Calcium-activated Neutral Protease Research Articles

Rapid communication: Linkage mapping of the porcine micromolar calcium-activated neutral protease 1 (mu-calpain) gene on SSC2.

Rapid communication: Linkage mapping of the porcine micromolar calcium-activated neutral protease 1 (mu-calpain) gene on SSC2.

Atomic force microscopic evidence for Z-band as a rigid disc fixing the sarcomere structure of skeletal muscle.

Atomic force microscopic images of single skeletal myofibrils showed periodical broad filamentous bands interspaced with narrow rigid bands corresponding to the sarcomere structures of skeletal muscle (Yoshikawa, Y., Yasuike, T., Yagi, A., and Yamada, T. 1999. Biochem. Biophys. Res. Comm., 256: 13-19). In order to identify the narrow rigid bands, comparative studies were made for intact single myofibrils and those treated with calcium-activated neutral protease by use of atomic force microscopy. It was found that (a) the periodical narrow rigid bands present in intact myofibrils were completely absent in myofibrils treated with calcium-activated neutral protease, and that (b) myofibrils treated with calcium-activated neutral protease were very fragile compared with intact myofibrils. As calcium-activated neutral protease selectively removes Z-bands of myofibrils (Reddy, M. K., Etlinger, J. D., Rabinowitz, M., Fischman, D. A., and Zak, R. 1975. J. Biol. Chem., 250: 4278-4284), these results clearly indicate that (a) the narrow rigid bands are the Z-bands, and that (b) the Z-bands are the essential disc supporting the sarcomere structure of skeletal muscle.

Calpain activity and expression increased in activated glial and inflammatory cells in penumbra of spinal cord injury lesion.

Following traumatic injury of the spinal cord, cells adjacent to the lesion are subject to ischemic cell death as a result of vascular disruption and secondary inflammatory responses. Proteases such as calcium-activated neutral proteinase (calpain) have been implicated in axon and myelin destruction following injury since they degrade structural proteins in the axon-myelin unit. To examine the role of calpain in cell death following spinal cord injury (SCI), calpain activity and translational expression were evaluated using Western blotting techniques. Calpain activity (as measured by specific substrate degradation) was significantly increased in and around the lesion site as early as 4 hr following injury with continued elevation at 48 hr compared to sham controls. Likewise, calpain expression was significantly increased in both the lesion site and penumbra at 4 and 48 hr after injury. Using double immunofluorescent labeling for calpain and cell-specific markers, this increase in calpain expression was found to be due in part to activated glial/inflammatory cells such as astrocytes, microglia, and infiltrating macrophages in these areas. Thus, since calpain degrades many myelin and axonal structural proteins, the increased activity and expression of this enzyme may be responsible for destruction of myelinated axons adjacent to the lesion site following SCI.

The role of calpain in neurofilament protein degradation associated with spinal cord injury.

AbstractCalpain is a calcium-activated neutral proteinase present in all mammalian tissues thus far studied (1,2). Ubiquitous calpain exists as μ-calpain and m-calpain isoforms which require μM and mM calcium levels for activation, respectively. In the central nervous system (CNS) μ-calpain is predominantly cytosolic while m-calpain is localized in both cytosolic and membrane fractions (2). Although μ-calpain is largely neuronal and m-calpain is present predominantly in glial cells, both calpain isoforms share similar substrate specificities and are inhibited by an endogenous inhibitor, calpastatin (2). A broad range of proteins in the CNS, including cytoskeletal and myelin proteins such as neurofilament proteins (NFPs), vimentin, micro-tubule-associated protein (MAP), and myelin basic protein (MBP) are known to be calpain substrates. The μ-and m-calpains also cleave protein kinase C (PKC), hormones, receptors, and histones (3).KeywordsSpinal Cord InjuryMyelin Basic ProteinMyelin ProteinLesion Spinal CordSpinal Cord TissueThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Purification and quantification of calcium-activated neutral proteases I and II and novel isoforms from cultured osteoblastic cells by ion-exchange fast protein liquid chromatography.

Purification and quantification of calcium-activated neutral proteases I and II and novel isoforms from cultured osteoblastic cells by ion-exchange fast protein liquid chromatography.

