Aberrant signal peptide cleavage of collagen X in Schmid metaphyseal chondrodysplasia. Implications for the molecular basis of the disease

Schmid metaphyseal chondrodysplasia results from mutations in the collagen X (COL10A1) gene. With the exception of two cases, the known mutations are clustered in the C-terminal nonhelical (NC1) domain of the collagen X. In vitro and cell culture studies have shown that the NC1 mutations result in impaired collagen X trimer assembly and secretion. In the two other cases, missense mutations that alter Gly(18) at the -1 position of the putative signal peptide cleavage site were identified (Ikegawa, S., Nakamura, K., Nagano, A., Haga, N., and Nakamura, Y. (1997) Hum. Mutat. 9, 131-135). To study their impact on collagen X biosynthesis using in vitro cell-free translation in the presence of microsomes, and cell transfection assays, these two mutations were created in COL10A1 by site-directed mutagenesis. The data suggest that translocation of the mutant pre-alpha1(X) chains into the microsomes is not affected, but cleavage of the signal peptide is inhibited, and the mutant chains remain anchored to the membrane of microsomes. Cell-free translation and transfection studies in cells showed that the mutant chains associate into trimers but cannot form a triple helix. The combined effect of both the lack of signal peptide cleavage and helical configuration is impaired secretion. Thus, despite the different nature of the NC1 and signal peptide mutations in collagen X, both result in impaired collagen X secretion, probably followed by intracellular retention and degradation of mutant chains, and causing the Schmid metaphyseal chondrodysplasia phenotype.     

Insight into Schmid metaphyseal chondrodysplasia from the crystal structure of the collagen X NC1 domain trimer

Collagen X is expressed specifically in the growth plate of long bones. Its C1q-like C-terminal NC1 domain forms a stable homotrimer and is crucial for collagen X assembly. Mutations in the NC1 domain cause Schmid metaphyseal chondrodysplasia (SMCD). The crystal structure at 2.0 A resolution of the human collagen X NC1 domain reveals an intimate trimeric assembly strengthened by a buried cluster of calcium ions. Three strips of exposed aromatic residues on the surface of NC1 trimer are likely to be involved in the supramolecular assembly of collagen X. Most internal SMCD mutations probably prevent protein folding, whereas mutations of surface residues may affect the collagen X suprastructure in a dominant-negative manner.     

Schmid's metaphyseal chondrodysplasia mutations interfere with folding of the C-terminal domain of human collagen X expressed in Escherichia coli.

Human collagen X contains a highly conserved 161-amino acid C-terminal non-triple helical domain that is homologous to the C-terminal domain of collagen VIII and to the C1q module of the human C1 enzyme. We have expressed this domain (residues 545-680) in Escherichia coli as a glutathione S-transferase fusion protein. The purified fusion protein trimerizes spontaneously in vitro, and after thrombin cleavage, the purified C-terminal domain trimer (46.2 kDa) is extremely stable and trypsin-resistant. Mutations within the C-terminal domain have been observed in patients with Schmid's metaphyseal chondrodysplasia (SMCD). Some of these mutations (Y598D, G618V, W651X, or H669X; X is the stop codon) were constructed by site-directed mutagenesis. Each mutation had identical consequences regarding the fusion protein: 1) absence of trimeric formation, 2) copurification of the approximately 60-kDa GroEL chaperone protein, and 3) sensitivity of the monomeric fusion protein to trypsin digestion. These results show that the C-terminal domain of collagen X is sufficient to produce a very stable and compact trimer in the absence of collagen Gly-X-Y repeats. Moreover, mutations causing SMCD interfere in this system with the correct folding of the C-terminal domain. The existence of a similar mechanism in chondrocytes might explain the relative homogeneity of phenotypes in SMCD despite the diversity of mutations.     

Collagen X chains harboring Schmid metaphyseal chondrodysplasia NC1 domain mutations are selectively retained and degraded in stably transfected cells.

Collagen X is a short chain, homotrimeric collagen expressed specifically by hypertrophic chondrocytes during endochondral bone formation and growth. Although the exact role of collagen X remains unresolved, mutations in the COL10A1 gene disrupt growth plate function and result in Schmid metaphyseal chondrodysplasia (SMCD). With the exception of two mutations that impair signal peptide cleavage during alpha1(X) chain biosynthesis, SMCD mutations are clustered within the carboxyl-terminal NC1 domain. The formation of stable NC1 domain trimers is a critical stage in collagen X assembly, suggesting that mutations within this domain may result in subunit mis-folding or reduce trimer stability. When expressed in transiently transfected cells, alpha1(X) chains containing SMCD mutations were unstable and presumed to be degraded intracellularly. More recently, in vitro studies have shown that certain missense mutations may exert a dominant negative effect on alpha1(X) chain assembly by formation of mutant homotrimers and normal-mutant heterotrimers. In contrast, analysis of cartilage tissue from two SMCD patients revealed that the truncated mutant message was fully degraded, resulting in 50% reduction of functional collagen X within the growth plate. Therefore, in the absence of data that conclusively demonstrates the full cellular response to mutant collagen X chains, the molecular mechanisms underlying SMCD remain controversial. To address this, we closely examined the effect of two NC1 domain mutations, one frameshift mutation (1963del10) and one missense mutation (Y598D), using both semi-permeabilized cell and stable cell transfection expression systems. Although able to assemble to a limited extent in both systems, we show that, in intact cells, collagen X chains harboring both SMCD mutations did not evade quality control mechanisms within the secretory pathway and were degraded intracellularly. Furthermore, co-expression of wild-type and mutant chains in stable transfected cells demonstrated that, although wild-type chains were secreted, mutant chains were largely excluded from hetero-trimer formation. Our data indicate, therefore, that the predominant effect of the NC1 mutations Y598D and 1963del10 is a reduction in the amount of functional collagen X within the growth cartilage extracellular matrix.     

Amino acid substitutions of conserved residues in the carboxyl-terminal domain of the alpha 1(X) chain of type X collagen occur in two unrelated families with metaphyseal chondrodysplasia type Schmid.

Type X collagen is a homotrimeric, short-chain, nonfibrillar extracellular-matrix component that is specifically and transiently synthesized by hypertrophic chondrocytes at the sites of endochondral ossification. The precise function of type X collagen is not known, but its specific pattern of expression suggests that mutations within the encoding gene (COL10A1) that alter the structure or synthesis of the protein may cause heritable forms of chondrodysplasia. We used the PCR and the SSCP techniques to analyze the coding and upstream promoter regions of the COL10A1 gene in a number of individuals with forms of chondrodysplasia. Using this approach, we identified two individuals with metaphyseal chondrodysplasia type Schmid (MCDS) with SSCP changes in the region of the gene encoding the carboxyl-terminal domain. Sequence analysis demonstrated that the individuals were heterozygous for two unique single-base-pair transitions that led to the substitution of the highly conserved amino acid residue tyrosine at position 598 by aspartic acid in one person and of leucine at position 614 by proline in the other. The substitution at residue 598 segregated with the phenotype in a family of eight (five affected and three unaffected) related persons. The substitution at residue 614 occurred in a sporadically affected individual but not in her unaffected mother and brother. Additional members of this family were not available for further study. These results suggest that certain amino acid substitutions within the carboxyl-terminal domain of the chains of the type X collagen molecule cause MCDS. These amino acid substitutions are likely to alter either chain recognition or assembly of the type X collagen molecule, thereby depleting the amount of normal type X collagen deposited in the extracellular matrix, with consequent aberrations in bone growth and development.     

Crystal structure of the collagen alpha1(VIII) NC1 trimer.

