Quantitation of tissue calpain activity after isolation by hydrophobic chromatography

A rapid and reliable method for quantitating tissue calpains (Ca2+-activated, neutral, thiol proteases) was developed using hydrophobic chromatography with phenyl-Sepharose. Calpains I and II isolated by this method are free of endogenous inhibitor(s) (calpastatin), activator(s), and nonspecific proteases. These calpains expose hydrophobic regions in the presence of Ca2+ and bind tightly to phenyl-Sepharose. Inactivation of bound calpain is prevented by the addition of leupeptin (20 microM). Calpains I and II bound initially by phenyl-Sepharose in a Ca2+-dependent manner are then eluted successively on the basis of their Ca2+-independent binding to phenyl-Sepharose. Because calpastatin may prevent binding of calpain to phenyl-Sepharose by forming a protease-inhibitor complex in the presence of Ca2+, preadsorbing the protease to a suspension of phenyl-Sepharose beads initially in the absence of Ca2+ separates most of the calpain present in tissue extracts from calpastatin. The isolated calpains obtained are assayed by casein digestion. This quantitation procedure is suitable for measuring calpain activity in various tissues and cells including erythrocytes.     

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.     

Hydrophobic association of calpains with subcellular organelles. Compartmentalization of calpains and the endogenous inhibitor calpastatin in tissues

Calpains I and II isolated from diverse tissues possess both Ca2+-independent, and Ca2+-dependent accessible hydrophobic regions. Possible subcellular organelle association of calpains involving these hydrophobic regions was studied. By homogenizing rat tissues directly in Ca2+ (50 microM), about 30-60% of the cytosolic calpain I and II activity reversibly associated with isolated subcellular fractions (microsomal greater than plasma membrane greater than nuclear). After binding to the particulate fraction, calpain II converted to a calpain I-like form exhibiting stronger Ca2+-independent binding to phenyl-Sepharose and a lower Ca2+ requirement for optimal activity. However, it retained its DEAE-cellulose chromatographic pattern, and precipitated with monospecific anti-calpain II antibodies. Although purified calpastatin (endogenous inhibitor) is known to form a Ca2+-dependent complex with calpains, it was not able to reverse the binding of calpains to the particulate fraction upon short incubation. It was, however, effective in blocking calpain binding when the isolated cytosolic fraction or a mixture of purified calpain and calpastatin was preincubated in the presence of Ca2+, and then added to the particulate fraction. Extraction of tissues under controlled conditions revealed that in fact calpains are already loosely associated with subcellular organelles even in the absence of Ca2+. This is the reason why in the crude homogenates with the addition of Ca2+, calpains strongly bind to the particulate fraction without interference by cytosolic calpastatin. Although calpastatin by complexing initially to calpain can prevent the association of this protease with subcellular organelles, it cannot dissociate calpains already bound to these subcellular fractions. By prior Ca2+-independent association with the hydrophobic proteins present in the subcellular fractions, calpains overcome the 3- to 30-fold inhibitory excess of calpastatin in tissues.     

A calpain-like activity insensitive to calpastatin in Drosophila melanogaster

Calpains are neutral Ca2+-dependent cysteine proteases. In this study, we utilized casein zymography to detect such a proteolytic activity in Drosophila melanogaster extracts throughout the life of this organism. One calpain-like activity that was sensitive to the general cysteine protease inhibitors, E64 and calpain inhibitor I, but insensitive to the human calpain-specific inhibitor, calpastatin, is demonstrated. The relevance of this finding is discussed with respect to the absence of a corresponding Drosophila gene, homologous to the vertebrate calpastatin genes, as concluded from our unsuccessful attempts to clone such a gene and our Blast searches using the FlyBase. The mechanisms of Drosophila calpain regulation require further investigation. However, we suggest that single chain, non-heterodimeric calpains may be insensitive to calpastatin and that Drosophila cystatin-like molecules may play a role in negatively regulating Drosophila calpain.     

