Expression and characterization of a human pyruvate carboxylase variant by retroviral gene transfer

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.