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J Biol Chem, Vol. 274, Issue 39, 27392-27398, September 24, 1999
1(I) Chains*
,, andFrom the Department of Paediatrics, Orthopaedic Molecular Biology Research Unit, University of Melbourne, Royal Children's Hospital, Parkville, Victoria 3052, Australia
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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We have previously shown that type I procollagen
pro- The major fibrillar collagens (types I, II, and III) are the
principal structural components of the extracellular matrix of many
tissues, forming characteristic architecturally precise fibrils (1).
They are synthesized as precursor molecules with a central triple-helical region containing a Gly-X-Y amino acid repeat motif, flanked by carboxyl- and amino-terminal propeptide globular domains (for review, see Ref. 2). Assembly of three individual pro- Intracellular degradation is an essential process for regulating the
levels of many proteins and an important "quality control" mechanism which minimizes the accumulation within cells and the secretion of mutant or malfolded proteins. Several cellular
compartments have been identified as sites for degradation, including
the lysosomes which contain acid hydrolases, a post-Golgi non-lysosomal
compartment, the ER, and the cytoplasm where the 26 S proteasome, a
large catalytic protease complex, is responsible for the degradation
(8, 9). While the molecular basis of intracellular collagen degradation has not been fully defined, three of these four cellular compartments have been implicated as the site of collagen proteolysis. In
fibroblasts, approximately 15% of normal procollagen is degraded
intracellularly by a process that has been termed basal degradation
(10, 11), and degradation is significantly increased in cells
synthesizing procollagens with structurally abnormal triple helical
domains (7, 12, 13). Degradation of both normal and structurally abnormal procollagen molecules can be inhibited by NH4Cl
(12-14), and brefeldin A (15), a drug which causes the cis-
and medial-Golgi to fuse with the ER and prevents further
intracellular transport. These results suggest that degradation occurs
in the distal region of the secretory pathway after the brefeldin A
block, in regions that are susceptible to NH4Cl inhibition
such as the trans-Golgi and secretory vesicles or in the
lysosomes. Recent studies have demonstrated directly the localization
of procollagen I in the lysosome/endosome system (16) identifying this
system as a site of collagen degradation, however, normal procollagen
degradation in I-cell disease (Mucolipidosis II) fibroblasts, which are
deficient in lysosomal hydrolases, suggests that trans-Golgi
and secretory vesicles also represent sites of procollagen degradation
(17).
Not all mutant collagen is degraded in the distal region of the
secretory pathway. Procollagens with mutations in the pro- Cell Culture--
Dermal fibroblast cultures from controls and a
patient with the lethal perinatal form of OI (OI26) were established
and maintained in culture as described previously (7). The molecular
defect in OI26 is a heterozygous single base insertion in the final
exon of COL1A1 (6). The codon reading frameshift generated
by the mutation alters the amino acid sequence of the pro- Production of Stably Transfected Mov13 Cell Lines Expressing an
Elongated Pro- Analysis of mRNA Expression--
Total RNA was isolated from
transfected Mov13 cells using the method of Wake and Mercer (30). RNA
samples (3 µg) were analyzed under denaturing conditions on 0.8%
(w/v) agarose gels and transferred to nitrocellulose filters. Filters
were pretreated at 42 oC with 50% (v/v) formamide, 5 × SSC (750 mM NaCl, 75 mM trisodium citrate),
0.02% (w/v) Ficoll, 0.02% (w/v) polyvinylpyrrolidone, 0.02% (w/v)
bovine serum albumin, 0.1% SDS, and 100 µg/ml denatured salmon sperm
DNA. Filters were then hybridized to
[ Collagen Biosynthetic Labeling--
Confluent cell cultures were
labeled with [3H]proline for 18 h as described
previously (7). Following labeling the cell layer and medium fractions
were treated separately (7, 32). Briefly, after disruption of the cell
layer by sonication, procollagens and collagens were precipitated from
the cell and medium fractions with ammonium sulfate at 25% saturation.
The precipitate was redissolved in 2 ml of 50 mM Tris-HCl,
pH 7.5, containing 0.15 M NaCl and the protease inhibitors
5 mM EDTA, 10 mM N-ethylmaleimide,
and 1 mM phenylmethylsulfonyl fluoride. Aliquots of
procollagens were precipitated with 75% ethanol and either analyzed
directly or subjected to limited pepsin digestion (100 µg/ml pepsin
in 0.5 M acetic acid, 4 oC, 16 h) to
determine if the collagens had formed pepsin-resistant triple helices.
