From the Department of Biochemistry, Duke University Medical
Center, Durham, North Carolina 27710
COS-7 cells expressing 1,360 residues from the
amino terminus of porcine submaxillary mucin were used to determine
whether this region, containing the D1, D2, and D3 domains, is involved in forming mucin multimers. Analysis of the proteins immunoprecipitated from the medium of transfected cells by reducing SDS-gel
electrophoresis showed a single N-glycosylated protein with
no indication of proteolytically processed forms. Without prior
reduction, only two proteins, corresponding to monomeric and
disulfide-linked trimeric species, were observed. The expressed protein
devoid of N-linked oligosaccharides also formed trimers,
but was secreted from cells in significantly less amounts than
glycosylated trimers. Pulse-chase studies showed that the
disulfide-linked trimers were assembled inside the cells no earlier
than 30 min after protein synthesis commenced and after the
intracellular precursors were N-glycosylated. Trimer
formation was inhibited in cells treated with brefeldin A, monensin,
chloroquine, or bafilomycin A1, although only brefeldin A
prevented the secretion of the protein. These results suggest that
trimerization takes place in compartments of the Golgi complex in which
the vacuolar H+-ATPase maintains an acidic pH. Coexpression
in the same cells of the amino-terminal region and the disulfide-rich
carboxyl-terminal domain of the mucin showed that these structures were
not disulfide-linked with one another. Cells expressing a DNA construct
encoding a fusion protein between the amino- and carboxyl-terminal
regions of the mucin secreted disulfide-linked dimeric and high
molecular weight multimeric species of the recombinant mucin. The
presence of monensin in the medium was without effect on dimerization, but inhibited the formation of disulfide-linked multimers. These studies suggest that disulfide-linked dimers of mucin are subsequently assembled into disulfide-linked multimers by the amino-terminal regions. They also suggest that the porcine mucin forms branched disulfide-linked multimers. This ability of the amino-terminal region
of mucin to aid in the assembly of multimers is consistent with its
amino acid identities to the amino-terminal region of human von
Willebrand factor, which also serves to form disulfide-linked multimers
of this protein.
 |
INTRODUCTION |
Porcine submaxillary mucin (GenBankTM accession number AF005273)
contains up to 13,288 amino acid residues arranged in different domains
characteristic of secretory mucins (1). A major central domain contains
90-135 81-residue tandem repeats rich in threonine, serine, glycine,
and alanine, with the exact number of repeats depending on the mucin
gene expressed (1). This central domain is flanked, at both ends, by
unique domains with amino acid compositions similar to the repeat
domain, but different in sequence from the repeat domain and one
another. All of the threonine and serine residues in the tandem repeat
and the unique domains appear to contain O-linked
oligosaccharides (2). Flanking the unique sequence domains are three
half-cystine-rich domains at the amino-terminal side and one (240 residues) disulfide-rich domain at the carboxyl-terminal side (1). The
half-cystine residues present at the carboxyl-terminal end of
submaxillary mucin are conserved in the corresponding regions of human
von Willebrand factor (3) and many secretory mucins, including frog
integumentary mucin FIMB.1 (4); rat intestinal mucin (5); bovine
submaxillary mucin (6); and human mucins MUC2 (7), MUC5B (8, 9), MUC5AC
(10), and MUC6 (11). Moreover, the 11 carboxyl-terminal half-cystine
residues in human von Willebrand factor and those in the above
secretory mucins are homologous to the 11 half-cystine residues in
Norrie disease protein (norrin) (12). It has been found that human von
Willebrand factor (13) and pig submaxillary mucin (14) form
disulfide-linked dimers through their respective carboxyl-terminal
domains, whereas norrin forms disulfide-linked oligomers (15). The
1,350 amino acid residues in the amino-terminal region of submaxillary
mucin are organized into three disulfide-rich homologous domains (1), named D1, D2, and D3, which share significant amino acid identities with the D-domains present in the amino-terminal regions of
human prepro-von Willebrand factor (GenBankTM accession number X04385) (3), frog integumentary mucin FIMB.1 (GenBankTM accession
number Y08296) (16), and human mucin MUC2 (GenBankTM
accession number L21998) (17). The D-domains in human pro-von
Willebrand factor mediate the formation of disulfide-linked multimers
of the protein (18), but the role of the corresponding D-domains in
secretory mucins remains unknown.
We report here expression studies in mammalian cells of plasmids
encoding the amino-terminal region of porcine submaxillary mucin. The
mucin region is N-glycosylated and forms disulfide-linked trimers prior to secretion from the cells. Trimer formation is not
dependent on N-linked oligosaccharides and takes place in acidic compartments of the Golgi complex. Expression studies of a
construct encoding the mucin amino-terminal region and the entire mucin
disulfide-rich carboxyl-terminal domain showed the formation of high
molecular weight disulfide-bonded multimeric species.
 |
EXPERIMENTAL PROCEDURES |
Antisera against the Mucin Domains--
Production of antiserum
3814 against the disulfide-rich carboxyl-terminal domain of mucin was
described earlier (14). The rabbit antisera specific for the
amino-terminal region of submaxillary mucin were obtained against mucin
polypeptides expressed in and purified from Escherichia coli
by a glutathione S-transferase fusion expression system. For
preparation of antiserum 4674, a cDNA fragment encoding amino acid
residues 266-1014 of the salivary mucin (1) was amplified by
polymerase chain reaction using Elongase (Life Technologies Inc.), a
cDNA encoding the entire amino-terminal region of the mucin (1) as
template, and the following primers:
5'-GAATTCCCGGGTCGACGTTGGACTCTGTGGTTCCTATAA-3' and
5'-CTCGAGTTCGAAAGCGGCCGCCGAGTTCACATTCCTTACTTTCTG-3'.
