title: 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 (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.
authors.affiliation: Department of Molecular Biology, University of Aarhus, DK-8000 Arhus, Denmark.
authors.forename: Dorte Aa
authors.initials: DA
authors.lastname: Olsen
authors.forename: Steen V
authors.initials: SV
authors.lastname: Petersen
authors.forename: Tim D
authors.initials: TD
authors.lastname: Oury
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Valnickova
authors.forename: Ida B
authors.initials: IB
authors.lastname: Thøgersen
authors.forename: Torsten
authors.initials: T
authors.lastname: Kristensen
authors.forename: Russel P
authors.initials: RP
authors.lastname: Bowler
authors.forename: James D
authors.initials: JD
authors.lastname: Crapo
authors.forename: Jan J
authors.initials: JJ
authors.lastname: Enghild
mesh_terms: D000595:Amino Acid Sequence; D001011:Aorta / Q000201:enzymology; D043424:Carboxypeptidase B / Q000737:chemistry; D002268:Carboxypeptidases / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D006031:Glycosylation; D006579:Heterozygote; D006801:Humans; D013058:Mass Spectrometry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013482:Superoxide Dismutase / Q000097:blood / Q000378:metabolism*; D013997:Time Factors
chemical_list: D018698:Glutamic Acid; C441642:SOD3 protein, human; D013482:Superoxide Dismutase; D002268:Carboxypeptidases; D043424:Carboxypeptidase B