©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Type X Collagen Multimer Assembly in Vitro Is Prevented by a Gly to Val Mutation in the 1(X) NC1 Domain Resulting in Schmid Metaphyseal Chondrodysplasia (*)

(Received for publication, October 25, 1994; and in revised form, December 16, 1994)

Danny Chan (1) William G. Cole (1)(§) John G. Rogers (2) John F. Bateman (1)(¶)

From the  (1)Orthopaedic Molecular Biology Research Unit, Department of Paediatrics, University of Melbourne, Royal Children's Hospital, and the (2)Murdoch Institute, Flemington Road, Parkville,Victoria 3052, Australia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Type X collagen is a homotrimer of alpha1(X) chains encoded by the COL10A1 gene. It is a highly specialized extracellular matrix component, and its synthesis is restricted to hypertrophic chondrocytes in the calcifying cartilage of the growth plate and in zones of secondary ossification. Our studies on a family with Schmid metaphyseal chondrodysplasia demonstrated that the affected individuals were heterozygous for a single base substitution in the COL10A1 gene, which changed the codon GGC for glycine 618 to GTC for valine in the highly conserved region of the carboxyl-terminal NC1 domain and altered the amino acid sequence in the putative oligosaccharide attachment site. Since hypertrophic cartilage tissue or cell cultures were not available to assess the effect of the mutation, an in vitro cDNA expression system was used to study normal and mutant type X collagen biosynthesis and assembly. Full-length cDNA constructs of the normal type X collagen sequence and also cDNA containing the specific Gly to Val NC1 mutation found in the patient were produced and expressed by in vitro transcription and translation. While the control construct produced type X collagen, which formed trimeric collagen monomers and assembled into larger multimeric assemblies, the mutant collagen was unable to form these larger aggregates. These experiments demonstrated that the mutation disturbed type X collagen NC1 domain interaction and assembly, a finding consistent with the abnormal disorganized cartilage growth plate seen in the patient. These studies provide the first evidence of the effect of a type X collagen mutation on protein structure and function and directly demonstrate the critical role of interactions between NC1 domains in the formation of type X collagen multimeric structures in vitro.


INTRODUCTION

Type X collagen is a ``short chain'' collagen with a restricted pattern of transient expression confined to terminally differentiated hypertrophic chondrocytes in the calcifying cartilage of the growth plate (1, 2, 3) and in zones of secondary ossification(3) . The molecule comprises three alpha1(X) chains encoded by a condensed gene (COL10A1) of three exons, one of which (exon 3) codes for the majority of the chain (4, 5, 6, 7) . The type X collagen homotrimer contains a short triple helix domain flanked by a small non-helical region at the amino terminus (NC2) and a larger highly conserved carboxyl-terminal (NC1) non-helical domain. While type X collagen cDNA and gene organization has been established in several species(4, 5, 6, 7) , little is known about the biosynthetic pathway or the molecular assembly and secretion of the molecule. Evidence from in vitro aggregation studies indicated that type X collagen could form a supramolecular hexagonal lattice structure(8) .

The function of type X collagen remains unknown, but its restricted expression to zones of calcifying or degrading cartilage suggested that type X collagen may play an important role in cartilage mineralization (9) . It has been suggested that the type X collagen lattice may provide an open structural support that allows vascular invasion, calcification, and remodeling to proceed to the next phase of bone development(8) . This specific localization and postulated function suggested that defects of type X collagen synthesis and organization could underlie chondrodysplasias with primary growth plate involvement. Direct evidence supporting this hypothesis came from studies where a dominant negative mutation of type X collagen was used to produce a transgenic mouse with a spondylometaphyseal dysplasia phenotype (10) . Subsequently, several type X collagen mutations have been identified in patients with Schmid metaphyseal chondrodysplasia (SMCD, (^1)MIM 156500), an autosomal dominant chondrodysplasia where patients are of short stature and exhibit coxa vara and a waddling gait due to skeletal deformities resulting from growth plate abnormalities(11) . The seven mutations defined to date have been clustered in the carboxyl-terminal NC1 domain and include deletions and point mutations(12, 13, 14, 15) . Although no biochemical evidence has been presented to date, the localization of the mutations in the NC1 domain led these workers (13, 15) to speculate that mutations may prevent assembly of the mutant chains into trimers by disturbing critical sequence domains in the highly conserved NC1 sequence, and thus prevent deposition of type X collagen in the matrix.

