The intracellular proteolytic processing of extracellular superoxide dismutase (EC-SOD) is a two-step event

Abstract

Extracellular superoxide dismutase (EC-SOD) is a tetramer composed of either intact (Trp1-Ala222) or proteolyticaliy cleaved (Trp1-Glu209) subunits. The latter form is processed intracellularly before secretion and lacks the C-terminal extracellular matrix (ECM)-binding region (210RKKRRRESECKAA222-COOH). We have previously suggested that the C-terminal processing of EC-SOD is either a one-step mechanism accomplished by a single intracellular endoproteolytic event cleaving the Glu209-Arg210 peptide bond or a two-step mechanism involving two proteinases (Enghild, J. J., Thogersen, I. B., Oury, T. D., Valnickova, Z., Hojrup, P., and Crapo, J. D. (1999) J. Biol. Chem. 274, 14818-14822). In the latter case, an initial endoproteinase cleavage occurs somewhere in the region between Glu209 and Glu216. A carboxypeptidase specific for basic amino acid residues subsequently trims the remaining basic amino acid residues to Glu209. A naturally occurring mutation of EC-SOD substituting Arg213 for Gly enabled us to test these hypotheses. The mutation does not prevent proteolysis of the ECM-binding region but prevents a carboxypeptidase B-like enzyme from trimming residues beyond Gly213. The R213G mutation is located in the ECM-binding region, and individuals carrying this mutation have an increased concentration of EC-SOD in the circulatory system. In this study, we purified the R213G EC-SOD variant from heterozygous or homozygous individuals and determined the C-terminal residue of the processed subunit to be Gly213. This finding supports the two-step processing mechanism and indicates that the R213G mutation does not disturb the initial endoproteinase cleavage event but perturbs the subsequent trimming of the C terminus.