The folding of human active and inactive extracellular superoxide dismutases is an intracellular event.
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.
Petersen
Steen V
SV
Center for Insoluble Protein Structures and Interdisciplinary Nanoscience Center, Departments of Molecular Biology and Chemistry, University of Aarhus, Aarhus, Denmark.
Kristensen
Torsten
T
Petersen
Jane S
JS
Ramsgaard
Lasse
L
Oury
Tim D
TD
Crapo
James D
JD
Nielsen
Niels C
NC
Enghild
Jan J
JJ
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
D004220:Disulfides; D013481:Superoxides; C441642:SOD3 protein, human; D013482:Superoxide Dismutase