From the
A heterozygous single base change in exon 49 of COL1A1, which
converted the codon for pro
The ``brittle-bone'' disease, osteogenesis imperfecta
(OI),
In this
study we have defined a point mutation which produces a cysteine for
tryptophan substitution at amino acid 1312
For
the pulse-chase analysis of procollagen assembly, confluent cultures
were incubated overnight in medium containing 10% (v/v) fetal calf
serum and 0.25 m
M ascorbic acid, preincubated for 2 h in
serum-free medium supplemented with 0.25 m
M ascorbic acid,
then labeled with 250 µCi/ml
L-[2,3,4,5-
For
biosynthetic labeling with [
To define the mutation, genomic DNA from the patient and from
the parents was amplified by the PCR and sequenced directly. The
patient was heterozygous for a single base change in exon 49 that
converted the codon for amino acid 1312 from tryptophan (TGG) to
cysteine (TGT). The mutation was not present in either of the
phenotypically normal parents (data not shown).
Intracellular accumulation of pro
The carboxyl-terminal propeptide mutations in OI64 and OI26
dramatically slowed the assembly of pro
In OI26, the frameshift mutation altered the amino acid
sequence of the carboxyl-terminal portion of the propeptide, but the
region containing the cysteines involved in intermolecular disulfide
bridges was unaffected
(6) . The finding of undisulfide bonded
pro
The tryptophan to cysteine mutation had no measurable
effect on mRNA stability. OI64 fibroblasts produced approximately equal
amounts of mutant and normal pro
Selective intracellular degradation is an important
``quality control'' mechanism preventing the accumulation
within cells and the secretion of unassembled and malfolded proteins
(41) . The pulse-chase procollagen assembly experiment indicated
that in control cells pro
Two observations led to
the view that pro
Together these data demonstrated that
mutant and excess normal chains were being degraded by separate
intracellular pathways. Mutant pro
The mechanisms by which chains or
molecules are targeted to different degradation pathways are unclear.
One targeting candidate, the endoplasmic reticulum-resident molecular
chaperone BiP, bound to the abnormal type I procollagen and was
coimmunoprecipitated in OI64 and OI26 but not control cells. BiP was
also found to bind to pro
The final aspect
of procollagen biosynthesis we examined was the effect of the
carboxyl-terminal propeptide mutations on collagen matrix formation.
When grown for an extended period in the continuous presence of
ascorbic acid both OI64 and OI26 cultures showed a marked reduction in
collagen accumulation. The deposited collagen was, however,
incorporated into a mature, cross-linked matrix since 80-90% was
solubilized only after pepsin digestion. The mature in vitro matrix collagen in OI64 and OI26 contained overmodified chains
demonstrating that at least some of the secreted mutant-containing
molecules were incorporated into the collagen fibrillar matrix. Poor
collagen matrix accumulation by OI26 fibroblasts closely resembled the
severely reduced matrices in the patients' dermis and bone which
contained only about 20% of the normal amount of collagen
(10) .
Incorporation of overmodified chains into the in vitro matrix
was consistent with the increased levels of hydroxylysine in collagen
from OI26 dermis and bone
(10) . This cell culture extracellular
matrix model system thus accurately reflected the in vivo biochemical phenotype of OI26. While no tissues were available
from OI64 for analysis, the in vitro data would predict a
major deficiency in the collagen content of the tissues.
The
pro
1(I) carboxyl-terminal propeptide
residue 94 from tryptophan (TGG) to cysteine (TGT) was identified in a
baby with lethal osteogenesis imperfecta (OI64). The C-propeptide
mutations in OI64 and in another lethal osteogenesis imperfecta cell
strain (OI26), which has a frameshift mutation altering the sequence of
the carboxyl-terminal half of the propeptide (Bateman, J. F., Lamande,
S. R., Dahl, H.-H. M., Chan, D., Mascara, T. and Cole, W. G. (1989)
J. Biol. Chem. 264, 10960-10964), disturbed procollagen
folding and retarded the formation of disulfide-linked trimers.
Although assembly was delayed, the presence of slowly migrating,
overmodified
1(I) and
2(I) chains indicated that mutant
pro
1(I) could associate with normal pro
1(I) and pro
2(I)
to form pepsin-resistant triple-helical molecules, a proportion of
which were secreted. Further evidence of the aberrant folding of mutant
procollagen in OI64 and OI26 was provided by experiments demonstrating
that the endoplasmic reticulum resident molecular chaperone BiP, which
binds to malfolded proteins, was specifically bound to type I
procollagen and was coimmunoprecipitated in the osteogenesis imperfecta
cells but not control cells. Experiments with brefeldin A, which
inhibits protein export from the endoplasmic reticulum, demonstrated
that unassembled mutant pro
1(I) chains were selectively degraded
within the endoplasmic reticulum resulting in reduced collagen
production by the osteogenesis imperfecta cells. This biosynthetic
deficiency was reflected in the inability of OI64 and OI26 cells to
produce a substantial in vitro collagenous matrix when grown
in the continuous presence of ascorbic acid to allow collagen matrix
formation. Both these carboxyl-terminal propeptide mutants showed a
marked reduction in collagen accumulation to 20% (or less) of control
cultures, comparable to the reduced collagen content of tissues from
OI26.