The resulting fragment was subcloned into a pGEX-4T vector
(Amersham Pharmacia Biotech) in frame with sequences encoding the
glutathione S-transferase. To obtain antiserum 5287, a mucin
cDNA (encoding amino acid residues 1059-1360) was similarly
amplified by polymerase chain reaction using the primers
5'-GAATTCCCGGGTCGACGGTCTTTGCGGAAACAATAATGG-3' and
5'-GCATGCCTCGAGTTCGAAAGCGGCCGCCGGTTGTGACACCCTCTGATAC-3', and the
resulting DNA fragment was subcloned into pGEX-4T. Both plasmids were
expressed in E. coli, and the fusion proteins were purified by affinity chromatography on glutathione-agarose following published procedures (19, 20). The purified proteins were injected into rabbits
(21) as fusion proteins (antiserum 5287) or after removing the
glutathione S-transferase sequences with thrombin (antiserum 4674) (20).
Construction of Mammalian Expression Vectors--
A DNA fragment
encoding six consecutive histidine codons was inserted at the 3'-end of
a cDNA encoding the entire amino-terminal region of submaxillary
mucin (amino acid residues 1-1360) cloned into the pFastBac vector
(1). The poly-His encoding fragment was made by annealing the following
complementary oligonucleotides: 5'-GGCCGCCATCACCATCACCATCACTAGC-3' and
5'-TCGAGCTAGTGATGGTGATGGTGATGGC-3'. The mucin cDNA fragments, with
or without the His tag coding sequence, were obtained as
SalI/XhoI fragments and subcloned into the
XhoI site of the pcDNA3.1(+) plasmid (Invitrogen) to
create pMN and pMNH, respectively. These plasmids were digested with
NheI and XhoI, and the mucin DNA fragments were
subcloned into the pSI vector (Promega) to create pSMN and pSMNH. The
pDMNH expression vector was made by digesting pMNH with SpeI
and XbaI and subcloning the resulting mucin DNA fragment
into the pcDNA1.1.Amp vector (Invitrogen). pMN and pSMN encode the
same amino-terminal region of porcine submaxillary mucin (amino acid
residues 1-1360) followed by nine additional residues (GGRFRTRGM) at
the carboxyl-terminal end. pMNH, pSMNH, and pDMNH encode a fusion
protein containing the amino-terminal region of submaxillary mucin
(residues 1-1360) followed by the sequence GGRHHHHHH at the carboxyl
terminus. To construct the pSMNCH expression vector, the pMCH plasmid
(14) was digested with SalI and SpeI, and the
resulting mucin DNA fragment was subcloned between the XhoI
and XbaI sites of the pSMN plasmid. The new vector directs
the expression of a fusion protein containing the amino-terminal region
of submaxillary mucin (residues 1-1360) and eight additional residues
(GGRFRTRP) followed by the disulfide-rich domain of the porcine mucin
(residues 13045-13288) and six histidine residues at the carboxyl
terminus. The expression vectors were verified by restriction nuclease
analysis, partial DNA sequencing using the Sequenase 2.0 system from
U. S. Biochemical Corp., and in vitro
transcription/translation assays with the T7-TNT coupled system
(Promega) as directed by the manufacturer.
Transfection of Mammalian Cells--
COS-7 cells were maintained
as described (14). MOP-8 cells were obtained from American Type Culture
Collection and maintained as directed by the provider. Transfections
were done with Fugene-6 (Boehringer Mannheim) according to the
manufacturer. Briefly, cells at 60-70% confluence in
75-cm2 culture flasks were incubated for 48 h in
culture medium containing 16 µl of Fugene-6 and 8 µg of DNA.
Metabolic Labeling, Purification, and Analysis of the Recombinant
Proteins--
Metabolic labeling with [35S]cysteine
(ICN), purification of the recombinant proteins by either
immunoprecipitation or absorption to TALON-IMAC beads
(CLONTECH), and analysis of the proteins by SDS-gel
electrophoresis and autoradiography were done essentially as described
earlier (14), except that cells were lysed in 50 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% SDS, 0.4% sodium
deoxycholate, 1% Nonidet P-40, 1 mM EDTA, 0.1 µM aprotinin, 1 µM pepstatin, and 5 µM leupeptin. 14C-Methylated proteins
(Amersham Pharmacia Biotech) used as molecular weight standards were
myosin (220,000), phosphorylase b (97,400-100,000), bovine
serum albumin (66,000), ovalbumin (46,000-50,000), carbonic anhydrase
(30,000), and lysozyme (14,300). 14C-Methylated plasma
fibronectin (Mr = 450,000 for the dimeric species) and thyroglobulin (Mr = 660,000 for the
unreduced form) were from Sigma.
 |
RESULTS |
Expression, N-Glycosylation, and Secretion of the Amino-terminal
Region of Mucin--
The pSMNH expression vector, which encodes the
entire amino-terminal region of submaxillary mucin (residues 1-1360)
plus a histidine tag (six residues) introduced at its carboxyl
terminus, was expressed in COS-7 cells. Forty-eight hours after
transfection, the cells were incubated in medium containing
[35S]cysteine in the presence or absence of tunicamycin.