Collagen VIII is a major component of Descemet's membrane and is also found in vascular subendothelial matrices. The C-terminal non-collagenous domain (NC1) domain of collagen VIII, which is a member of the C1q-like protein family, forms a stable trimer and is thought to direct the assembly of the collagen triple helix, as well as polygonal supramolecular structures. We have solved the crystal structure of the mouse alpha1(VIII)(3) NC1 domain trimer at 1.9 A resolution. Each subunit of the intimate NC1 trimer consists of a ten-stranded beta-sandwich. The surface of the collagen VIII NC1 trimer presents three strips of partially exposed aromatic residues shown to interact with the non-ionic detergent CHAPS, which are likely to be involved in supramolecular assemblies. Equivalent strips exist in the NC1 domain of the closely related collagen X, suggesting a conserved assembly mechanism. Surprisingly, the collagen VIII NC1 trimer lacks the buried calcium cluster of the collagen X NC1 trimer. The mouse alpha1(VIII) and alpha2(VIII) NC1 domains are 71.5% identical in sequence, with the differences being concentrated on the NC1 trimer surface. A few non-conservative substitutions map to the subunit interfaces near the surface, but it is not obvious from the structure to what extent they determine the preferred assembly of collagen VIII alpha1 and alpha2 chains into homotrimers.     

Competency for nonsense-mediated reduction in collagen X mRNA is specified by the 3' UTR and corresponds to the position of mutations in Schmid metaphyseal chondrodysplasia

Nonsense-mediated decay (NMD) is a eukaryotic cellular RNA surveillance and quality-control mechanism that degrades mRNA containing premature stop codons (nonsense mutations) that otherwise may exert a deleterious effect by the production of dysfunctional truncated proteins. Collagen X (COL10A1) nonsense mutations in Schmid-type metaphyseal chondrodysplasia are localized in a region toward the 3' end of the last exon (exon 3) and result in mRNA decay, in contrast to most other genes in which terminal-exon nonsense mutations are resistant to NMD. We introduce nonsense mutations into the mouse Col10a1 gene and express these in a hypertrophic-chondrocyte cell line to explore the mechanism of last-exon mRNA decay of Col10a1 and demonstrate that mRNA decay is spatially restricted to mutations occurring in a 3' region of the exon 3 coding sequence; this region corresponds to where human mutations have been described. This localization of mRNA-decay competency suggested that a downstream region, such as the 3' UTR, may play a role in specifying decay of mutant Col10a1 mRNA containing nonsense mutations. We found that deleting any of the three conserved sequence regions within the 3' UTR (region I, 23 bp; region II, 170 bp; and region III, 76 bp) prevented mutant mRNA decay, but a smaller 13 bp deletion within region III was permissive for decay. These data suggest that the 3' UTR participates in collagen X last-exon mRNA decay and that overall 3' UTR configuration, rather than specific linear-sequence motifs, may be important in specifying decay of Col10a1 mRNA containing nonsense mutations.     

Folding and assembly of type X collagen mutants that cause metaphyseal chondrodysplasia-type schmid. Evidence for co-assembly of the mutant and wild-type chains and binding to molecular chaperones

Schmid metaphyseal chondrodysplasia results from mutations within the COOH-terminal globular domain (NC1) of type X collagen, a short chain collagen expressed in the hypertrophic region of the growth plate cartilage. Previous in vitro studies have proposed that mutations prevent the association of the NC1 domain of constituent chains of the trimer based upon a lack of formation of a trimeric structure that is resistant to dissociation with sodium dodecyl sulfate. To examine the effect of mutations on folding and assembly within a cellular context, bovine type X cDNAs containing analogous disease causing mutations Y598D, N617K, W651R, and wild-type were expressed in semi-permeabilized cells. We assessed trimerization of the mutant chains by their ability to form a collagen triple helix. Using this approach, we demonstrate that although there is an apparent lower efficiency of association of the mutant NC1 domains, they can drive the formation of correctly aligned triple helices with the same thermal stability as the wild-type collagen. When epitope-tagged mutant and wild-type collagen were co-expressed, heterotrimers could be detected by sequential immunoprecipitation. Both wild-type and mutant type X chains were found in association with the molecular chaperones protein disulfide isomerase and Hsp 47. The implications of these findings on the likely mechanism of Schmid metaphyseal chondrodysplasia will be discussed.     

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

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.     

SSCP and segregation analysis of the human type X collagen gene (COL10A1) in heritable forms of chondrodysplasia.

