A secreted variant of cartilage oligomeric matrix protein carrying a chondrodysplasia-causing mutation (p.H587R) disrupts collagen fibrillogenesis

Authors

  • Uwe Hansen,

    1. University Hospital of Muenster, Muenster, Germany
    Search for more papers by this author
  • Nicole Platz,

    1. Center for Biochemistry and Center for Molecular Medicine Cologne, Medical Faculty, and Cologne Excellence Cluster on Cellular Stress Response in Aging-Associated Diseases, University of Cologne, Cologne, Germany
    Search for more papers by this author
  • Alexander Becker,

    1. Center for Biochemistry and Center for Molecular Medicine Cologne, Medical Faculty, and Cologne Excellence Cluster on Cellular Stress Response in Aging-Associated Diseases, University of Cologne, Cologne, Germany
    Search for more papers by this author
  • Peter Bruckner,

    1. University Hospital of Muenster, Muenster, Germany
    Search for more papers by this author
  • Mats Paulsson,

    1. Center for Biochemistry and Center for Molecular Medicine Cologne, Medical Faculty, and Cologne Excellence Cluster on Cellular Stress Response in Aging-Associated Diseases, University of Cologne, Cologne, Germany
    Search for more papers by this author
  • Frank Zaucke

    Corresponding author
    1. Center for Biochemistry and Center for Molecular Medicine Cologne, Medical Faculty, and Cologne Excellence Cluster on Cellular Stress Response in Aging-Associated Diseases, University of Cologne, Cologne, Germany
    • Center for Biochemistry, Medical Faculty, University of Cologne, Joseph Stelzmann Strasse 52, D-50931 Cologne, Germany
    Search for more papers by this author

Abstract

Objective

Mutations in human cartilage oligomeric matrix protein (COMP) cause multiple epiphyseal dysplasia or pseudoachondroplasia. Electron microscopic analyses of patient biopsy tissue have shown that, in most cases, mutated COMP is retained in granular or lamellar inclusions in the endoplasmic reticulum of chondrocytes. However, some mutations that do not interfere with protein trafficking, resulting in normal secretion of the mutated protein, have been identified. These mutations are likely to cause the chondrodysplasia phenotype, via events that occur after secretion. The aim of the present study was to identify such extracellular mechanisms associated with the pathogenesis of chondrodysplasias.

Methods

A mutated but secreted COMP variant, p.H587R, as well as wild-type COMP were recombinantly expressed and purified from cell culture supernatants. Since recent studies have shown that COMP can facilitate collagen fibrillogenesis in vitro, the effect of the p.H587R mutation on this process was determined by analyzing the kinetics of fibrillogenesis in vitro and determining the structure of the collagen fibrils formed by immunogold electron microscopy.

Results

Mutated p.H587R COMP accelerated fibril formation by type I collagen in vitro to a slightly greater extent than that with wild-type COMP. However, p.H587R COMP induced aggregation and disorganization of fibril intermediates and end products. Mixtures of cartilage collagens or of type XI collagen alone produced similar results. The addition of p.H587R COMP to preformed fibrils induced aggregation and fusion of the fibrils, whereas wild-type COMP had little effect.

Conclusion

The mutant COMP variant p.H587R generally interferes with normal collagen organization during fibrillogenesis. This constitutes a novel pathogenetic mechanism of COMP-associated chondrodysplasias.

Cartilage oligomeric matrix protein (COMP), also called thrombospondin 5, is a pentameric protein that, in initial studies, was isolated from cartilage tissue. COMP is predominantly expressed in all types of cartilage (1) but can also be expressed in tendons (2) and, to a lesser extent, by vascular smooth muscle cells (3). The role of COMP in cartilage matrix is not completely understood. COMP-deficient animals do not display any skeletal abnormalities or alterations in tendon structure (4). However, a variety of noncollagenous matrix molecules that interact with COMP have been identified. Binding proteins include types I, II, and IX collagen (5–8) as well as extrafibrillar matrix constituents such as matrilins (9), fibronectin (10), extracellular matrix protein 1 (11), and aggrecan (12).

