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 H(2)O(2)-oxidized bovine serum albumin (BSA) that are susceptible to proteolytic cleavage by oxidized protein hydrolase (OPH) were investigated. When oxidized BSA was treated with OPH, low-molecular-weight fragments (54, 46, 24, 22, 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 degradation. In the three-dimensional structure of BSA deduced by computer modeling, these cleavage sites were found to be located slightly inside the BSA molecule, in positions not easily accessible by OPH. The influence of oxidation on the tertiary structure of BSA was then investigated by hypothetically replacing all the four methionine and two tryptophan residues with their oxidized forms, methionine sulfoxide and N'-formyl-kynurenine, respectively. The three-dimensional structure of the hypothetically oxidized BSA indicated that all the three cleavage sites in the protein could become more exposed to the solvent than in unoxidized BSA. These results suggest that, upon oxidation of BSA, the amino acid sequences that are potentially cleavable by OPH but present inside the molecule become exposed on the surface and susceptible to proteolysis by OPH. This is the first report demonstrating the cleavage sites of oxidized protein by oxidized protein-selective protease, suggesting the possible mechanism of oxidized protein-selective degradation by the enzyme.
Fujino
T
T
School of Pharmacy and School of Life Science, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Kojima
M
M
Beppu
M
M
Kikugawa
K
K
Yasuda
H
H
Takahashi
K
K
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003198:Computer Simulation; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D020014:K562 Cells; D008715:Methionine; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010447:Peptide Hydrolases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry* / Q000378:metabolism*; D014364:Tryptophan
D011994:Recombinant Proteins; D012710:Serum Albumin, Bovine; D014364:Tryptophan; D008715:Methionine; D006861:Hydrogen Peroxide; D010447:Peptide Hydrolases; C038220:acylaminoacyl-peptidase