Subunit Of Calpain Research Articles

Mechanism of Action of a New Component of the Ca2+-Dependent Proteolytic System in Rat Brain: The Calpain Activator

Rat brain contains a calpain activator specific for the μ-form of the proteinase. We now report that this protein factor binds to the catalytic 80 kDa calpain subunit, promoting the dissociation of the heterodimer structure of the proteinase. The successive steps of the activation process, namely the two autoproteolytic steps producing the 78 kDa and the 75 kDa calpain forms, result in a 100 times faster rate. The activator competes with calpastatin and associates with the inner surface of plasma membranes. Based on its properties, the calpain activator can be visualised as the molecule indicating the sites for calpain activation at which the proteinase can also elude the negative control exerted by calpastatin.

Cloning of M-calpain 80 kD subunit from the axonal degeneration-resistant WLDs mouse mutant

Calpains are calcium-activated cysteine proteases that are involved in cellular degradation in models of neurodegeneration. Calpains are the effectors of cytoskeletal disruption during axonal degeneration, a pathological feature of many neurological disorders. The WLD(S) mouse mutant is resistant to axonal degeneration and demonstrates prolonged survival of the cytoskeleton after nerve injury. To investigate the possibility that a mutation in calpain or abnormalities in calpain protein expression is responsible for the resistance to axonal degeneration seen in the WLD(S) mouse mutant, we 1) cloned and sequenced the large subunit of the high calcium-requiring form of calpain (m-calpain) from nervous system tissues of WLD(S) and from wild-type C57BL/6 mice, and 2) generated polyclonal m-calpain antibodies for comparison of relative protein levels by Western blot. We found our sequence for mouse m-calpain to be almost identical to another recently published mouse sequence, and the wild-type and WLD(S) sequences to be identical. Our fusion protein and peptide polyclonal antibodies were specific for the 80 kD subunit and recognized appropriate protein bands from pure m-calpain, fusion protein, and in tissue. There was no apparent difference in m-calpain expression in nerve or spinal cord in noninjured adult animals. These data suggest that a defect in m-calpain 80 kD subunit does not likely underlie the WLD(S) phenotype, but raise questions about other subunits of calpain and possibly other proteases.

Evidence for the participation of the proteasome and calpain in early phases of muscle cell differentiation

Objectives were to investigate the role of the proteasome and m-calpain to muscle cell differentiation. Accordingly, we investigated the effects of lactacystin, a proteasome inhibitor, and calpain inhibitor-II (CI-II) on L8 muscle cell differentiation and assessed concentrations of proteasomal and calpain subunit mRNAs during differentiation. L8 myoblasts were induced to differentiate by culturing in mitogen-depleted medium. To assess the importance of the proteasome and calpain to differentiation, we examined effects of lactacystin and CI-II on creatine kinase (CK) activity. In the absence of inhibitor, CK activity was detectable within 48 h of mitogen depletion and myotubes were formed. Addition of lactacystin or CI-II to cultures drastically reduced CK activity and prevented formation of myotubes. Hence, proteasome and calpain are both necessary for differentiation. In order to identify which proteasomal subunits were regulated during differentiation, we examined the concentrations of two 20S core subunits (C8 and C9) and three 22S ATPases (MSS1, S4 and TBP1) during differentiation. Concentrations of m-calpain and β-tubulin mRNAs were also assessed. Differentiation was associated with slight increases (ca. 30%) in concentrations of mRNAs encoding the proteasomal 20S core subunits (C8 and C9) and with large increases (approximately 2-fold) in mRNAs encoding the regulatory subunit ATPases. m-calpain mRNA concentration also increased two-fold following mitogen depletion. β-tubulin mRNA concentration remained unchanged early in the differentiation process and thereafter declined. Of interest, changes in proteasomal and m-calpain mRNAs occurred within 6–24 h of mitogen depletion (i.e., at least 24–36 h prior to detectable changes in creatine kinase activity). These results indicate that changes in expression of proteasome and calpains subunits occur early in the differentiation process. These changes may be required for the normal course of differentiation to proceed. Differentiation is associated with larger changes in proteasomal ATPase mRNAs than in 20S core particle mRNAs indicating that either turnover rates of the 22S ATPase subunits are more rapid in differentiating cells than of the 20S core particles or that functions of the regulatory subunits become more important during muscle cell differentiation.

Oxidative Stress Inhibits Calpain Activity in Situ

In this study, the effects of oxidative stress on calpain-mediated proteolysis and calpain I autolysis in situ were examined. Calpain activity was stimulated in SH-SY5Y human neuroblastoma cells with the calcium ionophore, ionomycin. Calpain-mediated proteolysis of the membrane-permeable fluorescent substrate N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-7-amido-4-methylcouma rin, as well as the endogenous protein substrates microtubule-associated protein 2, tau and spectrin, was measured. Oxidative stress, induced by addition of either doxorubicin or 2-mercaptopyridine N-oxide, resulted in a significant decrease in the extent of ionophore-stimulated calpain activity of both the fluorescent compound and the endogenous substrates compared with control, normoxic conditions. Addition of glutathione ethyl ester, as well as other antioxidants, resulted in the retention/recovery of calpain activity, indicating that oxidation-induced calpain inactivation was preventable/reversible. The rate of autolytic conversion of the large subunit of calpain I from 80 to 78 to 76 kDa was decreased during oxidative stress; however, the extent of calpain autolysis was not altered. These data indicate that oxidative stress may reversibly inactivate calpain I in vivo.

Functional cleavage of the common cytokine receptor gamma chain (gammac) by calpain.

The small subunit of calpain, a calcium-dependent cysteine protease, was found to interact with the cytoplasmic domain of the common cytokine receptor gamma chain (gammac) in a yeast two-hybrid interaction trap assay. This interaction was functional as demonstrated by the ability of calpain to cleave in vitro-translated wild-type gammac, but not gammac containing a mutation in the PEST (proline, glutamate, serine, and threonine) sequence in its cytoplasmic domain, as well as by the ability of endogenous calpain to mediate cleavage of gammac in a calcium-dependent fashion. In T cell receptor-stimulated murine thymocytes, calpain inhibitors decreased cleavage of gammac. Moreover, in single positive CD4(+) thymocytes, not only did a calpain inhibitor augment CD3-induced proliferation, but antibodies to gammac blocked this effect. Finally, treatment of cells with ionomycin could inhibit interleukin 2-induced STAT protein activation, but this inhibition could be reversed by calpain inhibitors. Together, these data suggest that calpain-mediated cleavage of gammac represents a mechanism by which gammac-dependent signaling can be controlled.

Functional properties of recombinant calpain I and of mutants lacking domains III and IV of the catalytic subunit.

The catalytic subunit (L-microCANP) of human calpain I (muCANP, the high Ca2+ affinity form) and two of its mutants were expressed in Escherichia coli or using the baculovirus Sf9 system. The mutants lacked domain III (L-mu CANPDelta3) and the calmodulin-like domain IV (L-mu CANPDelta4), respectively. The bacterially expressed proteins were solubilized from the inclusion bodies and refolded with polyethylene glycol. In Sf9 cells, co-expression of the inhibitor calpastatin was necessary to prevent autolysis of L-muCANP, whereas co-expression of the regulatory subunit enhanced it. Only very low levels of mRNA of the truncated form L-mu CANPDelta4 were found in bacmid-transfected Sf9 cells, and it proved impossible to isolate this mutant using the baculovirus expression system. While the apparent Km(Ca2+) of freshly isolated human erythrocyte muCANP was about 60 microM, the recombinant monomeric forms L-mu CANP and L-mu CANPDelta3 required 65-215 and 400-530 microM Ca2+, respectively. Bacterially expressed L-mu CANPDelta4 was Ca2+-independent; the presence of inhibitors during its renaturation was necessary to prevent its autolysis. A chimeric form (L-mu mCANP) composed by domains I-III of muCANP and domain IV of calpain II (mCANP, the low Ca2+ affinity form) was also expressed in Sf9 cells. This mutant required less Ca2+ (about 50 microM) than native erythrocyte calpain for half-maximal activity and had the highest specific activity of all calpains tested. Domain III proved unnecessary for the activity of the recombinant catalytic subunit, but its absence raised the Km(Ca2+) and removed its inactivation at high Ca2+ concentrations. All recombinant proteins were active as monomers in polyethylene glycol-containing buffers; the in vitro association with the regulatory subunit enhanced only slightly the Vmax and the Ca2+ dependence of the expressed proteins. Activation by Ca2+ promoted the separation of the two subunits of the expressed recombinant proteins.

