Interaction of collagen alpha 1(X) containing engineered NC1 mutations with normal alpha 1(X) in vitro - Implications for the molecular basis of schmid metaphyseal chondrodysplasia

Collagen X is a short-chain homotrimeric collagen expressed in the hypertro phic zone of calcifying cartilage. The clustering of mutations in the carbo xyl-terminal nonhelical NC1 domain in Schmid metaphyseal chondrodysplasia ( SMCD) suggests a critical role for NCI in collagen X structure and function . In vitro collagen X DNA expression, using T7-driven coupled transcription and translation, demonstrated that although alpha 1(X) containing normal N C1 domains can form electrophoretically stable trimers, engineered SMCD NC1 missense or premature termination mutations prevented the formation of ele ctrophoretically stable homotrimers or heterotrimers when co-expressed with normal alpha 1(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 appr oach. Our studies show that alpha 1(X) chains containing SMCD mutations red uce the efficiency of normal al(X) trimer assembly, indicating that interac tions do occur between mutant and normal NC1 domains, which can impact on t he 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 d ominant interference is also possible if the mutant protein is expressed in vivo. Furthermore, we establish that a conserved 13-amino acid aromatic mo tif (amino acids 589-601) is critical for the interaction between the NCI d omains, suggesting that this region may initiate assembly and the other NC1 mutations interfered with secondary interactions important in folding or i n stabilizing the assembly process.