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.     

Mechanism of action of the calpain activator protein in rat skeletal muscle.

Rat skeletal muscle contains a calpain activator protein characterized by a high specificity for calpain II, the high Ca(2+)-requiring isoform of this class of proteinases. The activator protein increases the rate of intramolecular conversion of the native 80-kDa catalytic subunit of calpain into the autolysed 75-kDa forms with maximal rate at concentrations of calcium approximately 25 times lower than those required by the native proteinase. The activator protein interacts with native calpain II forming a 1:1 complex; interaction does not occur with the fully activated form, produced by autoproteolysis. Even after immobilization to membranes, the activator binds to calpain, which then undergoes sequential activation and release from its bound form. The activator is itself resistant to digestion by calpain II, whereas it increases the rate at which homologous calpastatin is degraded by the proteinase. Taken together, these results are indicative of the existence in rat skeletal muscle of an activating system specific for calpain II which is potentially involved in the regulation of the inhibitory efficiency of calpastatin, through modulation of its intracellular level.     

Binding to erythrocyte membrane is the physiological mechanism for activation of Ca2+-dependent neutral proteinase

In the presence of micromolar concentrations of Ca2+ the catalytic 80 kDa subunit of human erythrocyte procalpain binds to the cytosolic surface of the erythrocyte membrane. Binding is rapid, highly specific and is reversed by the removal of Ca2+. In the bound form the 80 kDa catalytic subunit undergoes a rapid conversion to calpain, the active 75 kDa Ca2+-requiring proteinase. The activated proteinase produces extensive degradation of membrane components, particularly of band 4.1 and 2.1 proteins. Binding to membranes may represent an obligatory physiological mechanism for the conversion of procalpain to calpain.     

Free calcium and calpain I activity

Activation of purified calpain I proceeds through a Ca(2+)-induced autolysis from the 80 kDa catalytic subunit to a 76 kDa form via an intermediate 78 kDa form, and from a 30 kDa form to a 18 kDa form as the result of two autocatalytic processes (intra and intermolecular). The minimum Ca2+ requirements for autolysis and proteolysis have been determined by physico-chemical and electrophoretic methods in the presence or absence of a digestible substrate. According to our results the activation process needs less free Ca2+ than the proteolysis of a digestible substrate, which means that proteolysis is really subsequent to activation. For very low Ca2+ levels, a digestible substrate does not initiate the calpain I activation process. In the presence of phospholipid vesicles, such as PI, PS or a mixture of PI (20%), PS (20%) and PC (60%), the apparent kinetic constants of activation are greatly increased without any change in the initial velocity of the substrate proteolysis. Thus, enzyme activation and substrate proteolysis are observed as independent phenomena. These results obtained from experiments using low free Ca2+ concentrations enable us to propose a hypothesis for the mechanism of regulation by which the enzyme could be activated in the living cell.     

Platelet factor XIII is activated by calpain

The action of calpain (EC 3.4.22.17; Ca2+-dependent cysteine proteinase) on platelet factor XIII has been studied. Calpain I activated platelet factor XIII up to 76% of the maximum level observed with thrombin. Activation was accompanied by the limited proteolysis of the a subunit of platelet factor XIII to produce a 76 kDa fragment which was comparable to the proteolytic product by thrombin. Activation of platelet factor XIII by calpain was inhibited by EDTA, leupeptin, and endogenous calpain-specific inhibitor calpastatin. These findings suggest that calpain is responsible for the intracellular activation of platelet factor XIII.     

Activation by hemoglobin of the Ca2+-requiring neutral proteinase of human erythrocytes: structural requirements

The proenzyme form of the Ca2+-requiring neutral proteinase of human erythrocytes (procalpain) is converted to the active proteinase (calpain) by low concentrations of Ca2+ in the presence of appropriate substrates such as beta-hemoglobin or heme-free beta-globin chains. Modification of these substrates by limited proteolysis with calpain abolishes their ability to promote the conversion of procalpain. A similar requirement for the presence of unmodified beta-hemoglobin or heme-free beta-globin chains is observed for the autocatalytic inactivation of calpain. The conversion of procalpain to calpain is accompanied by a small decrease in the molecular mass of the catalytic subunit, from 80 kDa to 75 kDa; however, the activation is not accelerated by the addition of a small quantity of calpain. The autocatalytic inactivation of active CANP is related to the disappearance of the 75 kDa subunit and the formation of smaller peptide fragments.     