Proteins were purified from the culture medium by immunoprecipitation
with antiserum 4674 prepared against amino acid residues 266-1014 of the mucin; with antiserum 5287 prepared against residues 1059-1360 of
the mucin; or with TALON-IMAC beads, which bind the poly-His tag
inserted at the carboxyl terminus of the mucin region. The purified
proteins were reduced with 2-mercaptoethanol and analyzed by SDS-gel
electrophoresis and autoradiography. As shown in Fig. 1A, irrespective of the
absorbent used, the amino-terminal region of mucin was secreted as a
single protein with Mr ~ 200,000. When the
addition of N-linked oligosaccharides was inhibited with
tunicamycin, the protein migrated on reducing SDS gels with
Mr ~ 165,000 (Fig. 1B), consistent
with the observed size of proteins obtained by in vitro
transcription/translation studies (data not shown). Digestion of the
protein purified from the medium of untreated cells with N-glycanase resulted in a reduction in size similar to that
found with protein expressed in the presence of tunicamycin (Fig.
1C). These results show that the amino-terminal region of
mucin, containing the D1, D2, and D3 domains, is secreted as a single
protein, which is N-glycosylated at several
Asn-X-(Thr/Ser) acceptor sites found in its sequence (1).
They also show that N-glycosylation is not required for
secretion of the mucin region, although the amounts of the protein
recovered from the medium of cells incubated in tunicamycin are
significantly lower than those secreted by untreated cells.

View larger version (55K):
[in this window]
[in a new window]
|
Fig. 1.
Expression, N-glycosylation, and
secretion of the amino-terminal region of mucin. A,
COS-7 cells transfected with the expression plasmid pSMNH were
metabolically labeled 48 h post-transfection with
[35S]cysteine for 4 h. Proteins from the medium were
immunoprecipitated with preimmune serum (lanes 1 and 3), antiserum 4674 (lane 2), or
antiserum 5287 (lane 4) or absorbed to TALON-IMAC
beads (lane 5) and analyzed by SDS-gel
electrophoresis in 2-mercaptoethanol and autoradiography. The molecular
weights of the standards are in thousands. B, COS-7 cells
expressing the pSMNH vector were incubated in
[35S]cysteine-containing medium for 4 h in the
absence (lane 1) or presence (lane
2) of 10 µg/ml of tunicamycin, and the proteins were
purified by absorption to and elution from TALON-IMAC beads and
analyzed as described for A. C,
35S-labeled proteins secreted into the medium by COS-7
cells transfected with vector pSMNH were purified with TALON-IMAC
beads, denatured by boiling in 2-mercaptoethanol in buffered SDS, and
incubated with buffer alone (lane 1) or
N-glycanase (lane 2) as reported
earlier (14). The digests were analyzed as described for
A.
|
|
Trimer Formation of the Amino-terminal Region of Mucin--
To
determine whether the amino-terminal region of mucin forms
disulfide-linked oligomers, the proteins expressed and secreted by
pSMNH-transfected COS-7 cells incubated in the presence of [35S]cysteine were purified by immunoprecipitation with
antiserum 5287 or absorption to TALON-IMAC beads and analyzed by
SDS-gel electrophoresis without prior reduction (Fig.
2A). Irrespective of the
absorbents employed, two proteins were observed. One corresponded to a
protein with Mr ~ 180,000, which is slightly
lower than the size of the reduced monomer (Mr ~ 200,000), and is thought to be unreduced monomer. The second
protein species that also entered the resolving gel migrated, as shown
in Fig. 2B (lane 1), slower than the myosin heavy
chain molecular weight standard (Mr = 220,000) and slightly slower than dimeric fibronectin (Mr = 450,000; lane 3), with Mr ~ 520,000. The second protein also migrated faster than unreduced
thyroglobulin (Mr = 660,000) (data not shown). These size estimates are consistent with the formation of
disulfide-linked trimers of the amino-terminal region containing the
three D-domains. These studies suggest that the amino-terminal region
of submaxillary mucin is secreted as monomeric and disulfide-linked
trimeric species. Fig. 2C shows that trimer formation was
not inhibited by treatment of the cells with tunicamycin, indicating
that N-glycosylation of the protein is not required for the
formation of disulfide-linked trimers.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 2.
Trimer formation of the amino-terminal region
of mucin. A, COS-7 cells transfected with the vector
pSMNH (lanes 1-3 and 5-7
) or pSMN (lanes 4 and 8) were
metabolically labeled as described in the legend to Fig. 1A,
and the proteins were purified from the culture medium by
immunoprecipitation with antiserum 5287 (lanes 1 and 5) or preimmune serum (lanes 2 and
6) or by absorption to TALON-IMAC resin (lanes
3, 4, 7, and 8). Proteins
were analyzed by SDS-gel electrophoresis in the presence
(lanes 1-4) or absence (lanes 5-8)
of 2-mercaptoethanol and by autoradiography. B, COS-7 cells
were transfected with plasmid pSMNH (lane 1) or
pSMNCH (lane 2), and the cells were incubated in
the presence of [35S]cysteine as described in the legend
to Fig. 1A. Proteins secreted into the medium were purified
by absorption to TALON-IMAC beads and analyzed by SDS-gel
electrophoresis without prior reduction. The migration pattern of
unreduced 14C-methylated plasma fibronectin is also shown
(lane 3). C, COS-7 cells
transfected with pSMNH were incubated for 4 h in
[35S]cysteine medium alone (lane 1)
or containing 10 µg/ml tunicamycin (lane 2).
