(Received for publication, December 29, 1995; and in revised form, February 7, 1996)
From the
COS-7 cells transfected with three different expression vectors
encoding the 240-amino acid residue, disulfide-rich domain at the
carboxyl terminus of porcine submaxillary mucin have been used to
determine the possible function of the domain in forming higher
oligomers of the mucin polypeptide chain. The domain is expressed as a
disulfide-bonded dimer, as shown by SDS-gel electrophoretic analysis of
the immunoprecipitated domain in the presence and absence of reducing
agent and the cross-linking agent bis(sulfosuccinimidyl) suberate.
Molecular weight determination by gel filtration on agarose columns in
6 M guanidine HCl confirmed dimer formation. However, the
domain expressed is heterogeneous as the result of different extents of
glycosylation. Pulse-chase studies with the S-labeled
domain show that dimer formation and secretion from cells occur very
rapidly. Moreover, dimer formation is not dependent on the N-linked oligosaccharides on the domain. Evidence is presented
that dimer formation most likely occurs in the endoplasmic reticulum
before complex-type oligosaccharide synthesis is completed. Neither
brefeldin A nor tunicamycin interferes with the rate of dimer
formation. These studies suggest that the disulfide-rich domain acts to
form dimers of the polypeptide chain of mucin. This role of the domain
is consistent with its amino acid sequence similarity to the
disulfide-rich domain of human prepro-von Willebrand factor, which also
serves to form dimers of this blood coagulation factor.
Porcine submaxillary mucin has a disulfide-rich domain of
240 residues at its carboxyl terminus(1) . This domain
contains 30 half-cystine residues and no free thiol groups, (
)and unlike the highly O-glycosylated portions of
the polypeptide backbone, it appears to be a globular structure. Such
disulfide-rich domains are characteristic of many mucins and have been
found in a bovine submaxillary gland mucin-like protein(2) ;
human intestinal mucin, designated MUC2(3) ; human
tracheobroncheal mucin(4) ; rat intestinal mucin(5) ;
and frog integumentary mucin(6) . Of these mucins, the complete
amino acid sequence has been established for only human intestinal
mucin. This intestinal mucin contains a single disulfide-rich domain at
its carboxyl terminus, which is similar in sequence to each of the
three contiguous disulfide-rich domains near its amino
terminus(7) . There is a high degree of identity in the amino
acid sequences among the disulfide-rich domains from different mucins,
especially in the location of the half-cystines. Moreover, there is a
striking sequence identity between the disulfide-rich domains of human
intestinal mucin and the four disulfide-rich domains of human
prepro-von Willebrand factor(7) . In addition, the four domains
of prepro-von Willebrand factor and human intestinal mucin are located
in the same regions of the molecule, one near the carboxyl terminus and
three near the amino terminus. Although the sequence identity between
the porcine submaxillary gland mucin domain and human prepro-von
Willebrand factor is only
20% (1) and thus of questionable
significance statistically, the location of the half-cystines in these
domains is very much the same.
The disulfide-rich domains of von
Willebrand factor have been shown to form interchain disulfide bonds
among von Willebrand factor monomers. The disulfide bonds between the
carboxyl-terminal domains of von Willebrand factor permit dimer
formation(8, 9) , whereas those in the amino terminus
permit multimer formation(8, 10, 11) . In
view of the structural similarities between von Willebrand factor,
human intestinal mucin, and porcine submaxillary mucin, it was of
interest to determine whether the mucin domains serve to form dimers or
oligomers. Dimer or oligomer formation in mucins would be difficult to
determine, however, because mucins have very high molecular weights
(>10) and high carbohydrate contents (75-90%
carbohydrate by weight) that prevent structural analysis by methods
such as gel electrophoresis or ultracentrifugation by sedimentation
equilibrium.
We report here studies showing that porcine submaxillary mucin can very likely form dimers between its carboxyl-terminal domains. This was determined by expression of the disulfide-rich domain in mammalian cells transfected with cDNA encoding the domain, but devoid of the other structures of mucin that interfere with gel electrophoretic analysis. By these means, it is shown that the disulfide-rich monomer is synthesized and converted rapidly to a disulfide-linked dimer in the endoplasmic reticulum before secretion of the dimer into the medium.
