A site-directed mutant of Cu,Zn-superoxide dismutase modeled after native extracellular superoxide dismutase.
The well-studied cytosolic Cu,Zn-superoxide dismutase (SOD) protects against reperfusion injury, although its short (6 min) plasma half-life and negative charge create undesirable pharmacokinetics. We have designed, cloned, and expressed a genetic variant of SOD with altered pharmacological properties. A fusion gene consisting of the entire coding region of human SOD followed by a positively charged carboxy-terminal (C-terminal) "tail" of eight glycine and six arginine residues was constructed. The tail was modeled after the extracellular SOD (EC-SOD) C-terminal 26-amino acid basic peptide. This EC-SOD tail binds to heparin-like proteoglycans on cell surfaces and contributes to the enzyme's very long (30 h) plasma clearance time. After expression in Escherichia coli, the mutant enzyme was purified and characterized. No differences in specific activity or UV absorption spectrum between the mutant and the native enzyme were found. The thermal stability of the fusion protein was greater than that of native SOD. Although native SOD has no affinity for heparin, the modified enzyme bound to a heparin-agarose column. A "designer" SOD able to bind to cell surfaces may aid in the prevention of superoxide-mediated endothelial damage.
Gao
B
B
Webb-Waring Institute for Biomedical Research, University of Colorado Health Sciences Center, Denver 80262, USA.
Flores
S C
SC
McCord
J M
JM
D000595:Amino Acid Sequence; D001120:Arginine; D001665:Binding Sites; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli; D014644:Genetic Variation; D005998:Glycine; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011509:Proteoglycans / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013482:Superoxide Dismutase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D011509:Proteoglycans; D011993:Recombinant Fusion Proteins; D001120:Arginine; D013482:Superoxide Dismutase; D005998:Glycine