Cloning and molecular characterization of calpain, a calcium-activated neutral proteinase, from different strains of Schistosoma japonicum

cDNA coding for calpain of Schistosoma japonicum were cloned and sequenced, and serological basis of host responses to calpain were analyzed. cDNA of calpain from S. japonicum of two different isolates, Yamanashi strain (Sj-J) and Hunan strain (Sj-C), were 2, 468 bp and 2, 465 bp in length, including the same number (2, 274) of open reading frame. Nucleotide sequence and amino acid sequence between the two calpains are 99.1% and 98.8% identity, respectively. Sj-J and Sj-C calpains were considered to be translated as a preproenzyme, and a 746-amino acid mature enzyme contains eight motifs without a signal peptide at the N-terminal based on the deduced amino acid sequences. mRNA for calpain were detectable in different developmental stages, however, sera obtained from mice immunized with recombinant calpain showed enhanced binding to cercarial antigen. Human sera from S. japonicum-infected individuals recognized the large subunit of schistosomal calpain, and light-infected sera showed stronger reactivities to the recombinant calpain than moderate/high infection cases. When we tested synthetic peptides, there were four common human B cell epitopes in schistosomal calpain, all of which are shared with S. mansoni. Together with these results, calpain of S. japonicum seems to be not only a vaccine candidate, but also a target antigen for immunodiagnosis of human schistosomiasis.

The calcium-activated neutral protease calpain I is present in normal foetal skin and is decreased in neonatal harlequin ichthyosis.

Calcium concentration is a critical factor for epidermal differentiation and is implicated in the expression and post-translational modification of numerous proteins in suprabasal cells of the epidermis. Calpains (calcium-activated neutral proteases) are believed to participate in signal transduction via highly regulated cytoplasmic protease activity. Here we investigate the expression of calpain I in normal human skin development and in neonatal harlequin ichthyosis (HI), a disorder of altered epidermal differentiation, especially the transition from the granular to the fully differentiated cornified layer. Calpain I was detected in developing foetal epidermis at 54 days estimated gestational age in the basal layer and the periderm of the developing foetal epidermis. By 125 days, calpain I was also detected in the granular layer. This pattern was maintained in newborn skin, but expression was significantly weaker in HI biopsies (n = 7). Reduced expression of calpain was specific to HI and was not observed in other skin diseases. Calpain was also normally expressed in the outer root sheath of hair follicles, in sebaceous glands and in sweat ducts and glands. Immunoblots of epidermal and keratinocyte extracts showed that the 78-kDa and 76-kDa active forms were generated via limited proteolysis of the 80-kDa inactive subunit; however, all forms were diminished in HI, consistent with findings in tissue sections. Our results show that calpain is present throughout the epidermis and is expressed from the early stages of development. These findings implicate calcium-mediated signalling events in the alteration of differentiation that occurs in HI.

Mutations of calpain 3 gene in patients with sporadic limb-girdle muscular dystrophy in Japan.

Mutations of the calpain 3 gene, an intracellular calcium-activated neutral protease, is one of the causes of limb-girdle muscular dystrophy (LGMD). We examined 14 Japanese patients with sporadic LGMD for calpain 3 mutations, and found four mutations in five patients. Three (R461C, D707G and R147P) were novel missense mutations, and one was a splice-site mutation (801+1g-->a) resulting in skipping of exons 4 and 5. Of the five patients, three patients with homozygous missense mutations showed later onset and slower progression than the other two patients with an exon skipping or mRNA loss of unknown cause. It would appear that the occurrence of calpain 3 gene mutations in sporadic LGMD in Japan may be quite high since all five patients with mutations in this gene were among the 14 patients without apparent family history, an incidence of 36%. These findings also suggest that calpain 3 deficiency occurs in both sporadic and familial LGMD and that direct analysis of the calpain 3 gene may be useful in the definitive diagnosis not only of the 15q-linked familial but also of sporadic cases of LGMD.