For the pulse-chase analysis of procollagen degradation, skin
fibroblasts and transfected Mov13 cells in 6-well plates were grown to
confluence and treated with 0.25 mM sodium ascorbate overnight. The cells were preincubated in 1 ml of Dulbecco's modified Eagle's medium without L-methionine and
L-cysteine (Life Technologies Inc.) for 1 h then
pulse-labeled for 1 h with 300 µCi of
L-[35S]methionine (Tran35S-label,
1032 Ci/mmol, ICN Pharmaceuticals Inc.). Cells were treated with the
proteasome inhibitors: carboxylbenzyl-leucinyl-leucinyl-leucinal (ZL3al) (Sigma),
carboxybenzyl-leucyl-leucyl-leucine-vinylsulfone (ZL3VS)
(kindly provided by H. Ploegh, MIT, Boston, MA (33)), clasto-lactacystin SDS-PAGE and Immunoblotting--
Type I procollagen and collagen
chains were analyzed by SDS-PAGE on 5% (w/v) polyacrylamide gels.
Where indicated, samples were reduced before electrophoresis by the
addition of dithiothreitol to a final concentration of 10 mM. Procollagen chains were also analyzed by
two-dimensional gel electrophoresis (35) which resolves the chains on
the basis of both charge and size. Radioactively labeled proteins were
detected by fluorography. For immunoblotting, procollagen chains
resolved by SDS-PAGE were electrophoretically transferred to
nitrocellulose filters. Blots were incubated with LF-67 at a dilution
of 1/10,000 and bound antibody detected using horseradish
peroxidase-conjugated Protein A (Bio-Rad) and an enhanced chemiluminescence kit (ECL, Amersham Pharmacia Biotech).
Stable Expression of the COL1A1 Frameshift Mutant Construct,
pWTCI-IAfs, in Mov13 Cells--
Mov13 cells are a unique model system
in which to study intracellular collagen degradation. Expression of
mutant COL1A1 genes in these cells allows the fate of the
resultant mutant pro- Mutant Frameshift Pro-
Despite the accumulation of small amounts of type I collagen in these
cultures over the 18-h labeling period, intracellular precursor
pro- Proteasome Inhibitors Protect the Frameshift Mutant Pro-
Recently, it has become apparent that a number of mutant proteins which
have been targeted to the ER and were thought to be degraded within
that compartment are, in fact, degraded in the cytoplasm by the
ubiquitin-proteasome pathway (18-23). To determine if this pathway was
also responsible for the degradation of the frameshift pro-
To further examine the role of proteasomes in degradation of
unassembled mutant procollagen, the effect of two other specific proteasome proteolytic activity inhibitors ZL3VS (33) and
clasto-lactacystin
In fibroblasts isolated from a patient with type I OI, the pro- OI26 Mutant Pro-
Many plasma membrane and secretory proteins that fail to fold correctly
within the ER are selectively degraded by a quality control process
known as ER-associated degradation. The 26 S proteasome has been shown
to be responsible for the ER-associated degradation of transmembrane
proteins such as the cystic fibrosis transmembrane conductance
regulator (21), connexin-43 (18), major histocompatibility complex
class I heavy chains (22), insulin receptors (23), and T-cell receptor
chains (19), as well as the secreted proteins
Although the mechanism of mutant procollagen chain removal to the
cytoplasm is unknown several ER-resident chaperones have been
implicated in mediating proteasomal degradation of abnormal proteins.
Mutant insulin receptors associate with heat shock protein 90 prior to
proteasomal degradation (23) and unassembled apolipoprotein B
associates with calnexin prior to ubiquitination and subsequent proteasomal destruction (39), suggesting multiple components are
involved in the recognition and export to the cytoplasm of proteins
with abnormal conformations. Recent work implicates the ER-resident
molecular chaperone BiP (40-42), one of several proteins known to be
associated with procollagen during folding and assembly, in the process
of "reverse translocation" (4, 43). Investigation of 20 OI patient
cell lines revealed that BiP was associated with type I procollagen in
three lines containing C-propeptide mutations that impaired chain
association but not with the remaining 17 lines that contained helix
mutations and assembled normally (43). Furthermore, we have previously
shown that BiP associates with mutant procollagen in OI26 cells and in
a second OI cell line that contains a C-propeptide Trp to Cys amino
acid substitution (4). In both these lines normal procollagen folding
is disturbed and the formation of disulfide-linked trimers is retarded
(4, 6), suggesting that BiP specifically associates with procollagen chains that contain C-propeptide mutations and have an impaired ability
to assemble but not with chains with mutations in the triple helix
which can assemble normally but have abnormal triple helices (43).
