title: The folding of human active and inactive extracellular superoxide dismutases is an intracellular event.
abstract: Human extracellular superoxide dismutase (EC-SOD) is a tetrameric glycoprotein responsible for the removal of superoxide generated in the extracellular space. Two different folding variants of EC-SOD exist based on the disulfide bridge connectivity, resulting in enzymatically active (aEC-SOD) and inactive (iEC-SOD) subunits. As a consequence of this, the assembly of the EC-SOD tetramers produces molecules with variable activity and may represent a way to regulate the antioxidant level in the extracellular space. To determine whether the formation of these two folding variants is an intra- or extracellular event, we analyzed the biosynthesis in human embryonic kidney 293 cells expressing wild-type EC-SOD. These analyses revealed that both folding variants were present in the intra- and extracellular spaces, suggesting that the formation is an intracellular event. To further analyze the biosynthesis, we constructed mutants with the capacity to generate only aEC-SOD (C195S) or iEC-SOD (C45S). The expression of these suggested that the cellular biosynthetic machinery supported the secretion of aEC-SOD but not iEC-SOD. The coexpression of these two mutants did not affect the expression pattern. This study shows that generation of the EC-SOD folding variants is an intracellular event that depends on a free cysteine residue not involved in disulfide bonding.
authors.affiliation: Center for Insoluble Protein Structures and Interdisciplinary Nanoscience Center, Departments of Molecular Biology and Chemistry, University of Aarhus, Aarhus, Denmark.
authors.forename: Steen V
authors.initials: SV
authors.lastname: Petersen
authors.forename: Torsten
authors.initials: T
authors.lastname: Kristensen
authors.forename: Jane S
authors.initials: JS
authors.lastname: Petersen
authors.forename: Lasse
authors.initials: L
authors.lastname: Ramsgaard
authors.forename: Tim D
authors.initials: TD
authors.lastname: Oury
authors.forename: James D
authors.initials: JD
authors.lastname: Crapo
authors.forename: Niels C
authors.initials: NC
authors.lastname: Nielsen
authors.forename: Jan J
authors.initials: JJ
authors.lastname: Enghild
mesh_terms: D019943:Amino Acid Substitution; D001483:Base Sequence; D002460:Cell Line; D004220:Disulfides; D004789:Enzyme Activation / Q000502:physiology; D005110:Extracellular Space / Q000201:enzymology*; D006801:Humans; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis / Q000502:physiology; D017510:Protein Folding; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism
chemical_list: D004220:Disulfides; D013481:Superoxides; C441642:SOD3 protein, human; D013482:Superoxide Dismutase