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INTRODUCTION |
The mucosal surface of the gastrointestinal tract is protected by
a visco-elastic mucus gel formed by high molecular mass (0.5-25 × 106) glycoproteins referred to as mucins. The protein
backbones of mucins are heavily substituted with O-linked
oligosaccharides attached to serine and/or threonine residues, and
these amino acids are, together with proline, typically enriched within
so-called mucin domains. Several mucin domains may occur in a single
mucin subunit and these regions are flanked by less heavily
glycosylated segments of the protein core. Large secreted mucins from
the respiratory tract, stomach, and endocervix have been shown to be
linear oligomers formed by subunits joined via disulfide bonds (1, 2).
However, less is known about the macromolecular architecture of
intestinal mucins.
The mucin genes MUC2, MUC3, MUC4,
MUC5B, and MUC6 are expressed in normal human colon
(3-7). MUC2 is believed to be the dominating mucus-forming species in
this tissue and is, so far, the only "large" mucin of which the
cDNA has been fully sequenced (8). The apoprotein contains two
mucin domains, which differ in length and are separated by a
cysteine-containing region (Fig. 1A). The longer domain,
which is referred to as the variable number tandem repeat
(VNTR)1 region is composed
largely of tandemly repeated 23 amino acid peptide units, which vary in
number between alleles and are rich in threonine and proline (9, 10).
The shorter mucin domain comprises a 347-amino acid-long irregular
repeat rich in threonine, serine, and proline. The regions flanking the
mucin domains each show a high degree of similarity to the N- and
C-terminal cysteine-containing D-domains of the prepro-von
Willebrand factor (8), and it has therefore been suggested that MUC2 is
processed and oligomerized in a fashion similar to this protein. In
support of this hypothesis, it has been shown that dimerization occurs
before O-glycosylation and the formation of larger
structures (11, 12). The temporal relationship between further
oligomerization and O-glycosylation is not clear, but data
from Sheehan et al. (13) suggest that there is a slow
assembly of what appear to be fully glycosylated units. Other
investigators have identified biosynthetic intermediates that have been
interpreted as being O-glycosylated monomers and dimers
(14). However, the presence of an O-glycosylated monomer population is in apparent conflict with dimerization preceding O-glycosylation.
The rat Muc2 homologue has been partially cloned and sequenced (15) and
shows significant homology to human MUC2, both in the N and C termini
(16, 17). Thiol reduction of rat and human small intestinal mucins
gives rise to a 118-kDa glycoprotein, previously referred to as the
"link" protein (18, 19). However, cDNA analysis has shown that
this structure is, in fact, part of the MUC2/Muc2 gene (17),
and the finding is explained by proteolytic cleavage within the
C-terminal domain of MUC2/Muc2. Proteolytic cleavage in the N-terminal
region of rat Muc2 has recently been reported (20).
Mucins isolated from rat small intestine are assembled into a complex
that cannot be solubilized by breaking noncovalent bonds; however,
reduction of disulfide bonds brings the complex into solution. Two high
molecular mass glycopeptides (650 and 335 kDa, respectively) were
isolated after reduction and trypsin digestion of the insoluble
glycoprotein complex from rat small intestine (21). The larger of these
glycopeptides apparently corresponds to a large mucin domain from rat
Muc2 (22). In the human colon, most of the mucins also appear as a
glycoprotein complex that is insoluble in guanidinium chloride
(23).
Here, we have isolated the insoluble glycoprotein complex from human
colon and shown that it contains virtually all the MUC2 mucin in this
tissue. Reduction of the complex revealed oligomeric forms of the MUC2
monomer that are insensitive to reduction and, in some samples,
proteolytic cleavage was found to occur in the C-terminal part of the
apoprotein. Preliminary communications have already been published (24,
25).
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EXPERIMENTAL PROCEDURES |
Materials--
Diisopropylphosphofluoridate was from Fluka,
dithiothreitol was from Merck, and N-ethylmaleimide and
trypsin (EC 3.4.21.4., type XIII, treated with
L-(1-tosylamido-2-phenyl) ethyl chloromethyl ketone) were
from Sigma. Guanidinium chloride (practical grade) was obtained from
Acros Organics (Geel, Belgium) and was treated with activated charcoal
and filtered through an Amicon PM-10 membrane before use. Synthetic
peptides conjugated to keyhole limpet hemocyanin were purchased from
Scandinavian Peptide Synthesis AB (Köping, Sweden). Sephacryl
S-500 HR was from Amersham Pharmacia Biotech, Immobilon-P
polyvinylidene difluoride membranes from Millipore, the ECL Western
detection kit was from Amersham Pharmacia Biotech, and Ultrapure
agarose was from Life Technologies, Inc.
Digoxigenin-3-O-succinyl-
-amido-caproic acid hydrazide
and alkaline phosphatase-conjugated anti-digoxigenin antibodies were
from Roche Molecular Biochemicals. Horseradish peroxidase and
alkaline phosphatase-conjugated swine anti-rabbit antibodies were
bought from Dako (Glostrup, Denmark). Bovine serum albumin (fraction V,
pH 7.0) was from Serva.
Analytical Methods--
Density was determined by weighing
aliquots. Total sialic acid was measured according to Davies et
al. (26) with a further modification to allow alkali treatment
(0.5 M NaOH for 5 min) of the samples on-line. Carbohydrate
was determined by slot-blotting aliquots onto nitrocellulose membranes
followed by staining with periodic acid-Schiff (PAS) (27) or as
periodate oxidisable structures with the Roche Molecular Biochemicals
digoxigenin glycan detection system (28). In the latter case, samples
were coated onto multiwell assay plates overnight at 4 °C, and
reactivity was expressed as absorbance at 405 nm.
MUC2 Polyclonal Antisera--
Keyhole limpet
hemocyanin-conjugated synthetic peptides with sequences present in the
MUC2 mucin (8) were used to raise polyclonal anti-MUC2 antibodies in
rabbits. The following peptides were used: CPKDRPIYEEDLKK (LUM2-2
antiserum), located in the D3 domain, NGLQPVRVEDPDGC (LUM2-3
antiserum), located N-terminal to the D4 domain, and
CIIKRPDNQHVILKPGDFK (LUM2-4 antiserum), located C-terminal to the D4
domain. Rabbits were injected intracutaneously with 350 µg of peptide
conjugate in Freund's complete adjuvant. A booster dose (250 µg of
peptide conjugate in Freund's incomplete adjuvant) was given after 3 weeks, and the animals were bled 2-3 weeks thereafter. The
specificities of the antisera were investigated using enzyme-linked
immunosorbent assay and immunohistochemistry.
