Expression of wild-type and mutant medium-chain acyl-CoA dehydrogenase (MCAD) cDNA in eucaryotic cells.
An effective EBV-based expression system for eucaryotic cells has been developed and used for the study of the mitochondrial enzyme medium-chain acyl-CoA dehydrogenase (MCAD). 1325 bp of PCR-generated MCAD cDNA, containing the entire coding region, was placed between the SV40 early promoter and polyadenylation signals in the EBV-based vector. Both wild-type MCAD cDNA and cDNA containing the prevalent disease-causing mutation A to G at position 985 of the MCAD cDNA were tested. In transfected COS-7 cells, the steady state amount of mutant MCAD protein was consistently lower than the amount of wild-type human enzyme. The enzyme activity in extracts from cells harbouring the wild-type MCAD cDNA was dramatically higher than in the controls (harbouring the vector without the MCAD gene) while only a slightly higher activity was measured with the mutant MCAD. The mutant MCAD present behaves like wild-type MCAD with respect to solubility, subcellular location, mature protein size and tetrameric structure. In immunoblot comparisons, the MCAD protein was present in normal fibroblasts, but essentially undetectable in patient fibroblasts homozygous for the prevalent mutation. We suggest that the MCAD protein carrying this mutation has an impaired ability to form correct tetramers, leading to instability and subsequent degradation of the enzyme. This finding is discussed in relation to the results from expression of human MCAD in Escherichia coli, where preliminary results show that production of mutant MCAD leads to the formation of aggregates.
Jensen
T G
TG
Institute of Human Genetics, University of Aarhus, Denmark.
Andresen
B S
BS
Bross
P
P
Jensen
U B
UB
Holme
E
E
Kølvraa
S
S
Gregersen
N
N
Bolund
L
L
D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000235:genetics*; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D004247:DNA; D005057:Eukaryotic Cells; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000235:genetics; D006720:Homozygote; D006801:Humans; D009154:Mutation; D011401:Promoter Regions, Genetic; D012313:RNA / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection
D012313:RNA; D004247:DNA; D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase