From the Department of Medical Biochemistry, Göteborg University, Medicinaregatan 9A, S-413 90 Gothenburg, Sweden
Received for publication, October 1, 2002, and in revised form, January 14, 2003
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ABSTRACT |
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During purification of a recombinant MUC2 C
terminus expressed in CHO-K1 cells, the protein was partly cleaved when
buffers with a pH of 6.0 were used. When buffers with higher pH values were used, less cleavage was found. Disulfide bonds held the two fragments generated together as these were only observed after reduction. Edman sequencing of the C-terminal 110-kDa fragment revealed
that the cleavage had occurred at an Asp-Pro bond, a site described
previously to generate the so-called "link peptide" after disulfide
bond reduction. In vitro studies on the conditions for
cleavage showed that it occurred in a time-dependent manner at a pH below 6.0. Furthermore, the reaction was not enzyme-mediated as
it occurred in pure preparations of the MUC2 C terminus and was not
inhibited by protease inhibitors. When expressed in the mucin producing
cell line LS 174T, the C terminus was cleaved to a higher extent
compared with the CHO-K1 cells. Neutralizing the secretory pathway with
either NH4Cl or bafilomycin A1 inhibited this cleavage.
Altogether, our results suggest that the cleavage is an autocatalytic
reaction that occurs in the acidic environment of the late secretory
pathway. Furthermore, the cleavage produced a new, reactive C terminus
that has the potential to attach the mucin to itself or other
molecules. Because a pH below 6 can be reached in the late secretory
pathway and on mucosal surfaces, the cleavage and possible
cross-linking are likely to be of biological importance.
Mucins are large glycoproteins with the carbohydrates accounting
for up to 90% of its molecular mass (1, 2). These sugars are mainly
O-linked to Ser and Thr residues localized to regions called
mucin domains. These domains contain tandemly repeated sequences (rich
in Ser, Thr, and Pro) that vary in number, length, and amino acid
composition depending on the particular mucin (3). Mucins account for
the major part of the mucous layers covering the epithelial surfaces
throughout the body, where they are believed to have several functions
including protection and lubrication of the underlying epithelia.
There are two major types of mucins, membrane-bound and secreted. In
human, eight membrane-bound (MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC13,
MUC16, and MUC17) (4-11) and five secreted mucins (MUC2, MUC5B,
MUC5AC, MUC6, and MUC7) (12-16) have been identified so far. Two
additional ones cannot be classified (MUC8 and MUC11) (7, 17). The
secreted mucins can be further sub-divided as being either gel-forming
(MUC2, MUC5B, MUC5AC, and MUC6) or not (MUC7). The ability to form gels
is dependent on the capacity of monomers to form polymeric structures.
The intermolecular interactions are of disulfide-bond nature and
mediated through the N- and C-terminal Cys-rich domains of mucins
(reviewed in Ref. 2). Both these domains show considerable sequence
homology to the human prepro-von Willebrand factor
(vWf)1 (18).
The most thoroughly studied mucin is the porcine submaxillary mucin
(PSM) (19). PSM has been shown to dimerize via its C-terminal domain in
the endoplasmic reticulum (ER) (20, 21) and oligomerize through its
N-terminal domain in the acidic compartments of the Golgi complex,
presumably making up a branched structure (22, 23).
In analogy with the results from the studies of PSM, we have reported
previously (24, 25) that the human MUC2 mucin forms disulfide-stabilized dimers in the ER. These studies were made on the
full-length MUC2, but due to the large size and high carbohydrate content of the mucins, it was hard to do further studies on its assembly by conventional methods. We have therefore expressed the last
981 amino acids of the human MUC2 mucin (corresponding to its
C-terminal Cys-rich domain) as a fusion protein with the mycTag epitope
and the green fluorescent protein (GFP) in Chinese hamster ovary cells
(CHO-K1) and in the human colon adenocarcinoma cell line LS 174T. With
this tool we could verify that the domain was capable of forming dimers
and that these dimers were secreted from the
cells.2 These results were
also supported by previous studies (26, 27) of the MUC2 homologue in
rat (Muc2).
In this study, we have studied a cleavage in the Cys-rich C-terminal
part of MUC2. The cleavage was first observed during attempts to purify
and study the glycosylation pattern of the recombinant C-terminal
Cys-rich domain expressed in CHO-K1 cells. We found that when buffers
of pH 6.0 were used during purification or glycosidase treatments, part
of the material was cleaved. The cleavage caught our attention and was
further analyzed, revealing that it was formed by an autocatalytic
mechanism triggered by low pH. Furthermore, the cleavage produced a
new, reactive C terminus that has the potential to link the cleaved
mucin to other compounds.
Antisera and Antibodies--
The polyclonal antiserum Recombinant Expression and Tissue Culture--
An expression
plasmid with the bases 12,622-15,708 of the human MUC2 sequence (12)
was generated from the plasmids SMUC41 (bases 12,243-13,083) and V5
(bases 12,880-15,708) (29). These were cloned into the eukaryotic
expression vector pEGFP-C1 (Clontech) where the
signal sequence had been replaced with the murine immunoglobulin Purification of Secreted MUC2 C Terminus from CHO-K1-pSMG-MUC2C
Cells--
After growing CHO-K1-pSMG-MUC2C cells until 3-4 days after
confluence, the cells were washed twice with phosphate-buffered saline
(PBS) and Iscove's modified Dulbecco's medium (Invitrogen) was added.
The spent culture media were harvested, and new media were added every
3rd day. After the addition of protease inhibitors (1 mM
phenylmethylsulfonyl fluoride (PMSF) (Sigma), 100 µM
leupeptin (Sigma), 100 KIE ml Metabolic Labeling--
Confluent cells were metabolically
labeled by preincubation in Met- and Cys-free minimal essential medium
(Invitrogen) containing 10% (v/v) fetal bovine serum, 100 IU
ml Preparation of Cell Lysates and Media--
Media were removed
and the cells washed twice with PBS. The cells were lysed in lysis
buffer (50 mM Tris-HCl, pH 7.9, 150 mM NaCl,
1% (v/v) Triton X-100) containing protease inhibitors and NEM
(concentrations as above) and sonicated (intensity 15) 3 times for
2 s (MSE Soniprep 100 sonifier), and the cell debris was removed
by centrifugation (16,000 × g for 10 min, 4 °C).
The media were supplemented with protease inhibitors and NEM
(concentrations as above) and centrifuged (1000 × g
for 10 min, 4 °C).
Immunoprecipitation--
Immunoprecipitations using the mAbs
SDS-PAGE--
Immunoprecipitated material was released from the
magnetic beads in sample buffer (25 mM Tris-HCl, 10% (v/v)
glycerol, 2.5% (w/v) SDS, pH 6.8) with or without 5% (v/v)
Western Blotting and Detection--
Transfer of the proteins to
PVDF membranes (Immobilon-P, 0.45 µm, Millipore) was done in a
Transfer-Blot SD-Dry Transfer Cell (Bio-Rad) at 2.5 mA/cm2.
The transfer buffer used contained 48 mM Tris, 39 mM glycine, 1.3 mM SDS, and 10% (v/v)
methanol. After blotting, the membranes were placed in blocking
solution (PBS containing 5% (w/v) milk powder, 0.1% (v/v) Tween 20, and 0.05% (w/v) NaN3) overnight at 4 °C and incubated
with either streptavidin-AP (diluted 1:1000) or the primary antibody
Characterization of the Cleavage Reaction--
Nonlabeled
material were immunoprecipitated with the Neutralization of the Secretory Pathway--
Confluent LS
174T-pSMG-MUC2C cells were used in these studies. When
NH4Cl was used, the cells were preincubated with media with
or without 25 mM NH4Cl for 12 h before the
1-h starvation and 8-h labeling period (both performed with 25 mM NH4Cl present). When bafilomycin A1 (a kind
gift from AstraZeneca, Mölndal, Sweden) was used, it was present
at 300 mM during the starvation (1 h) and labeling (6 h).
The cell lysates and media were immunoprecipitated using the Linking of a Biotinylating Agent to the Cleavage
Product--
Aliquots (114 ng) of purified recombinant MUC2 C terminus
secreted from CHO-K1 cells were incubated for 1.5 h at room
temperature in either a citric acid-Na2HPO4, pH
7.4 buffer, a citric acid-Na2HPO4, pH 5.4 buffer, or in the latter containing 1 µmol of biotin ethylenediamine hydrobromide (Sigma). The incubations above were terminated by addition
of SDS-PAGE sample buffer before analysis by SDS-PAGE and Western blotting.
Edman Sequencing--
Purified C terminus was dried (using a
SpeedVac concentrator), dissolved in 50 mM pyridine/acetic
acid, pH 5.5 buffer, and incubated overnight at 37 °C. The sample
was dried, dissolved in reducing SDS-PAGE sample buffer, separated by
SDS-PAGE, blotted to a PVDF membrane, and stained with Coomassie
Blue (BioSafe Coomassie G-250, Bio-Rad) for 1 h. The
membrane was destained in 50% (v/v) methanol, 10% (v/v) acetic acid,
and the band corresponding to the C-terminal cleavage fragment was cut
out and sequenced on a Procise 492 Protein Sequencer (Applied Biosystems).
Purification of Secreted MUC2 C Terminus from CHO-K1-pSMG-MUC2C
Cells--
The cell line CHO-K1-pSMG-MUC2C permanently expressed a
construct encoding the mycTag epitope at the N-terminal end followed by
GFP and the last 981 amino acids of the human MUC2 mucin. The murine
immunoglobulin
When the glycosylation of the recombinant MUC2 C terminus was studied,
using different exoglycosidases, two buffers with different pH values
were used (pH 6.0 and 7.0, respectively). When the samples were treated
in the pH 7.0 buffer, only the intact protein band was observed.
However, when the samples were treated in the pH 6.0 buffer, the two
cleavage products appeared (data not shown). These observations of a
cleavage occurring only when the sample had been treated at pH 6.0 led
to the hypothesis that this reaction was induced at low pH.
Purification of secreted MUC2 C terminus from CHO-K1-pSMG-MUC2C cells
grown under serum-free conditions, using ultrafiltration, ion exchange
chromatography, and gel filtration with buffers having a pH Localization of the Cleavage Site by Edman
Sequencing--
Purified SMG-MUC2C, from spent culture media, was
incubated at pH 5.5 overnight. After separation by SDS-PAGE and
transfer to a PVDF membrane, the 110-kDa band corresponding to the
C-terminal cleavage fragment was excised and subjected to N-terminal
amino acid sequencing. The sequence obtained, PHYVTF, is located at a
position compatible with the sizes of the two cleaved fragments (see
Fig. 1). The localization of this cleavage corresponds to the
N-terminal end of the previously found 118-kDa "link peptide" (32).
Characterization of the Cleavage Reaction--
To decipher the
identity of the cleavage products produced in the in vitro
studies, immunoprecipitated material from CHO-K1-pSMG-MUC2C cell
lysates and media was incubated at pH 6.0 and further analyzed by
SDS-PAGE, Western blotting, and immunodetection (Fig. 2B). The results showed that the N-terminal cleavage product migrated as
~50 kDa heavier after its secretion (130 relative to 80 kDa, left panel), whereas the C-terminal fragment only acquired
around 10 kDa after its secretion (110 relative to 100 kDa, right
panel). This caused the two bands to switch position relative to
the intact SMG-C monomer, when comparing cell lysate and media. This
implies that the modifications, leading to a secreted SMG-MUC2C with
lower mobility compared with the intracellular form, mainly are present in the N-terminal part of the protein.