Type X collagen is a homotrimeric, short chain, nonfibrillar collagen that is expressed exclusively by hypertrophic chondrocytes at the sites of endochondral ossification. The distribution and pattern of expression of the type X collagen gene (COL10A1) suggests that mutations altering the structure and synthesis of the protein may be responsible for causing heritable forms of chondrodysplasia. We investigated whether mutations within the human COL10A1 gene were responsible for causing the disorders achondroplasia, hypochondroplasia, pseudoachondroplasia, and thanatophoric dysplasia, by analyzing the coding regions of the gene by using PCR and the single-stranded conformational polymorphism technique. By this approach, seven sequence changes were identified within and flanking the coding regions of the gene of the affected persons. We demonstrated that six of these sequence changes were not responsible for causing these forms of chondrodysplasia but were polymorphic in nature. The sequence changes were used to demonstrate discordant segregation between the COL10A1 locus and achondroplasia and pseudoachondroplasia, in nuclear families. This lack of segregation suggests that mutations within or near the COL10A1 locus are not responsible for these disorders. The seventh sequence change resulted in a valine-to-methionine substitution in the carboxyl-terminal domain of the molecule and was identified in only two hypochondroplasic individuals from a single family. Segregation analysis in this family was inconclusive, and the significance of this substitution remains uncertain.     

Homologies between the non-collagenous C-terminal (NC1) globular domains of the alpha 1 and alpha 2 subunits of type-IV collagen

Many promoter-fusion vectors contain an intact beta-lactamase (BLA) gene (bla) to allow measurement of BLA activity as an internal control for plasmid copy number. This approach rests on the assumption that bla is constitutively expressed. To use such vectors for comparison of promoter activity at different growth rates it was necessary to confirm that this is the case under all physiological conditions. The relationship between plasmid copy number and BLA activity at different steady-state growth rates in Escherichia coli HB101 transformed with a ColE1-type plasmid (pBR325) was examined. Both BLA activity and plasmid copy number decreased in a parallel fashion as growth rate increased. This finding was tested further by measuring the growth-rate-regulated expression of the chloramphenicol acetyltransferase (CAT) gene under the control of the rrnB P1 promoter in a plasmid pKK231-1 fusion. The results indicate that BLA activity is a reliable indicator of copy number at a wide range of growth rates and that CAT/BLA ratios can be employed as a valid measure of promoter-specific activity in such plasmid fusions under these different physiological conditions.     

Central tolerance regulates B cells reactive with Goodpasture antigen alpha3(IV)NC1 collagen

Patients and rodents with Goodpasture's syndrome (GPS) develop severe autoimmune crescentic glomerulonephritis, kidney failure, and lung hemorrhage due to binding of pathogenic autoantibodies to the NC1 domain of the alpha3 chain of type IV collagen. Target epitopes are cryptic, normally hidden from circulating Abs by protein-protein interactions and the highly tissue-restricted expression of the alpha3(IV) collagen chain. Based on this limited Ag exposure, it has been suggested that target epitopes are not available as B cell tolerogens. To determine how pathogenic anti-GPS autoantibody responses are regulated, we generated an Ig transgenic (Tg) mouse model that expresses an Ig that binds alpha3(IV)NC1 collagen epitopes recognized by serum IgG of patients with GPS. Phenotypic analysis reveals B cell depletion and L chain editing in Tg mice. To determine the default tolerance phenotype in the absence of receptor editing and endogenous lymphocyte populations, we crossed Tg mice two generations with mice deficient in Rag. Resulting Tg Rag-deficient mice have central B cell deletion. Thus, development of Tg anti-alpha3(IV)NC1 collagen B cells is halted in the bone marrow, at which point the cells are deleted unless rescued by a Rag enzyme-dependent process, such as editing. The central tolerance phenotype implies that tolerizing self-Ag is expressed in bone marrow.     

The complete primary structure of mouse alpha 2(IV) collagen. Alignment with mouse alpha 1(IV) collagen

We have determined the nucleotide and amino acid sequences of mouse alpha 2(IV) collagen which is 1707 amino acids long. The primary structure includes a putative 28-residue signal peptide and contains three distinct domains: 1) the 7 S domain (residues 29-171), which contains 5 cysteine and 8 lysine residues, is involved in the cross-linking and assembly of four collagen IV molecules; 2) the triple-helical domain (residues 172-1480), which has 24 sequence interruptions in the Gly-X-Y repeat up to 24 residues in length; and 3) the NC1 domain (residues 1481-1707), which is involved in the end-to-end assembly of collagen IV and is the most highly conserved domain of the protein. Alignment of the primary structure of the alpha 2(IV) chain with that of the alpha 1(IV) chain reported in the accompanying paper (Muthukumaran, G., Blumberg, B., and Kurkinen, M. (1989) J. Biol. Chem. 264, 6310-6317) suggests that a heterotrimeric collagen IV molecule contains 26 imperfections in the triple-helical domain. The proposed alignment is consistent with the physical data on the length and flexibility of collagen IV.     