Enzyme-linked Immunosorbent Assay--
Samples (100 µl) were
coated onto multiwell assay plates overnight at 4 °C followed by
blocking (200 µl) with PBS (0.15 M NaCl, 5 mM
sodium phosphate buffer, pH 7.4) containing 0.5% bovine serum albumin
(w/v) and 0.05% (v/v) Tween 20 (blocking solution) for 1 h at
room temperature. The wells were incubated for 1 h with the
LUM2-2, LUM2-3, or LUM2-4 antiserum (1:1000) in blocking solution.
Alkaline phosphatase-conjugated secondary antibody (swine anti-rabbit
1:2000 in blocking solution) was added to the wells (1 h at room
temperature) and reactivity was detected as absorbance at 405 nm after
1 h of incubation with nitrophenylphosphate (2 mg/ml in 1 M diethanolamine-HCl buffer, pH 9.8, containing 0.5 mM MgCl2)
Immunohistochemistry--
Tissue sections (4 µm) of
formalin-fixed and paraffin-embedded human colonic and small intestinal
tissue on Superfrost Plus slides (Menzel-Gläser,
Braunschweig, Germany) were dewaxed, rehydrated, and treated with
10 mM sodium citrate buffer, pH 6, at 100 °C in a
microwave oven for 2 min. Sections were then treated with 3% (v/v)
hydrogen peroxide in water for 30 min at room temperature to inhibit
endogenous peroxidase activity, washed with 0.15 M NaCl,
0.1 M Tris-HCl buffer, pH 7.4 (Tris-buffered saline), and blocked with goat serum (1:5) in Tris-buffered saline for 1 h. After treatment with the LUM2-2 (1:1000), LUM2-3 (1:1000), or LUM2-4
(1:1000) anti-serum diluted in PBS containing 0.5% bovine serum
albumin (w/v) and 0.05% (v/v) Tween 20 for 1 h followed by the
StreptABComplex/HRP Duet kit (Dako), bound antibodies were visualized
using DAB (0.6 mg/ml) in Tris-buffered saline containing 0.03% (v/v)
hydrogen peroxide for 15 min. Sections were counterstained with Mayers hematoxylin.
Isolation and Purification of "Soluble" and "Insoluble
Mucins"--
Tissue specimens of human colon with a macroscopically
normal appearance, resected during cancer surgery, were frozen and stored at
20 °C until use. The tissue samples were sprinkled with
PBS containing 1 mM diisopropylphosphofluoridate and 5 mM N-ethylmaleimide and thawed on ice, and the
mucosa was removed using a microscope slide. This material is referred
to as "mucosal scrapings." Mucus secretions ("reservoir
washings" and "reservoir urine") were obtained from patients with
artificial urinary bladders constructed from proximal colonic tissue
(29). Reservoir washings were obtained by irrigating such
"neobladders" twice daily with a total of 150 ml of PBS during the
first 10 days postoperatively, when the ureters were catheterized and
the colonic mucosa exposed to a minimal amount of urine. The samples
were transferred to a vessel containing 0.1% w/v NaN3
(final concentration) and kept frozen until use. The material was
thawed and the gel phase obtained by centrifugation (Beckman J2-MC
centrifuge; JA 10 rotor; 5000 rpm; 10 °C; 60 min). Reservoir urine
samples were obtained from the urine of patients with established
neobladders. Sodium azide was added as above, and samples were stored
at 4 °C until centrifugation (Beckman J2-MC centrifuge; JA 20 rotor;
18,000 rpm; 60 min) was performed to recover the mucus gel.
Mucosal scrapings and the gel phase from reservoir washings and
reservoir urine were gently dispersed with a Dounce homogenizer in 6 M guanidinium chloride, 1 mM
diisopropylphosphofluoridate, 5 mM
N-ethylmaleimide, 10 mM sodium phosphate buffer,
pH 7.0, and left stirring at 4 °C overnight. After centrifugation
(Beckman J2-MC centrifuge; JA 20 rotor; 18,000 rpm; 4 °C; 60 min),
the soluble material was removed, and the pellets were re-extracted twice, as described above. The final extraction residues (insoluble glycoprotein complex) were solubilized by reduction in 6 M
guanidinium chloride, 10 mM dithiothreitol, 5 mM EDTA, 0.1 M Tris-HCl buffer, pH 8.0, at
37 °C overnight and then alkylated with 25 mM
iodoacetamide in the same buffer. After centrifugation (Beckman J2-MC
centrifuge; JA 20 rotor; 18,000 rpm; 4 °C; 60 min), the supernatant
was retained. The soluble phases (sols) obtained after the initial
centrifugation to recover the mucus gel from the reservoir washings and
reservoir urine were concentrated over a PM-10 membrane (Amicon).
All fractions (sols, material solubilized with guanidinium chloride
during extractions and material obtained after reduction/alkylation of
the insoluble glycoprotein complexes) were dialyzed against 6 M guanidinium chloride and subjected to isopycnic density
gradient centrifugation in 4 M guanidinium chloride/CsCl
(Beckman Optima L70 ultracentrifuge (15 °C for 90 h in a 50.2 Ti rotor at 36,000 rpm, starting density 1.37 g/ml or a 70.1Ti rotor at
40,000 rpm for 65 h). Fractions were collected from the bottom of the
tubes and analyzed for density, absorbance at 280 nm, sialic acid (with and without pre-treatment with 0.5 M NaOH), PAS reactivity,
and reactivity against the LUM2-2, LUM2-3, and LUM2-4 antisera.
