The calpain system in human placenta

The calpain system is involved in a number of human pathologies ranging from the muscular dystrophies to Alzheimer's disease. It is important, therefore, to be able to obtain and to characterize both mu-calpain and m-calpain from human tissue. Although human mu-calpain can be conveniently obtained from either erythrocytes or platelets, no readily available source of human m-calpain has been described. Human placenta extracts contain both mu-calpain and m-calpain in nearly equal proportions and in significant quantities (3-4 mg mu-calpain and 4-5 mg m-calpain/1000 g placenta tissue). Placenta also contains calpastatin that elutes off ion-exchange columns over a wide range of KCl concentrations completely masking the mu-calpain activity eluting off these columns and even partly overlapping m-calpain elution. Placenta mu-calpain requires 50-70 microM Ca2+ and placenta m-calpain requires 450-460 microM Ca2+ for half-maximal proteolytic activity. Western analysis of washed placenta tissue shows that placenta contains both mu- and m-calpain, although some of the mu-calpain in whole placenta extracts likely originates from the erythrocytes that are abundant in the highly vascularized placenta. Placenta calpastatin could not be purified with conventional methods. The most prominent form of calpastatin in Western analyses of placenta obtained as soon as possible after birth was approximately 48-51 kDa; partly purified preparations of placenta calpastatin also contained 48-51 and 70 kDa polypeptides. Human placenta extracts likely contain two different calpastatin isoforms, a 48-51 kDa "placenta calpastatin" and a 70 kDa erythrocyte calpastatin.     

Distribution of calpains and calpastatin in human blood cells

The occurrence and molecular sizes of calpains and calpastatin in the lysates of human erythrocytes, platelets, lymphocytes/monocytes, and polymorphonuclear cells were studied by immunoelectrophoretic blot analysis. The basic uniformity among these cells of the 85-kDa and 83-kDa heavy subunits of low- and high-Ca2+-requiring calpains I and II, respectively, and of the 29-kDa light subunit was confirmed. Molecular diversity of calpastatin species, ranging from 70 kDa to 107 kDa, among different blood cells was also shown. The obtained data are consistent with those known for other animal tissues, thus settling hitherto uncertain or rather controversial issues on the distribution of calpains and calpastatin in human blood cells.     

On the mechanism of binding of calpastatin, the protein inhibitor of calpains, to biologic membranes

Bovine myocardial calpastatin, the endogenous inhibitor of the calcium-dependent proteinases, calpains, could bind to sarcoplasmic reticulum preparations at neutral pH and low ionic strength. Even in the presence of 100 to 200 mM KCl, 4 to 5 micrograms of calpastatin was bound per mg of membrane. Although calpastatin is found associated with bovine myocardial sarcolemma, neither canine nor human erythrocyte calpastatins were present in isolated erythrocyte membrane preparations. The bovine myocardial calpastatin, but not human erythrocyte calpastatin, could associate with purified phospholipid vesicles at low ionic strength. Thus, phospholipids appear to be involved in the binding of calpastatin to membranes.     

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.     

Specific and common antigens of Clonorchis sinensis and Opisthorchis viverrini(Opisthorchidae,Trematoda)

The antigenic characterizations and serological reactions of human liver flukes, Clonorchis sinensis and Opisthorchis viverrini, were analyzed by immunoblot. The antigenic profiles of the crude extract of Clonorchis contained major proteins of 8, 26-28, 34-37, 43, and 70 kDa, and those of Opisthorchis 34-37, 43, 70, and 100 kDa. Of these, the 8, 26-28 and 34-37 kDa bands of Clonorchis and the 100 kDa of Opisthorchis were major components of each excretory-secretory antigen. The 8 and 26-28 kDa bands were specific to Clonorchis but the 100 kDa of Opisthorchis cross-reacted with the sera of clonorchiasis, and the 34-37, 70 and 100 kDa bands cross-reacted with sera of other helminthiases. The frequency and intensity of the immunoblot reactions were positively correlated with the intensity of the liver fluke infection.     

