The calpain small subunit gene is essential: its inactivation results in embryonic lethality.

Creation of transgenic (knockout) mice deficient in calpain small (30 kDa) subunit gene was undertaken to clarify the proposed role of the small subunit for calpain proteolytic activity and to gain insight into the importance of the gene in the whole animal. The gene was targeted and disrupted in embryonic stem cells by homologous recombination, and chimeric mice were generated. Heterozygous F1 generation mice were crossed to obtain F2 generation. Among F2 generation mice, we found only wild-type and heterozygous animals in the 80 pups genotyped to date; no homozygous mice have been found, although 20 were expected. The heterozygotes had no apparent phenotypic abnormalities. Analysis of their tissues revealed no significant difference in mRNA expression, protein content, or proteolytic activity in comparison with their wild-type littermates. Genotyping of fetuses at different stages of development also revealed only wild-type and normal heterozygous fetuses. No moribund embryos or resorption sites were observed in the uterine cavity. The results indicate that at least one normal allele is essential for postnatal survival. Disruption of both alleles appears to be lethal in very early fetal development.

Proteolysis of synaptobrevin, syntaxin, and SNAP-25 in alveolar epithelial type II cells.

Synaptobrevin-2, syntaxin-1, and SNAP-25 were identified in rat alveolar epithelial type II cells by Western blot analysis. Synaptobrevin-2 was localized in the lamellar bodies, and syntaxin-1 and SNAP-25 were found in 0.4% Nonidet P40-soluble and -insoluble fractions, respectively, of the type II cells. When the isolated type II cells were stimulated for secretion with calcium ionophore A23187 or with phorbol 12-myristate 13-acetate, these proteins were found to have been proteolyzed. Preincubation of cells with calpain inhibitor II (N-acetylleucylleucylmethionine), however, prevented the proteolysis. Treatment of the cell lysate with exogenous calpain resulted in a time-dependent decrease of these proteins. The data suggest that synaptobrevin, syntaxin, and SNAP-25 are subject to proteolytic modification by activated calpain in intact type II cells stimulated for secretion.

An antisense oligodeoxyribonucleotide to m-calpain mRNA inhibits secretion from alveolar epithelial type II cells.

We investigated the effect of translational suppression of m-calpain on [3H]-phosphatidylcholine (PC) secretion utilising an antisense oligodexoyribonucleotide (oligo) directed against mRNA encoding m-calpain catalytic subunit. Two types of oligo, sense (S) and antisense (AS), to a portion of exon 12 of rat m-calpain catalytic subunit mRNA were tested. Constitutive secretion was decreased by 23% by AS-oligo (1 microM) treatment, while S-oligo (1 microM) had no effect. TPA-stimulated secretion was inhibited about 50-60% by AS-oligo (1-3 microM) and the inhibition was concentration-dependent, while S-oligo (1 microM) only inhibited about 10% of TPA-stimulated secretion. Northern and Western blot analyses revealed that the AS-oligo treatment reduced m-calpain mRNA and protein levels by 32% and 78%, respectively. The data indicate that antisense strategy is effective in suppressing calpain expression and type II cell secretion.

Inhibition of lung surfactant secretion from alveolar type II cells and annexin II tetramer-mediated membrane fusion by phenothiazines.

We investigated the effects of phenothiazines on lung surfactant secretion from rat alveolar epithelial type II cells and on annexin II tetramer (Anx IIt)-mediated membrane fusion. Trifluoperazine and promethazine inhibited ATP-stimulated phosphatidylcholine (PC) secretion from type II cells in a dose-dependent manner. Concentrations that cause 50% inhibition (IC50) were approximately 3 and 25 microM for trifluoperazine and promethazine, respectively. Promethazine also inhibited PC secretion of type II cells stimulated by other secretagogues, including calcium ionophore A23187, phorbol 12-myristate 13-acetate, and terbutaline that are known to stimulate PC secretion via different signal transduction pathways. Since we have recently determined that Anx IIt is involved in PC secretion of type II cells, we examined whether phenothiazines influence Anx IIt's activity. Trifluoperazine and promethazine inhibited Anx IIt's ability to aggregate phosphatidylserine (PS) liposomes, to fuse PS/phosphatidylethanolamine (PE) liposomes, and to fuse PS/PE liposomes with lamellar bodies. These results suggest a relationship between lung surfactant secretion and Anx IIt-mediated membrane fusion.

Annexin II binds to the membrane of A549 cells in a calcium-dependent and calcium-independent manner.