Myocardial calpain 2 is inhibited by monoclonal antibodies specific for the small, noncatalytic subunit

Calpains (EC 3.4.22.17) are nonlysosomal intracellular proteinases which require calcium ion for activity. The calpains are heterodimers composed of a large catalytic subunit and a small subunit which may have a regulatory function during the catalytic cycle. However, whether calpains remain in the dimeric form or dissociate upon exposure to calcium is controversial. To resolve this issue, two monoclonal antibodies which specifically recognize the small calpain subunit were prepared using bovine calpain 2 heterodimer as the antigen. Both antibodies, designated P-1 and P-2, were capable of inhibiting bovine or canine calpain 2, and partially purified human erythrocyte calpain 1. However, neither could produce full inhibition. Further studies with P-1 and bovine calpain 2 indicated that the antibody decreased the calcium requirement for the proteinase. The Km for casein was increased and the Vmax was decreased. The addition of P-1 to the assay mixture several minutes after initiation of proteolytic activity resulted in a rapid inhibition. The P-1 antibody was also capable of decreasing the ability of the protein inhibitor of calpains (calpastatin) to inhibit bovine calpain 2. These studies indicate that the small subunit remains bound to the large subunit during catalysis and may influence its activity.     

Procalpain is activated on the plasma membrane and the calpain acts on the membrane

The mechanism of activation of human erythrocyte calpain was investigated using the immunoblotting technique with anticalpain monoclonal antibody. The purified calpain underwent a Ca2+-induced fragmentation of the 80 kDa subunit to 76 kDa and 36 kDa fragments. The behavior of the 76 kDa fragment in electrophoresis corresponded to the proteinase activity of calpain, whereas the behavior of the 80 kDa subunit and the 36 kDa fragment did not. When inside-out membrane vesicles were added to the reaction mixture of calpain and Ca2+ and the vesicles were separated from the supernatant solution by centrifugation, the 80 kDa subunit and 76 kDa fragment were found in the vesicle fraction. No other fragments were found in this fraction. On the other hand, the 80 kDa subunit and 36 kDa fragment were found in the supernatant fraction. When right-side-out membrane vesicles were added to the reaction mixture and the vesicles were separated from the supernatant fraction, no fragment was found in the vesicle fraction, while only the 36 kDa fragment was found in the supernatant fraction. These results indicate that the 80 kDa subunit of procalpain was bound in a Ca2+-dependent manner to the cytosolic surface of the plasma membrane and then underwent fragmentation to produce the 76 kDa fragment (active form) and that it expressed its proteinase activity at the surface of the membrane.     

The endoplasmic reticulum chaperone glycoprotein GRP94 with Ca(2+)-binding and antiapoptotic properties is a novel proteolytic target of calpain during etoposide-induced apoptosis.

GRP94 is a 94-kDa chaperone glycoprotein with Ca(2+)-binding properties. We report here that during apoptosis induced by the topoisomerase II inhibitor etoposide, a fraction of GRP94 associated with the endoplasmic reticulum membrane undergoes specific proteolytic cleavage, coinciding with the activation of the caspase CPP32 and initiation of DNA fragmentation. In vivo, inhibitors of caspases able to block etoposide-induced apoptosis can only partially protect GRP94 from proteolytic cleavage, whereas complete inhibition is observed with calpain inhibitor I but not with the proteasome inhibitor. In vitro, GRP94 is not a substrate for CPP32; rather, it can be completely cleaved by calpain, a Ca(2+)-regulated protease. The cleavage of GRP94 by calpain is Ca(2+)-dependent and generates a discrete polypeptide of 80 kDa. In contrast, calpain has no effect on other stress proteins such as GRP78 or HSP70. Further, immunohistochemical staining reveals specific co-localization of GRP94 with calpain in the perinuclear region following etoposide treatment. We further showed that reduction of GRP94 by antisense decreased cell viability in etoposide-treated Jurkat cells. Our studies provide new evidence that the cytoprotective GRP94, as in the case of the antiapoptotic protein Bcl-2, can be targets of proteolytic cleavage themselves during the apoptotic process.     

