Vogel B E, Minor R R, Freund M, Prockop D J
Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
J Biol Chem. 1987 Oct 25;262(30):14737-44.
Synthesis of type I procollagen was examined in fibroblasts from a proband with a lethal perinatal variant of osteogenesis imperfecta. After trypsin digestion of the type I procollagen, a portion of the alpha 1 (I) chains was recovered as disulfide-linked dimers. Digestion of the protein with vertebrate collagenase and mapping of cyanogen bromide peptides suggested that a new cysteine residue was present between residues 551 and 775 of the alpha 1 (I) chain. Sequencing of cloned cDNAs prepared using mRNA from the proband's fibroblasts demonstrated that some of the clones contained a single base mutation that converted the glycine codon in amino acid position 748 of the alpha 1 (I) chain to a cysteine codon. About 80% of the type I procollagen synthesized by the proband's fibroblasts had a decreased thermal stability. The results, therefore, were consistent with the conclusion that normal pro-alpha 1 (I) chains and pro-alpha 1 (I) chains containing a cysteine residue in the alpha chain domain were synthesized in about equal amounts and incorporated randomly into type I procollagen. However, only about 10% of the alpha 1 (I) chains generated by trypsin digestion were disulfide-linked. Further studies demonstrated a decreased rate of secretion of type I procollagen containing the new cysteine residue and decreased processing of the protein by procollagen N-proteinase in cultures of postconfluent fibroblasts. Both parents were phenotypically normal and their fibroblasts synthesized only normal type I procollagen. Therefore, the mutation in the proband was a sporadic one and is very likely to have caused the connective tissue fragility that produced the lethal phenotype.
对一名患有致死性围产期成骨不全变异型先证者的成纤维细胞中I型前胶原的合成进行了检测。用胰蛋白酶消化I型前胶原后，一部分α1（I）链以二硫键连接的二聚体形式回收。用脊椎动物胶原酶消化该蛋白并对溴化氰肽进行图谱分析表明，在α1（I）链的551和775位残基之间存在一个新的半胱氨酸残基。对使用先证者成纤维细胞的mRNA制备的克隆cDNA进行测序表明，一些克隆含有一个单碱基突变，该突变将α1（I）链氨基酸位置748处的甘氨酸密码子转换为半胱氨酸密码子。先证者成纤维细胞合成的约80%的I型前胶原热稳定性降低。因此，这些结果与以下结论一致：正常的前α1（I）链和在α链结构域含有一个半胱氨酸残基的前α1（I）链以大致相等的量合成，并随机掺入I型前胶原中。然而，胰蛋白酶消化产生的α1（I）链中只有约10%以二硫键连接。进一步的研究表明，在汇合后成纤维细胞培养物中，含有新半胱氨酸残基的I型前胶原分泌率降低，并且前胶原N蛋白酶对该蛋白的加工减少。父母双方表型正常，他们的成纤维细胞仅合成正常的I型前胶原。因此，先证者中的突变是散发性的，很可能导致了产生致死表型的结缔组织脆弱性。
