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+.     

The reversible activation by Mn2+ ions of the Ca2+-requiring neutral proteinase of human erythrocytes

Mn2+ (50 microM) satisfies the requirement for activity of the purified Ca2+-dependent neutral proteinase from human erythrocytes. Unlike the activation by Ca2+ [E. Melloni et al. (1984) Biochem. Int. 8, 477-489], the effect of Mn2+ is fully reversible and does not involve autodigestion of the native 80-kDa catalytic subunit. However, the native dimeric proenzyme (procalpain), which contains both the 80-kDa subunit and a smaller 30-kDa subunit, is not activated by Mn2+ alone but also requires the presence of micromolar concentrations of Ca2+. Under these conditions, 40% of the maximum activity is expressed without dissociation of the 80- and 30-kDa subunits. Mn2+, but not micromolar Ca2+, can also partially satisfy the metal requirement of the native 80-kDa subunit isolated after dissociation of the heterodimer. This activity is further enhanced by the addition of 5 microM Ca2+, which is ineffective in the absence of Mn2+. After procalpain is converted to active calpain by incubation with Ca2+ and substrate [S. Pontremoli et al. (1984) Biochem. Biophys. Res. Commun. 123, 331-337] full activity is observed with 5 microM Mn2+, which now substitutes completely for Ca2+. Activation of procalpain by Mn2+ represents a new mechanism for modulation of the Ca2+-dependent proteinase activity.     

Digestion of mu- and m-calpain by trypsin and chymotrypsin

Proteolytic digestion by trypsin and chymotrypsin was used to probe conformation and domain structure of the mu- and m-calpain molecules in the presence and the absence of Ca(2+). Both calpains have a compact structure in the absence of Ca(2+); incubation with either protease for 120 min results in only three or four major fragments. A 24-kDa fragment was produced by removal of the Gly-rich area in domain V of the 28-kDa subunit. The other fragments were from the 80-kDa subunit. Except for trypsin digestion of m-calpain, the region between amino acids 245 and 265 (human sequence) was very susceptible to cleavage by both proteases in the absence of Ca(2+); this region is in domain II (IIb of the crystallographic structure). Although no proteolytically active fragments could be isolated from either tryptic or chymotryptic digests, the calpain molecule can remain assembled in a proteolytically active complex even after the 80-kDa subunit has been completely degraded. The results suggest that interaction among different regions of the entire calpain molecule is required for its full proteolytic activity. In the presence of 1 mM Ca(2+), both calpains are degraded to fragments less than 40-kDa in less than 5 min. The C-terminal ends of both subunits, from amino acids 503 to 506 to the end of the 80-kDa subunit and from amino acids 85 to 88 to the end of the 28-kDa subunit, were resistant to degradation by either protease in the presence or in the absence of Ca(2+). Hence, this part of the calpain molecule is in a compact structure that does not change significantly in the presence of Ca(2+).     

Further characterization of calpain-mediated proteolysis of the human erythrocyte plasma membrane Ca2+-ATPase

The membrane-bound form and a solubilized and purified form of the Ca2+-ATPase from human erythrocyte have been proteolyzed under controlled conditions by highly purified Ca2+-dependent neutral cysteine-protease, calpain I, in the absence and in the presence of the calmodulin-calcium complex. In the absence of calmodulin the 136-kDa enzyme was transformed into a group of fragments of 125-124 kDa, followed by the slower formation of a second group of fragments of 82-80 kDa. These heterogeneous fragments were capable of forming an acylphosphate intermediate. The 125- and 82-kDa minor components of each heterogeneous group of fragments (125-124 and 82-80 kDa) were capable of binding calmodulin, whereas the 124- and the 80-kDa major components did not. In the presence of calmodulin, however, the native enzyme was transformed into a 127-kDa fragment followed by the slower formation of an 85-kDa fragment. Both fragments (127 and 85 kDa) formed an acylphosphate intermediate and were capable of binding calmodulin. The presence of calmodulin during calpain action effectively protected the Ca2+-ATPase from proteolytic activation (K.K.W. Wang, A. Villalobo, and B.D. Roufogalis (1988) Arch. Biochem. Biophys. 260, 696-704) and prevented the formation of the calmodulin-insensitive 124- and 80-kDa fragments. Smaller fragments not capable of forming the acylphosphate intermediate were also produced, in particular a 39-37 kDa doublet band retaining the capacity to bind calmodulin. In contrast to the membrane-bound form, the purified form of the Ca2+-ATPase was proteolyzed by calpain at a slower rate.     