Studies on Calpain Expression during Differentiation of Rat Satellite Cells in Primary Cultures in the Presence of Heparin or a Mimic Compound

We have synthesized dextran derivatives called RGTAs (for regenerating agents) that were designed to mimic some of the properties of heparin or heparan sulfate to interact with and protect heparin binding growth factors. Some of these growth factors have been described to be involved in myogenesis control. In previous studies, we have shown that muscle regeneration in adults could be greatly enhanced in vivo by treatment with RGTA. Since muscle regeneration occurs through the activation of satellite cells, in the present study we have used primary cultures of rat satellite cells and treated them with the heparan sulfate analogue RGTA or heparin in order to stimulate their growth and differentiation. We also studied the effect of these substances on calpain (calcium-activated neutral proteases) expression in these cultures. Indeed, several reports, principally based on fetal myoblast cultures or myogenic cell lines, have suggested that calpains might be involved in myoblast fusion during myogenic differentiation. We therefore studied the expression of microcalpain (μ-calpain), millicalpain (m-calpain), and calpain 3 in the course of differentiation of these satellite cell cultures in the absence or in the presence of heparin or of a mimic compound (the RGTA RG1282). RGTA and heparin were shown to have a dual effect on satellite cell proliferation and differentiation: RGTA stimulated proliferation with a maximum dose effect at 1 μg/ml. Heparin used at concentrations similar to those of RGTA was less efficient at stimulating proliferation. Both substances were shown, however, to induce precocious and enhanced differentiation of satellite cells. We showed by quantitative RT-PCR analysis that μ-calpain, m-calpain, and calpain 3 mRNAs were expressed in satellite cell cultures in proliferating myoblasts (day 3) and differentiating cultures (days 7 and 12). The level of μ-calpain mRNA was increased by a factor of 3 during differentiation of satellite cells, whereas the level of m-calpain mRNAs was slightly increased at day 12 only, and calpain 3 mRNA was slightly reduced in these differentiating cultures. Interestingly enough, RGTA and heparin, which both strongly increased differentiation, reduced the expression of the μ- and m-calpains and slightly increased that of calpain 3 in differentiating cultures. These results showed that there was no correlation between the extent of myoblast differentiation and the level of calpain expression in satellite cells grown in primary cultures and underscored the differences between these adult cells and fetal myoblasts.

A putative mechanism of demyelination in multiple sclerosis by a proteolytic enzyme, calpain.

In autoimmune demyelinating diseases such as multiple sclerosis (MS), the degradation of myelin proteins results in destabilization of the myelin sheath. Thus, proteases have been implicated in myelin protein degradation, and recent studies have demonstrated increased expression and activity of a calcium-activated neutral proteinase (calpain) in experimental allergic encephalomyelitis, the corresponding animal model of MS. In the present study, calpain activity and expression (at translational and transcriptional levels) were evaluated in white matter from human patients with MS and Parkinson's and Alzheimer's diseases and compared with that of white matter from normal controls. Western blot analysis revealed that levels of the active form of calpain and calpain-specific degradation products (fodrin) were increased by 90.1% and 52.7%, respectively, in MS plaques compared with normal white matter. Calpain translational expression was up-regulated by 462.5% in MS plaques compared with controls, although levels of the specific endogenous inhibitor, calpastatin, were not altered significantly. At the transcriptional level, no significant changes in calpain or calpastatin expression were detected by reverse transcription-PCR. Using double immunofluorescent labeling, increased calpain expression was observed in reactive astrocytes, activated T cells, and activated mononuclear phagocytes in and adjacent to demyelinating lesions. Calpain activity and translational expression were not increased significantly in white matter from patients with Parkinson's or Alzheimer's diseases compared with that of normal controls. Because calpain degrades all major myelin proteins, the increased activity and expression of this proteinase may play a critical role in myelinolysis in autoimmune demyelinating diseases such as MS.

