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INTRODUCTION |
Type X collagen is a small non-fibrillar collagen
(Mr 59,000) synthesized exclusively in the
epiphyseal growth plate during chondrocyte hypertrophy (1). The overall
domain structure of type X collagen is similar to the fibrillar
procollagens in that there are three distinct domains: a short
amino-terminal domain (NC2)1 of 38 amino acids, a collagenous triple helical domain of 463 amino acids,
and a non-collagenous carboxyl-terminal (NC1) domain of 161 amino acids
(2). However, unlike the fibrillar procollagens, the propeptides are
not cleaved following secretion into the extracellular matrix and are
thought to play a role in assembly of the individual molecules into
higher order structures (3).
Mutations within the COL10A1 gene result in Schmid metaphyseal
chondrodysplasia (SMCD), an autosomal dominant disorder of the osseous
skeleton. The majority of these mutations map to the NC1 domain and
include amino acid substitutions, nonsense mutations, and deletions
(4). The only exceptions so far found are at the junction of the signal
sequence and the NC2 domain (5) that may effect signal peptide
cleavage. Significantly no mutations within the triple helical domain
have been identified. Mutation within the NC1 domain has been proposed
to effect the initial stages in the folding and assembly of type X
collagen which are thought to occur in a similar fashion to the
fibrillar procollagens (6). Once fully translocated into the ER lumen,
the constituent chains of the trimer associate via their
carboxyl-terminal globular domains allowing nucleation of the triple
helix to occur at the carboxyl-terminal end (7) with subsequent
propagation of the helix in a zipper-like fashion from the COOH- to the
NH2-terminal direction (8). Therefore mutations in the NC1
domain would be predicted to disrupt the initial association of type X
chains and prevent nucleation of the triple helix.
This hypothesis has been supported by studies on the in
vitro transcription and translation of mutant and wild-type
cDNAs within a cell-free system (9). The ability of the NC1 domain to associate and form trimers was assayed by the resistance of the
trimer to denaturation by SDS. The wild-type trimer was shown to be
particularly resistant to dissociation by SDS, a property not shared by
the mutant chains. These results were taken to indicate that the mutant
chains did not assemble and therefore that the effect of the mutation
was to prevent correct folding of the mutant chain leading to its
retention within the ER and subsequent degradation. Thus attempts to
express mutant type X chains in cells grown in culture led to very poor
levels of expression with little or no secretion of the mutant chains
(10).
Lack of secretion of mutant chains would result in a reduction of the
extracellular type X available for assembly. Thus, the current model
for the mechanism of SMCD is haploinsufficiency. Here the phenotype
would be explained simply by a 50% reduction in the level of type X
secreted rather than a direct effect of the mutant chains on the
assembly of the wild-type chains. This model is supported by the
failure to detect, in one SMCD patient, any mutant type X protein in
the extracellular matrix due to the degradation of the mutant
transcript that contains a premature stop codon (11). This is
corroborated by analysis of transgenic mice that are null alleles for
type X that show some of the phenotypic changes associated with SMCD
(12). However, analogous transgenic null allele mice show no
significant phenotypic change (13).
Other evidence, however, raises the possibility that haploinsufficiency
may not be the only mechanism underlying SMCD. Transgenic mice
expressing chicken type X with a truncated helix show skeletal abnormalities (14), pointing toward a dominant negative interference of
the function of type X in the extracellular matrix. Trace amounts of
heterotrimer could be detected during cell-free co-expression of
wild-type and some point mutants (9) suggesting some co-assembly of
these chains could occur. Significantly, the distribution of mutations
within the NC1 domain are not random but clustered into three distinct
regions (4) which is counterintuitive to a general folding defect in
type X unless these regions alone are critical for assembly.
The expression studies of mutant and wild-type chains using cell-free
systems did not fully reconstitute the initial stages of collagen
assembly (9, 10). We have previously demonstrated that the complete
folding pathway of type X collagen can be reconstituted in
semi-permeabilized cells (SP cells) (15). This system was used here to
address the question of whether mutant type X collagen chains can
initiate triple helix formation within the ER lumen. We expressed type
X containing SMCD mutations within each of the three clusters: Y598D
(16), G617K (17), and W651R (18), and analyzed their effects on
folding, assembly, and triple helix formation (15). In contrast to
previous studies, we demonstrate that all three mutants can
individually associate to form homotrimers to form thermally stable
triple helices. We also show that heterotrimers can form between the
wild-type and mutant type X and that the pattern of binding of the
wild-type and mutant chains to molecular chaperones is identical.
