From the Centre for Immunology, Saint Vincent's Hospital and University of New South Wales, Victoria Street, Sydney, New South Wales 2010, Australia
Received for publication, November 2, 2000, and in revised form, February 14, 2001
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ABSTRACT |
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Macrophage inhibitory cytokine-1 (MIC-1) is a
divergent member of the transforming growth factor- Macrophage inhibitory cytokine-1
(MIC-1)1 is the first member
of a divergent group within the transforming growth factor- The MIC-1 protein is synthesized as a 308-amino acid polypeptide that
encompasses a 29-amino acid signal peptide, a 167-amino acid
propeptide, and a 112-amino acid mature region. The mature protein is
secreted as a disulfide-linked homodimer of 112 amino acids which is
released from the propeptide after intracellular cleavage at a typical
RXXR furin-like cleavage site (1). Importantly, unlike all
other TGF- To date, most of the studies that have examined the folding of TGF- No detailed studies on MIC-1 folding have yet been published, although
recently we reported that the propeptide has a role in the
intracellular quality control of MIC-1 secretion by targeting monomeric
precursor forms in the endoplasmic reticulum to the proteasome for
degradation (12). In the current paper we have addressed a number of
aspects of folding and secretion of MIC-1. In particular we have
determined sequence regions of the mature peptide which enable it to
fold and be secreted in the absence of the propeptide. We have also
identified a region of the MIC-1 propeptide which can assist in protein
folding/secretion.
Cell Culture--
Chinese hamster ovary cells (CHO-K1) were
maintained as recommended by the American Type Culture Collection in
Dulbecco's modified Eagle's medium/F-12 medium supplemented with 5%
fetal bovine serum. For collection of conditioned medium for
immunoprecipitation and Western blotting experiments, transfected cells
were cultured in the absence of fetal bovine serum (see below).
Antisera--
Anti-FLAG M2 antibody coupled to agarose used in
immunoprecipitations was purchased from Sigma. Polyclonal antisera to
MIC-1 (PAb 233) was raised by immunization of sheep with purified
recombinant human MIC-1 mature protein (13).
Preparation of cDNA Constructs--
The preparation of the
base FLAG-tagged, full-length "long" construct (i.e.
MIC-1 leader + propeptide + MIC-1 mature peptide; see Fig.
1, constructs A1-A3) and "short"
mature peptide only construct (FSH leader sequence + MIC-1 mature
peptide; Fig. 1, constructs F1-F3) has been described previously (1).
The FLAG epitope was engineered onto the amino terminus of the mature
peptide of all constructs (i.e. inserted immediately after
the furin-like cleavage sequence of propeptide region; see Fig. 1) to
facilitate immumoprecipitation of the secreted proteins. The relevant
constructs were cloned into the pOCUS-2 vector (Novagen) for the
construction of the chimeras and other mutants, and the pIRES2-EGFP
vector (CLONTECH) was used for transfection into
CHO cells.
Both the long and short constructs described previously were amplified
by polymerase chain reaction and either Pfu DNA polymerase (Promega) or Vent DNA polymerase (New England Biolabs) with the oligonucleotide primers 1 and 2 (for all primer sequences, see Table
I), which added an XhoI and
SacII + BglII sites onto their 5'- and 3'-ends,
respectively, for insertion into the pOCUS-2 vector (at the
XhoI and BglII sites) or pIRES2-EGFP (at the
XhoI and SacII sites). The various
mutant/chimeric constructs were made using a whole plasmid polymerase
chain reaction technique described previously (23). The primers for the
site 1, 2, and 3 chimeras have also been described previously (23). For
the propeptide deletion mutants (Fig. 1, constructs B-E), a single common reverse primer was used (primer 3) which corresponded to the
3'-end of the MIC-1 signal-peptide sequence, and the forward primers
(primer 4, deletion 1-28; primer 5, deletion 1-78; primer 6, deletion
1-41; primer 7, deletion 1-55) were positioned so that the 5'-end
started at the first base of the codon for the amino acid following the
region of the propeptide to be deleted. The glycosylation mutant in
which pro-Asn41 (asparagine residue 41 of the
propeptide) was changed to a serine (Fig. 1, constructs I)
mutant employed primers 8 and 9.
Plasmid DNA derived from individual colonies was sequenced
bidirectionally to confirm whether the correct construct had been created. For all of the constructs involving changes to the mature peptide region, the BssHII/SacII fragment from
the long, short, or both constructs in the pIRES2-EGFP vector was
replaced with the same fragment from the mutated construct in the
pOCUS-2 vector. For the propeptide deletion constructs, the
XhoI/EcoRI fragment of the pIRES2-EGFP long
construct was replaced with same fragment derived from the mutants in
the pOCUS-2 vector.
To create the constructs with the simian TGF- Cell Transfections--
Transient transfections were performed
in CHO-K1 cells grown in six-well plates to 60-80% confluence.
Plasmid DNA (1 µg), purified using a Qiaprep Spin purification kit
(Qiagen), was mixed with 9 µl of LipofectAMINE (Life Technologies,
Inc.) for 15-30 min at room temperature and then added to cells in a
total volume of 1 ml of serum-free Dulbecco's modified Eagle's
medium/F-12 medium. After overnight incubation at 37 °C, the cells
were washed with Dulbecco's modified Eagle's medium/F-12 medium
containing 5% fetal bovine serum and incubated for 6 h at
37 °C in the same medium. Cells were washed with serum-free medium
then maintained in 1 ml of the same medium for a further 48 h
before collection for immunoprecipitation and Western blot analysis.
