From the Orthopaedic Molecular Biology Research Unit, Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, Victoria 3052, Australia
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ABSTRACT |
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Collagen X is a short-chain homotrimeric collagen
expressed in the hypertrophic zone of calcifying cartilage. The
clustering of mutations in the carboxyl-terminal nonhelical NC1 domain
in Schmid metaphyseal chondrodysplasia (SMCD) suggests a critical role
for NC1 in collagen X structure and function. In vitro
collagen X DNA expression, using T7-driven coupled transcription and
translation, demonstrated that although The collagens are an extensive protein family defined by the
characteristic triple helix motif formed from three The critical importance of the NC1 domain in collagen X molecular
assembly and function has also been highlighted by the characterization of mutations in patients with Schmid metaphyseal chondrodysplasia (SMCD) (MIM 156500) (12, 13). Although these mutations include amino
acid substitutions, nonsense mutations and deletions resulting in
predicted protein truncation, in vitro expression, and
assembly studies suggested that a common molecular defect is that the
SMCD mutations compromise NC1 association and prevent the formation of
stable collagen X homotrimers (9, 10). These data suggested that SMCD
resulted from a functional haploinsufficiency of assembly-competent Collagen X interactions in in vitro expression studies have
been assessed by their stability to the denaturing conditions of
SDS-PAGE, conditions under which assemblies of other collagen types are
not stable. To overcome the obvious limitations of assessing potentially biologically important interactions in this harsh system,
we have developed a competition-based approach, which is designed to
allow the assessment of such SDS-stable assemblies, as well as weaker
interactions that may interfere with assembly but not produce
SDS-stable final products. We have used this in vitro
co-expression and assembly assay to examine in detail the molecular
consequences of SMCD mutations and to further dissect the role of NC1
domains in the assembly process. Our studies show that Construction of
Antisense primers, ter@610, ter@650, and ter@666 (Table
I), were designed to incorporate a stop
codon at the appropriate position with an additional overhang sequence
to include an AflII site for the ease of cloning. These
primers were paired with a sense primer, BX1 (Table I), to amplify
fragments of 118, 331, and 379 base pairs using a normal human
full-length collagen X cDNA construct (pTM1-h10wt) as the template
(9). The 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
resultant fragments were digested with EcoNI and
AflII. EcoNI is an endogenous site within the PCR
fragment, whereas AflII is an introduced site in the PCR product 3' to the termination codon. The appropriate restriction fragments were purified and cloned into the unique EcoNI and
AflII sites of pTM1-h10wt. In the construct with a
termination codon at position 650, the codon for the amino acid at
position 649 was changed from CAG (Gln) to CCT (Pro) to facilitate
cloning. All constructs were sequenced to ensure that there were no PCR errors.
Construction of Specific Deletions within the NC1 Domain--
To
explore the role of specific NC1 domains in assembly, three in-frame
deletions of the human collagen X NC1 domain were produced using
overlap extension PCR (10, 17). These deletions correspond to regions
A, B, and C in Fig. 1 (see "Results
and Discussion"). The mutant primer sets and their relative positions are shown in Table I. Primer sets A In Vitro Cell-free Transcription and Translation--
Cell-free
transcription/translation was performed as described previously in the
presence or absence of canine pancreatic microsomal membranes (Promega)
using the TNT T7 polymerase coupled transcription and translation
system (Promega) (9, 10). The conditions were modified by reducing the
amount of added plasmid DNA (25-50 ng), and the reaction volume of
6.25 µl was labeled with 5 µCi of translation grade
L-[35S]methionine (1000 Ci/mmol, NEN Life
Science Products). Co-translation experiments were performed in the
presence of microsomal membranes by aliquoting 5.25 µl from a master
mix containing 25 ng/5.25 µl of control plasmids, which were either
the wt full-length Although the characterization of collagen X mutations in SMCD
clearly demonstrates that it is an important component in cartilage development and growth, the precise molecular role of type X in the
hypertrophic cartilage extracellular matrix remains elusive (12).
Detailed ultrastructural analysis of cartilage from
Col10a1-null mice revealed subtle changes in the normal
distribution of proteoglycans and cartilage matrix vesicles in the
growth plate, suggesting that the collagen X matrix plays a role in the
organization of cartilage matrix structure during remodeling and
mineralization (18). The molecular interactions that initiate collagen
X assembly and facilitate the formation of the functional extracellular
assemblies are not yet well characterized. The definition of the
effects of SMCD mutations on protein assembly in vitro has
provided information on homotrimer assembly and identified the NC1
domain to be critical for the initiation of assembly (9, 10), but the
precise motifs within the NC1 domain that drive this process are not
known. Furthermore, because SMCD is a heterozygous dominant disorder,
important information is also missing about the effect of mutant
collagen X expression on the assembly and function of the normal
collagen X expressed in the growth plate cartilage.