In this study, we report a single base substitution that changes glycine 618 (^2)(GGC) to valine (GTC) in the conserved region of the type X collagen alpha1(X) NC1 domain in a family with SMCD. In vitro expression of mutant and normal alpha1(X) demonstrated that the mutation disturbed NC1 domain interactions and type X collagen assembly and provides the firstdirect evidence of the effect of a type X collagen mutation on protein structure and function.


EXPERIMENTAL PROCEDURES

Clinical Description

The proband presented as a 3.5-year-old with bowing of both the femur and tibia. Radiological examination of pelvis and limbs demonstrated coxa vara, irregularity, and widening of the epiphyseal plate, which was most marked in the proximal and distal femora and in the proximal tibial metaphysis. Histological examination of an iliac crest biopsy revealed an abnormality restricted to the hypertrophic zone of the growth plate cartilage, with an absence of identifiable hypertrophic chondrocytes and disorganized arrangement of the chondrocyte columns in the proliferative zone. The condition was diagnosed as SMCD. Two siblings and the mother were also affected. The father was clinically normal.

Amplification of COL10A1 Genomic Sequences

Genomic DNA was extracted from skin fibroblast grown in culture (16) or from whole blood(17) , and the entire coding sequence was PCR-amplified using sets of overlapping oligonucleotide primers based on the published human genomic sequence(18) . Exon 2 was amplified as a 287-bp product using the primers 5`-CTCATCTGTGAAACATGAGA-3` (intron 1) and 5`-CAAGCAACTTGTTAATAGAAC-3` (intron 2). Exon 3 was amplified using three sets of overlapping primers 5`-TCACTAACCATCCCCTTCTA-3` (intron 2) and 5`-GGTCCTCTTTCTCCCTTCAG-3` (bp 1031-1012) (^3)to generate a 850-bp fragment; 5`-AAAGGTGATAGAGGTTTTCC-3` (bp 796-815) and 5`-GCTTTGGAGAGAATAACAGT-3` (bp 1778-1759) to generate a 983-bp fragment; 5`-TAAAGGGGATCCAGGAAGTC-3` (bp 1491-1510) and 5`-CTTTTCAGCCTACCTCCATA-3` (bp 2235-2216) to generate a 745-bp fragment. Another set of primers that are internal to the 745-bp fragment 5`-CAGGGGGTAACAGGAATGCC-3` (bp 1726-1745) and 5`-TGAGAAAGAGGAGTGGACAT-3` (bp 2115-2096) was also used to generate a shorter fragment (390-bp) for cloning and sequencing. The polymerase chain reactions were carried out using the DNA amplification kit from Perkin-Elmer with the addition of 0.5 µl of [alpha-P]dCTP (3,000 Ci/mmol; DuPont NEN). The cycling conditions were 94 °C for 1.5 min, 62 °C for 1.5 min, and 72 °C for 2 min for 36 cycles. The amplified fragments were purified by gel electrophoresis on 2.5% (w/v) NuSieve agarose (FMC Bioproducts) and recovered using Geneclean (Bio 101 Inc.).

Amplification and Sequencing of the Coding Sequences

Genomic DNA from the patient was digested with restriction enzymes AccI and SacI, which cut in intron 2 and in the 3`-untranslated region, respectively, to produce a DNA fragment of 2.5 kilobases including the entire coding sequence of exon 3(18) . DNA fragments of 2.5 kilobases were size-selected and purified by electroelution. This fraction was used as template for the amplification of an exon 3 fragment using primers 5`-TCACTAACCATCCCCTTCTA-3` (intron 2) and 5`-CTTTTCAGCCTACCTCCATA-3` (bp 2235-2216). The amplification was carried out using a high fidelity thermally stable polymerase (Pfu, Stratagene) using cycling conditions of 95 °C for 1.5 min, 55 °C for 1.5 min, and 72 °C for 3.0 min over 35 cycles. The amplified 2034-bp fragment was purified and cloned into the SmaI site of pUC19 (New England Biolabs). Positive colonies were plasmid-purified and digested with BanI to determine the orientation of the inserts and to distinguish the normal from the abnormal allele. Inserts were cloned into M13 and sequenced using a Sequenase kit (U. S. Biochemical Corp.). The normal (pMC2N) and mutant (pMC2M) plasmids were also used in the construction of full-length cDNA constructs. The exon 2 coding sequence was amplified using the primers 5`-CTCATCTGTGAAACATGAGA-3` (intron 1) and 5`-CAAGCAACTTGTTAATAGAAC-3` (intron 2), purified, and sequenced(19) .

Preparation of Full-length Type X Collagen cDNA Constructs

The 5` cDNA sequences from the start of translation were generated by PCR of total mRNA extracted from chondrocytes isolated from human fetal hypertrophic cartilage using the rapid procedure of Gough et al.(20) . An oligonucleotide, 5`-CTTTCTGTCCATTCATACCA-3` (bp 661-642), was used as primer for cDNA synthesis and also used as the 3` primer in the PCR reaction. The conditions for reverse transcriptase PCR (Perkin Elmer) was as per manufacturer's protocol with 500 ng of mRNA. The cycling conditions were 95 °C for 5 min, 60 °C for 1.5 min, and 72 °C for 3.0 min for one cycle; then 95 °C for 1.5 min, 60 °C for 1.5 min, and 72 °C for 2.0 min over 35 cycles; and finished off with 7 min at 72 °C. The 5` primer AGAACATGTTGCCACAAATACCCT (bp 92-115) was designed with the introduction of an AflIII restriction enzyme site for cloning into the NcoI-compatible site of pTM1(21) . This change does not alter the resultant amino acid sequence of the translated protein. These primers were selected for the amplification of a 570-bp fragment, which contained sequences from the start of translation. This fragment was digested with restriction enzymes AflIII and XhoI to generate two major fragments of 208 and 357 bp. The 208-bp fragment (start of translation bp 97-304) was cloned into the NcoI and XhoI sites of pTM1. A positive colony (pTM1-H10a) was selected and sequenced to ensure there were no PCR errors. Full-length cDNA constructs of normal and mutant type X collagen were constructed by cloning a 1946-bp XhoI and SalI fragment (bp 305-2235) of type X collagen and 15 bp of pUC19 polylinker sequence (generated from the pUC19 plasmids pMC2N and pMC2M) into pTM1-H10a.

In Vitro Transcription and Translation

Type X collagen constructs were expressed using the TNT T7 polymerase-coupled recticulocyte lysate system (Promega) in a final reaction volume of 50 µl. 1 µg of the purified plasmid was incubated with 10-20 units of T7 polymerase in the presence of 40 units of ribonuclease inhibitor at 30 °C for 90 min. The translated products were labeled with translation grade L-[S]methionine (1000 Ci/mmol, DuPont NEN). In some experiments, equal amounts of normal and mutant plasmids were added to the same lysate mix and co-transcribed and translated as described above. Canine microsomal membranes (Promega) were added to the reaction mix in some experiments to promote post-translational modification of the translated products. The recommended protocols from the manufacturer were used.

Digestion with Bacterial Collagenase

5 µl of the translation mix were digested with 0.625 µg (geq300 units/mg) of highly purified Clostridium histolyticum collagenase (CLSPA, Worthington) in a final volume of 30 µl of 50 mM Tris/HCl (pH 7.5), containing 5 mM CaCl(2) and 5 mMN-ethymaleimide at 37 °C for 2.5 h. The digestion was terminated by the addition of an equal volume of a 2 times SDS-PAGE sample buffer and analyzed by SDS-PAGE.

Formation of Multimeric Structures

5 µl of the translation product was diluted to 30 µl with 50 mM Tris/HCl (pH 7.5), containing 5 mM CaCl(2) and 5 mMN-ethymaleimide and incubated at 37 °C. At the appropriate time points, the reaction was terminated by the addition of an equal volume of a 2 times SDS-PAGE sample buffer and analyzed by SDS-PAGE.

SDS-Polyacrylamide Gel Electrophoresis

Type X collagen chains were resolved on 7.5% (w/v) polyacrylamide separating gels with a 3.5% (w/v) stacking gel. The NC1 non-collagenous domains were resolved on 12.5% (w/v) polyacrylamide with a 4.5% (w/v) stacking gel. Samples were diluted with loading buffer to give a final concentration of 0.125 mM Tris/HCl, pH 6.8, containing 2% (w/v) SDS, 2 M urea, and 10 mM dithiothreitol and heated to 60 °C for 10 min prior to electrophoresis. Some samples were heated to 100 °C for 5 min, while others were not heated prior to electrophoresis. Electrophoresis conditions and fluorography of radioactive gels have been described previously(22, 23) . The distribution of radioactivity in specific type X collagen assemblies was quantified by excision of bands from the gels and scintillation counting(24) .


RESULTS AND DISCUSSION

Four PCR products spanning the entire COL10A1 gene coding sequence were analyzed for mutations by the chemical cleavage method(25) . Only one of the four amplification products contained a mismatch. The 390-bp fragment covering bases 1726-2115 of the COL10A1 cDNA sequence produced two unique cleavage products of 222 and 168 bp (data not shown). This result suggested that there was an allelic sequence variation within this region covering the NC1 protein domain. To identify the base change, this 390-bp PCR product was cloned and sequenced. Of the 10 clones sequenced, there were 6 normal clones and 4 clones with a base change of G to T resulting in the alteration of Gly (GGC) to Val (GTC) in the highly conserved NC1 domain of the alpha1(X) chain of type X collagen (Fig. 1).


Figure 1: Sequences of the normal and abnormal alleles prepared from SMCD fibroblast DNA. The amplified 390-bp PCR products containing the mismatch were cloned and sequenced (see ``Experimental Procedures'' for details). a, diagrammatic representation of the mutation relative to the alpha1(X) chain and COL10A1 gene structure. b, sequencing gel showing the normal and the abnormal sequences. The base substitution is indicated by arrowheads. The corresponding coding strand sequences and the deduced amino acid sequence with a box designating the abnormal codon sequence in the substitution of glycine 618 by valine.



The mutation deleted a BanI restriction enzyme cleavage site in the DNA sequence, and this was used to confirm the base change on multiple PCR products amplified from the patient and from other family members (data not shown). The altered restriction enzyme digestion pattern was also present in the affected mother and two other affected siblings but was not present in the clinically normal father, indicating that the mutation segregated with the disease in this small pedigree. This sequence change was not detected in the same PCR fragment from 60 alleles of unrelated individuals of the same ethnic origin (data not shown).

To ensure that there were no other sequence alterations undetected by chemical cleavage, the entire coding sequence of exon 3 of the COL10A1 gene was amplified, cloned, and sequenced. Sequencing of both the normal and abnormal alleles revealed that both carried the ACG(-) ATG (+) Thr Met polymorphism. The normal allele also carried an additional CCC deletion(-) polymorphism 6 bp downstream from the TGA termination codon. These polymorphisms have been reported previously by Sweetman et al.(19) . No other base changes were observed when compared with the published sequence(18) . Our data also confirmed the coding sequence deviations observed by Reichenberger et al.(18) from the previous published sequence(5) . Exon 2 from the patient was also amplified and sequenced directly. A silent polymorphism was detected in one of the alleles, which changed the codon for Gly in the NC2 domain (GGA GGT). Because of the nature of direct sequencing, we were unable to assign this polymorphism to a specific allele. All other sequences were in agreement with published data(18) .