We investigated the nature of annexin II binding to the biological membranes using a lung epithelium-derived cell line A549. The cytosolic and membrane fractions of A549 cells were separated in the presence of 5 mM EGTA. Both fractions contain annexin II monomer and tetramer as evaluated by western blots using specific monoclonal antibodies against p36 and p11 subunits of annexin II. A substantial amount of annexin II was associated with the membrane fraction even after extensive washing with EGTA buffer, indicating the presence of two pools of annexin II. The EGTA-resistant membrane-bound annexin II could be partially extracted by 1% Triton X-100 or 60 mM n-octyl-beta-D-glucopyranoside, and completely by 30 mM CHAPS or 0.1% deoxycholate. This fraction of annexin II was also extracted by 0.1 M Na2CO3, pH 11 and partitioned into the aqueous phase after being treated with Triton X-114, demonstrating that the EGTA-resistant annexin II is a peripheral membrane protein. When the cells were lysed in varying concentrations of Ca2+, annexin II translocated from cytosolic fraction to membrane fraction at 4-25 microM Ca2+. To identify proteins closely associated with annexin II the membrane fraction was treated with the bifunctional chemical cross-linker disulfosuccinimidyl tartarate, followed by western blot analysis using anti-p36 or anti-p11 antibodies. We find that both p36 and p11 were cross-linked to a 51 kDa protein. In addition, p11 also binds to several proteins with molecular mass of 91, 65, 40 and 36 kDa. Our results suggest that annexin II may bind to the A549 cell membranes via specific membrane-associated proteins.

Secretagogue-induced proteolysis of cAMP-dependent protein kinase in intact rat alveolar epithelial type II cells.

Stimulation of secretion from rat alveolar epithelial type II cells by the beta-adrenergic agonist terbutaline activates cAMP-dependent protein kinase (PKA). The same secretagogue also activates endogenous protease calpain in type II cells. In this study, we investigated the effect of calpain activation on PKA and its phosphorylation activity in stimulated type II cells. Type II cells were either pretreated with cell-permeable calpain specific inhibitor (N-acetyl-leucyl-leucyl-methioninal) or untreated, and subsequently stimulated with terbutaline. Stimulus-induced phosphorylation activity was assayed using the PKA-specific substrate Kemptide. Maximum PKA activity was observed within 1-3 min of stimulation. Peak activity of the untreated cells was 20-25% higher and longer than that of the inhibitor-treated cells. The stimulus-induced phosphorylation activity of both cell groups was suppressable by PKA-specific inhibitor. Concomitant photoaffinity labeling with radioactive 8-azido-cAMP revealed that a 39 kDa proteolytic fragment was generated in response to stimulation by terbutaline. Stimulus-induced activation of PKA resulted in the phosphorylation of two endogenous proteins, p112 and p47. Phosphorylation of p112 and p47 was modulated in cells pretreated with calpain inhibitor or in the presence of PKA inhibitor. Aggregate results indicate that stimulus-induced proteolysis of pKA occurs in type II cells, suggesting that limited proteolysis of PKA by endogenous calpain may convert an initial transient signal to sustained and augumented phosphorylation activity for secretion.

Involvement of annexin II in exocytosis of lamellar bodies from alveolar epithelial type II cells.

Annexins are a family of Ca(2+)- and phospholipid-binding proteins that have been implicated in exocytosis. In the present study, we investigated the participation of selected annexins in exocytosis of lamellar bodies by examining their liposome aggregation property and ability to reconstitute surfactant secretion from permeabilized rat lung alveolar type II cells. Annexins I, II, III, and VI were demonstrated in type II cells by immunoblot analysis, but annexin IV and V were not found. Annexins I-IV mediated liposome aggregation in the presence of 1 mM Ca2+. However, only annexin II tetramer had aggregation activity at 10 microM Ca2+. Annexins V and VI had negligible aggregation activity at any Ca2+ concentrations (up to 1 mM Ca2+). To study reconstitution of secretion by annexins, isolated type II cells were permeabilized with 40 microM beta-escin. Under these conditions, the permeabilized cells released approximately 30-40% lactic acid dehydrogenase into the medium. An underestimated fraction of cellular annexin content was lost during permeabilization. However, lamellar bodies in the permeabilized type II cells stained appropriately with the fluorescent dyes Nile red and quinacrine, indicating that they were intact. These permeabilized cells were secretion competent, since phosphatidylcholine (PC) secretion was stimulated by 0.2-1.0 microM Ca2+. Addition of an exogenous annexin mixture enhanced PC secretion from the permeabilized type II cells with maximal stimulation at 0.5 microM Ca2+. Of six purified annexins (I-VI) tested for their ability to reconstitute secretion from permeabilized cells, only annexin II was effective. Our results suggest that annexin II is not necessary for exocytosis of lamellar bodies.