To explore the consequences of NC1 mutations on collagen X homotrimer
assembly and assembly with normal chains to form heterotrimers, a range
of SMCD mutations was studied by co-expression and assembly in
vitro. In addition to point mutations G618V and Y598D (9, 10),
which are likely to be expressed at the protein level in vivo, mutations were generated, that resulted in the in
vitro production of truncated 1(X) containing normal NC1
domains can form electrophoretically stable trimers, engineered SMCD
NC1 missense or premature termination mutations prevented the formation of electrophoretically stable homotrimers or heterotrimers when co-expressed with normal
1(X). To allow the detection of more subtle
interactions that may interfere with assembly but not produce SDS-stable final products, we have developed a competition-based in vitro co-expression and assembly approach. Our studies
show that
1(X) chains containing SMCD mutations reduce the
efficiency of normal
1(X) trimer assembly, indicating that
interactions do occur between mutant and normal NC1 domains, which can
impact on the formation of normal trimers. This finding has important implications for the molecular pathology of collagen X mutations in
SMCD. Although we have previously demonstrated haploinsufficiency as
one in vivo mechanism (Chan, D., Weng, Y. M., Hocking,
A. M., Golub, S., McQuillan, D. J., and Bateman, J. F. (1998)
J. Clin. Invest. 101, 1490-1499), the current study
suggests dominant interference is also possible if the mutant protein
is expressed in vivo. Furthermore, we establish that a
conserved 13-amino acid aromatic motif (amino acids 589-601) is
critical for the interaction between the NC1 domains, suggesting that
this region may initiate assembly and the other NC1 mutations
interfered with secondary interactions important in folding or in
stabilizing the assembly process.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-chain subunits and the ability to associate into precise extracellular supramolecular assemblies (1, 2). Collagen X is a short-chain collagen expressed in
the hypertrophic zone of calcifying cartilage during skeletal
development and bone growth (3-6). The
1(X) homotrimer consists of
three distinct protein domains, a short triple helix (COL1) flanked by
a small amino-terminal nonhelical NC2 domain and a larger, more
conserved, nonhelical carboxyl-terminal
NC11 domain. By analogy with
the fibrillar collagens, the NC1 domain would be expected to associate
and initiate trimerization (7, 8). In vitro expression and
assembly studies have also shown that collagen X trimers can form
rapidly via NC1 interactions, which are stable to the dissociative
conditions of SDS-polyacrylamide gel electrophoresis (PAGE) in the
presence of urea (9, 10). These strong NC1 interactions are likely to
be largely hydrophobic in nature (8, 11).
1(X). The haploinsufficiency model was further supported by the characterization of an SMCD premature termination mutation that led to
mutant mRNA instability and a total absence of the
1(X) mutant
allele mRNA in patient growth plate cartilage tissue (14). However,
although the in vitro assembly data with other SMCD NC1 mutations demonstrated that the formation of electrophoretically stable
NC1 mutant homotrimers was prevented, these experiments did not exclude
the possibility that weaker or transient interactions may occur that
could impact on the formation of the collagen X trimer. Indeed, trace
amounts of heterotrimer assembly were detected with some
1(X) NC1
missense mutants expressed in vitro (10), suggesting that
some NC1 mutations may allow mutant/normal heterotrimer assembly or
interfere with the efficiency of normal
1(X) assembly and could thus
exert a dominant negative effect on collagen X assembly.
1(X) chains
containing SMCD mutations can interfere with the efficiency of normal
1(X) assembly during in vitro co-expression, demonstrating the potential of these mutations to exert a dominant negative effect if the mutant protein is expressed in vivo.
Furthermore, our studies demonstrate that deletion of the 13-amino acid
conserved aromatic motif (7) completely abrogates the ability of the
1(X) NC1 to interact with other normal or mutant chains, identifying this domain as a crucial sequence in collagen X assembly.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1(X) NC1 Domain Mutations--
The production
of full-length
1(X) cDNA expression constructs containing
specific SMCD NC1 missense mutations
(G618V,2 Y598D) and a
nonsense (premature termination) mutation (Y632X) predicting the
production of a truncated protein has been described previously (10).