Calpain activity and expression are increased in splenic inflammatory cells associated with experimental allergic encephalomyelitis

Since calcium-activated neutral proteinase (calpain) activity and expression are significantly increased in activated glial/inflammatory cells in the central nervous system of animals with autoimmune demyelinating diseases, this enzyme may also play a role in peripheral organ systems in these diseases. In this study, the activity and expression of calpain and the endogenous inhibitor, calpastatin, were evaluated at transcriptional and translational levels in spleens of Lewis rats with acute experimental allergic encephalomyelitis (EAE) prior to the onset of clinical symptoms. Calpain activity and translational expression were increased by 475.5% and 44.3% respectively, on day 4 post-induction in adjuvant controls and animals with EAE. These levels remained elevated compared to normal controls on days 8 and 12. Calpastatin translational expression was similarly increased at these time points although transcriptional expression was not significantly altered at any time following induction of EAE. Likewise, transcriptional expression of μ-calpain was unchanged following induction, while small increases in m-calpain transcriptional expression were observed on days 2 and 8. Most calpain expression was observed in activated splenic macrophages at day 8 post-induction even though activated T cells were also calpain positive. In spinal cords of animals with EAE, calpain expression was significantly increased in rats with severe disease compared to those exhibiting only mild symptoms at day 12 post-induction. Thus, prior to symptomatic EAE, increased calpain activity and expression in peripheral lymphoid organs may play an important role in T cell migration and subsequent disease progression.

Stabilizing neuromuscular contacts reduces motoneuron death caused by paralysis of muscles in neonatal rats

Transient paralysis of the soleus muscle in neonatal rats leads to permanent muscle weakness, loss of muscle fibres and motoneuron death. Application of leupeptin, an inhibitor of a calcium-activated neutral protease, to the neuromuscular junction is known to enhance the maintenance of neuromuscular contacts during development and axonal sprouting. Here, we show that treatment of soleus muscles with leupeptin as they recover from a period of paralysis rescues motoneurons that would otherwise die. The number of motoneurons to the soleus muscle was established by retrograde labelling with horseradish peroxidase eight to 10 weeks after recovery from paralysis. There were only 38.4 (±2.8 S.E.M., n=5) motoneurons innervating the soleus muscle that had been paralysed with α-bungarotoxin, compared to 58.2 (±3.1 S.E.M., n=5) to the control untreated soleus. Thus, the number of motoneurons to the soleus muscle on the α-bungarotoxin-treated side was 66.9% (±6.2% S.E.M., n=5) of the control side. In those animals where paralysis of the soleus muscle was followed three days later by treatment with leupeptin, the number of labelled motoneurons on the treated side of the spinal cord was 61.5 (±4.6 S.E.M., n=4) and that on the contralateral untreated control side was 59 (±3.8 S.E.M., n=4). This improvement in motoneuron survival in the leupeptin-treated animals is also confirmed by counts of the number of motor units in the soleus muscle obtained by recording muscle tension. In animals that had their soleus muscles paralysed at birth, only 21 (±0.7 S.E.M., n=5) motor units were present, compared to 30 motor units in control muscles. When the paralysed soleus muscle was subsequently treated with leupeptin, the number of remaining motor units in the muscle was 29.8 (±1.0 S.E.M., n=5). In addition, the force output of the soleus muscles that had undergone a period of neonatal paralysis was calculated for both the NaCl- and leupeptin-treated animals. The results showed that paralysis at birth results in a reduction in weight and force output of the soleus muscle, which is not improved following treatment with leupeptin. This study shows that application of leupeptin to the soleus muscle after α-bungarotoxin-induced paralysis rescues motoneurons to the soleus that would otherwise die. This effect is most likely due to stabilization of their neuromuscular junctions.

CAPN 8: Isolation of a New Mouse Calpain-Isoenzyme

Recent molecular biological approaches indicate that calpain, also named CANP for calcium-activated neutral protease and originally characterized as an intracellular cytoplasmatic nonlysosomal cysteine protease that requires calcium ions for activity, constitutes a large superfamily consisting of ubiquitous and tissue specific homologues, which are widely distributed in cells of various organisms from human to fungus. Due to the increasing number of substrates along with the involvement of calpain isoenzymes in mammalian diseases, especially in malignancies, members of the calpain superfamily seem to be important biomodulators in physiological as well as pathological cell function. Here we report the characterisation of a new calpain, named CAPN 8 with a different C-terminal domain, implicating a putative new regulatory mechanism. Northern blot analysis revealed an ubiquitous expression with different RNA levels in all tissues examined. Highest levels were found in brain, kidney, and digestive tract, suggesting a specific regulatory function of CAPN 8 in these tissues.

