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- Title
A Defect in the Mitochondrial Import of Mutant Mn-Superoxide Dismutase Produced in Sf21 Cells1.
- Authors
Fujii, Junichi; Ikeda, Yoshitaka; Watanabe, Toshiya; Kawasaki, Yoshimi; Suzuki, Keiichiro; Fujii, Chizuko; Takahashi, Motoko; Taniguchi, Naoyuki
- Abstract
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-Val4, 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.
- Subjects
MITOCHONDRIA; PROTOPLASM; IMINO acids; ORGANIC acids; SUPEROXIDE dismutase
- Publication
Journal of Biochemistry, 1998, Vol 124, Issue 2, p340
- ISSN
0021-924X
- Publication type
Article
- DOI
10.1093/oxfordjournals.jbchem.a022117