Three additional nonsense mutations at amino acid positions 610, 650, and 666 were generated using PCR to produce sequential protein
truncations of the NC1 domain from the carboxyl terminus (see
"Results and Discussion"). The terminations at positions 650 and
666 closely resemble nonsense mutations in SMCD patients reported at
positions 651 (15) and 665 (16), respectively.
Normal and mutant PCR primers for site-directed mutagenesis
-1 and A
-2, B
-1 and B
-2, and C
-1 and C
-2 were designed to delete regions A,
B, and C, respectively. The PCR reactions were
carried out as described above. Two independent PCR products were first
produced by using HX1 (sense) with primer set 2 (A
-2, B
-2, or
C
-2; antisense) and by using HX6 (antisense) with primer set 1 (A
-1, B
-1 or C
-1; sense) with each of the mutant primer sets
in the primary round of PCR using pTM1-h10wt (5 ng) as a template.
After purification, the appropriate fragment pairs were mixed and used
as templates in the second round of overlapping PCR with primers HX1
and HX6. The recombinant mutant PCR fragments were digested with
BamHI and NsiI, purified, and cloned into
corresponding sites within pTM1-h10wt. All mutant constructs were
sequenced to ensure there were no PCR errors.
1(X) or an assembly-competent helix deletion
(helix
)
1(X) reporter construct (10). To these reactions, 1 µl
containing 0, 6.25, 12.5, or 25 ng of NC1 mutant plasmids or a
luciferase control plasmid (Promega) were added prior to transcription
and translation at 30 °C for 90 min. Reactions were terminated by
adding 40 µl of sample buffer (10 mM Tris/HCl, pH 6.8, containing 2% SDS (w/v), 2 M urea, 10 mM
dithiothreitol, and 20% sucrose (w/v)) (9). In some experiments, a
sample buffer with milder denaturation properties was used (10 mM Tris/HCl, pH 6.8, containing 0.5% SDS (w/v) and 20%
sucrose (w/v)). Samples were incubated at room temperature for 10 min
prior to electrophoresis on a 7.5% SDS-polyacrylamide gel at 4 °C
for 16 h in the absence of urea. Radioactive bands were imaged and
quantified using a PhosphorImager (Molecular Dynamics).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1(X) chains terminating at amino
acids 610, 650, and 666. In conjunction with a previously identified nonsense mutation, ter@632 (14), these termination mutations allowed
us to explore the possible effect of sequentially truncated proteins on
collagen X assembly to address whether NC1 microdomains important for
assembly are identified by the concentration of SMCD amino acid
substitution mutations at residues 589-601, 614-618, and 644-652
(Fig. 1).
View larger version (31K):
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Fig. 1.
Schematic representation of the protein
products of normal and mutant human collagen X cDNA
constructs. The signal peptide (open box), the
amino-terminal globular domain (NC2), the triple helical
domain (COL1), and the conserved carboxyl-terminal domain
(NC1) of the normal (wt) collagen X protein
product are shown diagrammatically. Helix is an in-frame collagen X
helix deletion of 283 amino acids of the COL1 (10). Site-directed
mutations were created in the NC1 domain (see "Experimental
Procedures"), and the expected mutant protein products are shown
relative to the normal (wt) pre
1(X) chain.
Arrows indicate the position of the two amino acid
substitutions, Y598D and G618V (9, 10). Constructs labeled ter@666,
ter@650, ter@632, and ter@610 are nonsense mutations that
sequentially remove portions of region C, terminating the
NC1 at amino acids 666, 650, 632, and 610, respectively. Three large
in-frame NC1 deletions were also produced removing region A
(A
, amino acids 525-584), region B
(B
, amino acids 589-601), and region C
(C
, amino acids 605-667). Region B represents
the conserved aromatic motif described by Brass et al. (7).
Three regions with localized missense mutations in patients with SMCD
are numbered B, c1, and c2.
To study the contribution of other NC1 domains in assembly, three
in-frame deletions (A, B
, and C
) were produced (Fig. 1).
Region A (amino acids 525-584) represents a variable region that differs between species (19), in which no mutations have been
identified. Regions B (amino acids 589-601) and
C (amino acids 604-667) are conserved regions in the NC1
domain containing SMCD mutations. Region B also corresponds
to the conserved 13-amino acid aromatic motif proposed by Brass
et al. (7) to have functional importance in trimer association.