A recent study showed that COMP not only binds to collagens but also acts as an enhancer of collagen fibrillogenesis (13). COMP mediates not only interactions of soluble proteins, but also interactions of cartilage collagen fibrils with matrilin 3 and of type VI collagen microfibrils with cartilage fibrils via decorin binding (14). Therefore, it has been proposed that COMP is an adaptor protein that mediates the interactions and cohesion between the suprastructural compartments in cartilage matrix, such as fibrils, microfibrils, and proteoglycan aggregates (15).

These concepts are supported by the results of studies assessing the pathogenetic mechanisms of human diseases caused by COMP mutations. Pseudoachondroplasia (PSACH; MIM no. 177170) and multiple epiphyseal dysplasia (MED; MIM nos. 132400, 600204, 600969, 226900, and 607078) are 2 forms of autosomal-dominant chondrodysplasias and are characterized by severe-to-moderate disproportionate dwarfism and pronounced joint laxity. PSACH is known to be associated only with COMP mutations, whereas MED has been linked to the genes for COMP and matrilin 3, as well as each of the 3 chains of type IX collagen (16).

COMP is derived from a unique gene comprising 19 exons that encode an amino-terminal α-helical pentameric coiled-coil domain, followed by 4 epidermal growth factor–like domains (exons 1–7), 7 calcium-binding thrombospondin type 3 repeats, and a specific carboxy-terminal domain (exons 8–19). Even though more than 70 disease-causing mutations have been described in exons 8–19 of COMP, no mutations have been identified in exons 1–7. More than 85% of all mutations are localized in the calcium-binding thrombospondin 3–like repeats, but so far, no clear correlation between the localization of the mutation and severity of the disease has been established (17, 18). Electron microscopic analyses of patient biopsy tissue have shown that, at least in some cases, mutated COMP is retained in granular or lamellar inclusions in the endoplasmic reticulum (ER) of chondrocytes (19). The accumulation of COMP leads to coretention of COMP interaction partners, including type IX collagen and matrilin 3. Abnormal trafficking and secretion of type II collagen caused by COMP mutations remains a controversial subject (20–23). For these reasons, PSACH and MED are thought to be diseases caused by abnormal protein storage in the ER, resulting in an unfolded protein response and apoptosis (24, 25). A reduced viability of growth plate chondrocytes could explain the short-limb dwarfism in patients.

In animal experiments utilizing transgenic mice whose chondrocytes overexpressed COMP with the most common mutation found in patients (p.D469Δ), the ER was also dilated, most probably due to an impaired secretion of mutant COMP, thereby recapitulating the phenotype observed in patients. In addition, collagen fibrils with aberrant structure were observed in the growth plate cartilage (26). However, in mice harboring a mutation in the C-terminal domain of COMP (p.T583M) after homologous recombination (27, 28), the mutated protein was secreted into the extracellular matrix. Although COMP was not significantly retained within the ER, signs of an unfolded protein response were detected, chondrocyte proliferation was reduced, and apoptosis was increased and spatially dysregulated.

A variety of different mutations in COMP have been analyzed in patients, utilizing both in vitro and cell culture approaches (18, 20, 23, 29–31). Most disease-causing mutant proteins were retained by primary chondrocytes, but some of the mutant proteins were secreted (20, 29). This finding indicates that, in some cases, the extracellular events are sufficient to cause a disease. A prominent member of this subset of COMP mutations is p.H587R.

In the present study, we hypothesized that a dominant interference of mutated COMP with collagen fibrillogenesis in cartilage and tendons may, at least in some patients, explain the short-limb dwarfism, the early joint failure, and the ligament laxity seen in patients with PSACH and in those with MED. We therefore investigated the effect of p.H587R COMP, a mutant variant that, despite being properly secreted, leads to a rather severe form of PSACH, on collagen fibrillogenesis. We believe that studies of pathogenic mechanisms in rare forms of degenerative cartilage disease, e.g., those caused by mutations in cartilage matrix proteins such as COMP, are likely to provide significant information about disease mechanisms that also apply to more common disease forms, such as osteoarthritis.