Comparison of tryptic peptides from the heavy and light subunits of calpain I and calpain II by high performance liquid chromatography

Two different forms of Ca2+-dependent cysteine proteinase, low-Ca2+-requiring calpain I and high-Ca2+-requiring calpain II, are known to be heterodimers, each composed of one heavy (called 80K) and one light (called 30K) subunit. The most probable identity of the 30K and the substantial difference between the 80K subunits of porcine calpains I and II were clearly demonstrated by comparing the tryptic peptide maps obtained upon running a high performance liquid chromatography which permitted parallel detection of tryptophan-containing peptides by fluorometry. Comparison of the amino acid compositions of the two 30K and 80K subunits also confirmed this conclusion. The same chromatographical analysis also revealed close structural similarity of the human calpain I 30K subunit, and even some similarity existing between the calpain I 80K subunits of human and porcine origins.     

Molecular and catalytic characterization of intact heterodimeric and derived monomeric calpains isolated under different conditions from pig polymorphonuclear leukocytes

Evidence is presented of polymorphonuclear (PMN) cells derived from pig peripheral blood containing two molecular species of Ca2+-dependent cysteine endopeptidases, calpains I and II, which require low and high concentrations of Ca2+, respectively, for activation. Calpains I and II, purified from PMN homogenates, are heterodimers consisting of 83 plus 29 kDa and 80 plus 29 kDa subunits, respectively, which can be identified by using subunit-specific antibodies and which are identical with those of calpain species in other pig tissues and cells hitherto reported. However, a 70-kDa calpain can also be detected when pig PMN cells are disrupted by the nitrogen cavitation method under rather mild conditions, i.e., with minimal destruction of the lysosomes. Lines of evidence are presented showing that the 70-kDa species is devoid of the light subunit, that it is artificially derived from naturally occurring heterodimeric calpain I, and that the PMN cells before disruption contained no such monomeric form. The isolated 70-kDa calpain I, or monomeric artifact, requires only 1 microM Ca2+ for half-maximal activation, and it is less pH stable and much less heat stable than the parent heterodimeric calpain I. A possible mechanism for the production of this artifact is discussed.     

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.     

Calcium-activated neutral proteases (calpains) are carbohydrate binding proteins

Calcium-activated neutral proteases (calpain, EC 3.4.22.17) bind to agarose matrices (Bio-Gel A-150m, Sepharose 4B, and Ultrogel AcA 34) with high affinity in the presence of calcium. 6-O-beta-Galactopyranosyl-D-galactose, a disaccharide which closely resembles the repeating unit of the agarose matrices, completely blocks the binding of calpains and can release agarose-bound enzymes in the presence of calcium. At least 1 microM level of free calcium is required for binding. Other calcium binding proteins, including calmodulin, calpastatin, casein, and neurofilament proteins, fail to bind under the same conditions. Both calpain I and calpain II can be readily purified from crude enzyme preparations by agarose chromatography in the presence of calcium and leupeptin. Agarose-bound enzymes are eluted with calcium-free solutions or can be released in the presence of calcium by 1% Triton X-100, but not by 1 M urea or 20% ethylene glycol. Enzymes eluted from agarose are activated, as evidenced by the appearance of faster migrating forms (76 and 78 kDa) of the 80-kDa catalytic subunit of calpain I upon electrophoresis and by the increased sensitivity of calpain II to activation by micromolar levels of calcium. The electrophoretic migration of the 30-kDa regulatory subunit is, however, unaltered in enzyme fractions eluted from an agarose column. When the enzyme subunits are dissociated in 1 M NaSCN, only the 30-kDa subunit binds to the agarose matrix. Furthermore, neither calpain I nor calpain II binds to agarose when their 30-kDa subunit is autocatalyzed to an 18-kDa fragment, indicating that the NH2-terminal of the 30-kDa subunit is important for the binding of calpains to an agarose matrix.     