Protection against Schistosoma mansoni infection with a recombinant baculovirus-expressed subunit of calpain

Infections by human schistosomes, in particular Schistosoma mansoni, account for significant morbidity and mortality every year in tropical and sub-tropical areas. The eggs of the parasite induce pathological changes in the infected host; in chronic and heavy infections, these changes may lead to death. A well-designed anti-schistosomal vaccine, alone or in concert with existing control measures such as chemotherapy, may prove to be a safe, inexpensive, and effective means of reducing the occurrence of severe disease and death in S. mansoni infection. Previous studies have demonstrated the importance of the syncytial layer containing the apical plasma membrane (APM) of S. mansoni in both the survival of the parasite in the mammalian host and as a potential source of immunogens which may be utilized as vaccine candidates. In this paper, we present evidence for the protective capacity of several schistosomal antigen preparations, including a calcium binding protein of the APM, S. mansoni calpain (GenBank accession no. M74233). We have constructed and characterized expression of a recombinant baculovirus expressing the large subunit of S. mansoni calpain, Sm-p80. This recombinant Sm-p80 is recognized by IgA, IgM, IgG1, and IgG3 isotype antibodies found in S. mansoni-infected human sera and partially-purified recombinant Sm-p80 provided a 29–39% reduction in worm burden in immunized mice challenged with S. mansoni. Our data indicate that Sm-p80 may be a useful vaccine antigen for the reduction of the morbidity associated with S. mansoni infections of mammalian hosts.

Calcium-binding properties of human erythrocyte calpain.

The results presented provide more information on the sequential mechanism that promotes the Ca2+-induced activation of human erythrocyte mu-calpain under physiological conditions. The primary event in this process corresponds to the binding of Ca2+ to eight interacting sites, of which there are four in each of the two calpain subunits. Progressive binding of this metal ion is linearly correlated with the dissociation of the proteinase, which reaches completion when all eight binding sites are occupied. The affinity for Ca2+ in the native heterodimeric calpain is increased 2-fold in the isolated 80 kDa catalytic subunit, but it reaches a Kd consistent with the physiological concentration of Ca2+ only in the active autoproteolytically derived 75 kDa form. Binding of Ca2+ in physiological conditions, and thus the formation of the 75 kDa subunit, can occur only in the presence of positive modulators. These are represented by the natural activator protein, found to be a Ca2+-binding protein, and by highly digestible substrates. The former produces a very large increase in the affinity of calpain for Ca2+, and the latter a smaller but still consistent decrease in the Kd of the proteinase for the metal ion. As a result, both dissociation into the constituent subunits and the autoproteolytic conversion of the native 80 kDa subunit into the active 75 kDa form can occur within the physiological fluctuations in Ca2+ concentration. The delay in the expression of the proteolytic activity with respect to Ca2+ binding to native calpain, no longer detectable in the 75 kDa form, can be attributed to a Ca2+-induced functional conformational change, which is correlated with the accessibility of the active site of the enzyme.

Purification, crystallization and preliminary X-ray diffraction studies of recombinant calcium-binding domain of the small subunit of porcine calpain.

The calcium-binding domain of the small subunit of porcine calpain (domain VI) has been expressed in Escherichia coli, purified, and crystallized in the presence of Ca(2+). Two crystal forms have been obtained by the vapor-diffusion method using PEG 6000 as the precipitant. Crystal form I, belonging to trigonal space group P3(1)21 (or P3(2)21) with cell dimensions a = b = 79.8, c = 57.08 A, alpha = beta = 90.0 and gamma, = 120.0 degrees diffracted to 2.8 A. The second crystal form diffracts to 1.8 A and belongs to monoclinic space group P2(1) with cell dimensions a = 50.1, b = 79.7, c = 57.1 A and beta = 91.2 degrees.

