From the Department of Biochemistry, University of Hong Kong, Hong Kong SAR, China
Received for publication, April 18, 2000, and in revised form, November 26, 2000
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ABSTRACT |
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Schmid metaphyseal chondrodysplasia
results from mutations in the collagen X (COL10A1)
gene. With the exception of two cases, the known mutations are
clustered in the C-terminal nonhelical (NC1) domain of the collagen X. In vitro and cell culture studies have shown that the NC1
mutations result in impaired collagen X trimer assembly and secretion.
In the two other cases, missense mutations that alter Gly18
at the Collagen X is the most abundant extracellular matrix component
synthesized by hypertrophic chondrocytes during the transition from
cartilage to bone in endochondral ossification. It is classified as a
short-chain nonfibrillar collagen and consists of three distinct protein domains; a central, short triple helical COL1 domain (463 amino
acids) flanked by a small N-terminal nonhelical NC2 domain (38 amino
acids) and a larger, more conserved, nonhelical C-terminal NC1 domain
(161 amino acids). The chains are synthesized with an N-terminal signal
peptide, which is proteolytically removed from the pre- Mutations in the collagen X gene (COL10A1) result in Schmid
metaphyseal chondrodysplasia
(SMCD),1 an autosomal
dominant skeletal disorder characterized by short to normal stature,
bowed legs, coxa vara, and flaring of the metaphyses of long
bones. To date, all reported SMCD mutations in COL10A1, except two, are localized to the C-terminal NC1 domain of the protein
(see review by Chan and Jacenko, Ref. 3). These NC1 domain mutations
(COL10-NC1m) were proposed to affect the initial stages of
the folding and chain assembly of collagen X (4, 5), hindering
nucleation of the triple helix and, therefore, impairing secretion of
collagen X trimers.
The disruption of collagen X assembly as a mechanism underlying SMCD is
supported by studies using in vitro transcription and
translation of mutant and normal COL10A1 cDNAs in a cell-free system (1). In this system, the inability of COL10-NC1m
chains to trimerize was clearly demonstrated. Expression of
COL10-NC1m cDNAs in cells resulted in poor expression
levels, with little or no secretion of the mutant chains (6). Together,
these results indicate that the inability of mutant collagen However, there is increasing evidence suggesting that other mechanisms
could also underlie SMCD. For example, trace amounts of heterotrimers
can be detected during cell-free coexpression of normal and mutant
chains with amino acid substitutions in the NC1 domain (6). In
addition, the clustering of COL10A1 mutations in the NC1
domain, the absence of null mutations and the autosomal dominant
inheritance of SMCD in patients are more consistent with a
dominant-negative mechanism. That is, the expression of mutant collagen
X chains could impact on the assembly and secretion of normal chains as
well. We (8) and others (9, 10) have recently demonstrated through
in vitro approaches that a dominant-negative mechanism could
also underlie some SMCD mutations. Molecular modeling of the NC1 domain
based on the crystal structure of ACRP30 (9) showed that NC1 amino acid
substitutions in SMCD are localized to two regions of the folded domain
and that these mutations may not totally abolish the ability of the
mutant chains to form trimers.
Recently, two missense mutations in SMCD patients were identified in
the putative signal peptide of the molecule at nucleotide positions 148 and 149 (G148A and G149A)2
(11), altering Gly18 at the Construction of
PCR reactions were performed in 50 µl of 10 mM Tris/HCl,
pH 8.0, containing 1.5 mM MgCl2, 0.2 mM dNTPs, and 0.75 µM of each of the primers.
The reactions were carried out using the GeneAmp PCR system 2400 (Perkin-Elmer). Cycle one was performed at 96 °C for 2 min, 60 °C
for 1 min, and 72 °C for 1 min and then followed by 25 cycles at
96 °C for 20 s, 60 °C for 20 s, and 72 °C for 30 s. The reaction was terminated at 72 °C for 1 min. The
recombinant fragment was purified and digested with KpnI and
XhoI. A 337-base pair fragment containing the mutation was
cloned into appropriate sites of pTM1 (14). Two positive clones,
pTM1-G18R and pTM1-G18D were selected for sequencing to ensure that the
correct mutation was introduced and that there was no PCR error within
the 337-base pair KpnI/XhoI fragment. A
full-length cDNA was constructed by cloning a 3-kilobase fragment
generated by XhoI and SalI digestion of
pTM1-h10wt (6) into the XhoI site of pTM1-G18R or
pTM1-G18D.
