From the Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT United Kingdom
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ABSTRACT |
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Metaphyseal chondrodysplasia type Schmid (MCDS)
is caused by mutations in COL10A1 that are clustered in the
carboxyl-terminal non-collagenous (NC1) encoding domain. This domain is
responsible for initiating trimerization of type X collagen during
biosynthesis. We have built a molecular model of the NC1 domain trimer
based on the crystal structure coordinates of the highly homologous trimeric domain of ACRP30 (adipocyte complement-related protein of 30 kDa or AdipoQ). Mapping of the MCDS mutations onto the structure reveals two specific clusters of residues as follows: one on the surface of the monomer which forms a tunnel through the center of the
assembled trimer and the other on a patch exposed to solvent on the
exterior surface of each monomeric unit within the assembled trimer.
Biochemical studies on recombinant trimeric NC1 domain show that the
trimer has an unusually high stability not exhibited by the closely
related ACRP30. The high thermal stability of the trimeric NC1 domain,
in comparison with ACRP30, appears to be the result of a number of
factors including the 17% greater total buried solvent-accessible
surface and the increased numbers of hydrophobic contacts formed upon
trimerization. The 27 amino acid sequence present at the amino terminus
of the NC1 domain, which has no counterpart in ACRP30, also contributes
to the stability of the trimer. We have also shown that NC1 domains
containing the MCDS mutations Y598D and S600P retain the ability to
homotrimerize and heterotrimerize with wild type NC1 domain, although
the trimeric complexes formed are less stable than those of the wild
type molecule. These studies suggest strongly that the predominant
mechanism causing MCDS involves a dominant interference of mutant
chains on wild type chain assembly.
Type X collagen is a short chain, homotrimeric collagen
( The precise function of type X collagen remains to be determined
(14-16), but mutations in the COL10A1 gene cause
metaphyseal chondrodysplasia type Schmid (MCDS), an autosomal dominant
form of human skeletal dysplasia (17, 18). An intriguing finding is
that virtually all of the mutations causing MCDS occur within the
carboxyl-terminal non-collagenous NC1 domain of type X collagen (Ref.
19 and references therein). The only two MCDS mutations not found in
the NC1 domain affect the putative signal peptide cleavage site
upstream of the non-collagenous domain 2 in the molecule (20). The role
of the carboxyl-terminal non-collagenous domain of most collagens
(including type X) in initiating intracellular The crystal structure of the NC1-like domain of ACRP30, a protein
closely related to type X collagen, has been recently solved at 2.1-Å
resolution (24). We have therefore performed molecular modeling of the
NC1 domain, based on the ACRP30 crystal structure coordinates, to gain
greater insight into the structural basis of MCDS. The model reveals
that the MCDS mutations are localized to two specific regions of the
folded monomeric NC1 domain and that many of the MCDS mutations may not
totally abolish the ability of affected chains to trimerize.
Mutagenesis experiments, based on information obtained from the model,
demonstrate that NC1 domains containing specific MCDS mutations retain
the ability to form trimers and provide an explanation for the
unusually high thermal stability exhibited by the assembled NC1 trimer.
Alignments--
The amino acid sequence of the NC1 domain of
human type X collagen was used to probe EMBL, GenBankTM,
SwissProt and PIR data bases as well as data base updates using the
advanced BLAST 2.0 search (25). We used a cut-off expectancy value
(E) of 1 (such that no more than 1 match is expected to be
found merely by chance, according to the stochastic model of Karlin and
Altschul (26)) and a gapped alignment using the Blosum 62 matrix (27).
Multalin (28) was used to re-align the resulting 25 sequences using the
Blosum 62 matrix using a penalty of 12 for gap opening and 2 for gap
extending. None of the resulting aligned sequences have
three-dimensional structures in the Brookhaven data base.
Molecular Modeling--
All molecular modeling was performed on
an R5000 O2 Silicon graphics workstation using QUANTA® and CHARMm
23.1® programs. The three-dimensional model of the NC1 domain of type
X collagen was built based on the coordinates of human
ACRP30.2 The file containing
the coordinates was rewritten manually to make a Protein Data Base file
format that could be imported by QUANTA®. A homology model was built
by copying the coordinates of the backbone of the ACRP30 trimer and the
coordinates of identical residues in the type X collagen NC1 domain.
The remaining side chains were built in the Protein Design module using
the Ponder and Richards' rotamer library. Disordered loops in the
crystal structure of ACRP30 (loop G-H in M1 and loops G-H and A-A' in M2) were modeled where possible by overlaying the intact loop of a
corresponding part of one of the other two monomers. This left a gap
between Glu658 to Gly661 (corresponding to
Asp224 to Gly 228 in ACRP30) in each monomer.
The resulting trimer of the NC1 domain of type X collagen was
energy-minimized (using the steepest descents followed by
Newton-Raphson algorithm) to gradient convergence (<0.01 root mean
square) removing bad steric and electrostatic contacts. The water
molecules from the crystal structure were overlaid, and those internal
to the trimer were incorporated into the model. The Protein Health
module in QUANTA was used to check the integrity of the model using a Ramachandran map and to identify buried hydrophilic or exposed hydrophobic residues.
The solvent-accessible surfaces of monomers and assembled trimers were
calculated to estimate the surface area buried upon trimerization. The
local environment of each residue on the interfaces was examined to
locate those residues important for the trimer structure. The
interfaces are not symmetric, because of a slight twist and stagger to
the arrangement of the Preparation of the Constructs--
DNA
constructs were generated by PCR from the lambda genomic COL10A1 clone,
HX3 (5). DNA encoding the entire human type X collagen NC1
domain was amplified using the primers
5'-CCAGGTCAAGCACATATGCCTGAGGGT-3' (primer A, sense nucleotides
1550-1563), which incorporates an NdeI site (C (A/T)ATG)
in-frame with the Met521 codon of the NC1 domain, and
5'-GGGGTGTACTCACATTGGAGCCAC-3' (primer B, antisense nucleotides
2028-2052), which incorporates the stop codon and 9 base pairs of the
3'-untranslated region. To generate the extended reporter
construct, DNA encoding the entire NC1 domain and upstream DNA
encoding the 11 most carboxyl-terminal Gly-X-Y repeats
of the collagenous domain was generated by PCR. A sense oligonucleotide
of sequence 5'-CCAGCTCATATGGCAACTAAGGGCCTC-3' (nucleotides
1429-1455), which incorporates an NdeI site coding for an
in-frame Met codon at the amino terminus of the extended NC1 coding
region, was used together with the antisense primer B. To produce the
truncated NC1 translation construct, DNA encoding a portion of the NC1
domain from the Met549 codon to the stop codon was
generated by PCR using the sense primer 5'-GGGGTAACACATATGCCTGTGTCT-3'
(nucleotides 1632-1656) together with the antisense primer B. To
create constructs containing MCDS and related point mutations,
site-directed mutagenesis was carried out using the PCR-based
single overlap extension procedure described elsewhere (29) using
oligonucleotides A and B (described above) and mutagenic
oligonucleotides (nucleotides 1875-1899) sense,
5'-CCAGGAATATACTTTTTTTCATAC-3' and antisense,
5'-GTATGAAAAAAAGTATATTCCTGG-3' for Y598F; sense,
5'-CCAGGAATATACGCTTTTTCATAC-3' and antisense, 5'-GTATGAAAAAGCGTATATTCCTGG-3' for Y598A; 5'-CCAGGAATATACGATTTTTCATAC- 3' and antisense, 5'-GTATGAAAAATCGTATATTCCTGG-3' for Y598D; and (nucleotides 1881-2004) sense, 5'-ATATACGCTTTTCCATACCACGTG-3' and
antisense, 5'-CACGTGGTATGGAAAAGCGTATAT-3' for S600P. The
resulting PCR fragments were cloned into the T/A cloning vector pCR 2.1 (Invitrogen). Constructs generating sense RNA upon transcription from
the T7 promoter were identified by restriction mapping and sequenced.
Coupled in Vitro Transcription and Translation--
In
vitro expression was carried out using the TNT T7
polymerase-coupled rabbit reticulocyte lysate system (Promega). 1 µg of each purified construct was expressed in 40 µl of TNT premix containing 2 µl of [35S]methionine (1000 Ci/mmol, NEN
Life Science Products) made up to a final volume of 50 µl with
sterile distilled water. Reactions were incubated at 30 °C for 90 min. 5-µl aliquots of in vitro expression reaction were
added to 10 µl of sample buffer (50 mM Tris/HCl, pH 6.8 (room temperature), 50% (v/v) glycerol, 0.025% (w/v) bromphenol blue;
the concentration of SDS in the sample buffer was either 10% (w/v) or
0.75% (w/v) as indicated under "Results"). Where heating is
indicated, samples were overlaid with 20 µl of mineral oil and
incubated at the appropriate temperature for 5 min using Perkin-Elmer
480 thermal cycler prior to analysis by SDS-PAGE. All samples were
resolved on standard 12 or 16% PAGE gels containing 0.1% SDS
according to Laemmli (30). Gels were fixed (10% (v/v) acetic acid
containing 10% (v/v) methanol) for 30 min and washed twice in 30%
(v/v) methanol containing 3% (v/v) glycerol. Gels were dried and
exposed to Kodak Biomax Film, or, if quantitation was required,
phosphorimaged (Fuji-Bas).