To test the hypothesis of a pH-dependent cleavage,
radiolabeled material from CHO-K1-pSMG-MUC2C cell lysates and media was immunoprecipitated using
To investigate if the cleavage was induced at a low pH, aliquots of
immunoprecipitated SMG-MUC2C from cell lysate and media were treated in
citric acid-Na2HPO4 buffers with pH from 4.8 to 7.8 for 20 min at 37 °C (Fig. 3,
A and B). The cleavage was observed at a pH
around 6.0 and lower for both the intracellular and extracellular forms
of SMG-MUC2C. The cleavage products could be observed as weak bands
also at pH >6.0 in the secreted but not the intracellular form of
SMG-MUC2C. This indicated that the cleavage could occur prior to the
incubation and probably inside the cell. The intracellular SMG-MUC2C
has been localized to the ER,2 and thus this form has not
passed through the more acidic parts of the late secretory pathway. The
presence of some cleavage of the secreted SMG-MUC2C suggested that this
process occurred late in the secretory pathway.
To investigate if the cleavage reaction was time-dependent,
aliquots of immunoprecipitated SMG-MUC2C from cell lysate were incubated in a pH 6.0 buffer for different times (0-300 min) (Fig. 3C). The result from this experiment showed that cleavage
was time-dependent as an increased amount of the cleavage
products was observed with longer incubation times. However, cleavage
was not complete even after 5 h.
To explore if the cleavage was mediated by a protease, aliquots of
immunoprecipitated SMG-MUC2C from cell lysate were treated in a BisTris
buffer, pH 6.2 (gives pH 5.9 at 37 °C), with the addition of
protease inhibitors (Fig. 4). A wide
range of protease inhibitors including Ser, Cys, aspartic, and
metalloprotease inhibitors as well as an aminopeptidase inhibitor were
used. None of these inhibited the cleavage reaction to a significant
extent. This argues for the cleavage not being mediated by an enzyme
tightly attached to or being an integral part of the MUC2 C terminus
itself.
Neutralization of the Secretory Pathway--
The level of cleavage
in the secreted material from the CHO-K1-pSMG-MUC2C cells was very low.
This together with the fact that CHO-K1 cells are very different from
mucin-producing cells led us to test a more physiological relevant cell
line, LS 174T. This human colon adenocarcinoma cell line expresses
endogenous MUC2 and contains mucin granules and is thus a better
model for a goblet cell in studies of mucin biosynthesis. A stable cell line (LS 174T-pSMG-MUC2C) permanently expressing the same construct as
the CHO-K1-pSMG-MUC2C cells was generated and shown to secrete dimers
of SMG-MUC2C.2 After immunoprecipitations (using either the
To analyze if the cleavage observed in the secreted SMG-MUC2C was
dependent on the acidic pH in the late secretory pathway, this was
neutralized by treating the cells with either NH4Cl or bafilomycin A1. NH4Cl mediates its effect through ammonia
diffusing into the cells while bafilomycin A1 is an inhibitor of
H+-K+-ATPase pumps present in the secretory
pathway. The cells were radiolabeled, and cell lysates and media from
the treated cells were immunoprecipitated with the Pulse-Chase Studies of Cells Expressing the Recombinant MUC2 C
Terminus--
Bands migrating in the same positions as the noncleaved
and cleaved secreted material were observed in the lysates from the LS
174T-pSMG-MUC2C cell (Fig. 5), but not in the lysates from the
CHO-K1-pSMG-MUC2C cells. One possible explanation could be that the
SMG-MUC2C cells have a slower passage through the secretory pathway in
the LS 174T cells. To test if this was the case, both CHO-K1-pSMG-MUC2C
and LS 174T-pSMG-MUC2C cells were pulsed with 35S-labeled
Met and Cys for 15 min and chased for 40-110 min. Media from the
treated cells were immunoprecipitated with the mAb
As the total passage time was identical, another reason for a cleavage
to occur in the LS 174T, but not in CHO-K1 cells, could be that
SMG-MUC2C spends a longer time in the latter, and slightly acidic,
parts of the secretory pathway. To test this hypothesis, both cell
lines were pulsed for 5 min and chased for different times, and the
lysates were immunoprecipitated and analyzed as above. The fully
O-glycosylated protein was found also in the cell lysates of the LS
174T-pSMG-MUC2C cells from 30 min to the last chase time (70 min) (Fig.
6B). After long exposure of the film, the cleaved fragments
also could be found in the same fractions (not shown), in analogy with
those observed in Fig. 5. In contrast to the LS 174T cells, no bands
corresponding to the fully glycosylated SMG-MUC2C was found in the
CHO-K1 cell lysates. Only very faint bands could be observed in the
lysates at chase times 40-70 min after very long exposures of the film
(not shown). The results indicate that SMG-MUC2C actually spends a
longer time in the latter parts of the secretory pathway in the LS 174T
compared with the CHO-K1 cells, a fact that could explain why the
pH-dependent cleavage was observed in only in LS 174T cells.