MATERIALS AND METHODS

Cloning, expression, and purification of COMP.

Site-directed mutagenesis was performed on full-length rat COMP complementary DNA (GenBank accession number NM_012834.1) as described earlier (7). The mutated variant p.H587R as well as wild-type COMP were cloned into the episomal Strep II–tagged expression vector pCEP-Pu (N-terminal) in frame with the sequence of the BM-40 signal peptide.

The p.H587R COMP variant and the wild-type protein were recombinantly expressed in human embryonic kidney 293/Epstein-Barr nuclear antigen cells (Invitrogen). Cells were transfected using FuGENE HD (Roche). For protein purification, transfected cells were selected with 1 μg/ml puromycin and grown to confluence. The serum-containing cell culture supernatant containing N-terminally Strep II–tagged COMP was applied to a Streptactin column (1.5 ml; IBA) and eluted with 2.5 mM desthiobiotin in 10 mM Tris HCl, pH 8.0.

Collagen preparations and analysis of in vitro fibrillogenesis.

Types II, IX, and XI collagen were purified in a native and fibrillogenesis-competent form from cultures of chick embryo sternal chondrocytes grown in agarose gels, as previously described (32). Type I collagen was obtained from the tarsometatarsal tendons of 17-day-old chick embryos (33).

Solutions of pure type I collagen (200 μg/ml) and type XI collagen (100 μg/ml) were prepared in storage buffer (0.1M Tris HCl, 0.4M NaCl, pH 7.4). Mixtures of types II, IX, and XI collagen and of types II and XI collagen were prepared in storage buffer at a molar ratio of 8:1:1 and 8:1, respectively. Recombinant wild-type and p.H587R COMP (40 μg/ml) diluted in storage buffer was added. As negative control, an equal volume of storage buffer was added. Fibrillogenesis was initiated by dilution with an equal volume of distilled water, followed by immediate warming of the reaction mixtures to 37°C. Measurements were performed in microcuvettes (Multicell, light path, 1 cm; Beckman) in a spectrophotometer (Beckman UV 640, equipped with a Multicell holder, Micro Auto 12), connected to a water bath. The kinetics of fibrillogenesis were monitored by assessing turbidity development at 313 nm. Intermediate and end products were examined by electron microscopy at specific time points (33). In some experiments, these products were also analyzed by immunogold electron microscopy.

In order to exclude the possibility of formation of COMP aggregates in the absence of collagens, the recombinant proteins were treated in the same way (for 3 hours at 37°C) as in the in vitro fibrillogenesis experiments. Small aliquots of these mixtures were spotted onto sheets of Parafilm after 3 hours at 37°C and evaluated by immunogold electron microscopy.

In reconstitution experiments using mixtures of types II, IX, and XI collagen or of types II and XI collagen, recombinant COMP (40 μg/ml) was added after 3 hours, when collagen fibrils were already formed, and in vitro fibrillogenesis was continued for a further 3 hours. Storage buffer was added in control experiments. Finally, the reconstitution products were analyzed by electron microscopy.

Electron microscopy and immunogold labeling.

Aliquots of reconstitution products at different stages of fibril formation from in vitro fibrillogenesis experiments were spotted onto sheets of Parafilm. Nickel grids covered with Formvar/carbon were floated on the drops for 10 minutes to allow adsorption of material and subsequently washed with phosphate buffered saline (PBS) and treated for 30 minutes with 2% (weight/volume) dried skim milk in PBS. Next, the adsorbed material was allowed to react for 2 hours with a specific antibody against COMP (8), diluted 1:200 in PBS containing 0.2% dry milk. We confirmed by immunoblotting that the antibody used for gold labeling did not show any selective specificity for either COMP variant (results not shown).