Immunoprecipitation of NC1 Domains with the X34 Monoclonal
Antibody--
In vitro translations containing labeled NC1
domains were subjected to centrifugation at 109,000 × g for 1 h to remove particulates. 50 µl of the
supernatant was then dialyzed against NET buffer overnight (100 mM Tris/HCl, pH 7.4, 100 mM NaCl, 1% (v/v)
Tween 20, 1 mM EDTA). 5 µl of hybridoma medium containing
the X34 monoclonal antibody, which recognizes a
conformation-dependent epitope in the NC1 domain of native
type X collagen (31), was added to the dialyzed translation followed by
incubation for 16 h at 4 °C with gentle inversion. Following
the incubation, 10 µl of protein A-Sepharose (Amersham Pharmacia
Biotech) was added, and the incubation was allowed to proceed for a
further 4 h, after which the protein A-Sepharose was recovered by
centrifugation at 12,000 × g (control incubations were
carried out in the absence of X34). The pelleted protein A-Sepharose
was washed in 500 µl of 1× NET buffer three times followed by
resuspension in sample buffer containing 1% (v/v) Alignments--
The complete NC1 domain of human type X collagen
was aligned with the carboxyl-terminal non-collagenous domain of mouse
ACRP30 (Fig. 2). The type X collagen NC1
domain contains an extra 27 residues at the amino terminus that are not
present in ACRP30 and a deletion of ACRP30-Tyr219. The
globular domain of ACRP30 exhibits 40% identity and 65% similarity
with the equivalent region of the type X collagen NC1 domain.
Overlaying the secondary structural elements from the crystal structure
of ACRP30 on the alignment revealed that those residues forming
Molecular Model of Trimeric NC1 Domain--
A model of the
trimeric NC1 domain (Fig. 3) was built
using the three-dimensional coordinates of trimeric ACRP30 based on the
alignment shown in Fig. 2. The amino-terminal 27 residues of the NC1
domain of type X collagen were not included in the model since there is
no equivalent sequence in ACRP30 (Fig. 2). The 8 loop regions in the
NC1 domain were modeled on the ACRP30 structure since their lengths are
conserved, and they are anchored spatially by the
Upon trimerization (Fig. 3a), the mode of packing between
the NC1 monomer subunits produces a tight association contributed to by
the loss of solvent-accessible surface and hence providing a free
energy gain. The total buried solvent-accessible surface of the NC1
domain on trimerization is 6222 Å2, compared with 5324 Å2 for ACRP30. The three monomers pack together forming a
twisted tunnel that extends through the core of the structure. The
hydrophobic contacts that produce the close packing between the NC1
monomers are identical or show only conservative changes with respect
to ACRP30. These include Ala553, Ile557,
Leu575, Ile596, Val621,
Ile641, Phe675, and Ala678 in the
NC1 domain. However, in the NC1 domain additional residues (Pro550, Val551, Val668, and
Pro679) contribute to hydrophobic interfaces between
monomers. The equivalent residues in ACRP30 are Tyr114,
Arg115, Asn233 and His244, respectively.
Pro550, Val551, Ile641,
Phe675, and Pro679 form a hydrophobic plug at
the base of the NC1 trimer tunnel as viewed in Fig. 3b.
Moving up from the base, the trimer tunnel surface, contributed by all
three monomers, becomes more polar with Tyr598,
Ser600, Ser639, and Ser671 forming
a dense network of hydrogen bonds with each other and water molecules
(e.g. see Fig. 3c). These polar residues are
conserved across the entire family of type X collagen-related proteins
(Fig. 1).
Examining the monomer-monomer interfaces is complicated by the
asymmetric association of the three monomers (designated M1 Location of MCDS Point Mutations in the NC1 Trimer--
All NC1
domain residues that are affected by single amino acid substitutions
causing MCDS were displayed simultaneously on the monomer (Fig.
3d) and trimer (Fig. 3e) models revealing a striking clustering of position. The substituted residues are localized
to two distinct regions. The side chains of residues that are
substituted in MCDS either form the polar and hydrogen-bonded region
within the trimer tunnel (e.g. Tyr598 and
Ser600, see Fig. 3c) or form a patch on the
external surface of the monomer/trimer assembled from part of loop D-E
and part of Trimerization and Stability of Recombinant Human Type X Collagen
NC1 Domain--
The model was first used in an attempt to predict
motifs or residues responsible for the unusual stability of the NC1
domain of type X collagen (see below and "Discussion"). Expression
of the full-length NC1 domain in a reticulocyte lysate system resulted in detection of the monomeric protein (Mr = 20,000) and a complex of Mr = 45,000 that is
stable on SDS-PAGE in reducing conditions (Fig.
4a, lane
4). Similarly, expression of an "extended" NC1 construct
containing 11 Gly-X-Y repeats of the collagenous domain of
type X collagen (Fig. 4a, lane 3) and a
"truncated" construct lacking the amino-terminal 27 residues of the
NC1 domain (Fig. 4a, lane 1) produced monomers of
Mr = 28,000 (extended) and 17,000 (truncated) as
well as complexes of Mr = 60,000 and 35,000 (respectively). Based on Mr, the complexes
formed appeared too large to be dimers but too small to be trimers. In
order to resolve the molecular composition of the complexes,
co-expression studies were performed. Co-expression of the truncated
and extended constructs (Fig. 4a, lane 2) and full-length
and extended constructs (Fig. 4a, lane 5) produced the
expected NC1 monomer and complex bands as well as two extra complex
bands of intermediate Mr. The fact that 4 complex bands were formed in each co-expression reaction indicates clearly that the complexes are trimeric associations of the monomers running slightly faster on SDS-PAGE than would be predicted. It should
be noted that the data in Fig. 4a were produced adding the
translation mixes to sample buffer containing 10% SDS (final SDS
concentration 6.7%) and running on the SDS-PAGE gel without prior
heating.
