Gastrointestinal expression and partial cDNA cloning of murine
Muc2
B. Jan-Willem
Van Klinken1,
Alexandra W. C.
Einerhand2,
Louise
A.
Duits1,
Mireille K.
Makkink1,
Kristien M. A. J.
Tytgat1,
Ingrid B.
Renes2,
Melissa
Verburg2,
Hans A.
Büller2, and
Jan
Dekker2
1 Emma Children's Hospital,
Academic Medical Center, 1105 AZ Amsterdam; and
2 Pediatric Gastroenterology and
Nutrition, Department of Pediatrics, Sophia Children's Hospital,
Erasmus University Rotterdam, 3015 GE Rotterdam, The Netherlands
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ABSTRACT |
To help us investigate the role of mucin in the
protection of the colonic epithelium in the mouse, we aimed to identify
the murine colonic mucin (MCM) and its encoding gene. We isolated MCM,
raised an anti-MCM antiserum, and studied the biosynthesis of MCM in
the gastrointestinal tract. Isolated MCM resembled other mucins in
physicochemical properties. Anti-MCM recognized MCM as well as rat and
human MUC2 on Western blots, interacting primarily with
peptide epitopes, indicating that MCM was identical to murine Muc2.
Using anti-MCM and previously characterized anti-human and anti-rat
MUC2 antibodies, we identified a murine Muc2 precursor in the colon of
~600 kDa, which appeared similar in size to rat and human MUC2
precursors. Western blotting, immunoprecipitation of metabolically
labeled mucins, and immunohistochemistry showed that murine Muc2 was
expressed in the colon and the small intestine but was absent in the
stomach. To independently identify murine Muc2, we cloned a cDNA
fragment from murine colonic mRNA, encoding the 302 NH2-terminal amino acids of murine
Muc2. The NH2 terminus of murine
Muc2 showed 86 and 75% identity to the corresponding rat and human
MUC2 peptide sequences, respectively. Northern blotting with a murine
Muc2 cDNA probe showed hybridization to a very large mRNA, which was
expressed highly in the colon and to some extend in the small intestine
but was absent in the stomach. In situ hybridization showed that the
murine Muc2 mRNA was confined to intestinal goblet cells. In
conclusion, by two independent sets of experiments we identified murine
Muc2, which appears homologous to rat and human MUC2. Because Muc2 is
prominently expressed in the colon, it is most likely to be the
predominant mucin in the colonic mucus layer.
mucin; gastrointestinal tract; colon; intestine
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INTRODUCTION |
EPITHELIAL MUCINS ARE widely accepted to play
cytoprotective roles in many organs (9, 26, 37). Many epithelia express a variety of mucins (37); however, the human colonic epithelium expresses one mucin in very high amounts: MUC2 (28). Importantly, we
were able to show that in patients with ulcerative colitis the activity
of the mucosal inflammation correlates with a significant decrease in
MUC2 synthesis (32), implying an important role for MUC2 in colonic
cytoprotection. The colon of the mouse constitutes a very feasible
organ to test further the cytoprotective nature of mucins under
experimental pathophysiological conditions. The colonic epithelium has
to confront a particularly hostile environment, naturally necessitating
effective protection. Moreover, there are many colitis models in the
mouse that are well suited for testing the susceptibility of the
colonic epithelium toward luminal substances (reviewed in Ref. 8).
Murine mucins and their encoding genes have not been studied
extensively. Four murine mucins were identified so far. Muc1 is a
membrane-bound mucin that is very homologous to its human and rat
counterparts and is expressed at low levels in many epithelia but shows
little tissue-specific expression (25, 41). Murine Muc3 was cloned very
recently and was demonstrated to be specifically expressed in intestine
(23). Murine Muc5AC was cloned from stomach by Shekels et al. (24) and
was, like its human homologue, primarily expressed in stomach and
airways. Recently, a murine mucin was identified (confined to salivary
glands) that showed high homology to a rat salivary mucin (7). This
mucin shows striking resemblance in overall structure to the human
salivary mucin MUC7. However, this mucin still awaits inclusion into
the "MUC" nomenclature because it shows no sequence homology to
the human MUC7. Thus far, colonic mucins in the mouse were not
particularly well studied.
To help us investigate the role of murine colonic mucin (MCM) in the
protection of the epithelium of the colon, we aimed to identify the MCM
and its encoding gene by two independent approaches. First, we isolated
colonic mucin, prepared an antiserum against this, and studied the
nature of MCM by immunochemical techniques. Second, on the assumption
that MCM might be homologous to rat and human MUC2, we sought
to isolate a murine Muc2 cDNA fragment from murine colonic mRNA
using RT-PCR and studied the murine Muc2 mRNA expression in the
gastrointestinal tract. These approaches led to the same conclusion:
murine Muc2 appeared to be expressed in the mouse intestine and was
particularly abundant in the colon of the mouse.
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MATERIALS AND METHODS |
Unless otherwise indicated, chemicals were obtained from the following
manufacturers: Amersham (Buckinghamshire, UK), GIBCO BRL (Gaithersburg,
MD), Merck (Darmstadt, Germany), Sigma (St. Louis, MO), Bio-Rad
(Richmond, CA), Pharmacia (Uppsala, Sweden), BDH (Poole, UK), and
Boehringer (Mannheim, Germany).