'Oxidation Inhibits Substrate Proteolysis by Calpain I but Not Autolysis

In this study, the effects of oxidation on calpain I autolysis and calpain-mediated proteolysis were examined. Calpain I was incubated with increasing concentrations of free calcium in the presence or absence of oxidant, and autolytic conversion of both the 80- and 30-kDa subunits was measured by immunoblotting utilizing monoclonal antibodies which recognize both autolyzed and non-autolyzed forms of each subunit, respectively. Autolytic conversion of the 80-kDa subunit of calpain I was not detected until free calcium concentration was greater than 40 microM, whereas autolysis of the 30-kDa subunit did not occur until the free calcium concentration was greater than 100 microM. In addition, autolytic conversion of either the 80- or 30-kDa subunit was not inhibited by the presence of oxidant. Calpain I activity was measured using the fluorescent peptide N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-7-amido-4- methylcoumarin or the microtubule-associated protein tau as substrate. Calpain I was found to have proteolytic activity at free calcium concentrations below that required for autolysis. Calpain I activity was strongly inhibited by oxidant at all calcium concentrations studied, suggesting that proteolytic activity of both the non-autolyzed 80-kDa and autolyzed 76-kDa forms was susceptible to oxidation. Interestingly, whereas oxidation did not inhibit autolytic conversion, the presence of high substrate concentrations did result in a significant reduction of autolysis without altering calpain proteolytic activity. Calpain I activity that had been inhibited by the presence of oxidant was recovered immediately by addition of the reducing agent dithiothreitol.

Primary sequences of rat μ-calpain large and small subunits are, respectively, moderately and highly similar to those of human

cDNAs for rat μ-calpain large subunit and the calpain small subunit were cloned and sequenced. The large subunit encodes 713 amino-acid residues, which includes one deletion compared to that of human. The overall similarity is 89% to human μ-type, which is slightly lower than those compared between other types of calpain large subunits of rat and human (93–94%). On the other hand, the small subunit showed high conservation, being 94.0% identical to that of human. With these sequences, primary structures of all rat calpain subunits that have been considered to exist were completely elucidated.

Calpain Subunits Remain Associated during Catalysis

The Ca2+dependent cysteine proteases, calpains, are heterodimers containing a large (ca.80 kDa) catalytic subunit and a 25-30 kDa small subunit. Whether calpains remain dimers while catalyzing hydrolysis of protein substrates has been controversial. Now by doing subunit co-immunoprecipitation, we provide direct evidence to resolve this argument. In the presence of Ca2+concentrations which permit catalytic activity, both subunits of either m- or μ-calpain are co-immunoprecipitated by monoclonal antibodies directed against a single subunit. Furthermore, both subunits can be co-immunoprecipitated during calpain-catalyzed proteolysis of the substrate casein. These results indicate that both major calpain isozymes maintain their heterodimeric form during the catalytic cycle. Thus, small subunit might have a direct role in regulating the physiologic function of either major calpain isozyme.

Autolysis of human erythrocyte calpain produces two active enzyme forms with different cell localization

The 80 kDa human erythrocyte calpain, when exposed to Ca 2+, undergoes autoproteolysis that generates a 75 kDa species, with an increase in Ca 2+ affinity. It is demonstrated here that this proteolytic modification proceeds through an initial step producing a 78 kDa form which is rapidly converted to the 75 kDa one. In the presence of the calpain inhibitor E-64, the 78 kDa form accumulates and only small amounts of the 75 kDa polypeptide are formed. Following loading of erythrocytes with micromolar concentration of Ca 2+, in the presence of the ionophore A23187, the native 80 kDa calpain subunit is extensively translocated and retained at the plasma membrane, this process is accompanied by the appearance of only a small amount of the 75 kDa subunit which is released into the soluble fraction of the cells. Following exposure to μM Ca 2+, membrane-bound 80 kDa calpain is converted to the 78 kDa form, this conversion being linearly correlated with the expression of the proteinase activity. Taken together, these results demonstrate that the initial step in calpain activation involves Ca 2+-induced translocation to the inner surface of plasma membranes. In the membrane-bound form the native inactive 80 kDa subunit is converted through intramolecular autoproteolysis to a locally active 78 kDa form. Further autoproteolytic intermolecular digestion converts the 78 kDa to the 75 kDa form, no longer being retained by the membrane. This process generates two active forms of calpain, with different intracellular localisations.