In Vitro Cell-free Transcription and Translation--
Cell-free
transcription/translation was performed as described previously (1, 6),
in the presence or absence of canine pancreatic microsomal membranes
(Promega), using the TNT T7 polymerase-coupled transcription and
translation reticulocyte lysate system (Promega). The reactions were
carried out in a total volume of 12.5 µl and labeled with 10 µCi of
translation grade L-[35S]methionine (1000 Ci/mmol, PerkinElmer Life Sciences). To determine whether the mutant
chains can form heterotrimers with normal chains, cotranslation
experiments using an assembly-competent helix deletion
Coupled transcription and translation experiments were carried out at
30 °C for 90 min using a total of 100 ng of purified plasmids. When
required, the microsomes were separated from the reticulocyte lysate by
centrifugation and analyzed separately. To assay for the location of
the translation products, proteins external to the isolated microsome
vesicles were digested with trypsin and chymotrypsin at a final
concentration of 50 µg/ml each (15).
Samples for SDS-PAGE analysis were dissolved or mixed with 40 µl of
sample loading buffer (10 mM Tris/HCl, pH 6.8, containing 2% SDS (w/v), 2 M urea, 10 mM dithiothreitol,
and 20% sucrose (w/v)), and incubated at room temperature for 10 min
prior to electrophoresis on a 7.5% SDS-polyacrylamide gel.
Electrophoretic conditions and fluorography of radioactive gels have
been described previously (16). Radioactive bands were imaged and
quantified using a PhosphorImager (Molecular Dynamics).
Extraction of Proteins from Microsomal Vesicles with Sodium
Carbonate--
The sodium carbonate extraction procedure of Fujiki
et al. (17) was used, with minor modifications, to determine
whether the mutant chains remained as an integral component of the
lipid bilayer. Following cell-free synthesis, microsome vesicles were separated from the reticulocyte lysate by centrifugation at 14,000 rpm,
4 °C for 15 min, and washed with 0.5 ml of KHM buffer (110 mM KOAc, 20 mM HEPES, pH 7.2, 2 mM
Mg(OAc)2). Unbound proteins were extracted from the
microsome vesicles with 0.1 M
Na2CO3, pH 11.5, on ice for 30 min and then
centrifuged as described above. The lipid bilayer was washed with 0.5 ml of KHM buffer. The supernatants were neutralized to pH 7.5 with 1 M HCl and the extracted proteins precipitated with 75%
(v/v) ethanol. Proteins in both the
Na2CO3-soluble, and the membrane fractions were
dissolved in 40 µl of sample loading buffer, as described above,
prior to analysis on a 7.5% SDS-polyacrylamide gel.
Expression of Normal and Mutant Collagen X in Mammalian
Cells--
COL10A1 cDNA constructs were expressed in a
rat osteogenic sarcoma cell line, UMR 106-01 (American Type Culture
Collection, ATCC-CRL 1661), using the vaccinia-driven T7 bacteriophage
expression system previously described (6). Pulse-chase analysis was
used to study collagen X secretion and degradation. Transfected cells in 6-well plates were preincubated in 1 ml of Dulbecco's modified Eagle's medium without L-methionine (Life Technologies,
Inc.) for 1 h, then pulse-labeled for 2 h with 150 µCi of
L-[35S]methionine (1110 Ci/mmol, PerkinElmer
Life Sciences). For secretion studies, labeled cells were chased with
fresh medium containing excess unlabeled methionine over a period of
2 h. Collagen X from the cell and medium fractions was recovered
by immunoprecipitation using a specific antibody (gift from Dr. Olena
Jacenko) (7) and protein G-Sepharose (Roche Molecular Biochemicals) and
analyzed by 7.5% (w/v) SDS-PAGE. To study the formation of stable
triple helical molecules, aliquots of the cell fractions were subjected to limited pepsin digestion (16).