After washing 5 times with PBS, the grids were put on drops of 0.2% milk solution containing colloidal gold particles (18 nm) coated with goat antibodies to rabbit immunoglobulins (Jackson ImmunoResearch). Finally, the grids were washed with distilled water and negatively stained with 2% uranyl acetate for 7 minutes. Control experiments were undertaken with the first antibody omitted. Electron micrographs were obtained at 80 kV with a Philips EM 410 electron microscope.

RESULTS

Effects of wild-type and p.H587R COMP on the kinetics of type I collagen fibrillogenesis and induction of disorganized structures and aggregates by p.H587R COMP.

COMP is expressed not only in cartilage but also in tendons and ligaments, where the predominating collagen is type I. We therefore studied the effects of p.H587R and wild-type COMP on the kinetics of type I collagen fibrillogenesis, by monitoring the change in turbidity at 313 nm. Pure type I collagen at a concentration of 200 μg/ml produced sigmoidal turbidity curves with characteristic lag phases (Figure 1). In the presence of wild-type COMP (40 μg/ml), the length of the lag phase was comparable. However, the slope after the lag phase was steeper, and the final turbidity was slightly increased, suggesting a more efficient fibril formation in the presence of COMP. The addition of p.H587R COMP (40 μg/ml) led to a shortened lag phase but produced a similar gain in and final level of turbidity as that observed with wild-type COMP (Figure 1).

Figure 1.

Fibrillogenesis of type I collagen, as monitored by assessment of the kinetics of turbidity development at 313 nm. Fibrillogenesis of type I collagen was initiated in the absence of cartilage oligomeric matrix protein (COMP) (control) or in the presence of either wild-type (WT) COMP or mutant p.H587R (HR) COMP. The kinetics of fibril formation were then monitored according to changes in turbidity at 313 nm at 3 different time points. The concentration of type I collagen was 200 μg/ml and that of COMP was 40 μg/ml.

The final turbidity was consistently highest in the presence of mutant COMP. The turbidity depends not only on the protein amount in fibrils but also on the organization of the formed fibrils. Provided that an identical amount of protein is present, the higher final turbidity after addition of the COMP mutant might reflect a higher degree of disorganization. We therefore further analyzed the fibrils formed in analyses of the end products by electron microscopy.

In the absence of COMP, loose networks of very thin fibrils were visualized by transmission electron microscopy after 2 minutes (Figure 2A), whereas in the presence of wild-type COMP, more condensed structures were observed (Figure 2B). Already at these early stages, the reconstitution products after the addition of p.H587R COMP were much less organized (Figure 2C) than that with wild-type COMP (Figure 2B). Whereas maturation into thin flexible fibrils was observed after 10 minutes in the absence of COMP (Figure 2D), no obvious alterations in the aggregation products were seen between 2 minutes and 10 minutes in the presence of either wild-type COMP (Figure 2E) or p.H587R COMP (Figure 2F), with the degree of fibril disorganization being more prominent with mutant COMP. After 180 minutes, there was no apparent difference between the fibrils formed by type I collagen without (Figure 2G) or with (Figure 2H) wild-type COMP, in that fibrillogenesis in vitro produced very stiff fibrils with rather uniform diameters. In contrast, the fibrils formed in the presence of p.H587R COMP were distorted and tattered (Figure 2I).

Figure 2.

Reconstitution products at different stages of type I collagen fibrillogenesis. Collagen reconstitution products formed after 2 minutes (A–C), 10 minutes (D–F), and 180 minutes (G–I) in the absence of cartilage oligomeric matrix protein (COMP) (control) (A, D, and G) or presence of wild-type (WT) (B, E, and H) or mutant p.H587R (HR) COMP (C, F, and I) were visualized by electron microscopy. Bars at 2 minutes and 10 minutes = 100 nm; bars at 180 minutes = 200 nm.

