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J. Biol. Chem., Vol. 283, Issue 22, 15031-15036, May 30, 2008

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The Folding of Human Active and Inactive Extracellular Superoxide Dismutases Is an Intracellular Event^*

Steen V. Petersen, Torsten Kristensen, Jane S. Petersen, Lasse Ramsgaard, Tim D. Oury, James D. Crapo^¶, Niels C. Nielsen^||, and Jan J. Enghild¹

From the Center for Insoluble Protein Structures (inSPIN) and the Interdisciplinary Nanoscience Center (iNANO), Departments of Molecular Biology and ^||Chemistry, University of Aarhus, DK-8000 Aarhus, Denmark, the Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, and the ^¶Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado 80206

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.

Received for publication, February 26, 2008 , and in revised form, March 26, 2008.

^* This work was supported by the Danish National Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark. Fax: 45-8942-5063; E-mail: jje{at}mb.au.dk.

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