Binding of calpain fragments to calpastatin

Their cDNA-derived amino acid sequences predict that the 80-kDa subunits of the micromolar and millimolar Ca(2+)-requiring forms of the Ca(2+)-dependent proteinase (mu- and m-calpain, respectively) each consist of four domains and that the 28-kDa subunit common to both mu- and m-calpain consists of two domains. The calpains were allowed to autolyze to completion, and the autolysis products were separated and were characterized by using gel permeation chromatography, calpastatin affinity chromatography, and sequence analysis. Three major fragments were obtained after autolysis of either calpain. The largest fragment (34 kDa for mu-calpain, 35 kDa for m-calpain) contains all of domain II, the catalytic domain, plus part of domain I of the 80-kDa subunit of mu- or m-calpain. This fragment does not bind to calpastatin, a competitive inhibitor of the calpains, and has no proteolytic activity in either the absence or presence of Ca2+. The second major fragment (21 kDa for mu-calpain and 22 kDa for m-calpain) contains domain IV, the calmodulin-like domain, plus approximately 50 amino acids from domain III of the 80-kDa subunit of mu- or m-calpain. The third major fragment (18 kDa) contains domain VI, the calmodulin-like domain of the 28-kDa subunit. The second and third major fragments bind to a calpastatin affinity column in the presence of Ca2+ and are eluted with EDTA. The second and third fragments are noncovalently bound, so the 80- and 28-kDa subunits of the intact calpain molecules are noncovalently bound at domains IV and VI. After separation in 1 M NaSCN, the 28-kDa subunit binds completely to calpastatin, approximately 30-40% of the 80-kDa subunit of mu-calpain binds to calpastatin, and the 80-kDa subunit of m-calpain does not bind to calpastatin in the presence of 1 mM Ca2+.     

Autolysis of mu- and m-calpain from bovine skeletal muscle

The rate of autolysis of mu- and m-calpain from bovine skeletal muscle was measured by using densitometry of SDS polyacrylamide gels and determining the rate of disappearance of the 28 and 80 kDa subunits of the native, unautolyzed calpain molecules. Rate of autolysis of both the 28 and 80 kDa subunits of mu-calpain decreased when mu-calpain concentration decreased and when beta-casein, a good substrate for the calpains, was present. Hence, autolysis of both mu-calpain subunits is an intermolecular process at pH 7.5, 0 or 25.0 degrees C, and low ionic strength. The 78 kDa subunit formed in the first step of autolysis of m-calpain was not resolved from the 80 kDa subunit of the native, unautolyzed m-calpain by our densitometer, so autolysis of m-calpain was measured by determining rate of disappearance of the 28 kDa subunit and the 78/80 kDa complex. At Ca2+ concentrations of 1000 microM or higher, neither the m-calpain concentration nor the presence of beta-casein affected the rate of autolysis of m-calpain. Hence, m-calpain autolysis is intramolecular at Ca2+ concentrations of 1000 microM or higher and pH 7.5. At Ca2+ concentrations of 350 microM or less, the rate of m-calpain autolysis decreased with decreasing m-calpain concentration and in the presence of beta-casein. Thus, m-calpain autolysis is an intermolecular process at Ca2+ concentrations of 350 microM or less. If calpain autolysis is an intermolecular process, autolysis of a membrane-bound calpain would require selective participation of a second, cytosolic calpain, making it an inefficient process. By incubating the calpains at Ca2+ concentrations below those required for half-maximal activity, it is possible to show that unautolyzed calpains degrade a beta-casein substrate, proving that unautolyzed calpains are active proteases.     

