Calpain inhibitor in rabbit skeletal muscle: an immunochemical and histochemical study

Summary Calpastatin, the endogenous inhibitor of calcium-activated neutral proteases (calpains; EC 3.4.22.17), was studied in the rabbit vastus lateralis muscle by means of immunochemical and immunohistochemical techniques. Immunoaffinity chromatography using an antibody raised against the 34-kDa monomer of the 68-kDa dimeric inhibitor allowed us to isolate three main proteins (130-, 100- and 80-kDa). These proteins strongly inhibited calpain activity in muscle homogenate (I₅₀ at about 50 g/ml). Immunohistochemical experiments showed that calpastatin-related immunoreactivity was present in all fibre types (oxidative, glycolytic, oxidative-glycolytic) at both surface and cytoplasmic level. However, a few (20%) of the slow-twitch, oxidative fibres (5% of the total fibres), did not contain the cytoplasmic inhibitor. Specific immunoreactivity for calpastatin was also associated with the interstitial connective tissue. These results suggest that (i) calpastatin in skeletal muscle, as in other tissues, is present as a mixture of proteins of various molecular weights and (ii) the inhibitor may act not only in the cytoplasm but also at the surface or extracellular level.     

Degradation of skeletal muscle plasma membrane proteins by calpain

Summary Observations described here provide the first demonstration that calpain (Ca²⁺-dependent cysteine protease) can degrade proteins of skeletal muscle plasma membranes. Frog muscle plasma membrane vesicles were incubated with calpain preparations and alterations of protein composition were revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Calpain II (activated by millimolar concentrations of Ca²⁺) was isolated from frog skeletal muscle, but the activity of calpain I (activated by micromolar concentrations of Ca²⁺) was lost during attempts at fractionation. Calpain I obtained from skeletal muscle and erythrocytes of rats was tested instead, and exerted effects similar to those of frog muscle calpain on the membrane proteins. All of the calpain preparations caused striking losses of a major membrane protein of molecular mass of approximately 97 kDa, designated band c, and diminution of a thinner band of approximately 200 kDa. There were concomitant increases in 83-and 77-kDa polypeptides. These effects were absolutely dependent on the presence of free Ca²⁺, and were completely blocked by calpastatin, a specific inhibitor of calpain action. Frog muscle calpain differed only in being relatively more active at 0°C than were the calpains from rat tissues. Experimental observations suggest that calpain acts at the cytoplasmic surface of the plasma membrane.     

Failure to find Ca2(+)-dependent proteinase (calpain) activity in a plant species, Elodea densa

Five and nine-tenth kg of Elodea densa (Anacharis), a common aquarium plant, was extracted, and the extract was subjected to column chromatographic procedures that successfully purify the two Ca2(+)-dependent proteinases (calpains) and their protein inhibitor (calpastatin) from a variety of animal tissues. Although these procedures purified a protein having 55- and 16-kDa polypeptides, neither this protein nor any of the other chromatographic fractions contained detectable proteinase or calpastatin activity. Moreover, the purified 55- and 16-kDa polypeptides did not react on immunoblots with polyclonal antibodies that were monospecific for the calpains or calpastatin. We conclude that Elodea densa contains no calpain nor calpastatin at the level of 4 micrograms per g plant protein (1 part per 250,000), which was the sensitivity of our assay.     

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+.     

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.     

Studies on the distribution of calcitonin gene-related peptide-like and substance P-like immunoreactivities in rat hind limb muscles

Summary The tibialis anterior, extensor digitorum longus and soleus muscles in the rat were examined with respect to the presence of calcitonin gene-related peptide-like as well as substance P-like immunoreactivity. In some of the motor endplates in these muscles, identified by staining for acetylcholinesterase activity, calcitonin gene-related peptide-like immunoreactivity was detected, but in others it was not. Calcitonin gene-related peptide-like immunoreactivity was found to coexist with substance-P-like immunoreactivity in nerve fibres located outside and inside the capsule of the muscle spindles, as well as in nerve fibres located in nerve fascicles. These fibres presumably represent sensory nerve fibres. Calcitonin gene-related peptide-like immunoreactivity, but not substance P-like immunoreactivity, was also detected, in cap-like structures located on the surface of the intrafusal muscle fibres in the polar regions of the spindles, structures which are likely to correspond to motor plate endings. The observations suggest that calcitonin gene-related peptide is heterogeneously present in the endplates of rat hind limb muscles, and gives for the first time immunohistochemical evidence for the presence of calcitonin gene-related peptide and substance P in the innervation of muscle spindles.     

Characterization and identification of epididymal factors that protect ejaculated bovine sperm during in vitro storage.

The role of secretory epididymal factors on sperm survival and storage in bovine cauda epididymides is poorly understood. Thus, the effects of bovine epididymal epithelium fluid (BEEF) on frozen-thawed bovine sperm motility have been evaluated in vitro. Sperm motion parameters were assessed by computer-assisted sperm analysis. Compared with serum bovine proteins, BEEF efficiently sustained bovine sperm motility after a 6-h incubation period. The positive effect of BEEF on sperm motility was even more apparent using a fractionated BEEF extract (>10 kDa, 2 mg/ml). This beneficial effect was abolished when the BEEF active fraction was heat treated before incubation. A minimal 2-h BEEF preincubation period was necessary to maintain sperm motility activity and to protect sperm against oxidative injury caused by 150 microM hydrogen peroxide. The proteins from the BEEF >10-kDa fractions were biotinylated to identify the proteins that bind to the sperm surface. Five specific sperm-surface-binding proteins were revealed by Western blot analysis probed with avidin-horseradish peroxidase conjugate. These proteins were digested with trypsin for identification by matrix-assisted laser desorption ionization time-of-flight peptide mass spectrometric analyzer. Under reducing conditions, 5 bovine proteins were identified: the beta (36-kDa spot) and alpha (38-kDa spot) chains of clusterin, the beta-adrenergic receptor kinase 2 (48-kDa spot), and the antithrombin-III and the fibrinogen gamma-B chains, both corresponding to a doublet of about 50-52 kDa. These proteins are known to be present at the sperm surface in other species and could play a role in sperm protection in vivo. These results provide new insights to explain how secretory epididymal proteins sustain sperm motility during storage in vitro.     

Calcium Influx into Human Neuroblastoma Cells Induces ALZ-50 Immunoreactivity: Involvement of Calpain-Mediated Hydrolysis of Protein Kinase C

Abstract: Calcium influx into SH-SY5Y human neuroblastoma cells after ionophore treatment or transient permeabilization in calcium-containing medium increased ALZ-50 immunoreactivity markedly. This increase was prevented by inhibitors active against calpain or against protein kinase C (PKC), suggesting that both of these enzymes were required to mediate the effect of calcium influx on ALZ-50 immunoreactivity. Treatment with PKC activator TPA increased ALZ-50 immunoreactivity in the absence of calcium influx or after intracellular delivery of the specific calpain inhibitor calpastatin, indicating that the influence of PKC was downstream from that of calpain. Calcium influx also resulted in μ-calpain autolysis (one index of calpain activation) and the transient appearance of PKM (i.e., free PKC catalytic subunits, generated by calpain-mediated cleavage of the regulatory and catalytic PKC domains). Inhibition of calpain within intact cells resulted in a dramatic increase in steady-state levels of total τ (migrating at 46–52 kDa) but resulted in a relatively minor increase in 68-kDa ALZ-50-immunoreactive τ isoforms. Although calcium influx into intact cells resulted in accumulation of ALZ-50 immunoreactivity, total τ levels were, by contrast, rapidly depleted. Incubation of isolated fractions with calpain in the presence of calcium indicated that ALZ-50-immunoreactive τ isoforms were more resistant to calpain-mediated proteolysis than were non-ALZ-50 reactive τ isoforms. These data therefore indicate that calpain may regulate τ levels directly via proteolysis and indirectly through PKC activation. A consequence of the latter action is altered τ phosphorylation, perhaps involving one or more kinase cascades, and the preferential accumulation of ALZ-50-immunoreactive τ isoforms due to their relative resistance to degradation. These findings provide a basis for the possibility that disregulation of calcium homeostasis may contribute to the pathological levels of conversion of τ to A68 by hyperactivation of the calpain/PKC system.     

Ultrastructural localization of dystrophin in human muscle by using gold immunolabelling

Immunolabelling with a 5 nm gold probe was used to localize dystrophin at the ultrastructural level in human muscle. The primary antibody was monoclonal, raised against a segment (amino acids 1181-1388) from the rod domain of dystrophin. The antibody (Dy4/6D3) is specific for dystrophin and shows no immunoreactivity with any protein from mdx mouse muscle or from patients with a gene deletion spanning part of the molecule recognized by the antibody (Nicholson et al. 1989 a; England et al. 1990). Using this antibody, labelling was almost entirely confined to a narrow 75 nm rim at the periphery of the muscle fibres. Histograms of the distance from the gold probe to the cytoplasmic face of the plasma membrane and of the distance between gold probes (nearest neighbour in a plane parallel with the plasma membrane) displayed modes at approximately 15 nm and 120 nm, respectively. The distribution of the probe was the same in longitudinal and transverse sections of the muscle. These observations suggest that the rod portion of the dystrophin molecule is normally arranged close to the cytoplasmic face of the plasma membrane and that the molecules form an interconnecting network. Labelling was not associated with the transverse tubular system.     

Sepsis stimulates calpain activity in skeletal muscle by decreasing calpastatin activity but does not activate caspase-3

We examined the influence of sepsis on the expression and activity of the calpain and caspase systems in skeletal muscle. Sepsis was induced in rats by cecal ligation and puncture (CLP). Control rats were sham operated. Calpain activity was determined by measuring the calcium-dependent hydrolysis of casein and by casein zymography. The activity of the endogenous calpain inhibitor calpastatin was measured by determining the inhibitory effect on calpain activity in muscle extracts. Protein levels of mu- and m-calpain and calpastatin were determined by Western blotting, and calpastatin mRNA was measured by real-time PCR. Caspase-3 activity was determined by measuring the hydrolysis of the fluorogenic caspase-3 substrate Ac-DEVD-AMC and by determining protein and mRNA expression for caspase-3 by Western blotting and real-time PCR, respectively. In addition, the role of calpains and caspase-3 in sepsis-induced muscle protein breakdown was determined by measuring protein breakdown rates in the presence of specific inhibitors. Sepsis resulted in increased muscle calpain activity caused by reduced calpastatin activity. In contrast, caspase-3 activity, mRNA levels, and activated caspase-3 29-kDa fragment were not altered in muscle from septic rats. Sepsis-induced muscle proteolysis was blocked by the calpain inhibitor calpeptin but was not influenced by the caspase-3 inhibitor Ac-DEVD-CHO. The results suggest that sepsis-induced muscle wasting is associated with increased calpain activity, secondary to reduced calpastatin activity, and that caspase-3 activity is not involved in the catabolic response to sepsis.     

A calpain-like proteolytic activity produces the limited cleavage at the N-terminal regulatory domain of rabbit skeletal muscle AMP deaminase: evidence of a protective molecular mechanism

On storage at 4 degrees C, rabbit skeletal muscle AMP deaminase undergoes limited proteolysis with the conversion of the native 85-kDa enzyme subunit to a 75-kDa core that is resistant to further proteolysis. Further studies have shown that limited proteolysis of AMP deaminase with trypsin, removing the 95-residue N-terminal fragment, converts the native enzyme to a species that exhibits hyperbolic kinetics even at low K+ concentration. The results of this report show that a 21-residue synthetic peptide, when incubated with the purified enzyme, is cleaved with a specificity identical to that reported for ubiquitous calpains. In addition, the cleavage of a specific fluorogenic peptide substrate by rabbit m-calpain is inhibited by a synthetic peptide that corresponds to residues 10-17 of rabbit skeletal muscle AMP deaminase; this peptide contains a sequence (K-E-L-D-D-A) that is present in the fourth subdomain A of rabbit calpastatin, suggesting that the N-terminus of AMP deaminase shares with calpastatin a regulatory sequence that might exert a protective role against the fragmentation-induced activation of AMP deaminase. These observations suggest that a calpain-like proteinase present in muscle removes from AMP deaminase a domain that holds the enzyme in an inactive conformation and which also contains a regulatory region that protects against unregulated proteolysis. We conclude that proteolysis of AMP deaminase is the basis of the large ammonia accumulation that occurs in skeletal muscle subjected to strong tetanic contraction or passing into rigor mortis.     

EXTRA-LYSOSOMAL PROTEOLYSIS AND EXPRESSION OF CALPAINS AND CALPASTATIN IN CULTURED THYROID CELLS

Proteolysis at neutral pH in the soluble fraction of cultured pig thyroid epithelial cells was examined using a synthetic calpain substrate, succinyl-Leu-Tyr-7-amino-4-methylcoumarin. The Ca²⁺-independent proteolytic activity was largely inhibited by substances known to affect cysteine- and metalloproteases, whereas no or little effects were obtained with inhibitors affecting serine- and aspartic proteases. Addition of Ca²⁺did not significantly alter the rate of substrate degradation. Biochemical separation via hydrophobic interaction chomatography and Western blotting demonstrated the presence of both m-calpain (40% of total calpain) and μ-calpain (60%) in confluent thyrocytes. Determination of calpastatin activity indicated a 30 times higher level of the inhibitor as compared to total calpain activity. Western blotting showed the presence of a 110kD calpastatin form with additional low mol wt forms possibly representing fragmentation products. In immunofluorescent stainings, m-calpain had a diffuse cytoplasmic distribution whereas μ-calpain was located both in the cytoplasm and at the cell—cell contacts. Calpastatin immunoreactivity was mainly granular and located close to the nucleus, although a fibrillar distribution was also observed. The results show the presence of all components of the calpain/calpastatin system and indicate a strict control of calpain activity in cultured thyrocytes. The different subcellular distributions of calpains and calpastatin suggests that they are compartmentalized and require mobilization to interact.     

Cellular localization of insulin-like growth factor-II protein in the sea bass (Dicentrarchus labrax) from hatching to adult

The cellular localization of IGF-II protein was investigated during larval and postlarval developmental stages of sea bass (Dicentrarchus labrax) by immunohistochemistry using antisera raised against Sparus aurata IGF-II. At hatching, IGF-II immunoreactivity was already present in the skin, developing intestine and skeletal muscle. During larval life IGF-II protein was also observed in heart musculature, in kidney and gill epithelia as well as in liver. In fry skeletal muscle a moderate IGF-II immunostaining was detected in red fibres, whereas white muscle fibres exhibited a faint immunoreactivity. In adults, a marked IGF-II immunostaining was observed in red muscle fibres. A moderate immunoreactivity was also present in white fibres as well as in heart striated myocardial fibres. These results are in agreement with previous findings on the spatial localization of IGF-II and IGF type 1 receptor in S. aurata and Umbrina cirrosa, confirming the role of IGF system during development and growth of fish.     

Purification and characterization of calpain and calpastatin from rainbow trout, Oncorhynchus mykiss

Although the calpain system has been studied extensively in mammalian animals, much less is known about the properties of μ-calpain, m-calpain, and calpastatin in lower vertebrates such as fish. These three proteins were isolated and partly characterized from rainbow trout, Oncorhynchus mykiss, muscle. Trout m-calpain contains an 80-kDa large subunit, but the  26-kDa small subunit from trout m-calpain is smaller than the 28-kDa small subunit from mammalian calpains. Trout μ-calpain and calpastatin were only partly purified; identity of trout μ-calpain was confirmed by labeling with antibodies to bovine skeletal muscle μ-calpain, and identity of trout calpastatin was confirmed by specific inhibition of bovine skeletal muscle μ- and m-calpain. Trout μ-calpain requires 4.4 ± 2.8 μM and trout m-calpain requires 585 ± 51 μM Ca²⁺ for half-maximal activity, similar to the Ca²⁺ requirements of μ- and m-calpain from mammalian tissues. Sequencing tryptic peptides indicated that the amino acid sequence of trout calpastatin shares little homology with the amino acid sequences of mammalian calpastatins. Screening a rainbow trout cDNA library identified three cDNAs encoding for the large subunit of a putative m-calpain. The amino acid sequence predicted by trout m-calpain cDNA was 65% identical to the human 80-kDa m-calpain sequence. Gene duplication and polyploidy occur in fish, and the amino acid sequence of the trout m-calpain 80-kDa subunit identified in this study was 83% identical to the sequence of a trout m-calpain 80-kDa subunit described earlier. This is the first report of two isoforms of m-calpain in a single species.     

Immunochemical and immunohistochemical study of the 27- and 29-kDa calcium-binding proteins and related proteins in the porcine tooth germ

 Our previous report identified 27- and 29-kDa calcium-binding proteins in porcine immature dental enamel. In this study we revealed that the N-terminal amino acid sequences of the two proteins were identical: LLANPXGXIPNLARGPAGRSRGPPG. The sequence matches a portion of the amino acid sequence of the porcine sheath protein, sheathlin. Porcine tooth germs were investigated immunochemically and immunohistochemically using specific antibodies raised against synthetic peptide that included residues 13–25 of this sequence. The affinity-purified antibodies reacted with several proteins extracted from newly formed immature enamel in immunochemical analyses, especially protein bands migrating at 62, 35–45, 29, and 27 kDa in SDS-polyacrylamide gels. The largest protein detected was a weak band near 70 kDa. In immunochemical analyses of proteins extracted from the inner (old) immature enamel, the antibody reacted faintly with the 27- and 29-kDa proteins. In immunohistochemical preparations, the Golgi apparatus and secretory granules of the secretory ameloblast, and the surface layer of immature enamel showed immunoreactivity. The immunoreactivity of immature enamel just beneath the secretory face of the Tomes’ process was intense. No immunoreactivity was found in the Golgi apparatus of the maturation ameloblast. These results suggest that the 70-kDa protein, whose degradation might be very fast, is the parent protein of the 27- and 29-kDa proteins. Accepted: 20 January 1997     

Caspases cleave the amino-terminal calpain inhibitory unit of calpastatin during apoptosis in human Jurkat T cells

We have previously reported the activation of procalpain mu (precursor for low-calcium-requiring calpain) in apoptotic cells using a cleavage-site-directed antibody specific to active calpain [Kikuchi, H. and Imajoh-Ohmi, S. (1995) Cell Death Differ. 2, 195-199]. In this study, calpastatin, the endogenous inhibitor protein for calpain, was cleaved to a 90-kDa polypeptide during apoptosis in human Jurkat T cells. The limited proteolysis of calpastatin preceded the autolytic activation of procalpain. Inhibitors for caspases rescued the cells from apoptosis and simultaneously inhibited the cleavage of calpastatin. The full-length recombinant calpastatin was also cleaved by caspase-3 or caspase-7 at Asp-233 into the same size fragment. Cys-241 was also targeted by these caspases in vitro but not in apoptotic cells. Caspase-digested calpastatin lost its amino-terminal inhibitory unit, and inhibited three moles of calpain per mole. Our findings suggest that caspases trigger the decontrol of calpain activity suppression by degrading calpastatin.     

Atrial natriuretic peptide-dependent phosphorylation of smooth muscle cell particulate fraction proteins is mediated by cGMP-dependent protein kinase

The atrial natriuretic peptide (ANP) stimulates cGMP production and protein phosphorylation in a particulate fraction of cultured rat aortic smooth muscle cells. Three proteins of 225, 132, and 11 kDa were specifically phosphorylated in response to ANP treatment, addition of cGMP (5 nM), or addition of purified cGMP-dependent protein kinase. The cAMP-dependent protein kinase inhibitor had no effect on the cGMP-stimulated phosphorylation of the three proteins but inhibited cAMP-dependent phosphorylation of a 17-kDa protein. These results demonstrate that the particulate cGMP-dependent protein kinase mediates the phosphorylation of the 225-, 132-, and 11-kDa proteins. The 11-kDa protein is phospholamban based on the characteristic shift in apparent Mr from 11,000 to 27,000 on heating at 37 degrees C rather than boiling prior to electrophoresis. ANP (1 microM) increased the cGMP concentration approximately 4-fold in the particulate fractions, from 4.3 to 17.7 nM, as well as the phosphorylation of the 225-, 132-, and 11-kDa proteins. In contrast, the biologically inactive form of ANP, carboxymethylated ANP (1 microM), did not stimulate phosphorylation of any proteins nor did the unrelated peptide hormone, angiotensin II (1 microM). These results demonstrate the presence of the cGMP-mediated ANP signal transduction pathway in a particulate fraction of smooth muscle cells and the specific phosphorylation of three proteins including phospholamban, which may be involved in ANP-dependent relaxation of smooth muscle.     

The cytosol of human erythrocytes contains a highly Ca2+-sensitive thiol protease (calpain I) and its specific inhibitor protein (calpastatin)

The cytosol of human erythrocytes was found to contain a Ca2+-dependent thiol protease (calpain) and its specific inhibitor (calpastatin) by DEAE-cellulose chromatography at pH 8.0, although no proteolytic activity toward casein was detected in the unfractionated hemolysate. The protease required only 40 microM Ca2+ for 50% activation, indicating that it belongs to the highly Ca2+-sensitive type of calpain, namely, calpain I. It was not inactivated by heating at 58 degrees C for 10 min at pH 7.2, the optimal pH for its action on casein. The inhibitor comprised major and minor components, calpastatin H (Mr = 280,000) and caplastatin L (Mr = 48,000). Both were heat-stable proteins which were readily inactivated by tryptic digestion. The inhibition of erythrocyte calpain by erythrocyte calpastatin H or L was not due to sequestering of Ca2+ from the reaction medium by the inhibitor protein. The calpain preparation preferentially digests bands III and IVa of human erythrocyte membrane proteins, with little or no cleavage of the bands corresponding to spectrin.