For the experiment to determine whether propeptide could act both in
cis and in trans, mutant and wild type constructs
were transfected, as above, into a CHO-K1 cell line previously stably
transfected with the human MIC-1 propeptide alone (12). Transfection
efficiency was monitored using a fluorescent microscope that could
detect the enhanced green fluorescent protein, a product of the
pIRES2-EGFP vector, and was routinely in the order of 60-80%.
Comparison between supernatants was only made from wells in which the
cells were transfected to approximately the same degree.
Cell Lysis, Immunoprecipitation, SDS-PAGE, and Western
Blotting--
Conditioned medium was collected, and the cells were
washed with ice-cold phosphate-buffered saline and then lysed. To
perform lysis, cells were scraped off the wells in the presence of 0.5 ml of 50 mM Hepes, pH 7.0, containing 1% Triton X-100, 1 mM EDTA, and a protease inhibitor mixture (Roche Molecular
Biochemicals), and then incubated on ice for 30 min. The lysate was
collected after centrifugation at 4 °C, then either
immunoprecipitated with anti-FLAG coupled to agarose, as described
below, or 50 µl was analyzed directly by SDS-PAGE, performed under
reducing conditions, and Western blotting.
Immunoprecipitation of the FLAG-tagged proteins in the conditioned
medium or cell lysates was performed by adding 10 µl of anti-FLAG
antibody coupled to agarose and then incubating overnight at 4 °C.
The bound proteins were then washed three times with phosphate-buffered
saline, or in the case of the lysates with the lysis buffer itself, and
eluted by heating at 95 °C in SDS-PAGE (nonreducing or reducing)
sample buffer.
Western blot analysis was performed essentially as described previously
(1). Membranes were probed with either sheep anti-MIC-1 polyclonal
antiserum 233 (diluted 1:7,000) or anti-FLAG monoclonal antibody
followed by either biotinylated anti-sheep (1:1,000) (Sigma) or
anti-mouse antiserum (1:1,000) (Amersham Pharmacia Biotech),
respectively. Blots were then visualized on film after treatment with
strepavidin-horseradish peroxidase conjugate (Amersham Pharmacia
Biotech) and chemiluminescence reagents (PerkinElmer Life Sciences).
Rationale of MIC-1 Construct Design--
The three-dimensional
structures of the three mammalian TGF- The Propeptide of MIC-1 Enhances Secretion of MIC-1/TGF-
As indicated previously, MIC-1 is unique among the TGF- The MIC-1 Propeptide Carbohydrate Moiety Is Not Essential for
Secretion of the Site 2 MIC-1/TGF-
Both the wild type and site 2 chimera mature peptides were secreted
when expressed from constructs with either glycosylated or
unglycosylated propeptide (Fig. 4).
Although a lower level of secretion was observed for the site 2 chimera
mature peptide from the unglycosylated propeptide (Fig. 4, lane
4) construct compared with the glycosylated construct (Fig. 4,
lane 3), a corresponding lower level of synthesis was also
observed in the cell lysate for the same construct (Fig. 4,
lysate). It can therefore be concluded that the propeptide
carbohydrate does not play a role in facilitating secretion of the site
2 chimera.
Expression of the Correctly Processed and Assembled Site 2 Chimera
Only Occurs with the Human MIC-1 Propeptide--
To the best of our
knowledge, only one previous attempt has been made to interchange the
propeptides belonging to different TGF-
Proteins immunoprecipitated from the supernatant of CHO-K1 cells
transiently transfected with the wild type MIC-1 mature peptide sequence fused to either the murine MIC-1 and TGF-
In the case of the site 2 replacement, just a single major band of
~40 kDa (Fig. 5a, lane 2) or 55 kDa (Fig.
5b, lane 2), for the murine propeptide and
TGF- Residues 56-78 of the Propeptide Are Essential for Site 2 MIC-1/TGF-
Deletion of portions of the propeptide attached to the wild type mature
protein appeared to have little effect on the secretion of the wild
type MIC-1 (data not shown). In the case of the MIC-1/TGF-
These results therefore indicate that the region of the propeptide
necessary for folding and secretion of MIC-1, in which the The Propeptide Must Be in cis with the Site 2 Chimeric Mature
Peptide for Secretion to Occur--
To determine whether the MIC-1
propeptide could assist in secretion of the MIC-1/TGF- We have shown recently that the MIC-1 propeptide has a novel role
in proteasomal targeting (12). In addition, the MIC-1 mature peptide
was unique among TGF- Although all of the regions exchanged in the chimeras resulted in
reduced secretion of the mature peptide in the absence of the
propeptide, the sequence region that was most dependent on the
propeptide for secretion was the predicted major In the A potential clue as to why the propeptide is not required in MIC-1
folding/secretion may be found in hydrophilicity plots of the (TGF-
)
superfamily. While it is synthesized in a pre-pro form, it is unique
among superfamily members because it does not require its propeptide
for correct folding or secretion of the mature peptide. To investigate
factors that enable these propeptide independent events to occur, we
constructed MIC-1/TGF-
1 chimeras, both with and without a
propeptide. All chimeras without a propeptide secreted less efficiently
compared with the corresponding constructs with propeptide. Folding and secretion were most affected after replacement of the predicted major
-helix in the mature protein, residues 56-68. Exchanging the human
propeptide in this chimera with either the murine MIC-1 or TGF-
1
propeptide resulted in secretion of the unprocessed, monomeric chimera,
suggesting a specific interaction between the human MIC-1 propeptide
and mature peptide. Propeptide deletion mutants enabled identification
of a region between residues 56 and 78, which is important for the
interaction between the propeptide and the mature peptide.
Cotransfection experiments demonstrated that the propeptide must be in
cis with the mature peptide for this phenomenon to occur.
