Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M410758200 on February 4, 2005

J. Biol. Chem., Vol. 280, Issue 16, 15544-15552, April 22, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/16/15544    most recent
M410758200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wilson, R.
Right arrow Articles by Bateman, J. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wilson, R.
Right arrow Articles by Bateman, J. F.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Misfolding of Collagen X Chains Harboring Schmid Metaphyseal Chondrodysplasia Mutations Results in Aberrant Disulfide Bond Formation, Intracellular Retention, and Activation of the Unfolded Protein Response*

Richard Wilson{ddagger}, Susanna Freddi{ddagger}, Danny Chan§, Kathryn S. E. Cheah§, and John F. Bateman{ddagger}

From the {ddagger}Cell and Matrix Biology Research Unit, Murdoch Children's Research Institute, and the Department of Pediatrics, University of Melbourne, Royal Children's Hospital, Parkville, Victoria 3052, Australia and the §Department of Biochemistry, University of Hong Kong, Faculty of Medicine, Hong Kong, China

Received for publication, September 20, 2004 , and in revised form, January 20, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Collagen X is a short chain collagen expressed specifically by the hypertrophic chondrocytes of the cartilage growth plate during endochondral bone formation. Accordingly, COL10A1 mutations disrupt growth plate function and cause Schmid metaphyseal chondrodysplasia (SMCD). SMCD mutations are almost exclusively located in the NC1 domain, which is crucial for both trimer formation and extracellular assembly. Several mutations are expected to reduce the level of functional collagen X due to NC1 domain misfolding or exclusion from stable trimer formation. However, other mutations may be tolerated within the structure of the assembled NC1 trimer, allowing mutant chains to exert a dominant-negative impact within the extracellular matrix. To address this, we engineered SMCD mutations that are predicted either to prohibit subunit folding and assembly (NC1del10 and Y598D, respectively) or to allow trimerization (N617K and G618V) and transfected these constructs into 293-EBNA and SaOS-2 cells. Although expected to form stable trimers, G618V and N617K chains (like Y598D and NC1del10 chains) were secreted very poorly compared with wild-type collagen X. Interestingly, all mutations resulted in formation of an unusual SDS-stable dimer, which dissociated upon reduction. As the NC1 domain sulfhydryl group is not solvent-exposed in the correctly folded NC1 monomer, disulfide bond formation would result only from a dramatic conformational change. In cells expressing mutant collagen X, we detected significantly increased amounts of the spliced form of X-box DNA-binding protein mRNA and up-regulation of BiP, two key markers for the unfolded protein response. Our data provide the first clear evidence for misfolding of SMCD collagen X mutants, and we propose that solvent exposure of the NC1 thiol may trigger the recognition and degradation of mutant collagen X chains.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Collagen X is a major constituent of the pericellular matrix of hypertrophic chondrocytes within the cartilage growth plate (1), and the expression of collagen X during endochondral ossification is intimately linked to the onset of cartilage calcification and extracellular matrix remodeling (2). The absence of a functional collagen X network in mice is associated with displacement of proteoglycans, altered mineral deposition, compression of the growth plate, and hematopoietic changes (3–5). Based on these studies, it has been proposed that collagen X interactions within the cartilage extracellular matrix establish the correct microenvironment for matrix mineralization and subsequent bone development.

The human {alpha}1(X) collagen chain has a short collagenous domain flanked at the C and N termini by globular NC1 and NC2 domains. The NC1 domain plays a critical role in intracellular trimer formation (6, 7) and also contributes to the stability of extracellular collagen X networks (8, 9), probably involving aromatic residues exposed on the surface of each NC1 subunit (10). Mutations in the COL10A1 gene result in Schmid metaphyseal chondrodysplasia (SMCD),1 an autosomal dominant disorder of the osseous skeleton associated with growth plate deformities (11–13). Apart from two mutations that affect signal peptide cleavage (14), SMCD mutations are located in the NC1 domain (1, 15). Despite a lack of phenotypic variation in affected patients, the SMCD mutations identified to date range from amino acid substitutions to nonsense mutations and deletions resulting in premature termination codons. Analysis of the effect of SMCD mutations in vitro and in vivo has generated several alternative hypotheses for the underlying disease mechanism. In the case of nonsense mutations, analysis of cartilage from two SMCD patients with collagen X premature termination codons (W611X and Y632X) demonstrated complete nonsense-mediated decay of mutant mRNA (16, 17). Although cartilage has not been available from other patients with nonsense or frameshift mutations, recent studies using transfection of engineered mouse gene constructs into chondrocytes have shown that premature termination codon mutations throughout the gene region coding for the NC1 domain result in nonsense-mediated decay.2 However, premature terminations close to the normal stop codon (such as NC1del10) may escape degradation at the mRNA level and produce NC1 domains with short C-terminal truncations. These data strongly suggest that mRNA surveillance and degradation of transcripts with premature termination codons by nonsense-mediated decay, resulting in haploinsufficiency, are the most common disease mechanism in SMCD patients with nonsense or frameshift mutations.

Investigation of the effect of missense mutations on collagen X structure and function has relied on analysis of collagen X assembly and secretion in vitro. Both haploinsufficiency due to compromised collagen X assembly and dominant effects on collagen X function (either at the level of trimer formation or within the extracellular matrix) have been proposed as underlying disease mechanisms. Indeed, several SMCD missense mutants co-assemble with wild-type {alpha}1(X) collagen chains in vitro (18–20), consistent with a dominant-negative disease mechanism.

Careful analysis of the NC1 domain crystal structure and molecular modeling studies predict a degree of heterogeneity in the effect of missense mutations on NC1 domain folding and assembly (10, 20). Accordingly, mutations have been divided into three classes. Class I substitutions (C591R, G595E, Y597H, L614P, and L644R), which prohibit correct NC1 domain folding due to disruption of hydrophobic interactions within the subunit core, are likely to be excluded from collagen X assembly, resulting in haploinsufficiency. Class II mutations (S600P, S671P, and Y598D) are located within the solvent-filled region of the trimer center and may prevent the formation of stable NC1 trimers. However, class III mutations (N617K, G618V, D648G, and W651R) may be tolerated within the structure of the assembled NC1 trimer and perturb collagen X supramolecular assembly or interactions within the cartilage matrix.

Although supported by some experimental data, other results are inconsistent with these predictions. For example, in semipermeabilized cells, the N617K substitution results in marked reduction in trimer thermal stability compared with W651R, another class III mutation (18). In transfected cells and in a bacterial expression system capable of producing folded NC1 trimers, both Y598D and G618V mutations prevent collagen X assembly (21–23). Intriguingly, these data suggest that SMCD mutations that are theoretically tolerated within the native NC1 trimer, such as N617K and G618V, may still, in a cellular context, compromise trimer formation. This raises the possibility that the interaction of NC1 subunits may be altered by subtle conformational changes within structural elements of the NC1 domain apparently not directly involved in monomer or trimer stability.