Analytic procedures.
Rat colonic mucin (RCM) and human colonic mucin (HCM) and antisera
against these mucins, anti-RCM and anti-HCM, which recognize rat and
human MUC2, respectively, were obtained through earlier work (27, 28).
The monoclonal antibody WE9 against human MUC2, which also recognizes
rat Muc2, was characterized earlier (29). Protein samples were analyzed
on reducing PAGE in the presence of 0.1% SDS. Before SDS-PAGE
analyses, samples were boiled for 5 min in buffer containing 1%
(vol/vol) 2-mercaptoethanol (Bio-Rad) and 1% (wt/vol) SDS. SDS-PAGE
gels were stained with periodic acid-Schiff's reagent (PAS, Sigma) or
with Coomassie brilliant blue R-250 (Merck). Western blotting of
isolated mucins and gastrointestinal tissue homogenates was described
previously (27-29). To visualize radiolabeled bands in protein
samples, SDS-PAGE gels were incubated, after fixation in 10% acetic
acid-10% methanol, for 10 min with Amplify (Amersham) before drying
and analyzed by fluorography by exposing the gels for 1-4 wk at
70°C to Biomax MR films (Kodak). Prestained
high-molecular-mass markers were purchased from Bio-Rad (ranging from
49.5 to 205 kDa). For reference to very high-molecular-weight molecules, metabolically labeled, unreduced rat gastric mucin precursors were used (molecular weights of monomer and dimer = 300,000 and 600,000, respectively) (5). The density of CsCl gradient fractions
was measured by weighing 1 ml of each fraction using a calibrated
pipette. Hexose assay was performed using orcinol (Sigma) according to
François et al. (10) with galactose as standard. Monosaccharide
analysis was performed according to the method of Savage et al. (22).
Amino acid analysis was performed using the
o-phthalaldehyde (Pierce) derivative
technique and HPLC (35). For some analyses, purified mucins were
digested by proteinase K as described previously (28).
Isolation of colonic mucins and preparation of antiserum.
The mucosa of the entire colon was scraped off of 58 adult healthy mice
(27 males). MCM was isolated, and a polyclonal anti-MCM was elicited,
as described earlier for HCM and RCM (27, 28). Briefly, 2.3 g wet
weight of mucosal scrapings were homogenized in 50 ml buffer, pH 7.5, containing 6 M guanidinium · HCl (Sigma). All
procedures took place at 4°C. Mucin was chemically reduced to
enhance solubility by dithiothreitol (Sigma) and sulfhydryl groups were
carboxymethylated by iodoacetamide (Sigma). Mucins were purified by
equilibrium centrifugation using three consecutive CsCl (Boehringer)
density gradients. Mucin-containing fractions from each gradient were
pooled and run on the next gradient. In the first and second gradient,
CsCl was added to a density of 1.40 g/ml, with a
guanidinium · HCl concentration of 4 M. In the last
gradient, CsCl was added to a density of 1.50 g/ml, whereas the
guanidinium · HCl concentration was reduced to 0.2 M. Isopycnic density gradient centrifugation was performed in a Beckman
ultracentrifuge, Ti 60 rotor at 50,000 rpm for 66 h at 4°C. For
analysis, the fractions were dialyzed extensively against distilled
water at 4°C and stored at
20°C. Purified antigen was
mixed with Freund's complete adjuvant (Difco, Detroit, MI) and
injected subcutaneously in a New Zealand White rabbit. After booster
injections with Freund's incomplete adjuvant (Difco), the anti-MCM
serum was obtained.
Metabolic labeling of gastrointestinal tissue and
immunoprecipitation of mucins.
Metabolic labeling of tissue in vitro and immunoprecipitation of mucins
were performed as described previously (5, 29, 36). In brief, mucin
biosynthesis was studied by metabolic labeling with
35S-labeled amino acids
([35S]methionine/cysteine,
Pro-mix, Amersham), to label the polypeptides, or with
[35S]sulfate
(Amersham) to label mature mucins. Healthy adult female mice
(15-20 g) were killed by cervical dislocation. Tissue explants (10 mm3) of stomach, jejunum, or
colon were cultured and pulse-labeled with either Pro-mix for 30 min or
[35S]sulfate for 60 min, using 100 µCi of each label per 100 µl of medium per tissue
explant. In some experiments, chase incubations of 4 h were performed
after the pulse-labeling with
[35S]sulfate, after
which the tissue and the culture medium were collected. After the
respective pulse or chase experiments, explants were homogenized in, or
culture medium was mixed with, a Tris buffer containing 1% Triton
X-100 and 1% SDS and high concentrations of six protease inhibitors.
Mucins were immunoprecipitated from the homogenates overnight at
4°C with various antibodies. Immunocomplexes were precipitated
using Sepharose CL-4B-coupled protein A (Pharmacia). Immunoprecipitated
mucins were washed, separated by SDS-PAGE using a 3% stacking and 4%
running gel, and analyzed by fluorography. In some analyses,
immunoprecipitated mucins were digested by endoglycosydase H (endo H)
as described previously (28).