At higher magnifications, fibrils were observed to have the expected diameter, a well-defined surface, and the typical D-periodic banding, regardless of the presence or absence of wild-type COMP during fibril reconstitution (Figures 3A and B). Immunogold labeling showed that COMP was not specifically enriched at the fibril surface (Figure 3B), suggesting that COMP acted only as a transient component and was lost from the final, reconstituted type I collagen fibrils. In the presence of p.H587R COMP, the fibril diameter was more variable (Figures 3C and D). Moreover, the reconstitution products showed a clear but irregular banding (Figure 3C) and also contained variable amounts of amorphous nonfibrillar material diffusively attached to the fibril surfaces (Figure 3D). In this case, the gold particles labeling p.H587R COMP were mainly found at the fibril surface (Figure 3C) and in diffusely aggregated material (Figure 3D). This indicates that mutant COMP binds to type I collagen molecules but, unlike wild-type COMP, does not dissociate from mature fibrils. When p.H587R COMP alone was incubated under the same conditions, we did not detect any amorphous structures, indicating that such material is not a result of illegitimate precipitation of mutant protein (results not shown).

Figure 3.

End products of fibrillogenesis of type I collagen. A and B, In the absence of cartilage oligomeric matrix protein (COMP), formation of typical banded type I collagen fibrils was observed (A), and in the presence of wild-type COMP, the type I collagen fibrils formed were identical to those in the absence of COMP (B). Immunogold labeling using a polyclonal antibody against COMP revealed labeling (black particles) on the formed fibrils as well as beside the fibrils. C and D, In the presence of the mutant p.H587R COMP, disorganized type I collagen fibrils with an irregular banding pattern were formed (C), and amorphous nonfibrillar material (arrowhead in D) was often found, which was always absent in control experiments and in the presence of wild-type COMP. Bars = 200 nm.

Effects of p.H587R COMP on the fibrillogenesis of other cartilage collagen fibrils.

To determine whether the effect of p.H587R COMP is restricted to type I collagen, we performed similar experiments with mixtures of types II and XI collagen or of types II, IX, and XI collagen. Such mixtures reflect the composition of fibrils in mature cartilage, which is known to contain only small amounts of type IX collagen. Cartilage collagen fibrils are much thinner than those reconstituted from type I collagen and, hence, scatter light to a much lesser extent. Therefore, it was not possible to reliably monitor the kinetics of fibril formation through the assessment of turbidity. For this reason, fibrillogenesis of cartilage types II and XI collagen, with or without type IX collagen and COMP, was examined only by electron microscopy.

In electron microscopic analyses, we observed no obvious differences in the structure and diameter of reconstituted cartilage collagen fibrils containing types II and XI collagen or types II, IX, and XI collagen, formed with or without wild-type COMP (Figures 4A, B, D, and E). In contrast, fibrils formed in the presence of p.H587R COMP were banded but varied markedly in width. Some fibrils showed lateral fusion, and amorphous electron-dense material was frequently observed (Figures 4C and F).

Figure 4.

Reconstitution products of mixtures of types II and XI collagen, with or without type IX collagen. A and D, In control experiments, uniform and weakly banded collagen fibrils were formed by mixtures of types II and XI collagen (A) or types II, IX, and XI collagen (D). B and E, In the presence of wild-type (WT) cartilage oligomeric matrix protein (COMP), collagen fibrils formed in mixtures of types II and XI collagen (B) or types II, IX, and XI collagen (E) had uniform diameters and were identical to those observed in control experiments. Immunogold labeling for COMP revealed labeling (black particles) both on the formed fibrils and beside the fibrils. C and F, In the analysis of reconstitution products of mixtures of types II and XI collagen (C) and of types II, IX, and XI collagen (F) in the presence of mutant p.H587R (HR) COMP, the fibrils formed were more heterogeneous and thicker. Amorphous electron-dense material (arrowheads in C and F) was often found between the collagen fibrils. Bars = 100 nm.