A Primary Amine Reacts with the Cleaved MUC2 C
Terminus--
Previously, a mechanism for the cleavage at Asp-Pro
bonds at low pH has been proposed and involves the formation of an
internal anhydride between the two carboxylic groups of the generated
C-terminal Asp (34). Such anhydrides are relatively reactive with
primary amines, for example. In a first attempt to indicate the
presence of such an anhydride in the cleaved MUC2 C terminus, biotin
conjugated with a primary amine was used as a reagent. Purified
recombinant MUC2 C terminus, secreted from CHO-K1 cells, was incubated
in a citric acid-Na2HPO4 buffer at a pH 7.4 or
pH 5.4 with biotin ethylenediamine. Analysis by SDS-PAGE, Western
blotting, and detection with streptavidin revealed that only one band
was stained when incubation was done at pH 5.4 in the presence of the
reagent (Fig. 7). This band migrated at
the same position as expected for the N-terminal fragment (designated
MG-C1) and reacted with the It has been observed previously (32) that a 118-kDa glycopeptide
could be released from purified rat intestinal mucins by thiol
reduction. The peptide was called the "link" peptide, as it was
believed to be responsible for linking the mucin monomers together to a
polymeric gel structure. This glycopeptide was thoroughly studied
(35-37) but was questioned to be a degradation product. When the
cDNA encoding the link peptide was cloned, it was first believed to be a separate protein named mucin-like peptide (38), but it
was later shown to be an integral part of the rat Muc2. The N-terminal
amino acids of the link glycopeptide were sequenced, and it was
concluded that the peptide represented the last 689 amino acids of the
mucin and was generated by a cleavage at an Asp-Pro bond near the
N-terminal border of the mucins D4-domain, a cleavage site identical to
the one reported in the present study. The rat Muc2 mucin is the
homologue of the human MUC2 mucin and as such shows extensive sequence
similarities to its N- and C-terminal Cys-rich domains (12, 38-40). It
has been disputed whether the link peptide is a preparatory artifact or
not. The same cleavage fragment starting with the amino acids
Pro and His of GDPH have been found by Herrmann et al. (41)
in the human MUC2 mucin, prepared by rigorous methods, from the large
intestine. This further argues for the hypothesis that a cleavage
between Asp and Pro in GDPH occurs naturally. The results presented
here further suggest that this cleavage is due to an autocatalytic
mechanism triggered by low pH. It also argues for it being a natural
phenomenon occurring in MUC2 and suggests that the cleavage could be of
biological relevance as it takes place at a pH comparable with that of
the late secretory pathway. The cleavage occurred at pH 6 and below, and as the pH in the trans-Golgi network is slightly below 6.0 (42) and
can approach 5.2 in the secretory granules (43), the mucin is likely to
encounter a milieu in which this type of cleavage may occur. The use of
agents that could neutralize the secretory pathway inhibited the
cleavage found in LS 174T-pSMG-MUC2C cells, thus proving that the
cleavage can actually occur in the cell. Furthermore, the reaction was
not inhibited by any of the used protease inhibitors supporting the
hypothesis of it being a nonenzymatic process.
The GDPH sequence is also found in other proteins, but it has only been
studied in the pre- It is well known that Asp-Pro bonds are acid-labile, something that has
been used for chemical cleavage of proteins (48). However, the
conditions necessary for this type of protein fragmentation are far
harsher than the ones we used, typically including treatment in diluted
acids with pH Searches for GDPH sequences reveals that this is also present in other
mucins. Of the gel-forming mucins, this sequence is only found in the C
terminus of the MUC5AC but not in the other known gel-forming mucins
MUC5B, MUC6, and PSM (Fig. 8). The
absence of this sequence in the latter two is not surprising as both
lack the D4-domain, the region were the GDPH sequence is located. The sequence is also absent in the vWf, which is otherwise showing high
sequence similarities within the C terminus of the gel-forming mucins
and contains a D4-domain. When looking at the gel-forming mucins, it is
also interesting to notice that the ones that are normally expressed in
goblet cells (MUC2 and MUC5AC) contain the GDPH sequence, whereas the
ones normally expressed in glands (MUC5B and MUC6) do not (Fig. 8). In
addition to gel-forming mucins, the GDPH sequence is also found in the
transmembrane mucin MUC4 and its rat orthologue, the sialomucin complex
(SMC). SMC is encoded as a single protein but is found as a
heterodimeric glycoprotein complex composed of one extracellular and
one transmembrane subunit (50, 51). These two subunits are formed due
to an uncharacterized intracellular cleavage at an Asp-Pro bond located
in a GDPH sequence (52, 53). No proteases have been identified, and we
now suggest that this cleavage is also due to a pH-triggered
autocatalytic process.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-MUC2C2
has been described before (28). The
-mycTag monoclonal antibody
(mAb) was from spent culture media of the 1-9E10.2 hybridoma (ATCC
CRL-1729). Other antibodies used were
-GFP mAb
(Clontech), goat anti-mouse immunoglobulins coupled
to alkaline phosphatase (AP) (Dako), goat anti-rabbit immunoglobulins
coupled to alkaline phosphatase (Dako), and streptavidin coupled to
alkaline phosphatase (streptavidin-AP) (Dako).
-chain signal sequence from pSecTag A (Invitrogen) followed by the
mycTag (EQKLISEEDL).2 The resulting pSMG-MUC2C plasmid
encoding the
-chain signal sequence, the mycTag, GFP, and the MUC2 C
terminus was sequenced and transfected into the human colon
adenocarcinoma cell line LS 174T (ATCC CL-188) and Chinese hamster
ovary cells CHO-K1 (ATCC CCL-61). Both cell lines were selected for
permanent expression of the recombinant protein and named LS
174T-pSMG-MUC2C and CHO-K1-pSMG-MUC2C,2 respectively. These
were cultured as described earlier (24) for LS 174T with the addition
of 125 µg ml
1 G418 for LS 174T-pSMG-MUC2C and 250 µg
ml
1 G418 for CHO-K1-pSMG-MUC2C, respectively.
1 aprotinin (Trasylol,
Bayer), and 5 mM N-ethylmaleimide (NEM, Sigma)),
the media was centrifuged 1,000 × g for 10 min
(+4 °C), and 0.02% (w/v) NaN3 was added. 200 ml of
media were filtrated in an Amicon Stirred Cell model 8400 through an
Amicon Ultrafiltration Membrane YM-100 (Millipore). The volume was
reduced to 20 ml, filled up to 200 ml with 50 mM Tris-HCl,
pH 8.0, and reduced to 20 ml again. This procedure was repeated 3 times. The concentrate was filtered (0.22 µm Millex-GV, Millipore)
and loaded on a Mono Q HR 5/5 column (Amersham Biosciences) using a
flow rate of 1.0 ml min
1. After washing with 150 mM NaCl in 50 mM Tris-HCl, pH 8.0, bound components were eluted with a linear NaCl gradient (150-600
mM) in 50 mM Tris-HCl, pH 8.0, over 45 min. The
eluting components were monitored at 280 nm and collected in 1-ml
fractions. The fractions were individually analyzed by SDS-PAGE and
silver staining. Seven column fractions, eluting between 230 and 300 mM NaCl, containing the recombinant MUC2 C terminus were
pooled, concentrated to 200 µl by ultrafiltration (Vivaspin 2-ml
concentrator, 30,000 molecular weight cut-off RC, 3000 × g, 4 °C), loaded directly on a Superose 6 HR 10/30 column
(Amersham Biosciences) eluted with 150 mM ammonium acetate,
pH 8.5, at a flow of 250 µl min
1. The eluting
components were monitored at 280 nm and collected in 0.5-ml fractions.
