Abstract
Collagen X is a short-chain homotrimeric collagen expressed in the hypertrophic zone of calcifying cartilage. The clustering of mutations in the carboxyl-terminal nonhelical NC1 domain in Schmid metaphyseal chondrodysplasia (SMCD) suggests a critical role for NC1 in collagen X structure and function. In vitro collagen X DNA expression, using T7-driven coupled transcription and translation, demonstrated that although alpha1(X) containing normal NC1 domains can form electrophoretically stable trimers, engineered SMCD NC1 missense or premature termination mutations prevented the formation of electrophoretically stable homotrimers or heterotrimers when co-expressed with normal alpha1(X). To allow the detection of more subtle interactions that may interfere with assembly but not produce SDS-stable final products, we have developed a competition-based in vitro co-expression and assembly approach. Our studies show that alpha1(X) chains containing SMCD mutations reduce the efficiency of normal alpha1(X) trimer assembly, indicating that interactions do occur between mutant and normal NC1 domains, which can impact on the formation of normal trimers. This finding has important implications for the molecular pathology of collagen X mutations in SMCD. Although we have previously demonstrated haploinsufficiency as one in vivo mechanism (Chan, D., Weng, Y. M., Hocking, A. M., Golub, S., McQuillan, D. J., and Bateman, J. F. (1998) J. Clin. Invest. 101, 1490-1499), the current study suggests dominant interference is also possible if the mutant protein is expressed in vivo. Furthermore, we establish that a conserved 13-amino acid aromatic motif (amino acids 589-601) is critical for the interaction between the NC1 domains, suggesting that this region may initiate assembly and the other NC1 mutations interfered with secondary interactions important in folding or in stabilizing the assembly process.
Highlights
Collagen X is a short-chain homotrimeric collagen expressed in the hypertrophic zone of calcifying cartilage
In vitro collagen X DNA expression, using T7-driven coupled transcription and translation, demonstrated that ␣1(X) containing normal NC1 domains can form electrophoretically stable trimers, engineered Schmid metaphyseal chondrodysplasia (SMCD) NC1 missense or premature termination mutations prevented the formation of electrophoretically stable homotrimers or heterotrimers when co-expressed with normal ␣1(X)
Our studies show that ␣1(X) chains containing SMCD mutations reduce the efficiency of normal ␣1(X) trimer assembly, indicating that interactions do occur between mutant and normal NC1 domains, which can impact on the formation of normal trimers
Summary
(Received for publication, September 10, 1998, and in revised form, December 23, 1998). The critical importance of the NC1 domain in collagen X molecular assembly and function has been highlighted by the characterization of mutations in patients with Schmid metaphyseal chondrodysplasia (SMCD) (MIM 156500) (12, 13) These mutations include amino acid substitutions, nonsense mutations and deletions resulting in predicted protein truncation, in vitro expression, and assembly studies suggested that a common molecular defect is that the SMCD mutations compromise NC1 association and prevent the formation of stable collagen X homotrimers (9, 10). These data suggested that SMCD resulted from a functional haploinsufficiency of assembly-competent ␣1(X).
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