For analysis of intracellular collagen X degradation, cells were
treated with either a proteasome inhibitor (5 µM
clasto-lactacystin Mutations at Gly18 Prevent Cleavage of the Signal
Peptide--
Sequencing confirmed that the G148A and G149A mutations
had been introduced in the cDNA constructs (Fig.
1). In vitro transcription and
cell-free translation demonstrated that the mutant cDNAs were translated into pre-
Translation in the presence of microsomes promoted trimer association
for wt as well as G18R and G18D translation products (Fig.
2a, lanes 4-6). The homotrimers for G18R and
G18D chains migrated with a slightly increased molecular size,
suggesting that the mutant trimers contain chains with an unprocessed
signal peptide. When microsomes were separated from the reticulocyte lysate prior to analysis, both the mutant and wild-type translation products were found predominately in the microsome fraction (Fig. 2b). The radioactive trimer and monomer components of the
translation products were quantified using phosphor imaging. Trimer
quantity expressed as percentage of the total translation product
(trimers + monomers) was used as a measure of the efficiency of trimer assembly. The percentages of mutant pre- Mutant Chains Can Associate in Vitro via the NC1 Domain to Form
Heterotrimers with Signal Peptide-cleaved Chains--
Because SMCD is
an autosomal dominant disorder, an important consideration is whether
the mutant pre-
All translation products were localized to the microsomal membrane
fraction (Fig. 3). Cotranslation of the
SPm or wt transcripts with the helix
To assess trimer assembly efficiency, the trimer bands,
Mutant Chains with Uncleaved Signal Peptide Behave as Integral
Membrane Proteins--
Treatment of membrane vesicles at high pH
solubilizes nonmembrane proteins and leaves only integral membrane
proteins associated with the sedimentable lipid bilayers (18).
Following cell-free synthesis in the presence of microsomes, the
vesicles were treated with 0.1 M
Na2CO3, pH 11.5 and centrifuged to determine
whether the mutant chains remain integrated with the lipid bilayer.
This approach revealed that the majority of the monomers and trimers containing uncleaved SPm chains associate primarily with
the membrane pellet (Fig. 4a). Thus it appears that, in the absence of cleavage by signal peptidase, mutant pre- Mutations in the Signal Peptide Cleavage Site Impair Stable Triple
Helix Formation and Secretion from Cells--
The in vitro
cell-free system does not promote stable triple helix formation because
of the lack of appropriate post-translational modifying enzymes (6). To
address the issue of helix formation, secretion, and the fate of the
mutant chains, normal and mutant plasmids were transiently transfected
into cells. Expression studies in UMR-106-01 cells, a
collagen-producing osteoblast-like cell line, indicated that the wt
chains assembled as trimers (Fig. 5),
forming stable triple helical molecules (Fig.
6), which were secreted efficiently by
the cells (Fig. 5). In contrast, whereas the SPm chains
(G18R and G18D) associated into homotrimers intracellularly (Fig. 5),
they were sensitive to pepsin digestion (Fig. 6), demonstrating a lack
of helical configuration. In Fig. 6, the pepsin-resistant Unsecreted Mutants Chains Are Degraded Intracellularly in UMR106-01
Cells--
Even though SPm trimers or chains were not
secreted, the intracellular concentration decreases over the chase
period, suggesting that the mutant chains are being degraded
intracellularly (Fig. 5). To determine the likely degradative pathway
involved, cells were treated from the preincubation period with either
a proteasome inhibitor (clasto-lactacystin Signal peptide sequences of secreted proteins share common
features that include a net positive charge at the N terminus, a
central hydrophobic region, and a C-terminal region with small nonpolar
amino acids at positions Cell-free translations in the presence of microsomal membranes will
allow the interaction of the signal recognition particle with the newly
synthesized polypeptides, followed by docking and translocation into
the microsome vesicles, where cleavage of the signal peptide will occur
(20). This system was utilized to test whether the G18R and G18D
mutations in COL10A1 alter these events. When microsomes
were separated from the reticulocyte lysate following translation, the
translation products of wt, G18R, and G18D were found associated with
the microsome vesicles. Only small amounts of translation product
remained in the reticulocyte lysate thus showing no indication of a
preferential retention of mutant chains in the reticulocyte lysate when
compared with wt translation. This suggests that interaction with the
signal recognition particle and targeting to the translocons are not
affected, as the mutant chains are delivered to the microsome vesicles
during cell-free synthesis. Translocation into the lumen of the
microsome vesicles was also not affected because we were able to
demonstrate that the translation products are inside the microsomes
with a protease protection assay using exogenous trypsin and
chymotrypsin (data not shown). As the exogenous proteases cannot enter
the vesicle, the presence of intact pre- Our in vitro data suggest that signal peptidase failed to
recognize or cannot cleave the mutant sites. With no cleavage, the mutant pre- As shown by pulse-chase analysis in transiently transfected cells,
impaired secretion of the mutant collagen X chains is clearly evident
in the two cases reported here. That the nonsecreted SPm
chains are targeted for intracellular degradation is supported by the
continued decline of intracellular levels of SPm chains
over the chase period in the absence of secretion. The precise
mechanism(s) for this intracellular degradation is not clear at this
stage. Our preliminary study using inhibitors of lysosomal and
proteasome pathways suggests that both degradative pathways may be
involved. However, the more significant level of protection from
degradation shown by the proteasome inhibitor, clasto-lactacystin The ability of SPm chains to associate into homo- and
heterotrimers contrasts with our previous reports on SMCD mutations in the NC1 domain, where in vitro association of NC1 mutant
collagen X chains was impaired (1, 6). Although SPm chains
do associate into homo- and heterotrimers, there appears to be some
constraint on the nature of the trimers that are formed. In cell-free
translation experiments expressing SPm or wt chains, we
noted that the assembly of SPm pre- Collagen X triple helix formation is initiated via nucleation and
extension following the assembly of the C-terminal NC1 domains, so a
possible reason why heterotrimers are preferred is the physical constraint imposed by the anchoring of SPm chains to the
membrane. For the formation of homotrimers, only chains that are close
enough will be able to associate; however, this constraint will have a
lesser effect on the formation of heterotrimers containing two
SPm chains, and be smallest for heterotrimers containing
one SPm chain. We reason that whereas SPm
chains can associate into homo- and heterotrimers via the NC1 domain
in vivo, folding of SPm chain-containing
molecules into a triple helix is likely to be inefficient because the N
termini will be anchored to the ER membrane. A lack of stable triple
helical structure was demonstrated for SPm homotrimers by
the absence of intracellular pepsin resistance SPm collagen
X molecules in transfected cells.
The precise impact on the level of available normal molecules in
vivo is not clear. In transient transfected cells, it is difficult
to make reliable assessments of the yield of trimers because of the
variable transfection efficiency and the added complication of
intracellular degradation. However, we noted a reduction of ~60% in
the amount of collagen X molecules secreted in a cotransfection
experiment (data not shown), supporting a dominant-negative effect of
SPm chains that could affect the level of normal collagen X
molecules. We propose that SPm chains anchored to the
translocons impair triple helix formation, and the unfolded chains are
removed from the ER by retrograde translocation and targeted for
degradation via the proteasome pathway (22, 23).
It would appear that although the signal peptide mutations and the NC1
SMCD mutations reported previously (6) are at opposite ends of the
molecule and have different molecular effects on collagen X
biosynthesis, the net phenotype is similar, with intracellular retention and degradation of mutant collagen X. The fact that both
types of mutations should give rise to SMCD is consistent with a
similar molecular consequence for the mutations. Whereas an apparent
50% reduction in collagen X level has been shown in one patient to be
a mechanism for SMCD (7), this may not be the only mechanism. The
current finding together with other in vitro studies (8-10)
suggest that a dominant-negative effect may be a more common mechanism.
Intracellular retention and active degradation processes of collagen
molecules containing mutant chains could lead to deregulated cellular
metabolism, altering cell differentiation, proliferation, and
apoptosis. These are the critical cellular events in endochondral
ossification that induce precise alterations in the ECM. An imbalanced
ECM not only compromises matrix integrity but also alters cell-matrix
interactions, eliciting aberrant cellular responses with unknown
consequences. Further understanding of the impact of SPm
and COL10-NC1m SMCD mutations on the metabolism and
differentiation of chondrocytes in the growth plate will need to await
investigation of these mutations in animal models.
