Skeletal Muscle-specific Calpain Is an Intracellular Na+-dependent Protease

Abstract

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.

The N- and C-terminal autolytic fragments of CAPN3/p94/calpain-3 restore proteolytic activity by intermolecular complementation

“…4C, lanes 2 and 5 vs. lane 8). Consistent with the fact that CAPN3 is a Na + -dependent protease (35), these activities were accelerated by the presence of 0.1 M NaCl (Fig. 4C, lanes 3 and 6 vs. lane 9).…”

“…In line with such difference, the EF5-hand of CAPN3 is unique in that it is a binding site for Ca 2+ as well as an interacting site for homodimer formation of the PEF(L) domain (33). It was also shown that a CAPN3 mutant completely lacking IS2 and PEF(L) (CAPN3: S581X), expresses protease activity comparable to that of FL-CAPN3 (35). Further studies will be required to elucidate the precise role of the carboxyl-terminal region in reg-ulating CAPN3's autolysis and the function of iMOC.…”

“…6B, lane 6). These amino acid residues correspond to the Ca 2+ -binding site in PC1 (CBS1) in CAPN1 and CAPN2 and were shown to be involved in the Na + responsiveness of CAPN3 (35). The proteolytic product generated from FL:AHN (or FL:SHN) following iMOC with N-terminal competent fragments and detected by anti-C3IS2 antibodies, appeared to be identical to the C 58K fragment (Fig.…”

“…The effect of the ΔIS1 mutation was expected, as it compromises the structure of the CAPN3 autolytic sites. Because IS2 plays a role in the Ca 2+ -independent activity of CAPN3 (11,35), its deletion may change the structure of PC2. However, we demonstrated that the IS2 in C 58K was not required for the formation of the active CysPc through iMOC with the FL:CAA mutant (Fig.…”

Expanding Members and Roles of the Calpain Superfamily and Their Genetically Modified Animals

“…This autolysis is dependent on the presence of both the IS1 and the IS2 regions, and inhibitors of µ-and m-calpains such as calpastatin, E-64, and leupeptin have little effect on the autolysis. Surprisingly, this autolysis undergoes Na + -dependently even in the absence of Ca 2+ , showing that calpain-3/p94 is the first example of intracellular Na + -dependent enzyme [120]. Furthermore, calpain-3/p94 possesses a nuclear localization signal-like sequence in the IS2 region, and is sometimes localized in the nucleus in addition to the cytosol.…”