To check that the trimeric NC1 domains had adopted a native
conformation, translation reactions containing co-expressed full-length and extended NC1 domains were immunoprecipitated with the X34 monoclonal antibody which recognizes a
conformation-dependent epitope in the native trimeric NC1
domain but not monomeric NC1 (31). SDS-PAGE analysis of the
immunoprecipitated co-translation reactions revealed that the X34
monoclonal antibody recognized the trimeric but not monomeric NC1
domains present in the co-translation. The control immunoprecipitation
carried out in the absence of X34 revealed that low levels of monomeric
and trimeric NC1 domain bound nonspecifically to the protein
A-Sepharose (Fig. 4b, lane 3). Comparison of the control
immunoprecipitation with that carried out in the presence of the X34
antibody demonstrated that in addition to adopting a native structure,
all of the trimeric NC1 domain was stable in the conditions used for
SDS-PAGE analyses.
In order to investigate the stability of the trimers formed in the
system, translations of the full-length NC1 domain (Fig. 4c)
were heated for 5 min in 6.7% SDS at the temperatures indicated prior
to loading on the gel. The trimer formed is stable at 60 °C and,
while some denatures to monomer at 80 °C, a significant proportion
is still trimeric at 100 °C (see also Fig.
5a). The full-length monomer
appears unaffected by the exposure to increasing temperatures (Fig.
4c). ACRP30 lacks the amino-terminal 27 residues found in
the NC1 domain of type X collagen (Fig. 2) and also does not form a
trimer capable of withstanding SDS-PAGE (13). In order see whether the
amino-terminal NC1 sequence plays a role in determining stability, it
was deleted. In comparison with the full-length NC1 domain (Fig.
4c), the domain lacking the amino-terminal 27 residues
responded in a different fashion in two critical ways. First, as the
temperature increases above 40 °C, the monomer is seen to aggregate
into a high Mr complex that does not enter the resolving gel (Fig. 4d). Second, the trimer, which is stable
at 60 °C and unaffected by the aggregation process exhibited by the monomer, is completely denatured at 80 °C (Fig. 4d).
Assembly and Stability of NC1 Domains Containing MCDS and Related
Mutations--
In order to examine the role in determining trimer
stability of Tyr598, which forms a network of hydrogen
bonds in the trimer tunnel (Fig. 3c) and is mutated to Asp
in one case of MCDS (Table I), the stabilities of NC1 domains
containing Y598F, Y598A, and Y598D were examined (Fig. 5). Removal of
the OH group (Y598F) produced an approximately 10 °C drop in the
melting temperature of the trimer, and a further 10 °C decrease was
found for the trimer containing the Y598A mutation. Under these
conditions, in which the sample is placed in 6.7% SDS to prevent
"geling" of the translation mix during heating prior to loading on
the gel, a trimer could not be detected when expressing the
construct containing the MCDS mutation Y598D (see Fig. 5b).
Indeed, trimerization could not be detected for a second MCDS mutation,
S600P, under the same conditions (Fig. 5b).
To investigate whether the high level of SDS (6.7% final
concentration) to which samples were exposed prior to gel
electrophoresis was denaturing potential trimer complexes formed by
MCDS constructs, the concentrations of SDS in the sample buffer were
reduced. Translations separated under native conditions failed to
resolve in the gel, as did samples taken up in a final concentration of
0.1% SDS and run on standard SDS-PAGE gels (data not shown). However,
when samples were exposed to 0.5% SDS and (without heating) run on standard 0.1% SDS-PAGE gels, resolution of wild type monomers and
trimers was apparent (Fig. 5c, lanes 1 and
2). Analysis under the same conditions of NC1 domains
containing the Y598D and S600P MCDS substitutions (Fig. 5c,
lanes 3 and 5) demonstrated that each
formed a trimeric complex. When the same MCDS translations were exposed
to 1% SDS prior to running on the gel, the levels of detectable trimer
were reduced by more than 50% (results not shown). Co-translation of
each MCDS NC1 mutant construct with the extended wild type construct
led to the formation of heterotrimers (Fig. 5c,
lanes 4 and 6).
Structure and Assembly of the Wild Type Human NC1 Domain--
The
NC1 domain of type X collagen is a putative asymmetric trimer composed
of three 10
Previous studies on type X collagen assembly have demonstrated that
trimers generated by in vitro translation possess an
unusually high thermal stability (see Ref. 22 and references therein). We demonstrate here that this thermal stability is a consequence of the
stability of the NC1 domain (see Fig. 4). It is of interest to note
that the intact wild type NC1 domain trimerizes spontaneously forming a
native structure (Fig. 4, a and b), whereas when
translating the full-length type X collagen polypeptide in the same
system, either Ca2+ or microsomal membranes are required to
achieve trimerization (22). We assume that the presence of
Ca2+, or microsomal membranes (and associated chaperone
proteins), prevents the unfolded collagenous domain from interfering
with the folding and trimerization of the nascent NC1 domain.
