(Received for publication, October 25, 1994; and in revised form, December 16, 1994)
From the
Type X collagen is a homotrimer of 1(X) chains encoded by
the COL10A1 gene. It is a highly specialized extracellular matrix
component, and its synthesis is restricted to hypertrophic chondrocytes
in the calcifying cartilage of the growth plate and in zones of
secondary ossification. Our studies on a family with Schmid metaphyseal
chondrodysplasia demonstrated that the affected individuals were
heterozygous for a single base substitution in the COL10A1 gene, which
changed the codon GGC for glycine 618 to GTC for valine in the highly
conserved region of the carboxyl-terminal NC1 domain and altered the
amino acid sequence in the putative oligosaccharide attachment site.
Since hypertrophic cartilage tissue or cell cultures were not available
to assess the effect of the mutation, an in vitro cDNA
expression system was used to study normal and mutant type X collagen
biosynthesis and assembly. Full-length cDNA constructs of the normal
type X collagen sequence and also cDNA containing the specific Gly to
Val NC1 mutation found in the patient were produced and expressed by in vitro transcription and translation. While the control
construct produced type X collagen, which formed trimeric collagen
monomers and assembled into larger multimeric assemblies, the mutant
collagen was unable to form these larger aggregates. These experiments
demonstrated that the mutation disturbed type X collagen NC1 domain
interaction and assembly, a finding consistent with the abnormal
disorganized cartilage growth plate seen in the patient. These studies
provide the first evidence of the effect of a type X collagen mutation
on protein structure and function and directly demonstrate the critical
role of interactions between NC1 domains in the formation of type X
collagen multimeric structures in vitro.
Type X collagen is a ``short chain'' collagen with a
restricted pattern of transient expression confined to terminally
differentiated hypertrophic chondrocytes in the calcifying cartilage of
the growth plate (1, 2, 3) and in zones of
secondary ossification(3) . The molecule comprises three
1(X) chains encoded by a condensed gene (COL10A1) of three exons,
one of which (exon 3) codes for the majority of the chain (4, 5, 6, 7) . The type X collagen
homotrimer contains a short triple helix domain flanked by a small
non-helical region at the amino terminus (NC2) and a larger highly
conserved carboxyl-terminal (NC1) non-helical domain. While type X
collagen cDNA and gene organization has been established in several
species(4, 5, 6, 7) , little is
known about the biosynthetic pathway or the molecular assembly and
secretion of the molecule. Evidence from in vitro aggregation
studies indicated that type X collagen could form a supramolecular
hexagonal lattice structure(8) .
The function of type X
collagen remains unknown, but its restricted expression to zones of
calcifying or degrading cartilage suggested that type X collagen may
play an important role in cartilage mineralization (9) . It has
been suggested that the type X collagen lattice may provide an open
structural support that allows vascular invasion, calcification, and
remodeling to proceed to the next phase of bone
development(8) . This specific localization and postulated
function suggested that defects of type X collagen synthesis and
organization could underlie chondrodysplasias with primary growth plate
involvement. Direct evidence supporting this hypothesis came from
studies where a dominant negative mutation of type X collagen was used
to produce a transgenic mouse with a spondylometaphyseal dysplasia
phenotype (10) . Subsequently, several type X collagen
mutations have been identified in patients with Schmid metaphyseal
chondrodysplasia (SMCD, ()MIM 156500), an autosomal dominant
chondrodysplasia where patients are of short stature and exhibit coxa
vara and a waddling gait due to skeletal deformities resulting from
growth plate abnormalities(11) . The seven mutations defined to
date have been clustered in the carboxyl-terminal NC1 domain and
include deletions and point
mutations(12, 13, 14, 15) . Although
no biochemical evidence has been presented to date, the localization of
the mutations in the NC1 domain led these workers (13, 15) to speculate that mutations may prevent
assembly of the mutant chains into trimers by disturbing critical
sequence domains in the highly conserved NC1 sequence, and thus prevent
deposition of type X collagen in the matrix.
In this study, we
report a single base substitution that changes glycine 618 ()(GGC) to valine (GTC) in the conserved region of the type
X collagen
1(X) NC1 domain in a family with SMCD. In vitro expression of mutant and normal
1(X) demonstrated that the
mutation disturbed NC1 domain interactions and type X collagen assembly
and provides the firstdirect evidence of the effect of a type X
collagen mutation on protein structure and function.