Lung annexin II promotes fusion of isolated lamellar bodies with liposomes.

The role of annexin II in the secretion of lung surfactant was investigated using isolated lamellar bodies and/or liposomes as the model system for aggregation and fusion. We first compared membrane aggregation mediated by two forms of annexin II, annexin II monomer (Anx IIm) and annexin II tetramer (Anx IIt). Anx IIt required 20-fold less Ca2+ to mediate phosphatidylserine (PS) liposome aggregation compared to Anx IIm. Aggregation of lamellar bodies mediated by Anx IIt was 4-fold greater than that by Anx IIm at 1 mM Ca2+. These results suggest that Anx IIt may be the more active form in vivo. Fusion of lamellar bodies with PS liposomes was promoted by Anx IIt in a dose-dependent manner, with maximal fusion occurring at 10-15 micrograms/ml of Anx IIt. Fusion was dependent on Ca2+ and the phospholipid composition of liposomes. While the fusion of lamellar bodies with PS liposomes required 100 microM Ca2+, the fusion with PS/phosphatidylethanolamine (PE) (1:3) liposomes required only 10 microM Ca2+. Anx IIt-mediated lamellar body-liposome fusion was enhanced by arachidonic acid, a lung surfactant secretagogue and inhibited by 4.4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of lung surfactant secretion. The data suggest that Anx IIt may play a role in the fusion of lamellar bodies with plasma membranes during lung surfactant secretion.

Inhibition of secretion from isolated rat alveolar epithelial type II cells by the cell permeant calpain inhibitor II (N-acetyl-leucyl-leucyl-methioninal).

Although several signal transduction pathways, including activation of specific protein kinases have been proposed and studied for the secretory processes of lung surfactant from alveolar epithelial type II cells, the role of proteolytic processing by calpains (calcium-activated neutral proteases) in secretion has not been investigated. Therefore, we examined the effect of cell permeable calpain inhibitor I (N-acetyl-leucyl-leucyl-norleucinal) and II (N-acetyl-leucyl-leucyl-methioninal) on secretion to test the hypothesis that calpains participate in the secretory processes of alveolar epithelial type II cells. Calpain inhibitor I preferentially inhibits micro (mu)-calpain while inhibitor II inhibits milli (m)-calpain. Isolated type II cells were prelabelled with [3H]-choline for 18-24 h. To measure secretion, [3H]-labelled disaturated phosphatidylcholine (DSPC) released in the medium was monitored. Basal secretion of DSPC was maximally (87%) depressed by the presence of 10 microM inhibitor II. Secretagogue-stimulated secretion was also modulated by inhibitor II treatment. Stimulation with calcium ionophore A23187 enhanced secretion 3-fold. However, cells pre-exposed to inhibitor II displayed a 90% reduction of calcium-stimulated secretion. Terbutaline (10 microM) and ATP (1 mM) each increased secretion 2- and 4-fold, respectively. However, the inhibitor-treated cells, exposed to the same stimuli, attained only 53 or 62% of these increases. Calpain inhibitor I, on the other hand, inhibited neither basal nor stimulated secretion. The results suggest that m-calpain, the major isozyme of lung calpain requiring mM calcium for activity in vitro, is involved in the secretory pathways of alveolar epithelial type II cells.

Regulation of annexin I by proteolysis in rat alveolar epithelial type II cells.

Limited proteolysis of annexin I occurs endogenously in intact rat lung alveolar epithelial type II cells as revealed by immunoblot analysis. Proteolysis increases in the presence of phorbol 12-myristate 13-acetate (PMA), terbutaline, or ATP, agents that enhance secretion of lung surfactant from type II cells. Calpain, a calcium-dependent neutral protease, was activated in stimulated type II cells and cleaved purified annexin I at the N-terminus minus 26 amino acids. Liposome aggregation activity of truncated annexin I showed ten-fold increase in calcium sensitivity compared to the native form. The results suggest that proteolytic modification may be a regulatory mechanism for annexin I to mediate the secretory processes of alveolar type II cells at physiological calcium concentrations.

Annexins I, II and III are specific choline binding proteins.

