Rational design of a secreted enzymatically inactive mutant of extracellular superoxide dismutase

Abstract

Extracellular superoxide dismutase (SOD3) is a secreted enzyme that regulates levels of extracellular superoxide and protects the extracellular matrix from degradation by reactive species. The SOD3 protein contains a heparin-binding domain and resides in a microenvironment rich in other heparin-bound growth factors, raising the possibility that SOD3 may have some biological role independent of its catalytic activity. To begin to address this, we designed and created enzymatically inactive mutant constructs targeting either the copper coordinating (i.e. H96 and H98) or superoxide channeling (i.e. N180 and R186) amino acid residues of SOD3. All constructs expressed equal quantities of immature intracellular SOD proteins, but only the N180A, R186A, and combination N180A/R186A mutants produced fully processed and secreted extracellular protein. Furthermore, while SOD activity was significantly inhibited in the single N180A and R186A mutants, the activity was completely abrogated in the N180A/R186A double mutant. Overall, the use of this novel tool may have broad reaching impacts into various fields of biology and medicine, and will aid in the delineation of cellular processes that are regulated by solely the SOD3 protein, its reactive oxygen species substrates and products, or the combination of both.

Keywords:

• Antioxidants
• Free radicals
• Heparan sulfate proteoglycans
• Signaling proteins
• Oxidative stress

Acknowledgements

This work was supported by NIH grant CA115438 (FED). We thank the University of Iowa DNA facility for their help in plasmid sequencing. We also thank Dr Garry Buettner and Brett Wagner and the Radiation and Free Radical Research Core of the Holden Comprehensive Cancer Center supported by NIH P30 CA086862 for assistance with the EPR studies. J.E.H. and J.J.M. received salary support from T32CA148062. Finally, we thank Dr Apollina Goel for the use of a Tecan spectrophotometer.