Comparative studies on heat stability and autolysis of scallop (Patinopecten yessoensis) calpain II-like proteinase and rabbit calpain II.

1. The scallop calpain-like proteinase is about five times more labile than the rabbit calpain II upon heat treatment at 35 degrees C. 2. By autolysis of the scallop proteinase of two 100 kDa subunits, 90, 45 and 30 kDa fragments were formed. Thereby the activity decreased monophasically in the presence of millimolar order of Ca2+, but did not increase in the presence of micromolar order of Ca2+ unlike the rabbit calpain II.     

Purification and immunological characterisation of two calcium-activated neutral proteinases from rabbit skeletal muscle

Two Ca2+-activated neutral proteinases have been prepared to a high degree of purity from rabbit skeletal muscle. One, calpain I, is optimally activated by 100 microM Ca2+ and the other, calpain II, by 1 to 2 mM Ca2+. Both enzymes have two subunits of molecular weight 80 000 and 28 000. Antibodies have been raised against the native forms of both enzyme. It was found that the antibody to native calpain I reacted only with calpain I and not with calpain II, and similarly the antibody to native calpain II reacted only to calpain II. This suggested that the epitopes in the two enzymes are located in regions that are structurally different. However, immunoblotting of the denatured calpains after SDS-polyacrylamide-gel electrophoresis revealed cross-reaction between the two subunits for both enzymes. Therefore, although the denatured enzymes have common antigenic sites it would appear that these are not exposed equally in the native proteins.     

The calpain-calpastatin system in mammalian cells: properties and possible functions.

All mammalian cells contain a calcium-dependent proteolytic system, composed by a proteinase, calpain, and an inhibitor, calpastatin. In some cell types an activator protein has also been identified. Moreover, two calpain isoforms, distinguishable on the basis of a different calcium requirement, can be present in a single cell. Both calpain forms are heterodimers composed of a heavy subunit (80 kDa) that contains the catalytic site and a smaller (regulatory?) subunit (30 kDa). Calpain I expresses full activity at 10-50 microM Ca2+, whereas calpain II requires calcium concentrations in the millimolar range. The removal by autoproteolysis of a fragment from the N-terminus of both calpain subunits generates a proteinase form that can express catalytic activity at concentrations of Ca2+ close to the physiological range. This process is significantly accelerated in the presence of cell membranes or phospholipid vesicles. Calpastatin, the specific inhibitor of calpain, prevents activation and the expression of catalytic activity of calpain. It is in itself a substrate of the proteinase and undergoes a degradation process which correlates with the general mechanism of regulation of the intracellular proteolytic system. The natural calpain activator specifically acts on calpain II isoform, by reducing the Ca2+ required for the autoproteolytic activation process. Based on the general properties of the calpain-calpastatin system and on the substrate specificity, its role in the expression of specific cell functions can be postulated.     

Identification of both calpains I and II in nucleated chicken erythrocytes

Chicken erythrocytes were found to contain two species of calpains which differ in elution profile from DEAE-cellulose and in Ca2+ requirement. After partial purification, one of them was half-maximally activated by 10 microM Ca2+ and the other by 180 microM Ca2+. The low- and high-Ca2+-requiring proteases cross-reacted only with the respective monospecific antibodies for mammalian calpain I and calpain II, respectively. Approximately 5 times more calpain I than calpain II is present in chicken erythrocytes. By immunoelectrophoretic blot analysis, both calpains I and II from chicken erythrocytes were proved to be heterodimers composed of 76 and 28 kDa, and 80 and 28 kDa subunits, respectively. Our present finding that the heavy subunit of calpain I is smaller than that of calpain II is noteworthy, since the opposite is known to be true of various mammalian calpains. An immunological study has revealed that the calpain I newly found in chicken erythrocytes is not derived from calpain II. Thus, the co-existence of calpains I and II in one animal species also holds in chickens, contrary to the previously advocated notion that chickens have only one type of calpain.     