Proteins immunopurified from the medium with antiserum 5287 were
analyzed without prior reduction by SDS-gel electrophoresis and
autoradiography. D, MOP-8 cells were transfected with
plasmid pDMNH and, after 48 h, incubated for 4 h in medium
containing [35S]cysteine. Proteins secreted into the
medium were immunoprecipitated with antiserum 5287 and analyzed by
SDS-gel electrophoresis in the absence of 2-mercaptoethanol and by
autoradiography.
|
|
Secretion and trimer formation of the mucin amino-terminal region were
also found to occur in a different cell line. Fig. 2D shows
the electrophoretic pattern of proteins purified by immunoprecipitation with antiserum 5287 from the medium of mouse MOP-8 cells transfected with plasmid pDMNH. Only monomers and high molecular weight trimers of
the mucin region, identical to those protein species secreted by COS-7
cells, were observed. These results suggest that secretion and trimer
formation of the amino-terminal region of mucin do not depend on the
cell type.
Rate of Trimer Formation and Secretion of the Amino-terminal Region
of Mucin--
Pulse-chase studies with COS-7 cells transfected with
the pSMN vector were performed to measure the rates of formation and secretion of the trimers formed by the amino-terminal region. After
incubation (15 min) in medium containing [35S]cysteine,
the cells were incubated in complete medium with an excess of unlabeled
cysteine. At selected time periods, the cells were lysed with buffered
detergents, and proteins in the lysate and the medium were purified by
immunoprecipitation with antiserum 5287, reduced with mercaptoethanol,
and analyzed by SDS-gel electrophoresis and autoradiography. As shown
in Fig. 3A, the first
intracellular protein detected had a Mr of
~165,000 (lane 1), which is indistinguishable from the
mucin expressed and purified from tunicamycin-treated cells (Fig.
1B) or after digestion of the expressed mucin with N-glycanase (Fig. 1C). This molecular species is
thought to be protein synthesized in the endoplasmic reticulum but not
yet glycosylated. A second intracellular, less abundant protein with
Mr ~ 200,000 appeared after 30 min of
incubation in unlabeled medium (lane 3), just at the time
when the amount of the initial unglycosylated precursor started to
diminish, and reached a maximal amount at 60 min of chase (lane
4). This species is thought to be N-glycosylated mucin.
At longer chase times, both the unglycosylated and glycosylated proteins disappeared from the cells. These observations indicate that
N-glycosylation is a post-translational modification of the amino-terminal region of mucin. Fig. 3A also shows the
proteins purified from the medium of cells incubated for different
periods of time in unlabeled medium and analyzed after reduction in
2-mercaptoethanol. The N-glycosylated protein
(Mr ~ 200,000) appeared in the medium after 30 min (lane 10) and increased at longer chase times. Some unglycosylated precursors (Mr ~ 165,000) were
also observed in the medium, but in the same amounts in all the time
periods analyzed, including the pulse sample (lane 8). Thus,
these species may correspond, at least in part, to intracellular
precursors liberated into the medium from cells that were lysed during
the incubation with [35S]cysteine.

View larger version (80K):
[in this window]
[in a new window]
|
Fig. 3.
Biosynthesis of the amino-terminal region of
mucin. COS-7 cells were transfected with plasmid pSMN, and 48 h later, cells were metabolically labeled with
[35S]cysteine for 15 min and chased for the indicated
times in medium containing an excess of unlabeled cysteine. Proteins
from detergent extracts of cells (lanes 1-7 in
both panels) or medium (lanes 8-13 in both
panels) were immunoprecipitated with antiserum 5287 and analyzed by
reducing (A) or nonreducing (B) SDS-gel
electrophoresis and autoradiography.
|
|
Fig. 3B shows the same protein samples as in Fig.
3A but analyzed on gels without prior reduction. A protein
with Mr ~ 150,000 was the only species found
in cell lysates at the end of the pulse with
[35S]cysteine (lane 1). This material
corresponds to the unglycosylated monomeric precursors observed on SDS
gels after reduction in 2-mercaptoethanol. At longer chase times, the
latter protein gradually disappeared and was almost absent from the
cells following 2 h in unlabeled medium (lane 5). A
high molecular weight protein, with an electrophoretic migration
identical to that of the disulfide-bonded trimers purified from the
medium shown in Fig. 2, was clearly observed after 30 min of chase
(lane 3). These trimeric species increased slightly in
amount after 60 min in unlabeled medium (lane 4), but
decreased at longer chase times. This time course was indistinguishable from that followed by the N-glycosylated intracellular
protein observed on reducing SDS gels, suggesting that only
glycosylated domains are incorporated into trimers. Disulfide-linked
trimers appeared in the medium 30 min after the pulse (lane
10), at which time N-glycosylated monomers
(Mr = 180,000) were also observed, and both
species increased in the medium at longer chase times. Unglycosylated
monomers were also found in the medium, but in similar amounts
throughout the chase. Taken together, the above results suggest that
the amino-terminal region of mucin spends much of its intracellular
life as a monomeric, unglycosylated protein that resides in the
endoplasmic reticulum. As soon as the monomers are
N-glycosylated, they form disulfide-linked trimers that are rapidly secreted from the cells. However, a significant amount
of the N-glycosylated monomers do not form trimers, although they are also rapidly secreted into the medium.