Isovalerylcarnitine is a specific activator of the high calcium requiring calpain forms

Isovalerylcarnitine, a product of the catabolism of L-leucine, is a potent activator of rat calpains isolated from erythrocytes, kidney, liver, skeletal and heart muscle. Only calpains II, but not calpains I, are activated by IVC, with the only exception of rat erythrocyte calpain I, the only species present in these cells which has a Ca2+ requirement higher than that of most calpain I isoenzymes. Activation by IVC involves a dual effect: 1) a ten fold increase in the affinity of calpain for Ca2+, and 2) an increase in the Vmax 1.3-1.6 fold above the values observed with the native enzymes at saturating [Ca2+] as well as with the autolyzed fully active calpain form at 5 microM Ca2+. The increased affinity for calcium results in an increased rate of autoproteolysis of calpain II. Activation by IVC is additive to that promoted by interaction (or association) to phospholipids vesicles. Together these results suggest that IVC may operate as a selective activator of calpain both in the cytosol and at the membrane level; in the latter case in synergism with the activation induced by association of the proteinase to the cell membrane.     

Reversible activation of human neutrophil calpain promoted by interaction with plasma membranes

Human neutrophil calpain is a monomer of 85 kDa molecular weight. The proteinase shows an absolute requirement for Ca2+ with maximal catalytic activity at 0.1-0.2 mM Ca2+ and negligible activity at 1-5 microM Ca2+. At this concentration of Ca2+ neutrophil calpain becomes active and reaches 65% of its maximal catalytic activity following interaction with plasma membranes. The activation is fully reversible since the enzyme returns to its native, high Ca2+ requiring form following removal of the membranes. Membrane phospholipids appear to be the physiological compounds responsible for the promotion of such reversible activation. Unlike other Ca2+ dependent proteinases, neutrophil calpain does not undergo conversion to a low Ca2+ requiring form by limited autoproteolysis.     

Calpain I remains intact and intracellular during platelet activation. Immunochemical measurements with monoclonal and polyclonal antibodies.

As a step towards understanding the physiological function of calpain (Ca2+-activated neutral proteinase, EC 3.4.22.17) in blood platelets, and in view of some suggestions that calpain is transferred to the platelet external surface during platelet activation, the enzyme was studied with immunochemical methods in resting and thrombin-activated cells. (1) A mouse IgG1 monoclonal antibody was prepared which binds strongly only to the denatured large subunit of human calpain I, and weakly to that of human calpain II. A polyclonal antibody raised against rat calpain II was available which, apart from binding strongly to rat calpain II, binds to the large subunits of human calpain I and II about equally. (2) With these antibodies, it was found that calpain could be detected in fixed platelets in suspension only after permeabilization with 0.1% saponin, and could not be detected on the exterior surface of resting or of activated platelets, or in the supernatant media of these platelets. It was concluded that calpain is not significantly externalized during platelet activation. (3) Immunoblotting showed that conversion of the larger calpain I subunit from 80 kDa into 76-78 kDa occurred only when thrombin-activated platelets were stirred to permit aggregation, and did not occur during unstirred thrombin activation. Although an action of calpain in the 80 kDa form on possible platelet substrates such as cytoskeletal proteins cannot be excluded, calpain is certainly not present as the 76-78 kDa form, which is assumed to be its active form, until aggregation is initiated.     

Identification of calpain II in porcine sperm

The role that proteolytic enzymes may play in membrane-associated phenomena of sperm has been the subject of extensive investigation. In the present study, we have examined the possibility that a Ca2+-activated, neutral protease, calpain II, may be associated with sperm membranes. Using indirect immunofluorescence with primary antibodies, which are polyclonal and monoclonal antibodies directed against the 80 kDa subunit of calpain II, we have established the presence of this antigen in porcine sperm. Staining by anticalpain II (80 kDa subunit) of the apical segment of the acrosomal cap and basal body (centriolar) region was seen consistently. Variable staining of the sperm tail was also observed. These observations, combined with our positive identification of a 80 kDa protein in acrosomal membranes (via immunoblot), document the association of this protease with sperm membranes. The proximity of calpain II to the acrosome suggests a potential role for the protease in the Ca2+-mediation of the acrosome reaction.     