Immunohistochemistry.
Small segments of stomach, jejunum, and colon of mouse or rat colon
were fixed in 4% paraformaldehyde immediately after excision and
embedded and prepared for immunohistochemistry as described previously
(33). Anti-MCM and anti-RCM were applied at 1:3,000 and 1:500,
respectively. Immunoreaction was detected using the Vectastain Elite
ABC kit (Vector Labs, Burlingame, CA), and staining was developed using
diaminobenzidine. To enhance the signal, sections were either boiled in
10 mM citrate buffer (pH 6) for 10 min or treated with 20 µg/ml
proteinase K (Boehringer) for 7.5 min in PBS.
RT-PCR and sequence analysis.
Total RNA was isolated from mucosal scrapings of murine colon using
TRIzol (GIBCO BRL) following the manufacturer's protocol. One
microgram of total RNA was transcribed at 42°C into cDNA using Superscript RT (GIBCO BRL) in a total volume of 20 µl, following the
manufacturer's instructions. The final reaction conditions were as
follows: 20 mM Tris (pH 8.4), 50 mM KCl, 2.5 mM
MgCl2, 0.01% BSA, 10 mM
dithiothreitol, 500 nM random hexamers, 1 µg total RNA, and 500 µM
each of dATP, dCTP, dGTP, and dTTP. After a 1-h incubation, an RNase H
(GIBCO BRL) digestion was carried out for 10 min at 42°C. This was
followed by a PCR reaction in a total volume of 20 µl using 1 µl
cDNA as template in combination with the primers P70
(5'-CACCATGGGGCTGCCAC-3') and P62
(5'-AGCCGCTCTCCAGGTAC-3'), which correspond to nucleotides
24-40 and 945-961, respectively, of human MUC2 cDNA (15).
These primer sequences are perfectly conserved between rat and human
MUC2 (15, 20). Final PCR reaction conditions were as follows: 10 mM
Tris (pH 8.4), 50 mM KCl, 5 mM
MgCl2, 0.01% gelatin, 0.2 units
Taq polymerase, 200 nM of each primer,
cDNA template, and 200 µM each of dATP, dCTP, dGTP, and dTTP. The PCR
reaction was carried out as follows: 5 min at 95°C and 30 cycles of
1 min at 95°C, 1 min at 55°C, and 1 min at 72°C. After the
last cycle, a 10-min extension step at 72°C was done. The resulting
911-bp PCR product was isolated after analysis on a 1% agarose gel
using the Qiagen gel extraction kit. Subsequently, the purified PCR
product was double strandedly sequenced using the
Taq dye nucleotide cycle sequencing
kit with fluorescently labeled nucleotides (Applied Biosystems,
Norwalk, CT) and primers P62, P70, and other primers spanning the
entire PCR fragment (P71: 5'-GTCTGCAGCACCTGGGG-3', P72:
5'-CCCTCATGTGGAACCGGG-3', P73:
5'-AGTTTGGGAACATGCAGAAG-3', P75:
5'-GCACTGGCGGGAGAACTC-3', P76:
5'-CCCGGTTCCACATGAGGG-3', and P77:
5'-TGAGGTAGATGGTGTCATCC-3'). Sequence reactions were analyzed on an Applied Biosystems model 377 sequencer. Sequences were
analyzed using Macintosh Sequence Navigator and Autoassembler software. Nested primers P61
(5'-TAAGGTCGACACCATCTACCTCACC-3') and P63
(5'-GGAATTCTGCATGTTCCCAAACTC-3') were used to amplify and clone a fragment (244 bp) of the 911-bp PCR fragment in the Sal
I-EcoR I sites of pBluescript SK
(Stratagene, La Jolla, CA). This cloned fragment was double strandedly
sequenced as described above and used as a probe for Northern blot analysis.
Northern blot analysis.
Total RNA was isolated from murine stomach, small intestine, and colon
using TRIzol (GIBCO BRL) following the manufacturer's protocol. The
Northern blot analysis was essentially carried out as previously
described (30). Briefly, 10 µg of total RNA derived from each tissue
were separated on a 0.8% agarose gel containing 10 mM HEPES
(Sigma) (pH 7.5) and 2.2 M formaldehyde (Merck). Integrity of RNA was
assessed by analyzing the 28S and 18S ribosomal RNAs after
electrophoresis and staining with ethidium bromide. Capillary transfer
of RNA to Qiabrane (Qiagen) was carried out. The blot was hybridized to
a 32P-labeled 244-bp
Sal
I-EcoR I fragment (described above).
After exposure to Kodak X-Omat AR film, the probe was stripped from the
blot. The blot was reprobed with a
32P-labeled human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe as described
(33), and all RNA samples were found to contain intact GAPDH mRNA bands
of the expected size. The levels of hybridization on the Northern blots
to the murine Muc2 probe and the GAPDH probe were measured through
autoradiography using a PhosphorImager and ImageQuant software
(Molecular Imaging, Sunnyvale, CA), as described previously (33).
In situ hybridization.
