The protease core of the muscle-specific calpain, p94, undergoes Ca2+-dependent intramolecular autolysis

Limb girdle muscular dystrophy type 2A is linked to a skeletal muscle-specific calpain isoform known as p94. Isolation of the intact 94-kDa enzyme has been difficult to achieve due to its rapid autolysis, and uncertainty has arisen over its Ca2+-dependence for activity. We have expressed a C-terminally truncated form of the enzyme that comprises the protease core (domains I and II) along with its insertion sequence, IS1, and N-terminal leader sequence, NS. This 47-kDa p941-II mini-calpain was stable during purification. In the presence of Ca2+, p941-II cleaved itself within the NS and IS1 sequences. Mapping of the autolysis sites showed that NS and IS1 have the potential to be removed without damage to the protease core. Ca2+-dependent autolysis must be an intramolecular event because the inactive p941-II C129S mutant was not cleaved by incubation with wild-type p941-II. In addition, the rate of autolysis of p941-II was independent of the concentration of the enzyme. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.