Isolation and N-terminal Amino Acid Sequencing of a MUC2
C-terminal Cleavage Fragment--
Low density material reacting with
the LUM2-4 antiserum, partially separated from the major mucin band in
the density gradients, was pooled (see Fig. 3D, peak II) and
subjected to gel chromatography on a Sephacryl S-500HR column (1.6 × 50 cm) eluted with 4 M guanidinium chloride, 10 mM sodium phosphate buffer, pH 7, at a flow rate of 0.1 ml/min. Fractions (1 ml) were analyzed for sialic acid, as well as
reactivity against the LUM2-2, LUM2-3, and LUM2-4 antisera, and those
reacting with the LUM2-4 antibody were pooled, dialyzed against water,
freeze-dried, and dissolved in 62.5 mM Tris-HCl buffer, pH
6.8, containing 3% (w/v) SDS, 1 mM EDTA and 0.004% (w/v)
bromphenol blue. Samples (50 and 1.25 µg) were treated with 2%
mercaptoethanol (v/v) for 4 min at 100 °C and electrophoresed on a
4-10% gradient polyacrylamide gel (30). Protein bands were transferred to a polyvinylidene difluoride membrane by electroblotting for 2 h using a semi-dry blotter (Sartorius). After blotting, the
membrane was cut into halves, one of which was stained with 0.1%
Coomassie Brilliant Blue in 45% methanol, whereas the other was
incubated with the LUM2-4 antiserum as described for agarose gel
electrophoresis. The Coomassie-stained band from the 50 µg sample
corresponding to that revealed by the LUM2-4 antiserum was cut out and
subjected to Edman degradation using a pulsed liquid-phase sequencer
model 457A (Applied Biosystems) equipped with a 120A analyzer for
on-line detection of phenylthiohydantoin amino acids.
Isolation of Mucin Subunit Subfractions--
Mucin subunits were
pooled after density gradient centrifugation (see Fig. 3D, peak
I) and chromatographed on a Sephacryl S-500HR column (1.6 × 50 cm) eluted with 4 M guanidinium chloride buffer at a
flow rate of 0.1 ml/min. Fractions (1 ml) were analyzed for sialic acid
and reactivity against the LUM2-2, LUM2-3, and LUM2-4 antisera. In
order to obtain mucin subfractions for further analyses, the procedure
was repeated 10 times, and material was pooled as indicated in Fig.
7A (sub-I, sub-II, and sub-III) and concentrated over a
PM-10 filter (Amicon). In order to remove material that may have
detached from the column matrix and would interfere with further
analyses, the samples were finally subjected to density gradient
centrifugation in CsCl/4 M guanidinium chloride (70.1 Ti
rotor), and the mucin band was recovered.
Agarose Gel Electrophoresis and Western Blotting--
Mucin
subunits were dialyzed into 40 mM Tris acetate buffer, pH
8, containing 1 mM EDTA, and prior to electrophoresis, 10% (v/v) of a solution containing glycerol 30% (v/v), 1 mM
EDTA, 1% SDS, 0.002% bromphenol blue in 40 mM Tris
acetate buffer, pH 8, was added. Agarose gel electrophoresis was
performed in a Bio-Rad DNA subcell for 18 h at 30 V and room
temperature using 1.0% (w/v) agarose gels (15 × 15 cm) prepared
in 40 mM Tris acetate buffer, pH 8, containing 1 mM EDTA and 0.1% SDS (31). After electrophoresis, gels
were blotted onto nitro-cellulose membranes for 6 h at 40 mbar
with an Amersham Pharmacia Biotech LKB VacuGene XL vacuum blotter using
0.6 M NaCl, 0.06 M sodium citrate buffer, pH
7.0.
After blotting, membranes were blocked with 0.5% (w/v) bovine serum
albumin in PBS containing 0.05% (v/v) Tween 20 (blocking solution) for
1 h and incubated with the LUM2-2, LUM2-3, or LUM2-4 antiserum
(1:1000) diluted in blocking solution for 1 h. Bound antibody was
detected by incubation with horseradish peroxidase-conjugated swine
anti-rabbit antibody (1:2000 in blocking solution) for 1 h
followed by the ECL Western detection kit. All incubations were carried
out at room temperature.
Isolation of High Molecular Mass Tryptic
Glycopeptides--
Mucin subunits (see Fig. 3D, peak I)
were dialyzed against 0.1 M
NH4HCO3, pH 8.0, treated with trypsin (50 µg)
overnight at 37 °C, dialyzed against 4 M guanidinium
chloride, 10 mM sodium phosphate buffer, pH 7, and
subjected to gel chromatography on a Sephacryl S-500HR column (1.6 × 50 cm) eluted with the same buffer at a flow rate of 0.1 ml/min.
Fractions (1 ml) were collected and analyzed for sialic acid. The
material was pooled into two fractions, referred to as glycopeptides A
and B (see Fig. 5), and subjected to density gradient centrifugation in
CsCl/4 M guanidinium chloride as described for the mucin
subunits in order to ensure complete removal of peptide material and
contaminants from the column matrix. Finally, the two glycopeptide
populations were dialyzed against water and freeze-dried. A larger pool
of material was obtained using a column (2.6 × 93 cm) eluted with
the same buffer at a flow rate of 0.27 ml/min.
Amino Acid Analysis of the High Molecular Mass Tryptic
Glycopeptides--
Amino acid analysis was carried out on
approximately 2.5 µg of glycopeptides A and B according to Ref. 32
with modified gas-phase hydrolysis conditions. Samples were manually
hydrolyzed in a vessel with 500 µl of 5.7 M HCl and a
crystal of phenol at 110 °C for 24 h. The protein hydrolysate
was dissolved in 250 mM borate buffer (pH 8.5), and
analyzed with a GBC automated Aminomate HPLC system (GBC Scientific,
Australia). Amino acids were derivatized with Fmoc and separated on a
reversed-phase column (ODS-Hypersil, 5-µm packing, 150 × 4.6 mm
internal diameter, Keystone Scientific) at 38 °C. Amino acid Fmoc
derivatives were detected by fluorescence (LC1250 fluoro-detector
system; excitation wavelength, 270 nm; emission wavelength, 316 nm),
and peak analysis was controlled by the WinChrom chromatography data
system (GBC), version 1.2.
Monosaccharide Analysis of the High Molecular Mass Tryptic
Glycopeptides--
Monosaccharide analysis was carried out on mucin
aliquots from the same solution (2.5 µg/5 µl) as described above.