Crystal structure of calcium-free C-terminal domain of small subunit of rat calpain

Crystal structure of calcium-free C-terminal domain of small subunit of rat calpain

Calpain is the target antigen of a Th1 clone that transfers protective immunity against Schistosoma mansoni.

A CD4+ clone (clone B), characterized as Th1 based on its selective production of IFN-gamma and IL-2, was established from C57Bl/6 mice protectively immunized against Schistosoma mansoni by intradermal vaccination with soluble worm Ags, plus bacillus Calmette Guerin. In agreement with previous results demonstrating an IFN-gamma-dependent cell-mediated protective mechanism in this vaccination model, Ag-elicited peritoneal macrophages from syngeneic recipients of this clone were activated to kill schistosome larvae (schistosomula) in vitro. Moreover, recipients of clone B displayed significant resistance against cercarial challenge. By screening a battery of lambda(gt11) clones from an adult worm cDNA library, one recombinant (25B) was identified that stimulated clone B specifically. Analysis of the 25B cDNA insert revealed a nucleotide sequence identical with that of the large subunit of schistosome calpain, a Ca2+-activated neutral proteinase. By expressing the products of PCR subcloning, we identified a 146-amino acid region of the 25B gene containing immunologic activity equivalent to the whole polypeptide. Overlapping peptides spanning this region were synthesized, and a core epitope was identified with the sequence EWKGAWCDGS. Since clone B responds to supernatants from cultured schistosomula, we postulate that the recognition of calpain released by invading larvae and resulting induction of Th1 cytokines accounts for the protection mediated by the adoptively transferred clone. Our findings thus implicate calpain as a target of protective immunity in schistosomes and provide the first example of a candidate vaccine Ag for this parasite identified on the basis of T cell reactivity.

Maitotoxin Induces Calpain Activation in SH-SY5Y Neuroblastoma Cells and Cerebrocortical Cultures

Maitotoxin (MTX) is a highly potent marine toxin that activates both voltage-sensitive and receptor-operated calcium channels in the plasma membrane. This results in calcium overload that rapidly leads to cell death. We now report that maitotoxin (0.1–1 nM) induces calpain activation in both SH-SY5Y neuroblastoma cells and fetal rat cerebrocortical cultures. MTX-induced calpain activation was confirmed by the presence of autolytic fragmentation of both subunits of calpain. Secondly, the formation of calpain-produced α-spectrin breakdown products (150 and 145 kDa) was observed. We were also able to detect intracellular hydrolysis of a peptide substrate (succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin) by activated calpain in MTX-treated cells. Calpain inhibitors (calpain inhibitor I, MDL28170 and PD150606) inhibited spectrin breakdown and SLLVY-AMC hydrolysis in MTX-treated SY5Y cells. Our results suggest that (i) calpain is activated as a result of the maitotoxin-induced calcium influx; and (ii) coupling with thein situcalpain assays, maitotoxin would be a useful tool in investigating the physiologic and pathophysiologic roles of calpain in neuronal cells.

Investigation of the interaction of m-calpain with phospholipids: calpain-phospholipid interactions