In situ hybridization was performed by labeling double-stranded cDNA
fragments using 35S-labeled dCTP
and random priming, as described previously (21). To detect murine Muc2
mRNA, two probes were used: 1) the
244-bp Sal
I-EcoR I fragment of the murine Muc2
cDNA, described in RT-PCR and sequence
analysis, and 2) a
1.2-kb fragment of the rat Muc2 cDNA sequences that was isolated using
RT-PCR on rat colonic RNA, using primers that were based on the
published rat Muc2 cDNA sequence (42).
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RESULTS |
Isolation and characterization of MCM.
MCM was isolated by triple-density-gradient centrifugation. Mucins were
identified in the fractions of the first gradient with densities of
1.35-1.52 g/ml by orcinol assay after dialysis of aliquots of
these fractions (not shown). Analysis of these fractions by SDS-PAGE
demonstrated the presence of high-molecular-weight PAS-stainable
material, which entered the 4% running gels (not shown). The
mucin-containing fractions were rerun on a second- and third-density
gradient. All other proteins were removed by this procedure as judged
by SDS-PAGE and Coomassie blue staining of dialyzed aliquots of each fraction.
The mean buoyant density of the isolated MCM was 1.46 g/ml. Amino acid
analysis revealed a high content of serine and threonine residues,
together comprising ~37%, whereas the threonine content exceeded the
serine content (threonine-to-serine ratio of 1.69). The monosaccharide
analysis revealed a very high content of
O-linked oligosaccharides, whereas
mannose, an indicator of N-linked
glycans, was present in only low amounts (Table
1). In particular, the sialic acid content
was very high (22.9%; Table 1). The buoyant density of
the isolated MCM, the presence of a high percentage of hydroxylated
amino acids, and the presence of a high content of
O-linked glycans are hallmarks of
epithelial mucins (26).
Identification of MCM as murine Muc2.
When analyzed by SDS-PAGE and PAS staining, MCM, presented as a single
band just entering the 4% running gel, displayed a mobility very
similar to RCM and HCM (Fig. 1). Epithelial
mucins are known to display a characteristic resistance to enzymatic proteolysis, due to the very high number of
O-linked oligosaccharides (26). On
exhaustive digestion with proteinase K, the mobilities of MCM, RCM, and
HCM slightly increased in a similar manner (Fig. 1), indicating that a
large part of the molecules are indeed protected from digestion by
proteases.

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Fig. 1.
Specificity of the anti-murine colonic mucin (MCM) antiserum. Samples
of isolated MCM, human colonic mucin (HCM), and rat colonic mucin (RCM)
were analyzed on SDS-PAGE and stained with periodic acid-Schiff's
reagent (PAS) (left), before and
after digestion with proteinase K (pk). In parallel, these samples were
Western blotted using anti-MCM
(right). Arrowhead indicates the
border between the 3% stacking gel and the 4% running gel. At
left, the position of the 205-kDa
marker is indicated.
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A polyclonal anti-MCM antiserum was elicited, which was tested for its
ability to recognize intact and proteinase K-digested mucins on Western
blot (Fig. 1). Purified MCM was well recognized by anti-MCM, but after
proteolytic treatment nearly all recognition was lost, implying that
anti-MCM recognizes primarily peptide epitopes. Similarly, HCM and RCM
were recognized by anti-MCM before but not after incubation with
proteinase K (Fig. 1). We also found that anti-RCM and anti-HCM
recognized each of the three mucin preparations, whereas all
recognition was lost when digested with proteinase K (not shown). The
cross-reactivity of these antisera indicates that homology exists at
the polypeptide level among the colonic mucins of these three species.
Because we previously identified HCM and RCM as rat and human MUC2,
respectively (27, 28), it seems very likely that MCM is identical to
murine Muc2.
We took a second approach to try and identify MCM by immunoisolating
the MCM precursor. Mucin precursors were defined previously as the
primary translation products of mucin mRNAs, which are present in the
rough endoplasmic reticulum, that do not contain O-glycosylation (26). We
used anti-MCM and several established anti-MUC2 antibodies (i.e.,
anti-HCM, anti-RCM, and WE9) to immunoprecipitate mucin precursors from
metabolically 35S-labeled amino
acid colonic tissue homogenates (Fig.
2A).
Each of these four antibodies precipitated an ~600-kDa protein from the homogenate. A band with a very similar mobility was easily distinguished within the homogenate, strongly suggesting that this was
a prominently expressed protein. The polyclonal antisera, anti-MCM,
anti-RCM, and anti-HCM, showed precipitation of some additional bands.
These bands most likely represent nonspecific products because these
were absent from the sample precipitated with the monoclonal WE9. Also,
these extra bands were present to variable extents in the
immunoprecipitations with the antisera. Compare, e.g., the
immunoprecipitation with anti-MCM shown in A and
B of Fig. 2: Fig.
2A shows some additional bands, which are absent in the duplicate experiment shown in Fig.
2B. Given the cross-reactivity of the
previously characterized anti-MUC2 antisera with the 600-kDa band in
the murine colonic homogenates, it is very likely that the 600-kDa band
represents the murine Muc2 precursor. PAS staining of the
immunoprecipitated mucin also showed the precipitation of the mature
mucin, with a mobility slightly higher than that of the Muc2 precursor
(Fig. 2A).