We have isolated choline binding proteins from the plasma membrane fraction fraction of human lung epithelium-derived cell line (A549) by means of detergent solubilization, anion exchange and affinity chromatography. One of the affinity purified proteins had a specific choline binding activity of 44-57 pmol/mg, representing a two to three hundredfold enrichment relative to the specific activity of freshly prepared plasma membranes. The purified protein has a molecular mass of 38 kDa by SDS PAGE analysis and was identified as annexin II by N-terminal microsequencing. Annexin II, however, had not previously been known for choline binding activity. We therefore prepared a mixture of authentic annexins (I-V) from A549 cells. The mixture had a choline binding activity of 15 to 18 pmol/mg. The annexin mixture was subsequently affinity chromatographed on the choline-conjugated Sepharose 6B column. Analyses by SDS PAGE and immunoblot revealed that annexins I, II, and III are bound to the choline column while annexins IV and V did not. These results indicate that some of the annexins have specific choline binding activities.

An intramolecular disulfide bond is essential for annexin I-mediated liposome aggregation.

Dithiothreitol (DTT) inhibits the annexin I-mediated aggregation of phosphatidylserine (PS) liposomes, but has no effect on its binding to PS vesicles. Non-reducing SDS gel analysis indicates that intermolecular disulfide bonds between annexin I molecules are not involved in liposome aggregation. However, DTT causes changes in protein conformation of annexin I as monitored by hydrophobic fluorescent dye treatment. The results suggest that the reduction of the intramolecular disulfide bond leads to inhibition of annexin I-mediated liposome aggregation via protein conformational changes.

Secretagogue-induced proteolysis of lung spectrin in alveolar epithelial type II cells.

Incubation of isolated rat alveolar epithelial type II cells with secretagogues (calcium ionophore, ATP or terbutaline) resulted in rapid proteolysis of lung spectrin and appearance of multiple proteolytic products which showed immunoreactivity with an antibody against human erythrocyte spectrin. These proteolytic products were similar to those generated from erythrocyte spectrin or cultured lung tumor cells (A549 cells) incubated with purified calpain. Furthermore, incubation of alveolar type II cells with a calpain-specific inhibitor modulated the secretagogue-induced proteolysis of lung spectrin. Thus, stimulation of secretion appeared to activate endogenous calpain in type II cells, suggesting that calpain-mediated proteolysis of a submembranous cytoskeletal protein could play an important role in the secretory process.

Two-stage autolysis of the catalytic subunit initiates activation of calpain I.

Calcium-induced autolysis of bovine erythrocyte calpain I occurs in multiple stages. Initially, a 14 amino acid segment is cleaved from the N-terminus of the native 80 kDa catalytic subunit, yielding a 78 kDa form of the subunit. Then, an additional 12 amino acid segment is cleaved from the N-terminus, forming a 76 kDa subunit. The 76 kDa enzyme is the active form of the catalytic subunit that is able to proteolyze the 30 kDa regulatory subunit as well as exogenous substrates. While the initial autolytic step requires high calcium, the 76 kDa enzyme form is active in microM calcium and can cleave the amino termini of native 80 kDa and intermediate 78 kDa enzyme forms at low calcium. Both intramolecular and intermolecular proteolysis of the catalytic subunit appear to yield the same products.

Activation of calpain I and calpain II: a comparative study using terbium as a fluorescent probe for calcium-binding sites.

The present study demonstrates the activation of calpain I and calpain II by micromolar levels of terbium and has utilized the enhancement in the fluorescence of protein-bound terbium to study and compare the calcium binding sites of the two enzymes. Calpain I and calpain II were isolated from bovine erythrocytes and brain, respectively. While the rates of activation of calpain I by terbium and calcium are comparable, the rate of activation of calpain II was much greater in the presence of terbium than in the presence of calcium. Binding of terbium ions to calpains was monitored by the enhanced terbium fluorescence and by the changes in the intrinsic protein fluorescence of calpains. Stoichiometric titrations indicated that calpain I and calpain II bound four and six molar equivalents of terbium ion, respectively. During the titration, the intrinsic protein fluorescence of calpain II was successively quenched whereas that of calpain I showed an abrupt drop just prior to the saturation. The association constants (Ka) increased from 10(5) to 10(7) M-1 for calpain I and from 10(4) to 10(6) M-1 for calpain II with addition of increasing molar equivalents of terbium. Titration of enzymatic activities with calcium showed that the activation of calpain I required fewer molar equivalents of metal ions than were necessary for the activation of calpain II, in agreement with stoichiometric titration with terbium.

Calcium-activated neutral proteases (calpains) are carbohydrate binding proteins.

