A defect in the mitochondrial import of mutant Mn-superoxide dismutase produced in Sf21 cells.
Wild-type and several mutant human manganese superoxide dismutases (Mn-SODs) were produced in a baculovirus/insect cell system and characterized. The enzymatic activity of a homogenate of Sf21 cells, infected with baculovirus carrying wild-type Mn-SOD and grown in the conventional medium, was indistinguishable from that of control cells, but was augmented by supplementation with Mn2+. The protein produced was largely imported into the mitochondria, as judged from the enrichment in the mitochondrial fraction, the mobility of the protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the results of N-terminal processing, which was confirmed by sequencing of the purified enzyme. However, a significant amount of precursor was also detected by an antibody raised against the human Mn-SOD signal peptide. While both Mn2+ and Fe3+ stimulated Mn-SOD accumulation within mitochondria, the active form was produced in the presence of submillimolar Mn2+ only. Amino acid substitutions at a signal peptide-cleavage site, His-Ser-Leu4 to Pro-Met-Va14, in the mature Mn-SOD prevented the processing of the precursor protein, and thus resulted in the accumulation of the precursor protein within mitochondria, as judged on immunostaining with an anti-Mn-SOD antibody. Mutant Mn-SODs with a truncated signal peptide or carboxyl region (8, 13, and 42 amino acid residues in the mature form) were barely solubilized, even with a nonionic detergent, and exhibited no activity, suggesting inappropriate folding of these mutant SODs. They were also susceptible to proteolytic degradation, while the wild-type and precursor forms were resistant. Thus, the baculovirus/insect cell expression system appears to be adequate for the analysis of mitochondrial import using intact cells as well as for the large scale production of active Mn-SOD.
Department of Biochemistry, Osaka University Medical School, Suita, Osaka, 565-0871, Japan.
J
J
Fujii
Y
Y
Ikeda
T
T
Watanabe
Y
Y
Kawasaki
K
K
Suzuki
C
C
Fujii
M
M
Takahashi
N
N
Taniguchi
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D005455:Fluorescent Antibody Technique; D006801:Humans; D007313:Insecta / Q000166:cytology; D008345:Manganese / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D017354:Point Mutation; D021382:Protein Sorting Signals / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013347:Subcellular Fractions / Q000201:enzymology; D013482:Superoxide Dismutase / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D008345:Manganese; D013482:Superoxide Dismutase