In vitro transcription and cell-free translation
demonstrated that the mutant plasmid constructs generated in the
current study (ter@610, ter@650, ter@666, A, B
, and C
) were
translated into
1(X) chains of molecular weight consistent with the
introduced premature terminations or deletions when compared with the
normal (wt)
1(X) chains (Fig. 2). In
the presence of canine microsomal membranes, translocation of
pre-
1(X) chains into the microsomes was demonstrated by the removal
of the signal peptide, which resulted in smaller
1(X) chains for wt
1(X) chains and helix
1(X) chains (Fig. 2) and for all the
mutant
1(X) chains (data not shown). This efficient translocation of
normal and several SMCD
1(X) mutations (Y598D, G618V, Y632X,
1952delC, and 1963del10) has been demonstrated previously (10). Under
these conditions the wt
1(X) chains and helix
1(X) chains
containing normal NC1 domains assembled efficiently into homotrimers
that were electrophoretically stable (Fig. 2) (9, 10). In contrast, all
the
1(X) chains containing NC1 mutations (Y598D, G618V, Y632X,
ter@610, ter@650, ter@666, A
, B
, and C
) did not form
electrophoretically stable mutant homotrimers (Fig. 2). In addition,
co-translational expression studies also demonstrated that all these
NC1 mutant chains also did not associate stably with chains containing
a normal NC1 domain to form heterotrimers that were detectable
electrophoretically (Figs. 5-7). To determine whether the relatively
harsh denaturing conditions of the preparation for electrophoresis (2%
SDS and 2 M urea) masked the detection of mutant
1(X)
assembly, the analyses were repeated using sample loading buffer
containing 0.5% SDS and no urea under nonreducing conditions. Care was
taken to ensure that the samples were kept at room temperature or below
during preparation and electrophoresis. Even under these mild sample preparation conditions no electrophoretically stable mutant homotrimer or mutant/normal heterotrimer formation was detected (data not shown).
These results are also consistent with previous translation experiments
carried out with other SMCD NC1 mutant
1(X) chains (1952delC and
1963del10) (10).
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Although these data suggested that the likely molecular defect in SMCD
is a reduction of collagen X because of the inability of the mutant
chain to assemble, it is possible that some NC1 mutations may allow
mutant/normal heterotrimer assembly (10) or through weak and/or
transient interactions interfere with the efficiency of normal 1(X)
assembly and thus exert a dominant negative effect on collagen X
assembly. To address this issue, the in vitro expression and
assembly system was developed further to determine whether any
measurable interaction occurred between normal and NC1 mutant
1(X)
chains by determining if the presence of mutant chains altered the
ability of chains containing normal NC1 domains to trimerize. In these
studies the accurate measurement of the extent of normal NC1
trimerization is critically dependent on the electrophoretic
discrimination of the mutant chains from
1(X) chains with normal NC1
domains. For the truncation mutations this could be achieved by
co-translation with full-length wt
1(X) as a trimerization reporter.
For single amino acid substitutions, co-translation with full-length wt
1(X) as a trimerization reporter is not informative as the mutant
and wt
1(X) chains cannot be electrophoretically resolved. For these
mutations, an
1(X) with an internal helical deletion (helix
) was
used as a trimerization reporter because it has been shown to form
homotrimers with the same efficiency as wt
1(X) chains and trimerize
with wt
1(X) to form heterotrimers that are electrophoretically
distinguishable (Fig. 2) (10).
To maximize the detection of NC1 sequence-specific interactions and diminish any possible contribution of collagen helix sequences to the in vitro assembly process, the co-assembly experiments were performed using microsomes from pancreatic cells that do not produce collagen. These microsomes are deficient in prolyl hydroxylase, and thus post-translational proline hydroxylation and triple helix formation does not occur (20).
To determine the linear range of in vitro 1(X) synthesis
and assembly, increasing amounts of normal
1(X) plasmid were added, and the extent of synthesis and trimerization was determined (Fig. 3). Synthesis increased linearly from
12.5 to 50 ng of total plasmid DNA, and the efficiency of trimerization
was constant (determined as the trimer to monomer ratio) in this
plasmid concentration range. Hence, all subsequent
co-translation/assembly experiments were conducted with a total plasmid
concentration within this linear range. A similar result was obtained
with the
1(X) helix
construct (data not shown). Furthermore, to
ensure that co-transcription/translation of another DNA did not, in
itself, alter the efficiency of wild-type collagen X NC1 trimerization,
both the wt and helix
1(X) chains were co-translated with
increasing amounts of luciferase DNA (Fig. 4). As expected, NC1 trimerization of
both control
1(X) constructs was not affected by co-translation of
luciferase.
