title: Murine extracellular superoxide dismutase is converted into the inactive fold by the Ser195Cys mutation.
abstract: We have previously shown that human extracellular superoxide dismutase (EC-SOD) exists as two variants with differences in their disulfide bridge patterns: one form is the active enzyme (aEC-SOD), and the other is inactive (iEC-SOD). The availability of both active and inactive folding variants significantly reduces the specific activity of EC-SOD in vivo. Both forms are produced during biosynthesis, but the underlying folding mechanisms remain unclear. To address this issue, we expressed EC-SOD in heterologous systems that do not endogenously express iEC-SOD. Rodents express only aEC-SOD because they lack Cys195 (human EC-SOD sequence numbering), which is essential for the formation of iEC-SOD. However, cultured hamster cells and transgenic mice expressing human EC-SOD were able to produce both human a- and iEC-SOD variants, which led us to hypothesize that the folding was sequence-dependent rather than a property of the expression system. To substantiate this hypothesis, we expressed murine EC-SOD in a human cell line, and as expected, only aEC-SOD was produced. Significantly, when Cys195 was introduced, both murine aEC-SOD and a novel murine iEC-SOD were generated, and the specific activity of the murine EC-SOD was significantly reduced by the mutation. Collectively, these data suggest that Cys195 actuates the formation of iEC-SOD, independent of the expression system or host. In addition, the dual-folding pathway most likely requires biosynthesis factors that are common to both humans and rodents.
authors.affiliation: Center for Insoluble Protein Structures (inSPIN), Interdisciplinary Nanoscience Center (iNANO) and Department of Molecular Biology and Genetics, Aarhus University , DK-8000 Aarhus, Denmark.
authors.forename: Carsten
authors.initials: C
authors.lastname: Scavenius
authors.forename: Jane S
authors.initials: JS
authors.lastname: Petersen
authors.forename: Line R
authors.initials: LR
authors.lastname: Thomsen
authors.forename: Ebbe Toftgaard
authors.initials: ET
authors.lastname: Poulsen
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Valnickova-Hansen
authors.forename: Russell P
authors.initials: RP
authors.lastname: Bowler
authors.forename: Tim D
authors.initials: TD
authors.lastname: Oury
authors.forename: Steen V
authors.initials: SV
authors.lastname: Petersen
authors.forename: Jan J
authors.initials: JJ
authors.lastname: Enghild
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D051379:Mice; D008822:Mice, Transgenic; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D011994:Recombinant Proteins; C441642:SOD3 protein, human; C499090:Sod3 protein, mouse; D013482:Superoxide Dismutase; D003545:Cysteine