The Gly to Val mutation is one of eight mutations in the alpha1(X) NC1 domain leading to SMCD. Two deletion mutations of 13 bp (bp 1953-1965) and 10 bp (bp 1963-1972) (13, 14) occur in the sequence immediately adjacent within the NC1 domain. These were linked to the disease in large pedigrees and led to codon frameshifts and the theoretical generation of an abnormal carboxyl-terminal NC1 protein sequence. Deletions (1953 delC and 2088 delCT) result in the introduction of premature termination signals and NC1 truncation(12) . Three point mutations producing amino acid substitutions of Cys Arg(12) , Tyr Asp, and Leu Pro (15) have also been reported. The localization of all these mutations to within a relatively small region of the NC1 domain hinted at the critical importance of this domain. It was further speculated that the NC1 mutations may cause type X collagen malfunction by perturbing chain association(13, 15) , based on theoretical considerations and analogy to fibrillar collagen biochemistry(26) , but no experimental evidence has been provided to demonstrate a type X collagen protein abnormality resulting from the mutations.

In the SMCD case reported here, we undertook studies to directly demonstrate the effect of the mutation at the protein level. Since hypertrophic cartilage tissue or cell cultures were not available to isolate type X collagen in order to assess the effect of the mutation, an in vitro cDNA expression system was used to study normal and mutant type X collagen biosynthesis and assembly. Full-length cDNA constructs of the normal and the Gly Val mutant type X collagen alleles were produced by the PCR and cloning procedures. These constructs were expressed by in vitro transcription and translation using a cell-free system, which will perform transcription, translation, post-translational modification, and assembly of type X collagen(27) . Expression of the control or mutant constructs (Fig. 2a, lanes6 and 7) demonstrated that, in the absence of canine microsomes, the type X collagen chains were synthesized as pre-alpha1(X) chains with the intact hydrophobic leader sequence. If microsomal preparations were included in the translation (Fig. 2a, lanes1-5), the leader sequence was removed, generating the smaller processed alpha1(X) chain(27) . The striking feature of the control alpha1(X) was its ability to form stable associations of (alpha1(X))(3) during the translation reaction (Fig. 2a, lanes 2-4). In contrast, the mutant alpha1(X) was unable to form these multimeric structures (Fig. 2a, lanes 1 and 5). While the formation of these higher order structures was more efficient in the presence of microsomal membranes (compare lanes2-4 with lane6), they were not essential for the association process, since normal alpha1(X) cell-free translation products incubated in the absence of microsomal membranes also formed trimers and multimers when the translation mix was diluted in buffer containing 5 mM CaCl(2) and incubated at 37 °C for additional periods (Fig. 3a, lanes 1-4).


Figure 2: Cell free translation of normal and mutant type X collagen. The normal and mutant type X collagen cDNA constructs were transcribed and translated in a coupled cell-free translation system (see ``Experimental Procedures'' for details). The resultant [S]methionine-labeled products were analyzed by SDS-polyacrylamide gel electrophoresis. a, lanes2-4 and 6 are the products from the normal construct, while lanes1, 5, and 7 are the products from the mutant construct. Translations were carried out either in the presence (+) or the absence(-) of canine microsomal membranes. Prior to electrophoresis on 7.5% gels, the samples were denatured using different temperatures as indicated. b, the normal (N) and mutant (M) constructs were translated in the absence of microsomal membranes. The translated products were digested with bacterial collagenase at 37 °C for 2.5 h (see ``Experimental Procedures'' for details), and the resultant NC1 domain was denatured at 60 °C for 10 min and analyzed on a 12.5% gels. Lanes1 and 2 are the products after incubation in the absence of collagenase; lanes3 and 4 are the products after incubation with collagenase. The migration positions of alpha1(X) containing the hydrophobic signal sequence (pre-alpha1(X)), alpha1(X) after cleavage of this sequence, trimeric alpha1(X)(3), NC1 monomers, and trimers (NC1)(3) are indicated.




