From the Department of Medical Biochemistry, University of Göteborg, Medicinaregatan 9A, S-413 90 Gothenburg, Sweden
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ABSTRACT |
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Pulse-chase experiments in the colon cell line LS 174T combined with subcellular fractionation by sucrose density gradient centrifugation showed that the initial dimerization of the MUC2 apomucin started directly after translocation of the apomucin into the rough endoplasmic reticulum as detected by calnexin reactivity. As the mono- and dimers were chased, O-glycosylated MUC2 mono- and dimers were precipitated using an O-glycosylation-insensitive antiserum against the N-terminal domain of the MUC2 mucin. These O-glycosylated species were precipitated from the fractions that comigrated with the galactosyltransferase activity during the subcellular fractionation, indicating that not only MUC2 dimers but also a significant amount of monomers are transferred into the Golgi apparatus. Inhibition of N-glycosylation with tunicamycin treatment slowed down the rate of dimerization and introduced further oligomerization of the MUC2 apomucin in the endoplasmic reticulum. Results of two-dimensional gel electrophoresis demonstrated that these oligomers (putative tri- and tetramers) were stabilized by disulfide bonds. The non-N-glycosylated species of the MUC2 mucin were retained in the endoplasmic reticulum because no O-glycosylated species were precipitated after inhibition by tunicamycin. This suggests that N-glycans of MUC2 are necessary for the correct folding and dimerization of the MUC2 mucin.
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INTRODUCTION |
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The mucus layer on the epithelial surface of the mucous membrane is mainly made up of water and the gel-forming components, the mucus glycoproteins, or mucins, consisting of more than 50% O-linked oligosaccharides (1, 2). The peptide chain of mucins has domains with a high abundance of Ser, Thr, and Pro, usually in repetitive sequences (tandem repeats). The oligosaccharide chains are O-linked to Ser and Thr, thereby forming highly glycosylated domains or mucin domains.
The apoprotein of the human intestinal MUC2 mucin, which is fully sequenced, contains two mucin domains with large amounts of the amino acids Thr, Pro, and Ser (3, 4); the larger of these domains consists of well conserved 23-amino acid repeated sequences. The mucin domains are flanked by Cys-rich domains; one C-terminal, one N-terminal, and one central domain. The carboxyl and amino termini of the human MUC2 mucin and the blood coagulation factor, the von Willebrand factor (vWF),1 show sequence similarities in the positions of the cysteines. The vWF forms disulfide-bonded dimers between two C termini, and the N termini mediate further oligomerization (5). We have earlier shown that the human MUC2 apomucin forms dimers before being O-glycosylated (6). To study the initial assembly of the human MUC2 mucin in more detail, pulse-chase labeling and subcellular fractionation has been performed on LS 174T cells. An early dimerization was observed in the endoplasmic reticulum; there was no further oligomerization, and the dimerization was followed by O-glycosylation of the mono- and dimer in the Golgi apparatus. Tunicamycin treatment slowed down the dimerization rate, introduced formation of putative tri- and tetramers, and prevented transfer of the apomucin into the Golgi apparatus.
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MATERIALS AND METHODS |
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Antibodies--
A rabbit antiserum -MUC2TR (PH1900), against
a synthetic peptide based on the tandem repeat region of the human MUC2
apoprotein, was raised (6). The rabbit antiserum, PH1491 (later
referred to as
-MUC2N3), prepared against a synthetic peptide
(CPKDRPIYEEDLKK) based on amino acids 1167-1180 on the N terminus of
the human MUC2 apoprotein was prepared as follows. A New Zealand White
rabbit was immunized once with 500 µg of peptide conjugated to 400 µg of keyhole limpet hemocyanin in Freund's complete adjuvant and then twice with 250 µg of peptide conjugated to 200 µg of keyhole limpet hemocyanin in Freund's incomplete adjuvant.
Tissue Culture-- The colon adenocarcinoma cell line LS 174T (ATCC CL 188) was cultured as described (6).
