Identification of the cleavage sites of oxidized protein that are susceptible to oxidized protein hydrolase (OPH) in the primary and tertiary structures of the protein

Amino acid sequences in H2O2-oxidized bovine serum albumin (BSA) that are s usceptible to proteolytic cleavage by oxidized protein hydrolase (OPH) were investigated. When oxidized BSA was treated with OPH, low-molecular-weight fragments (54, 46, 24, 28, 20, and 8 kDa) were produced as analyzed by SDS -PAGE. N-Terminal amino acid sequence analysis of these fragments indicated that oxidized BSA was cleaved by OPH at three major sites, Leu218-Ser219, Tyr410-Thr411, and Phe506-Thr507, at an early stage of the proteolytic degr adation. In the three-dimensional structure of BSA deduced by computer mode ling, these cleavage sites were found to be located slightly inside the BSA molecule, in positions not easily accessible by OPH. The influence of oxid ation on the tertiary structure of BSA was then investigated by hypothetica lly replacing all the four methionine and two tryptophan residues with thei r oxidized forms, methionine sulfoxide and N'-formyl-kynurenine, respective ly. The three-dimensional structure of the hypothetically oxidized BSA indi cated that all the three cleavage sites in the protein could become more ex posed to the solvent than in unoxidized BSA. These results suggest that, up on oxidation of BSA, the amino acid sequences that are potentially cleavabl e by OPH but present inside the molecule become exposed on the surface and susceptible to proteolysis by OPH. This is the first report demonstrating t he cleavage sites of oxidized protein by oxidized protein-selective proteas e, suggesting the possible mechanism of oxidized protein-selective degradat ion by the enzyme.