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Co-expression of 1(X) chains containing either the Y598D or G618V
NC1 amino acid substitutions with the helix
1(X) trimerization reporter revealed that these mutations reduced the efficiency of normal
NC1 trimerization demonstrated by the reduced trimer/monomer ratio
(Fig. 5). This effect appeared to
demonstrate concentration dependence, although this is difficult to
assess in a semiquantitative assay of this type. It is of interest to
note that in our previous preliminary co-assembly experiments the G618V
mutation did not noticeably affect the level of normal trimer formation
(8). However, these experiments were not optimized for measuring
interactions and were performed in the nonlinear, saturated range of
plasmid concentration in the cell-free expression system (Fig. 3).
Competitive co-expression analysis of the NC1 premature termination
truncation mutations, ter@610, ter@632, ter@650, and ter@666,
demonstrated that these likewise reduced the efficiency of the
1(X)
reporter construct to trimerize (Fig. 6),
revealing that all the NC1 mutations must interact with the normal NC1
in this assay system.
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The implications of these in vitro data for the molecular
basis of SMCD are significant, suggesting the possibility of a dominant negative mechanism. Our previous studies where 1(X) with SMCD mutations were transfected into cells demonstrated that the mutant collagen chains were not assembled, and mutant homotrimers were not
secreted from the cells (10). The fate of these mutant chains was not
determined, but it was assumed that they were degraded intracellularly
by cellular collagen quality control mechanisms, such as those that
prevent the secretion of unassembled collagen I in osteogenesis
imperfecta (21-23), leading to a collagen X deficiency in the
extracellular matrix. Because collagen X with NC1 mutations can
interact with normal NC1 in vitro and compromise the
efficiency of trimerization of the normal
1(X), it is possible that
the functional levels of collagen X expression in SMCD are reduced to
less than that predicted from a haploinsufficiency model. Although our
data demonstrate that the interaction does not result in the formation
of electrophoretically stable in vitro heterotrimers, it is
not clear whether this in vitro association of normal/mutant heterotrimers would result in the formation of a stable heterotrimeric triple helical molecule in vivo. Even if small amounts of
heterotrimeric triple helical molecules can form in vivo and
are secreted, incorporation of these mutant molecules into the matrix
could contribute to matrix dysfunction. To determine the molecular
basis of SMCD, it is clear that more detailed studies on a spectrum of
mutations is required by both in vitro assembly analysis
and, more importantly, by in vivo analysis of growth plate
cartilage from SMCD patients.
In the only analysis of SMCD cartilage to date (14), a premature stop
codon (Y632X) resulted in mutant mRNA destruction by
nonsense-mediated mRNA decay rather than in the production of
mutant-truncated 1(X). Nonsense-mediated mRNA decay is a common finding in many diseases resulting from premature termination mutations
(24-28). Our data demonstrated that in this SMCD patient (14), the
mutant allele is functionally null, and the pathology results from
collagen X haploinsufficiency. Thus we would predict that in other
cases of SMCD resulting from nonsense mutations, the mutant mRNA
would be subjected to a similar degradation process in vivo,
leading to collagen X haploinsufficiency. However, the extent of this
degradation may be mutant-specific, because there are examples of
nonsense mutations leading to only partial mutant degradation (24, 25,
27, 28). As a result some truncated
1(X) expression may occur,
raising the possibility of a partial dominant negative phenotype
superimposed on the reduced mutant allele expression because of
nonsense-mediated mRNA decay.
Missense mutations of the NC1 may also result in haploinsufficiency of
1(X) by preventing assembly and targeting the mutant protein for
breakdown. However, our data suggest that the missense mutant
1(X)
chains also interact with normal
1(X) chains and thus may exert a
dominant negative effect further reducing the secretion and
supramolecular assembly of functional collagen X in the growth plate
matrix. Perhaps when the effects of a spectrum of SMCD mutations are
evaluated in vivo, the molecular pathology will be
comparable with that of collagen I in osteogenesis imperfecta where the
defects range from haploinsufficiency in mild dominant forms to
dominant negative mutations, which compromise matrix assembly in the
more clinically severe forms (23).
Because SMCD mutations compromise collagen X assembly in
vitro, the localization of the mutations in the NC1 domain may
provide us with important clues on the molecular basis of collagen X
association. For example, the three regions in which SMCD amino acid
substitutions are localized (Fig. 1) are likely to represent the
domains critical for assembly, possibly a series of microinteracting
regions involved in a sequential or cooperative multidomain folding and
assembly of the collagen X NC1 domain. The nonsense mutation cDNA
constructs were used in in vitro expression (where
nonsense-mediated mRNA decay does not occur) to produce sequential
truncations of the NC1 domain at amino acid residues 610, 632, 650, and
666 to experimentally address the role of these regions in in
vitro assembly. Specifically, ter@650 and ter@666 preserve all
three microregions with localized SMCD amino acid substitutions,
whereas ter@632 would preserve the first two of these regions, and
ter@610 would only contain the conserved aromatic motif (Fig. 1).
Interestingly, all truncated 1(X) chains interfered with wt
1(X)
assembly to a similar extent (Fig. 6) within the detection limitations
of this competition assay, suggesting that the truncated
1(X) chains
retained a common NC1 region responsible for the in vitro
interaction. Based on theoretical considerations a conserved aromatic
motif (amino acids 589-601) has been proposed as the putative site of
NC1-NC1 interaction (7), and this is consistent with our data because
the common NC1 sequence in all these truncated
1(X) chains is the
aromatic motif.
To directly test the role of the conserved aromatic motif in assembly,
a construct was produced where this sequence was deleted from the
otherwise normal NC1 (B construct). In vitro assembly studies with the B
protein demonstrated that not only was it unable
to associate into mutant homotrimers in vitro (Fig. 2), as
was the case with all NC1 mutations, but more importantly it had no
effect on the efficiency of normal
1(X) chain assembly (Fig.
7). Thus the removal of this specific NC1
domain completely abrogated the ability of the
1(X) chains to
associate in vitro, directly demonstrating the importance of
this sequence motif in assembly. In contrast, the more global deletions
of the variable region (A
), a domain also common among the truncated
1(X) chains, and region C (C
), where most of the SMCD
mutations are localized, maintained the ability to interact with normal
NC1 domains, reducing the efficiency of normal NC1 trimerization in
co-translation assembly to a similar extent as the SMCD mutations (Fig.
7).
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These studies provide the first biochemical evidence supporting the proposal by Brass et al. (7) that the conserved aromatic motif is critical for assembly, and it seems likely that this region represents the initial point of interaction necessary for the formation of a stable collagen X NC1 trimer. It is of interest that a point mutation in this conserved domain (Y598D) allowed some interaction with normal NC1 domains (Fig. 5), although the strength of the interaction was dramatically reduced and no electrophoretically stable trimers were formed. This ability to interact transiently or weakly with normal NC1 appears to be the common observation for all SMCD mutations studied, suggesting that the mutations disrupted or removed microinteracting regions critical in the folding or alignment of discontinuous motifs into a functional unit. Similarly, our data also suggest that other regions of the NC1, including the variable region (Fig. 1, region A), participate in the final interaction or perhaps allow the NC1 to attain a three-dimensional structure necessary for productive interactions. Based on these studies we propose that the assembly events of type X collagen resemble those of the fibrillar collagens3 (29, 30) and involve folding of individual NC1 domains into a functional three-dimensional conformation and the subsequent alignment and association of the three NC1 domains via the conserved aromatic motif.
The NC1 of collagen X has strong homology to the complement-1q protein
family, a member of which (ACRP30) has recently been crystallized, and
the protein structure has been solved (31). The superimposition of the
collagen X NC1 protein sequence on this structure will be of importance
and may reveal a three-dimensional clustering of SMCD mutations, which
will provide more insight into the molecular basis of the assembly
defects. However, because the in vivo process may be further
modulated by the participation of molecular chaperones (34) and by the
contribution of 1(X) chain helix formation, the consequences of the
SMCD NC1 mutations in this environment may be more complex, and the
analysis of collagen X interactions in experimental systems, which more
closely recreate the in vivo cellular environment, will be
required before the molecular pathology of the disease can be fully explained.
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ACKNOWLEDGEMENT |
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We thank Dr. Shireen Lamandé for critical evaluation of the manuscript.
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FOOTNOTES |
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* 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.
To whom correspondence should be addressed. Fax: 61-3-9345-6367;
Email: bateman{at}cryptic.rch.unimelb.edu.au.
2 Amino acid sequence assignment numbers are from the translation start site (32).
3 S. R. Lamandé and J. F. Bateman, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: NC1, carboxyl-terminal nonhelical domain of collagen X; PAGE, polyacrylamide gel electrophoresis; SMCD, Schmid metaphyseal chondrodysplasia; PCR, polymerase chain reaction; wt, wild type.
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