Intracellular Site for Trimer Formation of the Amino-terminal
Region of Mucin--
To determine the intracellular compartment for
trimer formation, COS-7 cells expressing the entire amino-terminal
region of mucin were incubated with brefeldin A, a compound knows to
disrupt the Golgi complex (22), during both the 15-min pulse with
[35S]cysteine and the subsequent 1-h chase in unlabeled
medium (Fig. 4). SDS-gel electrophoresis
of the proteins immunopurified with antiserum 5287 from cell lysates
and reduced with 2-mercaptoethanol showed two main bands in untreated
cells (lane 1), corresponding to
unglycosylated (Mr ~ 165,000) and
N-glycosylated (Mr ~ 200,000) monomers of the expressed protein. In the presence of brefeldin A
(lane 2), however, there was a single protein
with Mr ~ 185,000, thought to be an
N-glycosylated intermediate species retained in the
endoplasmic reticulum. Consistent with this conclusion, there was no
indication of any protein secreted into the medium in cells treated
with brefeldin A (lane 4), whereas untreated cells (lane 3) secreted the characteristic
N-glycosylated species (Mr ~ 200,000). Without prior reduction in 2-mercaptoethanol, the predominant
intracellular species expressed by the untreated cells (lane
5) included unglycosylated monomers
(Mr ~ 150,000) and N-glycosylated
disulfide-linked trimers. In contrast, brefeldin A-treated cells
(lane 6) synthesized one protein with a
Mr similar to those of the
N-glycosylated monomers observed on reducing SDS gels. These
results indicate that trimerization did not occur in the presence of
brefeldin A, suggesting that the formation of disulfide-linked trimers
of the amino-terminal region of mucin does not take place in the
endoplasmic reticulum, but in the Golgi complex.

View larger version (82K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of brefeldin A on the trimerization
and secretion of the amino-terminal region of mucin. COS-7 cells
transfected with plasmid pSMNH were incubated for 5 min in medium alone
(lanes 1, 3, 5, and
7) or in medium containing 1 µg/ml brefeldin A
(lanes 2, 4, and 6). All
cells were then incubated with [35S]cysteine for 15 min
and chased in unlabeled medium for 1 h in the absence
(lanes 1, 3, 5, and
7) or presence (lanes 2, 4, and 6) of 1 µg/ml brefeldin A. Proteins from cell lysates
(C ; lanes 1, 2,
5, and 6) or medium (M; lanes
3, 4, and 7) were immunoprecipitated
with antiserum 5287 and analyzed by reducing (lanes
1-4) or nonreducing (lanes 5-7)
SDS-gel electrophoresis and autoradiography.
|
|
Evidence for the involvement of specific Golgi complex compartments in
trimer formation of the mucin was obtained by incubating COS-7 cells
expressing the mucin amino-terminal region with agents that disrupt the
pH gradient of this organelle (reviewed in Ref. 23). As shown in Fig.
5A, incubation of the cells in
the presence of the ionophore monensin (lane 2)
or the lysosomotropic agent chloroquine (lane 3)
did not abrogate the secretion of the mucin region, although the latter
significantly reduced the amount of the protein in the medium. However,
in both cases, the secreted proteins exhibited a
Mr (175,000) on reducing SDS gels slightly lower
than those of protein species purified from the medium of untreated
cells (lane 1), suggesting that the processing of
the N-linked oligosaccharides has been altered by the above
compounds (23). Without prior reduction in 2-mercaptoethanol, only a
single species corresponding to a protein of Mr ~ 170,000 was observed on SDS gels of proteins purified from cells
incubated in the presence of monensin (lane 5) or
chloroquine (lane 6), whereas untreated cells
(lane 4) expressed and secreted both monomeric
and trimeric species. These results indicate that monensin and
chloroquine inhibited the formation of disulfide-linked trimers of the
mucin region, suggesting that trimer formation requires a low pH. This possibility was further confirmed by incubating pSMNH-transfected COS-7
cells in medium containing bafilomycin A1 (24), a specific inhibitor of the vacuolar H+-ATPase (reviewed in Ref. 25)
assumed to maintain a pH between 5.9 and 6.6 in the trans Golgi
compartments (e.g. Refs. 26-28). Fig. 5B shows
that on reducing SDS gels, the species secreted in the presence of
bafilomycin A1 (lane 2) presented a
slightly lower Mr than those secreted by
untreated cells (lane 1) and were indistinguishable from those secreted by cells incubated in the presence of monensin or chloroquine (Fig. 5A). Moreover,
bafilomycin A1 also inhibited the formation of
disulfide-linked trimers of the mucin region (lane
4). These results indicate that trimer formation requires
the continuous activity of the vacuolar H+-ATPase and
suggest that trimer formation occurs in acidic compartments of the
Golgi complex, which includes the trans cisternae and the trans tubular
network (29).

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 5.