Sialic acids were hydrolyzed from the mucin in 0.1 M
trifluoroacetic acid at 80 °C for 40 min in plastic screw-cap
Eppendorf centrifuge tubes. Neutral and amino sugars were determined
after hydrolysis in 2 M trifluoroacetic acid at 100 °C
for 4 h and 4 M HCl at 100 °C for 4 h,
respectively. The hydrolysates were dried under vacuum, dissolved in 50 µl of water and analyzed by high pressure anion exchange
chromatography (Dionex, CarboPac PA1 column, 4 × 250 mm) combined
with pulsed amperometric detection (Dionex DX 500). Neutral and amino
sugars were separated isocratically at 1 ml/min in 12 mM
NaOH, and N-acetyl neuraminic acid was eluted using a gradient of 0-200 mM sodium acetate in 250 mM
NaOH over 15 min. No N-glycolyl neuraminic acid was detected
even at a concentration of sodium acetate greater than 200 mM. Quantitation was performed using internal standards
(2-deoxyglucose for the neutral and amino sugars; lactobionic acid for
the sialic acids) with reference to the appropriate response factors.
Laser Light Scattering--
Reduced mucin subunits obtained from
the insoluble glycoprotein complex of reservoir urine after density
gradient centrifugation (see Fig. 3D, peak I), were dialyzed
against and then diluted with 6 M guanidinium chloride, 1 mM EDTA, 10 mM sodium phosphate buffer, pH 6.5. The intensity of light scattered at angles between 20° and 90° was
determined with a Malvern 4600 SM system equipped with a 25 mW HeNe
laser. The data were plotted in accordance with Zimm (33), and a
refractive index increment of 0.104 ml/g was used (34). The subunits
were studied at 37.5, 75, 150, and 300 µg/ml.
Analytical Ultracentrifugation--
Mucin subunits obtained by
density gradient centrifugation of the reduced insoluble glycoprotein
complex from reservoir urine (see Fig. 3D, peak I), subunit
subfractions (I, II, and III) obtained after gel chromatography (see
Fig. 7A), and glycopeptides A and B (see Fig. 5) were
dialyzed against 6 M ultrapure guanidinium chloride.
Sedimentation equilibrium experiments were performed in a Beckman
Optima XL-A analytical ultracentrifuge using six-channel charcoal
filled Epon centerpieces. Sedimentation distributions were, when
possible, studied at several concentrations using absorption optics at
230 nm. The weight-average molecular weight of the entire distribution
was determined by extrapolating the point-average value of M* to the
bottom of the cell (35), and the obtained values were then, when
possible, extrapolated to zero concentration. Partial specific volumes
of 0.66 and 0.65 ml/mg were used for subunits and high molecular mass
glycopeptides, respectively (36).
Southern Blot Analysis--
DNA-containing fractions obtained
after density gradient centrifugation of the guanidinium chloride
soluble material from the mucosal scrapings were pooled, desalted, and
subjected to digestion with the restriction enzyme HinfI.
The digest was fractionated on a 0.8% agarose gel, transferred to a
nylon membrane and hybridized with 32P-labeled SMUC41 probe
as described previously (10).
 |
RESULTS |
MUC2 Antibodies--
The LUM2-2, LUM2-3, and LUM2-4 antisera were
raised against three different sequences present in the C- and
N-terminal regions of MUC2 (Fig.
1A). Each antiserum reacted
only with the peptide used for immunization (Fig. 1B), and
no reactivity with the peptides used to raise the other antibodies was
observed (results not shown). All antisera reacted with mucin subunits
obtained after density gradient centrifugation (Fig.
1C).

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Fig. 1.
The MUC2 apoprotein and reactivity of the
MUC2 antisera with synthetic peptides and mucin subunits.
A, schematic picture of the MUC2 apoprotein. The open
boxes represent the domains with a high degree of homology to the
D-domains in the prepro-von Willebrand factor, and the
filled boxes illustrate the Ser/Thr/Pro-rich 347-amino acid
irregular repeat domain and the VNTR region with about 100 repeats of
23 amino acid sequence, rich in Thr and Pro. The locations of the
sequences used to raise antibodies are indicated by
arrowheads together with the name of the corresponding
antibody. B, the synthetic peptides (0.5 µg/ml) used to
raise the LUM2-2, LUM2-3, and LUM2-4 antisera were coated onto
multiwell assay plates and incubated with the MUC2 antisera at
different dilutions. C, mucin subunits (1.0 µg/ml)
obtained after density gradient centrifugation were coated onto
multiwell assay plates and incubated with LUM2-2 ( ), LUM2-3 ( ),
or LUM2-4 ( ) at different dilutions. Bound antibody was detected as
described under "Experimental Procedures" and expressed as
absorbance at 405 nm.
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Immunohistochemistry--
In human colon, staining with the LUM2-3
antiserum was seen over the goblet cells in the surface mucosa and
along the entire length of the crypt (Fig.
2A). Surrounding tissues were
not stained; however, reactivity was seen over secreted material at the
luminal surface of the mucosa. The LUM2-2 and LUM2-4 antibodies showed similar results (data not shown). Also in the small intestine, the
LUM2-3 antibody stained the goblet cells over the surface mucosa and
along the crypts, whereas the enterocytes showed no reactivity (Fig.
2B). Again, the other two antibodies provided similar
results (data not shown).

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Fig. 2.
Immunohistochemical staining of human colon
and small intestine. Sections (4 µm) of paraffin-embedded colon
(A) and small intestine (B) were stained with the
LUM2-3 antiserum and counterstained as described in the text. The
bars represent 100 µm.
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Isolation of MUC2 Mucin Subunits--
Repeated extractions of
human colonic mucins with 6 M guanidinium chloride
fractionated them into those that were soluble in this solvent and
those that resisted extraction. Density gradient centrifugation of the
guanidinium chloride soluble fraction (results not shown) showed that
little material was present at a density expected for mucins, although
some sialic acid-containing and PAS-reactive material banding at a
density 1.29-1.38 g/ml was detected. However, this material did not
react, or reacted only weakly, with the three MUC2 antisera. The sol
fractions from the reservoir urine and reservoir washings also
contained little MUC2.
Density gradient centrifugation of the reduced insoluble glycoprotein
complex revealed a single unimodal peak between 1.30 and 1.45 g/ml that
reacted strongly with the sialic acid and PAS analyses (Fig.