Structural characterization of the goat uterine estrogen receptor activation factor using an endogenous calcium activated neutral protease

An expedient method for the purification of a calcium activated neutral protease (CANP) of the goat uterus has been designed. This enzyme, purified to homogeneity, has been used as a tool in the structural characterization of the estrogen receptor activation factor II (E-RAF II) that dimerizes with an alternative form of estrogen receptor (ER), the non-activated estrogen receptor (naER). The enzyme cleaves the E-RAF into two fragments, α and β, of molecular mass 32 and 30 kDa, respectively. The α retains the DNA binding activity, as well as the capacity to dimerize with the naER. On the other hand, the cholesterol binding activity and the ATPase function are shared by both α and β fragments. The E-RAF domain that binds to the nuclear periphery appears to be localized on the β fragment. The β fragment, however, is incapable of entering the nucleus on its own.

Recombinant vaccinia viruses and gene gun vectors expressing the large subunit of Schistosoma mansoni calpain used in a murine immunization-challenge model

Schistosomiasis is a parasitic disease affecting over 200 million people every year in tropical regions of the world. Drug treatment and other existing control measures are costly and have failed to eliminate the incidence of infection, morbidity and mortality due to Schistosoma mansoni infection. Vaccination of susceptible individuals using recombinant vaccines encoding key S. mansoni antigens may be the most effective and least expensive means of controlling schistosomiasis. A candidate vaccine antigen is p80, the large subunit of the S. mansoni protease, calpain. In our vaccine studies, we have employed both the wild-type p80 and a mutant p80 (mut p80) in which an active site amino acid was genetically altered to create a less proteolytically-active enzyme. Two vaccine delivery approaches were implemented using p80 or mut p80 as vaccine antigen: recombinant vaccinia virus (RVV) inoculation and DNA immunization via the Accell® gene gun (GG) delivery system. RVV's expressing p80 and mut p80 were generated and tested for recombinant protein expression in vitro. These RVV's were tested for protective capacity in mouse challenge studies. Neither subcutaneous nor intranasal vaccinations with RVV-p80 or RVV-mut p80 were capable of significantly reducing the mean worm burdens of vaccinated mice. A GG-RVV combination immunization regime using WRG-vectors encoding p80 and mut p80 for GG priming and the RVV's for boosting prior to S. mansoni challenge infection was performed and no significant protection was obtained over two repeated studies. However, duplicate challenge studies involving GG immunization of mice with WRG-vectors encoding p80 or mut p80 revealed that 3 inoculations of mice with WRG-full5′ mut p80 (containing the full 5′ untranslated region of mut p80) provided 60% protection which was statistically significant ( p<0.05). These preliminary in vivo studies demonstrate the potential for further study of the protection afforded by gene gun-delivered WRG-full5′ mut p80 into subsequently-challenged mice. Such studies may pave the way to effective vaccination of humans using WRG DNA vectors expressing a schistosomal calcium-activated neutral protease.

Pathophysiological role of calpain in experimental demyelination.

Calcium-activated neutral proteinase (calpain) has been extensively studied over the past three decades such that many enzymatic and structural properties of this enzyme are well understood. However, the pathophysiological roles of calpain remain poorly defined. In addition to recent studies delineating a role for calpain in various pathological conditions, this proteinase has been implicated in the degradation of myelin proteins in autoimmune demyelinating diseases such as multiple sclerosis and experimental allergic encephalomyelitis (EAE). In EAE, calpain translational expression is significantly increased in activated glial/inflammatory cells that participate in myelinolysis while calpain substrates (axonal and myelin proteins) are lost. Thus, since all major myelin proteins are calpain substrates, early studies suggest calpain may play an important role in demyelination of the central nervous system.

