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 Ca(2+)-independent autolytic activity, p94delta was stably expressed in COS and insect cells. p94delta showed Ca(2+)-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.
Ono
Yasuko
Y
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan.
Kakinuma
Kazumi
K
Torii
Fukuyo
F
Irie
Akihiro
A
Nakagawa
Kazuhiro
K
Labeit
Siegfried
S
Abe
Keiko
K
Suzuki
Koichi
K
Sorimachi
Hiroyuki
H
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D019556:COS Cells; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002154:Calpain / Q000235:genetics / Q000378:metabolism; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D005453:Fluorescence; D051379:Mice; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D017384:Sequence Deletion
D002135:Calcium-Binding Proteins; C033668:calpastatin; D002154:Calpain; C078134:calpain p94