3, A-C). The material was
well separated from DNA and low buoyant density proteins that showed
strong absorbance at 280 nm at 1.47 g/ml and at the top of the gradient
respectively. Pretreatment with alkali before sialic acid analysis
increased the color yield as much as 15-fold in some, but not all,
preparations, suggesting the presence of O-acetylated sialic
acids. Reactivity against the LUM2-2 and LUM2-3 antisera coincided with
the mucin distribution as defined by sialic acid and PAS reactivity
(Fig. 3, D-F). The major part (>95%) of the MUC2 mucin
was present within the insoluble glycoprotein complex.

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Fig. 3.
Isopycnic density gradient centrifugation of
human colonic mucin subunits. Mucin subunits were obtained after
reduction of the insoluble glycoprotein complex from reservoir urine
(A and D), mucosal scrapings of colonic tissue
(B and E), and reservoir washings (C
and F) and subjected to density gradient centrifugation in 4 M guanidinium chloride/CsCl. Fractions were analyzed for
density ( ), sialic acid before ( ) and after ( ) pretreatment
with NaOH, PAS ( ), absorbance at 280 nm (- - -)
(A-C), and reactivity with the LUM2-2( ), LUM2-3( ),
and LUM2-4( ) antisera (D-F). The bars
indicate material that was pooled and subjected to further
analysis.
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The reactivity with the LUM2-4 antiserum varied between preparations.
In most, but not all, the reactivity appeared as two partially resolved
peaks, one appearing at the same density as the mucin subunits, the
second one on the lower buoyant density side of the main peak as would
be expected for a less glycosylated protein (Fig. 3F). In
some preparations, the reactivity against the LUM2-4 antibody appeared
as a peak with a density only slightly lower than that of the major
mucin band (Fig. 3, D and E), and in others, the
reactivity followed that of the main mucin distribution.
MUC2 C-terminal Peptide--
Low density material reacting with
the LUM2-4 antibody (Fig. 3D, peak II) was pooled and
subjected to gel chromatography on Sephacryl S-500HR. A peak reacting
with the LUM2-4 antiserum was well included on the column (Fig.
4), and virtually no reactivity with the
LUM2-2 or LUM2-3 antibodies was detected (results not shown). Similar
results were obtained with corresponding material from several other
preparations. The peak reacting with the LUM2-4 antiserum was pooled
and subjected to SDS-polyacrylamide electrophoresis followed by Western
blotting. A major band of approximately 120 kDa that reacted strongly
with both Coomassie Blue and the LUM2-4 antibody was detected (Fig. 4,
inset). This band was cut out and subjected to N-terminal
amino acid sequence analysis. The sequence PHYVTFD was obtained (with
the reservation that Y could possibly be V) corresponding to amino
acids 292-298 present within the C-terminal part of MUC2 as defined by
Gum et al. (37).

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Fig. 4.
Gel chromatography on Sephacryl S-500HR of
the putative C-terminal cleavage peptide obtained after reduction of
the insoluble glycoprotein complex. Low density, LUM2-4-reactive
material obtained after density gradient centrifugation (Fig. 3D,
peak II) was subjected to gel chromatography on a Sephacryl S-500
HR column (1.6 × 50 cm) eluted with 4 M guanidinium
chloride, 10 mM sodium phosphate buffer, pH 7.0. Fractions
(1 ml) were analyzed for absorbance at 280 nm (- - -) and
reactivity against the LUM2-4 antiserum ( ). The LUM2-4-reactive
material was pooled (as shown) and subjected to SDS-polyacrylamide
electrophoresis on a 4-16% gradient gel, blotted onto a
polyvinylidene difluoride membrane followed by Coomassie Blue staining
(C) or Western blotting (W) using the LUM2-4
antiserum (inset). The numbers indicate estimated molecular
weights as determined using high and low molecular weight markers
(electrophoresis calibration kits (Amersham Pharmacia Biotech)).
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High Molecular Mass Tryptic Glycopeptides--
When mucin subunits
(Fig. 3D, peak I) were subjected to trypsin digestion
followed by gel chromatography on Sephacryl S-500HR, two major peaks
(referred to as glycopeptides A and B, respectively) were detected with
the sialic acid assay (Fig. 5). Amino
acid analysis was performed on the two glycopeptides from two different individuals and compared with the deduced compositions of the VNTR
region and the irregular repeat domain (Table
I). Glycopeptide A contains high amounts
of threonine (approximately 45 mol %) and proline (approximately 20 mol %) but low amounts of serine (approximately 4 mol %), as expected
for the VNTR region. The amino acid composition of glycopeptide B was
close to that expected for the irregular repeat domain with high
amounts of threonine (approximately 38 mol %), serine
(approximately 10 mol %), and proline (approximately 28 mol
%).

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Fig. 5.
Gel chromatography on Sephacryl S-500 of
trypsin digested mucin subunits. Mucin subunits (Fig.
3D, peak I) were digested with trypsin and subjected to gel
chromatography on a Sephacryl S-500 HR column (1.6 × 50 cm)
eluted with 4 M guanidinium chloride, 10 mM
sodium phosphate buffer, pH 7.0. Fractions (1 ml) were analyzed for
sialic acid. The material was pooled into two fractions, A
and B.
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Table I
Amino acid composition of glycopeptides A and B isolated from two
individuals together with the amino acid composition of the VNTR region
and the irregular repeat domain
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The results of carbohydrate analysis of glycopeptides A and B are shown
in Table II. The monosaccharide
composition of the two glycopeptides is similar, in keeping with a
similar oligosaccharide substitution. Little mannose was found,
suggesting that no N-linked oligosaccharides occur within
these domains of the MUC2 mucin. N-acetyl neuraminic acid
but no N-glycolyl neuraminic acid was present.
Analytical ultracentrifugation showed that the molecular weights of
glycopeptides A and B are 930,000 and 180,000, respectively (Fig.
6). The ratio (5.2:1) of the molecular
weights for the large and small glycopeptides is similar to that
(5.9:1) expected from the differences in lengths of the VNTR region
predicted from the most common allele (100 repeats) and the invariant
irregular repeat domain, assuming a similar glycosylation of the two
protein regions. The ratio of the yields of glycopeptides A and B
(5.8:1) suggests that the two domains occur in equimolar proportions in
the molecule.

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Fig. 6.