Pro-inflammatory cytokine production by synoviocytes following exposure to protein I/II, a modulin from oral streptococci

We have tested the ability of protein I/II, an adhesin from oral streptococci, to stimulate the production of pro-inflammatory cytokines by synovial cells isolated from both rheumatoid arthritis and control patients. Protein I/II triggers synovial fluid cells to produce interleukin (IL)-6 and IL-8 while secretion of tumor necrosis factor-alpha (TNF-alpha) was less enhanced. Using fibroblast-like synoviocytes, we found that protein I/II also exerts an immunomodulatory effect (IL-6 and IL-8 release) on these cells. These findings indicate that, if it gains access to the joint cavity, protein I/II could participate in the initiation and/or perpetuation of rheumatic diseases, by stimulating pro-inflammatory cytokine release from various synovial cells.     

Changes in contents of calpain and calpastatin in rat liver during growth

Variation of calpain I, calpain II, and calpastatin in rat liver during growth from 0 to 14 weeks was studied by chromatographic fractionation of the liver cytosol and enzyme assays on the eluted fractions. When compared in terms of units per g wet liver, high-Ca2+-requiring calpain II always exceeded low-Ca2+-requiring calpain I in male and female rats. The level of calpain II in neonatal (0 week) rat liver was 1.9-2.9 times higher than that for the adults (7 to 14 weeks). The contents of calpastatin, calpain-specific inhibitor protein, were were always higher than those of calpain II in adult rat liver, but the difference was much less, or sometimes even reversed, in neonatal and young (1 and 2 weeks) animals. In general, the variation was more pronounced in female than in male rats.     

Calpains (intracellular calcium-activated cysteine proteinases): structure-activity relationships and involvement in normal and abnormal cellular metabolism

1. Calpains (calcium-activated cysteine proteinases) have evolved by gene fusion events involving calmodulin-like genes, cysteine proteinase genes and other sequences of unknown origin. 2. The enzymes are composed of two non-identical subunits, each of which contains functional calcium-binding sequences. 3. Calpains are inhibited by the endogenous protein inhibitor, calpastatin and some calmodulin antagonists are also inhibitors of calpain. A number of synthetic proteinase inhibitors also inhibit calpains. 4. Calpains can be activated by phospholipids, an endogenous protein activator and some amino acid derivatives. 5. Various protein substrates for calpains have been recognized in vitro, but the identity of in situ substrates remains unclear. 6. Proposals have been made for calpain function, including involvement in signal transduction, platelet activation, cell fusion, mitosis and cytoskeleton and contractile protein turnover. 7. Calpain and calpastatin expression is altered in a number of abnormal states including muscular dystrophy, muscle denervation and tenotomy, hypertension and platelet abnormalities.     

In vivo effect of a beta-adrenergic agonist on activity of calcium-dependent proteinases, their specific inhibitor, and cathepsins B and H in skeletal muscle

DEAE-Sephacel and phenyl-Sepharose chromatography were compared as methods for separating and quantitatively isolating calpain I, calpain II, and calpastatin from lamb muscle extracts. DEAE-Sephacel chromatography gave greater than 90% recovery of all three proteins, while phenyl-Sepharose gave only 70, 66, and 48% of the DEAE recovery of calpain I, calpain II, and calpastatin, respectively. Additionally, DEAE-Sephacel chromatography was shown to effectively separate calpastatin and calpain I. Consequently DEAE-Sephacel appears to be superior to phenyl-Sepharose for quantitative isolation of the components of the calcium-dependent proteinase system from muscle extracts. Dietary administration of beta-agonist (L-644, 969; Merck Sharpe & Dohme Research Laboratories) decreases extractable calpain I activity in lamb longissimus dorsi (LD) muscle by 10-14% (P less than 0.05), increases calpain II activity by 34-42% (P less than 0.001), and increases calpastatin activity by 59-75% (P less than 0.001). Additionally, net cathepsin B activity is reduced by 30% (P less than 0.05) in the LD of beta-agonist-treated lambs. Reduced activity of the calcium-dependent or catheptic proteinase systems may contribute to the increased protein accretion in muscles of beta-agonist-treated lambs.     