(
)
results from mutations in the type I
collagen genes (COL1A1 and COL1A2) in the vast majority of cases
studied to date
(1, 2) . The mutations include
insertions, deletions, gene rearrangements and, most commonly, point
mutations in the triple helical domains of the
1(I) and
2(I)
chains. These substitutions interrupt the obligatory Gly- X-Y repeat sequence and compromise the structural integrity of the
helix. Mutations outside the helical domain, in the carboxyl-terminal
propeptides, are infrequent but demonstrate the critical role of these
regions in directing chain assembly, trimerization, and the initiation
of helix formation. The severity of OI resulting from carboxyl-terminal
propeptide mutations is dependent on the extent to which the mutations
compromise procollagen assembly. Two frameshift mutations, one in
pro
1(I) and the other in pro
2(I), completely prevented
incorporation of the mutant pro
-chains into type I procollagen
heterotrimers resulting in a ``functionally null'' allele and
consequently relatively mild clinical phenotypes
(3, 4) . While the heterozygotes for the COL1A1 mutation
had OI type I, patients heterozygous for the COL1A2 frameshift mutation
were clinically normal, but the homozygote had a moderate OI type III
phenotype. However, four different carboxyl-terminal propeptide
mutations which disturbed, but did not prevent molecular assembly,
resulted in lethal OI
(5, 6, 7) .
(
)
of
the prepro
1(I) chain (carboxyl-terminal propeptide residue 94) and
causes a lethal form of OI. To further characterize the biosynthetic
consequences of carboxyl-terminal propeptide mutations, procollagen
molecular assembly, intracellular degradation, secretion, and
accumulation in the extracellular matrix were examined in this cell
strain (OI64) and also in OI26 cells, where a frameshift mutation
produced a change in the amino acid sequence beginning at residue 106
of the carboxyl-terminal propeptide and resulted in a truncated chain
(5, 6) . In both cell strains intermolecular
carboxyl-terminal propeptide disulfide-bond formation was disturbed,
and procollagen assembly was slowed. Furthermore, the endoplasmic
reticulum-resident molecular chaperone BiP was induced and bound to
type I procollagen in both cell strains. Although mutant pro
1(I)
chains were degraded within the endoplasmic reticulum, degradation was
not complete and some abnormal, overmodified molecules were secreted by
both OI cell lines. The formation of a collagenous matrix by the OI
cells was evaluated in an in vitro culture system where cells
were incubated for extended periods in the presence of ascorbate,
leading to the deposition and maturation of an extensive collagen
matrix in control cell cultures. As a consequence of defective
molecular assembly and increased intracellular degradation both cell
strains showed a dramatic collagen matrix deficiency.
The Patients
The first infant (OI64), was
delivered at 38 weeks gestation and died within 24 h. The ribs
contained healing fractures and the long bones showed numerous
metaphyseal and diaphyseal fractures but were not as wide and crumpled
in appearance as observed in many cases of OI type II
(8) . The
parents were unrelated and normal. The second patient (OI26) with a
previously defined pro1(I) propeptide frameshift mutation
(6) also had lethal OI with a strikingly similar radiological
appearance
(9) . OI and control fibroblast cultures were
established from dermal samples obtained at autopsy with parental
consent and the approval of the Ethics Committee of this hospital and
were maintained as described previously
(5, 10) .
Antibodies
Polyclonal antibodies to the
carboxyl-terminal propeptides of pro1(I) and pro
2(I) (PC),
and the carboxyl-terminal (LF-41, 11) and amino-terminal propeptides of
pro
1(I) (LF-9, 12) were kindly provided by Dr. Burton Goldberg
(University of Wisconsin School of Medicine) and Dr. Larry Fisher
(National Institute of Dental Research). Rat anti-BiP monoclonal
antibody
(13) was a generous gift from Dr. David Bole
(University of Michigan School of Medicine).
Biosynthetic Labeling and
Immunoprecipitation
Procollagens were biosynthetically labeled
with 5 µCi/ml
L-[5-H]proline (8.5
Ci/mmol, NEN Research Products) for 18 h in medium containing 10% (v/v)
dialyzed fetal calf serum and 0.25 m
M ascorbic acid. In some
experiments the ascorbic acid was replaced with 0.1 m
M
,
`-dipyridyl to prevent the post-translational
modification of proline and lysine residues, or 100 ng/ml brefeldin A
(Boehringer Mannheim) was added to prevent transport of secretory
proteins out of the endoplasmic reticulum
(14) . Cell layer and
medium fractions were harvested separately in the presence of the
proteinase inhibitors 5 m
M EDTA, 10 m
M N-ethylmaleimide, and 1 m
M phenylmethanesulfonyl
fluoride as described previously
(5, 10) . Procollagens
and collagens were precipitated with 25% saturated
(NH
)
SO
and resuspended in 50 m
M Tris-HCl, pH 7.5, containing 0.15
M NaCl, and proteinase
inhibitors. Aliquots of procollagens were precipitated with 75% (v/v)
ethanol and converted to collagen by limited pepsin digestion (100
µg/ml pepsin in 0.5
M acetic acid, 4 °C, 16 h).
H]proline (114 Ci/mmol, NEN
Research Products). After 30 min the medium was removed, the cells were
washed with medium containing 10% (v/v) fetal calf serum, 0.25 m
M ascorbic acid, and 10 m
M proline, and then incubated in
this chase medium for up to 6 h. Cell layer procollagens were harvested
in the presence of 20 m
M iodoacetamide to prevent further
disulfide bond formation and were precipitated with 30% (v/v) ethanol
(15) . In some experiments 100 ng/ml brefeldin A was included
during the preincubation, labeling, and chase periods.