Analytical ultracentrifugation of
glycopeptides A and B. The molecular mass of glycopeptides A ( )
and B ( ) obtained after trypsin digestion and gel chromatography
were determined using sedimentation equilibrium in the analytical
ultracentrifuge at 20 °C for at least 70 h. Samples were
monitored several times during the experiment to ensure that
equilibrium was achieved. Glycopeptide A was studied at 0.4, 0.5, 0.6, 0.8, and 1.0 mg/ml at 3000 rpm, and glycopeptide B was studied at 0.5, 0.6 0.8, and 1.0 mg/ml at 8000 rpm. The obtained values were
extrapolated to zero concentration.
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MUC2 Subunit Populations--
When MUC2 mucin subunits from
the insoluble glycoprotein complex (Fig. 3D, peak I) were
subjected to gel chromatography on Sephacryl S-500HR, a number of
partially separated populations reacting with the LUM2-2, LUM2-3 and
LUM2-4 antibodies were identified (Fig.
7A). The largest population
eluted close to the void volume of the column and the smallest one
appeared just ahead of the position for glycopeptide A. When individual
chromatographic fractions of the partially resolved MUC2 populations
were subjected to agarose gel electrophoresis, followed by Western
blotting using the LUM2-3 antiserum, a number of distinct bands were
obtained (Fig. 7A, inset). Molecules eluting first from the
column had a lower mobility on the agarose gel than those that were
more included and all bands reacted with all three MUC2 antisera
(results not shown). Subunits from several different preparations were
subjected to gel chromatography and agarose gel electrophoresis with
similar or identical results.

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Fig. 7.
Gel chromatography on Sephacryl S-500 of
reduced mucin subunits obtained from the insoluble glycoprotein
complex. A, mucin subunits obtained after density
gradient centrifugation (Fig. 3D, peak I) were subjected to
gel chromatography on a Sephacryl S-500 HR column (1.6 × 50 cm)
eluted with 4 M guanidinium chloride, 10 mM
sodium phosphate buffer, pH 7.0, at 0.1 ml/min. Fractions (1 ml) were
analyzed for sialic acid after pre-treatment with NaOH ( ),
absorbance at 280 nm (- - -), and reactivity with the LUM2-2
( ), LUM2-3 ( ), and LUM2-4 ( ) antisera. Individual fractions
containing mucin subunits were subjected to agarose gel electrophoresis
followed by Western blotting using the LUM2-3 antiserum
(inset). B, fractions were pooled into three
populations (sub-I, sub-II, and sub-III) as indicated in A
and re-run on the column as above; the fractions were analyzed for
carbohydrate (sub-I,  ; sub-II, - - -; sub-III,
· · · ·) using the glycan detection method. Agarose gel
electrophoresis followed by Western blotting using the LUM2-3 antiserum
of the three individual subunit populations is shown in the
inset.
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Corresponding fractions from 10 gel chromatography runs were pooled as
three populations referred to as sub-I, sub-II, and sub-III,
respectively, according to the bars shown in Fig.
7A, concentrated, and recovered using density gradient
centrifugation. The latter step was used to remove material that may
have detached from the column matrix and would interfere with further
analyses (see below). Subsequent gel chromatography and agarose gel
electrophoresis showed that the sub-III population contained mainly the
"fastest" band, whereas the sub-I and sub-II populations were
mixtures of the two "slowest" and the two fastest, respectively
(Fig. 7B).
Size of the MUC2 Subunit Populations--
MUC2 mucin subunits from
the insoluble glycoprotein complex (Fig. 3D, peak I) were
subjected to molecular weight determination using analytical
ultracentrifugation and laser light scattering. A Zimm plot of the data
obtained by laser light scattering (Fig. 8) reveals a molecular weight of 4.0 × 106 for the entire subunit population. The results
obtained using analytical ultracentrifugation (Fig.
9) for the same material provided a
molecular weight of 3.8 × 106, in good agreement with
that given by light scattering.

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Fig. 8.
Zimm plot analysis of light scattering data
for the mucin subunits. Mucin subunits were isolated after density
gradient centrifugation of the insoluble glycoprotein complex from
reservoir urine, solubilized by reduction (Fig. 3D, peak I).
Readings were taken at = 20°, 25°, 30°, 35°, 40°, 45°,
60°, 75°, and 90°, and the subunits were studied at 37.5, 75, 150, and 300 µg/ml at 20 °C. Extrapolation to = 0 was
performed from 20° to 45°.
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Fig. 9.
Analytical ultracentrifugation of the mucin
subunits and the three subpopulations thereof. Mucin subunits
isolated after density gradient centrifugation (Fig. 3D, peak I) ( )
and the three subfractions (sub-I, ; sub-II, ; sub-III, )
obtained after gel chromatography (Fig. 7A) were studied
with sedimentation equilibrium. Samples were run for approximately
70 h at 1500 rpm at 20 °C and monitored several times during
the experiment to ensure that equilibrium was achieved. The
unfractionated subunits were studied at 0.15, 0.20, 0.22, 0.35, and
0.47 mg/ml, whereas sub-I was studied at 0.21, sub-II at 0.20, and
sub-III at 0.17 mg/ml, respectively. The obtained values were
extrapolated to zero concentration. For sub-I, sub-II, and sub-III, the
same concentration dependence as for the unfractionated material was
used (- - -); for the sub-III population, also, the concentration
dependence obtained for glycopeptide A (Fig. 6) was used
(· · · ·).
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The molecular weights for the sub-I, sub-II, and sub-III populations
were determined using analytical ultracentrifugation, but due to the
small amounts of material available, the analysis could only be carried
out at a single concentration, and the concentration dependence could
not be assessed. However, the value for the sub-II population (2.5 × 106 at 0.20 mg/ml) falls on the line describing the
concentration dependence for the entire distribution, and assuming the
same concentration dependence, a molecular weight of 3.8 × 106 is obtained. Using the same concentration dependence,
the value for the sub-I population (3.4 × 106 at 0.21 mg/ml) was extrapolated to 4.7 × 106. Because the
concentration dependence is usually more pronounced for larger
molecules than for smaller ones, this value represents an
underestimation rather than an overestimation. When the value for the
sub-III population (1.4 × 106 at 0.17 mg/ml) was
corrected using the concentration dependence for the entire
distribution and for glycopeptide A (Fig. 9), values of 2.4 × 106 and 1.5 × 106, respectively, were
obtained. The two values are likely to represent over- and
underestimations, and the "true" value for the molecular weight of
the sub-III population is expected to fall between the two.
