Mutual inhibitory effect of calpastatins on calpains from carp muscle, carp erythrocytes and rat liver

The inhibitory effect of calpastatin (specific inhibitor for calpain) on calpain (Ca2+-dependent cysteine proteinase, EC 3.4.22.17) was examined using carp muscle, carp erythrocytes and rat liver preparations. A mutual inhibitory effect between calpains and calpastatins from different tissues and species was observed. The conservation of the inhibitory effect of calpastatin on calpain among vertebrates suggests that the calpain-calpastatin system may play a biologically fundamental and common role in various cells.     

cDNA cloning of human calpastatin: sequence homology among human, pig, and rabbit calpastatins

cDNA of human calpastatin, an inhibitor protein specific for calpain (EC 3.4.22.17; Ca2(+)-dependent cysteine proteinase) was isolated by screening of a library prepared from human liver mRNA with pig calpastatin cDNA fragment as a probe. The primary structure of human calpastatin was deduced from the nucleotide sequence of the cDNA and compared with that of pig and rabbit calpastatins already reported. Human calpastatin consisted of 673 amino acid residues and had 78% and 77% identity to pig or rabbit calpastatins, respectively. Human calpastatin had a domain structure with four internally repetitive sequences and one N-terminal non-homologous sequence like the other calpastatins. Human calpastatin had two deletions, 22 and 13 residues long in domain L and domain 1, respectively, compared to pig or rabbit calpastatins.     

Repetitive region of calpastatin is a functional unit of the proteinase inhibitor

A cDNA portion coding for one of the repetitive regions of pig heart calpastatin (107 kDa) was subcloned into E. coli plasmid pUC119 to express the portion of the proteinase inhibitor gene in bacteria. The expressed protein was a chimaeric protein whose calpastatin segment (130 amino acid residues) was fused with an amino-terminus portion (7 amino acid residues) of beta-galactosidase. The chimaeric protein could inhibit proteolytic activity of calpain (Ca2+-dependent cysteine proteinase), and maintained properties of the authentic calpastatin concerning inhibition specificity and heat stability. These findings led us to conclude that the repetitive region is a functional unit of the proteinase inhibitor.     

Identification of two calpastatin forms in rat skeletal muscle and their susceptibility to digestion by homologous calpains

Two forms of calpastatin, differing in their specificity for the homologous calpain isozymes I and II, have been separated from rat skeletal muscle extracts and purified to homogeneity. Calpastatin I, the first form to elute in chromatography on DE32, is more effective against calpain I, while calpastatin II is more effective as an inhibitor of calpain II. Based on their molecular mass (approximately 105 kDa) both calpastatin forms belong to the high molecular mass class found in muscles of other animal species (Murachi, T., 1989, Biochem. Int. 18, 263-294). For calpain I, which is active with low (mu-M) concentrations of Ca2+, maximum inhibition with either calpastatin form was observed over a wide range of Ca2+ concentrations. With calpain II, which requires high (mM) concentrations of Ca2+ for activity, maximum inhibition required Ca2+ concentrations above 1 mM. Both calpastatin forms were found to be highly sensitive to degradation by calpain II, but almost completely resistant to degradation by calpain I. Degradation of calpastatin by calpain II is competitively inhibited by the addition of a calpain substrate. Isovaleryl carnitine (IVC), an intermediate product of L-leucine catabolism, previously demonstrated to be a potent and specific activator of rat skeletal muscle calpain II (Pontremoli, S., Melloni, E., Viotti, P. L., Michetti, M., Di Lisa, F., and Siliprandi, N., 1990. Biochem. Biophys. Res. Commun. 167, 373-380) greatly enhances the rate of degradation of calpastatins by calpain II. IVC, which decreases the Ca2+ requirement for maximal calpain II activity, also decreases the concentration of Ca2+ required for digestion of the inhibitor. For calpain II, regulation by either calpastatins may occur only in the presence of high [Ca2+].     

Differential regulation of the calpain–calpastatin complex by the L-domain of calpastatin

Here we demonstrate that the presence of the L-domain in calpastatins induces biphasic interaction with calpain. Competition experiments revealed that the L-domain is involved in positioning the first inhibitory unit in close and correct proximity to the calpain active site cleft, both in the closed and in the open conformation. At high concentrations of calpastatin, the multiple EF-hand structures in domains IV and VI of calpain can bind calpastatin, maintaining the active site accessible to substrate. Based on these observations, we hypothesize that two distinct calpain–calpastatin complexes may occur in which calpain can be either fully inhibited (I) or fully active (II). In complex II the accessible calpain active site can be occupied by an additional calpastatin molecule, now a cleavable substrate. The consequent proteolysis promotes the accumulation of calpastatin free inhibitory units which are able of improving the capacity of the cell to inhibit calpain. This process operates under conditions of prolonged [Ca^2 +] alteration, as seen for instance in Familial Amyotrophic Lateral Sclerosis (FALS) in which calpastatin levels are increased. Our findings show that the L-domain of calpastatin plays a crucial role in determining the formation of complexes with calpain in which calpain can be either inhibited or still active. Moreover, the presence of multiple inhibitory domains in native full-length calpastatin molecules provides a reservoir of potential inhibitory units to be used to counteract aberrant calpain activity.     