Regulation of the Ca2+-dependent neutral proteinases from rabbit liver by an endogenous inhibitor

An endogenous inhibitor of neutral Ca2+-dependent proteinases has been isolated from rabbit liver cytosol. The inhibitor is a heat-stable, 240-kDa, tetrameric protein. It is dissociated into its 60-kDa subunits by high concentrations of Ca2+ (0.1-1 mM), but not by lower concentrations in the physiological range. Inhibition of the 150-kDa proteinase of rabbit liver [Melloni, E., Pontremoli, S., Salamino, F., Sparatore, B., Michetti, M. and Horecker, B.L. (1984) Arch. Biochem. Biophys. 232, 505-512] requires the monomeric form of the inhibitor, and occurs only at the high concentrations of Ca2+ which also cause dissociation of the dimeric 150-kDa proteinase into its 80-kDa subunits. The molecular weight of the inactive proteinase-inhibitor complex was estimated by the equilibrium gel penetration method to be 140 kDa, suggesting that it contains one subunit of proteinase and one of inhibitor. The mechanism of interaction of the inhibitor with the 200-kDa proteinase at high concentrations of Ca2+ is identical to that observed for the 150-kDa proteinase, namely dissociation of both proteinase and inhibitor into subunits and formation of an inactive 160-kDa proteinase-inhibitor complex. However, unlike the 150-kDa proteinase, which does not interact with the inhibitor at low Ca2+ concentrations, the 200-kDa proteinase is also inhibited at low concentrations of Ca2+. Under these conditions, the high-molecular-weight complex (greater than 400 kDa) formed between the tetrameric inhibitor and the dimeric proteinase prevents conversion of the 200-kDa proenzyme to the active, low-Ca2+-requiring form.     

Calpain and calpastatin in porcine retina. Identification and action on microtubule-associated proteins.

Two forms of Ca2+-dependent cysteine proteinase (calpain, EC 3.4.22.17) and their specific endogenous inhibitor (calpastatin) were partially purified from porcine retina: calpain I (low-Ca2+-requiring form) was half-maximally activated at 8 microM-Ca2+, and calpain II (high-Ca2+-requiring form) at 250 microM-Ca2+. Both calpain I and calpain II were inhibited by calpastatin. Calpain I from porcine retina was shown to be composed of 83 000- and 29 000-Mr subunits, and calpain II of 80 000- and 29 000-Mr subunits, by the use of monospecific antibodies. Calpains I and II were both found to hydrolyse microtubule-associated proteins 1 and 2 rapidly.     

Separation of fodrin subunits by affinity chromatography on calmodulin-Sepharose

Spectrin is composed of two nonidentical subunits, with the 240-kDa subunit of nonerythroid spectrin (fodrin) able to bind calmodulin (CaM) Ca2+-dependently. It was found that in the presence of chaotropic salts this binding site was still expressed, although the subunits of fodrin were dissociated. This has been exploited for separating the fodrin subunits rapidly and quantitatively by affinity chromatography on calmodulin-Sepharose. When bovine fodrin was dissolved in 2 M KI + 1 mM Ca2+ and applied to CaM-Sepharose the beta subunit (235-kDa) passed through unretarded whereas the alpha subunit (240-kDa) bound and could be eluted with ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid. These subunits would reform the intact molecule when mixed and dialyzed.     

