Department of Medical Biochemistry, University of Göteborg, Medicinaregatan 9, 413 90 Gothenburg, Sweden
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ABSTRACT |
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The MUC2 mucin is the major gel-forming mucin in the small and large intestine. Due to its sequence similarities with the von Willebrand factor, it has been suggested to dimerize in the endoplasmic reticulum and polymerize in the trans-Golgi network. Using an O-glycosylation-sensitive MUC2 antiserum, a dimerization has been shown to occur in the endoplasmic reticulum of LS 174T cells (Asker, N., Axelsson, M. A. B., Olofsson, S.-O., and Hansson, G. C. (1998) J. Biol. Chem. 273, 18857-18863). Using an antiserum immunoprecipitating O-glycosylated MUC2 mucin, monomers and dimers were shown to occur in soluble form in the lysate of LS 174T cells. The amount of O-glycosylated dimer was small, and no larger species were found even after long chase periods. However, most of the labeled MUC2 mucin was found in pelleted debris of the cell lysate. This insoluble MUC2 mucin was recovered by immunoprecipitation after reduction of disulfide bonds. Analysis by agarose gel electrophoresis revealed two bands, of which the smaller migrated as the O-glycosylated monomer and the larger migrated as the O-glycosylated dimer of the cell lysis supernatant. Mucins insoluble in 6 M guanidinium chloride could also be obtained from LS 174T cells. Such mucins have earlier been found in the small intestine (Carlstedt, I., Herrmann, A., Karlsson, H., Sheehan, J., Fransson, L.-Å., and Hansson, G. C. (1993) J. Biol. Chem. 268, 18771-18781). Reduction of the mucins followed by purification by isopycnic density gradient ultracentrifugation and analysis by agarose gel electrophoresis revealed two bands reacting with an anti-MUC2 tandem repeat antibody after deglycosylation. These bands migrated identically to the bands shown by metabolic labeling, and they could also be separated by rate zonal ultracentrifugation. These results suggest that the MUC2 mucin is forming nonreducible intermolecular bonds early in biosynthesis, but after initial O-glycosylation.
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INTRODUCTION |
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The mucosal surfaces comprise a 1000-fold larger interface between the external and internal milieu than the skin. At the same time, the chemical and microbiological challenges are more demanding. The most important protective component of the mucosal surfaces is the mucus layer, the gel properties of which are due to macromolecules called mucins. A majority of the mucins known today belong to this classical gel-forming type, although a few glycoproteins defined as mucins are membrane-bound with yet unknown physiological functions (1). The present definition of mucin includes all glycoproteins that consist of more than 50% O-linked oligosaccharides and that have a majority of these oligosaccharides localized to mucin domains. These domains have a high number of O-glycosylated Ser and Thr amino acids, often appearing in tandem repeat sequences. Gel-forming mucins are probably altered in several diseases. Thus, alterations in the mucus barrier are probably essential in the pathogenesis of, for example, infections, peptic ulcers, and inflammatory bowel disease. Diseases such as cystic fibrosis and chronic bronchitis, and also trivial infections, are characterized by increased mucus viscosity. Despite its medical interest, the biochemical nature of these altered mucin properties is still poorly understood, largely due to the difficulties associated with the large size of these molecules.
The gel-forming mucins are proposed to be disulfide bond-stabilized
linear polymers of highly glycosylated proteins (2), although other
models are discussed (3). A typical example of such a mucin is encoded
by the MUC2 gene, one of the few mucin genes fully sequenced
(4). The MUC2 mucin occurs in small and large intestine (5, 6) and
probably also in the airways upon epithelial stress, such as infection
(7). The primary translation product, the mucin apoprotein, has a size
of about 600 kDa, including N-glycans. It is composed of
five major regions; three of these, one N-terminal, one C-terminal, and
one central, are rich in Cys, whereas the two others are rich in Thr,
Ser, and Pro. The two latter regions are the mucin domains, also called tandem repeat domains, and they become heavily
O-glycosylated in the Golgi apparatus, thus obtaining
extreme proteolytic resistance. The Cys rich N- and C-terminal regions
show large similarities in the Cys positions to the corresponding parts
of the von Willebrand factor (4, 8). This protein is known to form
dimers stabilized by C-terminal disulfide bonds (9). The dimerization
of the von Willebrand factor takes place in the endoplasmic reticulum, whereas a further N-terminal oligomerization occurs in the late Golgi
compartments because it requires low pH (9). Due to the sequence
similarities, a similar assembly procedure has been proposed for the
MUC2 mucin. Using an antiserum (-MUC2TR) directed against protein
epitopes in the MUC2 tandem repeat, and thus only immunoprecipitating non-O-glycosylated MUC2 mucin species, we have previously
shown that non-O-glycosylated MUC2 dimers are formed in LS
174T cells (10). Because O-glycosylation starts in the
cis-Golgi or earlier, this indicates an endoplasmic
reticulum-based dimerization in analogy with the von Willebrand factor.