Murine erythroleukemia cell differentiation: possible involvement of a calcium dependent neutral proteinase

Murine erythroleukemia cells contain a single type of calpain classified, on the basis of its calcium requirement, as a type I proteinase. The enzyme is practically inactive at concentrations of calcium below 10 microM and expresses maximal activity in the presence of 0.12-0.15 mM Ca2+. The affinity for Ca2+ cannot be reduced by exposure of the enzyme to conditions known to promote autoproteolysis of calpain. Expression of catalytic activity at lower concentrations of Ca2+, is promoted by the interaction with phospholipid vesicles or plasma membranes. Conditions that promote activation of calpain, induce also the self-inactivation of the enzyme. During terminal differentiation of murine erythroleukemia cells induced by HMBA, the intracellular level of calpain activity undergoes significative reduction. Similar decrease in calpain activity progressively occurs during the loss of sensitivity to HMBA as a result of density growth arrest.     

Ca2+-dependent proteolysis of the Ah receptor

We previously reported (J. Biol. Chem. (1986) 261, 6352-6465) that the photoaffinity ligand for the Ah receptor, [125I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, upon incubation with the liver cytosol fraction from C57BL/6 mice, labeled in a 1:1 ratio two peptides that had apparent molecular masses of 95 and 70 kDa and similar proteolytic fragmentation patterns. In the cytosolic fraction of Hepa 1 cells, a cloned murine hepatoma cell line, the product of photoaffinity labeling is almost exclusively a 95-kDa peptide which is rapidly hydrolyzed by a Ca2+-dependent proteinase to a 70-kDa peptide as well as other fragments. Thus, the ligand binding unit of the Ah receptor in C57BL/6 mouse liver and Hepa 1 cell is a 95-kDa peptide, and the 70-kDa fragment is a proteolytic artifact. The Ca2+-dependent proteinase which hydrolyzes the 95-kDa peptide has the properties of calpain II: (i) an absolute requirement for Ca2+, with maximal activity at 0.5 to 1.0 mM Ca2+; (ii) a pH optimum of 7.5 to 8.0; (iii) inhibition by EDTA, iodoacetamide, leupeptin and L-trans-epoxysuccinylleucylamido(4-guanidino)butane, but not by soybean trypsin inhibitor, aprotinin, or phenylmethanesufonyl fluoride. Upon chromatographic separation of the liver cytosol of C57BL/6 mice on DEAE-Sephacel, Ca2+-dependent proteinase activity (using casein or the labeled 95-kDa peptide as substrates) elutes with 0.25 M NaCl, and a specific proteinase inhibitor elutes with 0.15 M NaCl. Ca2+-dependent proteinase activity that hydrolyzes the 95-kDa peptide is found in the liver cytosols of several mammalian species.     

Characterization of Brain Calpains

A new, simple one-step procedure [Karlsson et al. Biochem. J. 231, 201-204 (1985)] for the separation of calpains I and II was used prior to the characterization of these enzymes from rabbit brain, using alkali-denatured casein as the substrate. Enzyme activity was dependent on Ca2+ ions and free-SH groups and was maximal around pH 7.4. Incubation of calpains I and II with Ca2+ in the absence of substrate led to a rapid loss of enzyme activity. Enzyme activity was linear at room temperature and millimolar Ca2+ concentrations. However, when incubation of calpain I was performed with micromolar Ca2+ concentrations at room temperature proteolytic activity exhibited a lag period of approximately 10 min. This activation period was not as evident with calpain II.     

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+].     

Purification and characterization of a calpain activator from human platelets.

A calpain (Ca(2+)-activated neutral protease) activator was purified from human platelets by ammonium sulfate fractionation, gel-filtration, ion-exchange chromatography, followed by heat-treatment. The purified calpain activator with a Mr of 47.5 kDa was a heat-stable protein as demonstrated in other cells. The calpain activator did not change the Ca2+ sensitivity of calpain but activated calpain activity about 2-fold. This calpain activator may play an important role in the activation of the protease system leading to the Ca(2+)-mediated physiological process of platelets.