We and others have shown that certain mutant {alpha}1(X) collagen chains are selectively degraded and do not interfere with wild-type collagen X secretion (21, 23). This suggests that collagen X assembly is monitored by an efficient quality control mechanism that recognizes misfolded NC1 domains prior to any detrimental effect on normal chain folding. In this study, we have selected SMCD mutations that are predicted either to prohibit subunit folding and assembly (NC1del10 and Y598D, respectively) or to allow trimerization and secretion (N617K and G618V). We have expressed mutant and wild-type collagen X in both 293-EBNA and SaOS-2 cells and found that, like Y598D and NC1del10, the N617K and G618V mutations also prevented efficient secretion. Furthermore, all four SMCD mutations resulted in formation of aberrant disulfide bonds, and expression of mutant collagen X triggered the unfolded protein response. Therefore, this present study is the first to demonstrate a direct link between SMCD mutations and NC1 domain misfolding in the endoplasmic reticulum (ER).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of Wild-type and Mutant Collagen X Expression Constructs for Stable Transfection—For the stable expression of His6-tagged collagen X in transfected cells, we generated five expression constructs (pCEP4-WT-His, pCEP4-Y598D-His, pCEP4-G618V-His, pCEP4-N617K-His, and pCEP4-NC1del10-His) encoding {alpha}1(X) sequences flanked at the N termini by sequences encoding the BM40 signal peptide, a His6 tag, and an enterokinase cleavage site. The wild-type, Y598D, and NC1del10 constructs have been described previously (21). To generate the pCEP4-G618V-His construct, collagen X sequence encoding the NC1 domain containing the G618V mutation was excised from the plasmid pTM1-h10GV (23) by BamHI digestion. This 740-bp fragment was ligated into pCEP4-WT-His from which the equivalent wild-type sequence had been excised by BamHI digestion. The pCEP4-N617K-His construct was created by strand overlap extension PCR using primer set N617K-F (5'-CTGTATAAGAAGGGCACCCCTGTA-3') and HX6-NotI (5'-GCGGCCGCCTTTTCAGCCTACCTCCATA-3') or primer set N617K-R (5'-TACAGGGTGCCCTTCTTATACAG-3') and HX-SP-NotI (5'-GCGGCCGCGTGTTTTACGCTGAACGATA-3') to generate independent fragments with overlapping sequences. Additional sequences encoding NotI restriction enzyme sites were added to the primers (underlined) to enable subsequent cloning into the plasmid pCEP4-BM40-HisEK (24). All PCRs were performed at 60 °C for 35 cycles using 0.5 units of DeepVent polymerase (New England Biolabs Inc.), and PCR products were purified by agarose gel electrophoresis using QIAquick columns (Qiagen Inc.). Second round PCRs were carried out with primers HX-SP-NotI and HX6-NotI using 5 ng of the primary PCR products as template. The resulting 2-kb recombinant PCR product was digested with NotI, purified, and ligated with pCEP-4-WT-His from which the equivalent wild-type sequence had been excised by NotI digestion. The construct was sequenced to verify the presence of the N617K mutation and to ensure that no PCR errors were introduced.

Stable Transfection of 293-EBNA and SaOS-2 Cells—The human embryonic kidney 293-EBNA cell line (Invitrogen) and the SaOS-2 osteosarcoma cell line (HTB-85, American Type Culture Collection) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal calf serum. Cells were passaged at a 1:3 ratio ~24 h prior to transfection to achieve 70% confluency at the time of transfection. For each transfection, 6 µg of collagen X expression construct DNA was combined with 20 µl of FuGENE 6 (Roche Diagnostics) according to the manufacturer's protocol. After 48 h, cells were passaged at a 1:6 ratio into DMEM containing 10% (v/v) fetal calf serum and 250 µg/ml hygromycin B (Roche Diagnostics) to select for positive transfectants. Cells were maintained in selection medium as a pool of transfectants and seeded simultaneously into 6-well dishes for subsequent mRNA extraction, protein analysis, and collagen X biosynthetic labeling.

Northern Blotting and Hybridization Analysis—RNA was prepared from transfected and untransfected cells cultured in 6-well dishes at ~80% confluence using the RNeasyTM extraction kit (Qiagen Inc.). Electrophoresis, transfer of RNA to nitrocellulose filters, and preparation of a collagen X-specific 32P-labeled probe were performed as described previously (21). Filters were exposed to autoradiography film or visualized by phosphorimaging (STORMTM, Amersham Biosciences) where indicated (see "Results").

Biosynthetic Labeling and Immunoprecipitation—Transfected cells were grown to confluence in 6-well plates and incubated for 16 h with 1 ml of methionine- and serum-free DMEM (Invitrogen) containing 0.25 mM sodium ascorbate and 100 µCi of L-[35S]methionine (Tran35S-label, 1032 Ci/mmol; ICN Pharmaceuticals Inc.). In pulse-labeling experiments, cells were pretreated for 1 h with 1 ml of methionine- and serum-free DMEM prior to incubation with 100 µCi of L-[35S]methionine for 1 h. After metabolic labeling, the medium was removed, and cells were rinsed once with phosphate-buffered saline. Immunoprecipitation of {alpha}1(X) collagen chains in cell lysates and media samples was performed using a mouse monoclonal antibody raised against the His6 epitope tag (Roche Diagnostics) at a final concentration of 1 µg/ml as described previously (21).

SDS-PAGE and Immunoblotting—Samples were prepared for electrophoresis by heating to 65 °C for 10 min in an equal volume of SDS-PAGE sample buffer (50 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 20% glycerol and 0.025% (w/v) bromphenol blue) unless otherwise stated. In all experiments, samples were resolved by SDS-PAGE on 7.5% (w/v) acrylamide gels, and labeled proteins were visualized by fluorography. For analysis of cell lysates by immunoblotting, cells were grown to confluence in 6-well dishes, lysed in immunoprecipitation buffer (21), and clarified by centrifugation at 13,000 x g for 20 min. Soluble proteins were precipitated for 1 h on ice using 9 volumes of ethanol and then recovered by centrifugation at 13,000 x g for 10 min. Proteins were solubilized in 2% (w/v) SDS, and protein concentration measured using the BCA assay (Pierce) to ensure equal loading (0.5 µg of protein/lane). After SDS-PAGE, proteins were transferred to nitrocellulose and incubated for 1 h in phosphate-buffered saline containing 0.1% (v/v) Tween 20 (Sigma) in the presence of 5% (w/v) milk protein. Mouse anti-KDEL monoclonal antibodies (Stressgen Biotech Corp.) were used at a final concentration of 1 µg/ml, and primary antibodies were detected using a 1:10,000 dilution of horseradish peroxidase-conjugated rabbit anti-mouse IgG (Dako Corp.). Rabbit anti-collagen X polyclonal antibodies raised against human recombinant collagen X were used at a dilution of 1:2000 and detected using a 1:10,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG (Dako Corp.). Blots were developed by chemiluminescence (ECLTM, Amersham Biosciences) according to the manufacturer's protocol.

Reverse Transcription (RT)-PCR and Primer Extension Analysis of Spliced XBP1 Transcripts—Transfected and untransfected cells were grown to confluence in 6-well dishes, and following a 2-h incubation in fresh DMEM containing 0.25 mM sodium ascorbate, RNA was extracted using the RNeasyTM extraction kit. RNA samples were treated with DNase I (Ambion, Inc.) to remove traces of genomic DNA. To generate PCR products corresponding to the spliced and unspliced forms of the XBP1 transcript, a cDNA template was first synthesized from mRNA with reverse transcriptase and random hexamers (Roche Diagnostics) as primers using 100 ng of total RNA/reaction. With sense (5'-GGAGTTAAGACAGCGCTTGG-3', bp 401–420) and antisense (5'-ACTGGGTCCAAGTTGTCCAG-3', bp 648 to 629) primers spanning the XBP1 RNA processing sequence (GenBankTM accession number AB076383 [GenBank] ), we amplified PCR products corresponding to unspliced and spliced XBP1 (248 and 222 bp, respectively) for 35 PCR cycles using a primer annealing temperature of 60 °C. The products were resolved on 2.5% (w/v) agarose gels and visualized under ultraviolet light. To differentiate between spliced and unspliced XBP1 RNAs by primer extension analysis, we used an 18-mer oligonucleotide (5'-TCTGCTGAGTCCGCAGCA-3', bp 490–507), which is extended by a single cytosine when annealed to unspliced XBP1 cDNA and by two guanosine nucleotides when annealed to spliced XBP1 cDNA. Template for each reaction was prepared by RT-PCR using the conditions described above. Primer extension reactions were performed essentially as described (17) using 5 ng of each cDNA template and 2 µCi of either [{alpha}-33P]dGTP or [{alpha}-33P]dCTP (PerkinElmer Life Sciences). The products were resolved on a 15% (w/v) denaturing polyacrylamide gel containing 7 M urea, and the radioactivity of the primer extension products was quantified by phosphorimaging.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of SMCD Point Mutations on Collagen X Secretion in Transfected 293-EBNA Cells—We have recently shown that collagen X chains harboring the SMCD mutation Y598D assemble very poorly in stably transfected cells and are retained and degraded (21). Analysis of the NC1 domain crystal structure revealed that the substitution of Tyr598 with a charged Asp residue would compromise trimer stability by interfering with tight intersubunit assembly (10). To further investigate the molecular mechanism underlying SMCD, we have analyzed two more SMCD missense mutations, G618V and N617K, which, unlike Y598D, are predicted to permit trimerization of {alpha}1(X) collagen chains (10, 20). The G618V and N617K substitutions map to the surface of the assembled NC1 trimer and are therefore believed to interfere with subsequent collagen X supramolecular assembly and/or extracellular interactions. For initial transfection experiments, we elected to use the human embryonic kidney cell line 293-EBNA transfected with His6-tagged collagen X cDNAs. Collagenous and non-collagenous human recombinant proteins expressed in 293-EBNA cells undergo post-translational modifications such as N- and O-linked glycosylation, disulfide bond formation, oligomerization, and secretion (25–29).