Proteolysis of nuclear proteins by mu-calpain and m-calpain.

Purified calpains are capable of proteolyzing several high Mr nuclear proteins and solubilizing a histone H1 kinase activity from rat liver nuclei upon exposure to 10(-6) - 10(-5) M Ca2+. Major nuclear substrates displayed apparent molecular masses of 200, 130, 120, and 60 kDa on Coomassie Blue-stained SDS-PAGE gels. The nuclear proteins and the H1 kinase were released from Triton-treated nuclei following incubation with buffer containing 0.5 M NaCl. They therefore appeared to be internal nuclear matrix proteins. The nuclear H1 kinase activity solubilized by incubation with m-calpain was eluted in the void volume of a Bio-Gel A-1.5m column, indicating an apparent mass greater than 1,500 kDa. Treatment of the calpain-solubilized kinase with 0.5 M NaCl dissociated it to a form having an apparent mass of 300 kDa (Stokes radius = 5.6 nm), suggesting that the 300-kDa (Stokes radius = 5.6 nm), nuclei by calpain treatment as a large complex containing other internal matrix proteins. Purified human erythrocyte mu-calpain was capable of proteolyzing the nuclear matrix proteins at 10(-6) M Ca2+. In contrast, human erythrocyte multicatalytic protease complex produced little cleavage of the nuclear proteins. Proteolysis of nuclear proteins by either mu-calpain or m-calpain was inhibited by calpastatin. These experiments suggest a physiologic role for the calpains in the turnover of nuclear proteins.     

Identification of calpastatin and mu-calpain and studies of their association in pulmonary smooth muscle mitochondria

Using calpastatin antibody we have identified a 145 kDa major band along with two relatively minor bands at 120 kDa and 110 kDa calpastatin molecules in bovine pulmonary artery smooth muscle mitochondria. To the best of our knowledge this is first report regarding the identification of calpastatin in mitochondria. We also demonstrated the presence of micro-calpain in the mitochondria by immunoblot and casein zymogram studies. Immunoblot studies identified two major bands corresponding to the 80 kDa large and the 28 kDa small subunit of mu-calpain. Additionally 76 kDa, 40 kDa and 18 kDa immunoreactive bands have also been detected. Purification and N-terminal amino acid sequence analysis of the identified proteins confirmed their identity as mu-calpain and calpastatins. Immunoprecipitation study revealed molecular association between mu-calpain and calpastatin in the mitochondria indicating that calpastatin could play an important role in preventing uncontrolled activity of mu-calpain which otherwise may facilitate pulmonary hypertension, smooth muscle proliferation and apoptosis.     

Identification, purification, and partial characterization of a novel Mr 28,000 integral membrane protein from erythrocytes and renal tubules

A novel Mr 28,000 integral membrane protein ("28kDa") was identified in human erythrocytes and found entirely associated with the Triton X-100 insoluble membrane skeletons. Antibodies to 28kDa reacted strongly on immunoblots with 28kDa and a diffuse region of Mr 35,000-60,000 ("HMW-28kDa"). Selective proteolytic digestions of membranes demonstrated that HMW-28kDa has an extracellular domain, and both 28kDa and HMW-28kDa have intracellular domains. 28kDa and HMW-28kDa were purified to homogeneity. Quantitative immunoblots indicate that each erythrocyte contains 120,000-160,000 copies of 28kDa. Two-dimensional iodopeptide maps of 28kDa and HMW-28kDa were nearly identical; peptide-N-glycosidase digestion of purified HMW-28kDa demonstrated that it is the N-glycosylated form of 28kDa. When concentrated, 28kDa formed a series of larger oligomers which were stable in sodium dodecyl sulfate. Of several nonerythroid tissues studied with anti-28kDa immunoblots, only kidney displayed immunoreactive 28kDa. Purified rat kidney 28kDa was nearly identical to rat erythrocyte 28kDa when compared by two-dimensional iodopeptide mapping. Immunohistochemical staining of human kidney with anti-28kDa demonstrated prominent staining over the apical brush borders of proximal convoluted tubules. A novel integral membrane protein has been purified from erythrocyte and kidney membranes. This new protein may play a role in linkage of the membrane skeleton to the lipid bilayer.     

