Possible Regulation of the Conventional Calpain System by Skeletal Muscle-specific Calpain, p94/Calpain 3*
- Yasuko Ono‡§,
- Kazumi Kakinuma‡§,
- Fukuyo Torii‡,
- Akihiro Irie‡,
- Kazuhiro Nakagawa‡¶,
- Siegfried Labeit∥,
- Keiko Abe‡,
- Koichi Suzuki**‡‡ and
- Hiroyuki Sorimachi‡§§§
- ‡Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, §CREST, Japan Science and Technology, Kawaguchi 332-0012, Japan, ∥Institut für Anästhesiologie und Operative Intensivmedizin, Universitätsklinikum Mannheim, Mannheim 68167, Germany, and **Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, Japan
- ↵§§ To whom correspondence should be addressed: Laboratory of Biological Function, Dept. of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, the University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Tel./Fax: 81-3-5841-8118; E-mail: ahsori{at}mail.ecc.u-tokyo.ac.jp.
Abstract
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– (p94Δ), 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, p94Δ was stably expressed in COS and insect cells. p94Δ showed Ca2+-dependent caseinolytic and autolytic activities and an inhibitor spectrum similar to those of the conventional calpains. However, calpastatin did not inhibit p94Δ and is a substrate for p94Δ, 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 p94Δ, suggesting that it has potential to evaluate p94 activity in vivo and in the diagnosis of limb-girdle muscular dystrophy type 2A.
- Received August 8, 2003.
- Revision received October 1, 2003.
- The American Society for Biochemistry and Molecular Biology, Inc.
