Proteolytic specificity of cathepsin D on bovine F-actin

Proteolysis of bovine F-actin by cathepsin D (E.C. 3.4.23.5) in 50 mM Na acetate buffer, pH 5.5, at 37°C was investigated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and reverse-phase high performance liquid chromatography (RP-HPLC). Actin was hydrolyzed by cathepsin D during incubation to peptides detectable by RP-HPLC, although no degradation products were detected by SDS-PAGE. Peptides (2% trichloroacetic acid-soluble) from the hydrolyzate were isolated by RP-HPLC on a C₁₈ column using an acetonitrile/water gradient and identified from their N-terminal sequence and mass. Cathepsin D cleavage sites were identified at Cys₁₂-Asp₁₃, Gly₂₂-Phe₂₃, Arg₃₀-Ala₃₁, Thr₇₉-Asn₈₀, Ile₈₇-Trp₈₈, Thr₉₁-Phe₉₂, Phe₉₂-Tyr₉₃, Arg₉₇-Val₉₈, His₁₀₃-Pro₁₀₄, Leu₁₀₇-Thr₁₀₈, Thr₁₀₈-Glu₁₀₉, Lys₁₂₀-Met₁₂₁, Leu₁₄₄-Tyr₁₄₅, Ile₁₅₃-Val₁₅₄, Leu₁₅₅-Asp₁₅₆, Ile₁₆₇-Tyr₁₆₈, Leu₁₈₀-Asp₁₈₁, Met₁₉₂-Lys₁₉₃, Leu₁₉₅-Thr₁₉₆, Arg₂₀₈-Glu₂₀₉, Arg₂₁₂-Asp₂₁₃, Leu₂₂₃-Asp₂₂₄, Lys₂₄₀-Ser₂₄₁, Thr₂₆₂-Leu₂₆₃, Trp₃₄₂-Ile₃₄₃, Arg₃₄₉-Ser₃₅₀, Trp₃₅₈-Ile₃₅₉, and Lys₃₇₅-Cys₃₇₆. In general, cathepsin D preferentially cleaved bonds containing at least one hydrophobic amino acid residue. The results of this study showed that actin was degraded extensively by cathepsin D with peptides released from numerous locations in the protein molecule.