Enzymatic alteration of C1q, the collagen-like subcomponent of the first component of complement, leads to cross-reactivity with type II collagen

Native serum C1q, the collagenous-like subcomponent of the first component of complement, is not recognized by polyclonal anti-collagen type II antibodies. However, when purified C1q was subjected to limited proteolysis by collagenase it showed antigenic cross-reactivity with collagen type II. The same cross-reactivity was observed with hemolytically active C1q in synovial fluids of patients with rheumatoid arthritis (RA), whereas C1q from synovial fluids of patients with osteoarthritis (OA), villo-nodular synovitis and ankylosing spondylitis was not recognized by this antibody. However, incubation of synovial fluid C1q of OA patients with synovial fluid leucocytes from RA patients led to an alteration of OA-C1q which was now recognized by the anti-collagen type II antibody.     

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+].     

Inflammatory cytokines induced down-regulation of m-calpain mRNA expression in fibroblastic synoviocytes from patients with osteoarthritis and rheumatoid arthritis

Our previous reports revealed that calpain has proteoglycanase activity and exists in synovial fluid in osteoarthritis and rheumatoid arthritis. We examined the effects of cytokines on expression of the calpain-calpastatin system in fibroblastic synoviocytes (FLS). Primary cultures of human FLS from osteoarthritis (OA) and rheumatoid arthritis (RA) patients were stimulated with inflammatory cytokines and the amounts of m-calpain and calpastatin mRNAs expressed were determined by Northern blotting. Northern blots were subjected to computerized densitometer and band intensities were determined. Interleukin-1 (IL-1) down-regulated m-calpain and tissue-type calpastatin mRNA expression in OA and RA FLS. In RA FLS, although IL-6 did not alter m-calpain mRNA expression, IL-1 + tumor necrosis factor (TNF) and IL-1 + transforming growth factor (TGF) down-regulated m-calpain mRNA expression. These results provide new information about the effects of inflammatory cytokines on calpain and calpastatin system in OA and RA pathology.     

Association of the calpain/calpastatin network with subcellular organelles

The calcium-activated cysteine protease calpain is intimately involved in modulating cell adhesion and migration. The two ubiquitous isoforms of this protease, calpain I and II, are considered to be cytosolic proteins that can translocate to both focal complexes/adhesions or the plasma membrane. Using confocal microscopy and isopycnic density centrifugation, the results demonstrate that calpain I and II, the 30kDa regulatory subunit, and calpastatin associate with the endoplasmic reticulum and Golgi apparatus. Confocal microscopy reveals that calpain II colocalizes with the subcellular proteins calnexin and Rab6 in cells bound to laminin. To further verify this association, cell lysates prepared from laminin stimulated and unstimulated cells were subjected to isopycnic density centrifugation. The results reveal an increased association of calpain I, II, calpastatin, and the 30kDa regulatory subunit with the endoplasmic reticulum and Golgi apparatus as evidenced by their position in the gradient relative to calnexin, Rab6, caveolin, and beta1 integrin after laminin stimulation. This correlates with the accumulation of inducible calpain activity at the endoplasmic reticulum-Golgi apparatus interface. Further experiments established that calpain II colocalizes with phosphatidylinositol 4,5-bisphosphate. Finally, calpain II associates with membrane lipid rafts. These results provide new insights into how the calpain/calpastatin network is spatially and temporally regulated in cells binding to the extracellular matrix.     

Cardiac high molecular weight calmodulin-binding protein is homologous to calpastatin I and calpastatin II

Calpastatin is an endogenous inhibitor of calpain, which has been implicated in various physiological and pathological processes. In the present study we determined the molecular and inhibitory properties of HMWCaMBP, calpastatin I, and calpastatin II. Western blot analysis with antibodies raised against either full length HMWCaMBP or internal peptides that are common to all isoforms showed that all three homologs have common antigenic epitopes. However, additional Western blot analysis with N-terminal specific antibodies showed that all three proteins are different at the N-terminal end. HMWCaMBP is clearly different from two other homologues, calpastatin I and II, at the N-terminal end. In addition, HMWCaMBP also showed the same affinities for m-calpain as calpastatin I and calpastatin II. Our findings suggest that HMWCaMBP is the homolog of calpastatin and may be a CaM-binding form of calpastatin.     