Four PCR products spanning the entire COL10A1 gene coding
sequence were analyzed for mutations by the chemical cleavage
method(25) . Only one of the four amplification products
contained a mismatch. The 390-bp fragment covering bases
1726-2115 of the COL10A1 cDNA sequence produced two unique
cleavage products of 222 and 168 bp (data not shown). This result
suggested that there was an allelic sequence variation within this
region covering the NC1 protein domain. To identify the base change,
this 390-bp PCR product was cloned and sequenced. Of the 10 clones
sequenced, there were 6 normal clones and 4 clones with a base change
of G to T resulting in the alteration of Gly
(GGC) to Val (GTC) in the highly conserved NC1 domain of the
1(X) chain of type X collagen (Fig. 1).
Figure 1:
Sequences of the normal and abnormal
alleles prepared from SMCD fibroblast DNA. The amplified 390-bp PCR
products containing the mismatch were cloned and sequenced (see
``Experimental Procedures'' for details). a,
diagrammatic representation of the mutation relative to the 1(X)
chain and COL10A1 gene structure. b, sequencing gel showing
the normal and the abnormal sequences. The base substitution is
indicated by arrowheads. The corresponding coding strand
sequences and the deduced amino acid sequence with a box designating the abnormal codon sequence in the substitution of
glycine 618 by valine.
The mutation deleted a BanI restriction enzyme cleavage site in the DNA sequence, and this was used to confirm the base change on multiple PCR products amplified from the patient and from other family members (data not shown). The altered restriction enzyme digestion pattern was also present in the affected mother and two other affected siblings but was not present in the clinically normal father, indicating that the mutation segregated with the disease in this small pedigree. This sequence change was not detected in the same PCR fragment from 60 alleles of unrelated individuals of the same ethnic origin (data not shown).
To ensure that there were no other sequence alterations
undetected by chemical cleavage, the entire coding sequence of exon 3
of the COL10A1 gene was amplified, cloned, and sequenced. Sequencing of
both the normal and abnormal alleles revealed that both carried the
ACG(-) ATG (+) Thr
Met
polymorphism. The normal allele also carried an additional CCC
deletion(-) polymorphism 6 bp downstream from the TGA termination
codon. These polymorphisms have been reported previously by Sweetman et al.(19) . No other base changes were observed when
compared with the published sequence(18) . Our data also
confirmed the coding sequence deviations observed by Reichenberger et al.(18) from the previous published
sequence(5) . Exon 2 from the patient was also amplified and
sequenced directly. A silent polymorphism was detected in one of the
alleles, which changed the codon for Gly
in the NC2 domain
(GGA
GGT). Because of the nature of direct sequencing, we were
unable to assign this polymorphism to a specific allele. All other
sequences were in agreement with published data(18) .
The
Gly to Val mutation is one of eight mutations in the
1(X) NC1 domain leading to SMCD. Two deletion mutations of 13 bp
(bp 1953-1965) and 10 bp (bp 1963-1972) (13, 14) occur in the sequence immediately adjacent
within the NC1 domain. These were linked to the disease in large
pedigrees and led to codon frameshifts and the theoretical generation
of an abnormal carboxyl-terminal NC1 protein sequence. Deletions (1953
delC and 2088 delCT) result in the introduction of premature
termination signals and NC1 truncation(12) . Three point
mutations producing amino acid substitutions of Cys
Arg(12) , Tyr
Asp, and
Leu
Pro (15) have also been reported. The
localization of all these mutations to within a relatively small region
of the NC1 domain hinted at the critical importance of this domain. It
was further speculated that the NC1 mutations may cause type X collagen
malfunction by perturbing chain association(13, 15) ,
based on theoretical considerations and analogy to fibrillar collagen
biochemistry(26) , but no experimental evidence has been
provided to demonstrate a type X collagen protein abnormality resulting
from the mutations.
In the SMCD case reported here, we undertook
studies to directly demonstrate the effect of the mutation at the
protein level. Since hypertrophic cartilage tissue or cell cultures
were not available to isolate type X collagen in order to assess the
effect of the mutation, an in vitro cDNA expression system was
used to study normal and mutant type X collagen biosynthesis and
assembly. Full-length cDNA constructs of the normal and the Gly
Val mutant type X collagen alleles were produced by the
PCR and cloning procedures. These constructs were expressed by in
vitro transcription and translation using a cell-free system,
which will perform transcription, translation, post-translational
modification, and assembly of type X collagen(27) . Expression
of the control or mutant constructs (Fig. 2a, lanes6 and 7) demonstrated that, in the absence of
canine microsomes, the type X collagen chains were synthesized as
pre-
1(X) chains with the intact hydrophobic leader sequence. If
microsomal preparations were included in the translation (Fig. 2a, lanes1-5), the
leader sequence was removed, generating the smaller processed
1(X)
chain(27) . The striking feature of the control
1(X) was
its ability to form stable associations of (
1(X))
during the translation reaction (Fig. 2a, lanes 2-4). In contrast, the mutant
1(X) was unable
to form these multimeric structures (Fig. 2a, lanes
1 and 5). While the formation of these higher order
structures was more efficient in the presence of microsomal membranes
(compare lanes2-4 with lane6), they were not essential for the association process,
since normal
1(X) cell-free translation products incubated in the
absence of microsomal membranes also formed trimers and multimers when
the translation mix was diluted in buffer containing 5 mM CaCl
and incubated at 37 °C for additional periods (Fig. 3a, lanes 1-4).