Procollagen chains with abnormal triple helices are directed to
lysosomes for degradation (12, 13, 16), and our results demonstrate
that procollagens with an impaired ability to assemble as a consequence
of C-propeptide mutations are degraded by proteasomes. BiP binding may
thus play a critical role in recognizing and directing mutant
procollagen to proteasomes for degradation. BiP may bind to the
misfolded procollagen chains and then either escort them to the protein
translocation pore for reverse translocation into the cytoplasm, or
transfer them to other proteins that chaperone the abnormal procollagen
to the ER membrane.
1(I) chains from an osteogenesis imperfecta patient (OI26) with
a frameshift mutation resulting in a truncated C-propeptide, have
impaired assembly, and are degraded by an endoplasmic
reticulum-associated pathway (Lamandé, S. R., Chessler,
S. D., Golub, S. B., Byers, P. H., Chan, D., Cole,
W. G., Sillence, D. O. and Bateman, J. F. (1995)
J. Biol. Chem. 270, 8642-8649). To further explore
the degradation of procollagen chains with mutant C-propeptides, mouse Mov13 cells, which produce no endogenous pro-1(I), were stably transfected with a pro-1(I) expression construct containing a frameshift mutation that predicts the synthesis of a protein 85 residues longer than normal. Despite high levels of mutant mRNA in
transfected Mov13 cells, only minute amounts of mutant pro-1(I) could be detected indicating that the majority of the mutant
pro-1(I) chains synthesized are targeted for rapid intracellular
degradation. Degradation was not prevented by brefeldin A, monensin, or
NH4Cl, agents that interfere with intracellular transport
or lysosomal function. However, mutant pro-1(I) chains in both
transfected Mov13 cells and OI26 cells were protected from proteolysis
by specific proteasome inhibitors. Together these data demonstrate for
the first time that procollagen chains containing C-propeptide mutations that impair assembly are degraded by the cytoplasmic proteasome complex, and that the previously identified endoplasmic reticulum-associated degradation of mutant pro-1(I) in OI26 is mediated by proteasomes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-chains to form a triple helix occurs within the endoplasmic reticulum (ER),1 and is initiated by
interactions between the C-propeptides. Triple helix folding then
occurs sequentially from the COOH to the NH2 terminus, and
is essential for efficient secretion of the procollagen molecules (3).
Mutations in the pro-1(I) and pro-2(I) chains of type I collagen
which compromise initial chain association or disturb the folding of
the triple helix result in the brittle bone disease osteogenesis
imperfecta (OI) (4-7) and one of the important biosynthetic
consequences of these mutations is an increase in intracellular
collagen degradation (7).
1(I) C-propeptide which compromise chain association are degraded by a
process which is not prevented by brefeldin A, and was therefore assumed to occur within the ER (4). It has recently been shown that a
number of soluble and integral membrane proteins that have been
translocated into the ER and were thought to be degraded there, are in
fact, degraded by the cytoplasmic proteasome complex (18-23). This
process requires reverse transport of protein back to the cytoplasm, a
process which may be mediated by interaction of the protein with the
Sec61 complex, one of the major constituents of the translocation
apparatus (22), and could also involve molecular chaperones (24). In
this study we explore the role of proteasomes in the ER-associated
degradation of assembly impaired mutant type I procollagen. We examined
two pro-1(I) C-propeptide OI mutations. The first mutation, an
engineered frameshift mutation near the COOH-terminal end of the mouse
pro-1(I) chain, was analagous to a mutation defined in a patient
with OI type I (25). Fibroblasts from that patient contained both
mutant and normal pro-1(I) mRNA, but mutant protein could not be
detected in cells suggesting that it was rapidly and completely
degraded prior to assembly (25). In addition, we examine fibroblasts
from a patient (OI26) in which a heterozygous frameshift mutation
impaired, but did not prevent subunit assembly (4). In both cases the
use of specific proteasome inhibitors demonstrated a primary role for
cytoplasmic proteasomes in the selective degradation of procollagen
chains with mutations within the C-propeptide.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1(I)
C-propeptide and results in a chain which is 37 amino acids shorter
than normal (Fig. 1B). Mouse Mov13 cells (26) were provided
by Dr. R. Jaenisch (Whitehead Institute for Biomedical Research,
Cambridge, MA). Mov13 cells synthesize no endogenous pro-1(I) chains
since the transcription of both COL1A1 genes is blocked by a
retroviral insertion in the first intron (26, 27). Synthesis of
pro-2(I) is unaffected.