Effects of monensin, chloroquine, and
bafilomycin A1 on the trimerization and secretion of the
amino-terminal region of mucin. A, COS-7 cells were
transfected with plasmid pSMNH and, 48 h post-transfection,
incubated for 4 h in medium with [35S]cysteine alone
(lanes 1 and 4) or in medium
containing 10 µg/ml monensin (lanes 2 and
5) or 100 µM chloroquine (lanes
3 and 6). Proteins were purified from the medium
by absorption to and elution from TALON-IMAC beads and analyzed by
SDS-gel electrophoresis in the presence (lanes
1-3) or absence (lanes 4-6) of
2-mercaptoethanol. B, COS-7 cells transfected with plasmid
pSMNH were incubated for 4 h with
[35S]cysteine-containing medium in the absence
(lanes 1 and 3) or presence
(lanes 2 and 4) of 2.5 µg/ml
bafilomycin A1, and proteins secreted into the medium were
immunoprecipitated with antiserum 5287 and analyzed by reducing
(lanes 1 and 2) or nonreducing
(lanes 3 and 4) SDS-gel
electrophoresis and autoradiography.
|
|
Role of the Amino- and Carboxyl-terminal Domains in the Structure
of Mucin--
Previous studies (14) have shown that the
carboxyl-terminal domain of submaxillary mucin forms disulfide-linked
dimers between monomers. Therefore, it was important to determine
whether the amino- and carboxyl-terminal regions of the submaxillary
mucin form interchain disulfide bonds between one another. For this purpose, COS-7 cells were simultaneously transfected with pSMNH, which
encodes the amino-terminal region of submaxillary mucin with six
histidine residues at its carboxyl terminus, and pMC (14), a vector
encoding the entire disulfide-rich carboxyl-terminal domain of the
mucin. After incubation of the cells in medium with [35S]cysteine, the proteins secreted into the medium were
purified with antiserum 3814, which recognizes the carboxyl-terminal
domain; antiserum 5287, specific for the amino-terminal region; or
TALON-IMAC beads, which bind the amino-terminal region through the
poly-His tag at its carboxyl terminus. Fig.
6 shows the electrophoretic pattern
obtained after reduction of the purified proteins in 2-mercaptoethanol. Antiserum 3814 (lane 1) immunoprecipitated only
five proteins with Mr ranging from 33,000 to
47,000, which represent species of the mucin carboxyl-terminal domain
that differ in the extent of N-glycosylation (14). When the
secreted proteins were purified with antiserum 5287 (lane
2) or TALON-IMAC beads (lane 3),
protein species with the expected Mr (200,000)
for the amino-terminal region were the only proteins found. Therefore,
although both mucin regions were present in the culture medium, they
were not covalently associated with one another. These results suggest that dimer formation between the carboxyl-terminal domains (14) and
trimerization among the amino-terminal regions occur independently and
account for the permanent interchain disulfide bonds formed during
submaxillary mucin biosynthesis.

View larger version (86K):
[in this window]
[in a new window]
|
Fig. 6.
Coexpression of the disulfide-rich
carboxyl-terminal domain and the amino-terminal domain of mucin.
COS-7 cells were cotransfected with equal amounts of plasmid pMC and
pSMNH. After 48 h, the cells were labeled for 4 h with
[35S]cysteine, and the proteins from the medium were
purified by immunoprecipitation with antiserum 3814 (lane
1) or antiserum 5287 (lane 2) or by
absorption to TALON-IMAC beads (lane 3). The
proteins were reduced in 2-mercaptoethanol and analyzed by
SDS-gel electrophoresis and autoradiography.
|
|
Pulse-chase studies with COS-7 cells transfected with the pSMNCH vector
were performed to assess the formation of high molecular weight
disulfide-linked multimers. The pSMNCH expression vector encodes a
fusion protein containing the complete mucin amino-terminal region
followed by the entire disulfide-rich domain with six histidine residues at its carboxyl terminus. In these studies, transfected cells
were incubated for 15 min with [35S]cysteine-containing
medium and chased with unlabeled medium, and the proteins from cell
lysates and the medium were immunoprecipitated with antiserum 5287 and
then analyzed by SDS-gel electrophoresis. As shown in Fig.
7A, the predominant
intracellular species migrated without prior reduction as a broad band
centered at Mr ~ 430,000 (lane
6), representing disulfide-bonded dimers of the protein, which on reduction gave a single protein of Mr ~ 220,000. Monomeric forms were not observed, showing that dimer
formation of the recombinant mucin occurs very rapidly.
N-Glycosylation of the dimeric species very likely started
once the protein was translocated into the endoplasmic reticulum since
a second, less abundant intracellular protein, with
Mr ~ 250,000 on reducing gels, appeared after
15 min (lane 2) or longer chase times. Moreover,
this protein was not observed when the synthesis of the
N-linked oligosaccharides was inhibited with tunicamycin
(data not shown). The intracellular dimers almost disappeared from the
cells after 2 h of chase (lane 10). As we
have reported earlier (14), dimer formation by interchain disulfide
bonds between the carboxyl-terminal domains of porcine mucin to create
dimers occurs very rapidly in the endoplasmic reticulum and is
independent of the N-glycosylation of the monomers. Therefore, the predominant intracellular forms of the recombinant mucin
observed on nonreducing SDS gels (Fig. 7A) represent dimeric species linked by interchain disulfide bonds between their
carboxyl-terminal domains. Other, much less intense bands,
corresponding to species of higher molecular weight, were also observed
on the same nonreducing gels, suggesting the formation of
disulfide-linked oligomers and multimers of the mucin protein.

View larger version (57K):
[in this window]
[in a new window]
|
Fig. 7.