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EXPERIMENTAL PROCEDURES |
Construction of Recombinant Plasmids and
Mutagenesis--
Recombinant bovine type X maintained in pSKBX has
been previously described (15). Site-directed mutagenesis was carried out using the Quickchange kit (Stratagene Ltd., Cambridge, United Kingdom) using the accompanying protocol. Using pSKBX as a template, the following complementary primers were used to generate point mutations (underlined) in the NC1 domain of bovine type X (amino acids
are numbered from the translation start site of human
pre-
1(X)): 5'-GATTCCAGGGATATATGACTTCTACCAC-3' for
Tyr598
Asp (pSHM14);
5'-ACGACCAGGTAAGGCTCCAGCTG-3' for Trp651
Arg (pSHM15); and 5'-GTAGGTCTGTATAAGAAGGGCACCCCTGTA-3' for Asn617
Lys, (pS2). HA-tagged and Myc-tagged wild-type
and mutant type X molecules were generated by PCR overlap extension
using the principles outlined by Horton (19). PCR reactions (100 µl)
comprised template DNA (250 ng), oligonucleotide primers (100 pmol
each) in 20 mM Tris-HCl, pH 8.8, containing 10 mM KCl, 10 mM
(NH4)2SO4, 2-8 mM
MgSO4, 0.1% (v/v) Triton X-100, and 300 µM
each dNTP. Thirty rounds of amplification were performed in the
presence of 1 unit of Vent DNA polymerase (New England Biolabs,
Beverly, MA). Primary PCR products of approximately 300 base pairs
were generated using a 5'-amplification primer (5'-
CGCGCGTAATACGACTCACTATAG-3') which is complimentary to the T7 promoter
sequence upstream of the initiation codon of type X with either SMYCR2
(5'-CAAGTCCTCTTCAGAAATGAGCTTTTGCTCATAAAACACTCCAGAACCAAGT-3') or SMHAR2
(5'-AGCGTAATCGGGTACGTCGTAGGGATAAAACACTCCATGAACCAAGT-3') which are complimentary to the signal sequence and incorporate the
coding sequence for the Myc or HA-tag, respectively (underlined). Using
5'-amplification primers SMYCF1
(5'-GAGCAAAAGCTCATTTCTGAAGAGGACTTGACTGAGCGATACCAAACACC-3') or SMHAF1
(5'-CCCTACGACGTACCCGATTACGCTACTGAGCGATACCAAACACC-3'), which are complimentary to the start of the coding sequence for the mature type X with the 3'-amplification primer BXC
(5'-AGCTGGAGCCATACCTGGTCGT-3') which anneals within the coding region
for the NC1 domain, primary PCR products of approximately 1.9 kilobases
were generated. Primary PCR products were purified and used in overlap
extension reactions that generated 2.2-kilobase products which were gel
purified, EcoRV-PstI digested, and ligated into
pSKBX, pSHM14, pSHM15, and pS2 which had previously been digested with
EcoRV-PstI. All mutants and constructs were
verified by sequencing.
In Vitro Transcription--
Transcription reactions were carried
out as described by Gurevich et al. (20). Full-length
transcripts were obtained from 10 µg of pSKBX, pSHM14, pSHM15, or pS2
linearized with NotI. Transcription was initiated with T7
RNA polymerase (Promega, Southampton, UK) and carried out for 2 h
at 37 °C. RNA was purified on RNeasy spin columns (Qiagen, Dorking,
UK) using the supplied buffers, eluting with 50 µl of diethyl
pyrocarbonate-treated sterile distilled water containing 40 units of
RNasin (Promega) and 5 mM dithiothreitol.
