Isolation and characterization of monoclonal antibodies against calcium-activated neutral protease with low calcium sensitivity

Fifteen hybridomas secreting antibodies against calcium-activated neutral protease (CANP), especially those for rabbit muscle mCANP with low calcium sensitivity, have been produced by the cell fusion technique. Eight of the monoclonal antibodies belong to the class IgG1, one to the class IgG2a, and six to the class IgG2b. The antibodies from these clones were characterized with regard to their relative binding affinities to the large subunits (80K) and the small subunits (30K) of mCANP as well as mu CANP, which is another type of CANP with high calcium sensitivity. Fourteen antibodies bound only to the 80K subunit of mCANP and one antibody bound to the 80K subunit of both mCANP and mu CANP. These antibodies recognized rat mCANP but not chicken CANP, with the exception of one antibody. Examination of the effects of these antibodies on the enzyme activity of mCANP showed that six antibodies partially inhibited the enzyme activity and the others were noninhibitory. These monoclonal antibodies should be useful for analyzing the fine structure of CANPs and the mechanism of the activation of mCANP, and also for determining the intracellular localization of mCANP.     

Calcium-Activated Neutral Proteinase of Human Brain: Subunit Structure and Enzymatic Properties of Multiple Molecular Forms

Abstract Calcium-activated neutral proteinase (CANP) was purified 2,625-fold from postmortem human cerebral cortex by a procedure involving chromatography on diethylaminoethyl (DEAE)-cellulose, phenyl-Sepharose, Ultrogel AcA-44, and DEAE-Biogel A. The major active form of CANP exhibited a molecular weight of 94–100 kilodaltons (Kd) by gel filtration on Sephacryl 300 and consisted of 78-Kd and 27-Kd subunits. Two-dimensional gel electrophoresis resolved the small subunit into two molecular species with different isoelectric points. CANP degraded most human cytoskeletal proteins but was particularly active toward fodrin and the neurofilament protein subunits (145 Kd > 200 Kd > 70 Kd). The enzyme required 175 μMCa²⁺ for half-maximal activation and 2 mM Ca²⁺ for optimal activity toward [methl-¹⁴C]azocasein. Other divalent metal ions were poor activators of the enzyme, and some, including copper, lead, and zinc, strongly inhibited the enzyme. Aluminum, a neurotoxic ion that induces neurofilament accumulations in mammalian brain, inhibited the enzyme 47% at 1 mM and 100% at 5 mM A second CANP form lacking the 27-Kd subunit was partially resolved from the 100-Kd heterodimer during DEAE-Biogel A chromatography. The 78-Kd monomer exhibited the same specific activity, calcium ion requirement, pH optimum, and specificity for cytoskeletal proteins as the 100-Kd heterodimer, suggesting that the 27-Kd subunit is not essential for the major catalytic properties of the enzyme. The rapid autolysis of the 27-Kd subunit to a 18-Kd intermediate when CANP is exposed to calcium may explain differences between our results and previous reports, which describe brain mCANP in other species as a 76-80-Kd monomer or a heterodimer containing 76-80-Kd and 17-20-Kd subunits. The similarity of the 100-Kd human brain CANP to CANPs in nonneural tissues indicates that the heterodimeric form is relatively conserved among various tissues and species.     

A low-calcium-requiring calcium-activated neutral proteinase from human placenta

A low-calcium-requiring calcium-activated neutral proteinase (mu CANP) has been purified to homogeneity from human placenta. The purification procedure includes chromatography on DEAE-cellulose, Ultrogel AcA-22 and DEAE-Sephadex in succession. The purified mu CANP is a thiol proteinase and requires calcium for activity. Half-maximal activation occurs at 40 microM calcium. It is a heterodimer with subunits of 74 kDa and 32 kDa. (The placental mCANP has subunits of 70 kDa and 32 kDa.) Mn2+ or Sr2+, in combination with Ca2+, activates the enzyme synergistically. The presence of both mCANP and mu CANP in equal proportion in human placenta is reported for the first time. This will facilitate a comparative study of these two forms of human calcium-activated neutral proteinase, especially their physiological structural and functional interrelationship. Maximal activation of the autolysed mCANP occurs at a calcium concentration much higher than that for mu CANP; and this autolysed mCANP does not cross-react with antiserum against mu CANP, suggesting that the two forms of proteinase are independent species.     

Purification and characterization of a calcium-activated neutral protease from rabbit skeletal muscle which requires calcium ions of microM order concentration

One form of calcium-activated neutral protease (CANP) highly sensitive to calcium ions was purified by column chromatographic procedures to homogeneity. The purified enzyme required microM order Ca2+ (mu CANP), and the half-maximum activity was attained at 50 microM Ca2+. The electrophoretic mobility in a non-denaturing buffer showed that this enzyme is less acidic than another CANP which required mM order Ca2+ (mCANP). On SDS-polyacrylamide gel electrophoresis, the enzyme separated into two components with molecular weights of 79,000 and 28,000, respectively. Of these, the former was slightly larger than the counterpart of mCANP (Mr 76,000). Thus, mu CANP cannot be derived from mCANP by limited autolysis.     