This assumption has now been verified by subcellular fractionation
(11). By the help of an antiserum directed against protein epitopes
unaffected by O-glycosylation, the monomer and dimer were
identified in O-glycosylated form. However, the amount of
especially O-glycosylated dimers was small, suggesting that
these disappeared somewhere. The dimers were found to form insoluble
MUC2 mucin occurring in cell debris pelleted from cell lysate. The
oligomeric stage of this insoluble MUC2 mucin could not be analyzed, as
reduction was necessary for solubilization. When analyzing the reduced
insoluble MUC2 mucin, not only the monomeric forms expected from the
von Willebrand factor analogy were found. Up to half the amount of
monomers were linked together with nonreducible bonds and migrated as
O-glycosylated dimers on agarose gels.
Mucins insoluble in chaotropic solutions, such as 6 M guanidinium chloride, have earlier been reported in the small intestine (12, 13), and in the gall bladder bile of some patients (14). Here we report that the cell line LS 174T can also form mucins insoluble in 6 M guanidinium chloride. These were found to contain the MUC2 mucin with nonreducible bonds, migrating similarly to the metabolically labeled insoluble MUC2 mucin from cell debris pellets.
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MATERIALS AND METHODS |
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Antibodies and Cell Lines--
The polyclonal sera -MUC2TR
and
-MUC2N3 have been described (10, 11). An antiserum called
-MUC2C2 was raised in a rabbit against a synthetic peptide,
CIIKRPDNQHVILKPGDFK, based on amino acids 4995-5013 on the C terminus
of the human MUC2 apoprotein (4). A New Zealand White rabbit was
immunized once with 500 µg of peptide conjugated via Cys to 400 µg
of keyhole limpet hemocyanin in Freund's complete adjuvant, and twice
with 250 µg of peptide conjugated to 200 µg of keyhole limpet
hemocyanin in Freund's incomplete adjuvant. The interval between the
immunizations was 2 weeks. The specificity of the
-MUC2C2 antiserum
was tested as described for the
-MUC2N3 antiserum (11) using 1 mg of
immunizing peptide/25 µl of antiserum for inhibiting
immunoprecipitation. The colon adenocarcinoma cell line LS 174T (ATCC
CL 188), producing MUC2 mucin, was cultivated as described before
(10).
Pulse-Chase, Immunoprecipitation, and Electrophoresis
Procedures--
Newly confluent LS 174T cells were starved for 1 h in 2 ml of methionine-free minimum essential medium (Life
Technologies, Inc.) with 10% fetal bovine serum and 2 mM
glutamine per 27-cm2 Petri dish and metabolically labeled
using 150 µCi of [35S]methionine (Redivue Promix
[35S] Cell Labeling Mix, Amersham Pharmacia Biotech) per
Petri dish. Brefeldin A (Epicentre Technologies), 10 µg (solubilized
in 5 µl of ethanol) per ml of medium, was added 15 min before
metabolic labeling. Ammonium chloride (25 mM) was added at
the start of starvation. In pulse-chase experiments, the cells were
chased with culture medium, and when chase time exceeded 1 day, the
medium was replaced daily. The cells were washed twice in cold
PBS1 (137 mM
NaCl, 2.7 mM KCl, 1.8 mM
KH2PO4, 10.1 mM
Na2HPO4) before lysis on ice by the help of a
cell scraper in 1 ml (per Petri dish) of PBS with 50 mM
Tris-HCl, pH 7.4, 5 mM EDTA, 1% Triton X-100, 5 mM N-ethylmaleimide, 0.5 mM
phenylmethylsulfonyl fluoride (Sigma), 20 µg/ml aprotinin (Trasylol,
Bayer), 60 µg/ml leupeptin (Sigma), 3.8 µg/ml calpain inhibitor I
(Boehringer Mannheim), 0.7 µg/ml pepstatin (Boehringer Mannheim),
0.02% sodium azide. The lysate was sonicated three times for 2 s
each (intensity 15, MSE Soniprep 100) and clarified by centrifugation.