1(I) Chain--
The mutant mouse COL1A1
gene construct was a derivative of the previously described control
expression construct pWTCI-IA (28). This control construct contains a
functionally neutral Met822 
Ile
substitution2 within the
triple helix that allows the protein to be distinguished from wild-type
1(I) by its altered CNBr cleavage pattern (29), and a silent
Met1199 Ala substitution within the C-propeptide. A
clone was isolated that contained a 2-bp deletion within a
ClaI site 69 bases upstream of the translation stop signal
(data not shown). This deletion created a codon reading frameshift
which would direct the synthesis of a pro-1(I) chain 85 residues
longer than normal with an anomalous sequence covering the
COOH-terminal 109 amino acids (Fig. 1A). The final
reassembled 25-kilobase COL1A1 gene construct was named pWTCI-IAfs to indicate the amino acid substitutions in the protein product (the triple helical Met822 Ile substitution, a
silent C-propeptide Met1199-Ala alteration, and the 2-bp
deletion creating a codon reading frameshift). This frameshift mutation
is similar to one characterized in a patient with type I OI, where a
5-bp deletion predicted a pro-1(I) chain extended by 84 amino acids
(25). The mouse gene, pWTCI-IAfs, was stably transfected into Mov13
cells along with pSV2neo. Neomycin-resistant transfected cells were
selected in G418 (Life Technologies Inc.), and individual colonies
isolated and expanded into cell lines as described previously (28,
29).
-32P]dCTP-labeled mouse 1(I) and rat
glyceraldehyde-3-phosphate dehydrogenase (31)) cDNA probes for
16 h at 42 oC then washed in 0.1 × SSC, 0.1%
SDS at 65oC and exposed to x-ray film at
70 oC.
-lactone (Calbiochem), 1 µg/ml
brefeldin A (Roche Molecular Biochemicals), 10 µg/ml monensin (Sigma)
or 50 mM NH4Cl throughout the preincubation,
pulse labeling and chase periods. Cells were washed once with ice-cold
phosphate-buffered saline, scraped into 1 ml of phosphate-buffered
saline, and centrifuged briefly to pellet cells. Cells were lysed in
0.5 ml of lysis buffer (150 mM NaCl, 50 mM
Tris-HCl, pH 7.5, containing 5 mM EDTA, 20 mM
N-ethylmaleimide, 1 mM
4-(2aminoethyl)-benzenesulfonyl fluoride, 2 mM
iodoacetamide, 1% (v/v) Nonidet P-40) on ice for 30 min. Lysed cells
were centrifuged for 5 min at 10,000 × g to remove insoluble material and the supernatant precleared with 100 µl of 20%
Protein A-Sepharose (Amersham Pharmacia Biotech) at 4 oC
for 2 h. Type I procollagen pro-1(I) chains in the supernatant were immunoprecipitated with LF-67 (1/1000 dilution), a rabbit polyclonal antibody which recognizes the pro-1(I) carboxyl-terminal telopeptide of both human and mouse proteins (34) (kindly provided by
Dr. Larry Fisher, National Institute of Dental Research, Bethesda, MD),
and 100 µl of 20% Protein A-Sepharose at 4 oC
overnight. Immunoprecipitated complexes were washed twice for 30 min
each with 50% (v/v) lysis buffer, 50% (v/v) NET buffer (50 mM Tris-HCl, pH 7.5, containing 1 mM EDTA, 150 mM NaCl, and 0.1% Nonidet P-40), twice with NET buffer,
then once with 10 mM Tris-HCl, pH 7.5, containing 0.1%
Nonidet P-40. Immunoprecipitated chains were eluted into
electrophoresis loading buffer at 65 oC for 10 min.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1(I) chains to be easily followed without the
complications of endogenous pro-1(I) expression (28, 29). The mutant
mouse COL1A1 frameshift construct, pWTCI-IAfs (Fig.
1), contains a mutation which is similar to one characterized in a patient with type I OI, where a 5-bp deletion
predicted a pro-1(I) chain extended by 84 amino acids (25). While
mutant mRNA was present within the OI cells and could be translated
in an in vitro translation system, the protein was not
detected in cell culture suggesting that the aberrant protein was
rapidly degraded intracellularly. The predicted human and mouse
proteins show extensive sequence homology, including a highly
positively charged COOH terminus, and might be expected to share
similar metabolic fates. To examine the biochemical consequences of the
mouse frameshift mutation and address the question of procollagen subunit stability and the targeting of abnormal chains for
intracellular degradation raised by the human type I OI mutation, Mov13
cells were transfected with the mutant construct, pWTCI-IAfs.
Individual, stably transfected clones were selected in medium
containing G418 then screened for expression of pro-1(I) mRNA by
Northern blot analysis. In contrast to the untransfected Mov13 cells
which produced no pro-1(I) mRNA (Fig.
2, lane 1), high levels of
pro-1(I) mRNA of the correct size were apparent in two
transfected cell lines, Mov13-IAfs4 and Mov13-IAfs10 (Fig. 2,
lanes 5 and 6), and a third cell line,
Mov13-IAfs2, contained low levels of pro-1(I) mRNA (Fig. 2,
lane 4).