1 position of the putative signal peptide cleavage site were
identified (Ikegawa, S., Nakamura, K., Nagano, A., Haga, N., and
Nakamura, Y. (1997) Hum. Mutat. 9, 131-135). To study their impact on collagen X biosynthesis using in vitro
cell-free translation in the presence of microsomes, and cell
transfection assays, these two mutations were created in
COL10A1 by site-directed mutagenesis. The data suggest that
translocation of the mutant pre-
1(X) chains into the microsomes is
not affected, but cleavage of the signal peptide is inhibited, and the
mutant chains remain anchored to the membrane of microsomes. Cell-free
translation and transfection studies in cells showed that the mutant
chains associate into trimers but cannot form a triple helix. The
combined effect of both the lack of signal peptide cleavage and helical configuration is impaired secretion. Thus, despite the different nature of the NC1 and signal peptide mutations in collagen X, both
result in impaired collagen X secretion, probably followed by
intracellular retention and degradation of mutant chains, and causing
the Schmid metaphyseal chandrodysplasia phenotype.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1(X) chains
during biosynthesis (1, 2). In collagen biosynthesis, once the
polypeptide chains are translocated into the lumen of the ER, the
chains of the trimer associate via their C-terminal globular domains.
This allows nucleation and folding of the triple helix to occur
sequentially from this end of the molecule.
1(X)
chains to assemble can lead to their intracellular retention and
subsequent degradation. As a consequence, in SMCD, collagen X in the
matrix could be reduced, potentially to 50% of normal levels, because of haploinsufficiency. The absence of detectable mutant COL10A1 mRNA in extracts of growth plate cartilage from a SMCD patient with
a nonsense mutation in the NC1 domain, probably caused by nonsense-mediated mRNA degradation, is consistent with
haploinsufficiency as a mechanism in SMCD (7).
1 position of the signal
peptide cleavage site (2). The molecular consequences of these
mutations on collagen X biosynthesis are unknown but the predicted
consequence is that secretion of the molecules will be impaired as the
signal sequence plays a key role in this process (12). We have aimed to
obtain a better understanding of the molecular mechanisms underlying
SMCD in these patients by studying the impact of these two signal
peptide mutations (SPm) on collagen X biosynthesis and
assembly. Our data show that the G148A and G149A mutations do result in
impaired secretion of collagen X.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1(X) Signal Peptide Mutations--
Overlap
extension PCR (13) was used to reproduce the two human missense
mutations in collagen X identified by Ikegawa et al. (11).
Both mutations altered Gly18 at the
1 position of the
putative signal peptide cleavage site. These were the G148A and G149A
nucleotide substitutions which change the codon for Gly18
to codons for Arg (G18R) and Asp (G18D), respectively. To create these
changes, 5 ng of a plasmid pTM1-h10wt (6) containing a full-length
human collagen X cDNA was used as a template for the primary rounds
of PCR with primer pairs: sense, pTM1-1; antisense, G18R-2 or G18D-2;
and sense, G18R-1 or G18D-1; antisense, HX10 (Table
I) to generate independent fragments with
overlapping sequences. Second round PCR reactions were carried out with
primers pTM1-1 and HX10 and 5 ng of the two corresponding overlapping fragments from the primary PCR reactions.
Primers for site-directed mutagenesis using strand overlap
extension PCR
1(X) reporter
construct, helix
(6), were performed with either the G18R or G18D
mutant constructs.
-lactone, CalBiochem) or an ER-Golgi
transport inhibitor for the endosome/lysosome pathway (1 µg/ml
brefeldin A, Roche Molecular Biochemicals) throughout the
preincubation, pulse labeling and chase periods. Cells were harvested
after a 1-h chase, and collagen X was recovered by immunoprecipitation.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1(X) chains with the same molecular size as the
wild type (Fig. 2a, lanes
1-3). The lower molecular weight band in lanes 1-3
represents translation products initiated from the second methionine
residue. In the presence of microsomes (Fig. 2a, lanes
4-6), the initiation of translation was more accurate, with only
a single translation product for each of the constructs. A reduction in
the molecular size can be clearly demonstrated when wt pre-
1(X)
chains (Fig. 2a, lane 1) were processed to
1(X) chains (Fig. 2a, lane 4). In contrast,
the mutant translation products remained as pre-
1(X) chains with no
apparent reduction in the molecular size (Fig. 2a,
lanes 5 and 6).
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Fig. 1.
Sequences of mutant clones prepared by
site-directed mutagenesis. Sequencing gel showing the wild-type
and mutated sequences (G148A and G149A) created by overlapping
extension PCR (see "Experimental Procedures" for details). The base
substitution is indicated by white arrowheads. The
corresponding coding strand sequences and the deduced amino acid
sequence resulting from the mutations are shown in italics
designating the abnormal nucleotide codon sequence. The underlined
sequence represents the region of sequence listed on the corresponding
sequencing gel. The open arrow marks the putative signal
peptide cleavage site.
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Fig. 2.
Cell-free translation of normal and mutant
collagen X chains. The normal and mutant cDNA constructs were
transcribed and translated in a coupled cell-free translation system
(see "Experimental Procedures" for details). The resultant
[35S]methionine-labeled products were analyzed on a 7.5%
SDS-polyacrylamide gel. Translations were carried out either in the
presence (+) or the absence ( ) of microsomal membranes. In
panel a, the total translation mix was analyzed, whereas in
panel b, the reticulocyte lysate (L) and
microsomal membrane (M) fractions were separated prior to
electrophoresis. The identity of the lanes, the migration positions of
pre-
1(X) chains containing the signal sequence,
1(X) chains after
cleavage of this sequence, and trimeric
1(X)3 components
are indicated.
1(X) chains that assembled into homotrimers were determined to be 20 ± 4.5%
(n = 3) for G18R, 27 ± 5.5% (n = 3) for G18D, and 40 ± 6.5% (n = 3) for wild type.
1(X) chains are able to associate into heterotrimers
with normal
1(X) chains. To address this issue, reporter (helix
)
and SPm plasmids were cotranscribed and translated in the
presence of microsomes. The helix
plasmid is an
1(X) construct,
which contains a shortened triple helical domain but has normal NC1 and
NC2 domains, and a normal signal peptide sequence. This construct has
been used previously to generate a protein reporter, which allows the ability of mutant chains to assemble into heterotrimers in
cotranslation experiments to be assessed (6). If the SPm
chains can assemble with the helix
chains, the stoichiometry of
these complexes can readily be determined by assessing the electrophoretic migration of the multimers.
transcripts showed
that in addition to the trimeric components of each product, two
additional intermediate multimeric bands, labeled as a and
b, were also observed (Fig. 3, lanes 8-10). This
is consistent with band a containing heterotrimers of two
pre-
1(X) or wt chains and one helix
1(X) chain, and band
b containing heterotrimers of one pre-
1(X) or wt chain and two
helix
1(X) chains. When compared with the cotranslation products
of wt and helix
transcripts (Fig. 3, lane 8), bands a and b in lanes 9 and 10 (Fig.
3) migrated with a slight increase in their apparent molecular
sizes.
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Fig. 3.
Formation of heterotrimers in cell-free
cotranslations of normal and mutant collagen X. The reporter
plasmid (helix ) was cotranslated with normal
(wt) or mutant plasmids (G18R, G18D) in the presence of
microsomal membranes to promote in vitro trimer formation
(see "Experimental Procedures" for details). The samples were
separated into reticulocyte lysate and membrane fractions prior to
analysis on a 7.5% SDS-polyacrylamide gels. The identities of the
monomers and trimers are indicated. Bands labeled
(a) represent heterotrimers comprised of two full-length
molecules derived from the corresponding plasmid indicated for each
lane and one molecule from the helix
plasmid, whereas
bands labeled (b) are comprised of one molecule
from the full-length plasmid indicated by the lane and two molecules
from the helix
plasmid.