The influence of COMP on the fibrillogenesis of type XI collagen alone was also analyzed. Type XI collagen plays a key role as a nucleator in fibril formation. As judged on the basis of the electron microscopy images, wild-type COMP did not appear to alter the fibril suprastructures, including their D-periodic banding pattern (compare Figure 5A with Figure 5B), with the exception of the additional presence of very fine fibrils. However, mutant p.H587R COMP led to the introduction of broader fibrils, in addition to fibrils with a more typical smaller diameter (Figure 5C). All fibrils had almost indiscernible banding patterns (Figure 5C, inset).

Figure 5.

Reconstitution products of type XI collagen. Reconstitution products of pure type XI collagen (A), of mixtures of type XI collagen and wild-type cartilage oligomeric matrix protein (COMP) (B), and of mixtures of type XI collagen and mutant p.H587R COMP (C) were assessed by electron microscopy. Inset in C, Type XI collagen fibril without any banding pattern. Bars = 200 nm.

Induction of fusion and aggregation of preformed fibrils by mutant COMP.

We also assessed whether the addition of COMP could change the structure and arrangement of preformed fibrils. For this purpose, we allowed fibrils to form without COMP in mixtures of types II and XI collagen and of types II, IX, and XI collagen for 180 minutes, and then added COMP and analyzed the end products after another 180 minutes. Electron microscopy revealed that for both fibril types, the addition of wild-type COMP had only minor, if any, effects (Figure 6). In contrast, images obtained after the addition of p.H587R COMP showed fibril networks carrying an increased amount of attached amorphous material.

Figure 6.

Reconstitution products formed by the addition of cartilage oligomeric matrix protein (COMP) to preformed fibrils. The influence of COMP on already-formed cartilage fibrils was analyzed by transmission electron microscopy. Wild-type (WT) COMP (B and E) and mutant p.H587R (HR) COMP (C and F), or storage buffer as control (A and D), was added to reconstituted cartilage collagen fibrils containing either types II and XI collagen (A–C) or types II, IX, and XI collagen (D–F), followed by imaging after another 3 hours. Bars = 200 nm.

DISCUSSION

Attempts to directly elucidate the function of COMP in vivo by gene ablation in mice were inconclusive, because these animals did not display obvious abnormalities in their musculoskeletal system (4). The explanation for this, although still hypothetical, was that the absence of COMP is compensated for by other proteins, such as other members of the thrombospondin family, for example, thrombospondin types 1 and 4. These proteins are also expressed in cartilage and have been shown to interact with collagens (34, 35). However, the identification of COMP as a causative gene for the PSACH and MED forms of chondrodysplasia in humans points to an important role of COMP in the assembly and maintenance of the extracellular matrix in cartilage, tendons, and ligaments. Mutations in cartilage matrix proteins may retard their secretion or alter key interactions in the assembly of the extracellular matrix, thereby compromising the mechanical properties of joint cartilage. Nevertheless, the phenotype seen in these patients with chondrodysplasias is caused by the presence of a mutated protein rather than by its complete absence.

Studies of the extracellular matrix synthesized by chondrocytes isolated from patients with PSACH have shown that collagen fibril formation is abnormal and contributes to the disorganized matrix surrounding these chondrocytes (21). Furthermore, in electron micrographs of patient biopsy tissue, collagen fibrils appeared disorganized and sparse (31), indicating that a similar molecular mechanism may operate in vivo. Interestingly, gene-knockin mice harboring a mutation in the C-terminal domain of COMP (p.T585M) exhibit both a myopathy and a tendinopathy, most likely due to the presence of more fused and bifurcating collagen fibrils in tendons. The tendons from mutant animals were significantly thinner, leading to an increased laxity. This further underlines the importance of COMP in the biomechanical properties of musculoskeletal tissue (28).

In this study, we explored in detail the pathogenetic mechanisms underlying PSACH caused by the COMP mutation p.H587R. Unlike most of the other COMP proteins having mutations associated with PSACH, secretion of the p.H587R COMP from the producing cells is not decisively impaired, but normal incorporation into cartilage matrix is compromised. Therefore, we assumed that the mutated protein affects matrix assembly by normal cartilage proteins in a dominant-negative manner. We confirmed that fibrillogenesis of type I collagen in vitro with collagen was accelerated by recombinant wild-type COMP (13), and the same was true for the formation of heterotypic fibrils containing cartilage types II and XI collagen, with or without type IX collagen. From these results, we conclude that COMP alters fibril assembly by collagens in general, presumably by modulation of the nucleation/propagation pathway of fibrillogenesis. This was the case even in the presence of the minor type XI collagen, which is known to nucleate the formation and to strictly limit lateral growth of heterotypic cartilage fibrils (32).

We extended the previous observations of Halasz et al (13) by showing that the p.H587R COMP promoted aggregate formation of collagens even more strongly than the wild-type protein, by causing both an earlier onset and higher final plateau levels of turbidity development. However, examination by electron microscopy of the suprastructure of aggregates reconstituted in the presence of mutated COMP, but not wild-type COMP, revealed a profoundly disturbed fibril suprastructure. These observations are consistent with the notion that, unlike the wild-type protein, p.H587R COMP indeed causes PSACH by interfering with the normal formation of early intermediates of fibrillogenesis.

These dead-end products of fibril nucleation result in the formation of grossly aberrant prototypic cartilage fibrils. In the presence of wild-type COMP, nucleation occurs normally, albeit at an increased rate, and the formation of suprastructurally normal prototypic cartilage fibrils is followed by a lateral growth to a uniform limit diameter of ∼20 nm. After formation of malformed initial aggregates in the presence of p.H587R COMP, however, the subsequent assembly of a stable cartilage matrix may be further compromised by abrogation of crucial interactions between the aggrecan matrix and the altered fibril periphery (14). This represents a novel mechanism for the pathogenesis of musculoskeletal diseases in general, which departs from, for example, the protein suicide model of the pathogenesis of osteogenesis imperfecta. The model also differs from those describing the altered assembly of mature fibrils induced by the absence of proteoglycans with leucine-rich repeats, such as decorin, biglycan, or lumican. In this case, suprastructural abnormalities result from inappropriate and/or irregular fusion of prototypic precursors (36).

In summary, our results suggest a novel molecular mechanism for the role of COMP mutations in chondrodysplasias, particularly in describing how certain COMP mutations act by providing extracellular interference of collagen fibril formation. The disruption of proper collagen fibrillogenesis is likely to affect the extracellular matrix assembly and function in cartilage and tendons and could explain many of the phenotypic characteristics in patients.

The pathogenic mechanism of a mutation causing a rare skeletal disorder might also have implications for more common forms of degenerative cartilage disease, such as osteoarthritis. Interestingly, mutations in matrilin 3, similar to the mutations in COMP, can cause MED or predispose to the development of hand osteoarthritis, as has been observed in studies of the Icelandic population (37). The effects of the osteoarthritis-associated p.T303M mutation (in mouse p.T298M models) on the structure of matrilin 3 and on the role of matrilin 3 in extracellular matrix assembly were recently analyzed, and the results revealed that this mutation also allowed normal secretion (38), caused only minor changes in protein structure, and did not affect the affinity for collagens. However, a major impact on collagen fibrillogenesis with the formation of wider cartilage collagen fibrils was observed (39). This example confirms that studies of pathogenic changes in rare forms of cartilage degeneration are likely to provide significant insight into the pathogenetic mechanisms of more common forms of the disease, particularly since the sequence of molecular events seems to be largely independent of the factors that first initiated the disease (40).

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Zaucke had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Hansen, Bruckner, Paulsson, Zaucke.

Acquisition of data. Hansen, Platz, Becker, Zaucke.

Analysis and interpretation of data. Hansen, Bruckner, Paulsson, Zaucke.

Ancillary