Collagen X chains harboring Schmid metaphyseal chondrodysplasia NC1 domain mutations are selectively retained and degraded in stably transfected cells
Wilson, RR and Freddi, S and Bateman, JF, Collagen X chains harboring Schmid metaphyseal chondrodysplasia NC1 domain mutations are selectively retained and degraded in stably transfected cells, Journal of Biological Chemistry, 227, (15) pp. 12516-12524. ISSN 0021-9258 (2002) [Refereed Article]
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.