1(X)3/pre-
1(X)3, band a,
band b, and helix
1(X)3 in lanes
8-10, were quantified using phosphor imaging to estimate trimer
assembly preference (Table II). The data
showed that relative to the formation of helix
1(X)3,
the formation of trimers containing one or more SPm chains
in cotranslations with helix
is less efficient compared with
cotranslations of wt with helix
. In cotranslational experiments of
SPm and helix
chains, the assembly of heterotrimers
containing one or two SPm chains appears to be more favored
then the assembly of SPm homotrimers.
Relative trimer concentrations formed in cell-free co-translation
experiments
1(X)3/pre-
1(X)3, band a, band b, and helix
1(X)3, in lanes 8-10 of Figure 3 were quantified using
phosphor imaging to estimate trimer assembly preference. For each
co-translation experiment, the values were calculated relative to helix
1(X)3, which was set to unity. The values represent the
average of three experiments.
1(X) chains remain anchored to the translocons and behave
as integral membrane components. We observed a similar pattern for
cotranslation products of trimers containing SPm chains
with an uncleaved signal peptide, which were preferentially retained in
the membrane-bound fraction following treatment with Na2CO3 (Fig. 4b). These included
mutant pre-
1(X)3 homotrimers and the heterotrimers
labeled a and b described above (Fig.
4b, lanes 2 and 3). In contrast, the
homotrimers of the reporter (helix
), wt chains, and heterotrimers of
helix
and wt chains, were found predominantly in the soluble
fraction (Fig. 4b, lanes 4-6).
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Fig. 4.
Extraction of microsomes with sodium
carbonate. The wt, helix , and mutant cDNA constructs were
transcribed and translated in a coupled cell-free translation system
supplemented with microsomal membranes (see "Experimental
Procedures" for details). Panel a represents individual
translation products, and panel b shows products from
cotranslation experiments. Following the completion of translation, the
microsomes were isolated and treated with 0.1 M
Na2CO3 (18). The [35S]methionine
labeled translation products in the membrane-bound fractions
(MB) and the soluble fractions (S) from single or
cotranslation experiments were analyzed on a 7.5% SDS-polyacrylamide
gel. The identity of the lanes, the migration positions of pre-
1(X)
chains containing the signal sequence,
1(X) chains after cleavage of
this sequence, and trimeric
1(X)3 components are
indicated. The identities of bands labeled a and
b are as described in Fig. 3.
1(I) and
2(I) chains are endogenous collagen I expressed by the UMR106-01
cells and were present in control untransfected cells that were only
infected with the vTF7-3 virus. Secretion of the mutant chains was also
severely impaired as no immunoprecipitable products could be detected
in the medium fractions over the 2-h chase period (Fig. 5).
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Fig. 5.
Retention of SPm collagen X in
transfected UMR106-01 cells. Transfections and biosynthetic
labeling were performed as described under "Experimental
Procedures." Cells were pulse-labeled with
L-[35S]methionine for 2 h and chased for
0-2 h as indicated. Collagen X from cell and medium fractions was
recovered by immunoprecipitation and analyzed by SDS-PAGE. The
migration position of 1(X) trimers (
1(X)3)
and monomers (
1(X)) are indicated. Pepsin treated
collagens from human skin fibroblasts are included as standard
(std) molecular size references. The positions of the
1(I) and
2(I) chains of collagen I, and the trimer of unreduced
collagen III are shown.
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Fig. 6.
SPm collagen X homotrimers failed
to form stable triple helical molecules in transfected UMR106-01
cells. Transfections and biosynthetic pulse labeling with
L-[35S]methionine were performed as described
under "Experimental Procedures." Cell fractions after 1 h of
chase were harvested. Protein fractions were analyzed by SDS-PAGE with
(+ pepsin) or without ( pepsin) digestion by
pepsin. The position of collagen X trimers
(
1(X)3), and the pepsin-resistant
1(I) and
2(I) chains of collagen I and
1(X)P of collagen X are
indicated. The bands labeled as procollagen I
chains represent procollagen I
-chains and various processed
intermediates containing N- or C-propeptides.
-lactone) or a
vesicular transport inhibitor for the endosome/lysosome pathway
(brefeldin A). The concentration of mutant collagen X was compared
after 1 h of the chase period. Cells transfected with the G18R or
G18D constructs showed a similar response (Fig.