All the fractions contained in the symmetrical unimodal peak eluting at
Kav = 0.095 and positive for the recombinant MUC2 C terminus, as assessed by SDS-PAGE and silver staining, were
pooled and used for the subsequent studies.
1 penicillin, and 100 µg ml
1
streptomycin for 1-2 h followed by addition of labeling mix (Redivue Pro-mix L-35S in vitro labeling mix,
Amersham Biosciences) to a final concentration of 36-107 µCi
ml
1. After incubation for 6-16 h, cell lysates and media
were prepared. When pulse-chase studies were performed, the labeling
mix was removed after 5 or 15 min and the cells washed once and chased with normal cell culturing media with excess of Met and Cys (15 and 25 µg extra/ml, respectively).
-mycTag or
-GFP antibodies were performed by precoating these on
goat anti-mouse IgG-coupled magnetic beads (Dynabeads, Dynal). The
immunocomplexes were washed 4 times in 10 mM Tris-HCl, 2 mM EDTA, 0.1% (v/v) Triton X-100, 0.1% SDS (w/v), pH 7.4, and 1 time in lysis buffer.
-mercaptoethanol or 100 mM dithiothreitol for 5 min at
95 °C. Samples were analyzed by discontinuous SDS-PAGE using 3-10
or 3-15% gradient gels with 3% stacking gels or 5% linear gels with
3% stacking gels (30). Coomassie Blue staining was performed
according to the manufacturer (BioSafe Coomassie G-250,
Bio-Rad). After analysis of metabolically labeled samples, the gels
were fixed for 30 min in 30% methanol, 7% acetic acid, incubated in
Amplify (Amersham Biosciences) for 15 min, dried, and exposed to BioMax
MS films (Eastman Kodak Co.). The molecular markers used were the
High-Range Rainbow 14C-Methylated Protein Molecular Weight
Markers (Amersham Biosciences) and Precision Protein Standards
(Bio-Rad).
-mycTag mAb (diluted 1:10) or
-MUC2C2 (diluted 1:100) for
1.5 h at room temperature. The membranes were washed 3 times for 5 min with PBS-T (PBS containing 0.1% (v/v) Tween 20) and incubated with
secondary antibodies (either goat anti-mouse immunoglobulins coupled to
AP or goat anti-rabbit immunoglobulins coupled to AP 1:1000 in blocking
solution) for 1.5 h at room temperature. After another PBS-T wash
(3 times for 5 min) the membranes were developed with nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. In the cases where
biotin ethylenediamine hydrobromide were used (see below), another
blocking solution was used (10 mM Tris-HCl, pH 7.5, containing 2% (w/v) bovine serum albumin, 100 mM NaCl,
0.1% (v/v) Tween 20, and 0.05% (w/v) NaN3).
-mycTag mAb from
CHO-K1-pSMG-MUC2C cell lysates and media, incubated in citric
acid-Na2HPO4, pH 6.0, buffer (McIlvaine buffer)
(2.5 h, 37 °C) while still attached to the beads, analyzed by
SDS-PAGE, Western-blotted, and detected by immunostaining. The citric
acid-Na2HPO4 buffer system used here, as well
as below, is the one described by McIlvaine (31). Radiolabeled and
immunoprecipitated (
-mycTag mAb) material from CHO-K1-pSMG-MUC2C
cell lysates or media was treated as described below while still
attached to the magnetic beads. The pH dependence of the cleavage
reaction was studied by incubating the radiolabeled material from cell
lysates or media in citric acid-Na2HPO4 buffers
(McIlvaine buffers) with pH from 4.8 to 7.8 for 20 min at 37 °C.
Time dependence of the cleavage reaction was studied by incubating
aliquots of the lysate material in a citric
acid-Na2HPO4, pH 6.0 buffer (McIlvaine buffer),
at 37 °C for 0-300 min. The influence of different protease
inhibitors on the cleavage reaction was studied by incubating material
from cell lysates for 1 h at 37 °C in 50 mM
BisTris, pH 6.2 (pH 5.9 at 37 °C), containing one of the following
inhibitors: PMSF (1 mM), bestatin (10 mM)(Sigma), leupeptin (100 µM), pepstatin (1 µM) (Roche Molecular Biochemicals), aprotinin (2.15 µM), NEM (5 mM), EDTA (10 mM), or
a mixture of protease inhibitors (Complete mini-EDTA-free, Roche
Molecular Biochemicals). All incubations above were terminated by
removal of the incubation buffers and addition of SDS-PAGE sample
buffer before analysis by SDS-PAGE.
-GFP
antibody and analyzed by SDS-PAGE.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain signal sequence was used to direct the fusion
protein to the secretory pathway. The recombinant MUC2 C terminus
(SMG-MUC2C) formed dimers in the ER (with a molecular mass of about 330 kDa) and was later secreted as dimers of higher molecular mass (about
470 kDa), a difference partly due to glycosylation.2 These
cells were grown in the presence of 10% (v/v) fetal bovine serum, and
the secreted fusion protein was purified by ultrafiltration, gel
filtration, and ion exchange chromatography using a 50 mM BisTris, pH 6.0 buffer. The purified SMG-MUC2C was analyzed by SDS-PAGE
under nonreducing conditions revealing a single band with an
approximate molecular mass of 470 kDa, corresponding to the size of a
dimer (Fig. 1). When the same material
was analyzed under reducing conditions, three bands were observed (Fig.
1). The largest band corresponded to the expected monomer with an approximated molecular mass of about 250 kDa. The two additional bands
migrated at about 130 and 110 kDa, suggesting that these could be due
to a cleavage of the full-length SMG-MUC2C. Western blot analysis of
the nonreduced band revealed that this was immunoreactive with both the
-mycTag mAb and the
-MUC2C2 antiserum (Fig.
