title: 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.
authors: Free Radical and Radiation Biology Program, Department of Radiation Oncology, Holden Comprehensive Cancer Center, The University of Iowa, Iowa City, IA 52242, USA.
authors: Adam J
authors: AJ
authors: Case
authors: James J
authors: JJ
authors: Mezhir
authors: Brianne R
authors: BR
authors: O'Leary
authors: Jennifer E
authors: JE
authors: Hrabe
authors: Frederick E
authors: FE
authors: Domann
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D057809:HEK293 Cells / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D017510:Protein Folding; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C441642:SOD3 protein, human; D013482:Superoxide Dismutase