Molecular diversity of calpastatin in mammalian organs

The crude homogenates of various human and porcine organs were subjected to immunoelectrophoretic blot analysis using affinity-purified anti-calpastatin antibody which specifically reacts with human erythrocyte 70 kDa calpastatin. Multiple immuno-reactive bands were revealed which ranged from 100 to 50 kDa. The results indicated the diversity of monomeric calpastatin molecules. The band patterns were different from one organ to the other. Among them, lung, heart and skeletal muscle were characterized by the predominance of 90-100 kDa calpastatin, having a common antigenicity to erythrocyte 70 kDa calpastatin. Such molecular diversity of calpastatins was also substantiated by enzymatic and chromatographic analyses.     

Phosphorylation and subcellular distribution of calpastatin in human hematopoietic system cells

Calpastatin is an endogenous inhibitor protein acting specifically on calpain (EC 3.4.22.17; Ca2(+)-dependent cysteine proteinase). The phosphorylation of calpastatin was investigated in human hematopoietic system cell lines. Microheterogeneity of calpastatin was observed, in which 118- and 116-kDa forms were named calpastatin a and b, respectively. The phosphorylation of both calpastatins was identified in all cell lines examined and occurred mainly at serine residues with trace amounts of phosphothreonine in vivo. The incubation of cells with 12-O-tetradecanoylphorbol-13-acetate increased the incorporation of 32P-orthophosphate into calpastatin a. Two-dimensional maps of 32P-labeled phosphopeptide from both calpastatins were identical except for additional minor spots for calpastatin a. [35S]methionine-labeled calpastatins a and b were localized mainly in the cytosol, and only 6% of cellular calpastatins were detected in the membrane fraction. By contrast, more than 30% of the 32P-labeled calpastatins a and b were distributed in the membrane fraction. Thus, the phosphorylation of calpastatin may be involved in regulating the calpain-calpastatin protein kinase system by its subcellular distribution.     

Phosphorylation of rat brain calpastatins by protein kinase C.

Calpastatin, the natural inhibitor of calpain, is present in rat brain in multiple forms, having different molecular masses, due to the presence of one (low Mr form) or four (high Mr form) repetitive inhibitory domains. Recombinant and native calpastatin forms are substrates of protein kinase C, which phosphorylates a single serine residue at their N-terminus. Furthermore, both low and high Mr calpastatins are phosphorylated by protein kinase C at the same site. These calpastatin forms are phosphorylated also by protein kinase A, although with a lower efficiency. The incorporation of a phosphate group determines an increase in the concentration of Ca2+ required to induce the formation of the calpain-calpastatin complex. This effect results in a large decrease of the inhibitory efficiency of calpastatins. We suggest that phosphorylation of calpastatin represents a mechanism capable to balance the actual amount of active calpastatin to the level of calpain to be activated.     

Identification of calpastatin and mu-calpain and studies of their association in pulmonary smooth muscle mitochondria

Using calpastatin antibody we have identified a 145 kDa major band along with two relatively minor bands at 120 kDa and 110 kDa calpastatin molecules in bovine pulmonary artery smooth muscle mitochondria. To the best of our knowledge this is first report regarding the identification of calpastatin in mitochondria. We also demonstrated the presence of micro-calpain in the mitochondria by immunoblot and casein zymogram studies. Immunoblot studies identified two major bands corresponding to the 80 kDa large and the 28 kDa small subunit of mu-calpain. Additionally 76 kDa, 40 kDa and 18 kDa immunoreactive bands have also been detected. Purification and N-terminal amino acid sequence analysis of the identified proteins confirmed their identity as mu-calpain and calpastatins. Immunoprecipitation study revealed molecular association between mu-calpain and calpastatin in the mitochondria indicating that calpastatin could play an important role in preventing uncontrolled activity of mu-calpain which otherwise may facilitate pulmonary hypertension, smooth muscle proliferation and apoptosis.     

