Requirements of Different Subdomains of Calpastain for Calpain Inhibition and for Binding to Calmodulin-Like Domains1

Abstract

Calpain requires Ca2+ for both proteolysis of its substrates and interaction with its endogenous inhibitor, calpastatin. The mechanism of inhibition of calpain by calpastatin has remained unsolved, although Nishimura and Goll [J. Biol. Chem. 266, 11842-11850 (1991)] reported that autolyzed calpain fragments containing calmodulin-like domains (CaMLDs) bound to an immobilized calpastatin column. We investigated the correlation between CaMLD-binding and calpain inhibition using immobilized columns of gene-engineered CaMLDs derived from the human mu-calpain large subunit and various recombinant calpastatin mutants. Among the four internally repetitive inhibitory domains of calpastatin, each having conserved regions A, B, and C, only domains 1 and 4 showed the binding activity. The region B deletion mutant of domain 1, retaining the CaMLD-binding ability, no longer had the calpain inhibition activity, and became susceptible to proteolysis. In contrast, a synthetic oligopeptide of region B with moderate calpain inhibition activity did not bind to the column. Domain 3 acquired the binding ability on substitution of region A with that of domain 1. These results suggest that calpain inhibition and binding to the CaMLDs are not correlated or mediated by different subdomains of calpastatin.

Preference of calcium‐dependent interactions between calmodulin‐like domains of calpain and calpastatin subdomains

“…The above feature conforms to a basic amphiphilic c~-helical motif where hydrophobic and basic residues are localized on each side of the helical cylinder [25]. Likewise, although calpastatin shows little secondary structure in solution [10], the potential c~-helix regions found in the conserved sequences of regions A and C [8,20] may form amphiphilic c~-helical structures upon complexing with the CaMLDs. Indeed, the calpastatin domain 1 mutant, which had a substitution of one of the conserved hydrophobic Leu residues in region A with a helix breaker residue Pro, demonstrated a striking decrease in the binding ability of the L-CaMLD, and also showed reduced calpain inhibition activity [23].…”

“…The highly conserved internal repeat regions A, B and C are located on separate exons in the human calpastatin gene, suggesting distinct roles in inhibitory function [11,22]. Moreover, regions A and C have similar sequences [6,20]. Recently we showed that the L-CaMLD of/.t-calpain bound in a Ca2+-dependent fashion to calpastatin domain 1 but not to the region B oligopeptide, by using a Sepharose column immobilizing the L-CaMLD fused with glutathione-S-transferase [20].…”

“…Moreover, regions A and C have similar sequences [6,20]. Recently we showed that the L-CaMLD of/.t-calpain bound in a Ca2+-dependent fashion to calpastatin domain 1 but not to the region B oligopeptide, by using a Sepharose column immobilizing the L-CaMLD fused with glutathione-S-transferase [20]. Furthermore, the oligopeptide of D1AI9 bound specifically to the L-CaMLD [18].…”

“…The expression plasmid of S-CaMLD (pET-S-CaMLD) was constructed similarly to that of L-CaMLD (pET-p-L-CaMLD) [18,20]. Briefly, the cDNA fragment encoding CaMLD of the pig calpain small subunit (S-CaMLD) were prepared by the PCR method using the previously isolated cDNA clone as a template [13], and inserted into the BamHI site of plasmid pET-3d (a vector for expression by T7 RNA polymerase; from Novagen, Madison, USA).…”

Identification of novel single nucleotide polymorphisms in candidate genes for mutton quality in Indian sheep

“…Some candidate genes like calpain (CAPN4), calpastatin (CAST), diacylglycerol acyltransferase 1 (DGAT1) and fatty acid binding protein 3 (FABP3) have been implicated in muscle development and fat metabolism (Goll et al 1992;Calvo et al 2004;Scata et al 2009). The CAPN4 is a cytosolic endopeptidase that requires calcium for catalytic activity, whereas CAST is a specific endogenous inhibitor that acts on calpain (Ma et al 1993). These genes are involved in muscle development, growth (Nonneman and Koohmaraie, 1999) as well as in eating quality (tenderness) of the mutton (Byun et al 2009).…”