To compare the effects of the missense mutations Y598D, G618V, and N617K on assembly and secretion of {alpha}1(X) collagen chains, we transfected 293-EBNA cells with wild-type and mutant collagen X cDNAs. Transfected cells were metabolically labeled for 16 h, and collagen X chains were recovered from cell and media fractions by immunoprecipitation with anti-His6 antibodies. Consistent with previous results in stably transfected cells expressing wild-type and Y598D {alpha}1(X) collagen chains (21), the Y598D chains were secreted very poorly in comparison with wild-type chains (Fig. 1a, lanes 5 and 6). After disruption of collagen X immunocomplexes in 2% (w/v) SDS, secreted {alpha}1(X) collagen chains migrated as a mixture of trimers and monomers. To confirm that {alpha}1(X) collagen chains were secreted only as trimers, we analyzed the medium of 293-EBNA cells expressing wild-type collagen X by ethanol precipitation, followed by denaturation in 1% (w/v) SDS-PAGE buffer (Fig. 1c). We identified a single major protein band at the expected position of the collagen X trimer (lane 1), which dissociated into {alpha}1(X) collagen monomers in the presence of trichloroacetic acid (lane 2).



View larger version (65K):
[in this window]
[in a new window]
  		FIG. 1.
Effect of SMCD missense mutations on collagen X assembly and secretion in transfected 293-EBNA cells. cDNAs encoding His6-tagged wild-type collagen X (WT) and three SMCD missense mutations (Y598D, G618V, and N617K) were transfected into 293-EBNA cells and maintained as a pool of stable positive transfectants. Expression at the mRNA level was confirmed by Northern analysis of collagen X mRNA (see "Experimental Procedures" for details) (d). Transfected cells were biosynthetically labeled for 16 h, and collagen X chains were recovered from cell and media fractions by immunoprecipitation using anti-His6 antibodies. Immunocomplexes were denatured by heating to 65 °C for 10 min in 2% (w/v) SDS-PAGE buffer with (b) or without (a) 20 mM dithiothreitol, separated on 7.5% acrylamide gels, and visualized by fluorography. Conditioned medium from unlabeled 293-EBNA cells transfected with wild-type collagen X cDNA was analyzed to confirm that secreted {alpha}1(X) collagen chains were trimeric (c). Collagen X chains were precipitated by incubation of 400 µl of conditioned medium with 3 volumes of ethanol (lane 1) or 10% (v/v) trichloroacetic acid (lane 2) for 1 h on ice. Collagen X was recovered by centrifugation at 13,000 x g for 20 min, and the dried pellets were heated to 65 °C in 1% (w/v) SDS-PAGE buffer for 10 min. Samples were separated on a 7.5% polyacrylamide gel, and protein bands were visualized by Coomassie Blue staining.

 
Like Y598D, the N617K mutation resulted in very low levels of {alpha}1(X) collagen in the medium, and G618V chains were completely absent in the media fractions (Fig. 1a, lanes 6–8). We confirmed that this effect was not due to instability of the mutant transcripts, as comparable levels of expression were identified by Northern analysis (Fig. 1d). Therefore, our data indicate that all three missense mutations inhibit collagen X secretion in 293-EBNA cells.

Effect of SMCD Point Mutations on Collagen X Assembly in Transfected 293-EBNA Cells—In addition to the defect in collagen X secretion caused by the three missense mutations, we observed significant differences in trimer stability. In all transfections, a significant fraction of intracellular {alpha}1(X) collagen chains migrated as monomers (Fig. 1a, lanes 1–4). The remaining wild-type {alpha}1(X) collagen chains migrated as trimers (lane 1). In striking contrast, a very minor fraction of SMCD {alpha}1(X) collagen chains migrated as trimers, whereas significant amounts of mutant chains migrated at the expected position of {alpha}1(X) collagen dimers (lanes 2–4). To investigate the nature of the interactions contributing to NC1 multimer stability, samples were treated under conditions identical to used in the previous experiment, except for the addition of 20 mM dithiothreitol to the SDS-PAGE buffer. Whereas a similar banding pattern was observed for wild-type chains (Fig. 1, a and b, lane 1), the {alpha}1(X) collagen dimers were completely dissociated into monomers under reducing conditions (Fig. 1b, lanes 2–4). This indicates that correctly assembled NC1 subunits are stabilized by noncovalent forces, whereas the SMCD chains form dimers that are linked by disulfide bonds. Our results therefore provide strong evidence that the mutant {alpha}1(X) collagen chains misfold into a conformation not adopted by the native NC1 domain, leading to aberrant disulfide bond formation.

Misfolding of SMCD Mutant Collagen X Chains in SaOS-2 Cells—One potential limitation of recombinant protein expression in 293-EBNA cells is the very high expression level of the exogenous protein, which may compromise ER quality control. This has been shown in a recombinant collagen VI expression system in which unassembled {alpha}1(VI) and {alpha}2(VI) collagen chains are secreted (30). We therefore sought additional evidence that the disulfide bond formation associated specifically with the SMCD mutants was not an artifact of recombinant protein expression in 293-EBNA cells. We transfected SaOS-2 bone cells with wild-type and SMCD missense mutants and analyzed {alpha}1(X) collagen chain secretion by metabolic labeling as described above. Collagen X chains were recovered from cell and media fractions by immunoprecipitation, and samples were resolved by SDS-PAGE (Fig. 2a). In agreement with the pattern of collagen X secretion in 293-EBNA cells and our previously published data (21), Y598D, G618V, and N617K chains were not detected in media fractions (lanes 8–10). Because of the presence of protein bands in the media fraction that comigrated with the collagen X trimers, we analyzed media samples by immunoblotting using anti-collagen X antibodies (Fig. 2b). As expected, the medium from cells transfected with wild-type collagen X gave a strong signal at the position corresponding to a collagen X trimer (lane 2). A very weak collagen X band was detected in cells expressing the N617K mutant, whereas G618V and Y598D chains were absent (lanes 3–5). Although we detected some variation in transcript levels by Northern blotting, comparable expression was observed in cells transfected with the G618V and wild-type collagen X cDNA constructs, indicating that the poor secretion of mutant {alpha}1(X) collagen chains was not linked to low expression levels (Fig. 2c).



View larger version (43K):
[in this window]
[in a new window]
  		FIG. 2.
Analysis of wild-type and mutant collagen X assembly and secretion in transfected SaOS-2 cells. cDNAs encoding His6-tagged wild-type collagen X (WT) and three SMCD missense mutations (Y598D, G618V, and N617K) were transfected into SaOS-2 cells and maintained as a pool of stable positive transfectants. Expression at the mRNA level was confirmed by Northern analysis of collagen X mRNA (see "Experimental Procedures" for details) (c). Untransfected SaOS-2 cells and transfected cells were biosynthetically labeled for 16 h, and collagen X chains were recovered from cell and media fractions by immunoprecipitation using anti-His6 antibodies. Samples were denatured by heating to 65 °C in 2% (w/v) SDS-PAGE buffer, separated on 7.5% acrylamide gels, and visualized by fluorography (a). Conditioned media harvested from untransfected and transfected cells were also analyzed by immunoblotting using anti-collagen X antibodies to verify that the major labeled protein band in a corresponds to {alpha}1(X) collagen chains (b).

 
In contrast to the pattern of intracellular collagen X bands observed in 293-EBNA cells (Fig. 1a), we could not identify {alpha}1(X) collagen chain dimers in SaOS-2 cells transfected with mutant collagen X after 16 h of biosynthetic labeling. Our previous study demonstrated that mutant collagen X chains are degraded in SaOS-2 cells as part of secretion quality control (21). Therefore, at the end of an overnight metabolic labeling experiment, it is likely that the majority of misfolded or unassembled collagen X chains have been degraded. To investigate this possibility, we pulse-labeled SaOS-2 cells expressing wild-type and mutant collagen X for 1 h and isolated nascent collagen X chains from the intracellular fraction by immunoprecipitation. Samples were analyzed by SDS-PAGE under reducing and nonreducing conditions to allow detection of disulfide-bonded collagen X chains (Fig. 3). Wild-type {alpha}1(X) collagen chains migrated as a mixed population of monomers and trimers, which was unaltered by the addition of reducing agents (lanes 1 and 5). In contrast, an additional band with a faster electrophoretic mobility than that of the collagen X trimer was present in samples from cells expressing the Y598D, G618V, and N617K chains (lanes 2–4). Consistent with the migration pattern of SMCD mutants in 293-EBNA cells, a small fraction of Y598D and N617K trimers could be detected after pulse labeling. Under reducing conditions, most of the SMCD {alpha}1(X) collagen chains migrated as a single protein band corresponding to the collagen X monomer (lanes 6–8). Therefore, although SMCD chains appear to be less stable in SaOS-2 cells than in 293-EBNA cells, our data show that, in both transfection systems, SMCD missense mutations result in formation of disulfide-bonded chains, which are absent in cells transfected with wild-type collagen X.



View larger version (56K):
[in this window]
[in a new window]
  		FIG. 3.
Pulse labeling of SaOS-2 cells transfected with wild-type collagen X and SMCD missense mutants. Transfected cells were biosynthetically labeled for 1 h (see "Experimental Procedures"), and intracellular collagen X was recovered by immunoprecipitation using anti-His6 antibodies. Samples were denatured by heating to 65 °C in 2% (w/v) SDS-PAGE buffer with (lanes 5–8) or without (lanes 1–4)20 mM dithiothreitol (DTT), separated on 7.5% acrylamide gels, and visualized by fluorography. The migration positions of collagen X monomers, dimers, and trimers are indicated. WT, wild-type collagen X.

 
Effect of the SMCD Frameshift Mutation NC1del10 on Collagen X Assembly and Secretion in Transfected 293-EBNA Cells—The NC1del10 frameshift mutation is caused by a 10-bp deletion within the NC1 domain coding sequence, introducing nonsense sequence from residue 6233 to a premature termination at residue 673 (31). As the most C-terminal portion of the NC1 polypeptide chain is buried in the hydrophobic core, the introduction of nonsense sequence and premature termination is predicted to completely abrogate subunit folding (10). In agreement with this prediction, we and others have shown that this mutation prevents secretion of collagen X in transfected cells (21, 23). To ascertain experimentally whether NC1del10 subunits misfold, we expressed NC1del10 cDNAs in 293-EBNA cells. We observed comparable expression at the mRNA level by Northern analysis (Fig. 4c). Cells transfected with wild-type and NC1del10 collagen X cDNA constructs were biosynthetically labeled for 16 h, and {alpha}1(X) collagen chains were recovered from cell and media fractions by immunoprecipitation with anti-His6 antibodies (Fig. 4, a and b). As expected, very little mutant collagen X was evident in the media fraction compared with the level of wild-type collagen X secretion (Fig. 4a, lanes 3 and 4). Also in contrast to the wild-type {alpha}1(X) collagen chains, a population of intracellular NC1del10 chains migrated as dimers (lane 2). As shown for the Y598D, G618V, and N617K chains (Fig. 4b), the NC1del10 dimers dissociated under reducing conditions, thus apparently stabilized by disulfide bonds (lane 2).



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 4.
Effect of the NC1del10 nonsense mutation on collagen X assembly and secretion in transfected 293-EBNA cells. The NC1del10 mutation is a 10-bp deletion that introduces nonsense amino acid sequence from residue 623 to a premature termination at residue 673. The cDNA encoding NC1del10 chains was transfected into 293-EBNA cells, and Northern blotting was used to confirm expression at the mRNA level (c). Cells expressing wild-type (WT) and NC1del10 chains were biosynthetically labeled for 16 h, and {alpha}1(X) collagen chains were recovered by immunoprecipitation using anti-His6 antibodies. Immunocomplexes were denatured by heating to 65 °C in 2% (w/v) SDS-PAGE buffer with (b) or without (a) 20 mM dithiothreitol, separated on 7.5% acrylamide gels, and visualized by fluorography. The migration positions of collagen X monomers, dimers, and trimers are indicated.

 
A significant fraction of intracellular NC1del10 chains also appeared to migrate as SDS-stable trimers (Fig. 4a, lane 2). This was unexpected, considering the poor level of secretion and the deleterious effect that C-terminal truncation is predicted to have on subunit folding. However, whereas the wild-type {alpha}1(X) collagen trimers were resistant to reducing agents (Fig. 4, a and b, lane 1), the mutant NC1 trimer completely dissociated under reducing conditions (Fig. 4b, lane 2), indicating that these trimers were stabilized by disulfide bonds. The NC1del10 frameshift introduces nonsense sequence that codes for a second cysteine residue at position 647 (Fig. 5a). The presence of a second cysteine within the mutant NC1 domain therefore allows formation of a disulfide-bonded trimer, where both cysteine residues of either one or three NC1del10 chains participate in disulfide bond formation (Fig. 5b).



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 5.
Alignment of wild-type and NC1del10 collagen X sequences. a, amino acid sequences corresponding to the wild-type and NC1del10 mutant {alpha}1(X) collagen chains starting at residue 622. The 10-bp deletion within the COL10A1 mutant allele alters the nucleotide reading frame and changes residues 623–673 within the NC1 domain, resulting in the premature truncation of the mutant {alpha}1(X) polypeptide sequence by 8 amino acids. The substitution of Asn647 with Cys (marked by an asterisk) allows potential formation of a second and third interchain disulfide bond and therefore disulfide-bonded trimers, illustrated schematically in b.