S]methionine, the
cells were rinsed in methionine-deficient medium and then incubated for
3 h in methionine-deficient medium supplemented with 100 µCi/ml
[
S]methionine (>1000 Ci/mmol, Amersham
Corp.). Cells were lysed in an ATP-depleting non-ionic detergent buffer
(1% Nonidet P-40, 150 m
M NaCl, 50 m
M Tris, pH 7.5, 1
m
M phenylmethanesulfonyl fluoride, 8 units/ml hexokinase
(Sigma), 11 m
M glucose, and 24 units/ml apyrase (Sigma)).
After 30 min on ice, iodoacetamide (30 m
M, final) was added to
the lysates, and they were centrifuged briefly to remove nuclei and
insoluble material. Aliquots of cell extract were diluted with lysis
buffer, primary antibody was added, and samples mixed by end-over-end
rotation. After 1-2 h, protein G-Sepharose (Pharmacia) was added
to the reactions, and they were mixed for several hours more. The beads
were washed three times, 30 min each time, first in 50% lysis buffer
and 50% NET buffer (0.1% Nonidet P-40, 150 m
M NaCl, 1 m
M EDTA, 0.02% sodium azide, 50 m
M Tris-HCl, pH 7.5), then
twice with NET buffer. Precipitated protein complexes were examined by
SDS-PAGE. To verify the position of BiP, cell extracts from fibroblasts
treated overnight with tunicamycin (5 µg/ml) and labeled as above
with [
S]methionine were loaded on most gels
(16) .
Extraction of Collagen from the Extracellular
Matrix
Cell layers from cultures which had been supplemented
daily from confluence (day 0) with 0.25 m
M ascorbic acid were
sequentially extracted with 50 m
M Tris-HCl, pH 7.5, containing
0.15
M NaCl and proteinase inhibitors (neutral salt soluble
fraction), 0.5
M acetic acid (acetic acid soluble fraction),
and by digestion with pepsin (0.1 mg/ml pepsin in 0.5
M acetic
acid) as described previously
(17) . Cells were released from
the extracellular matrix by digestion with bacterial collagenase and
trypsin
(17) and the cell DNA content quantified using the
Hoescht 332558-specific fluorescence procedure
(18) .
SDS-Polyacrylamide Gel Electrophoresis
Procollagen
and collagen chains were resolved on 5% (w/v) polyacrylamide separating
gels with a 3.5% (w/v) stacking gel. Sample preparation,
electrophoresis conditions, Coomassie Brilliant Blue staining, and
fluorography of radioactive gels have been described elsewhere
(5, 10) . Coomassie-stained bands were quantitated by
densitometry in comparison to standard collagen samples loaded on each
gel. The radioactivity in the procollagen and collagen bands was
measured by scintillation counting of excised bands
(19) .
Procollagen chains labeled in culture or produced by cell-free
translation of mRNA were also analyzed by two-dimensional gel
electrophoresis
(20) which resolves the chains on the basis of
both charge and size.
Immunoblotting
Proteins were electrophoretically
transferred from 5% (w/v) SDS-polyacrylamide gels to nitrocellulose
filters (BA85, Schleicher and Schuell). Bound LF-41 antibody was
detected using horseradish peroxidase-conjugated protein A (Bio-Rad)
and an enhanced chemiluminescence kit (ECL, Amersham Corp.).
Detection of the OI64 RNA Mutation by Chemical Cleavage
of mRNA cDNA Heteroduplexes
Total RNA was purified from OI64
fibroblasts
(21) and hybridized to a 672-bp
XhoI- EcoRI fragment of the 1(I) cDNA clone Hf677
(22) which had been end-labeled with
[
-
P]dCTP (3000 Ci/mmol, Amersham Corp.).
Heteroduplex formation, hydroxylamine and piperidine treatment, and
electrophoretic analysis were performed as described previously
(6, 23) .
Preparation of Genomic DNA, PCR Amplification, and Direct
Sequencing
Genomic DNA was purified from cultured OI64
fibroblasts and whole blood from the parents
(24) and the
region of the COL1A1 gene containing the mutation amplified through 30
cycles by the PCR using the primers 5`-TACTGGATTGACCCCAACCA-3` and
5`-TGGTAGGTGATGTTCTGGGA-3` and Taq polymerase (Perkin-Elmer
Cetus). The 575-bp amplification product was gel purified and sequenced
directly using the 5` PCR primer
(25) .
Quantitation of pro
OI64 and control fibroblasts were grown to confluence
then supplemented daily with 0.25 m
M ascorbic acid for 3 days
to maximize type I collagen mRNA levels
(26) . Total RNA was
extracted and dilutions dotted onto duplicate nitrocellulose membranes.
cDNA probes for 1(I) and pro
2(I) mRNA
Ratios
1(I) (Hf677, 22) and
2(I) (Hf32, 27) were
labeled with [
-
P]dCTP (3000 Ci/mmol,
Amersham Corp.) by nick translation and the filters hybridized, washed,
and specific hybridization quantified as described previously
(28) .