Southern Blot Analysis and Comparison of the Purified MUC2
Subunits--
In order to assess the length of the VNTR region in the
various samples, DNA present in the density gradients of material soluble in guanidinium chloride was subjected to digestion with the
HinfI restriction enzyme followed by electrophoresis and
Southern blot analysis using the SMUC41 probe. Eleven different
individuals were tested. Eight of the samples contained a single band
of approximately 7-8 kilobases or two closely spaced bands
corresponding to the most common long allele(s) with a VNTR region
containing of the order of 100 repeats of the 23 amino acid peptide
unit. However, three individuals expressed both a long and a much
shorter (~4-kilobase) allele (Fig.
10). Agarose gel electrophoresis and
Western blotting of the mucin subunits from individuals homozygous for
two long alleles displayed a pattern of three major bands similar to
those shown above, whereas all individuals heterozygous for a long and a short allele showed a much more extensive ladder pattern (Fig. 10).
The putative MUC2 monomers derived from the short and long alleles are
referred to as
and
respectively, and 
and 
are
"homodimers" containing two short and two long monomers,
respectively. The band appearing between the 
and 
ones is
interpreted as a heterodimer (
) consisting of one short
and one long monomer.

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Fig. 10.
Western blot analysis of MUC2 reduced
subunits and Southern blot analysis of the VNTR region.
A, mucin subunits obtained after density gradient
centrifugation (mucosal scrapings) were subjected to agarose gel
electrophoresis followed by Western blotting using the LUM2-3
antiserum. B, DNA isolated from the extraction-soluble
fraction was subjected to digestion with HinfI and analyzed
by agarose gel electrophoresis followed by probing with SMUC41. Data
shown were obtained from one individual expressing both a long and a
short allele (lane 1) and one homozygous for long alleles
(lane 2). The putative structure of the isoforms is
indicated alongside the gel, where and represent the products
of the short and the long, allele respectively. The numbers indicate
estimated size in kilobases as determined by comparison with Raoul
molecular weight markers (Appligene, Durham, United Kingdom), the
1-kilobase ladder (Life Technologies, Ltd., Paisley, United Kingdom),
and lambda HindIII digest (Life Technologies, Inc.).
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DISCUSSION |
The antibodies raised against the three different peptide
sequences within the MUC2 apoprotein were shown to recognize both the
cognate peptide and the purified fully glycosylated mucin subunits
(Fig. 1, B and C). The peptide sequence recognized by the
LUM2-2 antibody is located N-terminal to the irregular repeat domain,
whereas LUM2-3 and LUM2-4 are directed to sequences C-terminal to the
VNTR region (Fig. 1A), and together the three antibodies flank the entire central glycosylated domain of MUC2. All three antibodies stained the goblet cells in human colon and small intestine (Fig. 2) but not normal human gastric mucosa or the submucosal glands
of normal human airway, suggesting that there is no cross-reactivity of
the antisera with MUC5AC, MUC5B, and MUC6.
Mucosal scrapings and the gel phase of colonic secretions were
subjected to extensive extractions using 6 M guanidinium
chloride, and the putative insoluble glycoprotein complex was finally
brought into solution using reduction. Density gradient centrifugation of the soluble material and the insoluble fraction after reduction revealed that most of the mucins were present in the insoluble glycoprotein complex, and virtually all of the MUC2 reactivity, as
determined by using the three antibodies, was confined to this fraction. It is concluded that the major part of human intestinal MUC2
occurs as an insoluble glycoprotein complex. Fresh specimens of colonic
tissue would be expected to provide the best source of colonic mucins,
but sufficiently large samples of tissue are difficult to obtain. For
this reason, secretions that can be obtained in relatively large
amounts from artificial urinary bladders constructed from colonic
segments, were also used. Previous studies have shown that such
secretions contain a major population of mucins that is insoluble in
guanidinium chloride but can be isolated as a distinct component
following reduction (38). In all respects studied here, the MUC2 mucin
from artificial bladders was similar to that obtained from mucosal
scrapings showing that artificial bladders are a good source of colonic MUC2.
The large secreted mucus-forming mucins from stomach, respiratory
tract, and cervix are long linear structures, formed by subunits linked
end-to-end by disulfide bonds. The major part of the macromolecules is
readily extracted using chaotropic salts such as guanidinium chloride.
In contrast, mucins from rat and human intestine resist extraction with
6 M guanidinium but can be isolated as an insoluble
glycoprotein complex following extensive treatment of the tissue with
this solvent (21, 23). The fact that the mucins cannot be solubilized
with guanidinium chloride suggests a larger involvement of covalent
bonds in the formation of intestinal mucus than in, for example,
gastric mucus and the gel phase of airway secretions. The physiological
relevance of this is not known but may be related to the need to
protect the intestinal surface with a highly resistant mucus gel that
is at the same time porous enough to allow transport from the lumen over the epithelium.
In many, but not all, preparations, the reactivity with the LUM2-4
antiserum in the density gradient of the subunits from the insoluble
MUC2 complex was present at a lower buoyant density than the main mucin
population as defined by carbohydrate analysis and the reactivity with
the two other antibodies. The low density LUM2-4 reactive material was
isolated using gel chromatography and SDS-polyacrylamide gel
electrophoresis and shown to be a distinct component with an apparent
molecular weight of approximately 120,000, similar to that (118,000) of
the so-called "link" glycoprotein that was first identified after
reduction of intestinal mucins (18, 19) and later shown to comprise the
C-terminal part of the MUC2/Muc2 apoprotein (17). Edman degradation
provided the sequence PHYVTFD for the 120-kDa component, corresponding
to the N-terminal amino acids of the putative link protein. The 120-kDa component identified here thus corresponds to this structure and is,
when cleaved, apparently at least in part left bound to the insoluble
MUC2 complex via disulfide bonds. It is not known at present whether
proteolytic cleavage in the C-terminal part of MUC2 has any biological
significance or merely reflects proteolysis that occurs during the
isolation procedure.