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Fig. 2.
Immunoprecipitation of mucin precursors from metabolically labeled
murine colonic tissue. A colonic segment from the middle of the colon
was incubated for 30 min with
35S-labeled amino acids and
homogenized. A: mucin precursors were
immunoprecipitated using anti-MCM, anti-HCM, and anti-RCM antiserum
antibodies and the monoclonal anti-MUC2 antibody WE9. Immunoisolates
were analyzed next to the colonic homogenate on SDS-PAGE, followed by
fluorography. PAS lane shows the PAS staining of the anti-MCM lane of
the adjacent fluorograph. B: mucin
precursor was immunoprecipitated using anti-MCM and incubated with or
without endoglycosydase H (endo H) as indicated. Arrowheads indicate
the borders between the 3% stacking gels and the 4% running gels. At
left of
A and
B, positions of the 300- and 600-kDa
markers are indicated.
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The identity of the alleged 600-kDa Muc2 precursor band was further
corroborated by digestion with endo H, which hydrolyzes high-mannose
N-linked glycans. The presence of
these glycans is a hallmark of proteins within the rough endoplasmic
reticulum. Endo H digestion of immunoprecipitated Muc2 precursor leads
to an increased mobility of the Muc2 precursor band (Fig.
2B), indicating that high-mannose
N-linked glycans are present and that
this protein indeed resides in the rough endoplasmic reticulum.
Muc2 is a secretory mucin, which is confined to intestinal goblet
cells.
We studied the expression of Muc2 in murine stomach, jejunum, and colon
by Western blotting (Fig. 3). PAS staining
of the 4% SDS-PAGE gel revealed very high-molecular-weight
glycoproteins in each of these organs, which most likely represent
mucins. Western blotting of these samples using anti-MCM revealed
extensive staining of a high-molecular-weight product in the colon. The
mobility and the appearance of this Muc2 band, stained with anti-MCM,
coincided with the PAS-stained band in the corresponding colon
homogenate, indicating that Muc2 is highly expressed in the colon.

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Fig. 3.
Region-specific expression of murine Muc2 in the gastrointestinal tract
by Western blotting. Segments of the stomach, jejunum, and colon of the
mouse were homogenized in the same buffer as used for
immunoprecipitation. Protein concentrations were measured and adjusted
to allow loading of equal amounts of protein in each lane. Samples of
each homogenate were analyzed on SDS-PAGE and stained with PAS
(A). In parallel, samples were
analyzed on Western blot using 1:500 diluted anti-MCM
(B). Arrowheads indicate the borders
between the 3% stacking gels and the 4% running gels. At
left of
A and
B, positions of the 205-kDa marker are
indicated.
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Staining by anti-MCM was largely absent from the stomach homogenate
(Fig. 3). In the jejunal sample, there was only limited staining
relative to the intense staining of the colonic sample, of material
just entering the running gel. The mobility on SDS-PAGE of this small
intestinal Muc2 was lower than the Muc2 in the colon. These findings
indicate that the major mucins stained by PAS in the stomach and the
jejunum are not identical to Muc2. Because no specific antibodies were
available that recognize murine mucins other than Muc2, we were not
able to identify further these gastric and small intestinal bands.
To investigate the cell type-specific expression of murine Muc2, we
performed immunohistochemistry using anti-MCM and anti-RCM on sections
of the stomach, jejunum, and colon. Anti-MCM and anti-RCM did not stain
stomach epithelium (not shown). Immunostaining of jejunal and colonic
sections revealed that anti-MCM stained goblet cells in both intestinal
segments in a highly specific manner (Fig.
4). Staining was largely confined to the
intracellular storage granules (Fig.
4B). However, particularly in the
jejunum, thin sheets of extracellular material were also stained by
both antisera (Fig. 4). Interestingly, immunostained material,
apparently streaming from the goblet cells, was sometimes observed to
be continuous with these extracellular sheets (Fig.
4A). The brush-border membranes of
the small intestinal enterocytes, which contain high amounts of various
glycoconjugates, were free of staining (Fig. 4,
A and B). The results obtained when using
anti-RCM were very similar to those obtained with anti-MCM (not shown).
The goblet cells in the upper part of the colonic crypts stained more
strongly with the anti-MCM antiserum than cells in the lower part of
the crypts (Fig. 4C), suggesting
that more Muc2 is present in the latter cells.

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Fig. 4.
Immunohistochemical detection of MCM in the murine intestine.
A: section from the middle of the
jejunum stained using anti-MCM. Large arrow points to a goblet cell
secreting mucin into the lumen. B:
higher magnification of jejunal epithelium. Smaller arrows (in
A and
B) point to the brush-border
membranes of the jejunal enterocytes, which are not stained with the
antiserum. C: section from the middle
of the colon that was stained using anti-MCM. Magnifications:
A = ×170;
B = ×850;
C = ×210.
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From the immunohistochemical studies, it seemed evident that the murine
Muc2 is secreted. To establish this, we performed pulse-chase
experiments using
[35S]sulfate-labeled
segments of jejunum and colon (Fig. 5).