Phosphatidyl inositol, phosphatidyl choline, phosphatidyl glycerol, phosphatidyl serine, phosphatidyl ethanolamine, phosphatidic acid and sphingomyelin were all found to be effective at reducing the Ca 2+ requirement for m-calpain autolysis. In the absence of phospholipid, pig kidney m-calpain required 1.4 mM Ca 2+ for 50% autolysis under the assay conditions used. Phospholipids caused a reduction in this Ca 2+ requirement to a value between 0.45 mM Ca 2+ for phosphatidyl glycerol and 1.1 mM Ca 2+ for phosphatidyl ethanolamine. Previous studies (Crawford, C., Brown, N.R. and Willis, A.C. (1990) Biochem. J. 265, 575–579) have shown that the most probable site for phospholipid interaction in calpain is the N-terminal region between residues 39 to 62 of the small subunit of calpain (G 17TAMRILGG). In this study we examine the possible role of this G 17TAMRILGG region. Three synthetic peptides corresponding to parts of this sequence were used to examine the phospholipid binding sequence. Analysis of the phospholipid vesicle binding properties of these peptides suggested that both the TAMRIL and polyglycine sequences were required for binding to phosphatidyl inositol vesicles.

Ca2+-DEPENDENT AND Ca2+-INDEPENDENT PROTEINASE CONTENTS IN THE SKELETAL MUSCLE IN SEPTIC RATS

This study assessed sepsis-induced changes in the contents of calpain and cathepsin B in rat soleus muscle. Sepsis was induced in rats by intra-abdominally implanting fecal pellets containing Escherichia coil and Bacteroides fragilis. Intact soleus muscles were isolated from non-operated control rats, and from rats sacrificed 1 and 2 days after they were implanted with bacteria-free (sterile implanted) or bacteria-laden (septic implanted) pellets. Western blot analyses of muscle homogenates were performed to identify and quantitate these proteinases using specific antibodies. No significant differences in cathepsin B contents were observed between the septic and nonseptic animals on days 1 and 2, post-implantation. Among the three distinct bands recognized by anti-calpain, two prominent bands of 80 and 76 kDa, representing calpain subunits, did not seem to be altered in septic rats compared to the nonseptic groups. The content of the 45-kDa subunit decreased in both the septic and sterile groups compared with non-operated control. These results along with our previous observations suggest that although Gram-negative sepsis does not appear to have an effect on Ca2(+)-insensitive lysosomal cathepsin B content or activity, it upregulates the activity of the Ca2(+)-dependent calpain but not its content in the skeletal muscle during sepsis.

Ca(2+)-binding domain VI of rat calpain is a homodimer in solution: hydrodynamic, crystallization and preliminary X-ray diffraction studies.

The 21-kDa calcium-binding domain (VI) of the small subunit of rat calpain II has been expressed in Escherichia coli, purified, and crystallized. Two orthorhombic crystal forms have been obtained: space group P2(1)2(1)2(1) with a = 50.3, b = 56.5, c = 141.3 A; and space group C222(1) with a = 69.4, b = 73.9, c = 157.4 A. Diffraction data have been collected to 2.4 A. Sedimentation equilibrium, dynamic light scattering, and gel-permeation chromatography indicate that domain VI exists as a homodimer in solution. In accordance with the protein's behavior in solution, each crystal form contains two molecules per asymmetric unit. Screening for heavy-atom derivatives is in progress. To decrease the sensitivity to mercurials and to aid in the search for useful derivatives, Cys-to-Ser mutants have been prepared, expressed, and crystallized.

Dopamine-stimulated changes in activated calpain I in rat hippocampal slices

Activated calpain I immunoreactivity (76 kDa band) was detected in membranes prepared from rat brain hippocampal slices using a polyclonal antiserum raised against an N-terminus peptide of the cleaved subunit of calpain I. While basal levels of activated calpain I were stable over incubation times, 1 nM dopamine (DA) produced an initial 32% increase (5 min) in the 76 kDa protein followed by a 53% decrease in this band at 20 min of incubation. The DA-induced changes in activated calpain I immunoreactivity were blocked by the calpain inhibitor peptide, N-acetyl-Leu-Leu-norleucinal(100 microM) or by EGTA. Basal levels of the 76 kDa band were not affected by the calpain inhibitor. These changes in activated calpain I, elicited by DA, are in accord with the DA-induced decreases in the levels of the calpain substrate, gamma PKC (Yurko-Mauro and Friedman; J Cell Biochem [Abstr] 180:80, 1994; J Neurochem 65: 1622-1630, 1995) and suggest that DA activates this Ca(++)-dependent protease in its regulation of neuronal signal transduction.