These results suggest a model for TGF-
superfamily protein folding.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TGF-
) superfamily (1, 2). It has also been reported as placental
transforming growth factor-
(3), prostate-derived factor (4),
growth/differentiation factor 15/MIC-1 (5), and as a placental bone
morphogenetic protein (6). The major function of the protein is
still uncertain although it has been variously described as being able
to inhibit tumor necrosis factor-
production from
lipopolysaccharide-stimulated macrophages (1), to induce cartilage
formation and the early stages of endochonadal bone formation (4), and
to inhibit proliferation of primitive hemopoietic progenitors (6). The
very high level of MIC-1 mRNA in the human placenta, relative to
other tissues (2, 4, 6-8), also suggests a role in embryo implantation
and placental function. Further, markedly elevated serum levels of
MIC-1 occur during pregnancy, suggesting a more generalized function in
this process (8). Finally, a number of recent reports have demonstrated that the MIC-1 promoter region is a target for the p53 tumor suppressor gene product (9-11) and that recombinant MIC-1 can suppress tumor cell
growth in a number of cell line lines including human breast cancer
cells (9). This last effect was a result of induction by MIC-1 of both
G1 cell cycle arrest and apoptosis in breast tumor cells.
superfamily members studied to date, the MIC-1 mature
peptide can be correctly folded and secreted without a propeptide (12,
13). This unique property of MIC-1 makes it a particularly suitable
molecule for the study of the factors that influence TGF-
superfamily protein folding. In the case of TGF-
1, activin A,
Müllerian inhibitory substance, and bone morphogenetic protein-2,
the propeptide region is known to be essential for the correct folding
and secretion of the mature peptide (14-16). For example, no TGF-
1
or activin A mature peptides were detected in the supernatants from
cells transfected with constructs with an in-frame deletion of their
propeptides, and analysis of the cell lysates indicated that the
activin A had formed large intracellular disulfide-linked aggregates
(16).
superfamily proteins have concentrated on the role of the propeptide.
Sha et al. (17) used deletion and insertion mutagenesis to
identify regions of the propeptide important for secretion of
biologically active mature TGF-
1 and determined that amino acids
between residues 50 and 80 interact with the mature peptide in the
latent complex form of TGF-
1. Elimination of all glycosylation sites
on the TGF-
1 and -
2 propeptide prevents secretion of any mature
protein (18, 19), and mutation of the TGF-
1 propeptide cysteine
residues results in the secretion of differently assembled and
processed forms (20). In addition, it has been shown that the
Müllerian inhibitory substance propeptide helps to maintain the
conformation of the Müllerian inhibitory substance mature peptide
after secretion by preventing aggregation (14). Only limited studies
have focused on the mature peptide region, where the role of the
cysteine residues has been examined for TGF-
1 and activin A, and all
were found to be essential for the secretion of a fully bioactive
mature protein (18, 21, 22).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Arrangement of constructs. The basic
elements of all constructs are represented. For each type of construct,
a separate construct with a wild type mature peptide was also made and
tested. In those construct types shown with more than one sequence
replacement, each region was actually exchanged independently.
Oligonucleotide sequences for primers used in polymerase chain
reactions for the creation of the various constructs
1 sequences added in the
construction of chimeras or the base pair change used in the
construction of the glycosylation mutant.
1 propeptide fused to
the MIC-1 mature (Fig. 1, construct H), the sim-TGF-
1 propeptide was
amplified with primers 10 and 11, which added XhoI and
EcoRI restriction enzyme sites to the 5'- and 3'-ends,
respectively. The template for this construct was sim-TGF-
1 with the
codons for Cys-223 and Cys-225 mutated to serine codons and was kindly provided by Ignacio Anegon (20). The XhoI/EcoRI
fragments from the long pIRES2-EGFP construct as well as the site 2 chimera cloned into the pIRES2-EGFP vector were then replaced with the
digested, amplified sim-TGF-
1 propeptide. The murine MIC-1
propeptide was added to the wild type or site 2 chimeric human MIC-1
following replacement of the Xho/EcoRI fragment with the
corresponding fragment of the long murine construct (Fig. 1, construct G).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
isoforms as well as bone
morphogenetic proteins-2 and -7 and glial cell line-derived
neurotrophic factor have all been solved (24-29). Comparison of
these structures indicates that they are very similar in the overall
fold of the subunits for each family member despite low amino acid
sequence homology between family groups (26% between human TGF-
1
and human bone morphogenetic protein-7 and 12% between human TGF-
1
and human glial cell line-derived neurotrophic factor). This
similarity suggested that it may be possible to construct chimeric
molecules in which sequence regions of one family member are exchanged
with the corresponding region of another family member. A similar
approach has been used for the identification of TGF-
1 and TGF-
2
receptor binding regions where chimeric molecules involving both
isoforms proved to be useful (30, 31). We therefore made a series of
constructs in which three distinct sequence regions of the mature
protein of MIC-1 were replaced with the corresponding regions of
TGF-
1. The three regions of MIC-1 selected were: residues 24-37
(site 1) which corresponds to part of the extended loop region also referred to as "finger 1" in TGF-
1; residues 56-68 (site 2), corresponding to the major
-helix (also called the "heel"
region) in TGF-
1; and residues 90-98 (site 3), which encompasses a
type II'
-turn and corresponds to the tip of "finger 2" of
TGF-
1 (for the structural location each region see Fig.
2a; for sequences, see Fig.
2b). Additional constructs were also made in which the propeptide region was either deleted or replaced with the murine MIC-1
or sim-TGF-
1 propeptide.
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Fig. 2.