When used to test their effect on the biosynthesis of the recombinant proteins, tunicamycin and brefeldin A (both from Sigma) were added to the culture medium before and during metabolic labeling at final concentrations of 10 and 5 µg/ml, respectively.
Figure 1:
Expression, N-glycosylation,
and secretion of the disulfide-rich domain of mucin. A, COS-7
cells transfected with expression plasmid pMC (lane 1), pMCH (lane 2), or pPA-MC2 (lane 3) were metabolically
labeled for 3 h, at 48 h post-transfection, with
[S]cysteine and
[
S]methionine (Tran
S-label).
Proteins were then immunoprecipitated from the medium with antiserum
3814 and analyzed by SDS-gel electrophoresis in
-mercaptoethanol
and by autoradiography. B, monolayers of COS-7 cells
transfected with plasmid pMC were incubated for 3 h in medium in the
absence (lane 1) or presence (lane 2) of 10 µg/ml
tunicamycin and labeled with Tran
S-label, and the secreted
proteins were isolated and analyzed as described for A. C, radiolabeled proteins from the medium of COS cells
transfected with pMC were isolated by immunoprecipitation and analyzed
on reducing SDS gels as describe for A. Lane 1 shows
the proteins without further treatment, and lane 2 shows the
proteins after digestion with 1 unit/ml N-glycanase. D, purified native submaxillary mucin was digested with 10%
pepsin at 37 °C for 18 h. A fraction was then reacted at pH 8.3
with 5 units/ml N-glycanase (lane 2) for 16 h at 37
°C. Proteins were separated by reducing SDS-gel electrophoresis,
blotted onto polyvinylidene difluoride membranes, reacted with an
antiserum against a peptide from the carboxyl-terminal mucin domain and
with a peroxidase-linked anti-rabbit antibody, and visualized by
enhanced chemiluminescence. Lane 1 is the peptic digest
without N-glycanase treatment.
Figure 2:
Dimer formation of the disulfide-rich
domain. A, COS-7 cells transfected with plasmid pMC were
incubated with TranS-label for 10 min and subsequently
chased in unlabeled medium for 45 min at 37 °C. A fraction of the
medium was reacted with 5 mM bis(succinimidyl) suberate (BS) for 15 min at room temperature, and the reaction was
stopped by adding an excess of glycine. The immunoprecipitated proteins
(antiserum 3814) were analyzed by SDS-gel electrophoresis in the
presence or absence of
-mercaptoethanol (ME) (lanes
1-3). Proteins precipitated by the preimmune serum are shown (lanes 4-6). B, COS cells were transfected with
plasmid pMCH and metabolically labeled as described for Fig. 1A. Proteins were purified from the medium by
absorption to IMAC resin and incubated for 30 min with 5 mM bis(succinimidyl) suberate (lane 2). Samples were then
analyzed by SDS-gel electrophoresis and autoradiography as described
for A. C, proteins from COS-7 cells expressing
plasmid pPA-MC2 were purified from the medium of
S-labeled
cells by absorption to IgG-agarose. Samples were analyzed by SDS-gel
electrophoresis in the presence (first lane) or absence (second lane) of
-mercaptoethanol.
Fig. 2B shows the same type of experiment as in Fig. 2A, except that the expression plasmid pMCH was
used to transfect COS cells and the expressed protein was isolated on a
TALON IMAC absorbent. The proteins expressed had about the same
molecular weights as those in Fig. 2A and clearly show
dimers under nonreducing conditions, monomers under reducing
conditions, and cross-linked dimers stable to reduction. Fig. 2C shows the SDS-gel electrophoretic patterns of
the proteins expressed by COS cells transfected with pPA-MC2, which
encodes the fusion protein of the domain and protein A. The major
protein on reducing gels is the monomeric species with an M of 86,000-90,000, whereas that on
nonreducing gels has an M
of 180,000. Thus, the
formation of dimers is not influenced by the non-mucin portion of the
fusion protein.
