Possible regulation of the conventional calpain system by skeletal muscle-specific calpain, p94/calpain 3

p94 (also called calpain 3) is the skeletal muscle-specific calpain and is considered to be a "modulator protease" in various cellular processes. Analysis of p94 at the protein level is an urgent issue because the loss of p94 protease activity causes limb-girdle muscular dystrophy type 2A. In this study, we enzymatically characterized one alternatively spliced variant of p94, p94: exons 6-15-16-(p94Delta), which lacks two of the p94-specific insertion sequences. In contrast to p94, which has hardly been studied enzymatically due to its rapid, thorough, and apparently Ca2+-independent autolytic activity, p94Delta was stably expressed in COS and insect cells. p94Delta showed Ca2+-dependent caseinolytic and autolytic activities and an inhibitor spectrum similar to those of the conventional calpains. However, calpastatin did not inhibit p94Delta and is a substrate for p94Delta, which is consistent with the properties of p94, presenting p94 as a possible regulator of the conventional calpain system. We also established a semi-quantitative fluorescence resonance energy transfer assay using the calpastatin sequence specifically to measure p94 activity. This method detects the activity of COS-expressed p94 and p94Delta, suggesting that it has potential to evaluate p94 activity in vivo and in the diagnosis of limb-girdle muscular dystrophy type 2A.