2A). This was also true for
the 250-kDa monomer found after reduction. The 130-kDa band only
reacted with the mycTag mAb, whereas the 110-kDa band reacted with the
-MUC2C2 antiserum raised against a peptide sequence in the
C-terminal end of MUC2. The results indicated that some of the
SMG-MUC2C had been cleaved, producing a 130-kDa N-terminal and a
110-kDa C-terminal fragment, and that these fragments were still held
together by disulfide bonds.
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Fig. 1.
Purification of secreted MUC2 C terminus from
CHO-K1-pSMG-MUC2C cells. Secreted MUC2 C terminus from
CHO-K1-pSMG-MUC2C cells was purified using ultrafiltration, ion
exchange chromatography, and gel filtration. The purified material was
separated on a 3-15% SDS-PAGE gel and visualized by Coomassie
Blue staining. NonPur, nonpurified material (used culture
media); Pur pH 8.0, material purified using buffers with
a pH
8.0 (as described under "Experimental Procedures");
Pur pH 6.0, material purified using a 50 mM
BisTris, pH 6.0 buffer; NonRed, nonreduced samples;
Red, reduced samples. Positions of molecular mass standards
are indicated to the left. A schematic picture of the
recombinant C terminus of MUC2 is given in the upper part of
the figure. The arrows indicate the cleavage site, and the
underlined sequence indicates the sequence obtained after
Edman sequencing of the C-terminal cleavage product (C2).
The N-terminal cleavage product is indicated by MG-C1 and
MUC2-C1 and MUC2-C2 corresponds to amino acids
4199-4486 and 4487-5179 of the MUC2 sequence (12), respectively.
-MUC2C2 indicates the localization of the epitope
detected by the antiserum with the same name.
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Fig. 2.
Identification of the cleavage products of
the MUC2 C terminus. A, MUC2 C terminus, secreted from
CHO-K1-pSMG-MUC2C cells and purified using a pH 6.0 buffer, was
separated on a 3-10% SDS-PAGE gel, blotted, and detected with either
-MUC2C2 antiserum or
-mycTag mAb. B, cell lysates and
media from CHO-K1-pSMG-MUC2C cells were immunoprecipitated (using the
-mycTag mAb), incubated for 2.5 h in a citric
acid-Na2HPO4, pH 6.0 buffer, separated by
SDS-PAGE (3-10%, reducing conditions), blotted, and detected by
either the
-MUC2C2 antiserum or the
-mycTag mAb. L,
material from cell lysates; M, material from the cell
culturing media; Di, position of the dimer of the MUC2 C
terminus; Mono, position of the monomer of the MUC2 C
terminus; MG-C1, the N-terminal cleaved fragment;
C2, the C-terminal cleaved fragment; NonRed,
nonreduced samples; Red, reduced samples. IB
indicates the antibody used for detection. Positions of molecular mass
standards are indicated to the left.
8.0, gave rise to a much lower extent of cleavage (Fig. 1), further
supporting this hypothesis.
-mycTag antibodies coupled to magnetic beads. While still attached to the beads, aliquots of the material were
treated under different conditions, and the labeled SMG-MUC2C was
analyzed by SDS-PAGE and autoradiography.
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Fig. 3.
pH requirement and time dependence of the
cleavage reaction of the MUC2 C terminus. Metabolically labeled
and immunoprecipitated (using the -mycTag mAb) material from
CHO-K1-pSMG-MUC2C cell lysates (A) or cell culture media
(B) was treated in citric
acid-Na2HPO4 buffers with pH from 4.8 to 7.8 for 20 min at 37 °C. Material from cell lysate was prepared in the
same way as above and treated in a citric
acid-Na2HPO4, pH 6.0 buffer at 37 °C for
different times (0-300 min) (C). All incubations above were
terminated by removal of the incubation buffers and addition of
SDS-PAGE sample buffer before analysis on a 5% SDS-PAGE gel under
reducing conditions. Mono, position of the MUC2 C terminus
monomer; MG-C1, the N-terminal cleaved fragment;
C2, the C-terminal cleaved fragment. Positions of molecular
mass standards are indicated to the left.
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Fig. 4.
Influence of different protease inhibitors on
the cleavage of the MUC2 C terminus. Metabolically labeled and
immunoprecipitated (using the -mycTag mAb) material from
CHO-K1-pSMG-MUC2C cell lysates was treated in a 50 mM
BisTris, pH 6.2 (gives pH, 5.9 at 37 °C) buffer for 1 h at
37 °C in the absence (Treated) or presence of different
protease inhibitors (PMSF (1 mM), bestatin (10 mM), leupeptin (100 µM), pepstatin (1 µM), aprotinin (2.15 µM), NEM (5 mM), EDTA (10 mM), and a mixture of protease
inhibitors (complete mini-EDTA-free)). All incubations above were
terminated by removal of the incubation buffers and addition of
SDS-PAGE sample buffer before analysis on a 5% SDS-PAGE gel under
reducing conditions. Mono, position of the MUC2 C terminus
monomer; MG-C1, the N-terminal cleaved fragment;
C2, the C-terminal cleaved fragment. Positions of molecular
mass standards are indicated to the left.
-mycTag or
-GFP antibodies), SDS-PAGE, and Western blot analysis,
a 130-kDa band immunoreactive with the
-MUC2C2 antiserum and a
210-kDa band immunoreactive with the
-mycTag mAb were revealed (Fig.
5A). These cleavage products
are slightly larger than the ones of the CHO-K1-pSMG-MUC2C cells, in
line with the observation that the secreted SMG-MUC2C is larger in this
cell line. The LS 174T cell line is derived from the intestine and
normally makes longer and more complex oligosaccharides compared with
CHO-K1 cells, suggesting that the differences are due to glycosylation.
One also observes that bands migrating in the same positions as the
secreted material as well as the cleavage products could be found also
in the LS 174T-pSMG-MUC2C cell lysates, something not observed in the
CHO-K1-pSMG-MUC2C cells.
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Fig. 5.