Fig. 3shows the estimated molecular weights
for the proteins expressed and secreted from transfected COS cells as
judged by gel filtration on Sepharose Cl-4B in the presence of
guanidine HCl. These analyses confirmed the molecular weight estimates
made from SDS-gel electrophoresis. Thus, the protein expressed by
pPA-MC2 under nonreducing conditions had an M of
150,000 as compared with an M
of 82,000 under
reducing conditions. Similarly, the protein from COS cells transfected
with pMCH had an M
of 66,000 in the absence of
reducing agent and an M
of 35,000 in the presence
of reducing agent. The molecular weight of pPROTA, the protein
expressed by the fusion protein vector without the disulfide-rich
domain insert, was about the same in the presence and absence of
-mercaptoethanol, which also indicates that dimer formation is not
dependent on the non-mucin portion of the fusion protein.
Figure 3:
Determination of the molecular weight of
the disulfide-rich domain of mucin by gel filtration on Sepharose
Cl-4B. The S-labeled domain was isolated from the
conditioned medium of COS cells transfected with pMCH, pPROTA, or
pPA-MC2. The purified domain was analyzed under nonreducing conditions
or after reduction in
-mercaptoethanol and alkylation with
iodoacetamide. The samples were applied and eluted in 6 M guanidine HCl, pH 5. The amount of eluate that emerged from the
column was measured from the weight of the fractions eluted.
Radioactivity (open circles) was used to detect the domains in
the eluates. The protein standards (closed circles) were
analyzed in the same way after reduction and alkylation with
iodoacetamide and were detected in the eluates by their absorbance at
280 nm. Further details are given under ``Experimental
Procedures.''
Figure 4:
Pulse-chase study of the synthesis and
secretion of the disulfide-rich domain of mucin. A, COS-7
cells, at 48 h post-transfection with plasmid pMC, were labeled with
TranS-label for 10 min at 37 °C and chased for the
indicated times in medium containing an excess of unlabeled methionine
and cysteine. Proteins from cell detergent extracts (C) and
medium (M) were immunoprecipitated with antiserum 3814 (lanes 1-7 and 10-16) and then analyzed
by nonreducing (lanes 1-9) or reducing (lanes
10-18) SDS-gel electrophoresis. Proteins absorbed by the
preimmune serum (PI) are shown in lanes 8, 9, 17, and 18. B, pMC-transfected
COS-7 cells were subjected to pulse and chase as described for A. The proteins in the cell lysates were immunoprecipitated with
antiserum 3814 and divided into two fractions, one of which was reacted
with 0.05 unit/ml endoglycosidase H (Endo H) (+ lanes) for 16 h at 37 °C. The proteins were analyzed by
reducing SDS-gel electrophoresis and
autoradiography.
As shown in Fig. 4B, the proteins expressed in the cells transfected with pMC were sensitive to endoglycosidase H digestion between 0 and 10 min under the conditions of the chase in unlabeled medium. This result suggests that dimer formation occurs before N-linked oligosaccharides are converted to the complex-type oligosaccharides that are endoglycosidase H-resistant. Because the enzymes that catalyze the conversion of endoglycosidase H-sensitive to endoglycosidase H-resistant species exist in the medial/trans-Golgi complex(22) , dimer formation must occur in the endoplasmic reticulum or the cis-Golgi apparatus. However, the endoplasmic reticulum is the main organelle acting in mucin dimer formation because cells transfected with pMC express dimers of the domain in the presence of brefeldin A (Fig. 5), a drug that disorganizes the Golgi apparatus(23) . Moreover, in brefeldin A, the dimers are not secreted into the medium, nor are their N-linked oligosaccharides converted to the complex type.