Furthermore, it is noteworthy that recombinant human NC1 domain
expressed in bacteria also spontaneously forms a thermally stable
native trimer that is specifically recognized by the
conformation-dependent X34 monoclonal antibody
(32). ACRP30 and other members of this family of related proteins, with the exception of type VIII collagen (34), do not appear to exhibit this level of thermal stability. The
modeling of the NC1 domain has revealed a number of features that may
help explain the differences in stability. First, the model revealed
that the packing of the aromatic side chains within the hydrophobic
core of the monomers of both NC1 and ACRP30 is organized in a
"herring bone" pattern similar to that associated with hydrophobic
cores of other stable proteins (34). However, the NC1 domain contains a
more densely packed hydrophobic core than ACRP30 because of the
presence of three additional hydrophobic side chains that may increase
stability of the monomer and trimer (35). Second, on trimerization, the
total buried solvent-accessible surface of the NC1 domain is 17%
greater than ACRP30, and the hydrophobic plug at the base of the NC1
domain has additional contributions from Pro550,
Val551, and Ile588 (corresponding residues in
ACRP30 are Tyr, Arg, and Gly, respectively). Third, sequence alignments
show that type X collagen has an additional 27 residues at the amino
terminus of the NC1 domain that are not present in ACRP30 or other
members of the family of proteins with the exception of the two chains
of type VIII collagen (Figs. 1 and 2).
To examine whether the additional 27-residue sequence stabilizes the
NC1 domain, we compared full-length and truncated NC1 domains. In the
absence of the 27 residues, monomeric NC1 forms high molecular weight
aggregates at temperatures above 40 °C (Fig. 4d) implying
that the monomer has a defined structure in SDS at temperatures below
40 °C that becomes denatured at higher temperatures leading to
aggregation. However, no aggregation is exhibited by the full-length
NC1 monomer, even at 100 °C (Fig. 4c), strongly suggesting that it has a folded structure that is resistant to denaturation. The trimer is still formed by the shortened NC1 protein
although its stability is reduced (compare Fig. 4, c and d) presumably because of the decreased stability of the monomer.
Structure and Assembly of NC1 Domains Containing MCDS and Related
Mutations--
Mapping MCDS point mutations onto the NC1 model
revealed a striking three-dimensional clustering of affected residues
into two distinct domains (Fig. 3, d and e). The
first cluster of residues substituted in MCDS is located on an external
patch that includes loop D-E and is known to play a role in molecular
recognition in structurally related molecules such as TNF and C1q as
described above. Since these residues are located on the external
surface of the trimer, it seems unlikely that the associated MCDS
mutations would prevent the trimerization process. The second cluster
of mutations affect the polar region of the trimer tunnel. It is significant that not one of the MCDS point mutations (see Table I)
affect the hydrophobic plug at the base of the trimer tunnel (Fig.
3b) or any of the monomer-monomer hydrophobic contacts that are likely to drive the trimerization process.
On the basis of the findings described above, we predicted that NC1
domains containing MCDS point mutations would trimerize but form less
stable complexes that may be difficult to detect on SDS-PAGE gels. In
order to test this hypothesis, the role of Tyr598 in
determining NC1 trimer stability was examined. Tyr598 not
only participates in a hydrogen bond network as shown in Fig.
3c but is also mutated to Asp in MCDS (Table I). Replacement of the Tyr598 by either Phe or Ala did not prevent the
formation of SDS-resistant trimers although their thermal stabilities
were considerably reduced (Fig. 5a). Under equivalent
conditions, in which the translation mix was dissolved in sample buffer
containing 6.7% SDS, no trimers could be detected when expressing NC1
domains containing MCDS Y598D or S600P mutations (Fig. 5b).
However, both Y598D and S600P mutant NC1 domains were shown to form
homotrimers, and heterotrimers with wild type NC1 domains, that are
detectable in low concentrations of SDS (Fig. 5c) but
denature as the level of SDS is increased (see Fig. 5b).
These data clearly demonstrate that the stabilities of trimers
containing MCDS mutant chains (in terms of sensitivity to the
concentration of SDS and temperature) are considerably reduced compared
with that of the wild type. Nevertheless, the fact that non-covalently
associated MCDS mutant trimers can be detected on SDS-PAGE indicates
that the stabilities of these mutant complexes are still greater than
equivalent non-covalent trimeric complexes formed during the assembly
of collagens with the exception of type VIII (33). We also have
preliminary evidence suggesting that NC1 domains containing frameshift
mutations causing MCDS also retain the ability to
trimerize.3 The MCDS mutant
NC1 domains examined here not only retain the ability to trimerize but
several
In conclusion, the study presented here has produced a
three-dimensional model of the NC1 domain of type X collagen that
reveals a clustering of MCDS mutations that is consistent with the
mutant chains retaining the ability to participate in the trimerization process. In addition, we have demonstrated that the NC1 domain forms an
unusually stable trimer and that NC1 domains containing MCDS mutations
retain the ability to trimerize. The modeling and protein assembly
studies suggest strongly that the predominant molecular mechanism
causing MCDS involves a dominant interference of mutant chains on wild
type assembly.