In Vitro Translation in Semi-permeabilized Human Fibroblasts
(HT1080)--
Cultured HT1080 fibroblasts (ATCC-CCL121, American Type
Culture Collection, Rockville, MD) were permeabilized with the
detergent digitonin (Calbiochem, Nottingham, UK) at a final
concentration of 40 µg/ml using a modification of the method of
Plutner et al. (21) as described in Ref. 22. RNA was
translated using a rabbit reticulocyte lysate (Flexi-Lysate, Promega)
at 30 °C in the absence of exogenous dithiothreitol. The translation
reaction (25 µl) contained 17.5 µl of reticulocyte lysate, 0.5 µl
of 1 mM amino acids (minus methionine), 0.5 µl of 2.5 M KCl, 1.5 µl of
L-[35S]methionine (10 mCi/ml, ICN Biomedicals
Ltd., Thane, Oxfordshire, UK), 1 µl of transcribed RNA, and 4 µl of
semi-permeabilized HT1080 cells. Ascorbate (Sigma, Poole, Dorset, UK)
was added to a final concentration of 0.25 mM. Translations
were incubated for 60 min at 30 °C. After translation,
N-ethylmaleimide was added to 20 mM and aliquots
were incubated on ice for 10 min. SP cells were pelleted by
centrifugation and resuspended in 1 ml of KHM buffer (110 mM KOAc, 20 mM Hepes, pH 7.2, 2 mM
MgOAc) and re-isolated prior to endoglycosidase H digestion, chemical
cross-linking, proteolysis, or electrophoresis.
Endo H Digestion--
Products of translation were digested with
endoglycosidase H as described previously (23)
Chemical Cross-linking--
After translation cell pellets were
resuspended in 100 µl of KHM buffer and dithiobis(succinimidyl
propionate) (DSP, Sigma) was added to 1 mM final
concentration from a 100 mM stock in dimethyl sulfoxide,
followed by incubation at room temperature for 10 min. The cross-linker
was quenched by addition of glycine to 40 mM, and
incubating for a further 5 min.
Prior to immunoprecipitation the cross-linked samples were denatured by
adding SDS to 0.5% (w/v) final concentration and heating to 100 °C
for 1 min. Immunoprecipitation buffer (50 mM Tris-HCl, pH
7.4, 150 mM NaCl, 5 mM EDTA, 0.25% (w/v)
gelatin, 0.10% (v/v) Nonidet P-40) was then added to 1 ml and samples
precleared by incubation with 40 µl of protein A-Sepharose (10%
(v/v) preincubated in NET-gel buffer containing 5% bovine serum
albumin) (Zymed Laboratories Inc., San Francisco, CA)
for 40 min at 4 °C. After removal of protein A-Sepharose-bound
material by centrifugation, the appropriate antiserum was added at a
dilution of 1:500 along with 40 µl of protein A-Sepharose and samples
incubated for 3 h at 4 °C. The goat anti-BiP polyclonal
antibody was supplied by Santa Cruz Biotechnology, Inc., Santa Cruz,
CA. The mouse anti-Hsp 47 monoclonal antibody was from Stressgen
Biotechnologies Corp., Victoria, Canada. The rabbit anti-protein
disulfide isomerase polyclonal antibody was raised to purified bovine
protein disulfide isomerase as described previously (24). After
incubation, pelleted complexes were washed three times with 1 ml of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% (v/v) Triton X-100.
Sequential Immunoprecipitations--
After translation cells
were solubilized in 1 ml of immunoprecipitation buffer and samples
precleared by incubation with 40 µl of 10%(v/v) protein A-Sepharose.
After removal of protein A-Sepharose-bound material by centrifugation,
mouse anti-Myc monoclonal antibody (9E10) (Calbiochem, Nottingham, UK)
was added at a 1:500 dilution along with 40 µl of 10% (v/v) protein
A-Sepharose. After incubation, pelleted complexes were washed three
times with 1 ml of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% (v/v) Triton X-100. The
material immunoprecipitated with the anti-Myc antibody was eluted from
the beads by addition of SDS to 1% (w/v) in a volume of 100 µl and
heating to 100 °C. A small volume (10 µl) was taken for gel
analysis while the rest of the sample was diluted to 1 ml in
immunoprecipitation buffer. Any HA-tagged products were then
immunoprecipitated with a rabbit anti-HA (12CA5) antibody diluted to
1:500 (Boehringer Mannheim, Lewes, E. Sussex, UK).
Chymotrypsin/Trypsin Digestion--
Cell pellets were
resuspended in CT/T buffer (50 mM Tris-HCl, pH 7.4, containing 0.15 M NaCl, 10 mM EDTA, 0.5% (v/v)
Triton X-100). Chymotrypsin and trypsin (Sigma) were added to 250 and 100 µg/ml final concentrations and samples incubated at room
temperature for 5 min. Further digestion was stopped by the addition of
soybean trypsin inhibitor to a final concentration of 5 mg/ml and 2.5 volume of boiling SDS-PAGE loading buffer containing 5% (v/v) 2-merceptoethanol, and the samples were boiled for 5 min.
Thermal Denaturation--
Pelleted SP cells were resuspended in
CT/T buffer and 16-µl aliquots were placed in a DNA thermal cycler. A
stepwise temperature gradient was set up from 25 to 47 °C with the
temperature being held for 2 min at 2 °C intervals. For a given
temperature at the end of 2 min the sample was assayed with
chymotrypsin/trypsin for 2 min as described above.
SDS-PAGE--
Samples were prepared for electrophoresis by
mixing with an equal volume 2 times SDS-loading buffer (0.125 M Tris-HCl, pH 6.8, 2% (w/v) SDS, 20% (v/v) glycerol, and
0.004% (w/v) bromphenol blue). When resistance to denaturation by
heating in the presence of SDS was assayed a lower final concentration
of SDS was used (0.5% w/v). Samples were reduced by addition of 5%
(v/v) 2-merceptoethanol. The samples were boiled for 5 min before
electrophoresis, unless otherwise stated. After electrophoresis, dried
gels were exposed to Kodak X-Omat AR film or analyzed on a Fuji BAS
2000TR PhosphorImager.
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RESULTS |
Analysis of the Ability of Wild-type and SMCD Mutants to Form
Trimers Resistant to Denaturation with SDS--
Previous experiments
carried out to determine the ability of wild-type and mutant type X
chains to form homotrimers involved expressing the RNA transcripts
coding for these proteins in a cell-free system. The ability to
assemble was assessed by the formation of a trimer that was resistant
to denaturation after heating to 60 °C in presence of 2% (w/v) SDS
and 2 M urea (9, 10). Under these conditions only the wild
type was able to form a resistant trimer suggesting that the effect of
the mutation was to prevent trimer formation. We wanted to determine
whether the mutant chains were able to form trimers at all and whether the trimers formed would be more stable under milder denaturing conditions.
For our experiments we used the bovine cDNA clone, the NC1 domain
of which shares over 90% identity with the human NC1 domain. All the
amino acids that have been identified as targets for mutations giving
rise to SMCD are identical. Thus we constructed three analogous SMCD
point mutations: Y598D which lies within a hydrophobic region of the
NC1 domain predicted to be critical for trimer association (25); G617K
which disrupts the potential N-glycosylation site (Asn-Gly-Thr) in the NC1 domain (17); and W651R which lies in the third
cluster of mutations in the NC1 domain (18). The cDNAs clones
coding for these mutants were transcribed in vitro and translated in a reticulocyte lysate supplemented with SP cells that
been shown to reconstitute the initial folding and assembly of
fibrillar collagens and type X collagen (15, 26). The ability of the
various mutants to assemble to form trimers was assayed by their
resistance to denaturation by heating in the presence of 0.5% (w/v)
SDS. As can be seen (Fig. 1) the
wild-type and mutant chains were all able to assemble to form trimers
which were resistant to denaturation when heated up to 60 °C but
which were fully denatured at 100 °C. The N617K mutant formed these
resistant trimers less efficiently than either the wild-type or other
mutant chains, but the fact that any resistant trimers are formed
demonstrates that the mutated NC1 domains can associate with themselves
to form homotrimers. We confirmed previous results (9) demonstrating that the trimerization of the wild-type and mutant chains to form SDS-resistant molecules was due to an association at the NC1 domain by
carrying out collagenase digestion (results not shown). At higher SDS
concentrations all the mutant chains were denatured when heated to
60 °C whereas the wild-type chain was not (results not shown),
confirming the previous results obtained with the human type X SMCD
mutant chains (9).

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Fig. 1.
Stability of wild-type and mutant chains of
type X collagen to denaturation after heating in the presence of
SDS. Type X collagen mRNA coding for either wild-type
(lanes 1-3) or mutated (lanes 4-12) chains was
translated in a reticulocyte lysate supplemented with SP cells for 60 min at 30 °C. Products of translation were separated by SDS-PAGE
through a 7.5% polyacrylamide gel after heating to the indicated
temperatures in the presence of 0.5% (w/v) SDS for 5 min and
visualized by autoradiography.
