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

Jane S. Petersen,Lasse Ramsgaard,Jan J. Enghild,Torsten Kristensen,Tim D. Oury,Niels C. Nielsen,James D. Crapo,Steen V. Petersen

doi:10.1074/jbc.m801548200

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.

Highlights

Human extracellular superoxide dismutase (EC-SOD2; SOD3) is present in the extracellular matrix of tissues, where it functions as a scavenger of the superoxide radical [1,2,3]
The majority of thiol isomerase enzymes are present in the endoplasmic reticulum, increasing evidence suggests that these proteins are present in the extracellular space [20]
Pseudo-aEC-SOD was generated by the substitution of Cys195 for Ser, and pseudo-iEC-SOD was generated by the substitution of Cys45 for Ser

Summary

Introduction

Human extracellular superoxide dismutase (EC-SOD2; SOD3) is present in the extracellular matrix of tissues, where it functions as a scavenger of the superoxide radical [1,2,3]. 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. 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.

Results

Conclusion