Quantitation of Normal and Mutant pro
The mutation deleted a RsaI site which
allowed PCR products derived from the normal and mutant alleles to be
discriminated. Total RNA from two separate preparations was amplified
by 25 PCR cycles using the same primers and conditions used for the
genomic DNA PCR. Two aliquots from each reaction were diluted 1:10 in
fresh PCR buffer and subjected to a final cycle in the presence of
[1(I) mRNA
Transcripts
-
P]dCTP. The products were digested with
RsaI, separated on an 8% non-denaturing polyacrylamide gel,
and the radioactivity in each band quantitated by excision and
scintillation counting. The final cycle eliminated heteroduplexes which
are resistant to restriction enzyme digestion and commonly form during
later PCR cycles when amplification has plateaued
(29) .
Cell-free Translation
Cell-free translation of
control and OI26 mRNA was performed in a rabbit reticulocyte lysate
system (Promega) labeling with
L-[2,3,4,5-H]proline.
Retarded Chain Association of Type I
Procollagen
When unhydroxylated procollagens, produced by
labeling cells in the presence of ,
`-dipyridyl, were analyzed
without reduction, the pro
1(I) and pro
2(I) chains in the
control were linked by disulfide bonds and migrated near the top of the
gel (Fig. 1, lane 2). In the OI26 and OI64 samples, there was
an additional band which had a similar migration to reduced
pro
1(I) chains (Fig. 1, lanes 4 and 6).
This band was identified as a pro
(I) chain by immunoblotting with
LF-41, an antibody which specifically recognizes the last 21 amino
acids at the carboxyl terminus of pro
1(I) (Fig. 1, lane
10). This suggested that, as in OI26 where an
1(I)
carboxyl-terminal propeptide mutation had been characterized
previously, pro
1(I) chains from OI64 contained a mutation which
interfered with chain association and the formation of interchain
disulfide bonds. Unreduced pro
1(I) monomers migrated faster than
fully reduced pro
1(I) indicating the presence of internal
disulfide bonds
(30) .
Figure 1:
Identification of
undisulfide-bonded pro1(I) chains in OI26 and OI64 cells.
Fibroblast cultures were labeled with [
H]proline
for 18 h in the presence of
,
`-dipyrridyl, and the
unhydroxylated procollagens from control ( C), OI26, and OI64
cultures were examined on 5% SDS-polyacrylamide gels either with
(+) or without (-) reduction with 10 m
M dithiothreitol ( DTT).
[
H]Proline-labeled proteins ( lanes 1-6) were detected by fluorography and quantitated
by scintillation counting as described under ``Experimental
Procedures.'' Lanes 2, 4, and 6 contain
twice the sample loading as lanes 1, 3, and
5. Proteins were transferred to nitrocellulose membranes and
pro
1(I) chains detected using the antibody LF-41 which recognizes
the last 21 amino acids of the pro
1(I) carboxyl-terminal
propeptide ( lanes 7-10). The migration
positions of the reduced unhydroxylated pro
1(I) and pro
2(I)
chains are indicated. Electrophoretic transfer of the large
disulfide-bonded procollagen molecules is inefficient, and this species
is therefore not routinely detected by
immunoblotting.
Molecular Characterization of a Carboxyl-terminal
Propeptide Mutation in OI64
The mutation was initially detected
and localized by chemical modification and cleavage of heteroduplexes
formed between mRNA extracted from OI64 fibroblasts and a control
1(I) 672 bp XhoI- EcoRI cDNA probe which spanned
the majority of the carboxyl-terminal propeptide.
Hydroxylamine/piperidine treatment resulted in cleavage of the probe
and the production of a 251-bp fragment (data not shown) indicating the
presence of a mismatched C in the probe in the region coding for
prepro
1(I) tryptophan 1312 (carboxyl-terminal propeptide residue
94).
Quantitation of OI64 Type I Collagen mRNA
Transcripts
Because the mutation deleted a RsaI
restriction site in cDNA we could assess the effect of the mutation on
mRNA expression. The normal transcript represented an average of 54% of
the total 1(I) mRNA indicating that the mutation did not
measurably affect steady-state mRNA levels. This was further confirmed
by comparing the levels of pro
1(I) and pro
2(I) mRNA. In
control cells the
1(I)/
2(I) ratio was 1.47 ± 0.08
(mean ± S.D., n = 8) compared with 1.46 in OI64
cells (average of determinations on mRNA preparations from two separate
cultures). These values were not corrected for the probe size and
specific activity.
Analysis of Interchain Disulfide Bond Formation in OI64
and OI26
To characterize the effect of the point mutation in
OI64 and a frameshift mutation in OI26
(6) on interchain
disulfide bond formation and molecular assembly, control, OI64, and
OI26 cells were pulse-labeled for 30 min and then chased for up to 6 h
prior to analysis of the procollagen by SDS-PAGE under non-reducing
conditions. In control cells almost all of the pro1(I) chains were
incorporated into disulfide-linked trimers by 40 min of chase (Fig.
2 a). In contrast, monomeric pro
1(I) chains were present
in OI64 cells for more than 120 min and in OI26 cells even after 6 h of
chase (Fig. 2, c and d) indicating that the
mutations slowed the formation of interchain disulfide bonds. In
control cells monomeric pro
2(I) chains disappeared more slowly
than free pro
1(I) chains, after 1-2 h of chase, suggesting
that they were synthesized in excess (Fig. 2 a).