Neutralization of the secretory pathway
inhibits the cleavage of the MUC2 C terminus. A, cell
lysates and media from LS 174T-pSMG-MUC2C cells were immunoprecipitated
(using either the -GFP or
-mycTag antibodies), separated by
SDS-PAGE (3-10%, reducing conditions), blotted, and detected by
either the
-MUC2C2 antiserum (when immunoprecipitation with
-GFP)
or
-mycTag mAb (when immunoprecipitation with
-mycTag mAb).
L, material from cell lysates; M, material from
the spent culture media. Mono, position of the monomer of
the MUC2 C terminus (in B only indicated in the right
part); MG-C1, the N-terminal cleaved fragment;
C2, the C-terminal cleaved fragment. IB indicates
the antibody used for detection. Positions of molecular mass standards
are indicated to the left. B, confluent LS
174T-pSMG-MUC2C cells were preincubated with media with or without 25 mM NH4Cl for 12 h. After washing the cells
with PBS they where starved (1 h) and metabolically labeled (8 h) in
the presence of the same concentration of NH4Cl as during
the preincubation. In the case where bafilomycin A1 was used as the
neutralizing agent, 300 mM bafilomycin A1 was present
during the starvation (1 h) and labeling steps (6 h). After the
labeling cell lysates and media were prepared, and immunoprecipitations
(using the
-GFP antibody) and SDS-PAGE were performed (5%
separation gel, reducing conditions). The mAb
-GFP and
-mycTag
gave identical results.
-GFP mAb followed
by SDS-PAGE (Fig. 5B). Both treatments blocked the cleavage
in the secreted material more or less completely as the C2 fragment
almost disappeared from the media. The intracellular SMG-MUC2C
precursor found at around 210 kDa was as expected not affected. These
observations support the idea that the cleavage of the SMG-MUC2C can
occur in vivo and that it is triggered by the lower pH in
the late secretory pathway. It also argues against the cleavage being
an experimental artifact. One can also observe that the secreted
SMG-MUC2C acquires a slightly higher mobility after both treatments.
This is in agreement with previous studies (33) showing that a
neutralization of the secretory pathway, using these compounds, leads
to a redistribution of glycosyltransferases and a subsequent decrease
in O-glycan chain length.
-mycTag, and the
precipitated material was analyzed by SDS-PAGE and autoradiography. The
results show that SMG-MUC2C cells appear in the media after 60 min from
both cell lines, indicating that there is no difference in the passage
rate through the secretory pathway (Fig.
6A).
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Fig. 6.
Pulse-chase studies of cells expressing the
recombinant MUC2 C terminus. CHO-K1 and LS 174T cells expressing
the C terminus of the MUC2 mucin was metabolically labeled with Redivue
Pro-mix L-35S in vitro labeling mix
for 15 (A) or 5 min (B) and chased in
nonradioactive medium with excess of Met and Cys for different times.
Cell lysates and media were prepared and immunoprecipitated by
-mycTag mAb coupled to magnetic beads. Samples were analyzed by
SDS-PAGE on 5% gels under reducing conditions. A, media
(40-110 min) from CHO-K1 (upper panel) and LS 174T cells
(lower panel) expressing the C terminus of MUC2.
B, cell lysates (0-70 min) and media (70 min) from CHO-K1
(upper panel) and LS 174T cells (lower panel)
expressing the C terminus of MUC2. Monoec and
Monoic, positions of the monomeric MUC2 C termini in
media and cell lysates, respectively; MG-C1, the N-terminal
cleaved fragment; C2, the C-terminal cleaved fragment.
Positions of molecular mass standards are indicated to the
left.
-mycTag mAb. That the noncleaved and the
C-terminal fragment (C2) was not labeled indicated that the reaction
with biotin ethylenediamine was specific. The only difference between
the cleaved fragment MG-C1 and the intact protein and C1 fragment is
the newly produced C terminus. This make it likely that the biotin
ethylenediamine did not react with carboxyl groups, for example, and
that it has reacted with the Asp C terminus as suggested from the
hypothesis of an anhydride formed during the cleavage (34). This should mean that the MUC2 mucin might have a possibility to covalently cross-link with other molecules.
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Fig. 7.
Biotin with a primary amine react with a MUC2
C terminus cleavage product. Aliquots of purified recombinant MUC2
C terminus secreted from CHO-K1 cells were incubated for 1.5 h at
room temperature in either a citric
acid-Na2HPO4, pH 7.4 buffer, a citric
acid-Na2HPO4, pH 5.4 buffer, or in the latter
containing 1 µmol of biotin ethylenediamine hydrobromide. After
SDS-PAGE on a 3-10% gel, under reducing conditions, the proteins were
blotted and detected by either the -mycTag mAb or streptavidin.
Mono, the monomer of the MUC2 C terminus; MG-C1,
the N-terminal cleaved fragment; C2 with an
arrow, the position of the C-terminal cleaved fragment (not
shown here). IB indicates the antibody used for detection.
Positions of molecular mass standards are indicated to the
left.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-inhibitor, which has shown a similar type of
cleavage as we report here. The pre-
-inhibitor is a serum protein
secreted from rat liver and whose physiological function is still not
understood. This protein consists of the two polypeptides, bikunin and
heavy chain 3 (H3), covalently linked to each other by a chondroitin
sulfate chain (44). H3 is synthesized as a precursor that, like the
MUC2 mucin, is cleaved between an Asp and a Pro in a GDPH sequence
(45). After the cleavage, H3 is esterified, via the
-carbon of its
C-terminal Asp, to C-6 of an internal N-acetylgalactosamine
of the glycosaminoglycan chain attached to bikunin (44). The cleavage
has been shown to be induced by the low pH in the late Golgi and
mediated through an intramolecular process, suggested to generate a
reactive Asp anhydride (46), which could be the case for MUC2 as well.
Mutation studies of H3 have shown that the presence of the Asp and the Pro in the GDPH sequence are absolutely essential for the cleavage to
occur (47). However, other residues surrounding these two amino acids
as well as residues located further C-terminal seem to be of importance
for the cleavage reaction (46). The sequences flanking the GDPH
sequences in MUC2 and H3 show limited sequence similarity (Fig. 6),
suggesting there might be differences in how the cleavage takes place
and its consequences.