The cell debris pellet thus obtained was washed several times in PBS,
and its mucin content was solubilized by reduction in 200 µl of PBS,
100 mM Tris-HCl, pH 8, 50 mM dithiothreitol
(Merck) for 1 h at 37 °C under agitation. Material not
solubilized by the reduction was pelleted by centrifugation and
discarded, and the supernatant was incubated with 150 mM
iodoacetamide (Sigma) for 30 min at room temperature under agitation in
the dark, and then diluted with PBS to 1 ml. This solution and the
clarified cell lysate were then handled in similar ways. They were
incubated with 25 µl of preimmune rabbit serum for 30 min followed by
30 min with 300 µl of 10% (v/v) immunoprecipitin (Life Technologies,
Inc.). After pelleting the immunoprecipitin by centrifugation, the
supernatant was incubated under agitation for 2 h at room
temperature or overnight at 4 °C with 50 µl of -MUC2N3, 50 µl
of
-MUC2C2, or 25 µl of
-MUC2TR. 300 µl of immunoprecipitin
was then added before further incubation for 30 min. The
immunoprecipitates were pelleted by centrifugation and washed four
times in washing solution (10 mM Tris-HCl, pH 7.4, 2 mM EDTA, 0.1% Triton X-100, 0.1% SDS). The samples were
released from the immunoprecipitin for 5 min at 95 °C in 50 mM Tris-HCl, pH 6.8, 20% glycerol, 5% SDS and, if
reducing conditions denoted, also 5%
-mercaptoethanol and 10 mM dithiothreitol. The immunoprecipitin was pelleted, and
bromphenol blue (0.015%) was added to the supernatant prior to
electrophoresis. Agarose gel electrophoresis (1.5 mm thick) was run on
a vertical gel apparatus (140 × 160-mm gels, Hoefer) casted on
agarose gel support medium (Gel Bond Film, FMC). The separation gel was
1% Ultrapure (Life Technologies, Inc.) and 1% Sea Plaque low gelling
temperature (FMC) or 2% Ultrapure (Life Technologies, Inc.). The
buffer contained 0.378 M Tris-HCl, pH 8.8, and 0.1% SDS.
The stacking gel was made of 0.8% agarose (SeaKem Gold, FMC) in 0.126 M Tris-HCl, pH 6.8, and 0.1% SDS. The electrode buffer at
the cathode was 0.05 M Tris, 0.384 M glycine,
and 0.1% SDS, and at the anode 0.025 M Tris, 0.192 M glycine, and 0.05% SDS. The current for electrophoresis was about 10 mA. Gels were fixed for 3 h in 30% ethanol and 10% acetic acid, soaked in Amplify fluorographic reagent (Amersham Pharmacia Biotech) with 5% glycerol for 30 min, dried on slab gel
dryer for 2 h at 50 °C, and exposed at
80 °C to a Biomax MS film (Kodak).
Purification of Insoluble Mucins Followed by Western
Blot--
LS 174T cells were cultured in roller bottles for 10 days
with daily medium changes and washed twice in cold PBS. The
purification was performed as described before (13). In short, the
attached cells were extracted with guanidinium chloride (6 M guanidinium chloride, 5 mM EDTA, 10 mM NaH2PO4, pH 6.5, 5 mM N-ethylmaleimide, and 0.5 mM
phenylmethylsulfonyl fluoride), homogenized in a Dounce homogenizer
(loose pestle), and incubated for 1 h at room temperature under
agitation. Insoluble material was pelleted by centrifugation for 25 min
at 40,000 × g, and the pellet was washed and
centrifuged six times in similar guanidinium chloride buffer. The
pellets were brought into solution by reduction of disulfide bonds in reduction guanidinium chloride (6 M guanidinium chloride,
100 mM Tris, 5 mM EDTA, 10 mM
freshly added dithiothreitol, pH 8.0) for 5 h at 37 °C under
gentle agitation. Cysteine groups were alkylated by the addition of 150 mM iodoacetamide (powder) followed by incubation overnight
at room temperature under agitation in the dark. The mucins were then
purified by three rounds of isopycnic density gradient
ultracentrifugation (15), two with 4 M guanidinium chloride
and one with 0.2 M guanidinium chloride. After unloading the gradient into fractions, the mucin peaks were identified by periodic acid-Schiff slot blot, dialyzed against water, lyophilized, redissolved, and boiled for 5 min at 95 °C in 50 mM
Tris-HCl, pH 6.8, 20% glycerol, and 5% SDS. After electrophoresis
performed as above (without Gel Bond Film and without stacking gel),
the proteins were electrophoretically transferred (1 mA/cm2
for 3 h at +4 °C, Sartoblot II-S) to a nylon blotting membrane (Immobilon-P, Millipore) using 48 mM Tris, 39 mM glycine, 0.0375% SDS, and 10% methanol (pH 8.3) as
transfer buffer. The membrane was washed several times in water after
blotting and dried before deglycosylation with gaseous hydrogen
fluoride at room temperature overnight (16). The membrane was then
blocked in saturated casein (Sigma) in 150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 0.1% Tween 20 for 1 h at room
temperature and stained with 1% -MUC2TR antiserum for 1 h at
room temperature, followed by incubation for 1 h at room
temperature with peroxidase-conjugated anti-rabbit antibody (DAKO)
diluted 1:1000. Both antibody incubations were in 10% of saturated
casein in 150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 0.01% Tween 20. The assay was developed by the ECL reagent (Amersham Pharmacia Biotech), according to the manufacturers recommendations and
using Biomax MS film (Kodak).
Rate Zonal Ultracentrifugation-- MUC2 mucin insoluble in 6 M guanidinium chloride was purified as described above, lyophilized, and redissolved in water (2 µg/µl). 20 µl of this solution was layered on top of a linear 22-43% (w/w) sucrose gradient in 150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 0.02% NaN3, with a volume of 5 ml in a 13 × 51-mm ultracentrifuge tube (Beckman). The ultracentrifugation was performed in a Beckman swinging bucket rotor (SW55Ti) for 15 h at 40,000 rpm at +5 °C. Fractions (200 µl) were recovered from the top, and the water was evaporated in a vacuum centrifuge (Heto, Allerød, Denmark). The material was dissolved and boiled in 50 mM Tris-HCl, pH 6.8, 20% glycerol, 5% SDS and subjected to SDS-agarose gel electrophoresis, Western blotting, hydrogen fluoride treatment, and assay as described above. The intensity of the bands obtained on the film was measured by video densitometry using a video camera from KAPPA Messtechnik (Gleichen, Germany) and software from Bildanalys (Stockholm, Sweden).
Subcellular Fractionation-- Subcellular fractionation was performed as described elsewhere (11). An alternative homogenization technique was also used. Cells were resuspended in ice-cold 130 mM KCl, 25 mM Tris-HCl, pH 7.5, and protease inhibitors (11) and homogenized by passing through syringe needles (5 × 22 G/0.7 mm, 5 × 25 G/0.5 mm, 3 × 27 G/0.4 mm).
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RESULTS |
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Migration of Non-O-glycosylated and O-Glycosylated MUC2 Species on
SDS-Agarose Gel Electrophoresis--
We have previously shown that the
-MUC2TR antiserum precipitates only non-O-glycosylated
MUC2 species (10), whereas the
-MUC2N3 antiserum also precipitates
species migrating slower on SDS-agarose gels. These were regarded as
O-glycosylated also because they occurred in the Golgi
apparatus but not in the endoplasmic reticulum, of LS 174T cells (11).
In order to further relate these different bands on SDS-agarose gel
electrophoresis to each other, partially O-glycosylated MUC2
molecules, with intermediate sizes between
non-O-glycosylated and O-glycosylated species,
were obtained by Brefeldin A treatment of the LS174T cells and analyzed by SDS-agarose gel electrophoresis. An additional antiserum, supposed to immunoprecipitate both non-O-glycosylated and
O-glycosylated MUC2, called
-MUC2C2, was also used to
further verify that the different bands were indeed the MUC2 mucin.
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Larger MUC2 Species than O-Glycosylated Dimer Are Insoluble and
Contain Nonreducible Intermolecular Bonds--
No MUC2 species larger
than O-glycosylated dimer were observed on the agarose gels
irrespective of the labeling and chase times used. To find out where
the predicted larger forms of MUC2 were lost, the cell debris pelleted
from the metabolically labeled cells, lysed with detergent and
ultrasonicated, was reduced by dithiothreitol. The obtained solubilized
material was immunoprecipitated with the -MUC2N3 antiserum, followed
by agarose gel electrophoresis (Fig.