Calcium-activated neutral proteases (calpain, EC 3.4.22.17) bind to agarose matrices (Bio-Gel A-150m, Sepharose 4B, and Ultrogel AcA 34) with high affinity in the presence of calcium. 6-O-beta-Galactopyranosyl-D-galactose, a disaccharide which closely resembles the repeating unit of the agarose matrices, completely blocks the binding of calpains and can release agarose-bound enzymes in the presence of calcium. At least 1 microM level of free calcium is required for binding. Other calcium binding proteins, including calmodulin, calpastatin, casein, and neurofilament proteins, fail to bind under the same conditions. Both calpain I and calpain II can be readily purified from crude enzyme preparations by agarose chromatography in the presence of calcium and leupeptin. Agarose-bound enzymes are eluted with calcium-free solutions or can be released in the presence of calcium by 1% Triton X-100, but not by 1 M urea or 20% ethylene glycol. Enzymes eluted from agarose are activated, as evidenced by the appearance of faster migrating forms (76 and 78 kDa) of the 80-kDa catalytic subunit of calpain I upon electrophoresis and by the increased sensitivity of calpain II to activation by micromolar levels of calcium. The electrophoretic migration of the 30-kDa regulatory subunit is, however, unaltered in enzyme fractions eluted from an agarose column. When the enzyme subunits are dissociated in 1 M NaSCN, only the 30-kDa subunit binds to the agarose matrix. Furthermore, neither calpain I nor calpain II binds to agarose when their 30-kDa subunit is autocatalyzed to an 18-kDa fragment, indicating that the NH2-terminal of the 30-kDa subunit is important for the binding of calpains to an agarose matrix.

Clustering of phosphorylated amino acid residues in neurofilament proteins as revealed by 31P NMR.

The state of phosphorylation in neurofilament (NF) proteins is studied by the 31P NMR technique. The 31P NMR spectrum of intact NF proteins at pH 7.0 is comprised of a major resonance at 4.18 ppm and a minor resonance at 3.53 ppm. The chemical shifts of the major and minor resonances are strongly dependent on pH and have pKa values for phosphoserine of 5.85 and for phosphothreonine of 6.00, respectively. 31P NMR spectra of isolated NF polypeptides show nonequivalent phosphoserine clusters in NF150 and in NF200. Their chemical shifts are very similar in both polypeptides, but the intensities of homologous resonances are different. NF68 has no detectable 31P resonance signal. Phosphate-specific monoclonal antibodies to NF200 can distinguish phosphates of various clusters. Microtubule proteins can also produce specific alteration of the 31P resonances of NF200. NF proteins digested by calcium-activated neutral protease (CANP) show relatively little change in 31P resonances.

Characterization of calcium-activated neutral protease (CANP)-associated protein kinase from bovine brain and its phosphorylation of neurofilaments.

Calcium-activated neutral protease with low affinity for calcium (CANP II, Mr 76,000) can be purified to apparent homogeneity by casein affinity chromatography but contains cyclic-AMP dependent protein kinase activity. CANP II-associated kinase from bovine brain copurifies with protease activity through multiple chromatographic procedures but can be separated by cyclic-AMP affinity chromatography. Isolated protein kinase has subunits of Mr 80,000, 53,000 and 42,000. The kinase preferentially "autophosphorylates" CANP II, but histones, phosphorylase b and neurofilament proteins are also good substrates. The concentrations for half-maximal phosphorylation activity (Km) of cyclic-AMP, (32P)ATP and Mr 150,000 neurofilament protein substrate are 0.2, 6.0 and 0.5 microM, respectively. The specific activity of CANP II associated kinase in phosphorylating neurofilament proteins is intermediate between that of neurofilament- and MAPs 2-associated kinases.

An immunoblot study of neurofilament degradation in situ and during calcium-activated proteolysis.

The degradation of neurofilament (NF) proteins was examined by immunoblot methods to identify, characterize, and monitor the appearance of immunoreactive breakdown products during the loss of NF triplet proteins. Individual NF proteins and their breakdown products were identified using polyclonal and monoclonal antibodies to NF proteins. NF degradation was compared during calcium-activated proteolysis of isolated rat NF, during an experimental influx of calcium into excised rat spinal nerve roots, and during NF breakdown in transected rat peripheral nerve. These different experimental conditions produced similar patterns of NF fragmentation, including the transient appearance of NF immunobands between Mr 150,000-200,000 and 110,000-120,000 as well as the appearance and accumulation of NF immunobands between Mr 45,000 and 65,000. Most immunoreactive NF fragments remained Triton-insoluble. Low levels of the same immunoreactive fragments were present in control neural tissues, suggesting that calcium-activated proteolysis may be operative in the turnover and/or processing of NF proteins in vivo. Very similar patterns of NF degradation during experimental calcium influxes into different CNS and PNS tissues are indicative of the widespread distribution of calcium-activated NF protease in neural tissues.