Localization of m-calpain and calpastatin and studies of their association in pulmonary smooth muscle endoplasmic reticulum

Calpain and calpastatin have been demonstrated to play many physiological roles in a variety of systems. It, therefore, appears important to study their localization and association in different suborganelles. Using immunoblot studies, we have identified 80 kDa m-calpain in both lumen and membrane of ER isolated from bovine pulmonary artery smooth muscle. Treatment of the ER with Na(2)CO(3) and proteinase K demonstrated that 80 kDa catalytic subunit and 28 kDa regulatory subunit (Rs) of m-calpain, and the 110-kDa and 70-kDa calpastatin (Cs) forms are localized in the cytosolic side of the ER membrane. Coimmunoprecipitation studies revealed that m-calpain is associated with calpastatin in the cytosolic face of the ER membrane. We have also identified m-calpain activity both in the ER membrane and lumen by casein-zymography. The casein-zymogram has also been utilized to demonstrate differential pattern of the effects of reversible and irreversible cysteine protease inhibitors on m-calpain activity. Thus, a potential site of Cs regulation of m-calpain activity is created by positioning Cs, 80 kDa and 28 kDa m-calpain in the cytosolic face of ER membrane. However, such is not the case for the 80-kDa m-calpain found within the lumen of the ER because of the conspicuous absence of 28 kDa Rs of m-calpain and Cs in this locale.     

Spectrin-like proteins in green algae (Desmidiaceae)

Immunochemical detection of actin as well as spectrin-like proteins have been carried out in the green algae Micrasterias denticulata, Closterium lunula, and Euastrum oblongum. In these algae, actin is detected on Western blots at 43 kDa with antibodies to actin from higher plant and animal origin. By use of antibodies to human and chicken erythrocyte spectrin a cross-reactivity with desmid proteins is found at about the molecular mass of 220 kDa, where also human erythrocyte spectrin is detected. Additional bands are present at 120 kDa and 70 kDa, which are probably breakdown products. An antibody against chicken alpha-actinin, a small protein of the spectrin superfamily, recognizes bands at 90 kDa, where it is expected, and 70 kDa, probably the same breakdown product as mentioned for spectrin. Isoelectric focusing provides staining at pI 4.6 with antibodies against spectrin. Immunogold labelling of spectrin and alpha-actinin antigens on high-pressure frozen, freeze-substituted Micrasterias denticulata cells with the same antibodies exhibits staining, especially at membranes of different populations of secretory vesicles, at dictyosomes, and the plasma membrane. However, no clear correlation to the growth pattern of the cell could be observed. Taken together, our results demonstrate the presence of spectrin-like proteins in desmid cells which are probably functional in exocytosis.     

Characterization of β-lactoglobulin from buffalo (Bubalus bubalis) colostrum and its possible interaction with erythrocyte lipocalin-interacting membrane receptor

Lipocalins form a widespread class of proteins involved in the transport of weakly soluble vitamins, hormones or hydrophobic molecules. β-lactoglobulin (BLG-col), a major lipocalin present in whey was purified and characterized from buffalo colostrum. The molecular weight of BLG-col as determined by Liquid chromatography -electrospray ionization mass spectrometry (LC-ESI-MS) was 18.257 kDa and the peptide mass fingerprint of the purified protein revealed 67% sequence homology to buffalo milk β-lg. The N-terminal-IIVTQ and LC-ESI-collision-induced dissociation-Electron transfer dissociation mass spectrometry/mass spectrometry analyses of doubly (m/z 1156(+2)) and triply (m/z 546(+3)) charged ion pairs corresponding to VYVEELKPTPEGDLEILLQK (41-60) and TPEVDDEALEKFDK (125-138) sequences confirmed the identity of BLG-col. Using these peptide sequences, the location of a gene encoding for BLG-col was identified on chromosome 11 at 11q28 loci of bovine genome. The unique property of the BLG-col isolated from buffalo colostrum was its strong and specific haemagglutinating activity with 'O' blood of human erythrocytes with 10,309 HAU/mg protein. The cell surface localization of BLG-col on human erythrocytes was confirmed by immunocytochemistry and the specificity of interaction was established by immunoblot analysis of human erythrocyte membrane proteins. Based on these observations, we suggest the presence of lipocalin receptor (70 kDa) on human erythrocyte membrane and the multiple sequence alignment supported structural diversity among lipocalin receptors.     

Isolation and characterization of acetylcholinesterase from Drosophila

The purification and characterization of acetylcholinesterase from heads of the fruit fly Drosophila are described. Sequential extraction procedures indicated that approximately 40% of the activity was soluble and 60% membrane-bound and that virtually none (less than 4%) corresponded to collagen-tailed forms. The membrane-bound enzyme was extracted with Triton X-100 and purified over 4000-fold by affinity chromatography on acridinium resin. Hydrodynamic analysis by both sucrose gradient centrifugation and chromatography on Sepharose CL-4B revealed an Mr of 165,000 similar to that observed for dimeric (G2) forms of the enzyme in mammalian tissues. In contrast, the purified enzyme gave predominant bands of about 100 kDa prior to disulfied reduction and 55 kDa after reduction on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, values that are significantly lower than those reported for purified G2 enzymes from other species. However, the presence of a faint band at 70 kDa which could be labeled by [3H]diisopropyl fluorophosphate prior to denaturation suggested that the 55-kDa band as well as a 16-kDa species arose from proteolysis. This was confirmed by reductive radiomethylation and amine analysis of the 70-, 55-, and 16-kDa bands. All three contained ethanolamine and glucosamine residues that are characteristic of a C-terminal glycolipid anchor in other G2 acetylcholinesterases. The catalytic properties of the enzyme were examined by titration with a fluorogenic reagent which revealed a turnover number for acetylthiocholine that was 6-fold lower than eel and 3-fold lower than human erythrocyte acetylcholinesterase. Furthermore, the Drosophila enzyme hydrolyzed butyrylthiocholine much more efficiently than these eel or human enzymes, an indication that the fly head enzyme has a substrate specificity intermediate between mammalian acetylcholinesterases and butyrylcholinesterases.     

Agglutination of mouse erythrocytes by binding of non-choline phospholipids to a 70 000-Dalton protein

The structure of some phospholipids that cause agglutination of mouse erythrocytes has been studied. Haemagglutination is a property of non-choline-containing phospholipids; the phosphate group is essential and unsaturated fatty acids optimal. A protein of Mr 70 000 was isolated from mouse erythrocyte membranes which completely inhibited phospholipid-mediated erythrocyte agglutination. It is proposed that this protein is the phospholipid binding site on mouse erythrocytes and the ligand for the human B-lymphocyte receptor for mouse erythrocytes. Preliminary investigations suggest that a similar inhibitor of phospholipid-mediated agglutination is found in serum. Agglutination of mouse erythrocytes by phospholipid and specific inhibition by the 70 kDa membrane protein constitute a simple system for studying the interaction of phospholipid with protein.     

Solubilization of the binding protein from Ehrlich ascites cells and erythrocytes to Pseudomonas aeruginosa cytotoxin.

The binding protein for pore-forming Pseudomonas aeruginosa cytotoxin was solubilized from Ehrlich ascites cell plasma membranes and rabbit and bovine erythrocyte ghosts using nonionic and zwittergent detergents. Analysis of solubilized plasma membranes from Ehrlich cells by a ligand-blot technique after separation by SDS-PAGE/electrophoretic transfer to nitrocellulose or affinity chromatography showed a protein of 70 kDa molecular mass, which binds to cytotoxin. The binding protein solubilized from rabbit erythrocyte ghosts showed a molecular mass of 50 kDa and that from bovine ghosts 55 kDa according to the former test. The binding proteins could be characterized as acidic. They contain a glycan moiety which is, however, not involved in the interaction of cytotoxin with the binding site.     

Guanine nucleotide regulation of phospholipase C activity in permeabilized rabbit neutrophils. Inhibition by pertussis toxin and sensitization to submicromolar calcium concentrations.

Calpastatin, the inhibitor protein acting specifically on calpain (EC 3.4.22.17; Ca2+-dependent cysteine proteinase), is known to be widely distributed in mammalian and avian cells. Two different molecular species of calpastatin were isolated and purified to homogeneity from pig heart muscle and from pig erythrocytes, and shown to be of 107 kDa and 68 kDa respectively on SDS/polyacrylamide-gel electrophoresis. Both calpastatins had very similar amino acid compositions when expressed as mol per cent of the residues, differed by only 0.1 pH unit in their isoelectric points, and showed immunological cross-reactivity. One molecule of the 107 kDa species could bind approx. 8 calpain molecules, whereas the 68 kDa inhibitor could bind approx. 5 calpain molecules. These findings suggest similar protein structures of the 107 kDa and 68 kDa calpastatins, each being composed of extended multidomains, with unit inhibitor domains aligned along the polypeptide chain of the molecule. The present study does not conclude, however, whether or not the 68 kDa calpastatin found in erythrocytes is a derived product from the 107 kDa species, which is present as such in heart muscle.     

Factors influencing the binding of calpain I to human erythrocyte inside-out vesicles

The mechanism for binding of human erythrocyte calpain I to human erythrocyte inside-out vesicles was studied by immunoelectrophoretic blot analysis. Binding of calpain I to inside-out vesicles was observed both in the absence and presence of Ca2+. Moreover, in the absence of Ca2+, acidic proteins like casein, ovalbumin and calpastatin suppressed while basic proteins like arginase and lysozyme did not affect the binding of calpain I to inside-out vesicles. Here, we propose a model for the binding of calpain to the membrane.     

Plasmodium vivax: malarial proteins associated with the membrane-bound caveola-vesicle complexes and cytoplasmic cleft structures of infected erythrocytes

The identification of antigens of parasite origin associated with the altered membrane of Plasmodium vivax-infected erythrocytes was undertaken in this study. The 125I-lactoperoxidase catalyzed surface radiolabeling of trophozoite-infected erythrocytes revealed new bands of 95 and 70 kDa not labeled in normal erythrocytes. Erythrocyte membrane-enriched preparations from [35S]methionine biosynthetically labeled-infected erythrocytes also indicated that in addition to bands at 95 and 70 kDa, several other parasite proteins were possibly membrane associated. Five monoclonal antibodies (Mabs) reactive with P. vivax produced an immunofluorescent pattern of numerous small dots scattered over the entire infected erythrocyte. This pattern mimics that of Schuffner's stippling; small red dots seen in Giemsa-stained P. vivax-infected erythrocytes, which represent accumulations of dye in caveola-vesicle complexes (CVC). Four of the monoclonal antibodies immunoprecipitated a Triton X-100 detergent-insoluble 95-kDa parasite protein which was localized by immunofluorescent assay and immunoelectron microscopy exclusively to the CVC. Two of these Mabs were immunofluorescence reactive with the surface of intact infected erythrocytes in suspension. The fifth Mab, which also localized exclusively to the CVC structures, immunoprecipitated a Triton X-100 extractable protein of 70 kDa. Two other monoclonal antibodies reacted exclusively with the numerous membranous cleft structures found in the cytoplasm of infected erythrocytes. This cleft-associated parasite antigen was 28 kDa in size. Some of these Mabs recognize epitopes and produce similar IFA patterns on erythrocytes infected with P. cynomolgi, P. knowlesi, and P. ovale parasites, but not with P. falciparum- or P. brasilianum-infected erythrocytes.     

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+.