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Fig. 1.
Comparison of normal and mutant
pro- 1(I) C-propeptides. A, a
2-bp deletion (underlined) results in a codon reading
frameshift that predicts a protein which extends beyond the normal
translation stop signal by 85 amino acids. Amino acid sequence numbers
are shown on the right. The additional cysteine residues
(bold) and the second consensus sequence for
N-linked oligosaccharide addition (bold and
underlined) are shown. The translation stop codon is indicated by
the asterisks (***). B, diagram of the normal
pro-1(I) C-propeptide, the mutant mouse C-propeptide
([fs]pro-1(I) C-propeptide), and the OI26 mutant pro-1(I)
C-propeptide. The propeptide regions (boxes) are drawn to
scale and extend from the C-proteinase cleavage site (P) to
the carboxyl terminus. The locations of cysteines in the predicted
amino acid sequence are denoted by S and the proposed normal
intrachain disulfide bonds indicated by the dotted lines.
Potential N-linked oligosaccharide attachment sites are
designated by CHO. The location of the frameshift mutations
in the mouse pro-1(I) C-propeptide, and in the OI26 C-propeptide are
shown by the arrow and arrowhead, respectively.
The mutant amino acid sequences are indicated by the shaded
areas.
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Fig. 2.
Expression of mutant
pro- 1(I) mRNA in transfected Mov13
cells. Approximately 3 µg of total RNA was fractionated on a
0.8% agarose gel and transferred to nitrocellulose. The filter was
hybridized simultaneously to [32P]dCTP-labeled mouse
1(I) and rat glyceraldehyde-3-phosphate dehydrogenase
(GADPH) cDNAs and exposed to x-ray film. The migration
positions of the two major pro-1(I) mRNAs and the
glyceraldehyde-3-phosphate dehydrogenase mRNA are indicated.
Lane 1, parental untransfected Mov13 cells (Mov). Lane
2, Mov13 cells transfected with a wild-type COL1A1 gene
(WT). Lanes 3-6, Mov13 cells transfected with the
frameshift mutant construct pWTCI-IAfs, Mov13-IAfs8, -IAfs2, -IAfs4,
and -IAfs10.
1(I) Are Degraded Intracellularly in
Transfected Mov13 Cells--
To examine the ability of the frameshift
mutant pro-1(I) mRNA to be translated and the mutant pro-1(I)
chains to assemble into functional collagen molecules, stably
transfected cells were biosynthetically labeled with
[3H]proline for 18 h and cell and medium fractions
analyzed by SDS-PAGE after digestion with pepsin. Pepsin removes the
NH2- and COOH-terminal globular domains but leaves the
triple helical domain intact. Thus the presence of pepsin-resistant
collagen indicates that stable collagen trimeric assembly has occurred.
In untransfected Mov cells no pepsin-resistent collagen was present
(Fig. 3, lanes 1 and
2) and in cells transfected with the wild-type collagen gene, high levels of pepsin-resistant collagen were present (Fig. 3,
lanes 3 and 4). In cells expressing mutant
mRNA, only minute amounts of pepsin-resistant triple-helical
collagen were present (Fig. 3, lanes 7-12). While these
data indicate that the mutant procollagen is not able to assemble to
form significant amounts of stable collagen trimer, it was important to
demonstrate that this small amount of collagen trimer was derived from
the transfected mutant gene and not from low-level transcription of the
endogenous inactivated COL1A1. The mutant frameshift
construct, pWTCI-IAfs, also carried the silent reporter
Met822-Ile amino acid change within the helix. This marker
allows normal and transfected mutant 1(I) chains to be distinguished
because it deletes a cyanogen bromide cleavage site in the protein (28, 29). The presence of the larger distinctive peptide in CNBr-digested pepsin-resistant collagen produced by cells transfected with the frameshift construct confirmed that the 1(I) chains were synthesized from the mutant gene and were not the result of low-level transcription of the inactivated endogenous COL1A1 in these clonal Mov13
cell lines (data not shown). From these results we conclude that
although the mutant mRNA is transcribed at levels at least as high
as that of the control COL1A1 transfectants which produce
abundant collagen heterotrimers, only a very small proportion of the
mutant pro-1(I) chains can associate with endogenous pro-2(I) and
form triple-helical molecules which can be secreted from the cell.
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Fig. 3.
