Pack M, Constantinou C D, Kalia K, Nielsen K B, Prockop D J
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
J Biol Chem. 1989 Nov 25;264(33):19694-9.
Recent reports have demonstrated that a series of probands with severe osteogenesis imperfecta had single base mutations in one of the two structural genes for type I procollagen that substituted amino acids with bulkier side chains for glycine residues and decreased the melting temperature of the triple helix. Here we demonstrate that the type I procollagen synthesized by cultured fibroblasts from a proband with a severe form of osteogenesis imperfecta consisted of normal molecules and molecules over-modified by post-translational reactions. The thermal stability of the intact type I collagen was normal as assayed by protease digestion under conditions in which a decrease in thermal stability was previously observed with eight other substitutions for glycine in the alpha 1(I) chain. In contrast, the thermal stability of the one-quarter length B fragment generated by digestion with vertebrate collagenase was decreased by 2-3 degrees C under the same conditions. Nucleotide sequencing of cDNAs and genomic DNA established that the proband had a substitution of A for G in one allele of the pro alpha 1(I) gene that converted the codon for alpha 1-glycine 844 to a codon for serine. The results also established that the alpha 1-serine 844 was the only mutation that could account for the decrease in thermal stability of the collagenase B fragment. There are at least two possible explanations for the failure of the alpha 1-serine 844 substitution to decrease the thermal stability of the collagen molecule whereas eight similar mutations decreased the melting temperature. One possibility is that the effects of glycine substitutions are position specific because not all glycine residues make equivalent contributions to cooperative blocks of the triple helix that unfold in the predenaturation range of temperatures. A second possible explanation is that substitutions of glycine by serine have much less effect on the stability of protein than the substitutions by arginine, cysteine, and aspartate previously studied.
最近的报告表明，一系列患有严重成骨不全症的先证者在I型前胶原的两个结构基因之一中存在单碱基突变，这些突变用侧链更庞大的氨基酸取代了甘氨酸残基，并降低了三螺旋的解链温度。在此，我们证明，来自一名患有严重成骨不全症先证者的培养成纤维细胞合成的I型前胶原由正常分子和经翻译后反应过度修饰的分子组成。在先前观察到α1（I）链中其他八个甘氨酸替代物热稳定性降低的条件下，通过蛋白酶消化测定，完整I型胶原的热稳定性正常。相比之下，在相同条件下，用脊椎动物胶原酶消化产生的四分之一长度B片段的热稳定性降低了2 - 3摄氏度。对cDNA和基因组DNA进行核苷酸测序确定，该先证者的proα1（I）基因的一个等位基因中发生了A被G替代的情况，这将α1 - 甘氨酸844的密码子转换为丝氨酸密码子。结果还确定，α1 - 丝氨酸844是唯一能够解释胶原酶B片段热稳定性降低的突变。对于α1 - 丝氨酸844替代未能降低胶原分子热稳定性而八个类似突变降低了解链温度，至少有两种可能的解释。一种可能性是甘氨酸替代的影响具有位置特异性，因为并非所有甘氨酸残基对在变性前温度范围内展开的三螺旋协同结构域都有同等贡献。另一种可能的解释是，与先前研究的精氨酸、半胱氨酸和天冬氨酸替代相比，丝氨酸替代甘氨酸对蛋白质稳定性的影响要小得多。