Expression of calpain I messenger RNA in human renal cell carcinoma: Correlation with lymph node metastasis and histological type

Calpain, also named CANP (for calcium-activated neutral protease), is an intracellular cytoplasmatic non-lysosomal cysteine endopeptidase that requires calcium ions for activity. Many substrates of the calpain isoenzymes, such as the transcription factors c-Fos and c-Jun, the tumor supressor protein p53, protein kinase C, pp60c-src and the adhesion molecule integrin, have been implicated in the pathogenesis of different human tumors, suggesting an important role of the calpains in malignant diseases. We now report differential expression of the calpain I gene (CL 1) in a variety of tumors, extending our study to a larger series of renal cell carcinomas. Using Northern-blot analysis, we studied calpain I expression in 30 renal cell carcinomas as compared with matched healthy tissues. Tumor samples were classified according to their histological type: 21 clear cell carcinomas, 4 chromophobe carcinomas, 3 papillary carcinomas and 2 oncocytomas. In renal tumor samples, calpain I gene mRNA was expressed at highly variable levels, significantly depending on the different histological types. Moreover, there was a correlation of higher calpain I expression with increased malignancy: within the clear cell carcinoma subset, tumor samples with advanced nodal status (N1 and N2) showed a significantly higher calpain I expression than tumors without metastasis to regional lymph nodes. Our data suggest an important role of calpain isoenzymes in carcinogenesis and tumor progression. Int. J. Cancer (Pred. Oncol.) 84:6–9, 1999. © 1999 Wiley-Liss, Inc.

Expression of calpain I messenger RNA in human renal cell carcinoma: correlation with lymph node metastasis and histological type.

Calpain, also named CANP (for calcium-activated neutral protease), is an intracellular cytoplasmatic non-lysosomal cysteine endopeptidase that requires calcium ions for activity. Many substrates of the calpain isoenzymes, such as the transcription factors c-Fos and c-Jun, the tumor supressor protein p53, protein kinase C, pp60c-src and the adhesion molecule integrin, have been implicated in the pathogenesis of different human tumors, suggesting an important role of the calpains in malignant diseases. We now report differential expression of the calpain I gene (CL I) in a variety of tumors, extending our study to a larger series of renal cell carcinomas. Using Northern-blot analysis, we studied calpain I expression in 30 renal cell carcinomas as compared with matched healthy tissues. Tumor samples were classified according to their histological type: 21 clear cell carcinomas, 4 chromophobe carcinomas, 3 papillary carcinomas and 2 oncocytomas. In renal tumor samples, calpain I gene mRNA was expressed at highly variable levels, significantly depending on the different histological types. Moreover, there was a correlation of higher calpain I expression with increased malignancy: within the clear cell carcinoma subset, tumor samples with advanced nodal status (N1 and N2) showed a significantly higher calpain I expression than tumors without metastasis to regional lymph nodes. Our data suggest an important role of calpain isoenzymes in carcinogenesis and tumor progression.

Calpain-induced proteolysis of β-spectrins

The calcium-activated neutral protease calpain is activated in several pathological conditions. Calpain usually hydrolyses one or only a few peptide bonds in its substrate. One prominent substrate for calpain is spectrin and it has been shown that α-spectrin is the preferred substrate. We now show that the β-chain of spectrin is also a substrate for calpain proteolysis, and that the cleavage site in each β-subunit is located at the very C-terminal part of the molecule. Surprisingly, βIΣ1-spectrin is cleaved at a different site than βIΣ2- and βIIΣ1-spectrins despite their high degree of sequence identity.

Calpain activity and translational expression increased in spinal cord injury

Calpain, a calcium-activated neutral proteinase, has been implicated in myelin and cytoskeletal protein degradation following spinal cord injury. In the present study, we examined the activity and transcriptional expression of calpain in spinal cord injury lesions via Western blotting analysis and RT-PCR, respectively. No increases in transcriptional expression of calpain or calpastatin, the endogenous inhibitor, were observed in the lesion at 1, 4, 24, and 72 h following injury. However, calpain activity (as measured by calpain-specific degradation of the endogenous substrate fodrin) was marginally increased at 4 h and significantly increased by 129.8% at 48 h compared to sham controls after injury. Calpain translational expression was localized in injured spinal cords using double immunofluorescent labeling which revealed increased calpain expression in astrocytes compared to sham controls. These results suggest that calpain produced by astrocytes located in or near spinal cord injury lesions may participate in myelin/axon degeneration following injury.