Differential compartmentalization of the calpain/calpastatin network with the endoplasmic reticulum and Golgi apparatus

Calpain, a calcium-activated cysteine protease, is involved in modulating a variety of cell activities such as shape change, mobility, and apoptosis. The two ubiquitous isoforms of this protease, calpain I and II, are considered to be cytosolic proteins that can translocate to various sites in the cell. The activity of calpain is modulated by two regulatory proteins, calpastatin, the specific endogenous inhibitor of calpain, and the 28-kDa regulatory subunit. Using velocity gradient centrifugation, the results of this study confirm and greatly expand upon our previous finding that the calpain/calpastatin network is associated with the endoplasmic reticulum and Golgi apparatus in cells. Moreover, confocal microscopy demonstrates that calpain II colocalizes with specific proteins found in these organelles. Additional experiments reveal that hydrophobic rather than electrostatic interactions are responsible for the association of the calpain/calpastatin network with these organelles. Treatment of the organelles with Na2CO3 or deoxycholate reveal that calpain I, 78-kDa calpain II, and the regulatory subunit are "embedded" within the organelle membranes similar to integral membrane proteins. Proteinase K treatment of the organelles shows that calpain I and II, calpastatin, and the regulatory subunit localize to the cytosolic surface of the organelle membranes, and a subset of calpain II and the regulatory subunit are also found within the lumen of these organelles. These results provide a new and novel explanation for how the calpain/calpastatin network is organized in the cell.     

Activities of dipeptidyl peptidase II, dipeptidyl peptidase IV, prolyl endopeptidase, and collagenase-like peptidase in synovial membrane from patients with rheumatoid arthritis and osteoarthritis.

We examined the activities of peptidases in the synovial membrane from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Dipeptidyl peptidase II (DPP II), prolyl endopeptidase (PEP), and collagenase-like peptidase (CLP) activities were higher in knee joint synovial membrane from patients with RA than in that from patients with OA. DPP II and PEP activities in knee joint synovial membrane of patients with RA increased in parallel with the increase in joint fluid volume, whereas DPP IV activity decreased in parallel with the increase in joint fluid volume. These results suggest that these peptidases in the synovial membrane may play some role in immunological disturbances in the joints of patients with RA. Measurement of these peptidases in synovial membrane may be useful in the diagnosis of the severity of local joint inflammation.     

Calpain and calpastatin levels in dystrophic hamster skeletal muscles

1. Two fast-twitch skeletal muscles from normal and dystrophic hamsters were analysed for their calpain and calpastatin contents. 2. Assays of wide-specificity calpain II showed that the activity levels in the two muscles were increased 1.5 and 1.6 times in dystrophic animals. 3. Analysis of calpastatin levels showed that the respective dystrophic muscles had activity levels of 2.2 and 2.8 times those of control muscles. 4. These results contrast with previous studies on denervated hamster muscles which showed that denervation causes an increase in calpain levels but a decrease in calpastatin levels.     

Evidence for clonal expansion of T cell receptor V gamma II+ T cells in the synovial fluid of patients with arthritis.

We have demonstrated among synovial fluid T cells a unique profile of V gamma II sequences likely arising from clonally expanded T cells. We have determined the junctional diversity associated with each expressed V gamma family by resolving amplified fragments of cDNA into component parts on large denaturing gels. Among synovial fluid T cells we frequently find dominant fragments of a unique size clearly smaller than the dominant band observed with peripheral blood T lymphocytes. In some cases the dominant bands are 12 or 15 nucleotides smaller than the corresponding most abundant band from peripheral blood T lymphocytes. Patterns of lower m.w. species not typical of a polyclonal population argues that clones of T cells expressing the V gamma II family are expanding in the joint and that a high proportion of these cells do not express the V gamma IIJP sequence typical of peripheral blood but rather express V gamma II in combination with a shorter J fragment, JP1, JP2, J1, or J2. In addition by examining joint effusions from the left and right knees from the same individual we have shown that the profiles of V gamma II sequences derived from the fluids are identical to each other but clearly distinct from that of peripheral blood. We have, in addition, quantitated with a series of synthetic internal standards the relative usage of each V gamma family expressed by T cells in the synovial fluid and peripheral blood of seven patients with arthritis including six patients who were either children or adolescents and one adult patient. All patients showed a reduction in the relative expression of V gamma II in synovial T cells relative to peripheral blood T lymphocytes and a corresponding increase in the expression of V gamma I or V gamma III or both. We did not detect expression of V gamma IV in either lymphocyte population.     

Carboxypeptidase N (kininase I) activity in blood and synovial fluid from patients with arthritis

Carboxypeptidase N (CPN, kininase I) and kininase II (angiotensin converting enzyme) activities were measured simultaneously in blood plasma and synovial fluid in patients suffering from rheumatoid arthritis (RA), psoriatic arthritis (PA) and osteoarthritis (OA) and in the plasma of normal volunteers. CPN levels (defined as the rate of hydrolysis of furylacryloyl-Ala-Lys) in blood were modestly increased and correlated with erythrocyte sedimentation rate in RA and PA. Based on the hydrolysis of synthetic substrates, CPN activity was much higher than kininase II activity in synovial fluid (SF). SF kininase activities were always inferior to the blood levels in all patients and were correlated with the logarithm of SF leukocyte counts, an indicator of the intensity of inflammation. In addition, CPN and albumin levels in SF were highly correlated when expressed as a percent of the plasma concentrations. Biochemical properties of CPN in crude SF confirmed its similarity to blood CPN. Polymorphonuclear leukocytes derived from inflammatory SF did not release CPN. It is concluded that kininases diffuse from the blood into SF through increased vascular permeability and that CPN could be a major metabolic pathway for kinins in this form of exudate. CPN leads to the formation of des-Arg kinins, selective agonists of the B1 receptors for kinins.     

The Calpain–Calpastatin System and Cellular Proliferation and Differentiation in Rodent Osteoblastic Cells

The calpain–calpastatin system, which consists of calpains I and II (two ubiquitously distributedcium-activated pa-like cysteine proteases), as well as calpastatin (the endogenous calpain inhibitor), plays an important role in cell proliferation and differentiation in many tissues. However, its contribution to the regulation of osteoprogenitor or pluripotent stem cell proliferation and differentiation into osteoblasts remains poorly defined. In these studies, rat pluripotent mesodermal cells (ROB-C26) and mouse MC3T3-E1 preosteoblasts were induced to differentiate into osteoblasts by long-term culture or in response to bone morphogenetic protein (BMP). The occurrence and distribution of calpain–calpastatin system proteins were determined by immunofluorescent microscopy, measurement of calcium-dependent proteolytic activity, and Western blotting. Treatment of intact MC3T3-E1 cells with an irreversible, membrane-permeable cysteine protease inhibitor attenuated proliferation and alkaline phosphatase upregulation under differentiation-enhancing conditions. Calpain II activity increased during differentiation of MC3T3-E1 cells in postconfluent culture. When ROB-C26 cells were maintained in long-term culture, neutral protease, calpain I, and calpain II activities increased 2- to 3-fold in the absence of BMP. In the presence of partially purified native BMP, neutral protease and calpain I activities also increased similarly, but calpain II activity increased by 10-fold in 3 days. The maximal increase in alkaline phosphatase occurred 4 to 11 days after the calpain II activity had peaked. Induction of differentiation in long-term MC3T3-E1 cultures was associated with higher calpain II and 70- and 110-kDa calpastatin protein levels and lower 17-kDa calpastatin degradation product levels. In conclusion, cysteine protease activity is essential for preosteoblastic proliferation and differentiation. The calpain–calpastatin system is regulated during osteoprogenitor proliferation and differentiation, as it is in other cells, and bone morphogenetic protein is a specific regulator of calpain II.     

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

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