Ca²⁺ Dependency of Calpain 3 (p94) Activation

Abstract

Calpain 3, commonly called p94 in the literature, is the abundant skeletal muscle-specific calpain that is genetically linked to limb girdle muscular dystrophy type 2A. Recently, we showed that p94's insertion sequence 1 (IS1) is a propeptide that must be autoproteolytically cleaved to provide access of substrates and inhibitors to the enzyme's active site. Removal of IS1 from the core of p94 by recombinant methods produced a fully active enzyme. Here we have resolved the discrepancies in the literature about the Ca(2+) requirement of p94 using the protease core. Even at substoichiometric levels of Ca(2+), and in competition with EDTA, autoproteolyzed enzyme slowly accumulated. Because the initial autoproteolytic cleavage is an intramolecular reaction, transient binding of two Ca(2+) ions to the core would be sufficient to promote the reaction that is facilitated by having the scissile peptide lying close to the active site cysteine. The second autolytic cleavage was much slower and required higher Ca(2+) levels, consistent with it being an intermolecular reaction. Other metal ions such as Na(+), K(+), and Mg(2+) cannot substitute for Ca(2+) in catalyzing the intramolecular autoproteolysis of the p94 core or in the subsequent hydrolysis of exogenous substrates. These metal ions increase moderately the activity of this enzyme but only at very high concentrations. Thus, the proteolytic activity of the core of p94 and its deletion mutant lacking NS and IS1 was shown to be strictly Ca(2+)-dependent. We propose a two-stage model of activation of the proteolytic core of p94.

The effects of gender and muscle type on the mRNA levels of the calpain proteolytic system and beef tenderness during post-mortem aging

“…Thus, higher levels of calpain-3 could also mean reduced degradation of myofibrillar proteins in vivo, which may support increased muscle growth. This, coupled with the fact that calpastatin does not inhibit calpain-3, may allow this protease to cleave titin when activators such as the concentration of Ca 2+ (García Díaz et al, 2006) are in sufficient quantities, as in post-mortem muscle. Apart from this eminent suicide relationship between calpain-3 and titin, calpain-3 may degrade calpastatin (Ono et al, 2004) which would further enhance post-mortem proteolysis and tenderization.…”

A New Insight into the Role of Calpains in Post-mortem Meat Tenderization in Domestic Animals: A review

“…At the same time, CAPN3 has some unique domains distinguishing it from the ubiquitous CAPNs, including its NH2-terminal domain I (contains 20 to 30 additional amino acids) and two “insertion sequences” which contain 62 and 77 amino acids at the COOH-terminal regions of domain II and III, called IS1 and IS2, respectively (Goll et al, 2003). iii) It is thought that CAPN3 is activated at the nanomolar calcium concentration (Garcia Diaz et al, 2006), although Ca 2+ -dependency of CAPN3 is not clear. iv) CAPN3 is very unstable and undergoes fast autoproteolytic degradation (Sorimachi et al, 1993; Kinbara et al, 1998).…”

Various Jobs of Proteolytic Enzymes in Skeletal Muscle during Unloading: Facts and Speculations

“…Skeletal muscles contain also calpain 3 (p94), which has common structure with calpains 1 and 2. Ca 2+ dependence of p94 was proved only in 2006 [36]. At the early stages of functional unloading, calpains are activated and redistributed from cytoplasm fraction to the membrane in slow and fast muscles [37].…”