Pulse labeling resulted in the accumulation of high-molecular-weight [35S]sulfate-labeled
material that was partly secreted into the media of both jejunal and
colonic segments during the 4-h chase period. This product could be
immunoprecipitated by anti-MCM from both tissue and medium samples of
jejunum and colon homogenates and was thus identified as secretory
Muc2. The jejunal Muc2 had a lower mobility on SDS-PAGE, as previously
noted in Fig. 3, and labeled less intense with
[35S]sulfate than the
colonic Muc2.

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Fig. 5.
Muc2 is secreted from jejunum and colon. Segments of jejunum and colon
were pulse incubated for 60 min with
[35S]sulfate and chase
incubated for 4 h in the absence of radiolabeled sulfate. Tissue was
then homogenized in 1 ml buffer, and the medium was collected and
diluted with homogenization buffer to 1 ml; 15 µl of each homogenate
were loaded on SDS-PAGE and analyzed by fluorography. Muc2 was
immunoprecipitated by anti-MCM from each homogenate, analyzed by
SDS-PAGE, and fluorographed. p, Samples of pulse-labeled tissue; c,
samples of chase-incubated tissue; m, medium samples. Arrowhead
indicates the border between the 3% stacking gel and the 4% running
gel. At left, positions of the 300- and 600-kDa markers are indicated.
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Isolation and sequencing of a partial murine Muc2 cDNA.
Part of the murine Muc2 cDNA was cloned by RT-PCR on murine colonic
RNA, representing the 5'-region of the murine Muc2 mRNA. A
fragment of 911 bp was amplified and double strandedly sequenced, following the strategy shown in Fig.
6A, and
was deposited in GenBank under accession number AF016695. It was found
to encode a fragment of 302 amino acids of the
NH2 terminus of murine Muc2. The
cloned Muc2 sequence was highly conserved among species: compilation of
the murine, rat, and human MUC2 sequences showed 73% identical amino
acids (Fig. 6B). Conservation
between rat and murine Muc2 appeared highest with 86% identity. The
cloned murine Muc2 sequence contains one putative
N-glycosylation site at N159, which is
conserved in the rat and human MUC2 sequences. Also, all of the 17 cysteine residues present in murine Muc2 were conserved in the rat and human MUC2 sequences. Furthermore, homology (30% identity of amino acids) was observed with the D domain of the human von Willebrand factor, as found earlier for rat and human MUC2 (15, 20).

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Fig. 6.
The NH2-terminal amino acid
sequence of murine Muc2 and sequence comparison with rat and human
MUC2. A: sequence strategy for the
911-bp murine Muc2 (mMUC2) cDNA fragment, which was isolated using
RT-PCR on murine colonic RNA. In B,
sequence of the 302-amino-acid
NH2-terminal mMuc2 polypeptide
fragment is aligned to the
NH2-terminal sequences of human
MUC2 (hMUC2) and rat Muc2 (rMUC2). The 3 sequences are 73% identical;
mismatches between mMuc2 and rMUC2 or hMUC2 are indicated by
asterisks.
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Murine Muc2 mRNA is highly expressed in the colonic goblet cells.
Part of the amplified 911-bp murine Muc2 cDNA sequence was cloned and
used as a probe to detect Muc2 mRNA in RNA samples from nine regions of
the murine gastrointestinal tract (Fig.
7A).
Stomach RNA showed very little hybridization to the Muc2 cDNA probe.
However, each RNA sample from small intestine as well as colon showed
hybridization to a very high-molecular-weight band. In addition to this
band, a smear was noted with an intensity that corresponded to the
intensity of the band, suggesting that this smear resulted from
degradation of the Muc2 mRNA present in the high-molecular-weight band.
It should be noted, in general, that the detection of this type of polydispersed signal for mucin mRNAs on Northern blots is a commonly observed phenomenon (see, e.g., Refs. 11-13). However, the rRNA bands on the gel and the bands detected for GAPDH on the Northern blot
showed discrete bands (not shown). The hybridization signal found with
the Muc2 probe was quantified relative to the signal observed for GAPDH
mRNA in each lane (Fig. 7B). Muc2
mRNA was particularly abundant in proximal and middle colon, indicating that Muc2 mRNA was most abundant in these segments. The signal in the
distal colon was relatively low; expression level of Muc2 mRNA was 26%
of that found in the other colonic segments.

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Fig. 7.
Expression of murine Muc2 mRNA in the gastrointestinal tract.
A: RNA was isolated from the indicated
9 regions of the gastrointestinal tract of the mouse and analyzed by
Northern blotting using the
32P-labeled, 244-bp,
Sal
I-EcoR I Muc2 cDNA fragment. The 18S
and 28S rRNA bands and slots (arrowhead) are indicated to the
right.
B: quantitative analysis of the
Northern blot results, in which the signal obtained with the murine
Muc2 probe in each lane was expressed relative to the signal obtained
with the glyceraldehyde-3-phosphate dehydrogenase probe in the same
lane.
|
|
To localize the Muc2 mRNA in colonic tissue, in situ hybridization was
performed. The number and localization of cells with positive signal
for Muc2 mRNA were very similar to the number and distribution of the
goblet cells as stained for Muc2 protein in immunohistochemistry (Fig.