 
Expression of SMCD Mutant Collagen X Is Associated with Activation of the Unfolded Protein Response—Our results demonstrate that the NC1del10 {alpha}1(X) collagen frameshift mutation and the missense mutations Y598D, G618V, and N617K dramatically impair correct intersubunit interactions and subsequent progression of collagen X through the secretory pathway. Although it is unlikely that the different mutations cause identical conformational changes, the formation of non-native disulfide bonds is evidence that all the SMCD mutations analyzed in this study cause a degree of NC1 domain misfolding. The cellular response to misfolded proteins in the ER is activation of a complex signal transduction pathway called the unfolded protein response (UPR) (reviewed in Refs. 32 and 33). The UPR, which has evolved to limit the potential damaging effects of unfolded protein on cellular homeostasis, culminates in the induction of ER stress response genes by two potent transcription factors, activating transcription factor-6 and XBP1 (X-box DNA-binding protein-1) (34, 35). In cells undergoing ER stress, active XBP1 is generated by excision of a 26-nucleotide sequence from the XBP1 transcript by IRE1 endonuclease, one of three ER transmembrane "stress sensors" (36). Interestingly, accumulation of mutant collagen X within hypertrophic chondrocytes of transgenic mice expressing the del13 mutation (a 13-bp deletion that is equivalent to the human NC1del10 SMCD mutation) triggered the UPR, as illustrated by enhanced splicing of XBP1.4

To determine whether the UPR is associated specifically with the del13 mutation or whether missense mutations also elicit the UPR, we analyzed the relative proportions of spliced and unspliced XBP1 mRNAs in 293-EBNA and SaOS-2 cells transfected with wild-type, Y598D, N617K, G618V, and NC1del10 cDNAs. Cells were grown to confluence and incubated in fresh medium for 2 h prior to RNA extraction to avoid exposure of the cells to ER stress induced by glucose starvation. After reverse transcription of mRNA, XBP1 sequences were amplified using primers flanking the target sequence for IRE1 endonuclease, and PCR products were resolved on 2.5% agarose gels (Fig. 6, a and b). In 293-EBNA cells expressing wild-type collagen X, we observed only a very faint band corresponding to processed XBP1, which was also present in untransfected 293-EBNA cells (Fig. 6a, lanes 1 and 2), indicating that synthesis and secretion of normal collagen X do not induce ER stress. In contrast, we found that, in cells expressing mutant collagen X chains, a significant fraction of XBP1 PCR products migrated as the processed form (lanes 3–6), indicating that the IRE1-induced splicing of XBP1 mRNA can be triggered by collagen X chains harboring frameshift and missense mutations. However, in SaOS-2 cells, we found little evidence of XBP1 processing in any of the transfectants (Fig. 6b, lanes 2–6).



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 6.
Analysis of markers for the UPR in 293-EBNA and SaOS-2 cells transfected with wild-type and mutant collagen X. Untransfected (Control) and collagen X-transfected 293-EBNA and SaOS-2 cells were seeded into 6-well dishes and grown to confluence prior to RNA extraction. XBP1 mRNA splicing in 293-EBNA cells (a) and SaOS-2 cells (b) was analyzed by RT-PCR using a primer pair flanking the IRE1 target site within the XBP1 transcript. The PCR products were separated on 2.5% agarose gels, and the two bands corresponding to spliced (Xbp1s) and unspliced (Xbp1us) XBP1 mRNAs are indicated by arrows. To quantify the relative levels of spliced and unspliced XBP1 mRNAs, we performed primer extension analysis of XBP1 processing in untransfected (Control) and collagen X-transfected 293-EBNA cells (c) and SaOS-2 cells (d). Template for each reaction was generated by RT-PCR (see "Experimental Procedures"). The 18-mer oligonucleotide designed to anneal specifically to XBP1 at a site 5' to the IRE1 processing site and the two expected primer extension products are illustrated above c. G* and C* represent 33P-labeled guanosine and cytidine, respectively. Extension with [{alpha}-33P]dGTP (lanes 2, 4, 6, 8, 10, and 12) resulted in a radioactively labeled 20-mer that corresponds to the level of spliced XBP1 template (Xbp1s). Extension with [{alpha}-33P]dCTP (lanes 1, 3, 5, 7, 9, and 11) resulted in a radioactively labeled 19-mer that represents the level of unspliced XBP1 template (Xbp1us). The level of XBP1 processing is represented as the ratio of spliced and unspliced XBP1 primer extension products (Xbp1s:Xbp1us), annotated under c and d. A second set of 293-EBNA dishes was used to analyze intracellular levels of BiP, a key downstream target of activated XBP1. Intracellular proteins extracted from untransfected (Control) and collagen X-transfected 293-EBNA cells were separated on a 7.5% SDS-polyacrylamide gel, and proteins were transferred to nitrocellulose. The filter was initially probed with anti-KDEL antibodies (e) and then with anti-collagen X antibodies (f) to ascertain the level of intracellular collagen X under steady-state conditions. WT, wild-type collagen X.

 
Although RT-PCR can provide semiquantitative analysis of XBP1 processing, we elected to use a more sensitive technique to accurately quantify the ratios of spliced and unspliced XBP1 present in 293-EBNA and SaOS-2 cells expressing wild-type and SMCD mutant collagen X. Therefore, we performed primer extension with an 18-mer oligonucleotide primer complementary to the XBP1 sequence 5' to the splice site. Following synthesis of cDNA template using RT-PCR amplification of XBP1 sequences, equal amounts of template were added to duplicate sets of primer extension reactions including either radioactive dGTP or radioactive dCTP to differentiate spliced and unspliced XBP1. The primer is extended by two guanosines when annealed to template corresponding to spliced XBP1 and by a single cytidine when annealed to unspliced XBP1 template. Resolution of primer extension products on a 15% denaturing acrylamide gel identified radioactive bands corresponding to 19- and 20-mer products (Fig. 6, c and d). In 293-EBNA cells expressing wild-type collagen X, we detected spliced and unspliced XBP1 primer extension products at a ratio of 0.25:1, similar to the value of 0.17:1 in untransfected cells (Fig. 6c, lanes 1–4). However, in 293-EBNA cells expressing the collagen X mutants, the ratio of spliced and unspliced XBP1 ranged from 0.75:1 to 1.10:1 (lanes 5–12), representing a 4–6-fold increase in XBP1 processing relative to untransfected cells. In SaOS-2 cells, we detected a 2–4-fold increase in XBP1 processing in cells expressing mutant collagen X relative to untransfected cells (Fig. 6d, lanes 1, 2, and 7–12). It is possible that the more modest stress response we observed in SaOS-2 cells reflects a difference in mutant collagen X processing. This is consistent with our biosynthetic labeling experiments suggesting more rapid degradation of mutant collagen X chains in SaOS-2 cells. Taken together, our RT-PCR data and the results from primer extension analysis demonstrate that misfolded {alpha}1(X) collagen chains trigger the processing of XBP1, a key marker for cells undergoing ER stress.

The activation of XBP1 results in up-regulation of ER-resident molecular chaperones and components of the ER-associated degradation machinery (37, 38). To investigate whether the enhanced processing of XBP1 associated with mutant collagen X produces a physiological response within 293-EBNA cells, we analyzed the levels of BiP (immunoglobulin heavy chain-binding protein), a key downstream target of XBP1 (39). 293-EBNA cells were seeded into 6-well dishes and grown to confluence, and equal amounts of cell extracts were separated by SDS-PAGE and transferred to a nitrocellulose filter. The filter was probed initially with anti-KDEL antibodies and subsequently with antibodies raised against collagen X. In comparison with untransfected 293-EBNA cells, expression of wild-type collagen X resulted in a slight increase in BiP levels (Fig. 6e, lanes 1 and 2). However, we detected significantly greater levels of intracellular BiP in cells expressing Y598D, G618V, and N617K collagen X mutants (lanes 3–5) and slightly higher levels of BiP in cells expressing NC1del10 chains (lane 6). Analysis of intracellular collagen X revealed that the steady-state level of collagen X mutants was lower than that of wild-type chains (Fig. 6f, lanes 2–6), indicating that up-regulation of BiP is specifically related to mutant collagen X expression and is not a function of protein load within the ER of the transfected cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The results of this study provide new insights into how a broad range of SMCD mutations affect collagen X stability. One striking aspect of our data is that all three missense mutations caused the same secretion defect, regardless of their location within the NC1 domain three-dimensional structure. Molecular modeling of collagen X NC1 domain trimers and resolution of their crystal structure revealed that SMCD mutations could affect collagen X assembly either at the level of individual trimer formation or at the supramolecular level (10, 20). Mutations that introduce buried charge at the NC1 subunit interface are likely to compromise assembly and be unstable within the ER. Hence, Y598D chains were secreted very poorly in stably transfected cells (Figs. 1 and 2), consistent with our previous results (21). Mutations located on the exterior of the NC1 trimer, such as N617K and G618V, are predicted to allow trimerization, but to compromise supramolecular assembly (10). However, in this study, we have provided substantial evidence that neither N617K nor G618V {alpha}1(X) collagen chains form native NC1 domains in a cellular context. The poor assembly and secretion of G618V chains are not surprising given the trace levels of secretion detected in transiently transfected cells (23). These data are supported by in vitro studies clearly demonstrating that the G618V mutation weakens the NC1-NC1 interactions involved in trimer formation (40). The impaired secretion of N617K chains was not predicted from the NC1 domain three-dimensional structure model, as a lysine residue at this position of exterior loop DE is theoretically permitted (10). Although Asn617 is conserved among human, mouse, bovine, and chicken collagen X sequences, it is substituted with a lysine in ACRP30. However, the sequence context is considerably divergent between human collagen X (Asn617-Gly-Trp-Pro-Val-Met) and human ACRP30 (Lys617-Asp-Lys-Ala-Met-Leu), suggesting that the local side chain interactions within this region of the trimer surface may differ between ACRP30 and collagen X. There is also experimental evidence that the N617K substitution in bovine collagen X significantly reduces trimer stability in the ER (18). Therefore, although the G618V and N617K substitutions are not predicted to affect trimer stability, we have shown that NC1 domain folding and assembly are also dependent upon interactions involving residues ultimately located on the trimer exterior.