In order to assess the size of the MUC2 monomers and to investigate
whether or not the insoluble glycoprotein complex contains other major
components than MUC2, the subunit population obtained using density
gradient centrifugation was subjected to trypsin digestion followed by
gel chromatography. Two major glycopeptide populations (A and B) with
similar carbohydrate compositions were identified, and the low amount
of mannose present suggests that most of the oligosaccharides are
O-linked structures. The amino acid compositions and the
relative sizes of glycopeptides A and B are in good agreement with
those expected from the VNTR region and the irregular repeat domain,
respectively, strongly suggesting that they correspond to these two
domains of MUC2 and that no other glycoproteins contribute
significantly to the subunits obtained from the insoluble complex.
The molecular weights of glycopeptides A (930,000) and B (180,000) are
in keeping with those estimated from structures containing 2300 amino
acids (100 repeats of 23 amino acids as for the most common allele) and
347 amino acids, respectively, assuming approximately 70-80%
carbohydrate, realistic values for epithelial mucins. It should be
pointed out that the entire VNTR region has not been sequenced and that
the amino acid composition predicted by the SMUC41 and SMUC42 clones
(3) shows that lysine residues, and thus trypsin-sensitive sites, may
appear in this part of the mucin. If so, the size of the VNTR region
determined as the molecular weight of glycopeptide A may be an
underestimation. However, the amino acid sequence for the irregular
repeat domain is defined, and the size of the smallest possible tryptic
fragment can thus be predicted. Because the ratios of the molecular
weights (5.2:1) and of the predicted lengths of the protein cores
(5.9:1) are similar, it appears likely
assuming a similar
glycosylation of the two glycopeptides
that glycopeptide A represents
the major part of the VNTR region, if not the entire region. A more
detailed chemical characterization of these glycosylated regions is
currently in progress. Finally, the ratio of the yields of
glycopeptides A and B (5.8:1) compared with that of the molecular
weights (5.2:1) suggests that the two fragments occur in equimolar
proportions as would be expected for the VNTR region and the irregular
repeat domain. From the values of the molecular weights of
glycopeptides A and B, the size of the C-terminal cleavage fraction
obtained by SDS-polyacrylamide gel electrophoresis and the size of the N-terminal domain (approximately 1400 amino acids; molecular weight, 150,000), the molecular weight of the MUC2 monomer from human colon is
predicted to be on the order of 1.5 × 106.
Gel chromatography and gel electrophoresis were used to show that the
MUC2 subunits comprise a mixture of well defined populations. Gel
chromatography would suggest that the subpopulations are of different
size and, as expected, molecules with a larger hydrodynamic volume were
more retarded on agarose gel electrophoresis than the smaller ones. The
possibility that the bands represent proteolytically truncated species
was dismissed because all bands reacted with all three antibodies that
together "cover" the major part of the MUC2 apoprotein. For
example, the C-terminal cleavage that occurs in some, but not all, MUC2
subunits does not give rise to bands that lack reactivity with the
LUM2-4 antiserum, showing that the removal of domains of this size does
not influence the size of the molecules to the extent that it affects
the electrophoretic mobility and/or the chromatographic behavior.
Furthermore, the molecular weight obtained by both laser light
scattering (4 × 106) and analytical
ultracentrifugation (3.8 × 106) for the entire
subunit population is very high compared with that (1.5 × 106) predicted for the MUC2 monomer on the basis of data
obtained for the MUC2 fragments.
To further investigate the size of the MUC2 subunits, populations
enriched in the various bands were isolated using gel chromatography and subjected to molecular weight determinations. The molecular weight
for the most included, smallest (sub-III) population, which represents
almost entirely the fastest moving band was estimated to be greater
than 1.5 × 106 but less than 2.4 × 106: significantly smaller than that (approximately 4 × 106) for the entire population, but close to the value
predicted for a MUC2 monomer (see above). The molecular weight for the
sub-II (3.8 × 106) and sub-I (4.7 × 106) populations would accommodate two and three units of
sub-III, respectively, considering the fact that they both represent
mixtures of larger and smaller species. The interpretations are
complicated by the fact that the concentration dependence of the
subpopulations of the subunits can only be estimated. However, the size
differences between the entire subunit population and that obtained for
the smallest population is so large that it is safe to conclude that the MUC2 population obtained after reduction is not a true monomer but
must comprise a series of oligomers joined by linkages that are not
sensitive to reduction. This conclusion is strongly supported by the
electrophoretic patterns obtained for heterozygous individuals with two
MUC2 alleles of very different size, which displayed a much more
extensive ladder pattern. The data obtained from such individuals
indicate that both alleles are expressed and that both may form dimers
(
, 
, and 
) and possibly also higher oligomers in all
possible permutations, as indicated in Fig. 10.
While this work was in progress, a paper was published that suggested
the presence of reduction-insensitive MUC2 dimers in LS174T cells (39).
After reduction of insoluble mucins from this source, two highly
polydisperse bands were observed with agarose gel electrophoresis. The
components corresponding to the two bands were partially separated
using rate-zonal centrifugation in a sucrose gradient, and the
differences in sedimentation rates were interpreted as being consistent
with the behavior of a monomer and a dimer. The authors suggest that
the smeared appearance of the individual bands following agarose gel
electrophoresis reveals glycosylation differences; however, the range
of migration rates covered by the smears is in many cases of the same
order of magnitude as the differences in migration rate between the
putative monomer and the surprisingly sharp dimer bands. Furthermore,
the broad and overlapping distributions of the putative monomer and
dimer revealed using rate-zonal centrifugation suggest that both
populations encompass a range of s values that is actually
larger than the difference in s value obtained from the
"peak positions," i.e. each population may contain
molecules that differ in size by a factor of more than 2. It is thus
not clear from these experiments, without the support of independent
data, which differences in migration rates may be interpreted as
glycosylation polydispersity and which as "oligomerization."
Nevertheless, the interpretations made by Axelsson et
al. (39) are consistent with our findings.
In summary, we have shown that the MUC2 mucin from human colon is
present as an insoluble glycoprotein complex and that this glycoprotein
complex is mainly composed of MUC2. The complex is assembled from MUC2
"subunits" using disulfide bonds. The subunits obtained after
reduction occur as monomer and a series of oligomers apparently joined
by a novel, reduction-insensitive linkage, the nature of which is
currently unknown. High molecular weight glycopeptides corresponding to
the VNTR region and the irregular repeat domain were identified
following trypsin digestion, and it was shown that proteolytic cleavage
may occur in the C-terminal part of MUC2, leaving a 120-kDa fragment
attached to the insoluble complex with disulfide bonds.