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The fact that the N617K mutant forms a trimer resistant to denaturation
under these conditions less efficiently than the other chains could be
due to this mutation abolishing the consensus sequence for
glycosylation. This in turn could effect the solubility of the monomer
and therefore the assembled trimer. As there is some confusion as to
whether this glycosylation site is actually utilized in type X collagen
we carried out endoglycosidase H digestion of the wild-type and mutant
chains following translation in the presence of SP cells to determine
the glycosylation status. As a control we translated tissue-type
plasminogen activator which contains three sites for N-link
glycosylation (27). Clearly the tissue-type plasminogen activator
translation product is susceptible to digestion with endoglycosidase H
(Fig. 2, lanes 1 and
2) whereas there is no difference in mobility of the
wild-type or N617K translation products after digestion (Fig. 2,
lanes 3 and 4, lanes 5 and 6). There
is also no difference in mobility between the wild-type and N617K
mutant which would be expected if the glycosylation site was occupied.
These results clearly demonstrate that this site is not occupied in
type X collagen. The actual sequence in type X collagen has a proline
residue following the glycosylation site. A proline in this position
has previously been shown to inhibit glycosylation (28) providing a
reason for the lack of occupancy of this site in type X collagen.

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Fig. 2.
Sensitivity of wild-type and N617K mutated
type X collagen chains to digestion with endoglycosidase H. Translation of mRNA coding for tissue-type plasminogen activator
(tPA) (lanes 1 and 2) type X collagen
(lanes 3 and 4) of N617K mutant type X collagen
(lanes 5 and 6) was carried out in a reticulocyte
lysate in the presence of SP cells for 60 min at 30 °C. Products of
translation were denatured and incubated either in the absence
(lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of 1 milliunit of endoglycosidase H for
16 h at 37 °C. Treated translation products were then separated
by SDS-PAGE through a 10% polyacrylamide gel and visualized by
autoradiography.
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Can Mutant Chains Assemble to Form Correctly Folded Triple Helical
Molecules?--
Once we had shown that the type X chains containing
point mutations associated with SMCD could associate to form
homotrimers, we then wanted to determine whether the triple helical
domains within these chains would fold correctly. Translation was
carried out in the presence of SP cells and ascorbate to ensure
hydroxylation of proline residues and the formation of a triple helix
was assayed by resistance to digestion by a combination of trypsin and
chymotrypsin. The results (Fig.
3A) show that
protease-resistant fragments corresponding to the collagenous domain of
type X were generated after treatment of both wild-type and mutant
chain with proteases. The efficiency of triple helix formation was
determined by quantification of the translation products before and
after protease treatment. The results (average over three experiments)
illustrated that the wild-type chain folded efficiently (94%) whereas
the mutant chains folded less efficiently than the wild-type (Y598D,
55%; N617K, 63%; W651R, 70%). The thermal stability of the triple
helical domains formed by the wild-type and mutant chains was also
assayed (Fig. 3B). The denaturation temperatures of the
triple helix formed were very similar indicating that the extent of
hydroxylation of the type X collagen is not influenced by these
mutations in the NC1 domain. Thus the mutant chains are able to
associate to form trimers and once these trimers have formed they can
fold correctly to form a stable triple helix.

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Fig. 3.
Protease treatment of products of translation
to assay for triple helix formation. Type X collagen mRNA was
translated in a reticulocyte lysate supplemented with SP cells 60 min
at 30 °C. Isolated cells were lysed and either analyzed directly
(lanes 1, 3, 5, and 7) or digested with
chymotrypsin and trypsin (lanes 2, 4, 6, and 8)
and analyzed by 7.5% SDS-PAGE and autoradiography (A). The
intensities of protease-digested bands remaining after digestion of the
products at varying temperatures were quantified by PhosphorImaging and
plotted relative to the 25 °C sample (100%) (B).
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Can the Mutant Chains Co-assemble with Wild-type
Chains?--
Having established that mutant chains can form
homotrimers we then went on to determine if the mutant chains could
co-assemble with wild-type chains to form heterotrimers. The approach
we took was to add one epitope tag (HA) to the wild-type chain and a
different epitope tag (Myc) to a mutant chain (for these experiments we used the Y598D mutant). After co-translation of the two chains in the
presence of SP cells we first immunoprecipitated the mutant chains with
an anti-Myc antibody. Any wild-type chains that co-assembled with the
mutant chains would also be immunoprecipitated under these conditions.