7). Incubation with brefeldin A resulted
in a 4-fold increase in intracellular mutant chains compared with
untreated cells and an approximately 8-fold increase was observed in
the presence of clasto-lactacystin
-lactone.
View larger version (51K):
[in a new window]
Fig. 7.
Degradation of SPm collagen X
chains in UMR106-01 cells is prevented by proteasome and vesicular
transport inhibitors. Transfections and biosynthetic pulse
labeling with L-[35S]methionine were
performed as described under "Experimental Procedures." Cells were
treated throughout the preincubation, pulse, and chase periods with (+)
or without ( ) inhibitors for intracellular protein degradation; 5 µM clasto-lactacystin
-lactone
(Lac) for the proteasome, or 1 µg/ml brefeldin A
(BFA) for the endosome/lysosomal pathways. Cells were
harvested after 1 h of chase and collagen X recovered by
immunoprecipitation and analyzed on a 7.5% SDS-polyacrylamide gel. A
standard (std) of pepsin-treated collagens from human skin
fibroblasts was included as a molecular size reference. The position of
the
1(I) and
2(I) chains of collagen I and the trimer of the
unreduced collagen III are shown. Radioactive bands were imaged and
quantified using a phosphor imager. The quantified value for the bands
indicated as collagen X trimers and monomers were combined as a measure
of total collagen X present in the cell fractions. The degree of
protection against intracellular degradation by the addition of
inhibitors was estimated relative to the corresponding untreated
cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and
3 from the cleavage site (12). The
1 position of the putative signal peptide in collagen X is
Gly18 (2). In view of the common requirement for signal
peptide sequences, the G18R and G18D mutations are significant,
changing the nonpolar glycine residue at the
1 position to the highly charged arginine or aspartate residues. Based on reports of signal sequence mutations in other secretory proteins (15, 19), the most
likely consequence of these mutations is impaired cleavage of the
signal peptide and inhibition of collagen X secretion.
1(X) chains implies that
they are inside the microsomes (15).
1(X) chains would not be released from the translocons and remain anchored to the microsomal membranes. The molecular consequence of mutations involving the signal peptide has not been
characterized in the collagen family of proteins or other matrix
components. However, there are a number of reports characterizing signal peptide mutations in secreted proteins that are associated with
several diseases. For example, in human coagulation factor X deficiency
(15) and diabetes insipidus (19, 21), signal peptide mutations have
been identified in factor X and prepro-vasopressin, respectively. For
these nonmatrix-secreted proteins, failure to cleave the signal peptide
resulted in intracellular retention of the mutant products, with a
severe reduction in the amount of circulating protein and protein activity.
-lactone, suggests this could be the
major degradative pathway.
1(X) chains into
homotrimers was less efficient relative to the assembly of wt
homotrimers. When cotranslated with helix
1(X) chains,
SPm chains showed a preference for assembly into
heterotrimers over SPm homotrimers, with heterotrimers
containing one SPm chain most favored.
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ACKNOWLEDGEMENT |
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The authors thank Dr. David Smith for a critical reading of the manuscript.
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FOOTNOTES |
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* This work was funded in part by a Committee on Research Conference grant from the University of Hong Kong and RGC Grant HKU 7285/99M from Hong Kong (to D. C.).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: Dept. of Biochemistry,
University of Hong Kong, Li Shu Fan Bldg., 5 Sassoon Rd., Hong Kong.
Fax: 852-28551254; E-mail: chand@hkusua.hku.hk.
Published, JBC Papers in Press, December 13, 2000, DOI 10.1074/jbc.M003361200
2 Positions of nucleotides and amino acids are numbered from the start of transcription (24) and translation (2), respectively.
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ABBREVIATIONS |
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The abbreviations used are: SMCD, Schmid metaphyseal chondrodysplasia; COL10-NC1m, a collective term for mutations within the collagen X NC1 domain; PAGE, polyacrylamide gel electrophoresis; SPm, signal peptide mutant; wt, wild type; PCR, polymerase chain reaction; ER, endoplasmic reticulum.
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