Structural location and sequences of the
regions interchanged in the chimeras. (a), the
three-dimensional structure for the TGF- 1 carbon backbone as
determined by multinuclear magnetic resonance spectroscopy (26) is
represented using the Rasmac version 2.6 software and coordinates
obtained from the Protein Data Bank. The black shaded
regions correspond to the three regions interchanged between MIC-1 and
TGF-
1. (b), the primary sequences (single letter code)
for the MIC-1 and TGF-
1 mature pepitde were aligned using the
MegAlign program in the DNA Star suite of programs. Boxed
sequences indicate the three regions, site 1, 24-37; site 2, 56-68;
and site 3, 90-98, interchanged between the proteins in the
construction of the chimeras. Note that a one-amino acid gap was
inserted in the alignment in site 1, therefore the phenylalanine
residue at position 24 of TGF-
1 was included in the chimeric
molecule constructed to ensure that regions of equal length were
interchanged.
Chimeras--
To determine whether each of the sequence substitutions
described above would be tolerated and result in the secretion of a
correctly processed dimeric molecule, the three MIC-1/TGF-
1 chimeric
constructs with associated MIC-1 propeptide (see Fig. 1, constructs
A1-A3) were transfected into CHO-K1 cells. Supernatants were collected
and immunoprecipitated with anti-FLAG-coupled agarose and analyzed by
Western blotting. Bands of the correct apparent molecular mass
(30 kDa) for dimeric, FLAG-tagged MIC-1 were observed for each
construct (Fig. 3, a-d,
lanes 1), indicating that the mature protein was correctly
assembled, processed, and secreted. Furthermore, the level of secretion
of the chimeras was similar to that of the wild type mature peptide. No
similar bands were observed in the immunoprecipitated supernatant from
cells transfected with the vector only (Fig. 3d, lane
3). Various additional higher molecular mass bands observed in
lane 1 at ~33, 40, and 50 kDa correspond to aberrantly or
differentially processed forms of MIC-1 which have been characterized
previously (12).
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Fig. 3.
Expression of MIC-1/TGF-
chimeras with and without propeptides. Upper
panel, constructs consisting of either the (a) wild
type mature peptide or (b) site 1, (c) site 2, or
(d) site 3 MIC-1/TGF-
chimeras were made with the human
MIC-1 propeptide (lane 1) or no propeptide (lane
2) and then transiently transfected into CHO cells. Supernatants
were collected 48 h post-transfection, immunoprecipitated with
anti-FLAG-coupled agarose, and then bound proteins were electrophoresed
on 15% SDS-PAGE gels under nonreducing conditions and immunoblotted.
Equal volumes of immunoprecipitated supernatant were loaded onto
lanes 1 and 2 of gel (a), whereas in
gels (b)-(d) 4-fold more supernatant was loaded
onto lane 2, i.e. from cells transfected with
chimeric constructs without propeptides. The blots were probed with an
anti-MIC-1 polyclonal antiserum. Supernatant from cells transfected
with the vector only is shown in (d), lane 3. Lower panel, cell lysates generated from cells transfected
with either the full-length (+propeptide) or truncated (
propeptide)
wild type (lane 2) or chimeric (lane 3, site 1;
lane 4, site 2; lane 5, site 3) constructs were
electrophoresed on 15% SDS-PAGE gels under reducing conditions and
immunoblotted with anti-MIC-1 polyclonal antiserum. The band
shown in each case corresponds to the correct size of reduced
FLAG-tagged MIC-1 precursor (full-length constructs) or mature protein
(truncated constructs). The lysate for cells tranfected with the vector
only is shown in lane 1.
superfamily
members studied to date in that it can be secreted from a construct in
which the propeptide is deleted. To identify sequence regions of the
mature MIC-1 protein which enable it to fold correctly without
association or interaction with its propeptide, the three chimeras were
expressed using constructs in which the propeptide had been deleted
(Fig. 1, constructs F1-F3). An FSH leader sequence was fused to these
proteins to enable secretion. This adds three extra amino acids to each
subunit resulting in slower migration of the expressed proteins on
SDS-PAGE compared with those expressed from the full-length constructs.
Bands corresponding to the molecular mass of dimeric MIC-1 were
observed for both the wild type MIC-1 and the site 1 and site 3 replacements (Fig. 3, a, b, and d,
lanes 2) following immunoprecipitation and immunoblotting of
the supernatants from CHO cells transfected with these constructs. This
indicates that these proteins were correctly assembled and secreted
despite the deletion of the propeptide. However, the level of secretion of the site 1 and site 3 chimeras was consistently less than the corresponding constructs with a propeptide. In the case of the site 2 replacement, no measurable protein was secreted from the expressed
construct without propeptide (Fig. 3c, lane 2),
demonstrating that this protein cannot be folded and/or secreted
without the presence of the propeptide. Analysis of the cell lysates
for all truncated constructs demonstrated that the mutant proteins were synthesized at approximately the same level (Fig. 3, lower
panel), confirming that the markedly lower level of protein
observed in the supernatant for the site 2 chimera in the absence of
its propeptide is caused by its inefficient folding/secretion. These
combined results therefore suggest that the MIC-1 propeptide, although not essential, can assist in folding/secretion. Furthermore, it appears
that the site 2 (
-helix) sequence is one region of MIC-1 which
enables it to be secreted in the absence of the propeptide.
Chimera--
The carbohydrate
moieties on the propeptide of the TGF-
1 and -
2 propeptides are
important for the secretion of biologically active TGF-
mature
peptides. Glycosylation of the MIC-1 propeptide occurs at a single
N-linked glycosylation site at residue pro-Asn41
(residue asparagine 41 of the propeptide) (12). Therefore, it was of
interest to determine whether the carbohydrate moiety played a role in
MIC-1 folding and secretion and in particular to determine whether it
was essential for secretion of the site 2 MIC-1/TGF-
chimera. To
achieve this, pro-Asn41 of the propeptide was mutated to a
serine residue on constructs with both the wild type and site 2 chimeric mature peptides (Fig. 1, construct I).
