Campeau E, Dupuis L, León-Del-Rio A, Gravel R
Department of Human Genetics, McGill University-Montreal Children's Hospital Research Institute, 4060 Ste-Catherine West, Montréal, Québec, H3Z 2Z3, Canada.
Mol Genet Metab. 1999 May;67(1):11-22. doi: 10.1006/mgme.1999.2850.
Propionic acidemia is a rare autosomal recessive disorder of intermediary metabolism. It is caused by a deficiency of the mitochondrial enzyme propionyl-CoA carboxylase (PCC, EC 6.4.1.3), a heteropolymeric protein composed of two subunits, alpha and beta. PCC requires ATP and biotin as cofactors for the reaction, the latter enzymatically added onto the alpha subunit. We investigated coding sequence mutations in the alpha subunit of PCC by analyzing fibroblast RNA from propionic acidemia patients deficient in alpha subunit function by single-strand conformation polymorphism and direct sequencing. Five missense mutations and one short in-frame deletion were found among different patients. Four mutations were located in the putative biotin carboxylase domain, whereas the two others were within the 67-amino-acid C-terminal domain previously shown to be required to obtain biotinylation of the alpha subunit. We analyzed fibroblast extracts for the presence of a biotinylated alpha subunit by Western blot analysis using streptavidin coupled to alkaline phosphatase. Four of five cell lines failed to show a biotinylated alpha subunit, regardless of the position of the mutations within the coding sequence. Two mutations located in the biotinylation domain were expressed in an Escherichia coli-based system and shown to abolish biotinylation of the domain. The results suggest that most mutations have a severe impact on the stability or the functionality of the alpha subunit.
丙酸血症是一种罕见的常染色体隐性中间代谢紊乱疾病。它由线粒体酶丙酰辅酶A羧化酶（PCC，EC 6.4.1.3）缺乏引起，PCC是一种由α和β两个亚基组成的杂聚蛋白。PCC需要ATP和生物素作为反应的辅因子，后者通过酶促作用添加到α亚基上。我们通过单链构象多态性和直接测序分析来自α亚基功能缺陷的丙酸血症患者的成纤维细胞RNA，研究了PCCα亚基中的编码序列突变。在不同患者中发现了五个错义突变和一个短的框内缺失。四个突变位于假定的生物素羧化酶结构域，而另外两个在先前显示为获得α亚基生物素化所必需的67个氨基酸的C末端结构域内。我们使用与碱性磷酸酶偶联的抗生物素蛋白通过蛋白质印迹分析来分析成纤维细胞提取物中生物素化α亚基的存在。五个细胞系中的四个未能显示出生物素化的α亚基，无论编码序列内突变的位置如何。位于生物素化结构域的两个突变在基于大肠杆菌的系统中表达，并显示消除了该结构域的生物素化。结果表明，大多数突变对α亚基的稳定性或功能有严重影响。