4C), indicating that the Muc2 mRNA was confined to goblet cells in the colon (Fig.
8). With the use of a cDNA probe,
representing rat Muc2 mRNA, on murine colonic sections, very similar
results were obtained (not shown). Also, when the murine Muc2 cDNA
probe was used on rat colonic tissue, the signal was confined to rat
colonic goblet cells, which could also be stained with anti-rat Muc2
antibody in immunohistochemistry (not shown). It should be noted that
goblet cells in the lower part of the colonic crypts hybridize less
strongly to the Muc2 cDNA probe than the cells in the upper part of the
crypts (Fig. 8), suggesting that less Muc2 mRNA is produced in the
goblet cells of the lower crypt region. Because these cells also stain
less intensely with the anti-Muc2 antibodies (Fig.
4C), it seems that these goblet
cells produce less Muc2 than the goblet cells of the upper crypt
region.

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Fig. 8.
Cellular expression of the murine Muc2 mRNA. In situ hybridization was
performed on a section of the middle of the colon using a
35S-labeled, 244-bp fragment of
the murine cDNA, as described in MATERIALS AND
METHODS. L, lumen of the colon; S, serosal side of the
tissue.
|
|
 |
DISCUSSION |
In this study, we were able to show that the major colonic mucin from
the mouse is considered to be identical to murine Muc2, as is evident
from the following six considerations.
Consideration 1.
The physicochemical characteristics of MCM are similar to rat and human
MUC2. The threonine plus serine content is around 40% of the amino
acids for all three mucins, and characteristically the threonine
content exceeds the serine content. The monosaccharide composition is
also similar for all three mucins, with low levels of fucose and
mannose and particularly high sialic acid contents. The buoyant density
of the mucins, which is a measure for the chemical composition of the
mucins, is similar, ranging from 1.45 to 1.50 g/ml. Also, the behavior
of the isolated mucins on SDS-PAGE is very similar, yielding bands with
mobilities corresponding to molecular masses of ~600 kDa. This
behavior of these colonic mucins on SDS-PAGE is characteristic yet
anomalous with respect to their molecular masses, as will be discussed
separately below.
Consideration 2.
The identity of MCM as murine Muc2 was also corroborated by the
cross-reactivities between MCM and previously characterized anti-MUC2
antibodies, which were demonstrated to recognize the polypeptides of
rat and human MUC2 (29). Also the cross-reactions of anti-MCM with rat
and human MUC2 indicate that homology at the polypeptide level exists
among the colonic mucins of these three species. The cross-reactivity
between the antibodies and the mucins from these three species was
noted in all techniques used: Western blotting of mature mucins,
immunoprecipitation of both mature mucins and mucin precursors, and
immunohistochemistry. Because these cross-reactivities have been
demonstrated to be primarily based on polypeptide recognition, it is
very likely that MCM is identical to murine Muc2.
Consideration 3.
After metabolic labeling of murine colonic explants with radioactive
amino acids, an ~600-kDa band was immunoprecipitated using either
anti-MCM or a variety of anti-MUC2 antisera, which most likely
represent the murine Muc2 precursor. The human MUC2 cDNA was completely
sequenced by Gum et al. (11, 14, 15), revealing that the encoded human
MUC2 precursor has a molecular mass of ~600 kDa. In line with this
expected molecular mass, we identified the human MUC2 precursor as an
~600-kDa band in the human colon and small intestine and a colonic
cell line (28, 36, 39). In the rat colon, we were able to identify a
very similar 600-kDa band that we could independently identify as the rat Muc2 precursor (27). On the basis of its estimated molecular mass
as well as on the cross-reactivity with previously characterized anti-MUC2 antisera, it is very likely that the 600-kDa band represents the murine Muc2 precursor.
Consideration 4.
Murine Muc2 mRNA appeared very large, as would be expected when
encoding a polypeptide precursor of ~600 kDa. The size of this mRNA,
as for other mucins, is very difficult to estimate due to the low
resolution of agarose gels for these high-molecular-mass molecules and
the lack of appropriate markers. Nevertheless, similar very large mRNAs
were detected by Northern blotting for rat and human MUC2 (30, 42),
which were consistent with the sizes of the very large MUC2 precursors
that these respective mRNAs encode.
Consideration 5.
Independently, the NH2-terminal
sequence of murine Muc2 was determined using RT-PCR on murine colonic
RNA. The deduced sequence of the 302 NH2-terminal amino acids was very
similar in all aspects to the rat and human MUC2 sequences (15, 20).
Particularly, all 17 cysteine residues and the single
N-glycosylation site were conserved in
all three sequences, indicating that the three-dimensional structure of
this part of the polypeptide is likely to be conserved. This high level
of similarity, which may also involve other regions of the polypeptide,
likely explains the extensive cross-reactivities of the anti-MUC2
antisera with the MUC2 molecules among these three species.
Consideration 6.
The tissue and cell type-specific expression of MCM, as shown by
Western blotting, immunohistochemistry, Northern blot, and in situ
hybridization is similar to rat and human MUC2, since MCM expression is
1) high in the colon,
2) low in the small intestine, 3) confined to intestinal goblet
cells, but 4) undetectable in the
stomach. Similar observations were made for human MUC2 expression (1-4, 11, 14, 15, 36) as well as for the expression of rat Muc2
(12, 18, 20, 42, 43).
We set out to identify the mucins in the murine colon that are involved
in cytoprotection through the mucus layer. The colonic mucus is copious
and could consist of a mixture of mucins. Our strategy for the
identification of the MCMs would enable us to isolate a potential
mixture of mucins, because our isolation was based on a buoyant density
around 1.4 g/ml, a ubiquitous characteristic of secretory mucins (9,
26). As indicated above, it seems very likely that MCM is identical to
murine Muc2. Nevertheless, we cannot exclude the possibility that other
mucins are present in small amounts in the colon of the mouse, which
remain as yet undetected in our studies.
That the interactions of anti-MCM are primarily limited to polypeptide
recognition was substantiated by three observations. 1) All epitopes of MCM that are
recognized by anti-MCM were protease sensitive, whereas protease
treatment left intact the major part of the molecule, carrying
virtually all glycosylation. This implies that anti-MCM recognizes
primarily peptide epitopes, as was previously found for a large number
of anti-mucin antisera, which were prepared following an identical
protocol (29). 2) Anti-MCM was able
to recognize and immunoprecipitate the murine Muc2 precursor, which very likely contains no
O-glycosylation. Therefore,
it is very likely that the anti-serum is primarily directed against
peptide epitopes. 3) Recognition of
mucins at the histological level was limited to intracellular granules
of intestinal goblet cells and extracellular material. In contrast, the
brush border and the Golgi apparatus of enterocytes, which lies
characteristically in a supranuclear position in enterocytes, are
completely devoid of any staining. Because both of these cellular
structures contain high amounts of very diverse glycoconjugates, it
seems very unlikely that anti-MCM would recognize carbohydrate structures.
On SDS-PAGE, homogenates of the stomach, jejunum, and colon showed
similar amounts of PAS-stainable, high-molecular-weight mucin. With
Western blotting of these samples, staining by anti-MCM was absent from
the stomach, whereas staining was low in jejunum relative to the very
intense staining of mucin in the colon. Moreover, the mobility of the
Muc2 in jejunum samples, as detected by anti-MCM, was dissimilar from
the mobility of the PAS-stainable mucin band. These findings therefore
indicate that the major mucins stained by PAS in the stomach and the
small intestine are very likely not identical to Muc2. The major murine
stomach mucin has been identified as murine Muc5AC, explaining why
anti-MCM fails to recognize the murine stomach mucin. In line with
this, neither anti-MCM nor anti-RCM stained gastric epithelium
immunohistochemically, and also the Muc2 mRNA was virtually absent from
stomach RNA using Northern blot. In the small intestine of rat, human,
and mouse, a second major mucin has been identified: Muc3 (13, 18, 19, 23, 36, 40). Therefore, the PAS-stained band, which was not stained
using anti-MCM, most likely represents mature murine Muc3. Because no
antibodies are available that recognize mature murine Muc3, we were
unable to identify further this small intestinal mucin band.
The mature Muc2 that was detected, by Western blotting and
immunoprecipitation, in the small intestine displayed a significantly lower mobility on SDS-PAGE than the colonic Muc2. As discussed at
length previously (31), the mobilities of mature mucins are generally
anomalous and are dependent on the intrinsic negative charge of the
mucins, which is imposed by the high sialic acid content. Also, the
presence of sulfate esters has been shown to have dramatic effects on
the mobility of some mucins on gel (6, 34); therefore, potential
differences in sulfation may also contribute to the observed
differences in mobility between the small intestinal and colonic MUC2.
Thus the Muc2 in the small and large intestine of the mouse may differ
in their glycan structure and composition. Similarly, differently
glycosylated forms of rat and human MUC2 were detected in various parts
of their respective gastrointestinal tracts (17, 36).
Taking all data together, it is evident that Muc2 is expressed
throughout the mouse intestine and that Muc2 is very likely the
prominent colonic mucin in the mouse. Studies are now underway to
quantify the Muc2 synthesis in various colitis models in mice, to
evaluate the role of Muc2 in the cytoprotection of the colonic epithelium against luminal threats.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge the help of George Jörning (Dept. of
Exp. Internal Medicine, Academic Medical Center, Amsterdam) for the
skillful amino acid analyses. We thank Carolien Koeleman (Dept. Medical
Chemistry, Free University, Amsterdam) for the excellent determination
of the monosaccharide compositions.
 |
FOOTNOTES |
Our work was made possible through the financial support from ASTRA
Pharmaceuticals (B. J.-W. Van Klinken), Nutricia BV Zoetermeer (M. Verburg and I. B. Renes), and the Netherlands Foundation for Scientific
Research (K. M. A. J. Tytgat).
A preliminary report was made in abstract form (38).
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. §1734 solely to indicate this fact.
Address for reprint requests: J. Dekker, Pediatric Gastroenterology and
Nutrition, Laboratory Pediatrics, Rm Ee-1571b, Erasmus Univ. Rotterdam,
Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands (E-mail:
dekker{at}kgk.fgg.eur.nl).
Received 23 July 1998; accepted in final form 13 October 1998.
 |
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