Expression of 1(I)
chains in transfected Mov13 cells. SDS-polyacrylamide gel
electrophoresis of [3H]proline-labeled pepsin-resistant
collagens from the cell layer (C) and medium (M)
fractions of Mov13 cell cultures. Lanes 1 and 2,
untransfected Mov13 cells (Mov); lanes 3 and 4,
Mov13 cells transfected with a wild-type COL1A1 gene (WT);
lanes 5-12, Mov13 cells transfected with the frameshift
construct, pWTCI-IAfs, Mov13-IAfs8, -IAfs2, -IAfs4, and -IAfs10. The
protein loadings are equivalent in all lanes and the gels were exposed
for the same times. The migration positions of type 1 collagen 1(I)
and 2(I) chains and type V collagen 1(V) and 2(V) chains are
indicated.
1(I) chains could not be detected by proline radiolabeling and
fluorography (data not shown). This failure to detect intracellular frameshift mutant pro-1(I) chains using standard radiolabeling procedures indicated that degradation was rapid, almost complete, and
the unassembled subunits did not accumulate within the cells. A more
sensitive method of protein detection was thus required to allow the
fate of the mutant protein to be investigated further. Cell layer
proteins produced by untransfected Mov13 cells, and cells transfected
with wild-type and mutant COL1A1 genes, were separated by
SDS-PAGE, electrophoretically transferred to nitrocellulose, and the
filter probed with antibody LF-67. Mov13-IAfs10 cells synthesized a
pro-1(I) subunit that migrated more slowly than control pro-1(I)
(Fig. 4, lane 3). This was
consistent with the prediction that the mutant protein would be 85 amino acids larger than normal and may be substituted with an
additional N-linked oligosaccharide group. The mutant
pro-1(I) subunits migrated as monomers when analyzed without
reduction (Fig. 4, lane 5), indicating that interchain
disulfide bonds had not formed and suggesting that trimer assembly is
severely impaired.
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Fig. 4.
Detection of intracellular frameshift mutant
pro- 1(I) chains. Procollagens from the
cell layer were separated on SDS-polyacrylamide gels either with (+) or
without () reduction with 10 mM dithiothreitol
(DTT). Proteins were transferred to nitrocellulose and
pro-1(I) chains detected with the polyclonal antibody LF-67 which
recognizes the pro-1(I) C-telopeptide. The migration positions of
normal pro-1(I) chains and the larger frameshift mutant
[fs]pro-1(I) chains are indicated. Lane 1,
untransfected Mov13 cells; lane 2, Mov13 cells transfected
with a wild-type COL1A1 gene; lanes 3-5,
Mov13-IAfs10 cells.
1(I)
Chains from Intracellular Degradation--
To determine the site of
intracellular degradation, Mov13-IAfs10 cells were treated with several
protease and vesicular traffic inhibitors and the relative levels of
mutant protein compared. Cells were pretreated for 1 h with
inhibitors, metabolically labeled with [35S]methionine
for 2 h, chased for 30 min in the presence of inhibitor, and the
intracellular pro-1(I) chains immunoprecipitated with LF-67. In
these untreated cells, mutant pro-1(I) was not detected, indicating
that complete degradation of the mutant protein produced during the 2-h
pulse had occurred within the 30-min chase period (Fig.
5A, lane 1). The mutant
subunit was also not detected in cells treated with brefeldin A, which
blocks protein transport into the Golgi and results in redistribution
of most of the Golgi into the ER (36), monensin, a carboxylic ionophore
which blocks intracellular traffic within the trans-Golgi,
or NH4Cl, which raises the pH of acidic compartments such
as the trans-Golgi and lysosomes (Fig. 5A, lanes
2-4). Since these agents inhibit intracellular vesicular traffic,
or inhibit lysosomal function, degradation of the mutant frameshift
pro-1(I) does not occur in the distal regions of the secretory
pathway. This suggests that, as in lethal OI patients with C-propeptide
mutations (4), the degradation pathway may be "ER-mediated."
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Fig. 5.
Effect of protease and vesicular transport
inhibitors on degradation of mutant
[fs]pro- 1(I) collagen. A,
Mov13-IAfs10 cells were labeled for 2 h with
[35S]methionine then chased for 30 min and lysed.
Pro-1(I) chains were immunoprecipitated with LF-67 and then
separated on SDS-polyacrylamide gels. Cells were labeled without
treatment () (lane 1) or were treated for 1 h prior
to and during pulse-chase with the following agents; 1 µg/ml
brefeldin A (BFA) (lane 2), 10 µg/ml monensin
(mon) (lane 3), 50 mM NH4Cl
(lane 4), and 20 µM ZL3al
(lane 5). B, identification of pro-1(I) chains
protected by the proteasome inhibitor ZL3al. Mov13-IAfs10
cells either without treatment () (lane 1) or treated with
20 µM ZL3al for 2 h were lysed and
electrophoresed on an SDS-polyacrylamide gel. Pro-1(I) chains were
identified by immunoblotting with LF-67. The migration positions of
[fs]pro-1(I) chains, and a smaller species, [fs]pro-1(I)*,
are indicated.
1(I)
chain, Mov13-IAfs10 cells were treated for 1 h with the specific
proteasome inhibitor, ZL3al, labeled with [35S]methionine for 1 h, chased for 30 min and the
intracellular pro-1(I) chains immunoprecipitated with a
pro-1(I)-specific antibody, LF-67 (Fig. 5, lane 5).
Incubation with this proteasome inhibitor prevented degradation of the
frameshift mutant pro-1(I) chain. The identity of the
protected protein was confirmed by immunoblotting (Fig.
5B, lane 2).
-lactone (37) were studied (Fig.
6). In these experiments cells were
preincubated for 1 h with the inhibitor, labeled for 1 h (Fig. 6A) and chased for up to 1 h (Fig.
6B). Both these proteasome inhibitors also protected mutant
procollagen from degradation (Fig. 6A, lanes 7 and
8) while in control cells transfected with wild-type
pro-1(I) (Fig. 6A, lanes 1-4) similar procollagen band intensities in untreated and proteasome inhibitor-treated cells strongly suggested that normal collagen is not degraded by a
proteasomal pathway. Interestingly, two bands which were not present in
untreated samples were seen in the presence of the proteasome
inhibitors and both were shown to be pro-1(I) chains by
immunoblotting with LF-67 (Fig. 5B). The larger minor band
migrated at the same position as the mutant chains previously detected
by immunoblotting (Fig. 4), but the major protected pro-1(I) form
was somewhat smaller. One possibility is that these two bands represent
glycosylated and deglycosylated variants of the mutant pro-1(I)
protein. Several glycoproteins have been shown to undergo
deglycosylation prior to proteasomal degradation, such as the heavy
chain of major histocompatibility complex class I molecules (22) and
ribophorin I (38). There are two N-linked oligosaccharides
addition sites within the mutant pro-1(I) C-propeptide (Fig.
1B) which offer possible targets for deglycosylation.
However, treatment of the cells during the preincubation and labeling
period with tunicamycin did not significantly alter the mobility of
either band (data not shown) indicating that the deglycosylation cannot
account for the difference in mobility of the two bands. In a
pulse-chase experiment in the presence of clasto-lactacystin
-lactone the upper band representing the full-length frameshift
mutant protein was converted to the smaller species almost completely
after a 1-h chase period (Fig. 6B). These data suggest that
the smaller form of the pro-1(I) is derived from the upper band by
the action of a non-proteasomal ER or cytoplasmic protease, and it is
the smaller form that is degraded by proteasomes since it is protected
from degradation by proteasome inhibitors. Importantly, pro-2(I)
chains did not coimmunoprecipitate with LF-67 even when the mutant
pro-1(I) chains were protected from degradation (Fig. 6A,
lanes 6-8). This suggested either that the mutant C-propeptide
sequence rendered the chains completely incompetent for assembly, or
that the mutant pro-1(I) chains were no longer within the ER but had
been transported to the cytoplasm by reverse translocation and were
therefore unable to interact with pro-2(I). Together these data
demonstrate that the vast majority of the frameshift mutant pro-1(I)
chains have an impaired ability to assemble into trimers and are
rapidly degraded in transfected Mov13 cells by proteasomes.
View larger version (33K):
[in a new window]
Fig. 6.
Degradation of mutant
pro- 1(I) chains in Mov13 cells is prevented by
proteasome inhibitors. A, effect of proteasome
inhibitors on degradation of wild-type and [fs]pro-1(I) collagen.
Mov13 cells transfected with a wild-type COL1A1 gene (WT)
(lanes 1-4) and Mov13-IAfs10 cells (lanes 5-8)
were labeled for 1 h with [35S]methionine and
pro-1(I) chains immunoprecipitated with LF-67 and electrophoresed
under reducing conditions. Cells were labeled without treatment ()
(lanes 1 and 5), or were treated for 1 h
prior to and during labeling with the proteasome inhibitors 20 µM ZL3al (lanes 2 and
6), 50 µM ZL3VS (lanes
3 and 7), or 5 µM
clasto-lactacystin -lactone (Lac) (lanes 4 and
8). B, pulse-chase analysis of Mov13-IAfs10 cells
treated with clasto-lactacystin -lactone. Mov13-IAfs10
cells were labeled for 1 h with [35S]methionine then
chased for up to 1 h in Dulbecco's modified Eagle's medium
containing 10% fetal calf serum. Pro-1(I) chains were
immunoprecipitated with LF-67 and electrophoresed under reducing
conditions. Untreated cells (Lac) were immediately lysed (lane
1) or chased for 30 min (lane 2). Cells treated with 5 µM clasto-lactacystin -lactone during the
preincubation and pulse-chase periods (+Lac) were chased for
0 (lane 3), 20 (lane 4), 30 (lane 5),
or 60 min (lane 6). Migration positions of normal
pro-1(I) and pro-2(I), fibronectin (FN),
[fs]pro-1(I), and the smaller chain [fs]pro-1(I)* are
indicated.
1(I)
chain contains a COOH-terminal frameshift mutation (25), which is very
similar to the engineered pro-1(I) mutant presented here. mRNA
levels for the mutant chain were normal but mutant procollagen
chains were not detectable suggesting that the mutant protein was
rapidly degraded (25). The similarity of these OI mutant procollagen
chains to the engineered pro-1(I) mutation expressed in Mov13 cells,
suggested that degradation of the OI mutant may also be
proteasome-mediated.
1(I) Is Also Protected by Proteasome
Inhibitors--
To determine if proteasomal degradation of procollagen
chains with C-propeptide mutations is a general mechanism or is
specific to the engineered mutation, we examined the degradation of
mutant pro-1(I) chains in OI26 cells, derived from a patient with
lethal OI. In OI26 a heterozygous frameshift mutation within the
C-propeptide results in the synthesis of a truncated pro-1(I)
containing an altered C-propeptide sequence (Fig. 1B) (4,
6). This mutation slowed, but did not completely prevent, assembly of
the mutant chains into triple helical molecules. Mutant unassembled
pro-1(I) chains were selectively degraded in OI26 cells and this
degradation was not prevented by brefeldin A, suggesting an
ER-associated degradation pathway (4). Because OI mutations are
heterozygous it is normally not possible to discriminate the fates of
normal and mutant allele products. However, the frameshift mutation in OI26 results in the synthesis of a more basic pro-1(I) chain, and
the normal and mutant chains can be resolved by two-dimensional gel
electrophoresis, which separates the proteins on the basis of both
charge and size (4, 6). We were able to take advantage of this unique
feature of the mutant protein to further explore its intracellular
degradation. OI26 fibroblasts were incubated with the proteasome
inhibitor ZL3al, labeled with
[35S]methionine, and the pro-1(I) chains
immunoprecipitated with LF-67 and resolved by two-dimensional gel
electrophoresis. In untreated cells only normal pro-1(I) chains were
seen, however, mutant and normal pro-1(I) chains were present in
comparable amounts in samples treated with the proteasome inhibitor
(Fig. 7). These data demonstrate that, as
in transfected Mov13 cells, pro-1(I) chains with a mutant
C-propeptide are degraded by proteasomes in OI26 fibroblasts.
View larger version (24K):
[in a new window]
Fig. 7.
Proteasomal degradation of mutant
pro- 1(I) chains in OI26 cells. OI26 cells
were labeled for 2 h with [35S]methionine and
pro-1(I) chains immunoprecipitated with LF-67, and analyzed by
two-dimensional non-equilibrium pH-gradient gel electrophoresis which
separates by charge in the first dimension and molecular weight in the
second. Top panel, untreated OI26 cells; bottom
panel, OI26 cells incubated with 40 µM
ZL3al for 2 h prior to and during metabolic labeling.
Migration of normal pro-1(I), pro-2(I), and mutant pro-1(I)
are indicated.
1-antitrypsin (20) and apolipoprotein B (39). The
experiments here demonstrate for the first time that proteasomes can
also degrade extracellular matrix molecules during biosynthesis, and are involved in the ER-associated degradation of type I collagen pro-1(I) chains with mutant C-propeptide domains.
| |
FOOTNOTES |
|---|
* This work was supported by grants from the National Health and Medical Research Council of Australia and the Royal Children's Hospital Research Institute.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.
Contributed equally to the results of this work.
§ To whom correspondence should be addressed: Dept. of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville 3052, Victoria, Australia. Fax: 61-3-9345-7997; E-mail: bateman@cryptic.rch.unimelb.edu.au.
2 Amino acids are numbered from the start of the triple helix.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: ER, endoplasmic reticulum; C-propeptide, carboxyl-terminal propeptide; OI, osteogenesis imperfecta; PAGE, polyacrylamide gel electrophoresis; ZL3al, sulfate carboxybenzyl-leucinyl-leucinyl-leucinal; ZL3VS, carboxybenzyl-leucyl-leucyl-leucine-vinylsulfone; bp, base pair(s); BiP, immunoglobulin heavy chain-binding protein.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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