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 ؊ (p94⌬), 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, p94⌬ was stably expressed in COS and insect cells. p94⌬ showed Ca 2؉ -dependent caseinolytic and autolytic activities and an inhibitor spectrum similar to those of the conventional calpains. However, calpastatin did not inhibit p94⌬ and is a substrate for p94⌬, 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 p94⌬, suggesting that it has potential to evaluate p94 activity in vivo and in the diagnosis of limb-girdle muscular dystrophy type 2A.Calpain (EC 3.4.22.17, clan CA, family C2) is a Ca 2ϩ -requiring cysteine protease representing one of the most important families of the cysteine proteases (1-9). To date, various molecules showing significant similarity to the calpain protease domain have been identified in almost all kinds of living organisms and constitute the "calpain superfamily" (6). Two representative members, -and m-calpains, the so-called "conventional" calpains, are ubiquitously expressed and have been well characterized. These two calpains consist of a distinct larger catalytic subunit containing a protease domain (-or m-calpain large subunit, abbreviated as CL 1 or mCL, respectively) and a common smaller regulatory subunit (abbreviated as 30K according to its molecular weight). On the basis of amino acid similarities, the large and small subunits have been described as consisting of four and two domains, respectively, which agrees with the recently resolved three-dimensional structure of m-calpain (10, 11) (Fig. 1A).Conventional calpain has a specific endogenous proteinaceous inhibitor, calpastatin (12). Calpastatin contains four repetitive inhibitory units, each of which inhibits equimolar amounts of conventional calpain. The conserved reactive site interacts with the calpain protease domain, whereas the flanking ␣-helical regions bind to domains IV and VI of the large and small subunits, respectively. Synthetic oligopeptides (see Fig. 4D) corresponding to the calpastatin-reactive site specifically inhibit conventional calpain efficiently, although their inhibitory activity is weaker than that of the full-length inhibitory unit.The primary structure of p94 (also called calpain 3) is very similar to those of CL ...

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 ␤-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.Calpains (named after the first discovered and best characterized member, Ca 2ϩ -dependent and papain-like protease, EC 3.4.22.18, clan CA, family C2) correspond to a diverse gene family whose members all share the characteristic "calpain protease domain" and comprise a unique branch of the cysteine proteases (1, 2). To date, calpains have been identified in many different mammalian tissues and in almost all types of living organism (3).Studies on calpain protease activity, from enzymatic characterization to clarification of its roles in cellular phenomena, have mainly advanced knowledge of the conventional calpains, the -and m-calpains. These ubiquitously expressed calpains can be conveniently assayed in vitro using established and reproducible methods, such as casein hydrolysis assay, facilitating development of more specific inhibitors as well as more sensitive substrates.In contrast, the skeletal muscle-specific calpain, p94/calpain 3, has remained poorly characterized with regard to its protease activity. The expression of p94 predominates over other calpain species in skeletal muscle, and a defect in p94 proteolytic activity originating from gene mutations causes muscular dystrophy (4 -6). In this context, p94 exemplifies the critical importance of tissue-spe...

Because intracellular [Na+] is kept low by Na+/K+-ATPase, Na+ dependence is generally considered a property of extracellular enzymes. However, we found that p94/calpain 3, a skeletal-muscle-specific member of the Ca2+-activated intracellular “modulator proteases” that is responsible for a limb-girdle muscular dystrophy (“calpainopathy”), underwent Na+-dependent, but not Cs+-dependent, autolysis in the absence of Ca2+. Furthermore, Na+ and Ca2+ complementarily activated autolysis of p94 at physiological concentrations. By blocking Na+/K+-ATPase, we confirmed intracellular autolysis of p94 in cultured cells. This was further confirmed using inactive p94:C129S knock-in (p94CS-KI) mice as negative controls. Mutagenesis studies showed that much of the p94 molecule contributed to its Na+/Ca2+-dependent autolysis, which is consistent with the scattered location of calpainopathy-associated mutations, and that a conserved Ca2+-binding sequence in the protease acted as a Na+ sensor. Proteomic analyses using Cs+/Mg2+ and p94CS-KI mice as negative controls revealed that Na+ and Ca2+ direct p94 to proteolyze different substrates. We propose three roles for Na+ dependence of p94; 1) to increase sensitivity of p94 to changes in physiological [Ca2+], 2) to regulate substrate specificity of p94, and 3) to regulate contribution of p94 as a structural component in muscle cells. Finally, this is the first example of an intracellular Na+-dependent enzyme.

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