Autoproteolysis of the small subunit of calcium-dependent protease II activates and regulates protease activity

Calcium-dependent protease II (CDP-II) from bovine heart is a heterodimer with subunit molecular weights of 80,000 and 26,000. Previous studies have demonstrated that the protease requires 350 microM Ca2+ for half-maximal activity and that the large subunit contains both the catalytic and Ca2+ binding functions of the enzyme. The function of the small subunit has been unclear. We have examined the effect of Ca2+ on structural and catalytic properties of CDP-II in the presence and absence of substrate proteins. When incubated with Ca2+ in the absence of substrate, CDP-II undergoes a series of autoproteolytic cleavages that sequentially reduce the small subunit's molecular weight from 26,000 to 24,000 to 22,000 to 17,000. During this time there is no detectable change in the 80-kDa subunit, which remains associated with the autolyzed small subunit. The rate of autoproteolysis is dependent on temperature and on the concentration of Ca2+ (half-maximal rate at approximately 600 microM Ca2+). The first cleavage appears to be unimolecular because its rate is unaffected by CDP-II concentration or by the presence of exogenous protein substrates. Subsequent cleavages result in the formation of the 80-kDa/17-kDa heterodimer and appear to occur by bimolecular reactions; rates of these reactions were slowed by decreasing CDP-II concentrations and by the presence of protein substrates. Autoproteolysis of the small subunit has two distinct functional consequences, each of which is associated with different forms of the autolyzed protease. Our results indicate that the 80-kDa/26-kDa form of CDP-II represents an inactive proenzyme and that the initial Ca2+-dependent cleavage of the 26-kDa subunit results in activation of the protease. The activated enzyme hydrolyzes protein substrates with a Ca2+ concentration requirement of 350 microM for half-maximal rates. The further autoproteolysis, which results in the formation of the 80-kDa/17-kDa heterodimer, serves to reduce the Ca2+ concentration requirement for protease activity by 25-fold. Thus, these results provide evidence for specific roles of the small subunit in the regulation of CDP-II activity.     

Intracellular localization of two distinct Ca2+-proteases (calpain I and calpain II) as demonstrated by using discriminative antibodies

Intracellular localization of two molecular species of calpain (Ca2+-dependent cysteine proteinase) was studied by immunocyto- and histochemical methods employing antibodies strictly monospecific for the respective antigens. Apparent immunological cross-reactivity between the larger subunits of calpain I (low Ca2+-requiring form) and calpain II (high Ca2+-requiring form) was calculated to be 15-17%, and two steps of affinity chromatography were needed to obtain antibodies which can discriminate between the two proteases. Indirect immunofluorescent staining of cultured PK 15 cells revealed diffuse staining of the cytoplasm with both antibodies against calpain I and calpain II. Preincubation with Ca2+-ionophore had no effect on the staining patterns. Sections of porcine kidney were stained by the avidin-biotinylated peroxidase complex method. The proximal and distal tubules and collecting duct were stained, but the glomerulus, macula densa, and vascular vessels were not stained by either anti-calpain I or anti-calpain II antibodies.     

Isolation and sequence analysis of cDNA clones for the small subunit of rabbit calcium-dependent protease

We have isolated and sequenced cDNA clones for the small subunit (30-kDa subunit) of rabbit calcium-dependent protease (Ca2+-protease) using synthesized oligodeoxynucleotide probes based on the partial amino acid sequence of the protein. A nearly full-length cDNA clone containing the total amino acid coding sequence was obtained. From the deduced sequence, the following conclusions about possible functions of the protein are presented. The kDa subunit comprises 266 residues (Mr = 28,238). The N-terminal region (64 residues) is mainly composed of glycine (37 residues) and hydrophobic amino acids and may interact with the cell membrane or an organelle. The sequence of the C-terminal 168 residues is highly homologous to the corresponding C-terminal region of the large subunit (80-kDa subunit) which has been identified as the calcium-binding domain. This region of the 30-kDa subunit contains four E-F hand structures and presumably binds Ca2+, as in the case of the 80-kDa subunit. Thus, the 30-kDa subunit may play important roles in regulating enzyme activity and/or possibly in determining the location of the Ca2+-protease. The marked sequence homology of the C-terminal regions of the two subunits may indicate that the calcium-binding domains have evolved from the same ancestral gene.     

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.     

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.