Figure 2:
Cell free translation of normal and mutant
type X collagen. The normal and mutant type X collagen cDNA constructs
were transcribed and translated in a coupled cell-free translation
system (see ``Experimental Procedures'' for details). The
resultant [S]methionine-labeled products were
analyzed by SDS-polyacrylamide gel electrophoresis. a, lanes2-4 and 6 are the products from
the normal construct, while lanes1, 5, and 7 are the products from the mutant construct. Translations
were carried out either in the presence (+) or the
absence(-) of canine microsomal membranes. Prior to
electrophoresis on 7.5% gels, the samples were denatured using
different temperatures as indicated. b, the normal (N) and mutant (M) constructs were translated in the
absence of microsomal membranes. The translated products were digested
with bacterial collagenase at 37 °C for 2.5 h (see
``Experimental Procedures'' for details), and the resultant
NC1 domain was denatured at 60 °C for 10 min and analyzed on a
12.5% gels. Lanes1 and 2 are the products
after incubation in the absence of collagenase; lanes3 and 4 are the products after incubation with collagenase.
The migration positions of
1(X) containing the hydrophobic signal
sequence (pre-
1(X)),
1(X) after cleavage of this sequence,
trimeric
1(X)
, NC1 monomers, and trimers (NC1)
are indicated.
Figure 3:
Multimer formation by cell-free
translation products. The normal and mutant constructs were translated
in the absence (a) or presence (b) of
microsomal membranes. The resultant products were tested for their
ability to form multimers by incubation at 37 °C in an appropriate
buffer for periods up to 60 min (see ``Experimental
Procedures'' for details). Samples were denatured at 60 °C for
10 min prior to electrophoresis on a 7.5% SDS-polyacrylamide gel. a, lanes 1-4, products for transcription and
translation of the normal construct; lanes5-8,
product of the mutant construct; lanes 8-12, products of
an equal mixture of normal and mutant constructs. b, lane1, transcription and translation of 1 µg of normal
plasmid; lane2, transcription and translation of a
mixture of 0.5 µg of normal and 0.5 µg of mutant plasmid. The
migration positions of monomeric pre-1(X),
1(X), and trimeric
assemblies of pre-
1(X)
and
1(X)
are
indicated. pre-
1(X)
designates the presumed
larger multimeric assemblies.
The stability
of the type X collagen trimers formed during cell-free translation was
reduced by increasing the temperature of sample denaturation prior to
electrophoresis (Fig. 2a). If the samples were not
heated, but allowed to stand at 20 °C before gel loading, the
predominant species was 1(X)
(Fig. 2a, lane2). When samples were denatured at 60 °C for
10 min (Fig. 2a, lane4), the
proportion of
1(X)
, while reduced, was still the
predominant species. However, denaturation at 100 °C for 5 min (Fig. 2a, lane3) significantly
denatured the
1(X)
complex so that the majority of
type X collagen migrated as an
1(X) monomer. These data support
the suggestion that type X collagen multimers are held together by
strong non-covalent bonds, probably predominantly hydrophobic
interactions within the NC1
domains(1, 26, 28) . To further study the
interactions of the non-collagenous NC1 domains of the normal and
mutant type X collagen, collagenase digestion was used to remove the
collagenous helix-forming domain (Fig. 2b). These data
demonstrate that while the normal NC1 domains associate into higher
order (NC1)
components (Fig. 2b, lane3), the mutant NC1 domain is not able to associate into
multimers (Fig. 2b, lane4). The
slight change in migration of the mutant NC1 domain relative to the
control may reflect subtle changes in secondary structure or variation
in SDS binding.