2C). Two major MUC2 bands were
found, of which the larger showed a partial separation into two. As a
comparison, nonreduced MUC2 immunoprecipitated in the normal way from
the clarified cell lysate is shown (Fig. 2B). This contains
O-glycosylated monomer (O-M) and
O-glycosylated dimer (O-D). A corresponding
reduced lane was also included to demonstrate that the dimer
(O-D) is reducible disappearing upon thiol reduction (Fig.
2A). The monomer (O-M) and dimer (O-D)
correspond in size to the two main bands of the reduced insoluble
portion. It seems therefore reasonable to believe that the upper band
from the insoluble portion (Fig. 2C, O-X)
consists of two monomers held together with bonds that are nonreducible
by mercaptoethanol and dithiothreitol. This nonreducible material
(O-X) is different from the in vivo-occurring
reducible dimer (O-D) and will therefore be referred to as
X-dimer in this paper. Bands migrating to the same positions as monomer
and X-dimer could also be precipitated from the debris pellets by the
-MUC2C2 antiserum or by the H. pomatia lectin (not
shown). No material at all in the reduced debris pellets could be
precipitated using the
-MUC2TR antiserum (not shown). This shows
that non-O-glycosylated MUC2 species are soluble and not
incorporated into insoluble material. It should also be noted that no
signs of nonreducible bonds have ever been observed in
non-O-glycosylated MUC2 species (10, 11).
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MUC2 Mucin, Which Was Insoluble in 6 M Guanidinium
Chloride, Also Contained Nonreducible Bonds--
Mucins insoluble in 6 M guanidinium chloride have earlier been reported from
small intestine (12, 13), where the major part in the rat has been
shown to be due to Muc2 (6, 8). Cultivated LS 174T cells were extracted
with 6 M guanidinium chloride, and the insoluble mucin
complexes were pelleted by centrifugation and washed six times. The
material was solubilized by dithiothreitol reduction and purified by
three rounds of isopycnic density gradient ultracentrifugation in
guanidinium chloride (15). The periodic acid-Schiff slot blot intensity
of the fractions from the third preparative ultracentrifugation step,
in 0.5 M guanidinium chloride, is shown in Fig.
2E, having a peak within the density interval between 1.53 and 1.49 g/ml, as expected for mucins (15). The material from these
fractions was pooled and analyzed by SDS-agarose gel electrophoresis to
compare the electrophoresis pattern to that obtained by
immunoprecipitation. The mucins were Western blotted onto nylon
membranes and deglycosylated by gaseous hydrogen fluoride before
staining with the tandem repeat antiserum -MUC2TR. Two bands were
found, migrating to the same positions on the gel as monomer and
X-dimer, respectively (Fig. 2D). No staining at all was
found with
-MUC2TR on a non-deglycosylated control membrane (not
shown). Because this antiserum does not bind O-glycosylated MUC2, this finding is further evidence that the species were
O-glycosylated. This procedure also confirms that the bands
at monomer and X-dimer position on the gels are indeed the MUC2 mucin,
because the deglycosylation exposed the epitopes detected by the
-MUC2TR antiserum.
Separation of Monomer and X-dimer by Rate Zonal
Ultracentrifugation--
As an alternative molecular size separation
method to SDS-agarose gel electrophoresis, not dependent on electrical
charge, the monomer and X-dimer were also subjected to rate zonal
ultracentrifugation. MUC2 mucin insoluble in 6 M
guanidinium chloride solubilized by reduction was layered on top of a
sucrose gradient, which, after the centrifugation, was recovered as 25 fractions run on agarose gel and assayed by Western blot (Fig.
3A). The intensity of the obtained monomer and X-dimer bands in every fraction was measured by
video densitometry (Fig. 3B). As expected for
heterogeneously glycosylated molecules, both monomer and X-dimer showed
a broad distribution over the gradient. Some of the material migrated to the bottom of the ultracentrifuge tube, probably due to aggregation, but not by covalent linkages because it could be dissolved by boiling
in SDS prior to electrophoresis. Such aggregation might also have
contributed to the broad distribution over the gradient. Both species,
however, showed fairly distinct intensity maxima, probably representing
similar glycosylation stages of nonaggregated molecules. These maxima
were localized at 29.25% sucrose for the monomer and 31.5% sucrose
for the X-dimer, giving sedimentation values of 14 S and 19 S,
respectively, as calculated by the method of McEwen (18). As shown in
Fig. 2E, both species had the same density, about 1.51 g/ml,
as expected for O-glycosylated mucins (15). If the diffusion
constant of the O-glycosylated MUC2 monomer is similar to
that of monomer units from human cervical mucins in water (19),
4.7 × 108 cm2/s, our results would give
a mass of the MUC2 monomer of about 2.7 × 106. The
diffusion constant of the X-dimer is not known, making a calculation of
its absolute mass impossible. However, together with the SDS-agarose
gel electrophoresis results, the rate zonal ultracentrifugation makes
it likely that the X-dimer indeed is a dimer. It also shows that the
separation observed on the agarose gels is due to size differences and
not, for example, charge.