In this study, we have identified novel disulfide-bonded collagen X species that are associated specifically with SMCD mutations and not wild-type collagen X. Native collagen X trimers are stabilized by strong noncovalent interactions that are not affected by reducing agents (7, 9, 41). Recent analysis of the NC1 domain crystal structure has confirmed that correctly folded NC1 domains are precluded from disulfide bond formation (10). The Cys591 sulfhydryl group projects into the subunit interior and forms part of a hydrogen bond network that stabilizes the hydrophobic subunit core (Fig. 7b). Importantly, the Y598D, G618V, and N617K mutations do not directly affect side chain interactions involving Cys591 (Fig. 7a). To expose the Cys591 thiol group to solvent, these mutants clearly adopt a non-native conformation that dramatically alters the overall subunit structure. This is consistent with the observation that the G618V and Y598D mutations cause increased susceptibility of the NC1 domains to proteolysis (22). Therefore, our data indicate that, within the ER of mammalian cells, missense mutations and the NC1del10 nonsense mutation result in serious NC1 domain folding defects.



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 7.
Structural representation of the collagen X NC1 domain. The NC1 domain structure is shown in ribbon format, with the locations of the three SMCD missense mutations used in this study (Tyr598, Asn617, and Gly618) and the position of Cys591 highlighted in ball-and-stick representation (a). To illustrate the orientation of the Cys591 side chain and the close proximity of Cys591 and Ile593 side chains within the subunit interior, the base of the subunit is shown at magnification x4 (b), with the Cys591 sulfur and Ile593 oxygen atoms shown in black. The molecular model of the collagen X NC1 domain (Molecular Modeling Database accession number 18672) (10) was visualized and rendered using MOLMOL and POV-ray.

 
Although not involved in disulfide bond formation, Cys591 of collagen X is highly conserved. One possible role is the intracellular retention of misfolded {alpha}1(X) collagen chains. Our data are consistent with a model in which NC1 misfolding causes a conformational switch that unmasks the Cys591 thiol group, leading to targeting of mutant {alpha}1(X) collagen chains for degradation. A similar mechanism exists for the removal of both unassembled (42) and misfolded (43–45) major histocompatibility complex heavy chains, which also undergo aberrant disulfide bond formation due to exposure of a reactive thiol group. In 293-EBNA cells, after biosynthetic labeling, the intracellular mutant {alpha}1(X) collagen chains exist as a mixed population of monomers, dimers, and, in the case of NC1del10 chains, disulfide-bonded trimers. It is unclear whether mutant {alpha}1(X) collagen multimers are reduced prior to disposal via the same mechanism as unassembled chains or whether a separate degradation pathways exists. The results from our previous study indicate that either scenario is possible, as multiple pathways are involved in the degradation of SMCD chains (21). We also reported that mutant chains interact stably with protein-disulfide isomerase, an ER chaperone capable of retaining unassembled or misfolded proteins within the ER. In contrast, mutant NC1 domains expressed in bacteria specifically copurify with GroEL, the bacterial homolog of BiP (22). As part of this study, we were unable to identify stable interaction of mutant collagen X chains with BiP either by chemical cross-linking or by co-immunoprecipitation (data not shown). Another potential candidate for ER retention of mutant collagen X is ERp44, which has been shown to interact with exposed unpaired thiol residues (46–49). Additional studies are in progress to elucidate whether Cys591 acts as an ER retention signal and whether ERp44 has a role in the quality control of collagen X secretion.

The consequences of {alpha}1(X) collagen chain misfolding in the growth plates of SMCD patients are likely to depend on the ability of hypertrophic chondrocytes to efficiently degrade mutant collagen X prior to any dominant effects. Our data illustrate that the response to misfolded collagen X may vary in different cell lines, as SMCD mutants trigger a more dramatic UPR in 293-EBNA cells than in SaOS-2 cells. One explanation is that unstressed SaOS-2 cells express higher constitutive levels of ER chaperones compared with 293-EBNA cells (data not shown). It is also possible that SaOS-2 cells are equipped with more efficient mechanisms for recognition and targeting of misfolded {alpha}1(X) collagen chains for degradation, consistent with the observation that mutant {alpha}1(X) collagen dimers are less stable in the ER of SaOS-2 cells. Whether the chondrocytes of SMCD patients become overloaded with misfolded collagen X is likely to depend upon their capacity to sort and degrade misfolded collagen X. The clearest example of a chondrodysplasia in which ER function is compromised is pseudoachondroplasia (50). The intracellular retention of cartilage oligomeric matrix protein (COMP) in the chondrocytes of pseudoachondroplasia patients leads to massive distortion of the ER, induction of the UPR, and increased apoptosis (51, 52). In the absence of SMCD-affected tissue samples, we cannot determine whether the chondrocytes of SMCD patients exhibit a similar phenotype. Although SMCD and pseudoachondroplasia are both caused to some extent by impaired protein secretion, the fun-damental mechanisms underlying the two diseases are likely to be very different. First, the emerging model for the molecular basis of pseudoachondroplasia is the retention of large protein aggregates within the ER due to the heterotypic interaction of COMP with collagen IX and matrilins (51, 53, 54). Interaction studies from our laboratory have shown binding of collagen X to several members of the small leucine-rich proteoglycan family,5 although if these interactions are physiological, they are more likely to take place in the extracellular matrix than in the ER. Second, in all cases of pseudoachondroplasia described to date (reviewed in Ref. 55), the mutant COMP molecules contain normal coiled-coil oligomerization domains, thus allowing co-assembly of wild-type and mutant COMP subunits into pentamers and secretion of mutant COMP into the extracellular matrix (56). Our data suggest that SMCD mutations prevent NC1 domains from folding correctly, reducing the likelihood of stable heterotrimer formation and secretion of mutant {alpha}1(X) collagen chains (21).

The SMCD phenotype results from functional haploinsufficiency in at least two patients, due to nonsense-mediated decay of alleles with premature stop codons (16, 17). In contrast, no disease-causing mutations that introduce premature stop codons in COMP have been described to date, and the COMP-null mouse has no apparent skeletal phenotype (57), consistent with a gain-of-function mechanism underlying pseudoachondroplasia. Therefore, our data support a model in which, in most cases of SMCD, misfolding of collagen X chains results in selective intracellular retention and degradation of mutant chains by quality control mechanisms within the ER. Whether expression of mutant collagen X generates ER stress and has a wider impact on chondrocyte function in vivo is the target for future work.