Formation of multimeric species of the
mucin. A, COS-7 cells were transfected with pSMNCH,
metabolically labeled with [35S]cysteine for 15 min, and
chased for the indicated times in medium containing an excess of
unlabeled cysteine. Cells were lysed by buffered detergents, and
proteins in the cell lysates were immunoprecipitated with antiserum
5287 and analyzed by reducing (lanes 1-5) or
nonreducing (lanes 6-10) SDS-gel electrophoresis
and autoradiography. B, COS-7 cells expressing the pSMNCH
vector were pulse-labeled with [35S]cysteine for 15 min
in medium alone (lanes 1-9) or with monensin
(lane 10) and chased in unlabeled medium for the
indicated times in the absence (lanes 1-9) or
presence (lane 10) of monensin. Proteins from the
medium were immunoprecipitated by antiserum 5287 and analyzed by
reducing (lanes 1-5) or nonreducing (lanes
6-10) SDS-gel electrophoresis and autoradiography.
|
|
As shown in Fig. 7B, on reducing SDS gels, the
N-glycosylated species of Mr ~ 250,000 were the major proteins purified from the medium of
pSMNCH-transfected cells. Some unglycosylated species were also
observed, but they disappeared from the medium after 2 h of chase
(lane 5), suggesting that they were degraded.
Without prior reduction, N-glycosylated dimeric species were
the predominant proteins in the medium, although significant amounts of
high molecular weight species that migrated in the interface between
the stacking and resolving gels appeared in the medium with the same
kinetics as the former. These high molecular weight forms very likely
represented disulfide-linked multimers of the dimeric species that were
assembled by the formation of interchain disulfide bonds among their
amino-terminal regions. Consistent with this interpretation, when
monensin was present in the medium (lane 10), the
cells secreted only dimeric species, with no indication of multimeric
forms (Fig. 7B), showing that formation of the latter needs
a low pH. Formation of interchain disulfide bonds between the
carboxyl-terminal domains occurs in the endoplasmic reticulum (14), and
therefore, monensin is without effect on the synthesis of dimeric
species. Formation of disulfide-linked multimers requires the
cross-linking of the amino-terminal regions in the acidic compartments
of the Golgi complex, a reaction that can be disrupted by monensin
(Fig. 5).
Taken together, these observations suggest that dimer formation via the
carboxyl-terminal domains is an early processing step that occurs
rapidly after the synthesis of the mucin in the endoplasmic reticulum,
consistent with studies reported earlier (14). Multimer formation via
the amino-terminal domains, on the contrary, occurs in COS-7 cells once
the dimers are N-glycosylated and shortly before the
recombinant mucin is secreted into the medium since intracellular
accumulation of multimeric species was not observed (Fig.
7A). These results also show that whereas all the precursors form dimeric species in the endoplasmic reticulum, only a fraction of
the dimers are assembled into disulfide-linked multimers in the distal,
acidic compartments of the Golgi complex. Thus, dimers and multimers,
but not monomers, are secreted into the medium.
 |
DISCUSSION |
The studies reported here show that the amino-terminal region of
porcine submaxillary mucin is secreted from transiently transfected cells as a single protein that forms disulfide-linked trimers. Two
different antisera against polypeptides from the amino-terminal region
of the mucin, in addition to absorption of the carboxyl-terminal poly-His tag with TALON-IMAC beads, failed to show proteins of lower
molecular weight (Fig. 1). The homologous amino-terminal region of
human pro-von Willebrand factor is proteolytically processed during its
biosynthesis (reviewed in Ref. 30), which results in the secretion of
two proteins, the propolypeptide, which includes the D1 and D2 domains,
and mature von Willebrand factor. Formation of mature von Willebrand
factor involves the cleavage of the peptide bond between Arg-763 and
Ser-764 by furin (30, 31), a ubiquitously expressed subtilisin-like
serine endoprotease. Proteolytic processing of human pro-von Willebrand
factor has been shown to occur not only in endothelial cells, but in
different cell types including COS cells (30). These observations,
together with the absence of proteolytic forms of the amino-terminal
region of the mucin expressed and secreted by COS-7 cells or MOP-8
cells (Figs. 1 and 2) and the fact that none of the predominant
consensus recognition sequences for furin
(Arg-X-Arg/Lys-Arg; e.g. Ref. 31) are found in
the submaxillary mucin (1), strongly argue against a role for furin
during the biosynthesis of the porcine mucin.
The amino-terminal region of the mucin expressed in COS-7 cells or
MOP-8 cells forms disulfide-linked oligomers (Fig. 2). The molecular
weight of these oligomers could not be determined exactly, but by
SDS-gel electrophoresis, they migrated slightly slower than dimeric
fibronectin (Fig. 2B) with an estimated
Mr (520,000) indicating the formation of
disulfide-linked trimers. Consistent with this conclusion, the trimeric
species also migrated slightly slower than the dimeric species of a
fusion protein between the same amino-terminal region and the
disulfide-rich carboxyl-terminal domain of mucin, which migrated at a
similar position as dimeric fibronectin (Fig. 2B). The mucin
amino-terminal region does not form interchain disulfide bonds with the
disulfide-rich carboxyl-terminal domain (Fig. 6), indicating that
dimerization and trimerization are independent processes during the
biosynthesis of mucin. Indeed, dimer formation via the
carboxyl-terminal domains of the mucin occurs in the endoplasmic
reticulum (14) and precedes multimerization through the amino-terminal
domains (Figs. 3 and 7). Disulfide-linked multimers of submaxillary
mucin are assembled in the Golgi complex (Fig. 4) and very likely in a
compartment with a pH between 5.9 and 6.6, which is characteristic of
the distal/trans compartments of the Golgi complex (Figs. 5 and
7B). These roles of the amino- and carboxyl-terminal domains
of the mucin in multimer formation are consistent with their amino acid
sequence similarities to the corresponding amino- and carboxyl-terminal
regions of human von Willebrand factor (1). This human blood-clotting
factor also forms multimers by interchain disulfide bonds among the
amino-terminal regions of dimeric subunits (3, 30). Moreover, multimer
formation of pro-von Willebrand factor requires a low pH and seems to
occur in distal compartments of the Golgi complex. Human von Willebrand factor catalyzes its own multimerization, which depends on the presence
of the D1 and D2 domains (32). Three CGLCG sequence motifs, two in the
D1 and D2 domains and one in the D3 domain, resemble the active site of
protein-disulfide isomerase and are thought to play an important role
in the catalysis of interchain disulfide bond formation in human
pro-von Willebrand factor (33). Porcine submaxillary mucin contains two
CGLCG sequence motifs, one in the D1 domain and the other in the D3
domain (1), suggesting that they may also function in mucin
multimerization. All the interchain disulfide bonds in mature von
Willebrand factor multimers are between D3 domains on different
subunits, in which half-cystine 379 (34) and one or more of the
half-cystines at residues 459, 462, and 464 (35) are forming interchain
disulfide bonds. These half-cystines are conserved in the corresponding
D3 domain of porcine submaxillary mucin (1), and they may also form
interchain disulfide bonds.