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Pulse-Chase Analysis of the Appearance of Soluble and Insoluble
MUC2 Mucin--
In order to study the appearance of
non-O-glycosylated and O-glycosylated soluble
MUC2 species, immunoprecipitation with the -MUC2N3 antiserum (Fig.
4A) serially followed by
immunoprecipitation with the
-MUC2TR antiserum (Fig. 4B)
was performed on the soluble parts of the lysates from LS 174T cells
that had been pulse-chased for up to 3 days. The
O-glycosylated molecules appeared after about 1 h and
reached maximum radiolabeling after about 2 h (Fig. 4A). The amounts of both species then quickly decreased to
levels that still remained constant in the cells after three days. As shown previously (10, 11), the non-O-glycosylated precursors appeared within 0.5 h and showed maximum intensity after 1 h, as is also shown in Fig. 4B. The
non-O-glycosylated monomer decreased to low levels after
only a few hours, whereas the dimer, formed from the monomer, was still
present in reasonable amounts after 24 h.
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Subcellular Fractionation-- In an attempt to localize the cellular location for the formation of the insoluble MUC2 molecules, subcellular fractionation was performed, using two different types of homogenization. However, all of the insoluble material after homogenization was recovered in the pellet obtained by centrifugation at 1,400 × g prior to subcellular fractionation (not shown). No nonreducible X-dimers were found in the subcellular fractions. This means that the appearance of insoluble MUC2 could not be studied by subcellular fractionation.
Nonreducible Bond Formation Does Not Require Low pH-- The N terminus of the MUC2 mucin shows similarities in the localizations of the Cys residues with the von Willebrand factor. Because the von Willebrand factor dimers are known to form polymers stabilized by N-terminal disulfide bonds by a process that requires an acidic pH (9), a similar mechanism has been proposed for the MUC2 mucin. No disulfide-stabilized MUC2 species larger than dimers were detected in this study. To study whether a lowered pH could influence the formation of the nonreducible bonds in MUC2, LS 174T cells were cultured in the presence of ammonium chloride. This neutralizes the acidic pH in the distal Golgi apparatus and secretory vesicles (20). The ratio between monomer and X-dimer was constant after ammonia treatment (Fig. 5B), although the amount of both species was decreased, which we have noticed before.2 This indicates that the formation of the nonreducible bonds in MUC2 is not pH-sensitive.
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DISCUSSION |
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The striking similarities in the localizations of the Cys residues
between the N and C termini of MUC2 and the von Willebrand factor made
it likely that the assembly followed similar pathways. The present
results show that this is not the case, and instead an insoluble form
of MUC2 is formed early via intermolecular bonding that cannot be
reduced. The present results are dependent on the specificity of the
anti-MUC2 antisera available. The antisera -MUC2N3 and
-MUC2C2
were able to precipitate glycosylated MUC2 monomers and reducible
dimers from the soluble cell lysate portion and monomers and
nonreducible X-dimers from reduced insoluble cell debris pellet. The
conclusion that the immunoprecipitated species were
O-glycosylated was based on the observations that they (i)
were larger than the non-O-glycosylated species and the incompletely glycosylated species produced upon Brefeldin A treatment (Fig. 1), (ii) appeared later in time during pulse-chase than the
non-O-glycosylated species (Fig. 4A), (iii)
showed nonreducible size polydispersity on the gels, most probably due
to heterogeneous glycosylation, (iv) were localized to the Golgi
apparatus according to subcellular fractionation (11), (v) reacted with
the H. pomatia lectin (11), and (vi) were not
immunoprecipitated by
-MUC2TR (Fig. 1). In addition, monomers and
X-dimers from the guanidinium chloride insoluble preparation were
regarded as O-glycosylated because they (vii) reacted with
the
-MUC2TR antiserum on Western blot only after deglycosylation
(Fig. 2D), (viii) had a density of 1,51 g/ml (Fig.