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Fig. 4.
Expression of the site 2 chimera with the
unglycosylated MIC-1 propeptide. Constructs consisting of either
the wild type MIC-1 propeptide (lanes 1 and 3) or
MIC-1 propeptide in which the glycosylation site was mutated
(lanes 2 and 4) were made with both the wild type
MIC-1 mature peptide (lanes 1 and 2) and
MIC-1/TGF- 1 site 2 chimera (lanes 3 and 4) and
then transiently transfected into CHO cells and the supernatants
analyzed as described previously. The Western blot of the
immunoprecipitated cell lysate for the site 2 chimera constructs with
or without mutated glycosylation site is shown below. Lane V
corresponds to the lysate for cells transfected with vector only. The
band shown in the lysates corresponds to the correct size
for the unprocessed precursor proteins.
superfamily members (16).
This earlier study involved exchanging the activin A propeptide for the
TGF-
propeptide. The results demonstrated that the mature protein
was secreted, albeit with low efficiency, when the non-native
propeptide was introduced. As MIC-1 is secreted without a propeptide,
it was of interest to establish whether the protein would undergo
correct assembly, processing, and secretion after replacement of the
MIC-1 propeptide with that of the closely related murine MIC-1
propeptide (65% identical) or the more distantly related and quite
dissimilar TGF-
1 propeptide. More importantly, we also wanted to
establish whether these other propeptides could facilitate folding and
secretion of the site 2 chimeric mature peptide. It should be noted
that in these experiments we utilized the simian TGF-
1 propeptide, which has the cysteine residues at positions 223 and 225 mutated to
serine residues. Use of this mutated propeptide has been shown to
enable secretion of a mature TGF-
1 peptide without associated propeptide, i.e. in a non-latency-associated peptide complex
form (20).
1 propeptides (Fig. 1, constructs G and H) were analyzed by Western blotting. A band
of 30 kDa corresponding to the molecular mass of the dimeric mature
MIC-1 peptide was observed in both cases (Fig.
5, a and b,
lanes 1). Additional high molecular mass bands of ~75,
100, and 100-200 kDa, most probably corresponding to unprocessed (and possibly differentially glycosylated) chimeric monomers, hemidimers, and full-length dimers, were also observed with the TGF-
propeptide construct.
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Fig. 5.
Expression of the site 2 chimera with the
murine MIC-1 and TGF- 1 propeptides.
Upper panel, constructs consisting of (a) the
murine MIC-1 propeptide or (b) the TGF-
1 propeptide with
either the wild type mature peptide (lane 1) or
MIC-1/TGF-
1 site 2 chimera (lane 2) were transiently
transfected into CHO cells and the supernatants analyzed as described
previously (see Fig. 4 legend), except the blot was probed with the M2
anti-FLAG monoclonal antibody. Lower panel, the cell lysates
corresponding to each of the constructs above were immunoprecipitated
and analyzed by Western blot using the polyclonal anti-MIC-1 antiserum.
The band shown corresponds to the correct size for the
unprocessed precursor proteins. Lane V corresponds to the
lysate for cells transfected with the vector only.
propeptide constructs, respectively, was observed. These bands
correspond to the unprocessed, and unassembled (i.e.
monomeric) full-length proteins. Furthermore, the cell lysates for each
of these types of construct indicate equal levels of synthesis of the
chimera compared with the wild type precursor proteins (Fig. 5,
a and b, lower panels, lanes
1 and 2). The presence of the murine or TGF-
1
propeptides therefore does not prevent folding, processing, or
secretion of wild type MIC-1. However, these propeptides do prevent
processing and assembly of the site 2 chimera.
Chimera Expression--
The MIC-1/TGF-
site 2 chimera
also appeared to be a useful molecule for studying the sequence regions
of the propeptide which allowed the protein to be folded/secreted. To
examine this, a series of constructs was made in which progressively
larger regions were deleted from the amino terminus of MIC-1
propeptide. These were linked to either the wild type mature MIC-1
peptide or the MIC-1/TGF-
site 2 chimera (Fig. 1, constructs
B-E).
site 2 chimera, deletion of residues 1-28, 1-41 (which includes the
glycosylation site), and 1-55 resulted in only a marginal progressive
decrease in the level of secretion observed (Fig. 6, upper panel, lanes
1-3), The deletion of residues 1-78, however, essentially
prevented secretion of any chimeric protein (Fig. 6, lane
4). The cell lysates for each of these constructs indicated approximately equal levels of synthesis of each precursor protein (Fig.
6, lower panel, lanes 1-4).
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Fig. 6.
Expression of propeptide deletion
mutants. Constructs consisting of the truncated MIC-1 propeptides
with the MIC-1/TGF- 1 site 2 chimera were transiently transfected
into CHO cells. Supernatants (upper panel) and cell lysates
(lower panel) were collected 48 h post-transfection,
immunoprecipitated with anti-FLAG-coupled agarose, then bound proteins
were electrophoresed on SDS-polyacrylamide gels under nonreducing
conditions and Western blotted. The blots were then probed with an
anti-MIC-1 polyclonal antiserum. Lane 1, MIC-1 propeptide
del 1-28; lane 2, MIC-1 propeptide del 1-41; lane
3, MIC-1 propeptide del 1-55, lane 4, MIC-1 propeptide
del 1-78. The band shown for the lysates corresponds to the
correct sizes for the unprocessed precursor proteins, which
progressively decreases because of the deletions. No corresponding
bands were observed in the lysate of cells transfected with vector
only.