Figure 5:
Synthesis of the disulfide-rich domain of
mucin in cells incubated with brefeldin A. A, COS cells
transfected with plasmid pMC were incubated for 5 min in medium
containing 5 mg/ml brefeldin A, pulsed-labeled for 10 min with
TranS-label as described for Fig. 4A, and
chased for the indicated times in unlabeled medium in the presence of
brefeldin A. Proteins from cell lysates (C) or conditioned
medium (M) were immunoprecipitated with antiserum 3814 (lanes 1-3) and analyzed by nonreducing SDS-gel
electrophoresis and autoradiography. Proteins absorbed by the preimmune
serum are shown (lanes 4-6). B, cell lysates
from cells chased for 60 min in the presence of brefeldin A were
immunoprecipitated with antiserum 3814 and analyzed before (lane
1) and after (lane 2) digestion with 0.05 unit/ml
endoglycosidase H (Endo H; as described for Fig. 4B) by reducing SDS-gel electrophoresis and
autoradiography.
Dimer formation is
not specific to expression in COS-7 cells since stable transfectants of
3T3 cells with plasmid pMC also express dimers, as shown in Fig. 6. In the pulse-chase study shown, dimers (M = 66,000) were observed in cell lysates between 0 and 10
min of chase, but had disappeared from the cells after 120 min when
they were observed in the medium. Fig. 6also shows the large
band at the interface between the stacking and running gels and another
band at M
55,000. These bands also appeared
with preimmune serum as in Fig. 2, Fig. 4, and Fig. 5, but are of unknown nature.
Figure 6: Synthesis and secretion of the disulfide-rich domain of mucin in 3T3 cells. 3T3 fibroblasts were cotransfected with pMC and a plasmid conferring resistance to G418. Positive colonies were pooled and expanded. Subconfluent cells were analyzed by pulse-chase and immunoprecipitation with antiserum 3814 as described for Fig. 4A. Purified proteins from cell lysates (C) or medium (M) were analyzed under nonreducing conditions by SDS-gel electrophoresis and autoradiography.
The observation that
tunicamycin (Fig. 1B) does not inhibit the secretion of
the disulfide-rich domain from COS-7 cells indicates that the N-linked oligosaccharides are not required for either
intracellular trafficking or secretion from the cell. The pulse-chase
studies in Fig. 7show that the N-linked
oligosaccharides are also not required for dimer formation. In these
studies, transfected COS-7 cells were incubated with tunicamycin and
then pulsed for 10 min with S-labeled medium and chased
with unlabeled medium. The nonglycosylated proteins expressed
intracellularly and secreted into the medium were dimers (M
= 54,000) on nonreducing gels and
monomers (M
= 33,000) on reducing gels. As
in Fig. 4Fig. 5Fig. 6, the major band on
nonreducing gels at the interface between the stacking and running gels
was also observed with preimmune serum.
Figure 7:
Synthesis and secretion of the
disulfide-rich domain of mucin in the presence of tunicamycin. COS-7
cells transfected with plasmid pMC were incubated for 3 h with 10
µg/ml tunicamycin in the culture medium and then subjected to
pulse-chase analysis as described for Fig. 4A in the
presence of tunicamycin. Proteins from cell lysates (C) or
medium (M) were immunoprecipitated with antiserum 3814 and
analyzed by SDS-gel electrophoresis in the absence (lanes
8-14) or presence (lanes 1-7) of
-mercaptoethanol.
The studies reported here show that the disulfide-rich domain
at the carboxyl terminus of porcine submaxillary mucin is synthesized
rapidly as a dimer in transiently expressed COS-7 cells (Fig. 1)
and stably expressed 3T3 cells (Fig. 6) transfected with
expression vectors encoding the domain. The domain is expressed as
disulfide-bonded dimers as judged by SDS-gel electrophoresis in the
presence and absence of -mercaptoethanol and the cross-linking
agent bis(sulfosuccinimidyl) suberate (Fig. 2). The domain
contains four Asn-X-Thr/Ser sequences that are N-glycosylated to different extents during dimer formation (Fig. 1). Although native mucin contains N-linked
oligosaccharides in the disulfide-rich domain (Fig. 1D), the oligosaccharides are apparently not
involved in dimer formation. The differential glycosylation of the
domain observed in COS-7 cells is not necessarily a feature of domain
formation and likely reflects the high rate of expression of the domain
and overloading the glycosylation capacity of transfected COS cells.