Tissue distribution of an endogenous inhibitor of calcium-activated neutral protease and age-related changes in its activity in rats

1. The distribution of an endogenous inhibitor of calcium-activated neutral protease (CANP) and age-related changes in its activity were studied in male and female rats of different ages by a fluorometric assay on tissue extracts after heat treatment. 2. Ubiquitous distribution of CANP inhibitor in brain, cardiac muscle, lung, spleen, liver, skeletal muscle, kidney and testis and its abundance in spleen, liver and kidney was demonstrated. 3. Comparison in terms of units/ml of crude extracts showed that the level of CANP inhibitor exceeded that of CANP in most tissues and that the relative content of CANP inhibitor to mCANP and microCANP differed greatly among tissues. 4. Sex and species differences in CANP inhibitor activity in each tissue were of little significance. 5. Changes in CANP inhibitor during aging from 6 to 12 months was not obvious but senescent rats showed a tendency toward increased inhibitor activity. This increase was especially evident in the testis.     

Tissue distribution of calcium-activated neutral proteinases in rat

A simple separation method involving a minimum number of essential steps was established to separate the two kinds of calcium-activated neutral proteinase (CANP) activity from each other and from endogenous CANP inhibitor activity. Determination of CANP activity in rat tissues using this method revealed a ubiquitous distribution of two CANPs. The level of CANP activity, however, differs between tissues. Low-calcium-requiring CANP (microCANP) is plentiful in spleen and kidney, while high-calcium-requiring CANP (mCANP) is plentiful in lung and brain. In all of the tissues examined, mCANP activity predominates over microCANP activity.     

Specificity of calcium-activated neutral proteinase (CANP) inhibitors for human μCANP and mCANP

We investigated the relative inhibition of purified human CANP and mCANP by five cysteine proteinase inhibitors including N-acetyl-Leu-Leu-nor-leucinal (C-I) and N-acetyl-Leu-Leu-methioninal (C-II), calpeptin, E64, and leupeptin. Based on IC₅₀ measurements, calpeptin and C-I were stronger inhibitors by one to two orders of magnitude than C-II, leupeptin or E64. None of the five inhibitors, however, exhibited greater specificity for human CANP or mCANP. These results indicate that, although the inhibition of a given cellular event by these compounds may suggest CANP involvement, effects on CANP cannot be discriminated from those on mCANP.     

The regional and subcellular distribution of calcium activated neutral proteinase (CANP) in the bovine central nervous system

Calcium-activated neutral proteinase (CANP) activity was determined in subcellular fractions and in different regions of bovine brain. The CANP specific activity in spinal cord and corpus callosum, areas rich in myelin, were almost six-fold greater than cerebral cortex and cerebellum. Treatment of whole homogenate and myelin with 0.1% Triton X-100 increased the CANP activity by tenfold. Subcellular fractions were prepared from bovine brain gray and white matter. Most of the CANP activity (70%) was in the primary particulate fractions P₁ (nuclear), P₂ (mitochondrial) and P₃ (microsomal). On subfractionation of each particulate fraction, the majority of the activity (greater than 50%) was recovered in the myelin-enriched fractions (P₁A, P₂A, P₃A) which separate at the interphase of 0.32 M- and 0l85 M-sucrose. The distribution of activity was P₂A>P₁A>P₃A. Further purification of myelin (of P₂A) increased the specific activity over homogenate by more than three-fold. The same myelin fractions contained the highest proportion (60%) and specific activity (five-fold increase) of CNPase. The enzyme activity in different regions of brain and in subcellular fractions was increased by 20–39% after the inhibitor was removed. Electron microscopic study confirmed that the myelin fractions were highly purified. The cytosolic fraction contained 20–30% of the total homogenate CANP activity. Other fractions contained low enzyme activity. CANP was identified in the purified myelin fraction by electroimmublot-technique. It is concluded that the bulk of CANP in CNS is tightly bound to the membrane, may be masked or hidden and is intimately associated with the myelin sheath.Abbreviations Used CANP calcium-activated neutral proteinase - CNPase adenosine-2, 3-cyclic nucleotide 3-phosphohydrolase     

Regulation of the calcium-activated neutral proteinase (CANP) of bovine brain by myelin lipids