INTRODUCTION
Top
Abstract
Introduction
References
1(X)3) expressed specifically by hypertrophic
chondrocytes in the endochondral growth plate (1). The expression of
type X collagen is also re-activated during fracture repair and in
osteoarthritis (2-4). The human
1(X) collagen chain consists of a
short amino-terminal non-collagenous domain 2 of 37 amino acids
followed by a triple helix-forming collagenous domain with 154 Gly-X-Y repeats and a carboxyl-terminal non-collagenous
domain (NC1)1 of 161 amino
acids (5). The
1(X)3 molecule is thought to assemble
into a hexagonal lattice within the extracellular matrix in a fashion
similar to that of type VIII collagen (6, 7). Type X collagen is part
of a family of collagen-like proteins sharing a condensed gene
structure, a collagen triple helical domain, and in particular, a
highly conserved carboxyl-terminal non-collagenous (NC1-like) domain
(8, 9). This family includes collagen types X and VIII, C1q component
of complement, hibernation proteins (10), cerebellin (11, 12), and
ACRP30, an abundant serum protein implicated in energy homeostasis and
obesity (13).
-chain selection,
assembly, and helix formation is well established (Refs. 8 and 21 and
references therein). Assembly studies based on cell-free translation of
recombinant RNA encoding wild type and MCDS transcripts of type X
collagen suggest that mutant chains do not interfere with the
trimerization of the wild type protein based on SDS-PAGE assays (22).
These findings have led to the suggestion that the phenotype of MCDS is
best explained by haplo-insufficiency. In support of this hypothesis,
in one individual with MCDS, only wild type mRNA for type X
collagen could be detected in the growth plate, suggesting that the
mutant transcript is unstable and rapidly degraded (23). However,
haplo-insufficiency is in discord with the clustering of both point and
frameshift mutations in the NC1 domain of type X collagen; in
haplo-insufficiency one would expect to find MCDS-causing frameshift
mutations to be randomly distributed through the gene (19). The
non-random clustering of MCDS mutations in the NC1 domain is more
consistent with a mechanism involving dominant interference in which
the mutant chains retain the ability to trimerize. It is possible that
previous investigations have failed to detect trimerization of chains
containing MCDS mutations due to the harsh assay conditions, such as
SDS-PAGE, that have been employed.
EXPERIMENTAL PROCEDURES
-sandwich monomers (24). Particular attention
was given to the position of those residues in the NC1 model trimer
that differed in ACRP30 and related proteins (see Fig.
1).
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Fig. 1.
Sequence alignment of C1q-like domains with
a BLAST 2.0 E value of less than 0.015. BLAST 2.0 was
used to probe the data bases with the NC1 domain of type X collagen
sequence and retrieved 58 sequences with an E value less
than 10. After discarding redundancy we aligned 25 sequences in
Multalin using the Blosum 62 matrix and default penalty values. Those
residues in red are conserved throughout the alignment;
those in blue are conserved in at least 70% of sequences,
and those highlighted in yellow show the MCDS point
mutations. -Strand topology is taken from ACRP30 and TNF
nomenclature; ca1a_human, human type X collagen (Swiss-Prot accession
number Q03692); ca1a_bovin, bovine type X collagen (Swiss-Prot
accession number P3206); ca1a_mouse, murine type X collagen (Swiss-Prot
accession number Q00780); ca1a_chick, gallus gallus type X
collagen (Swiss-Prot accession number P08125); ca28_mouse, murine
2
type VIII collagen (Swiss-Prot accession number P25318); ca28_human,
human
2 type VIII collagen (Swiss-Prot accession number P25067);
ca18_rabbit, rabbit
1 type VIII collagen (Swiss-Prot accession
number P14282); ca18_human, human
1 type VIII collagen (Swiss-Prot
accession number P27658); ca18_mouse, murine
1 type VIII collagen
(Swiss-Prot accession number Q00780); acr3_human, human adipocyte
complement-related protein (Swiss-Prot accession number Q15848);
acr3_mouse, murine adipocyte complement-related protein (Swiss-Prot
accession number Q60994); cole_lepma, lepomis macrochirus
inner ear-specific collagen (Swiss-Prot accession number P98085);
hp27_tamas, chipmunk hibernation-associated protein (Swiss-Prot
accession number Q06577); hp25_tamas, chipmunk hibernation-associated
protein (Swiss-Prot accession number Q06576); hp20_tamas, chipmunk
hibernation-associated protein (Swiss-Prot accession number Q06575);
c1qc_mouse, murine C1q C chain (Swiss-Prot accession number Q02105);
c1qc_human, human C1q C chain (Swiss-Prot accession number P02747);
c1qb_mouse, murine C1q B chain (Swiss-Prot accession number P14106);
c1qb_rat, rat C1q B chain (Swiss-Prot accession number P31721);
c1qb_human, human C1q B chain (Swiss-Prot accession number P02746);
c1qa_mouse, murine C1q A chain (Swiss-Prot accession number P98086);
c1qa_human, human C1q A chain (Swiss-Prot accession number P02745);
cerl_rat, rat cerebellin (Swiss-Prot accession number P23436);
cerb_human, human cerebellin 1 (Swiss-Prot accession number P23435);
multimerin_h, human multimerin (Swiss-Prot accession number
A57384).
