Abstract
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.
Highlights
Collagen X is a short-chain collagen expressed at sites of endochondral ossification during normal skeletal development and under conditions that involve new bone growth, such as fracture repair or osteoarthritis [1,2,3]
We closely examined the effect of two NC1 domain mutations, one frameshift mutation (1963del10) and one missense mutation (Y598D), using both semipermeabilized cell and stable cell transfection expression systems
Human Collagen X Chains Harboring Schmid metaphyseal chondrodysplasia (SMCD) Mutations Y598D and NC1⌬10 Assemble Less Efficiently Than Wild-type Chains When Expressed in Semi-permeabilized Cells—In this study we used an in vitro translation system supplemented with semi-permeabilized human fibroblasts (HT 1080 cells) as a starting point to analyze the effect of SMCD mutations on assembly of the full-length human ␣1(X) chain
Summary
Collagen X is a short-chain collagen expressed at sites of endochondral ossification during normal skeletal development and under conditions that involve new bone growth, such as fracture repair or osteoarthritis [1,2,3]. The formation of SDS-stable trimers, it was initially proposed that SMCD is caused by exclusion of mutant chains from ␣1(X) assembly, resulting in a 50% reduction in functional collagen X within the growth plate [14]. Missense mutations are unlikely to reduce mRNA stability, and studies have shown that ␣1(X) chains harboring certain point mutations can form stable homotrimers and co-assemble with the normal chain when expressed in semi-permeabilized cells [16]. Expression of collagen X in transiently transfected mammalian cells demonstrated that wild-type ␣1(X) chains were secreted efficiently whereas SMCD mutant ␣1(X) chains were present in only trace amounts within the media [17]. We find that human ␣1(X) chains carrying the Y598D and NC1⌬10 mutations assemble less efficiently than the wild-type chain when expressed in semi-permeabilized cells. Our results show that only trace amounts of mutant collagen X chains could be detected in the media, indicating that the predominant effect of these mutations is a 50% reduction in functional collagen X secretion
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