The extent of multimer formation of the control
construct translation products increased with time of incubation at 37
°C in buffer containing CaCl, first by the formation of
trimers and then the subsequent formation of aggregates that were too
large to migrate into the stacking gel (Fig. 3a, lanes 1-4). No significant formation of
1(X)
trimers or larger multimers were detected with products of the
mutant construct translation, even after 60 min of incubation in
vitro (Fig. 3a, lanes 5-8). To
determine whether the mutant pre-
1(X) chain interferes with the
molecular assembly of normal pre-
1(X) chain in vitro,
equal amounts of normal and mutant construct DNA were mixed and
co-transcribed and translated (Fig. 3a, lanes
9-12). The amount of trimeric and larger multimeric
structures formed by the normal pre-
1(X) was comparable to that
formed in the absence of the mutant product (compare lanes
9-12 with lanes 1-4), demonstrating that in vitro normal pre-
1(X) assembly is not impeded by the
presence of mutant pre-
1(X). The persistence of monomeric
pre-
1(X) (compare lane12 with lane4) confirms that the mutant pre-
1(X) remains as
unassembled monomeric chains.
Since in vitro assembly was
more efficient in the presence of microsomal membranes, it was
suggested that the structural organization imposed by the microsomes,
or the presence in the extracts of accessory or chaperoning proteins
may facilitate assembly(27) . Thus it was important to study
the effect of mutant type X on normal type X collagen assembly in the
presence of microsomes to determine if, under these more favorable
assembly conditions, mutant collagen interacted with normal collagen
and affected association and multimer formation. In this experiment,
the coupled transcription and translation of 1 µg of normal plasmid (Fig. 3b, lane1) was compared with
the co-transcription and translation of a mixture of 0.5 µg of
normal and 0.5 µg of mutant plasmid (Fig. 3b, lane2). Quantification of the monomer and trimer
bands demonstrated the expected amount of normal 1(X)
and increased accumulation of monomeric
1(X) corresponding
to unassociated mutant. These data further confirm that mutant
1(X) synthesis does not affect the efficiency of normal
(X)
chain multimer assembly in vitro.
These experiments
directly demonstrated, for the first time, the effect of a type X
collagen mutation on protein assembly. The NC1 Gly
Val mutation prevented the in vitro assembly of type X
collagen trimers by interfering with the association of NC1 domains.
The molecular mechanisms of type X assembly are not known, but by
analogy with the more extensively studied fibril-forming collagens, it
is thought that assembly is initiated by nucleation of three
-chains at the carboxyl-terminal end of the molecule with
propagation of the helix toward the amino terminus(29) . Our
data demonstrate the critical role of these NC1 domains in this
assembly process. The initial assembly of the
1(X) chains is an
efficient process, even under the non-optimal in vitro conditions used, and denaturation studies (Fig. 2)
demonstrated that, once associated, the multimers are very stable. The
association of the NC1 domains into multimeric components has been
observed previously in chicken type X collagen and appears to be
important for the end-to-end association in the formation of hexagonal
lattice structures(8) .
The mechanism by which the mutation
perturbs assembly is not known, but the clustering of known mutations
causing SMCD in the NC1 domain emphasizes the critical role of this
region. Gly is conserved in type X collagen sequences
from all the species published so far, including chicken(4) ,
mouse(7) , bovine(6) , and human(5) . The
mutation may affect the initial NC1 hydrophobic interactions directly,
since a Gly
Val substitution would increase the hydrophobicity,
or by altering critical protein secondary structure. This mutation also
changed the amino acid sequence of the putative oligosaccharide
attachment site from Asn-Gly-Thr to Asn-Val-Thr. It is not known if
this site is normally glycosylated, but the sequence change may affect
its ability to be N-glycosylated or alter the structure of the
oligosaccharide attachment(30, 31) .
While it is
likely that the same forces drive type X collagen assembly in
vivo, and the mutation will also prevent initial association,
trimerization, and matrix assembly in vivo, it is possible
that special localized conditions or factors may allow some mutant
association. If a proportion of the mutant chain can associate in
vivo, it may exert a dominant negative effect. To fully resolve
this important question, cell transfection studies are in progress. The
more likely scenario, supported by our in vitro data, is that
the NC1 mutation leads to haploinsufficiency by preventing assembly,
and by analogy with mutations that completely prevent type I collagen
assembly in osteogenesis imperfecta(32, 33) , would
prevent type X collagen secretion and target the mutant 1(X)
chains for rapid intracellular degradation. This would result in a type
X collagen deficiency in the cartilage growth plate and dramatically
reduce the type X collagen open matrix structure, which is thought to
be critical for hypertrophic chondrocyte function leading to the
disorganization of the growth plate seen in patients with SMCD.
Recently, type X collagen-null transgenic mice have been generated by
gene targeting in embryonic stem cells(34) . These mice show no
obvious phenotypic abnormalities in long bone growth and development.
The reason for this surprising finding has not yet been established but
may reflect species-specific differences in type X collagen function or
anatomical or developmental factors which result in haploinsufficiency
of type X collagen having a more severe phenotype in humans than mice.