-mercaptoethanol.
Prior to SDS-PAGE analysis, the samples were heated for 2 min at
68 °C.
RESULTS
-strands of ACRP30 were mostly conserved in the NC1 domain, whereas
differences were predominantly localized to the loop regions (Fig. 2).
Therefore, the secondary structural elements are highly conserved.
Within the
-strands, those residues forming the hydrophobic core of
the monomer of ACRP30 (see below) are particularly conserved. This
conservation strongly indicates that the tertiary fold of the NC1
domain is identical to the
-sandwich of ACRP30. In addition, the
conservation of key interface residues between monomers (see below and
Fig. 2) indicates that the quaternary structures of these two proteins
are similar. This pattern of conservation, based on alignments, also
holds across the complete family of related proteins including collagen
types X and VIII, C1q, ACRP30, hibernation protein and cerebellin (see
Fig. 1).
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Fig. 2.
Alignment of mouse ACRP30 (Swiss-Prot
accession number Q60994, acr3_mouse) and human type X
collagen (Swiss-Prot accession number Q03692, ca1a_human)
from the end of the triple helical domains to their carboxyl
termini. Conserved residues are shown in white font
with black shading; * indicates the position of
amino acid substitutions causing MCDS; -strands are indicated by
shaded boxes and labeled according to ACRP30 and TNF-
nomenclature (24). The alignment was calculated in Multalin (28) with
default parameters then adjusted by hand in the triple helical
region.
-strands.
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Fig. 3.
a, type X collagen non-collagenous
carboxyl-terminal domain (Met548 to Met682)
trimer in protein cartoon representation. Modeled using the crystal
structure of ACRP30 trimer (24) in QUANTA®. Each monomer is a
different color. Both the amino and carboxyl termini of this domain
emerge at the base; the amino terminus has an additional 27 residues
followed by the triple helical domain. The asymmetry can be seen
particularly with the (blue) loop of M3 near the apex of the
trimer. b, type X collagen NC1 trimer viewed from the base
looking down the tunnel formed in the center by the association of the
three monomers. The backbone is represented in pale green;
hydrophobic residues at the base of the tunnel are shown in red
licorice. c, Tyr598 and Ser600
shown from each monomer as viewed looking down the tunnel from the
base. A putative network of hydrogen bonds is formed with water
molecules and direct monomer-monomer contacts between these side
chains. The distances indicated by black dotted lines are
all within 2.3 Å. Residues are shown in licorice with
different colors for each monomer with the water molecules in
pale blue in a Van der Waals representation. d,
monomer backbone in green with residues that are mutated in
MCDS shown in purple with a Connolly surface as viewed from
the side of the trimer. Shown in the same orientation as M1 in
a. e, trimer backbone in green viewed
from below (same orientation as b). Residues mutated in MCDS
are shown with side chains in purple with a Connolly
surface.
M3; see
Fig. 3a). Significant monomer-monomer contacts include a
backbone hydrogen bond between the carbonyl oxygen of
Tyr623 and the amide nitrogen of His669
(between M2-M3 and M3-M1). M1-M2 has a different backbone contact involving the carbonyl oxygen of Gly631 and the amide
nitrogen of Leu633. Examination of the NC1 trimer model
revealed that the hydrophobic side chains of Pro568,
Pro570, Ile588, Trp611, and
Trp651 are exposed to solvent on the external surface of
the trimer.
-strand G (Figs. 1 and 2). A detailed summary of the
location and orientation of amino acid residues substituted within the
NC1 domain in MCDS is presented in Table
I.
Predicted location and orientation of NC1 domain amino acid
substitutions causing MCDS
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Fig. 4.