Figure 3: Multimer formation by cell-free translation products. The normal and mutant constructs were translated in the absence (a) or presence (b) of microsomal membranes. The resultant products were tested for their ability to form multimers by incubation at 37 °C in an appropriate buffer for periods up to 60 min (see ``Experimental Procedures'' for details). Samples were denatured at 60 °C for 10 min prior to electrophoresis on a 7.5% SDS-polyacrylamide gel. a, lanes 1-4, products for transcription and translation of the normal construct; lanes5-8, product of the mutant construct; lanes 8-12, products of an equal mixture of normal and mutant constructs. b, lane1, transcription and translation of 1 µg of normal plasmid; lane2, transcription and translation of a mixture of 0.5 µg of normal and 0.5 µg of mutant plasmid. The migration positions of monomeric pre-alpha1(X), alpha1(X), and trimeric assemblies of pre-alpha1(X)(3) and alpha1(X)(3) are indicated. pre-alpha1(X) designates the presumed larger multimeric assemblies.



The stability of the type X collagen trimers formed during cell-free translation was reduced by increasing the temperature of sample denaturation prior to electrophoresis (Fig. 2a). If the samples were not heated, but allowed to stand at 20 °C before gel loading, the predominant species was alpha1(X)(3) (Fig. 2a, lane2). When samples were denatured at 60 °C for 10 min (Fig. 2a, lane4), the proportion of alpha1(X)(3), while reduced, was still the predominant species. However, denaturation at 100 °C for 5 min (Fig. 2a, lane3) significantly denatured the alpha1(X)(3) complex so that the majority of type X collagen migrated as an alpha1(X) monomer. These data support the suggestion that type X collagen multimers are held together by strong non-covalent bonds, probably predominantly hydrophobic interactions within the NC1 domains(1, 26, 28) . To further study the interactions of the non-collagenous NC1 domains of the normal and mutant type X collagen, collagenase digestion was used to remove the collagenous helix-forming domain (Fig. 2b). These data demonstrate that while the normal NC1 domains associate into higher order (NC1)(3) components (Fig. 2b, lane3), the mutant NC1 domain is not able to associate into multimers (Fig. 2b, lane4). The slight change in migration of the mutant NC1 domain relative to the control may reflect subtle changes in secondary structure or variation in SDS binding.

The extent of multimer formation of the control construct translation products increased with time of incubation at 37 °C in buffer containing CaCl(2), first by the formation of trimers and then the subsequent formation of aggregates that were too large to migrate into the stacking gel (Fig. 3a, lanes 1-4). No significant formation of alpha1(X)(3) trimers or larger multimers were detected with products of the mutant construct translation, even after 60 min of incubation in vitro (Fig. 3a, lanes 5-8). To determine whether the mutant pre-alpha1(X) chain interferes with the molecular assembly of normal pre-alpha1(X) chain in vitro, equal amounts of normal and mutant construct DNA were mixed and co-transcribed and translated (Fig. 3a, lanes 9-12). The amount of trimeric and larger multimeric structures formed by the normal pre-alpha1(X) was comparable to that formed in the absence of the mutant product (compare lanes 9-12 with lanes 1-4), demonstrating that in vitro normal pre-alpha1(X) assembly is not impeded by the presence of mutant pre-alpha1(X). The persistence of monomeric pre-alpha1(X) (compare lane12 with lane4) confirms that the mutant pre-alpha1(X) remains as unassembled monomeric chains.

Since in vitro assembly was more efficient in the presence of microsomal membranes, it was suggested that the structural organization imposed by the microsomes, or the presence in the extracts of accessory or chaperoning proteins may facilitate assembly(27) . Thus it was important to study the effect of mutant type X on normal type X collagen assembly in the presence of microsomes to determine if, under these more favorable assembly conditions, mutant collagen interacted with normal collagen and affected association and multimer formation. In this experiment, the coupled transcription and translation of 1 µg of normal plasmid (Fig. 3b, lane1) was compared with the co-transcription and translation of a mixture of 0.5 µg of normal and 0.5 µg of mutant plasmid (Fig. 3b, lane2). Quantification of the monomer and trimer bands demonstrated the expected amount of normal alpha1(X)(3) and increased accumulation of monomeric alpha1(X) corresponding to unassociated mutant. These data further confirm that mutant alpha1(X) synthesis does not affect the efficiency of normal alpha(X) chain multimer assembly in vitro.