2E), as expected for O-glycosylated mucins (15),
and (ix) had higher sedimentation coefficients on ultracentrifugation than (SDS-treated) non-O-glycosylated species, as for
monomers 14 S (Fig. 3B) and 8 S (11), respectively. It
should be noted that the
-MUC2TR antiserum is much more efficient in
immunoprecipitating MUC2 than the
O-glycosylation-insensitive antisera, as shown in Fig. 1.
This makes immediate kinetic comparisons between the amounts of
O-glycosylated and non-O-glycosylated MUC2 mucin,
immunoprecipitated with the different types of antisera, impossible.
The reason for the difference in effectiveness is not totally clear,
but it might include different antibody titers and a large consumption
of O-glycosylation-insensitive antibodies by epitopes on
nonlabeled MUC2 mucin stored in the cells. The
O-glycosylation-insensitive antisera are directed against nonrepeated protein epitopes, whereas the epitopes of
-MUC2TR are
tandemly repeated about 100-fold in every molecule. This might also
contribute to the differences wherein the MUC2TR antiserum should be
less sensitive for the peptide conformation as caused by, for example,
the high number of probable Cys-linkages in the nontandem repeat
regions. Because the MUC2 apomucin is very large, repeated binding
sites might also minimize mechanical dissociation from the
immunoprecipitin during the washing steps.
The two bands observed on the agarose gels of reduced insoluble MUC2 mucin were regarded as a monomer and a dimer (called X-dimer), because they migrated similarly on the gel to the monomer and reducible dimer of the soluble MUC2 mucin. Because mobility comparisons are uncertain when dealing with broad bands caused by heterogeneous O-glycosylation, and because separation could be due also to charge, rate zonal ultracentrifugation was performed to verify that the monomer and X-dimer were indeed of different size. This analysis suggested a mass of 2.7 MDa for the monomer using a previously published diffusion coefficient for human cervical mucins (19). With a predicted size for the non-O-glycosylated MUC2 monomer of 600 kDa, this suggests that the mass is made up of about 80% O-glycans, which is within the usual range of mucin glycosylation (13, 21). The lack of information on the diffusion constant for the X-dimer makes it impossible to calculate its mass and state that it is indeed a dimer. For human cervical mucins, which have MUC5AC, MUC5B, MUC2, and MUC6 as known gel-forming components (22, 23), it has been shown that oligomers, with the average size of four monomer units, have a sedimentation coefficient about twice that of the monomers (40.4 for oligomers and 19.2 S for monomers in water) (19, 21). The sedimentation coefficient of the X-dimer was only 1.3-1.4 times larger than that of monomer, and if oligomerization does not affect the diffusion properties of MUC2 and human cervical mucins in very different ways, these figures may suggest that the X-dimer consists of two or, less likely, three monomer units. The ratio between sedimentation coefficients for non-O-glycosylated, SDS-treated MUC2 dimer and monomer was just above 1.2, as was the ratio between apoB100 and apoB53/48 (11). Dimerization of O-glycosylated and non-O-glycosylated MUC2 apomucins may affect the diffusion constants in proportional ways, if O-glycans and bound SDS, respectively, cause similar stretching of the tandem repeat regions. If this is the case, one should thus expect identical ratios. The small differences found for the ratios, together with the results from the SDS agarose gel electrophoresis support the interpretation that the X-dimer is indeed a dimer.
The finding of nonreducible bonds associated with MUC2 mucin
insolubility was an unexpected phenomenon. Because the insoluble MUC2
portion had to be solubilized by reduction prior to analysis, the rate
of disulfide bond-stabilized oligomerization could not be investigated.
It is therefore impossible to say whether nonreducible links between
disulfide bond-stabilized dimers are enough to give the insolubility or
whether the mucins are further oligomerized by disulfide bonds before
the nonreducible links are assembled. If the von Willebrand factor
analogy is correct, no further oligomerization than dimerization should
occur until the trans-Golgi network, because such
oligomerization requires low pH. It seems possible that the
nonreducible bonds are assembled earlier because their formation does
not require low pH, as shown by ammonium chloride treatment (Fig.