Degradation of skeletal muscle plasma membrane proteins by calpain

Summary Observations described here provide the first demonstration that calpain (Ca²⁺-dependent cysteine protease) can degrade proteins of skeletal muscle plasma membranes. Frog muscle plasma membrane vesicles were incubated with calpain preparations and alterations of protein composition were revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Calpain II (activated by millimolar concentrations of Ca²⁺) was isolated from frog skeletal muscle, but the activity of calpain I (activated by micromolar concentrations of Ca²⁺) was lost during attempts at fractionation. Calpain I obtained from skeletal muscle and erythrocytes of rats was tested instead, and exerted effects similar to those of frog muscle calpain on the membrane proteins. All of the calpain preparations caused striking losses of a major membrane protein of molecular mass of approximately 97 kDa, designated band c, and diminution of a thinner band of approximately 200 kDa. There were concomitant increases in 83-and 77-kDa polypeptides. These effects were absolutely dependent on the presence of free Ca²⁺, and were completely blocked by calpastatin, a specific inhibitor of calpain action. Frog muscle calpain differed only in being relatively more active at 0°C than were the calpains from rat tissues. Experimental observations suggest that calpain acts at the cytoplasmic surface of the plasma membrane.     

Decreased expression of calpain and calpastatin mRNA during development is highly correlated with muscle protein accumulation in neonatal pigs

It is well known that rapid gain of muscle mass in neonatal pigs is highly related to protein synthesis. However, the role of protein degradation in muscle gain of the neonatal period has not been well established. Calpains and their endogenous inhibitors, calpastatins, play a significant role in early-stage myofibrillar protein degradation. To investigate the role of calpain–calpastatin system in muscle protein accumulation, we studied the expressions of their mRNA in muscle tissue sampled at days 1, 4, 6, 12, 20 and 28 from a total of 36 neonatal pigs. The steady-state mRNA levels of calpains 1A, 2 and 3A, calpastatin types 1, 2 and 3, obtained by quantitative real-time PCR analysis, decreased by 2–4 folds at the age of 4 to 6 days compared to 1-day-old piglets. Then, the relatively low expression level was maintained through 28 days of age. Expressions of calpains 1A, 3A and calpastatin type 1 were significantly correlated with the measurements of muscle protein accumulations such as muscle protein content and RNA/protein ratio. Expressions of calpain 1A, calpastatin types 1 and 3 were negatively correlated with birth weight and fractional rate of growth. The levels of calpains 1A and 2 mRNA were correspondent to their protease activities. In conclusion, decreased levels of calpain and calpastatin expressions over development in neonatal pigs are associated with high protein accumulations, suggesting that dramatic muscle growth during the neonatal period may be partially controlled by down-regulated calpain–calpastatin system.     

Analysis of structure-function relationship of pig calpastatin by expression of mutated cDNAs in Escherichia coli

Structure-function relationships in pig calpastatin were investigated. Calpastatin is an endogenous inhibitor protein specifically acting on calpains (Ca2+-dependent cysteine endopeptidases). We recently cloned and sequenced the cDNA for pig heart calpastatin and determined the amino acid sequence of the molecule from the nucleotide sequence. Various deletion mutants in one of the four internally repetitive domains (Domain 3, approximately 140 amino acid residues) were created by in vitro site-directed mutagenesis of a cloned cDNA fragment and expressed in Escherichia coli. Deletion of a conserved region on either the amino-terminal or carboxyl-terminal side caused a drastic loss of inhibitory activity against calpain I (low Ca2+-requiring form) and, to a lesser degree, against calpain II (high Ca2+-requiring form). Inhibitory activities were below the detectable level in mutants deleted further toward the central region. Substitution of two amino acids in the latter region of the wild-type Domain 3 protein caused a drastic loss of activity against both calpains. The creation of lowered affinity inhibitors enabled us to perform a conventional kinetic analysis which showed the mode of inhibition to be competitive. Prediction of the secondary structure of Domain 3 suggests that both the amino- and carboxyl-terminal conserved regions form alpha-helical structures, which are largely located in the interior of the calpastatin molecule, whereas the central region does not form alpha-helix or beta-structure. The central region contains a 12-residue consensus sequence common to Domains 1, 2, and 4, and this portion is predicted to be located on the surface of the calpastatin molecule. These results suggest that the central conserved region of each domain of calpastatin is an area for direct interaction either with the active center of calpain or a region in close proximity, and the rest of the domain is a region stabilizing the functionally important tertiary structure of the domain.     