The co-assembled wild-type chains could then be identified by first
denaturing the anti-Myc immunoprecipitate and then immunoprecipitating
with anti-HA. As a positive control for co-assembly, we tagged
wild-type chains with Myc and co-translated Myc-tagged along with
HA-tagged wild-type chains. We first established that the antibodies
could specifically recognize the variously tagged chains by translating
them individually. The wild-type chain tagged with the HA epitope was
not immunoprecipitated with anti-Myc and consequently no radiolabeled
product was immunoprecipitated with anti-HA from the anti-Myc
immunoprecipitate (Fig. 4, lanes 1 and 2). In contrast, the Y598D chain tagged with the
Myc epitope was immunoprecipitated with anti-Myc but no radiolabeled
protein was immunoprecipitated with anti-HA from the anti-Myc
immunoprecipitate (Fig. 4, lanes 3 and 4). These
results demonstrate that there is no nonspecific immunoprecipitation of
chains using this sequential immunoprecipitation approach. When the
wild-type chain tagged with the HA epitope was co-translated with
either the wild-type chain or the Y598D chain tagged with the Myc
epitope, radiolabeled material was present in the anti-Myc
immunoprecipitate (Fig. 4, lanes 5 and 7).
Between 5 and 10% of this material was subsequently immunoprecipitated
by anti-HA (Fig. 4, lanes 6 and 8) demonstrating that the HA-tagged wild-type or the HA-tagged Y598D chains co-assembled with the Myc-tagged wild-type chains. We carried out a similar experiment by first immunoprecipitating with the anti-Myc antibody, carrying out a second immunoprecipitation with the anti-HA antibody, and obtained similar results (results not shown). These results demonstrate that within the environment of the ER both wild-type and
chains containing SMCD mutations can co-assemble to form
heterotrimers.

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Fig. 4.
Co-translation and sequential
immunoprecipitation of wild-type and mutated type X collagen
chains. Type X collagen mRNA was translated in a reticulocyte
lysate in the presence of SP cells for 60 min at 30 °C. RNA coding
for wild-type tagged with HA epitope was translated alone (lanes
1 and 2) or co-translated with either wild type
(lanes 5 and 6) or Y598D (lanes 7 and
8) tagged with the Myc epitope. Y598D RNA was also
translated alone (lanes 3 and 4). Products of
translation were first immunoprecipitated with an antibody to the Myc
epitope (lanes 1, 3, 5, and 7). The Myc
immunoprecipitate was denatured and after dilution re-precipitated with
an antibody specific for the HA epitope (lanes 2, 4, 6, and
8). Immunoprecipitates were analyzed by SDS-PAGE through a
10% gel and radiolabeled proteins visualized by autoradiography.
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Interaction of Type X Collagen Chains with Molecular
Chaperones--
The main conclusions that could be drawn from the
preceding experiments was that both wild-type and mutant chains can
assemble and fold to form homo- or heterotrimers. The trimers formed,
however, differed in their sensitivity to heat denaturation in the
presence of SDS which would suggest that the NC1 domain formed from
mutant chains adopted a more open conformation than the NC1 domain
formed from wild-type chains. One consequence of this change in
conformation could be a more stable interaction with chaperone proteins
within the ER, which in turn would lead to retention of the mutant
protein within the cell. Alternatively mutant chains could interact
with a different set of chaperones than the wild-type chains resulting in differential retention. To assess this point we determined whether
there were any differences in the types of molecular chaperones that
the wild-type or mutant chains interacted with in our SP cells. We
translated both the wild-type and mutant chains individually and after
isolation of the cells from the translation mixture cross-linked
translated chains to interacting proteins using the bifunctional
cross-linking reagent DSP. Essentially we obtained the same results
irrespective of the type of chain we translated (Fig.
5). We have previously shown that the two
main proteins that interact with type X are protein disulfide isomerase
(Fig. 5, lanes 1-4) and Hsp 47 (Fig. 5, lanes
9-12). Previous work on mutation within the C-propeptide of
fibrillar procollagens has shown that mutations that disrupt folding of
the monomer result in an interaction of the misfolded chain with BiP
(29). We could not detect any interaction of either the wild-type or
mutant chains with BiP using this approach even though we have
previously shown that the anti-BiP antibody can immunoprecipitate BiP
following this cross-linking treatment (30). Thus our results do not
suggest any significant differences in the way in which the main
collagen binding chaperones interact with wild-type and mutant
chains.

