Interaction of collagen α1(X) containing engineered NC1 mutations with normal α1(X) in vitro. Implications for the molecular basis of Schmid metaphyseal chondrodysplasia

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 α1(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 α1(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 α1(X) chains containing SMCD mutations reduce the efficiency of normal α1(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 589601) 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.link_to_subscribed_fulltex