Identification of an endogenous activator of calpain in rat skeletal muscle

An additional component of the regulatory system of rat skeletal muscle calpain has been identified. It exerts a potent activating effect on calpain activity and is a heat stable small molecular weight protein. Of the two calpain isozymes present in muscle, the activator is specific for calpain II, being uneffective with calpain I. It promotes activation of the proteinase by reducing 50 fold, from 1 mM to of 20 microM, the requirement of Ca2+ for maximum catalytic activity of the proteinase. However in the presence of the activator calpain II expresses a consistent fraction of the maximum activity even at significantly lower concentrations of Ca2+ (below 5 microM Ca2+). The activator effect follows kinetics that are consistent with the presence of specific binding sites on the calpain molecules. The activator not only removes in a dose dependent fashion the inhibition of calpain by calpastatin, but also prevents inhibition of the proteinase upon the addition of calpastatin. Competition experiments revealed that the proteinase contains distinct sites for the activator and the inhibitor, and that both ligands can bind to calpain with the formation of an almost fully active ternary complex.     

The calpain—calpastatin system in vascular smooth muscle

Vascular smooth muscle contains large amounts of the Ca²⁺-dependent protease calpain II. In this study, we compared bovine aortic muscle (muscle phenotype) to cultured bovine aortic cells of smooth muscle origin (modulated phenotype) with respect to major constituents of the calpain—calpastatin system. Bovine aortic muscle contained only calpain II by activity measurements, Western blot of tissue extracts and Northern blot of poly(A)⁺ RNA. On the other hand, using the same methodologies, both calpains I and II as well as the 110 kDa inhibitor protein, calpastatin, were identified in cultured bovine aortic cells of smooth muscle origin. We conclude that the phenotypic state of smooth muscle cells is associated with differential expression of major components of the calpain—calpastatin system. Moreover, bovine aortic muscle is the only tissue identified to date that contains calpain II exclusively.     

On the mechanism of binding of calpastatin, the protein inhibitor of calpains, to biologic membranes

Bovine myocardial calpastatin, the endogenous inhibitor of the calcium-dependent proteinases, calpains, could bind to sarcoplasmic reticulum preparations at neutral pH and low ionic strength. Even in the presence of 100 to 200 mM KCl, 4 to 5 micrograms of calpastatin was bound per mg of membrane. Although calpastatin is found associated with bovine myocardial sarcolemma, neither canine nor human erythrocyte calpastatins were present in isolated erythrocyte membrane preparations. The bovine myocardial calpastatin, but not human erythrocyte calpastatin, could associate with purified phospholipid vesicles at low ionic strength. Thus, phospholipids appear to be involved in the binding of calpastatin to membranes.     

Characterization of the calpastatin defect in erythrocytes from patients with essential hypertension

In erythrocytes of patients with essential hypertension the level of calpastatin activity was found to be significantly lower than in red cells of normotensive subjects (1). We now demonstrate, by Western blot analysis, that the decreased inhibitory activity is due to a corresponding decrease in the amount of the inhibitor protein. This is also supported by the observation that calpastatins isolated and purified from erythrocytes of normotensive and hypertensive patients, have identical specific activity. Data are presented indicating that the decreased level of calpastatin cannot be ascribed to an accelerated decay of the inhibitor during the erythrocyte life span. Taken together the previous and present results further emphasize that an umbalanced proteolytic system may represent one of the molecular mechanisms responsible for those membrane abnormalities underlying the development of essential hypertension and its clinical complications.     

A new human calpastatin skipped of the inhibitory region protects calpain-1 from inactivation and degradation

Several human acute and chronic diseases involve calpain over-activation. However, the mechanistic linkages between the etiology and the progression of cell damages are not yet completely understood. Here we show that different human cells and tissues, including brain tumor specimens, cell lines of nerve origin, breast tumor samples and peripheral blood mononuclear cells from healthy donors, express a calpastatin form that lacks all the exons coding for the domains responsible of calpain inhibition. The open reading frame of this new form of calpastatin, named hcast 3-25, starts inside the L-domain (exons 2 and 3) and continues with the exons from 25 to 29 that code for the conserved C-terminal tail shared by all the full-length calpastatins. We have here observed that unlike the other calpastatins forms, that are predominantly Δ3 splice variants, hcast 3-25 is endowed with exon 3. At a functional level, recombinant hcast 3-25 operates as a positive modulator of calpain-1 in vitro by preventing 1) calpain-1-mediated proteolytic degradation of the activated enzyme and 2) binding to calpain-1 of inhibitory calpastatins that contain the L-domain. Thus hcast 3-25 can be considered as a novel member and possible modulator of the calpain/calpastatin system acting by a mechanism alternative to inhibition.