The Intracellular Proteolytic Processing of Extracellular Superoxide Dismutase (EC-SOD) is a Two-step Event

• Published in: The Journal of biological chemistry Vol. 279; no. 21; pp. 22152 - 22157
• Main Authors: Olsen, Dorte Aa, Petersen, Steen V., Oury, Tim D., Valnickova, Zuzana, Thøgersen, Ida B., Kristensen, Torsten, Bowler, Russel P., Crapo, James D., Enghild, Jan J.
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 21.05.2004
• Subjects: Amino Acid Sequence; Aorta - enzymology; Carboxypeptidase B - chemistry; Carboxypeptidases - chemistry; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Extracellular Matrix - metabolism; Glutamic Acid - chemistry; Glycosylation; Heterozygote; Humans; Mass Spectrometry; Models, Chemical; Molecular Sequence Data; Mutation; Protein Binding; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Spectrometry, Mass, Electrospray Ionization; Superoxide Dismutase - blood; Superoxide Dismutase - metabolism; Time Factors
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M401180200

Extracellular superoxide dismutase (EC-SOD) is a tetramer composed of either intact (Trp 1 –Ala 222 ) or proteolytically cleaved (Trp 1 –Glu 209 ) subunits. The latter form is processed intracellularly before secretion and lacks the C-terminal extracellular matrix (ECM)-binding region ( 210 RKKRRRESECKAA 222 –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 Glu 209 –Arg 210 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 Glu 209 and Glu 216 . A carboxypeptidase specific for basic amino acid residues subsequently trims the remaining basic amino acid residues to Glu 209 . A naturally occurring mutation of EC-SOD substituting Arg 213 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 Gly 213 . 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 Gly 213 . 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.