Metabolic Labeling-- Cells were seeded at a concentration of about 50 × 106 cell/28-cm2 Petri dish the day before labeling. Cells were preincubated in methionine-free medium for 1 h followed by radiolabeling with 150 µCi 35S methionine (Redivue Promix [35S] labeling mix, Amersham Pharmacia Biotech)/dish. When cells were pulse-labeled for 2 min, 500 µCi of labeling mix/dish was added. In pulse-chase experiments cells were chased with culture medium supplemented by 15 µg of Met/ml of medium and 25 µg of Cys/ml of medium. Cells were washed and lysed as described (6), with the addition of 5 mM N-ethylmaleimide (NEM) in the lysis buffer. Inhibition of N-glycosylation was performed by incubating cells in 20 µg of tunicamycin (Calbiochem)/ml of methionine-free medium 1 h prior to labeling, as well as during pulse and chase.
Subcellular Fractionation--
Cells were washed twice with 5 ml
of 250 mM sucrose and twice in 5 ml 50 mM
sucrose. Cells were harvested in 500 µl of 50 mM sucrose
including protease inhibitors (110 µg/ml phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 60 µg/ml leupeptin, 3.8 µg/ml
calpain inhibitor I) using a cell scraper. The cell suspension was
homogenized with 15 strokes in a Dounce homogenizer using a tight
pestle, and the sucrose concentration was adjusted to 250 mM followed by an additional 5 homogenization strokes.
Washing and homogenization were performed on ice. The homogenized
suspension was centrifuged in a microtube at 4200 rpm and +4 °C for
10 min, and the supernatant was recovered. The cell pellet was washed
twice in 250 µl of 250 mM sucrose, and all supernatants
were pooled and carefully placed on a sucrose gradient in a centrifuge
tube containing a 4-ml gradient of 35-50% sucrose on top of a
400-µl 65% sucrose cushion. All sucrose solutions used contained 3 mM imidazole and were at pH 7.4. Ultracentrifugation was
performed 50,000 rpm at 12 °C for 3 h in a Beckman vertical
rotor (Vti, 65.2). Fractions were collected from the bottom.
Phosphate-buffered saline was added to a final volume of 1.2 ml, and
300 µl of lysate buffer (250 mM Tris-HCl, pH 7.4, 750 mM NaCl, 25 mM EDTA, 5% (v/v) Triton X-100),
including protease inhibitors (as above) and 5 mM NEM, was
added. Samples were sonicated three times for 2 s each (intensity
15) on a MSE Soniprep 100. The fractions collected were analyzed for
NADPH cytochrome c reductase activity and
galactosyltransferase activity (7, 8). Fractions were also analyzed by
12% SDS-PAGE (9) followed by Western blot, and calnexin was visualized
using a monoclonal -calnexin antibody (Transduction Laboratories,
Lexington, KY) and ECL (Amersham Pharmacia Biotech), followed by video
densitometry.
Immunoprecipitation--
Immunoprecipitation was performed as
described (6) using 25 µl of the -MUC2TR antisera, 40 µl of the
-MUC2N3 antiserum, or 1 µg of Helix pomatia followed by
10 µl of a rabbit
-H. pomatia antiserum (Serotec).
N-Glycosidase Digestion--
Immunoprecipitated samples were
washed and incubated in 100 µl of 0.2 M phosphate buffer,
pH 8.0, containing 1% SDS and 10 mM dithiothreitol for 5 min at 95 °C. Samples were centrifuged, and the SDS concentration in
the supernatant was adjusted to 0.1% with 0.2 M phosphate
buffer. N-Octylglycoside was added at an amount 10-fold
greater than the amount of SDS followed by the addition of 2 units of
PNGase F (Boehringer Mannheim). Samples were incubated at 37 °C for
20 h, and iodoacetamide was added at an amount 2.5-fold greater
than the amount of dithiothreitol, followed by incubation for 30 min in
the dark. Samples were then reimmunoprecipitated with the -MUC2TR
antisera as described above.