   FOOTNOTES
 
* This work was supported by Grant 114118 from the National Health and Medical Research Council of Australia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 61-3-9345-6367; Fax: 61-3-9345-7997; E-mail: john.bateman{at}mcri.edu.au.

1 The abbreviations used are: SMCD, Schmid metaphyseal chondrodysplasia; ER, endoplasmic reticulum; DMEM, Dulbecco's modified Eagle's medium; RT, reverse transcription; UPR, unfolded protein response; COMP, cartilage oligomeric matrix protein. Back

2 J. F. Bateman, S. Freddi, S. Golub, and K. M. Bell, unpublished data. Back

3 The base pairs in the COL10A1 coding sequence are numbered from the translation start site (58). Back

4 K. Y. Tsang, D. Chan, D. Cheslett, W. C. W. Chan, K. M. Kwan, E. B. Hunziker, J. F. Bateman, K. M. C. Cheung, and K. S. E. Cheah, unpublished data. Back

5 R. Wilson and J. F. Bateman, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Drs. James Horne and Katrina M. Bell for molecular modeling of the collagen X NC1 domain.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Chan, D., and Jacenko, O. (1998) Matrix Biol. 17, 169–184[CrossRef][Medline] [Order article via Infotrieve]
  2. Schmid, T. M., Popp, R. G., and Linsenmayer, T. F. (1990) Ann. N. Y. Acad. Sci. 580, 64–73[Medline] [Order article via Infotrieve]
  3. Gress, C. J., and Jacenko, O. (2000) J. Cell Biol. 149, 983–993[Abstract/Free Full Text]
  4. Jacenko, O., Roberts, D. W., Campbell, M. R., McManus, P. M., Gress, C. J., and Tao, Z. (2002) Am. J. Pathol. 160, 2019–2034[Abstract/Free Full Text]
  5. Kwan, K. M., Pang, M. K., Zhou, S., Cowan, S. K., Kong, R. Y., Pfordte, T., Olsen, B. R., Sillence, D. O., Tam, P. P., and Cheah, K. S. (1997) J. Cell Biol. 136, 459–471[Abstract/Free Full Text]
  6. Zhang, Y., and Chen, Q. (1999) J. Biol. Chem. 274, 22409–22413[Abstract/Free Full Text]
  7. Schmid, T. M., and Linsenmayer, T. F. (1983) J. Biol. Chem. 258, 9504–9509[Abstract/Free Full Text]
  8. Kwan, A. P., Cummings, C. E., Chapman, J. A., and Grant, M. E. (1991) J. Cell Biol. 114, 597–604[Abstract/Free Full Text]
  9. Frischholz, S., Beier, F., Girkontaite, I., Wagner, K., Poschl, E., Turnay, J., Mayer, U., and von der Mark, K. (1998) J. Biol. Chem. 273, 4547–4555[Abstract/Free Full Text]
  10. Bogin, O., Kvansakul, M., Rom, E., Singer, J., Yayon, A., and Hohenester, E. (2002) Structure (Lond.) 10, 165–173[Medline] [Order article via Infotrieve]
  11. Warman, M. L., Abbott, M., Apte, S. S., Hefferon, T., McIntosh, I., Cohn, D. H., Hecht, J. T., Olsen, B. R., and Francomano, C. A. (1993) Nat. Genet. 5, 79–82[CrossRef][Medline] [Order article via Infotrieve]
  12. Wallis, G. A., Rash, B., Sweetman, W. A., Thomas, J. T., Super, M., Evans, G., Grant, M. E., and Boot-Handford, R. P. (1994) Am. J. Hum. Genet. 54, 169–178[Medline] [Order article via Infotrieve]
  13. Wallis, G. A., Rash, B., Sykes, B., Bonaventure, J., Maroteaux, P., Zabel, B., Wynne-Davies, R., Grant, M. E., and Boot-Handford, R. P. (1996) J. Med. Genet. 33, 450–457[Abstract/Free Full Text]
  14. Chan, D., Ho, M. S., and Cheah, K. S. (2001) J. Biol. Chem. 276, 7992–7997[Abstract/Free Full Text]
  15. Bateman, J. F., Freddi, S., McNeil, R., Thompson, E., Hermanns, P., Savarirayan, R., and Lamande, S. R. (2004) Hum. Mutat. 23, 396–402[Medline] [Order article via Infotrieve]
  16. Chan, D., Weng, Y. M., Graham, H. K., Sillence, D. O., and Bateman, J. F. (1998) J. Clin. Investig. 101, 1490–1499[Medline] [Order article via Infotrieve]
  17. Bateman, J. F., Freddi, S., Nattrass, G., and Savarirayan, R. (2003) Hum. Mol. Genet. 12, 217–225[Abstract/Free Full Text]
  18. McLaughlin, S. H., Conn, S. N., and Bulleid, N. J. (1999) J. Biol. Chem. 274, 7570–7575[Abstract/Free Full Text]
  19. Chan, D., Freddi, S., Weng, Y. M., and Bateman, J. F. (1999) J. Biol. Chem. 274, 13091–13097[Abstract/Free Full Text]
  20. Marks, D. S., Gregory, C. A., Wallis, G. A., Brass, A., Kadler, K. E., and Boot-Handford, R. P. (1999) J. Biol. Chem. 274, 3632–3641[Abstract/Free Full Text]
  21. Wilson, R., Freddi, S., and Bateman, J. F. (2002) J. Biol. Chem. 277, 12516–12524[Abstract/Free Full Text]
  22. Dublet, B., Vernet, T., and van der Rest, M. (1999) J. Biol. Chem. 274, 18909–18915[Abstract/Free Full Text]
  23. Chan, D., Weng, Y. M., Hocking, A. M., Golub, S., McQuillan, D. J., and Bateman, J. F. (1996) J. Biol. Chem. 271, 13566–13572[Abstract/Free Full Text]
  24. Bengtsson, E., Aspberg, A., Heinegard, D., Sommarin, Y., and Spillmann, D. (2000) J. Biol. Chem. 275, 40695–40702[Abstract/Free Full Text]
  25. Fichard, A., Tillet, E., Delacoux, F., Garrone, R., and Ruggiero, F. (1997) J. Biol. Chem. 272, 30083–30087[Abstract/Free Full Text]
  26. Tillet, E., Mann, K., Nischt, R., Pan, T. C., Chu, M. L., and Timpl, R. (1995) Eur. J. Biochem. 228, 160–168[Medline] [Order article via Infotrieve]
  27. Elamaa, H., Snellman, A., Rehn, M., Autio-Harmainen, H., and Pihlajaniemi, T. (2003) Matrix Biol. 22, 427–442[CrossRef][Medline] [Order article via Infotrieve]
  28. Klatt, A. R., Nitsche, D. P., Kobbe, B., Macht, M., Paulsson, M., and Wagener, R. (2001) J. Biol. Chem. 276, 17267–17275[Abstract/Free Full Text]
  29. Piecha, D., Wiberg, C., Morgelin, M., Reinhardt, D. P., Deak, F., Maurer, P., and Paulsson, M. (2002) Biochem. J. 367, 715–721[CrossRef][Medline] [Order article via Infotrieve]
  30. Tillet, E., Wiedemann, H., Golbik, R., Pan, T. C., Zhang, R. Z., Mann, K., Chu, M. L., and Timpl, R. (1994) Eur. J. Biochem. 221, 177–185[Medline] [Order article via Infotrieve]
  31. Dharmavaram, R. M., Elberson, M. A., Peng, M., Kirson, L. A., Kelley, T. E., and Jimenez, S. A. (1994) Hum. Mol. Genet. 3, 507–509[Free Full Text]
  32. Rutkowski, D. T., and Kaufman, R. J. (2004) Trends Cell Biol. 14, 20–28[CrossRef][Medline] [Order article via Infotrieve]
  33. Zhang, K., and Kaufman, R. J. (2004) J. Biol. Chem. 279, 25935–25938[Free Full Text]
  34. Lee, A. H., Iwakoshi, N. N., and Glimcher, L. H. (2003) Mol. Cell. Biol. 23, 7448–7459[Abstract/Free Full Text]
  35. Yoshida, H., Matsui, T., Yamamoto, A., Okada, T., and Mori, K. (2001) Cell 107, 881–891[CrossRef][Medline] [Order article via Infotrieve]
  36. Calfon, M., Zeng, H., Urano, F., Till, J. H., Hubbard, S. R., Harding, H. P., Clark, S. G., and Ron, D. (2002) Nature 415, 92–96[CrossRef][Medline] [Order article via Infotrieve]
  37. Kaufman, R. J. (1999) Genes Dev. 13, 1211–1233[Free Full Text]
  38. Yoshida, H., Matsui, T., Hosokawa, N., Kaufman, R. J., Nagata, K., and Mori, K. (2003) Dev. Cell 4, 265–271[CrossRef][Medline] [Order article via Infotrieve]
  39. Kozutsumi, Y., Segal, M., Normington, K., Gething, M. J., and Sambrook, J. (1988) Nature 332, 462–464[CrossRef][Medline] [Order article via Infotrieve]
  40. Chan, D., Cole, W. G., Rogers, J. G., and Bateman, J. F. (1995) J. Biol. Chem. 270, 4558–4562[Abstract/Free Full Text]
  41. Schmid, T. M., and Linsenmayer, T. F. (1984) Biochemistry 23, 553–558[CrossRef][Medline] [Order article via Infotrieve]
  42. Capps, G. G., Robinson, B. E., Lewis, K. D., and Zuniga, M. C. (1993) J. Immunol. 151, 159–169[Abstract]
  43. Mear, J. P., Schreiber, K. L., Munz, C., Zhu, X., Stevanovic, S., Rammensee, H. G., Rowland-Jones, S. L., and Colbert, R. A. (1999) J. Immunol. 163, 6665–6670[Abstract/Free Full Text]
  44. Dangoria, N. S., DeLay, M. L., Kingsbury, D. J., Mear, J. P., Uchanska-Ziegler, B., Ziegler, A., and Colbert, R. A. (2002) J. Biol. Chem. 277, 23459–23468[Abstract/Free Full Text]
  45. Allen, R. L., O'Callaghan, C. A., McMichael, A. J., and Bowness, P. (1999) J. Immunol. 162, 5045–5048[Abstract/Free Full Text]
  46. Anelli, T., Alessio, M., Mezghrani, A., Simmen, T., Talamo, F., Bachi, A., and Sitia, R. (2002) EMBO J. 21, 835–844[CrossRef][Medline] [Order article via Infotrieve]
  47. Isidoro, C., Maggioni, C., Demoz, M., Pizzagalli, A., Fra, A. M., and Sitia, R. (1996) J. Biol. Chem. 271, 26138–26142[Abstract/Free Full Text]
  48. Fra, A. M., Fagioli, C., Finazzi, D., Sitia, R., and Alberini, C. M. (1993) EMBO J. 12, 4755–4761[Medline] [Order article via Infotrieve]
  49. Anelli, T., Alessio, M., Bachi, A., Bergamelli, L., Bertoli, G., Camerini, S., Mezghrani, A., Ruffato, E., Simmen, T., and Sitia, R. (2003) EMBO J. 22, 5015–5022[CrossRef][Medline] [Order article via Infotrieve]
  50. Dinser, R., Zaucke, F., Kreppel, F., Hultenby, K., Kochanek, S., Paulsson, M., and Maurer, P. (2002) J. Clin. Investig. 110, 505–513[CrossRef][Medline] [Order article via Infotrieve]
  51. Vranka, J., Mokashi, A., Keene, D. R., Tufa, S., Corson, G., Sussman, M., Horton, W. A., Maddox, K., Sakai, L., and Bachinger, H. P. (2001) Matrix Biol. 20, 439–450[CrossRef][Medline] [Order article via Infotrieve]
  52. Hashimoto, Y., Tomiyama, T., Yamano, Y., and Mori, H. (2003) Am. J. Pathol. 163, 101–110[Abstract/Free Full Text]
  53. Maddox, B. K., Keene, D. R., Sakai, L. Y., Charbonneau, N. L., Morris, N. P., Ridgway, C. C., Boswell, B. A., Sussman, M. D., Horton, W. A., Bachinger, H. P., and Hecht, J. T. (1997) J. Biol. Chem. 272, 30993–30997[Abstract/Free Full Text]
  54. Mann, H. H., Ozbek, S., Engel, J., Paulsson, M., and Wagener, R. (2004) J. Biol. Chem. 279, 25294–25298[Abstract/Free Full Text]
  55. Briggs, M. D., and Chapman, K. L. (2002) Hum. Mutat. 19, 465–478[CrossRef][Medline] [Order article via Infotrieve]
  56. Delot, E., Brodie, S. G., King, L. M., Wilcox, W. R., and Cohn, D. H. (1998) J. Biol. Chem. 273, 26692–26697[Abstract/Free Full Text]
  57. Svensson, L., Aszodi, A., Heinegard, D., Hunziker, E. B., Reinholt, F. P., Fassler, R., and Oldberg, A. (2002) Mol. Cell. Biol. 22, 4366–4371[Abstract/Free Full Text]
  58. Thomas, J. T., Cresswell, C. J., Rash, B., Hoyland, J., Freemont, A. J., Grant, M. E., and Boot-Handford, R. P. (1991) Biochem. Soc. Trans. 19, 804–808[Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 BloodHome page
G. Bianchi, L. Oliva, P. Cascio, N. Pengo, F. Fontana, F. Cerruti, A. Orsi, E. Pasqualetto, A. Mezghrani, V. Calbi, et al.
The proteasome load versus capacity balance determines apoptotic sensitivity of multiple myeloma cells to proteasome inhibition
Blood, March 26, 2009; 113(13): 3040 - 3049.
[Abstract] [Full Text] [PDF]

