Interaction of collagen α1(X) containing engineered NC1 mutations with normal α1(X) in vitro -: Implications for the molecular basis of schmid metaphyseal chondrodysplasia
Interaction of collagen α1(X) containing engineered NC1 mutations with normal α1(X) in vitro -: Implications for the molecular basis of schmid metaphyseal chondrodysplasia
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DOI：
10.1074/jbc.274.19.13091
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发表时间：
1999-05-07
影响因子：
4.8
通讯作者：
Bateman, JF
中科院分区：
文献类型：
--
作者：
Chan, D;Freddi, S;Bateman, JF
胶原X是在钙化软骨的肥大区中表达的短链同源三聚体胶原。施密德干骺端软骨发育不良（SMCD）羧基末端非螺旋NC 1结构域的突变聚类表明NCI在胶原X结构和功能中起关键作用。使用T7驱动的偶联转录和翻译的体外胶原X DNA表达证明，尽管含有正常NC 1结构域的α 1（X）可以形成遗传稳定的三聚体，但工程化的SMCD NC 1错义或过早终止突变阻止了与正常α 1（X）共表达时遗传稳定的同源三聚体或异源三聚体的形成，为了检测可能干扰组装但不产生SDS稳定的最终产物的更微妙的相互作用，我们开发了基于竞争的体外共表达和组装方法。我们的研究表明，含有SMCD突变的α 1（X）链降低了正常α 1（X）三聚体组装的效率，表明突变体和正常NC 1结构域之间确实发生了相互作用，这可能影响正常三聚体的形成。这一发现对SMCD中胶原X突变的分子病理学具有重要意义。D，，Weng，Y，M，，Hocking，A. M.，Golub，S.，McQuillan，D，J，和Bateman，J，F，（1998）J，Clin. Invest，101，1490-1499），目前的研究表明，如果突变蛋白在体内表达，显性干扰也是可能的。此外，我们建立了一个保守的13个氨基酸的芳香基序（氨基酸589-601）的NCI结构域之间的相互作用是至关重要的，这表明该地区可能会启动组装和其他NC 1突变干扰折叠或稳定的组装过程中重要的二级相互作用。
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 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 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 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 approach. Our studies show that alpha 1(X) chains containing SMCD mutations reduce the efficiency of normal al(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 NCI 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.