-helix
region has been replaced with the corresponding region of TGF-
1,
lies between residues Pro56 and Pro78.
1 site 2 chimera in trans as well as in cis, the site 2 chimeric construct (without associated propeptide) was transfected into
a cell line previously stably transfected with the (FLAG-tagged) MIC-1
propeptide (12). The supernatants were then analyzed as previously. No
secretion of the chimeric protein into the supernatant was observed
(Fig. 7, upper panel, lane 3) although, as would be expected, secretion of the
wild type MIC-1 mature peptide did occur (Fig. 7, upper
panel, lane 2). The cell lysates were also analyzed to
confirm synthesis of the propeptide, and a band of ~28 kDa,
consistent with the correct size of theMIC-1 propeptide, was present in
all samples (Fig. 7, lower panel, lanes 2 and
3). No similar band was seen in the lysate of untransfected
CHO cells (Fig. 7, lower panel, lane 1). These
results demonstrate that the propeptide must be in cis with the mature peptide region to enable secretion of the site 2 chimera.
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Fig. 7.
Independent expression of the site 2 chimera
and propeptide. The wild type MIC-1 mature peptide (lane
2) or MIC-1/TGF- 1 site 2 chimera was transfected into a cell
line previously stably transfected with the FLAG-tagged MIC-1
propeptide. Normal CHO cells were also transfected with the site 2 chimera (lane 1). The supernatants (upper panel)
and cell lysates (lower panel) were immunoprecipitated with
anti-FLAG-coupled agarose, then bound proteins were electrophoresed on
a 15% SDS-PAGE gel under nonreducing conditions (supernatants) or
reducing conditions (lysates) and immunoblotted using either anti-MIC-1
polyclonal antiserum (supernatants) or the M2 anti-FLAG monoclonal
antibody (lysates).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
superfamily members studied to date in that
the propeptide region was neither required for the correct folding and
assembling of subunits, nor was it necessary for secretion of the
mature protein. Indeed, MIC-1 secreted from constructs without the
propeptide is both biologically active (32) and immungenically
identical to MIC-1 produced from full-length constructs (13). There is
also no evidence that the MIC-1 propeptide associates with the mature
peptide to regulate activity, as does the propeptide of TGF-
in the
latency-associated peptide complex. Nor is there any evidence to date
that MIC-1 secretion is associated with the interaction with other
binding proteins, as occurs with TGF-
(12). The key result in this
present paper is that by interchanging specific regions of the MIC-1
mature peptide with the corresponding regions of the structurally
related TGF-
1, the protein is converted into a molecule that is now
dependent on its propeptide for efficient folding and secretion. This
demonstrates that the MIC-1 propeptide can facilitate mature peptide
folding despite not actually being required.
-helix (residues 56-68). It is likely, therefore, that it is the physicochemical properties of this region which substantially contribute to the ability
of MIC-1 to be secreted without its propeptide. The corollary of this
is that in members of the TGF-
superfamily which cannot be secreted
without their propeptides, it may be the properties of their
-helical region which are, at least in part, responsible for their
requirement for the propeptide to achieve a correctly folded and
secreted mature protein.
-helical region in question, at least six (mainly
hydrophobic) amino acids from this region of TGF-
1, -
2, -
3,
and bone morphogenetic protein-7 are present at the dimer interface and
make contact with amino acids on the opposite subunit. At least one of
these residues participates in hydrogen bonding with residues on the
other subunit (24-27). Therefore, in the case of the site 2 chimera,
it is possible that the propeptide region may play a role in ensuring
that the appropriate intersubunit contacts can be made and/or that the
subunits only align in the correct orientation. Alternatively, or
concomitantly, the propeptide may also act to ensure that other
inappropriate contacts do not occur. This is discussed further below.
Consistent with these hypotheses is the previous finding of Gray and
Mason (16) who used dimer-specific antisera to determine that
intracellular dimer formation of activin A was dependent on the
presence of the propeptide. Our data, which showed that replacement of
the human propeptide with either the murine MIC-1 or TGF-
1
propeptide resulted in the secretion of just the unprocessed monomeric
site 2 chimera, also supports this idea.
-helix
region of various TGF-
superfamily proteins (Fig. 8). Compared with other proteins that
have been shown to require their propeptides for folding and secretion,
the MIC-1 helix is significantly more hydrophilic across the entire
region. This perhaps suggests that it may be possible to predict other
family members that do not require propeptides for folding and
secretion based upon the hydrophilicity in this region
View larger version (49K):
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Fig. 8.
Hydrophilicity plots of superfamily
-helices. Kyte-Doolittle hydrophilicity plots
were generated for the region corresponding to the major
-helix of
various TGF-
superfamily members using the Protean module of the DNA
Star suite of programs. BMP-7, bone morphogenetic protein-7; MIS,
Müllerian inhibitory substance.
The apparent capacity of the MIC-1 propeptide to facilitate protein
folding is of particular interest as it appears that the mature peptide
region has evolved independently of the propeptide region such that the
propeptide is no longer required for this particular function. An
important practical consequence of this finding is that it may be
possible to engineer other TGF- superfamily proteins to be secreted
without the necessity of their propeptides by making mutations within
specific sequence regions. This is currently under investigation in our laboratory.
The fact that the site 2 chimera is only secreted from a construct with
the propeptide made it a useful model protein for the study of which
regions of the propeptide are important in the folding process.
Deletion mutants were used to determine that amino acids 56-78 of the
propeptide are essential for secretion of the mature chimeric peptide.
Furthermore, the secretion of the correctly assembled and processed
site 2 chimera was prevented after replacement of the human MIC-1
propeptide with the murine propeptide or TGF-1 propeptides,
indicating a specific interaction between the human propeptide and
mature peptide regions. Of the 23 residues in the region 56-78, six
(Thr61, Gly69, His71,
His73, Ile76, and Ser77) are not
conserved between the mouse and human propeptides, suggesting that at
least one of these residues is important for interaction with the
mature peptide. In addition, this interaction is not dependent on the
presence of the carbohydrate moiety on the propeptide, unlike both
TGF-
1 and TGF-
2, in which elimination of all propeptide glycosylation sites prevents mature peptide secretion (18, 19). Although it is somewhat difficult to compare regions of the MIC-1 and
TGF-
propeptides because of the significant difference in lengths
(the TGF
1 propeptide is 82 residues longer), it is noteworthy that
Sha et al. (17) have demonstrated that a region comparable to MIC-1 56-78 (in terms of position relative to the amino terminus) is essential for TGF-
1 propeptide interaction with the mature peptide region.
Unlike another study of this superfamily, the MIC-1 propeptide
only facilitated folding in cis. In the case of TGF-1 and activin A, the independent cotransfection of the propeptide and mature
peptide regions of both of these proteins rescued mature peptide
secretion (16). The level of protein observed in the supernatant,
however, was very low (2.4% or 9% of that secreted from a wild type,
full-length TGF-
1 or activin A construct, respectively), and hence
the difference between the studies may simply reflect a difference in
the sensitivities of the assays used for detection (enzyme-linked
immunosorbent assay and bioactivity for TGF-
1 and activin A compared
with immunoprecipitation followed by Western blotting for MIC-1).
In this paper we demonstrated that the MIC-1 propeptide is a
multifunctional domain that, in addition to its previously reported role in proteasomal targeting, can also facilitate protein folding and
secretion. We believe that the mechanism of propeptide-mediated folding
in MIC-1 may provide some important insights that could be applicable
to other superfamily members. One model that we propose is that the
propeptide may act similarly to an intramolecular chaperone. In this
context the propeptide acts by masking particular regions along the
mature peptide which may form inappropriate interactions during the
folding process. For TGF- superfamily members other than MIC-1, the
data from the present study suggest that the major
-helix in
particular may be a specific region that requires masking. Because the
TGF-
propeptide itself was unable to facilitate folding/secretion of
the site 2 MIC-1/TGF-
1 chimera, it is likely that an additional
specific interaction between the MIC-1 propeptide and the mature
peptide region (outside of the
-helix) occurs. A schematic
representation of how this may occur is presented in Fig.
9. Herein we indicate at least two
contacts between the propeptide and mature peptide: an undefined specific interaction involving residues outside of the
-helix and
amino acids in the propeptide; and a second, perhaps less specific
(hydrophobic) interaction, which provides the masking and occurs
between the mature peptide
-helix and the propeptide. In at least
one of these interactions, residues between 56-78 of the propeptide
are important. Additional contacts and masking of regions other than
the
-helix not shown in the figure are also likely. Further studies
will be required to confirm the validity of this model, and these are
currently in progress in this laboratory.
|
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FOOTNOTES |
---|
* This work was funded in part by grants from St. Vincent's Hospital and by Meriton Apartments Proprietary Ltd. through a research and development syndicate arranged by Macquarie Bank Ltd., and by a New South Wales health research and development infrastructure grant.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.:
61-2-8382-2590; Fax: 61-2-8382-2830; E-mail:
s.breit@cfi.unsw.edu.au.
Published, JBC Papers in Press, February 26, 2001, DOI 10.1074/jbc.M010000200
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ABBREVIATIONS |
---|
The abbreviations used are:
MIC-1, macrophage
inhibitory cytokine-1;
TGF-, transforming growth factor-
;
CHO, Chinese hamster ovary;
PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Bootcov, M. R.,
Bauskin, A. R.,
Valenzuela, S. M.,
Moore, A. G.,
Bansal, M.,
He, X. Y.,
Zhang, H. P.,
Donnellan, M.,
Mahler, S.,
Pryor, K.,
Walsh, B. J.,
Nicholson, R. C.,
Fairlie, W. D.,
Por, S. B.,
Robbins, J. M.,
and Breit, S. N.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
11514-11519 |
2. | Fairlie, W. D., Moore, A. G., Bauskin, A. R., Russell, P. K., Zhang, H. P., and Breit, S. N. (1999) J. Leukocyte Biol. 65, 2-5[Abstract] |
3. | Lawton, L. N., Bonaldo, M. F., Jelenc, P. C., Qiu, L., Baumes, S. A., Marcelino, R. A., de Jesus, G. M., Wellington, S., Knowles, J. A., Warburton, D., Brown, S., and Soares, M. B. (1997) Gene (Amst.) 203, 17-26[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Paralkar, V. M.,
Vail, A. L.,
Grasser, W. A.,
Brown, T. A.,
Xu, H.,
Vukicevic, S.,
Ke, H. Z.,
Qi, H.,
Owen, T. A.,
and Thompson, D. D.
(1998)
J. Biol. Chem.
273,
13760-13767 |
5. | Bottner, M., Suter-Crazzolara, C., Schober, A., and Unsicker, K. (1999) Cell Tissue Res. 297, 103-110[CrossRef][Medline] [Order article via Infotrieve] |
6. | Hromas, R., Hufford, M., Sutton, J., Xu, D., Li, Y., and Lu, L. (1997) Biochim. Biophys. Acta 1354, 40-44[Medline] [Order article via Infotrieve] |
7. | Yokoyama-Kobayashi, M., Saeki, M., Sekine, S., and Kato, S. (1997) J. Biochem. (Tokyo) 122, 622-626[Abstract] |
8. |
Moore, A. G.,
Brown, D. A.,
Fairlie, W. D.,
Bauskin, A. R.,
Brown, P. K.,
Munier, M. L. C.,
Russell, P. K.,
Salmonsen, L. A.,
Wallace, E. M.,
and Breit, S. N.
(2000)
J. Clin. Endrinol. Metab.
85,
4781-4789 |
9. |
Li, P.-X.,
Wong, J.,
Ayed, A.,
Duc, N.,
Brade, A. M.,
Arrowsmith, C.,
Austin, R. C.,
and Klamutt, H. J.
(2000)
J. Biol. Chem.
275,
20127-20135 |
10. |
Tan, M.,
Wang, Y.,
Guan, K.,
and Sun, Y.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
109-114 |
11. | Kannan, K., Amariglio, N., Rechavi, G., and Givol, D. (2000) FEBS Lett. 470, 77-82[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Bauskin, A. R.,
Zhang, H.-P.,
Fairlie, W. D.,
Russell, P. K.,
Moore, A. G.,
Brown, D. A.,
Stanley, K. K.,
and Breit, S. N.
(2000)
EMBO J.
19,
2212-2220 |
13. | Fairlie, W. D., Zhang, H.-P., Brown, P. K., Russell, P. K., Bauskin, A. R., and Breit, S. N. (2000) Gene (Amst.) 254, 67-76[CrossRef][Medline] [Order article via Infotrieve] |
14. | Wilson, C. A., di Clemente, N., Ehrenfels, C., Pepinsky, R. B., Josso, N., Vigier, B., and Cate, R. L. (1993) Mol. Endocrinol. 7, 247-257[Abstract] |
15. | Israel, D. I., Nove, J., Kerns, K. M., Moutsatsos, I. K., and Kaufman, R. J. (1992) Growth Factors 7, 139-150[Medline] [Order article via Infotrieve] |
16. | Gray, A. M., and Mason, A. J. (1990) Science 247, 1328-1330[Medline] [Order article via Infotrieve] |
17. | Sha, X., Yang, L., and Gentry, L. E. (1991) J. Cell Biol. 114, 827-839[Abstract] |
18. | Brunner, A. M., Lioubin, M. N., Marquardt, H., Malacko, A. R., Wang, W. C., Shapiro, R. A., Neubauer, M., Cook, J., Madisen, L., and Purchio, A. F. (1992) Mol. Endocrinol. 6, 1691-1700[Abstract] |
19. | Lopez, A. R., Cook, J., Deininger, P. L., and Derynck, R. (1992) Mol. Cell. Biol. 12, 1674-1679[Abstract] |
20. |
Brunner, A. M.,
Marquardt, H.,
Malacko, A. R.,
Lioubin, M. N.,
and Purchio, A. F.
(1989)
J. Biol. Chem.
264,
13660-13664 |
21. | Mason, A. J. (1994) Mol. Endocrinol. 8, 325-332[Abstract] |
22. |
Amatayakul-Chantler, S.,
Qian, S. W.,
Gakenheimer, K.,
Bottinger, E. P.,
Roberts, A. B.,
and Sporn, M. B.
(1994)
J. Biol. Chem.
269,
27687-27691 |
23. | Fairlie, W. D., Russell, P. K., Wu, W. M., Moore, A. G., Zhang, H.-P., Brown, P. K., Bauskin, A. R., and Breit, S. N. (2001) Biochemistry 40, 65-73[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Griffith, D. L.,
Keck, P. C.,
Sampath, T. K.,
Rueger, D. C.,
and Carlson, W. D.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
878-883 |
25. | Daopin, S., Piez, K. A., Ogawa, Y., and Davies, D. R. (1992) Science 257, 369-373[Medline] [Order article via Infotrieve] |
26. | Hinck, A. P., Archer, S. J., Qian, S. W., Roberts, A. B., Sporn, M. B., Weatherbee, J. A., Tsang, M. L., Lucas, R., Zhang, B. L., Wenker, J., and Torchia, D. A. (1996) Biochemistry 35, 8517-8534[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Mittl, P. R.,
Priestle, J. P.,
Cox, D. A.,
McMaster, G.,
Cerletti, N.,
and Grutter, M. G.
(1996)
Protein Sci.
5,
1261-1271 |
28. | Scheufler, C., Sebald, W., and Hulsmeyer, M. (1999) J. Mol. Biol. 287, 103-115[CrossRef][Medline] [Order article via Infotrieve] |
29. | Eigenbrot, C., and Gerber, N. (1997) Nat. Struct. Biol. 4, 435-438[Medline] [Order article via Infotrieve] |
30. | Burmester, J. K., Qian, S. W., Ohlsen, D., Phan, S., Sporn, M. B., and Roberts, A. B. (1998) Growth Factors 15, 231-242[Medline] [Order article via Infotrieve] |
31. |
Burmester, J. K.,
Qian, S. W.,
Roberts, A. B.,
Huang, A.,
Amatayakul-Chantler, S.,
Suardet, L.,
Odartchenko, N.,
Madri, J. A.,
and Sporn, M. B.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
8628-8632 |
32. |
Strelau, J.,
Sullivan, A.,
Bottner, M.,
Lingor, P.,
Falkenstein, E.,
Suter-Crazzolara, C.,
Galter, D.,
Jaszai, J.,
Krieglstein, K.,
and Unsicker, K.
(2000)
J. Neurosci.
20,
8597-8603 |
33. | Jones, W. K., Richmond, E. A., White, K., Sasak, H., Kusmik, W., Smart, J., Oppermann, H., Rueger, D. C., and Tucker, R. F. (1994) Growth Factors 11, 215-225[Medline] [Order article via Infotrieve] |