Home page
 J BiochemHome page
R. Iuliano, C. Raso, A. Quintiero, I. L. Pera, F. Pichiorri, T. Palumbo, D. Palmieri, A. Pattarozzi, T. Florio, G. Viglietto, et al.
The Eighth Fibronectin Type III Domain of Protein Tyrosine Phosphatase Receptor J Influences the Formation of Protein Complexes and Cell Localization
J. Biochem., March 1, 2009; 145(3): 377 - 385.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
A.-S. Dhaunchak and K.-A. Nave
A common mechanism of PLP/DM20 misfolding causes cysteine-mediated endoplasmic reticulum retention in oligodendrocytes and Pelizaeus Merzbacher disease
PNAS, November 6, 2007; 104(45): 17813 - 17818.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
M. S.P. Ho, K. Y. Tsang, R. L.K. Lo, M. Susic, O. Makitie, T. W.Y. Chan, V. C.W. Ng, D. O. Sillence, R. P. Boot-Handford, G. Gibson, et al.
COL10A1 nonsense and frame-shift mutations have a gain-of-function effect on the growth plate in human and mouse metaphyseal chondrodysplasia type Schmid
Hum. Mol. Genet., May 15, 2007; 16(10): 1201 - 1215.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Hozumi, N. Suzuki, P. K. Nielsen, M. Nomizu, and Y. Yamada
Laminin {alpha}1 Chain LG4 Module Promotes Cell Attachment through Syndecans and Cell Spreading through Integrin {alpha}2beta1
J. Biol. Chem., October 27, 2006; 281(43): 32929 - 32940.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/16/15544    most recent
M410758200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wilson, R.
Right arrow Articles by Bateman, J. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wilson, R.
Right arrow Articles by Bateman, J. F.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement