The preferential cleavage sites of a myosin heavy chain degrading protease

Myosin heavy chain degrading protease (MHDE) appears to be an undescribed protease in skeletal muscle with high specificity for myosin heavy chain. It does not degrade actin, tropomyosin, troponin, casein or myoglobin nor does it degrade myosin-V. Conditions for maximum activity are similar to calpains, requiring Ca2+, neutral pH and a reducing agent. However, optimum temperature is 50°C to 60°C. To clarify identity of the protease the cleavage sites of MHDE were determined using myosin-ll as a substrate. Proteolysis conditions were: 0.6 mg/ml of purified myosin and 0.06 mg/ml of MHDE in 10 mM Tris-HCI (pH 7.2) containing 0.5 M NaCI, 5 mM CaClj, 5 mM MgCI2, and 0.5 mM DTT at 50°C for 90 min. Myosin fragments were separated on 5%T SDS-PAGE gels and then electrotransfered to polyvinylidene difuoride membrane. Ten amino-terminal residues were determined on fragments using an Applied Biosystems 477A Sequencer. MHDE cleaved myosin heavy chain at residues 1183, 1250, 1337, and 1418 resulting in eight major bands visualized on gels after staining. MHDE preferentially hydrolyzed the carboxyl side of GLU, GLN, or LYS with LEU at position P2 or P3. This preferential cleavage site of MHDE distinguishes it from other known proteases capable of degrading myosin heavy chain including trypsin, chymotrypsin, calpains, papain and cathepsins.