2.5 at 40 °C for at least 24 h. The milder conditions used for the in vitro experiments in this
study may explain why the cleavage reaction does not come to
completion. The mild acidic environment that proteins experience on
their way through the secretory pathway is probably also the reason for
the low extent of cleavage seen in the secreted material. Furthermore,
the in vitro studies show that the time the proteins spend
in the acidic environment is an important factor for the extent of
cleavage. The SMG-MUC2C protein analyzed here was continuously secreted
from both the LS 174T-pSMG-MUC2C and CHO-K1-pSMG-MUC2C cells. Although
the overall passage rate of SMG-MUC2C through the secretory pathway was
similar in both cell lines, the time it spent in the latter and
slightly acidic parts seemed to be longer in the LS 174T cells. This
might explain the higher cleavage seen in this cell line as compared
with the CHO-K1 cells. A fast passage through the latter part of the
secretory pathway will only expose the protein to the acidic pH for a
rather short period. However, in vivo the normal full-length
MUC2 mucin is directed to the regulated secretory pathway and stored in
secretory granules for extended periods (49). This means that
the extent of cleavage may be more pronounced as the protein encounters
a more acidic environment and for a longer time. One can thus suggest
that the cleavage observed in both rat and human MUC2 is natural and
that the extent of cleavage could vary.
View larger version (22K):
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Fig. 8.
Sequence comparisons of selected mucins and
other proteins. Selected proteins are aligned with the sequence
surrounding the GDPH sequence of the human MUC2 mucin. The residue
numbers in the figure do not necessarily refer to the actual number
from the true N terminus because all proteins in the figure have not
been fully sequenced. The residue numbers for MUC5AC and MUC5B are from
the assembly made by Perez-Vilar and Hill (2) and are marked with * in
the figure. The secretory mucins that contain the GDPH sequence has
this located in their early D4-domain. Both MUC5B and the vWf lack the
GDPH sequence in their D4-domains, whereas MUC6 and PSM lack the whole
D4-domain and are therefore not represented in the figure. The GDPH of
hemolectin is located in a D-domain, corresponding to the
D2-domain on the mucins. The GDPH sequences of MUC4, SMC, and H3 are
not located in any D-domain, and these proteins show little
sequence homology with the others. The amino acids within the GDPH
sequence are printed as white on black, and the
other amino acids matching the MUC2 sequence are highlighted with a
gray background.
The GDPH sequence is also found in proteins other than mucins. The H3 protein has already been discussed, but another case is the recently cloned protein hemolectin from Drosophila melanogaster (52). This protein is synthesized in the hemocytes at the larvae stage and shows sequence similarities to the vWf and the gel-forming mucins. The hemolectin has been shown to be sensitive to acidic cleavage (54). The exact localization of the cleavage site has not been defined, but it has been proposed to be located in the area around its D2-domain. Interestingly, hemolectin contains a GDPH sequence as well as other similarities to MUC2 in the region surrounding this sequence (Fig. 8). It is thus likely that this protein also is cleaved at the Asp-Pro linkage and that this occurs by a similar mechanism as that for MUC2.
The consequence of the pH-dependent cleavage for the
properties of the MUC2 mucin is presently not known. One possibility is
that it leads to conformational changes giving the protein new
properties that, for instance, could affect its viscosity. There is no
experimental support for such a hypothesis, but there is for the idea
that the cleavage could give the mucin an opportunity to link to other
components. The present results suggest that the GDPH cleavage
of MUC2 generates a reactive Asp (an anhydride) at the C-terminal end
because it was possible to label this fragment with biotin substituted
with a primary amine. As discussed above, this generation of an Asp
anhydride has been shown for H3, where the anhydride reacts with the
C-6 hydroxyl group of an internal GalNAc in the chondroitin sulfate
chain attached to bikunin, generating the pre--inhibitor (44, 45).
Such an ester linkage could theoretically be formed between the
reactive Asp produced after cleavage of MUC2 and any of the abundant
O-glycans of mucins. The previously observed covalent bond
connecting MUC2 monomers even after reduction of the disulfide bonds
(28, 41) could be due to such a linkage. Although it is not known to
date if the cleavage of GDPH in MUC2 is accompanied by the formation of any covalent bond to another molecule or not, the potential appearance of such a linkage could be physiologically very important. An example
where this might be relevant is the disease cystic fibrosis, characterized by viscous mucus and impaired clearance of the mucous layer. Recent studies (55, 56) suggest that a nonfunctional cystic
fibrosis transmembrane conductance regulator, as in cystic fibrosis,
causes a lowered pH value of the secretory pathway as well as a more
acidic extracellular milieu. If either or both of these assumptions are
correct, this lower pH value could lead to a higher degree of cleavage
of MUC5AC in the lungs and MUC2 in the intestine and lungs followed by
cross-linking to itself or other molecules. It could be speculated that
this is one explanation for the observed, but still not explained,
mucous aberration of this disease. However, these ideas have to be
confirmed by biochemical proof for the presence of such cross-links and
a deeper understanding of the pH-dependent autocatalytic
cleavage at the C-terminal domain of the MUC2 mucins.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Fredrik Olson for help with the Edman sequencing.
![]() |
FOOTNOTES |
---|
* This work was supported by Swedish Research Council Grant 7461 and by the IngaBritt and Arne Lundberg Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 46-31-773-34- 88; Fax: 46-31-41-61-08; E-mail: gunnar.hansson@medkem.gu.se.
Published, JBC Papers in Press, February 11, 2003, DOI 10.1074/jbc.M210069200
2 M. E. Lidell, M. E. V. Johansson, M. Mörgelin, N. Asker, C. J. R. Gum, Jr., Y. S. Kim, and G. C. Hansson, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: vWf, von Willebrand factor; PSM, porcine submaxillary mucin; ER, endoplasmic reticulum; GFP, green fluorescent protein; mAb, monoclonal antibody; AP, alkaline phosphatase; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; NEM, N-ethylmaleimide; H3, heavy chain 3; SMC, sialomucin complex; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; PVDF, polyvinylidene difluoride.
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