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.