in GalNAcα-O-benzyl-treated cells.
As mentioned above, GalNAcα-O-benzyl treatment did not
affect incorporation of GlcNAc or sulfate into mucins, sug-
1284 G. Huet and others
gesting that the subcellular compartment in which the enzymes
responsible for this transfer are located is different from the
compartment where α-2,3-ST(O) is present. This possibility
would be in agreement with the concept that O-glycosylation
proceeds in a stepwise manner according to the subcompartmentalization of the enzymes that participate in it (Egea et al.,
1993). In fact, sialylation and sulfation of the rat mammary
sialomucin have been reported to occur in separate compartments (Carraway and Hull, 1989). GalNAcα-O-benzyl may be
of use in dissecting these steps of mucin maturation. Further
studies may help to determine the influence of the compartmentalization of ST and sulfotransferases upon the post-translational processing of these molecules.
Taken together our findings indicate that sialylation and sulfatation of mucins and proteoglycans are important post-translational steps that may be modulated by the state of differentiation of HT-29-derived cell populations. Furthermore,
aryl-N-acetyl-α-galactosaminides can impair the action of α2,3-ST(O) in the O-glycosylation process of glycoproteins and
this effect may be useful in investigating the activity of this
enzyme and the biological effects of its products.
This work was supported in part by grant no. 2209 from the Association pour la Recherche sur le Cancer and grant 93/1228 from the
Fondo de Investigaciones Sanitarias (Madrid, Spain). The authors
thank Drs S. I. Hakomori and K. O. Lloyd for providing antibodies,
and Mrs E. Delaleau, M. T. Maillard and F. Roussez for typing the
manuscript.
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(Received 27 July 1994 - Accepted 15 November 1994)