Figure 2:
Delayed procollagen assembly in OI26 and
OI64 cells. Control, OI26, and OI64 fibroblasts were pulse-labeled for
30 min with [H]proline then chased in
proline-supplemented medium for up to 360 min. Cell layer procollagens
were separated without reduction on 5% SDS-polyacrylamide gels.
a, control cells; c, OI26; d, OI64. In each
case lanes 1-7 show procollagens from cells chased for
0, 20, 40, 60, 120, 240, and 360 min, respectively. The migration
positions of pro
1(I) and pro
2(I) chains and the
disulfide-bonded pro
chain dimers and trimers are indicated. The
protein migrating above pro
2(I) ( arrow in a)) is
unknown. b, control cells were preincubated, pulse labeled,
and chased in the presence of brefeldin A which prevents protein
transport out of the endoplasmic reticulum.
Intracellular Accumulation of Excess pro
The excess synthesis of
pro2(I) Chains
in Brefeldin A-treated Cells
2(I) chains relative to pro
1(I) was highlighted when
brefeldin A was included during the preincubation, pulse labeling, and
chase periods. Brefeldin A is a fungal metabolite which inhibits
protein secretion by causing the cis-Golgi to fuse with the
endoplasmic reticulum, preventing further protein transport
(14) . While pro
1(I) chain disulfide-bonding may have been
slightly slowed in the presence of brefeldin A, incorporation of
pro
1(I) monomers into disulfide-bonded trimers was complete by 60
min of chase (Fig. 2 b), and the most obvious effect of
brefeldin A was to almost completely prevent the disappearance of
undisulfide-bonded pro
2(I) chains. These chains migrated more
slowly with increasing chase time consistent with the proposal that
they were excess non-helical subunits and continued to be substrates
for post-translational enzymatic hydroxylation and glycosylation
(31) .
2(I) chains was
also observed when control, OI64, and OI26 cells were continuously
labeled for 18 h in the presence of brefeldin A. About 25% of the total
pro
2(I) chains from control cells migrated as undisulfide-bonded
monomers when exit from the endoplasmic reticulum was blocked by
brefeldin A (Fig. 3, lanes 1 and 2);
however, in brefeldin A-treated OI64 cells, undisulfide-bonded
pro
2(I) chains represented a larger proportion, about 40%, of the
total pro
2(I) (Fig. 3, lanes 3 and
4). In contrast, brefeldin A-treated OI64 cells did not
accumulate extra undisulfide-bonded pro
1(I) chains; approximately
10% of the pro
1(I) chains migrated as monomers in both
,
`-dipyridyl and brefeldin A-treated OI64 cells
(Fig. 1, lanes 5 and 6; Fig. 3,
lanes 3 and 4). OI26 cells exhibited a similar
pattern of intracellular procollagen chain accumulation. Brefeldin
A-treated OI26 cells accumulated excess undisulfide-bonded pro
2(I)
chains to a greater extent than control cells (Fig. 3, lanes 5 and 6), while the proportion of
undisulfide-bonded pro
1(I) chains was not increased compared with
,
`-dipyridyl-treated OI26 cells (Fig. 1, lanes 3 and 4; Fig. 3, lanes 5 and 6). This pattern of additional pro
2(I)
accumulation by brefeldin A-treated OI26 and OI64 cells was consistent
in three separate experiments.
Figure 3:
Electrophoretic analysis of intracellular
procollagens from brefeldin A-treated fibroblasts. Control
( C), OI26, and OI64 fibroblasts were labeled with
[H]proline for 18 h in the presence of brefeldin
A to prevent protein transport out of the endoplasmic reticulum, as
described under ``Experimental Procedures.'' Intracellular
procollagens were analyzed with (+) and without (-)
reduction with 10 m
M dithiothreitol ( DTT) on 5%
SDS-polyacrylamide gels. [
H]Proline-labeled
proteins were detected by fluorography and quantitated by scintillation
counting as described under ``Experimental Procedures.''
Lanes 2, 4, and 6 contain twice the sample
loading as lanes 1, 3, and 5. The migration
positions of the reduced pro
1(I) and pro
2(I) chains are
shown.
Selective Intracellular Degradation of Mutant
pro
The extra
accumulation of excess, monomeric pro1(I) Chains within the Endoplasmic Reticulum
2(I) chains in brefeldin
A-treated OI26 and OI64 cells when compared to control cells suggested
that relatively fewer pro
1(I) chains were available for assembly
with pro
2(I) subunits in the OI cells probably because of
increased intracellular pro
1(I) degradation. The frameshift
mutation in OI26 results in the synthesis of a more basic pro
1(I)
chain which is 37 amino acids shorter than normal. Mutant and normal
OI26 pro
1(I) chains were resolved by two-dimensional gel
electrophoresis, which separates proteins on the basis of both charge
and size, and this allowed the intracellular fate of these chains to be
directly compared (Fig. 4). The identity of pro
1(I) chains was
confirmed by immunoblotting (data not shown). Mutant and normal
pro
1(I) chains were almost equally abundant in samples produced by
cell-free translation of fibroblast mRNA (Fig. 4 b)
indicating that the steady-state levels of normal and mutant mRNA were
similar. In contrast, the mutant chain was significantly reduced
relative to normal pro
1(I) in cells labeled for 18 h in the
presence of
,
`-dipyridyl (Fig. 4 d),
demonstrating that it was selectively degraded. Degradation of the
mutant pro
1(I) chain was not inhibited by brefeldin A
(Fig. 4 f) consistent with an endoplasmic
reticulum-localized degradation pathway.
Figure 4:
Two-dimensional gel electrophoresis of
control and OI26 procollagens. [H]Proline-labeled
procollagens were produced by cell-free translation of control
( a) and OI26 ( b) fibroblast mRNA, and by incubation
with
,
`-dipyridyl ( c and d) or brefeldin A
( e and f). Pro
1(I) and pro
2(I) chains are
designated in a by 1 and 2, respectively.
The arrowhead indicates the migration position of the mutant,
more basic pro
1(I) chain produced by OI26 cells and by cell-free
translation.
Normal and abnormal OI64
pro1(I) carboxyl-terminal propeptides could not be resolved on
two-dimensional isoelectric focusing gels (data not shown) so it was
not possible to determine the relative proportions of normal and mutant
pro
1(I) chains remaining in the cells in these experiments.
Coprecipitation of BiP and Type I Procollagen
The
endoplasmic reticulum-resident protein BiP has been shown to bind to
malfolded secretory proteins
(32) . When type I procollagen was
immunoprecipitated with an antibody to its carboxyl-terminal propeptide
(that recognizes both normal and abnormal procollagen molecules), BiP
coprecipitated in extracts from OI64 and OI26 cells but not from
control cells (Fig. 5), demonstrating BiP binding to the abnormal
procollagen produced by the OI cells. BiP has ATPase activity and,
in vitro, dissociates from proteins to which it is bound in
the presence of ATP and Mg(33, 34) .
When immunoprecipitated type I procollagen from OI64 and OI26 cells was
incubated with ATP, BiP was released from the precipitated protein
complex, (Fig. 5, a, d, and e),
verifying the specificity of BiP-pro
chain binding in both OI26
and OI64. BiP synthesis increased in both OI64 and OI26 cells after the
addition of ascorbate to the culture medium. After 20-24 h of
ascorbate treatment, BiP protein content was substantially greater in
OI64 and OI26 cells than in control cells (data not shown).
Figure 5:
BiP binds type I procollagen in OI26 and
OI64 cells. OI26, OI64, and control cells ( C) were treated
overnight with ascorbate, labeled for 3 h with
[S]methionine, harvested in ATP-depleting lysis
buffer, and analyzed by SDS-PAGE under reducing ( a and
b) and non-reducing ( c and d) conditions.
a, OI26 cell extracts were immunoprecipitated with a
polyclonal antibody to the carboxyl termini of pro
1(I) and
pro
2(I) ( PC). The right lane shows the effect of
ATP on the binding of BiP to pro
chains from OI26. Between washes,
the precipitated protein complexes were incubated for 30 min at 23
°C in NET buffer without EDTA and with either 10 m
M MgCl
alone (-) or with 10 m
M MgCl
and 4 m
M ATP (+). ATP specifically disrupted
BiP-procollagen chain binding. b, OI64 cell extracts
immunoprecipitated with antibodies to the carboxyl-terminal and
amino-terminal propeptides of type I procollagen. BiP coprecipitated
with type I procollagen in lysates from OI64 cells. c, OI26
proteins were precipitated with either the monoclonal antibody to BiP
( B), or the polyclonal antibody to the
1(I) and
2(I)
carboxyl-terminal propeptides ( PC). The type I pro
chains
that coprecipitated with BiP from OI26 cells migrated as monomers under
non-reducing conditions. d, OI64 proteins were
immunoprecipitated, washed, and treated with (+) or without
( -) 4 m
M ATP as in a. e, a
longer exposure of the region of the gel containing the coprecipitated
proteins. BiP was bound to type I procollagen molecules from OI64 cells
and was released by treatment with ATP. The protein that coprecipitated
with type I procollagen from control cells is unknown. The positions of
the type I pro
chains, BiP, type I procollagen trimers , and fibronectin ( FN) are
indicated.
Secretion of Mutant pro
To further compare the fate of pro1(I) Chains by OI64 and OI26
Fibroblasts
1(I) chains
with mutations in the carboxyl-terminal propeptide domain, collagens
from OI26 and OI64 were biosynthetically labeled with
[
H]proline and aliquots from the medium were
examined for the presence of undisulfide-linked pro
1(I) chains.
Some undisulfide-linked pro
1(I) chains were seen in the medium of
OI26 cells (Fig. 6 a, lane 2); however, OI64
cells did not secrete undisulfide-bonded pro
1(I)
(Fig. 6 a, lane 3). The presence of
slowly migrating
1(I), pN
2(I), and
2(I) chains in the
medium of both cell stains (Fig. 6 b, lanes 2 and 4) confirmed that some procollagen
molecules containing mutant chains were secreted. No undisulfide-bonded
pro
2(I) chains were secreted by either control or OI cells (Fig.
6 a) indicating that the excess pro
2(I) chains synthesized
were degraded within the cell.