Indeed, the dimers formed in stably transfected 3T3 cells (Fig. 6) are more extensively glycosylated than those in COS
cells. Although tunicamycin inhibits the oligomerization, maturation,
and secretion of rat gastric mucin(24) , there is no evidence
for such effects in dimer formation of the expressed disulfide-rich
domain.
Pulse-chase studies (Fig. 4Fig. 5Fig. 6) indicate that the dimers of the disulfide-rich domain are formed rapidly once protein synthesis commences. Dimer formation most likely occurs in the endoplasmic reticulum since the first dimers detected (Fig. 4B) are susceptible to endoglycosidase H digestion. This result is consistent with the observation that rat gastric mucin (24) oligomers form in the endoplasmic reticulum, where most secretory proteins are folded and assembled into oligomers (reviewed in (25) ). That dimer formation is an early event in the endoplasmic reticulum is consistent with other secreted mucins such as human gastric mucin (26) and human gall bladder mucin(27) . However, studies on these mucins are difficult to interpret because the high molecular weights of dimers and their precursors are difficult to measure accurately by gel electrophoresis, which is not a problem in the present studies that examine dimer formation of the disulfide-rich domain in the absence of other parts of the mucin polypeptide chain.
The molecular weights of the domains determined by gel electrophoresis are inexact because the mobilities of proteins are influenced markedly by the presence or absence of disulfide bonds, covalent cross-links, and N-linked oligosaccharides. However, the estimated values reported here are sufficiently accurate to leave little doubt that the domains are synthesized as disulfide-bonded dimers. Nevertheless, gel filtration in 6 M guanidine HCl was used as an independent method to check the values obtained by gel electrophoresis.
The SDS gels used to detect monomers and dimers also showed immunoreactive proteins of high molecular weight accumulating at the interface between the stacking and running gels. The amounts of protein at the interface varied somewhat from one gel to another under nonreducing conditions, and under reducing conditions, the interfacial protein was much reduced or absent. Moreover, the interfacial proteins were also observed when preimmune serum was used to precipitate the expressed proteins from cells or medium. These observations suggest that the interfacial proteins are disulfide-bonded proteins that nonspecifically react with components of rabbit serum, but not with the anti-mucin antibodies. The exact nature of the interfacial protein remains to be determined, but it may be aggregates of the domain that form nonspecifically in cells. Of interest is the fact that no evidence for very high molecular weight protein comparable to the interfacial protein was observed on gel filtration of purified proteins in 6 M guanidine HCl.
The carboxyl-terminal disulfide-rich domain of porcine mucin is marginally identical in its complete amino acid sequence to the carboxyl-terminal disulfide-rich domain of human prepro-von Willebrand factor(1) . Nevertheless, the half-cystine residues have a high degree of identity. Moreover, the analogous domains from other types of human mucin that show more significant sequence identity to prepro-von Willebrand factor (7) also show statistically significant sequence identity to the porcine disulfide-rich domain. Thus, since dimer formation of von Willebrand factor is the result of disulfide bonding among its carboxyl-terminal disulfide-rich domains, a similar function for the corresponding domain in mucins is not particularly surprising. Nevertheless, proof of dimer formation in mucins would be very difficult to establish by direct analysis of native mucins because their high carbohydrate contents, and high molecular weights, prevent direct analysis by gel electrophoretic or physicochemical methods for examining proteins in solution.
Human prepro-von Willebrand factor
and human intestinal mucin (MUC2) (7) have three additional
disulfide-rich domains in their amino-terminal regions. These domains
act in human prepro-von Willebrand factor to form disulfide-bonded
oligomers. Similar domains are present in porcine submaxillary mucin, ()but further studies will be necessary to determine whether
they serve to form oligomers in mucins.