Possible Regulation of the Conventional Calpain System by Skeletal Muscle-specific Calpain, p94/Calpain 3

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 ؊ (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 ...

Insertion Sequence 1 of Muscle-specific Calpain, p94, Acts as an Internal Propeptide

“…Very recently, a new splice variant of p94, which lacks IS1 and the lysine-rich sequence in IS2, has been found in human peripheral blood cells and is active in casein zymograms (18). Moreover, an alternatively spliced variant of mouse skeletal muscle p94 that lacks IS1 and IS2 (p94⌬) has been successfully expressed in mammalian cells and shown to be proteolytically active (21).…”

“…Furthermore, leupeptin and calpain inhibitors I and II effectively inhibit the splice variant of p94 present in peripheral blood mononuclear cells that lacks IS1 (18). Leupeptin, E-64, and calpeptin, among other calpain inhibitors, inhibited the proteolytic activity of mouse skeletal muscle p94 that lacks IS1 and IS2 (p94⌬) (21). Our results show that complete inhibition of proteolytic activity in p94I-II (and its deletion mutant p94I-II ⌬NS) by E-64 and leupeptin can only be detected after several hours of Ca 2ϩ -dependent autolysis, suggesting that accessibility to the active site is hindered by the presence of IS1 in non-autolyzed p94I-II.…”

“…Furthermore, a recombinant construct of Lp82 was stably produced in insect cells, purified, and characterized (20). Very recently, an alternatively spliced variant of mouse skeletal muscle p94 that lacks IS1 and IS2 (p94:exon 6 Ϫ 15 Ϫ 16 Ϫ , p94⌬) has been successfully expressed in mammalian cells and shown to be proteolytically active (21). Introduction of IS1 into Lp82 destabilized the protein and caused a loss of enzymatic activity, whereas substitution of the shorter Nterminal sequence (AX1) present in Lp82 by NS or introduction of IS2 did not affect stability (22).…”

Endogenous Calpain-3 Activation Is Primarily Governed by Small Increases in Resting Cytoplasmic [Ca2+] and Is Not Dependent on Stretch

“…It has been shown that the IS2 region of calpain-3 is important not only because it enables binding at the N2A line of titin (19) but also because its presence affects the Ca 2ϩ sensitivity of the autolytic activation (11,41). The combined excision of the IS1 and IS2 regions greatly reduces the Ca 2ϩ sensitivity of calpain-3 (41), and mutation of acidic amino acids in the IS2 region seemingly has a comparable effect (11).…”

μ-Calpain binds to lipid bilayers via the exposed hydrophobic surface of its Ca2+-activated conformation

“…The initial Ca 2q concentrations for autolysis in vitro are comparable to those needed for proteolytic activity of the unautolysed enzyme. Additional mechanisms have been proposed to account for the reduction in activation threshold by Ca 2q in vivo: association with membrane phospholipids (Pontremoli et al, 1985), activator molecules (Melloni et al, 1998), phosphorylation by protein kinases (Glading et al, 2004), and calpain activation cascades (Tompa et al, 1996;Ono et al, 2004). The existing reports on how these factors contribute to calpain activation are often controversial and their modes of action remain largely unexplored (Goll et al, 2003).…”