Earlier studies (14, 36-38) and the studies presented here reveal the
major steps that occur during submaxillary mucin biosynthesis, as
summarized diagramatically in Fig. 8.
Porcine apomucin is synthesized in the endoplasmic reticulum of mucus
cells (36) and rapidly forms disulfide-linked dimers between its
carboxyl-terminal domains (14) including 11 half-cystines (37). Among
the latter, one or more of half-cystines 13223, 13244, and 13246 are
required for forming interchain disulfide bonds (37).
N-Glycosylation of the amino- and carboxyl-terminal domains
of the mucin seems to occur post-translationally (Figs. 3A
and 7A). N-Glycosylation is not required for
dimer formation (14) or, later, for multimerization (Fig. 2), although
the extracellular half-life of the unglycosylated mucin domains seems
to be decreased (Figs. 1B and 7A).
O-Glycosylation commences in the cis Golgi compartments (36,
38) and continues in the medial and trans Golgi compartments. Upon
reaching the distal/trans compartments of this organelle,
multimerization through the amino-terminal regions containing the
D-domains is very likely triggered by the intraluminal acidic pH (Fig.
5A). Indeed, the action of the vacuolar
H+-ATPase is critical for trimer formation of the mucin
D-domains expressed in COS-7 cells (Fig. 5B).
Multimerization does not affect all the dimeric species (Fig.
7B), and eventually, multimers and dimers are constitutively
secreted into the medium, the only route in COS-7 cells, or presumably
accumulated in secretory granules of mucus cells (36). It is possible
that secreted dimers of the mucin are assembled into disulfide-linked
multimers following changes in the extracellular pH.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 8.
Schematic representation of the sequence of
events in porcine submaxillary mucin biosynthesis. Step
1, porcine apomucin is synthesized in endoplasmic reticulum-bound
polyribosomes and then translocated and folded in the endoplasmic
reticulum. Step 2, immediately after its
synthesis, the apomucin molecules dimerize in the endoplasmic reticulum
through interchain disulfide bonds between the carboxyl-terminal
domains including 11 half-cystines. Step 3, disulfide-bonded
dimers are N-glycosylated and transported to the Golgi
complex. Step 4, N-glycosylated
dimeric mucins are initially O-glycosylated in the cis Golgi
compartments. The biosynthesis of O- and N-linked
oligosaccharides continues in the medial and trans Golgi compartments.
Step 5, upon reaching the trans Golgi compartments,
multimeric species of the mucin are assembled by interchain disulfide
bonds involving one or more of the amino-terminal D-domains of the
dimeric species. Step 6, presumably, some
of the glycosylated dimeric and multimeric species of the mucin are
taken in the trans Golgi network by small transport vesicles, whereas
the majority are concentrated in large secretory granules. Steps
7 and 8, transport vesicles and mucin granules are
secreted by constitutive and regulated exocytotic pathways,
respectively, into the extracellular space.
|
|
As a direct consequence of dimerization via the carboxyl-terminal
domains and trimer formation through the amino-terminal regions, it can
be predicted that the porcine mucin is able to form branched multimers
(Fig. 8). Multimeric species of human von Willebrand factor are built
by the independent dimerization of the amino- and carboxyl-terminal
regions, resulting in the formation of unbranched linear structures
(39). Moreover, electron microscopy studies on purified cervical,
respiratory, and gastric mucins (e.g. Ref. 40) show that
these mucins form flexible, long filaments of variable length.
Unfortunately, the primary structure of the latter mucins is
incomplete. These observations suggest that the mechanisms for the
formation of disulfide-linked multimers in von Willebrand factor,
porcine submaxillary mucin, and likely other mucins are similar, but
not necessarily identical.
The present studies and those on the multimerization of human
prepro-von Willebrand factor clearly show that the D-domains found in
the amino-terminal regions of these proteins serve to form
disulfide-linked multimers. It can be anticipated that similar D-domains present in the amino-terminal regions of frog integumentary mucin FIMB.1 (16) and human mucins MUC2 (17) and MUC5AC (41, 42) also
serve to form interchain disulfide bonds among the respective proteins.
Indeed, other studies have suggested that human MUC2 mucin forms
disulfide-linked multimers (43).