These experiments directly demonstrated, for the first time, the effect of a type X collagen mutation on protein assembly. The NC1 Gly Val mutation prevented the in vitro assembly of type X collagen trimers by interfering with the association of NC1 domains. The molecular mechanisms of type X assembly are not known, but by analogy with the more extensively studied fibril-forming collagens, it is thought that assembly is initiated by nucleation of three alpha-chains at the carboxyl-terminal end of the molecule with propagation of the helix toward the amino terminus(29) . Our data demonstrate the critical role of these NC1 domains in this assembly process. The initial assembly of the alpha1(X) chains is an efficient process, even under the non-optimal in vitro conditions used, and denaturation studies (Fig. 2) demonstrated that, once associated, the multimers are very stable. The association of the NC1 domains into multimeric components has been observed previously in chicken type X collagen and appears to be important for the end-to-end association in the formation of hexagonal lattice structures(8) .

The mechanism by which the mutation perturbs assembly is not known, but the clustering of known mutations causing SMCD in the NC1 domain emphasizes the critical role of this region. Gly is conserved in type X collagen sequences from all the species published so far, including chicken(4) , mouse(7) , bovine(6) , and human(5) . The mutation may affect the initial NC1 hydrophobic interactions directly, since a Gly Val substitution would increase the hydrophobicity, or by altering critical protein secondary structure. This mutation also changed the amino acid sequence of the putative oligosaccharide attachment site from Asn-Gly-Thr to Asn-Val-Thr. It is not known if this site is normally glycosylated, but the sequence change may affect its ability to be N-glycosylated or alter the structure of the oligosaccharide attachment(30, 31) .

While it is likely that the same forces drive type X collagen assembly in vivo, and the mutation will also prevent initial association, trimerization, and matrix assembly in vivo, it is possible that special localized conditions or factors may allow some mutant association. If a proportion of the mutant chain can associate in vivo, it may exert a dominant negative effect. To fully resolve this important question, cell transfection studies are in progress. The more likely scenario, supported by our in vitro data, is that the NC1 mutation leads to haploinsufficiency by preventing assembly, and by analogy with mutations that completely prevent type I collagen assembly in osteogenesis imperfecta(32, 33) , would prevent type X collagen secretion and target the mutant alpha1(X) chains for rapid intracellular degradation. This would result in a type X collagen deficiency in the cartilage growth plate and dramatically reduce the type X collagen open matrix structure, which is thought to be critical for hypertrophic chondrocyte function leading to the disorganization of the growth plate seen in patients with SMCD. Recently, type X collagen-null transgenic mice have been generated by gene targeting in embryonic stem cells(34) . These mice show no obvious phenotypic abnormalities in long bone growth and development. The reason for this surprising finding has not yet been established but may reflect species-specific differences in type X collagen function or anatomical or developmental factors which result in haploinsufficiency of type X collagen having a more severe phenotype in humans than mice.


FOOTNOTES

*
This work was supported by grants from the National Health and Medical Research Council of Australia and the Royal Children's Hospital Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Division of Orthopedics, The Hospital for Sick Children, Toronto M5G 1X8, Canada.

To whom correspondence should be addressed: Dept. of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, Victoria 3052, Australia. Fax: 61-3-345-5789; bateman{at}cryptic.rch.unimelb.edu.au.

(^1)
The abbreviations used are: SMCD, Schmid metaphyseal chondrodysplasia(11) ; PAGE, polyacylamide gel electrophoresis; PCR, polymerase chain reaction; bp, base pair(s).

(^2)
Amino acids are numbered from the translation start site of pre-alpha1(X)(5

(^3)
The base pairs in the COL10A1 coding sequence are numbered from the transcription start site(18) .


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