SDS-Agarose and Autoradiography--
Immunoprecipitated samples
were dissolved in nonreducing or reducing sample buffer at 95 °C for
5 min as described (6). Samples were analyzed on SDS-agarose gels using
0.8% stacking gel of SeaKem Gold (FMC) and a separation gel of 1.3%
Ultrapure agarose (Life Technologies, Inc.) and 1.3% Sea Plaque
agarose (FMC) or 2% Ultrapure agarose (Life Technologies, Inc.).
Electrophoresis was performed at 10 mA for 18 h using a
discontinuous buffer system (6, 9). Gels were fixed in 30% ethanol and
10% acetic acid, incubated in Amplify (Amersham Pharmacia Biotech),
dried, and exposed to film at 70 °C as described (6).
Two-dimensional gel electrophoresis was performed by analyzing the
first dimension under nonreducing conditions. The lane was cut out, and
the strip was incubated for 30 min in 0.5 M Tris-HCl, 0.4%
SDS, pH 6.8, containing 50 mM dithiothreitol and then
placed on top of another gel. The strip was overlaid with 0.8% (w/v)
agarose gel containing 10 mM dithiothreitol.
Rate Zonal Ultracentrifugation--
Metabolically labeled LS
174T cells were immunoprecipitated with the -MUC2TR antiserum, and
samples were dissolved in phosphate-buffered saline containing 2% SDS
at 95 °C for 5 min. Samples were layered on top of a 5-ml linear
10-40% (w/w) sucrose gradient containing 0.1% SDS.
Ultracentrifugation was performed at 50,000 rpm at 20 °C for 14 h in a Beckman swing-out rotor (SW 55 Ti). Fractions were collected
from the bottom of the tube and analyzed by 3-5% SDS-PAGE. McA-RH7777
cells expressing apoB-100 (512 kDa) and apoB-48 (246 kDa) or the
recombinant apoB-80 (410 kDa), apoB-72 (369 kDa), and apoB-53 (271 kDa)
were metabolically labeled, and the cell supernatants were collected,
immunoprecipitated with an
-apoB antiserum, subjected to rate zonal
ultracentrifugation as described above, and analyzed by 3-15% PAGE
(10, 11). The sedimentation coefficients for the MUC2 mono- and dimer
and apoB-variants were estimated according to the method of McEwen
(12).
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RESULTS |
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Sedimentation of the Non-O-glycosylated Mono- and Dimer by Rate
Zonal Ultracentrifugation--
By immunoprecipitation of LS 174T cells
using the -MUC2TR antiserum directed against the large tandem repeat
of the MUC2 mucin, we have shown that the human MUC2 apomucin forms a
disulfide-stabilized oligomer that is interpreted as a dimer (6). To
confirm that the oligomeric species was a dimer, sedimentation of
immunoprecipitated mono- and dimers were analyzed by rate zonal
ultracentrifugation. The gradient was recovered form the bottom of the
tube into 34 fractions, the sucrose concentration was measured, and
each fraction was analyzed by SDS-PAGE. The intensity of the mono- and
dimer bands was estimated by densitometry and plotted in Fig.
1. The monomer banded at 29% sucrose,
and the dimer banded at 31.5% sucrose, giving estimated sedimentation
coefficients of about 8.3 and 10.2 Svedberg units, respectively, using
the method of McEwen (12). The ratio of the sedimentation coefficients
for the dimer and monomer were estimated to just above 1.2. Analysis of
different apoB species with known masses by the same method showed a
sedimentation coefficient of 6.0 S for apoB-100 (512 kDa) and a ratio
of S values of about 1.2 between apoB-100 and apoB-48/53. That this
ratio is similar to the one for the two MUC2 species suggests that the MUC2 monomer forms a dimer and not a trimer or larger oligomeric species at the initial biosynthetic step.
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O-Glycosylated Monomers and Dimers of the Human MUC2 Mucin--
An
antiserum (-MUC2N3), raised against a peptide from the
non-O-glycosylation N terminus parts of MUC2, has now been
used to study the MUC2 biosynthesis also after initiation of
O-glycosylation. Immunoprecipitation of metabolically
labeled LS 174T cells using this antiserum followed by analysis with
agarose (2% Ultrapure, Life Technologies, Inc.) gel electrophoresis
under nonreducing conditions revealed two MUC2 bands with a lower
mobility than the non-O-glycosylated monomer (Fig.
2, M) and dimer
(D). Another antiserum reacting with
O-glycosylated MUC2 (
-MUC2C2), described in an
accompanying article (13), gave similar results to the
-MUC2N3
antiserum. Preincubation of the
-MUC2N3 antiserum with the peptide
used for immunization inhibited the precipitation of these two MUC2
species (results not shown). Bands migrating to the same positions were
also found when the N-acetylgalactosamine binding lectin
from H. pomatia was used for precipitation. That these bands
were not precipitated with the O-glycosylation-sensitive antiserum,
- MUC2TR, but rather with the H. pomatia
lectin suggested that the two MUC2 bands were
O-glycosylated. The upper and weaker of the two
-MUC2N3-precipitated bands disappeared upon reduction of the
disulfide bonds, whereas the lower band remained at the same position.
A probable explanation for the disappearance of the larger band upon
reduction was that the upper band was a disulfide-stabilized dimer of
the lower band. To further illustrate this, a two-dimensional gel
electrophoresis was performed analyzing the sample under nonreducing conditions in the first dimension and under reducing conditions in the
second (Fig. 2B). The upper band migrated to the same
position as the lower band after reduction, supporting the
interpretation that the upper band was a dimer of the lower. The bands
were diagonally oriented on the two-dimensional gel, reflecting a
nonreducible size polydispersity, probably due to
O-glycosylation heterogeneity.
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Biosynthesis of the MUC2 Apomucin as Studied by Subcellular Fractionation-- To determine the intracellular localization of the non-O-glycosylated and O-glycosylated MUC2 species, subcellular fractionation by sucrose density gradient centrifugation was performed on LS 174T cells. The gradient was recovered from the bottom in 14 fractions, which were analyzed for calnexin (marker for the rough endoplasmic reticulum), NADPH cytochrome c reductase activity (marker for the smooth endoplasmic reticulum), and galactosyltransferase activity (marker for the Golgi complex) (Fig. 3). The sucrose gradient was adjusted to obtain endoplasmic reticulum fractions that were depleted of galactosyltransferase activity. Fractions containing galactosyltransferase activity also contained NADPH cytochrome c reductase activity, suggesting an incomplete separation of the Golgi complex from the smooth endoplasmic reticulum.
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Dimerization of the Human MUC2 Apomucin in the Endoplasmic
Reticulum--
To study the intracellular localization of the MUC2
apomucin dimerization in more detail, LS 174T cells were pulse-labeled for 2 min and chased for 0, 10, and 20 min (Fig.
5, A, B, and C,
respectively), followed by sucrose density gradient centrifugation, under the conditions described in Fig. 3. The tubes were unloaded into
14 fractions, pooled in pairs, followed by immunoprecipitations using
the O-glycosylation-sensitive -MUC2TR antiserum and
analysis under nonreducing conditions by SDS-agarose gel
electrophoresis. After only 2 min of labeling, small amounts of the
dimer were observed in the heaviest parts of the gradient, containing
the markers for the endoplasmic reticulum. With time, the amount of dimer increased, and mono- and dimers were observed also in fractions with lower sucrose densities.
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Oligomerization of Non-N-glycosylated MUC2 Apomucins--
The
importance of N-glycans in dimerization and further
processing of the MUC2 mucin in LS 174T cells was studied. Cells were treated with increasing amounts of tunicamycin, and an optimal concentration completely inhibiting N-glycosylation (20 µg
of tunicamycin/ml of culture medium) was chosen for the subsequent experiments (results not shown). Metabolically labeled LS 174T cells
were treated with (Fig. 6A, lanes
2 and 4) and without (lanes 1 and
3) tunicamycin, followed by immunoprecipitation using the -MUC2TR antiserum. Two major and two minor bands were observed when
analyzing samples from tunicamycin-treated cells under nonreducing conditions (Fig. 6A, lane 2). When the sample
from tunicamycin-treated cells was analyzed under reducing conditions,
only one band was shown (lane 4), migrating as the lowest
band observed under nonreducing conditions (lane 2). The
reduced band migrated as the monomer treated with PNGase F, an enzyme
eliminating most of the N-glycans form the protein core
(lane 5). This also made it likely that the inhibition of
N-glycosylation by tunicamycin was complete. The lowest band
upon tunicamycin treatment (lane 2) was thus the non-N-glycosylated MUC2 monomer (Fig. 6A,
non-N-M), and the second smallest showed a corresponding
relative migration to the N-glycosylated dimer, suggesting
that it was the non-N-glycosylated dimer
(non-N-D).
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The Rate of Dimerization and O-Glycosylation in Tunicamycin-treated
Cells--
To follow the rate of dimerization, further
oligomerization, and O-glycosylation of the MUC2 apomucin in
normal and tunicamycin-treated cells, LS 174T cells were metabolically
labeled for 30 min followed by a chase for 0-5 h. Serial
immunoprecipitations were performed on the cell lysates starting with
the -MUC2TR antiserum (Fig. 7A), followed by precipitation
with the
-MUC2N3 antiserum (Fig. 7B) and analysis under
nonreducing conditions by SDS-agarose gel electrophoresis. In the
nontreated cells, the formation of N-glycosylated mono- and
dimers during the first hour was followed by a decrease in the amount
during the next hours. As the non-O-glycosylated mono- and
dimers of MUC2 started to disappear after about 3 h, the
-MUC2N3 antiserum started to precipitate O-glycosylated
MUC2 mono- and dimers. In tunicamycin-treated cells, formation of
non-N-glycosylated mono- and dimers was also observed, but
the rate was considerably slower than in the nontreated cells. With
increased chase times, also, further oligomerization of the
non-N-glycosylated MUC2 was observed. No
O-glycosylated bands were observed on the autoradiograms when immunoprecipitation was performed using the
-MUC2N3 antiserum. These results suggest that no non-N-glycosylated MUC2
apomucins were transported into the Golgi apparatus. Chase times up to
10 h were tested and did not show any O-glycosylated
species precipitated with the
-MUC2N3 antiserum (results not
shown).
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Tunicamycin-dependent Oligomerization in the
Endoplasmic Reticulum--
To determine the intracellular localization
of the non-N-glycosylated MUC2 oligomeric forms, subcellular
fractionation was performed (Fig. 8).
Tunicamycin-treated cells were labeled for 30 min and chased for 2 h, followed by subcellular fractionation according to Fig. 3. After
centrifugation, the tube was unloaded into 14 fractions, pooled in
pairs, followed by immunoprecipitation using the -MUC2TR antiserum
and analysis under nonreducing conditions by SDS-agarose gel
electrophoresis. Already, the heaviest endoplasmic reticulum fraction
revealed not only the non-N-glycosylated mono- and dimers,
but also the aggregates corresponding to putative trimers
(non-N-A1) and tetramers (non-N-A2). The
tunicamycin-dependent oligomerization thus occurs early in
the endoplasmic reticulum. The intensity of the four
non-N-glycosylated bands decreased and was very weak over
the fractions corresponding to the Golgi fractions, suggesting that
these were not transported to the Golgi apparatus. This was also shown
by the inability to precipitate any O-glycosylated homologues from these fractions by using the
-MUC2N3 antiserum (not
shown).
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DISCUSSION |
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In earlier studies on the biosynthesis of the human MUC2 apomucin in the colon carcinoma cell line LS 174T (6), we showed that the apomucin forms a non-O-glycosylated oligomer, as determined by two-dimensional gel electrophoresis. This oligomeric form was interpreted as a dimer based on the size estimated by SDS-PAGE. Sedimentation of the non-O-glycosylated mono- and dimer by rate zonal ultracentrifugation has now been performed on SDS-treated samples. The estimated sedimentation coefficients of the mono- and dimer were 8.3 and 10.2 S, respectively. These sedimentation coefficients cannot be compared with sedimentation coefficients of globular proteins and can thus only be used to compare the mass between the two species. Because the sedimentation coefficient of a particle is proportional to the mass, assuming a similar diffusion constant of the di- and monomer, the relationship between their sizes is estimated to be just above 1.2. The ratio of the S values for SDS-treated apoB-100 and apoB-48/52, having a relation in mass of 1:2, was also determined and found to be about 1.2, suggesting a similar ratio for a doubled mass for other proteins also. These results, together with the migration in SDS-PAGE and SDS-agarose gel electrophoresis, strongly suggest that the monomer of the human MUC2 mucin forms a dimer in the initial step of the biosynthesis.
To study the initial biosynthesis of the human MUC2 mucin in more detail, subcellular fractionation by sucrose gradient centrifugation on LS 174T cells has now been performed. Fractions containing the rough endoplasmic reticulum were well separated from the Golgi apparatus as judged from the presence of calnexin and the absence of galactosyltransferase activity (Fig. 3). On the other hand, less dense parts of the endoplasmic reticulum, containing NADPH cytochrome c reductase activity, were not completely separated from the fractions containing galactosyltransferase activity. Within the first minutes of biosynthesis, both the MUC2 mono- and dimers were observed in the endoplasmic reticulum. This showed that the dimerization occurs directly after translation of the apoprotein. Both the monomer and dimer of the MUC2 apomucin are found in fractions containing lighter parts of endoplasmic reticulum, proposing that the process of folding and dimerization might continue when MUC2 is moved within the reticulum.
Studies on the biosynthesis of the non-O-glycosylated mono-
and dimers of the MUC2 apomucin showed that the amounts of these had
their maximum 30 min to 1 h after labeling and that both declined simultaneously as the chase time increased. This is in accord with the
biosynthesis of the vWF, in which dimers are the only oligomers formed
before transport to the Golgi apparatus (14). To follow the MUC2
apomucin into the Golgi apparatus, an
O-glycosylation-insensitive antiserum (-MUC2N3) against
the N-terminal parts of the MUC2 mucin protein core was used. As the
amount of mono- and dimers decreased, larger molecules were
precipitated with the
-MUC2N3 antiserum. These two species were
interpreted as O-glycosylated and found in the subcellular
fractions containing markers for the Golgi apparatus. The
-MUC2TR
antiserum was not able to precipitate these species, probably due to
hiding of the epitopes by the added O-glycans. H. pomatia, however, precipitated species migrating to the same
position on the gel. The two O-glycosylated MUC2 species were interpreted as mono- and dimers because they appeared when the
non-O-glycosylated mono- and dimer started to disappear in the endoplasmic reticulum. It was also verified by two-dimensional gel
electrophoresis that both O-glycosylated species migrate to the same positions under reducing conditions (Fig. 2B). The
monomeric band seems upon reduction to divide into two adjacent bands
(Fig. 2A, lane 1), a phenomenon that has not yet been
explained but that is probably due to glycosylation heterogeneity. The
tiny band under the O-glycosylated monomer (lane
1) is the non-O-glycosylated monomer, with an identical
position on the gel as to the position of this species precipitated by
-MUC2TR.
The O-glycosylated monomers and dimers appeared at the same time, indicating that monomers and dimers from the endoplasmic reticulum are O-glycosylated in parallel. The presence of an O-glycosylated monomer in the Golgi apparatus is surprising. The only known function of the MUC2 mucin, gel formation, is thought to require long polymers, the precursors of which, according to the von Willebrand factor analogy, should be dimers. It is, of course, possible that dimers are formed from monomers both in the endoplasmic reticulum and in the Golgi apparatus, but the retaining of labeled glycosylated monomers in the cells at a substantial amount 3 days after labeling (13) contradicts the idea that such a process is of great importance. This fact also suggested the possibility that the O-glycosylated monomers in the Golgi apparatus were not destined for degradation. It might be, instead, that single monomers comprise a specific physiological function different from forming mucus. Another possibility could be that single monomers in the Golgi apparatus/secretory vesicles should terminate polymer elongation and that the ratio monomer/dimer released from the endoplasmic reticulum could determine the final polymer length.
A comparison of the amount of non-O-glycosylated mono- and
dimer being transferred from the endoplasmic reticulum to the Golgi apparatus is difficult to make because the -MUC2N3 antiserum is less
efficient in precipitating MUC2 compared with the
-MUC2TR antiserum.
This means that these immunoprecipitations could not be used to
determine the total amount of MUC2 apomucin transported into the Golgi
system. However, further studies of the MUC2 apomucin assembly show the
formation of larger and more complex forms of the
O-glycosylated MUC2 apomucins in the Golgi apparatus (13). The relatively low intensity of the bands representing the
O-glycosylated forms of the MUC2 apomucin precipitated by
the
-MUC2N3 antiserum could then not only depend on this antiserum
but also on the transformation of O-glycosylated species
into larger, insoluble MUC2 species not found using the present
approach.
By treating LS 174T cells with tunicamycin, the importance of addition
of N-glycans to the growing MUC2 apomucin was analyzed. By
the mobility shift observed on the gel between the apomucins precipitated from tunicamycin-treated and nontreated cells (Fig. 6A), it was evident that the N-glycosylation of
the MUC2 apomucin was inhibited. Studies on the biosynthesis of the
human MUC2 apomucin after treatment with tunicamycin have been reported
earlier (15, 16), but none of them showed any difference in mobility
between the N-glycosylated and the
non-N-glycosylated MUC2 apomucins. Despite the lack of
N-glycans, the human MUC2 apomucin formed dimers in the
endoplasmic reticulum. Pulse-chase experiments showed that this
dimerization was delayed compared with dimerization in the nontreated
cells. The lower rate of dimerization in tunicamycin-treated cells
could be due to misfolding of the protein, thus allowing for the
formation of incorrect disulfide bonds. As observed in Fig.
6A, large amounts of non-N-glycosylated MUC2
mono- and dimers still appear after a 5-h chase as compared with a
rapid decrease of mono- and dimer after a 2-h chase in nontreated
cells. The human non-N-glycosylated MUC2 apomucins were not
transported out of the endoplasmic reticulum because no
O-glycosylated MUC2 mono- and dimers were found in
tunicamycin-treated cells, proposing the idea that the mono- and dimers
formed were not correctly folded and trapped in the endoplasmic
reticulum control machinery. In a pulse-chase experiment with a 10-h
chase, the amount of mono- and dimers was lower than it was after a 5-h
chase, but still no MUC2 species were precipitated with the -MUC2N3
antiserum (results not shown). These observations suggest that
non-N-glycosylated MUC2 apomucins slowly degraded in the
endoplasmic reticulum and that they were never transported to the
Golgi apparatus.
The similarities in the cysteine position in the C-terminal of the vWF with several mucins, including the human MUC2 and the porcine submaxillary mucin, suggests similarities in the dimer formation of these proteins. Expression of the C-terminal domain of the porcine submaxillary mucin in COS cells (17) proposed that the dimerization of the C-terminal domain occurred irrespective of N-glycosylation inhibition. The inhibition of N-glycan formation did not inhibit further transport of the porcine submaxillary mucin dimers into the Golgi apparatus. Inhibition of N-glycosylation of the human vWF in human umbilical vascular endothelial cells inhibited dimerization and further transport into the Golgi complex (18). The different biosynthetic patterns for the non-N-glycosylated human MUC2 apomucin, the vWF, and the porcine submaxillary mucin could be the result of studies performed in different cell lines. However, different mechanisms could be involved in controlling the folding and the transport of the different proteins through the cell, despite similarities in the localization of the disulfide bonds. Alignment of the C-terminal amino acid sequences of the MUC2 apomucin, the porcine submaxillary mucin, and the vWF according to the positions of the cysteine domains found in the vWF showed no similarity in the possible N-glycosylation sites of the far C-terminal end postulated to be involved in dimerization. Some similarity in the possible N-glycosylation sites was observed upstream of the C-terminal domain but never involving all three sequences. This suggests that inhibition of N-glycans could have different effects during the biosynthesis of the three proteins.
Not only dimers, but also two fainter bands larger in size than the
dimers, were observed in the endoplasmic reticulum when tunicamycin-treated cells were precipitated using the -MUC2TR antiserum. These two faint bands were interpreted as disulfide bond-stabilized putative tri- and tetramers of the
non-N-glycosylated MUC2 apomucin as seen by studies done
with two-dimensional gel electrophoresis (Fig. 6B). In the
first dimension, the mono- and dimers and the two oligomeric bands were
separated form each other under nonreducing conditions. In the second
dimension, under reducing conditions, all bands migrated to the same
position. Because the lysis buffer contained NEM, inhibiting in
vitro disulfide bond formation, the disulfide-stabilized oligomers
must be formed in the cells. The bands migrating as putative tri- and
tetramers could also be interpreted as the
non-N-glycosylated mono- and dimers destined for degradation
and covered with chaperones causing the apparent larger size. This is
unlikely, however, because the same large amount of chaperones must
then be bound. Also, the interaction between chaperones and proteins is
not known to be resistant to boiling in 5% SDS. Two faint bands larger
in size than the dimer could also be shown without tunicamycin
treatment, but in much smaller amounts than in treated cells because
these could be observed only after weeks of exposing the gel to x-ray film.
Comparing the biosynthesis of the human MUC2 mucin with studies on other mucins would indicated if there is a common biosynthetic pathway for all secreting mucins. The biosynthetic studies on rat gastric mucins by Dekker et al. (19, 20) showed the formation of not only dimers but also trimers and possibly tetramers before O-glycosylation of the apomucin. Studies on human gastric mucin and human gallbladder mucin by Klomp et al. (21, 22) showed that both apomucins form only one oligomer species in the endoplasmic reticulum, interpreted as a tri- or tetramer based on the size determined by SDS-PAGE. Our finding of glycosylated monomers is also in contrast to the biosynthesis of oligomeric mucins, as described for the rat gastric mucin (19, 20). Oligomerization was interpreted as a prerequisite for the transport of this rat gastric mucin into the Golgi apparatus. However, for the human MUC2 mucin, the N-glycans are interpreted as required for the correct folding of MUC2 monomer and dimer, and only these are allowed to pass the endoplasmic reticulum control machinery. The finding of Perez-Vilar et al. (17) of the transport of a recombinant truncated C terminus of the porcine submaxillary mucin through the whole secretory machinery of COS cells could propose another biosynthetic pathway or cell type specific control processes for this mucin compared with others. One may then speculate that there is not one common biosynthetic pathway for all gel-forming mucins, but instead biosynthetic differences between different mucins, as well as between mucin-producing cells.
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FOOTNOTES |
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* This work was supported by Swedish Medical Research Council Grants 7142, 7461, and 10443 and the IngaBritt and Arne Lundbergs Stiftelse.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.
To whom correspondence should be addressed. Tel.: 46-31-7733488;
Fax: 46-31-416108; E-mail: gunnar.hansson{at}medkem.gu.se.
1 The abbreviations used are: vWF, von Willebrand factor; NEM, N-ethylmaleimide; PAGE, polyacrylamide gel electrophoresis.
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