Recombinant NC1 domain of human type X
collagen forms a stable SDS-PAGE-resistant trimeric complex in
vitro. DNA constructs encoding either the full-length NC1
domain (F), or an extended NC1 domain possessing the first
11 Gly-X-Y repeats at the amino terminus (E), or
an NC1 domain truncated by 27 residues from the amino terminus
(T) were transcribed and translated in the presence of
[35S]methionine by an in vitro expression
system. a, autoradiographs of translated samples exposed to
6.7% SDS and then resolved by standard SDS-PAGE analysis under
reducing conditions without prior heating. Lane 1,
expression of truncated (T) NC1 construct; lane
2, co-expression of truncated and extended (E) NC1
constructs; lane 3, expression of the extended NC1
construct; lane 4, expression of the full-length
(F) NC1 construct; lane 5, co-expression of
full-length and extended NC1 constructs. b, 12% SDS-PAGE
analysis of immunoprecipitations of translations containing normal
length and extended NC1 domains. Lane 1, whole
co-translation reaction; lane 2, material immunoprecipitated
by monoclonal antibody X34; lane 3, negative
control, material bound nonspecifically by the protein A-Sepharose
only. c and d, full-length (F in
c) and truncated (T in d) NC1
constructs were analyzed exactly as described in a except
that the samples were heated at the temperatures indicated
above the gels for 5 min prior to loading. Top
indicates the boundary between the stacking and resolving gels.
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Fig. 5.
a, analysis of the thermal stability of
normal, Y598F, and Y598A NC1 domain trimers. Samples were analyzed as
described in Fig. 4, b and c, having been heated
in 6.7% SDS prior to loading on the gel. The relative proportion of
trimer: total labeled NC1 domain was quantified in each case by
phosphorimaging the gel. The data plotted represents the mean ± S.E. (n = 3). The autoradiograph to the
right of the graph shows radiolabeled normal
(N), Y598F, and Y598A monomers (NC11) and
trimers (NC13) as separated by SDS-PAGE after
incubation for 5 min at 20 °C in the above conditions. b,
SDS-PAGE analysis of NC1 homotrimers harboring MCDS point mutations.
5-µl aliquots of translation reactions containing
35S-labeled normal NC1 domain (N) and NC1
domains harboring Y598D and S600P substitutions were mixed with sample
buffer (6.7% SDS final concentration) and separated, without prior
heating, by SDS-PAGE followed by autoradiography. The positions of
monomers (NC11) and the normal trimer
(NC13) are indicated. c, detection of NC1
homotrimers and heterotrimers harboring MCDS mutations in low SDS
conditions. Constructs encoding NC1 domains with the Y598D and S600P
substitutions were expressed alone and co-expressed with the construct
encoding the extended normal NC1 domain. 5-µl aliquots of translation
reactions containing 35S-labeled NC1 domains were mixed
with sample buffer (0.5% SDS final concentration) and separated,
without prior heating, by SDS-PAGE. Lane 1, expression of
normal full-length NC1 construct; lane 2, co-expression of
normal full-length and extended constructs; lane 3,
expression of Y598D construct; lane 4, co-expression of
Y598D and extended normal constructs; lane 5, expression of
S600P construct; lane 6, co-expression of S600P and normal
extended constructs. The positions of monomers
(NC11) and trimeric complexes
(NC13) are indicated.
DISCUSSION
-strand jelly roll motifs based on modeling of the
ACRP30 crystal structure, which is remarkably similar to the structure
of the TNFs (24). In both TNF and C1q, the sequences involved in
molecular recognition (e.g. receptor and IgG binding,
respectively) have been shown to include loop D--E and loop
A-A' (see Ref. 24 and references therein). These sequences map to two
specific exposed patches on each monomer within the assembled trimer.
Interestingly, the residues forming these loops differ in composition
and in length across the whole family of type X collagen-like proteins
as shown in Figs. 1 and 2. These data suggest strongly that the
equivalent regions in type X collagen are involved in molecular
recognition events such as supramolecular assembly (6) or interaction
with cells.
1(X) chains containing similar MCDS substitutions have
recently been shown to fold stable collagenous triple
helices.4
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ACKNOWLEDGEMENTS |
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We thank Larry Shapiro for the ACRP30 coordinates, Klaus Von der Mark for providing the X34 monoclonal antibody, and Mike Grant and Neil Bulleid for constructive discussions and suggestions.
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FOOTNOTES |
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* This work was supported by Grants B0547 and G0520 from the Arthritis Research Campaign and Grant 019512 from The Wellcome Trust.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.
These authors contributed equally to the data presented in this paper.
§ To whom correspondence should be addressed: University of Manchester, 2.205 Stopford Bldg., Oxford Rd., Manchester, M13 9PT United Kingdom. Tel.: 44 161 275 5097; Fax: 44 161 275 5082; E-mail: ray.boot-handford{at}man.ac.uk.
The abbreviations used are: NC1, non-collagenous carboxyl-terminal domain of type X collagen; MCDS, metaphyseal chondrodysplasia type Schmid; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; TNF, tumor necrosis factor; nt, nucleotide(s).
2 L. Shapiro, personal communication from Albert Einstein College of Medicine, New York.
3 D. S. Marks, C. A. Gregory, G. A. Wallis, A. Brass, K. E. Kadler, and R. P. Boot-Handford, unpublished observations.
4 S. McLaughlin and N. Bulleid, personal communication from School of Biological Sciences, University of Manchester, United Kingdom.
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REFERENCES |
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