Identification of collagen alpha1(I) trimer and normal type I collagen in a polyoma virus-induced mouse tumor.

A bone- and cartilage-forming mouse tumor, induced by transforming salivary epithelial cells with polyoma virus, contained large quantities of collagen. Two types of collagen molecules were isolated which had different solubilities in salt. One was type I collagen with a chain composition [α1(I)]₂ α2 and the other was an unusual form of type I collagen with a chain composition [α1(I)]₃. This would appear to be the first in vivo demonstration of α1 type I trimer.     

Copolymerization of normal type I collagen with three mutated type I collagens containing substitutions of cysteine at different glycine positions in the alpha 1 (I) chain.

Previous observations with type I collagen from a proband with lethal osteogenesis imperfecta demonstrated that type I collagen containing a substitution of cysteine for glycine alpha 1-748 copolymerized with normal type I collagen (Kadler, K. E., Torre-Blanco, A., Adachi, E., Vogel, B. E., Hojima, Y., and Prockop, D. J. (1991) Biochemistry 30, 5081-5088). Here, three preparations containing normal type I procollagen and type I procollagen with a substitution of cysteine for glycine alpha 1-175, glycine alpha 1-691, or glycine alpha 1-988 were purified from cultured skin fibroblasts from probands with osteogenesis imperfecta. The procollagens were then used as substrates in a system for assaying the self-assembly of type I collagen into fibrils. The cysteine-substituted collagens in all three preparations were incorporated into fibrils. The cysteine alpha 1-175 and cysteine alpha 1-691 collagens were shown to increase the lag time and decrease the propagation rate constant for fibril assembly. All three preparations containing cysteine-substituted collagens formed fibrils with diameters that were two to four times the diameter of fibrils formed under the same conditions by normal type I collagen. Also, the three preparations containing cysteine substituted collagens had higher solubilities than normal type I collagen. The results, therefore, demonstrated that the three cysteine-substituted collagens copolymerized with normal type I collagen. The effects of the mutated collagens on fibril assembly can be understood in terms of a recently proposed model of fibril growth from symmetrical tips by assuming that the mutated monomers partially inhibit tip growth but not lateral growth of the fibrils. Of special interest was the observation that the Cys alpha 1-175 collagen from a proband with a non-lethal variant of osteogenesis imperfecta had quantitatively less effect on several parameters of fibril assembly at 37 degrees C than cysteine-substituted collagens from three probands with lethal variants of the disease.     

Crystal structure of NC1 domains. Structural basis for type IV collagen assembly in basement membranes

Type IV collagen, which is present in all metazoan, exists as a family of six homologous alpha(IV) chains, alpha1-alpha6, in mammals. The six chains assemble into three different triple helical protomers and self-associate as three distinct networks. The network underlies all epithelia as a component of basement membranes, which play important roles in cell adhesion, growth, differentiation, tissue repair and molecular ultrafiltration. The specificity of both protomer and network assembly is governed by amino acid sequences of the C-terminal noncollagenous (NC1) domain of each chain. In this study, the structural basis for protomer and network assembly was investigated by determining the crystal structure of the ubiquitous [(alpha1)(2).alpha2](2) NC1 hexamer of bovine lens capsule basement membrane at 2.0 A resolution. The NC1 monomer folds into a novel tertiary structure. The (alpha1)(2).alpha2 trimer is organized through the unique three-dimensional domain swapping interactions. The differences in the primary sequences of the hypervariable region manifest in different secondary structures, which determine the chain specificity at the monomer-monomer interfaces. The trimer-trimer interface is stabilized by the extensive hydrophobic and hydrophilic interactions without a need for disulfide cross-linking.