Figure 6:
Mutant pro1(I) chains are secreted
and deposited into the extracellular matrix by OI26 and OI64
fibroblasts. a, control, OI26 , and OI64 fibroblasts
were labeled with [
H]proline for 18 h
and procollagens prepared as described under ``Experimental
Procedures .'' Samples from the medium were analyzed
without reduction on 5% SDS-polyacrylamide gels. Undisulfide-bonded
pro
1(I) chains were present in the medium from OI26 cells
( lane 2) but were not secreted by control ( lane 1) or OI64 fibroblasts ( lane 3). The
migration positions of pro
1(I), pN
1(I),
1(I),
pN
2(I) , and
2(I) chains are indicated. The expected
migration position of pro
2(I) chains is shown by the
arrow. b, control, OI26 , and OI64 cells were
grown from confluence(day 0) in the presence of ascorbic acid
and labeled with [
H]proline for 18 h on day 8.
Labeled collagens from the medium, neutral salt, and pepsin extracted
fractions were analyzed by SDS-PAGE and detected by fluorography (see
``Experimental Procedures'' for details). Slowly migrating,
overmodified
1(I) and
2(I) chains were present in the medium
and neutral salt soluble fractions, as well as the mature pepsin
extracted matrix from OI26 ( lanes 2, 6, and
10) and OI64 ( lanes 4, 8, and
12). Collagens from the medium, neutral salt soluble
fraction , and pepsin -extracted matrix of control
cells were loaded in alternate lanes to facilitate comparison of chain
migrations ( lanes 1, 3, 5, 7,
9, and 11)
The Effect of Carboxyl-terminal Propeptide Mutations on
Matrix Formation by OI64 and OI26 Fibroblasts
When cultured in
the continuous presence of ascorbic acid human fibroblasts accumulate
an extensive, insoluble, collagenous matrix
(26) . This culture
system was exploited to examine and compare the effects of these
1(I) carboxyl-terminal propeptide mutations on the deposition and
maturation of an in vitro extracellular matrix. The matrix
deposited by control and OI cells grown for up to 12 days
post-confluence was sequentially extracted with a neutral salt buffer
to remove the newly synthesized soluble collagens, acetic acid to
extract the acid-labile cross-linked collagens, and pepsin to
solubilize the more mature, covalently cross-linked collagen matrix.
The total collagen accumulated in these three extracted fractions was
related to the DNA content of the cells and is shown in Fig. 7. Both
OI26 and OI64 showed a striking deficiency in matrix production. Slowly
migrating, overmodified
1(I) and
2(I) chains were apparent,
not only in the readily soluble fraction (Fig. 6 b,
lanes 6 and 8), but also in the mature,
pepsin-extracted matrix (Fig. 6 b, lanes 10 and 12). The distribution of the collagen among the
neutral salt, acetic acid, and pepsin extracts was similar in all three
cultures with 80-90% of the total collagen present in the pepsin
extracted fraction (data not shown).
1(I) chains into
disulfide-linked trimers. Procollagen assembly is initiated by
interactions between the carboxyl-terminal propeptides, and although
the details of the folding pathway and intermediates are not known, the
carboxyl-terminal propeptides of individual pro
chains fold and
form intrachain disulfide bonds before the chains associate and are
linked by interchain disulfide bonds
(35) . Disulfide bond
formation during protein folding involves catalysis by protein
disulfide isomerase, the
-subunit of prolyl-4-hydroxylase
(36, 37) , and the formation and interchange of
disulfide bond intermediates
(38) . The additional cysteine in
the OI64 mutant pro
1(I) chain may disturb these folding pathways
by inappropriately pairing with other cysteines thus stabilizing
non-native folding intermediates and slowing the formation of
assembly-competent pro
1(I) chains. Mutations which impair
carboxyl-terminal propeptide folding and retard chain association may
also have an effect on chain selection. Since the retardation of
propeptide association will also result in delayed folding of the
triple helix, and consequently expose the component
-chains to
increased post-translational lysyl hydroxylation and hydroxylysyl
glycosylation, the presence of overmodified
-chains was used to
determine the
-chain composition of mutant-containing trimers.
Slowly migrating, overmodified
1(I) and
2(I) chains were
present in the cell layer and the medium of OI64 and OI26 cells, thus
it is apparent that although assembly was delayed, mutant pro
1(I)
could associate with normal pro
1(I) and pro
2(I) to form
pepsin-resistant triple-helical molecules, a proportion of which were
secreted.
1(I) chains in the medium of OI26 provided further evidence for
the secretion of mutant chains by this cell line. For secretion these
chains have presumably associated and folded into a triple helix
(39, 40) . Thus, the correct formation of the
intermolecular disulfide bonds which stabilize carboxyl-terminal
propeptide association is not a prerequisite for helix formation.
Recent studies using site-directed mutagenesis to perturb
carboxyl-terminal propeptide disulfide bonding confirm that assembly
and secretion can occur in the absence of normal interchain disulfide
bonding.
(
)
Other non-covalent interactions,
possibly involving the telopeptides and the amino-terminal half of the
carboxyl-terminal propeptide, must be sufficient for nucleation of
helix folding.
1(I) mRNAs, and the ratio of total
pro
1/pro
2 mRNA was the same as in control cells. Cell-free
translation of OI26 mRNA also demonstrated that mutant and normal mRNA
was present in equal amounts. It is likely that in both OI64 and OI26
type I collagen was synthesized in equal amounts by both normal and
mutant alleles, and the reduction in net collagen production in OI64 to
around 30% of control values
(28) , and to 50% in OI26
(10) , reflected increased degradation. In OI26 increased
intracellular collagen degradation was directly demonstrated
(10) .
2(I) chains were synthesized in excess of
the amount required for stoichiometric assembly with two pro
1(I)
chains. Excess monomeric pro
2(I) chains are not secreted and are
not normally seen within fibroblasts during longer biosynthetic
labeling experiments probably because they are continually being
degraded and thus represent only a small proportion of the total
intracellular pro
2(I) pool. However, pro
2(I) chain monomers
were present in brefeldin A-treated cells which indicated that excess,
unassembled pro
2(I) are normally transported out of the
endoplasmic reticulum before degradation. Similarly, the monomeric
pro
2(I) chains synthesized by Mov13 cells which do not produce
pro
1(I) subunits are not secreted
(42) but are degraded
intracellularly by a mechanism which is sensitive to inhibition by
brefeldin A,
further confirming a post-endoplasmic
reticulum site for pro
2(I) degradation.
1(I) chains with carboxyl-terminal propeptide
mutations are selectively degraded in the endoplasmic reticulum. When
degradation of excess pro
2(I) chains was inhibited by brefeldin A
treatment, unassembled pro
2(I) monomers represented a larger
proportion of the total pro
2(I) in the OI cells than in the
control. Since the pro
chains were synthesized initially in the
correct ratios, the increased accumulation of pro
2(I) monomers
suggested that fewer pro
1(I) chains were available for assembly
with pro
2(I). However, there was no extra accumulation of
undisulfide-bonded pro
1(I) chains in brefeldin A-treated OI64 or
OI26 cells. This indicated that, in contrast to degradation of the
excess pro
2(I) chains, degradation of pro
1(I) chains was not
prevented when transport out of the endoplasmic reticulum was blocked.
Direct evidence for an endoplasmic reticulum-localized mechanism which
recognizes and selectively degrades pro
1(I) chains with
COOH-terminal propeptide mutations that interfere with subunit assembly
was provided by two-dimensional electrophoresis of OI26 procollagens,
where the more basic mutant chains could be resolved from normal
pro
1(I). The mutant protein was present in similar amounts to the
normal pro
1(I) in cell-free translation products but was
dramatically reduced relative to the normal pro
1(I)
intracellularly. Mutant pro
1(I) was not protected from degradation
by brefeldin A treatment, consistent with a degradation pathway located
in the endoplasmic reticulum.
1(I) chains were selectively
degraded within the endoplasmic reticulum, while the excess normal
pro
2(I) was degraded after exiting the endoplasmic reticulum. Two
distinct intracellular pathways for collagen degradation have been
described previously. Approximately 10-15% of the collagen
synthesized by cultured fibroblasts is degraded prior to secretion in a
process known as basal degradation
(43) , and this intracellular
degradation is increased when cells are induced to synthesize
structurally abnormal, non-helical collagen by incubation with the
proline analogue cis-hydroxyproline
(44) . Although
both degradation processes occur in the distal region of the secretory
pathway, beyond the brefeldin A block
(45) , they differ in
their sensitivity to lysomotrophic agents; the degradation of
cis-hydroxyproline-containing collagen is mediated by
lysosomal proteases
(43, 44) while basal degradation
occurs in an acidic, but non-lysosomal compartment, possibly the
trans-Golgi
(45, 46) . Continued degradation of
the abnormal OI26 and OI64 pro
1(I) in the presence of brefeldin A
identifies the endoplasmic reticulum as an additional site of collagen
degradation and indicates that the mechanisms for degrading
unincorporated structurally normal chains and molecules with defective
helices may differ from those for disposing of mutant chains which are
unable to assemble efficiently.
chains from three other cell strains in
which pro
1(I) carboxyl-terminal propeptide mutations interfered
with chain association and interchain disulfide bonding
(47) .
The main function of BiP is thought to be the promotion of
intracellular protein assembly
(38, 48) as it binds
transiently to many secretory proteins before they have folded or
assembled into their mature form and remains associated with aberrant,
misfolded proteins
(12, 32, 48, 49) . In
OI26 and OI64 cells, the synthesis of BiP was increased by ascorbate
treatment. Ascorbate addition rapidly increases the rate of type I
collagen production by human fibroblast cultures
(26) . Since
BiP levels are elevated when abnormal proteins accumulate in the
endoplasmic reticulum
(16) it is likely that BiP induction
reflected the presence of increased amounts of the mutant protein in
ascorbate-treated cells. In contrast to defects in the
carboxyl-terminal propeptide, mutations in the triple-helical domain of
type I procollagen chains that delay folding of that region do not
result in binding of the abnormal molecules to BiP or in increased
synthesis of BiP even though the molecules can be quantitatively
retained in the endoplasmic reticulum
(50) .
1(I) carboxyl-terminal propeptide mutations in these patients
delay, but do not prevent, the assembly of the mutant pro
1(I) into
procollagen trimers. The combination of endoplasmic reticulum
degradation of mutant pro
1(I) chains and further intracellular
degradation of malfolded mutant-normal molecules results in a collagen
biosynthetic deficiency much more severe than that produced by a null
allele. Since collagen matrix deposition is a cooperative process
(26) , where a pre-existing matrix induces more efficient
collagen deposition, the effect of the collagen deficiency on matrix
formation is amplified. In both OI26 and OI64, some mutant-containing
trimers escape intracellular degradation, and these
post-translationally overmodified collagens are incorporated into the
extracellular matrix. The incorporation of these abnormal molecules
into collagen fibers may disturb matrix architecture and thus
contribute to the severe phenotype.
1(I).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.