Abstract
p94/calpain 3 is a skeletal muscle-specific member of the Ca(2+)-regulated cytosolic cysteine protease family, the calpains. Defective p94 protease activity originating from gene mutations causes a muscular dystrophy called calpainopathy, indicating the indispensability of p94 for muscle survival. Because of the existence of the p94-specific regions IS1 and IS2, p94 undergoes very rapid and exhaustive autolysis. To elucidate the physiological relevance of this unique activity, the autolytic profiles of p94 and the effect of the p94 binding protein, connectin/titin, on this process were investigated. In vitro analysis of p94 autolysis showed that autolysis in IS1 proceeds without immediate disassembly into fragments and that the newly identified cryptic autolytic site in IS2 is critical for disassembling autolyzed fragments. As a genetic system to assay p94 autolysis semiquantitatively, p94 was expressed in yeast as a hybrid protein between the DNA binding and activation domains of the yeast transcriptional activator Gal4. Transcriptional activation by the Gal4-p94:WT hybrid protein is precluded by p94 autolysis. Complete or partial loss of autolytic activity by C129S active site mutation, limb girdle muscular dystrophy type 2A pathogenic missense mutations, or PCR-based random mutagenesis could be detected by semiquantitative restoration of Gal4-dependent beta-galactosidase gene expression. Using this system, the N2A connectin fragment that binds to p94 was shown to suppress p94 autolytic disassembly. The proximity of the IS2 autolytic and connectin-binding sites in p94 suggested that N2A connectin suppresses IS2 autolysis. These data indicate the importance of p94-connectin interaction in the control of p94 functions by regulating autolytic decay of p94.
Highlights
Highly conserved residues in the calpain superfamily; boldface type, conserved among p94, CL, and mCL; italic type, conserved among human, mouse, chicken, and fugu p94s; in quotation marks, a residue position where missense mutations are found in LGMD2A patients; underlined, a pathogenic missense mutation found in LGMD2A patients
Using the in vitro reconstituted autolytic reaction of fulllength p94, it was suggested that p94 retains functional conformation as a protease during autolysis
We have developed a genetic method of studying the autolytic properties of p94
Summary
CDNA Constructs—The cDNAs for human and mouse p94/calpain 3 were kind gifts from Drs Muriel Herasse and Isabelle Richard. The reaction was performed in 50 l of the solution (0.2 mM dGTP, 1 mM dATP/dCTP/dTTP, 400 nM each primer, 3 mM MgCl2, 0.5 mM MnCl2 in 1ϫ rTaq buffer (Takara Bio)) with 50 ng of template DNA (human wild type p94 cDNA) and 1.25 units of rTaq using the following cycles: 94 °C for 5 min; 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 120 s ϫ 25 cycles; 72 °C for 7 min. Cells transfected with expression constructs for mouse p94 with or without a C-terminal Myc-His tag were harvested in lysis buffer (50 mM Tris-HCl, pH 7.5, 0.15 M KCl, 1 mM EDTA-K, 1% (v/v) Nonidet P-40, and 0.5% Triton X-100) containing protease inhibitors as follows: 1.5 M aprotinin, 1 mM phenylmethylsulfonyl fluoride, 50 M leupeptin, 50 M 1-chloro-3-tosylamido-7-amino2-heptanone, and 2 mM iodoacetoamide. An anti-p94-C terminus antibody was elicited using C-NVLEWLQLTMYA, corresponding to the C terminus of rat p94, conjugated to KLH
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