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Fig. 5.
Cross-linking of type X collagen chains to
molecular chaperones. Type X mRNA was translated as described
in the legend to Fig. 4. After translation the SP cells were isolated
and resuspended in KHM buffer prior to cross-linking with DSP.
Cross-linked products were immunoprecipitated with antibodies to
protein disulfide isomerase (PDI) (lanes 1-4),
BiP (lanes 5-8), or Hsp 47 (lanes 9-12).
Cross-links were disrupted by reduction with dithiothreitol prior to
separating the samples by SDS-PAGE through a 10% gel and visualizing
the radiolabeled proteins by autoradiography.
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DISCUSSION |
The main objective of this study was to ascertain whether the
phenotype underlying SMCD, which has been suggested to be due to a
reduction in the level of secretion of mutant chains, could be
explained by an intracellular folding and transport defect. The
previous studies on the synthesis and folding of type X collagen chains
containing point mutations associated with SMCD clearly demonstrated a
difference in the resistance of the protein to dissociation by heating
in the presence of SDS and urea (9, 10). Here we show that this
observation does not mean that the mutant chains cannot assemble to
form trimers, only that the trimers once formed are more susceptible to
dissociation than the wild-type under the conditions of this assay.
Indeed the mutant chains were not only able to form homotrimers but
were capable of nucleating triple helix formation to form a thermally
stable triple helix. Interestingly the mutant chains were also able to
co-assemble with the wild-type chains to form heterotrimers. These
conclusions do not rely on the formation of NC1 domain trimers
resistant to heating in the presence of SDS. Thus by three independent
assays we have shown that point mutations associated with SMCD do not prevent association of the mutant chains with themselves or with wild-type chains. These results prove that the molecular mechanism underlying SMCD cannot be exclusively explained by haploinsufficiency as the mutant chains can co-assemble with wild-type chains. There is,
however, at least one case where a 50% reduction of the level of
expression of type X collagen leads to a SMCD phenotype (11).
Clearly the mutations do alter the conformation of any homotrimers
formed and would be predicted to have varying effects on the
conformation of heterotrimers. This could lead to retention of trimers
containing mutant chains within the ER with the consequence of a
reduction in the level of type X chains secreted. Such a mechanism of
ER "quality control" prevents the secretion of incompletely assembled or malfolded proteins and can be mediated either by glycoprotein-specific chaperones such as calnexin and calreticulin (31), by interaction with BiP or other ER-resident proteins (32), or by
a thiol-mediated process (33). Attempts to evaluate whether homotrimers
containing mutant chains could be secreted from intact cells have been
hampered by their low levels of expression (10). This low level of
secretion in itself could be due to an efficient mechanism of retention
of mutant chains leading to an increase in the intracellular
degradation of the mutant homotrimers. No attempts to express both
wild-type and mutant chains in the same cell have yet been reported and
it would indeed be interesting to determine if heterotrimers of mutant
and wild-type chains could be secreted. In this scenario the SMCD
phenotype would be explained not by a lack of folding and assembly of
the mutant chains but rather by a decrease in their stability with the
overall result that fewer type X chains would be secreted and be
available for assembly.
The alternative explanation to intracellular degradation of trimers
containing mutant chains is that these molecules are secreted normally
but interfere with the normal function of type X collagen outside the
cell. The most persuasive argument for such a mechanism to explain the
SMCD phenotype arises from the fact that only these specific mutations
give rise to SMCD. Other mutations within the NC1 domain that could
effect folding of the monomer or the association of the monomers to
form trimers would also be degraded and lead to a reduction in the
levels of type X collagen secreted and should give rise to a SMCD
phenotype. Thus the main characteristic of at least the point mutations
studied here which cause SMCD is that they do not cause a general
folding defect. This would argue that the effect of the point mutation
is on either the assembly of the protein outside the cell or on an
interaction with other extracellular matrix proteins as has been
suggested previously (4). The crucial experiment to verify such a
mechanism would be the isolation of type X molecules containing mutant
chains in the extracellular matrix of affected individuals.