Carbone Mary Anna, Robinson Brian H
Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8.
Biochem J. 2003 Feb 15;370(Pt 1):275-82. doi: 10.1042/BJ20021397.
Type A pyruvate carboxylase (PC) deficiency presents mainly in the Amerindian population, specifically the Ojibwa, Cree and Micmac tribes of the Algonquin-speaking peoples. The gene for PC contains a homozygous founder mutation (G1828-->A) that results in an Ala610-->Thr amino acid substitution in Ojibwa with Type A PC deficiency. The mutation is located in the highly conserved pyruvate-binding domain of PC. The present paper describes a retroviral expression system for human PC used to analyse the effects of this mutation. We show, through immunoblot analysis, PC enzyme activity assays, reverse-transcription PCR and mitochondrial-import experiments, that this mutation is disease-causing in the Ojibwa population owing to its decreased catalytic activity, decreased steady-state levels of expression and inefficient import into the mitochondria. Our data suggest that this mutation may affect the stability of the protein, resulting in decreased steady-state levels of expression, and that it may also affect the secondary structure of the protein during the import process, thereby inhibiting proper translocation into the mitochondria, where PC is active.
A型丙酮酸羧化酶（PC）缺乏症主要出现在美洲印第安人群体中，特别是讲阿尔冈昆语的奥吉布瓦族、克里族和米克马克族部落。PC基因存在一个纯合的始祖突变（G1828→A），该突变导致奥吉布瓦族中患A型PC缺乏症的个体出现丙氨酸610→苏氨酸的氨基酸替换。该突变位于PC高度保守的丙酮酸结合结构域。本文描述了一种用于分析该突变影响的人PC逆转录病毒表达系统。我们通过免疫印迹分析、PC酶活性测定、逆转录PCR和线粒体导入实验表明，由于其催化活性降低、表达的稳态水平降低以及导入线粒体的效率低下，该突变在奥吉布瓦人群体中具有致病性。我们的数据表明，该突变可能影响蛋白质的稳定性，导致表达的稳态水平降低，并且在导入过程中可能还会影响蛋白质的二级结构，从而抑制其正确转运到PC发挥活性的线粒体中。