5B). As already mentioned, no nonreducible bonds could be
seen using -MUC2TR either in supernatant (10, 11) or pellet (not
shown), showing that no non-O-glycosylated species were
linked with such bonds. This indicates a localization of the
nonreducible bond formation in the Golgi rather than in the endoplasmic
reticulum. Attempts to verify this theory by two different subcellular
fractionation protocols failed, as the insoluble MUC2 mucin was found
already in the cell debris routinely pelleted after homogenization, and
thus was excluded from the ultracentrifugation step. It is not clear
why the insoluble mucins were pelleted in these experiments when they
should have been encapsulated into membrane vesicles. One possibility
for this could be that they gave the whole vesicle a density high
enough for pelleting; another possibility is that they in some way
caused the vesicles to rupture. However, vesicles containing Golgi
markers were obtained at subcellular fractionation and contained
soluble O-glycosylated mono- and dimers (11). This might
suggest that there could exist different vesicles containing soluble
and insoluble MUC2 mucins.
The nature of the nonreducible bonds is presently unknown. The fact that the linkages are nonreducible and survive 6 M guanidinium chloride or boiling in 5% SDS suggests an uncomplicated covalent nature. This linkage could be between amino acids within the primary MUC2 sequence. Another possibility is that the bonding is via smaller linking proteins or peptides. Mucins have previously been proposed to be polymerized via a linking peptide, but an isolated linking peptide has been shown to be encoded by the C terminus of Muc2 (24, 25).
The X-dimers, obtained by reduction, are obviously soluble because they can be immunoprecipitated. Our results contradict that such soluble particles exist in vivo in substantial amounts (Fig. 2A). Thus, nonreducible bonds between monomers seem to be absent or rare in vivo. If monomers are not forming nonreducible links, this suggests a conformational change of the MUC2 molecule upon disulfide-stabilized dimer formation, allowing the formation of the nonreducible bond. This would imply that the sites for the two different kinds of bonds are localized near each other on the molecule, in the C terminus if the von Willebrand factor analogy is correct. If the nonreducible bonds are C-terminal and if they are formed in the Golgi, one may speculate on the presence of a specific enzyme system catalyzing their formation. Attempts were made to move an eventual bond formation catalyzing enzyme system from the Golgi to the endoplasmic reticulum by the help of Brefeldin A. This did not, however, give rise to any nonreducible bonds after 30 min either in the soluble or insoluble portion of the cell lysate (not shown). If the formation of the nonreducible bonds takes place in the early Golgi, the C terminus heparin binding domain on the MUC2 mucin could participate in some way, because this domain is cleaved off already in the cis-Golgi apparatus (26). Such a function of this domain might be to bind heparan sulfate and thus orient the MUC2 dimers in a way facilitating nonreducible bond formation.
In Fig. 2B, the X-dimer band consists of two equally strong adjacent bands. The presence and intensity of the upper of these is, in our experience, highly variable, and often only one X-dimer band can be found. However, the intensity of this additional band had a tendency to increase upon prolonged pulse-chase times (Fig. 5A). The variable additional bands indicate that some MUC2 monomer residues might be involved in more than one nonreducible bond and that the formation of the second bond could be delayed. The presence of nonreducible bonds within MUC2 from the intestine has been proposed by Carlstedt et al. (27). They have shown that reduced MUC2 from intestinal tissue can form a ladder of bands upon agarose gel electrophoresis, suggesting that more than two nonreducible bonds can be formed in vivo.
Mucins that are insoluble in 6 M guanidinium chloride are consistently found in the small intestine (12, 13) and, to a more variable extent, in for example the gall bladder (14). The physiological function of mucin insolubility could be to improve the mucus barrier in water-filled lumen organs. The finding that both guanidinium chloride insoluble MUC2 mucins and the metabolically labeled water-insoluble MUC2 mucins contain the unexpected X-dimers suggests that water and guanidinium chloride insolubilities are reflecting an identical phenomenon. This could propose that one of the key events in the formation of the very resistant and insoluble MUC2 is the formation of nonreducible bonds. How the bonds give rise to insolubility is not clear. It could also be the case, of course, that they are not vital for insolubility but are only synchronized with disulfide bond-stabilized polymerization and/or oligosaccharide elongation, yielding the insolubility. An attractive hypothesis, however, could be that the nonreducible bonds serve as cross-links between linear MUC2 oligomers.
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FOOTNOTES |
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* This work was supported by Swedish Medical Research Council Grants 7461 and 10443, the IngaBritt and Arne Lundbergs Stiftelse, the Göteborg Medical Society, and the Swedish Cystic Fibrosis Foundation.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 abbreviation used is: PBS, phosphate-buffered saline.
2 M. A. B. Axelsson, N. Asker, and G. C. Hansson, unpublished data.
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