Since calcium-activated neutral proteinase (CANP; calpain) activation occurs at the plasmalemma and the enzyme is found in myelin, we examined myelin lipid activation of brain CANP. Purified lipids were dried, sonicated and incubated with purified myelin CANP. The CANP was assayed using [14C]azocasein as substrate and the Ca2+ concentration ranged from 2 microM for muCANP to 5 mM for mCANP. Phosphatidylinositol (PI), phosphatidylserine (PS) and dioleoylglycerol stimulated the mCANP activity by 193, 89 and 78%, respectively. PI stimulated both m- and muCANP in a concentration-dependent manner, while phosphatidylcholine was least effective. Cerebroside and sulfatide at higher concentrations (750 microM) were stimulatory. The phospholipid (PL)-mediated activation was inhibited by the PL-binding drug trifluoperazine. PI reduced the Ca2+ requirement for CANPs significantly (20-fold). These results suggest that acidic lipids and particularly acidic phospholipids activate membrane CANP.     

Fragmentation of an endogenous inhibitor upon complex formation with high- and low-Ca2+-requiring forms of calcium-activated neutral proteases

The interaction of an endogenous inhibitor for the calcium-activated neutral protease (CANP or calpain EC 3.4.22.17) with CANP was examined by SDS-polyacrylamide gel electrophoresis, immunoblot analysis, and gel filtration. Fragmentation of the inhibitor (Mr 110K) by mCANP, a high-Ca2+-requiring form, was shown only in the presence of Ca2+ ions of millimolar order, with decreased inhibitor activity recovered from gel extracts in the 110-kDa area. This fragmentation took place even when the inhibitor could completely inhibit the caseinolytic activity of mCANP. The fragmented inhibitor retained considerable inhibitor activity after the CANP-inhibitor complex was dissociated by the addition of EDTA, and 69% of the initial activity was recovered from the mixture reacted with excess mCANP lacking the 110-kDa band. A C-terminal fragment of CANP inhibitor produced in Escherichia coli (Mr 40K) was also hydrolyzed by mCANP in the presence of Ca2+. The interaction of both forms of the inhibitor with mu CANP, a low-Ca2+-requiring form, led to the same phenomena in the presence of micromolar levels of Ca2+. CANP inhibitor could not completely inhibit the autolysis of mCANP and mu CANP, indicating that these were intramolecular events. Gel filtration analysis revealed that the mass of the smallest fragment with inhibitor activity was about 15,000 daltons. These results suggest that CANP inhibitor may act in the manner of a suicide substrate.     

Comparison of calcium-activated neutral proteases from skeletal muscle of rabbit and chicken

Calcium-activated neutral proteases (CANPs) were purified from rabbit skeletal muscle and chicken skeletal muscle, and compared as to their electrophoretic properties, metal requirements, subunit amino acid compositions and immunological cross-reactivities. Two kinds of CANPs (mu CANP and mCANP) were isolated from rabbit but the chicken tissue lacked one corresponding to mu CANP. They were acidic in the order of chicken mCANP, rabbit mCANP, and rabbit mu CANP but the difference between the former two was very small. All of them were composed of two subunits, so-called 80K and 30K subunits. The molecular weight of the 30K subunit was the same for these CANPs (28K) but those of the 80K subunit were different (79K for rabbit mu CANP, 75K for rabbit mCANP and 81K for chicken mCANP). The calcium-sensitivity of chicken mCANP was very high when compared with that of rabbit mCANP and close to that of rabbit mu CANP. Antisera against chicken CANP and those against rabbit CANP cross-reacted with rabbit CANP and chicken CANP, respectively, when examined by immunoelectrotransfer blot techniques.     

Ganglioside-Modulated Proteolysis by Ca2+-Activated Neutral Proteinase (CANP): A Role of Glycoconjugates in CANP Regulation

We examined ganglioside modulation of the activity of the millimolar Ca2(+)-sensitive form (mCANP) of calcium-activated neutral proteinase (CANP), which is enriched in myelin, from brain. GM1, GD1a, GT1a, GM2, and GM4 produced a concentration-dependent increase of mCANP activity. GD1a stimulated the greatest increase of enzyme activity (107%), followed by GT1a, whereas GD1b was inhibitory (56%). GM1, GM2, and GM4 stimulated but less so than GD1a and GT1a. Free N-acetylneuraminic acid, asialo-GM1, GM3, and a ganglioside mixture containing GM1, GD3, GD1a, and GD1b had no effect. The ganglioside-mediated modulation was not affected by trifluoperazine and chlorpromazine (phospholipid-binding antagonists). The mCANP Ca2+ requirement was significantly reduced in the presence of stimulatory gangliosides, and this increased sensitivity varied (10-50-fold) with ganglioside structure. Gangliosides may interact with membrane mCANP and modulate its proteolytic action.     

Limited autolysis of calcium-activated neutral protease (CANP): reduction of the Ca2+-requirement is due to the NH2-terminal processing of the large subunit

Calcium-activated neutral protease (rabbit mCANP), composed of large and small subunits, was converted to a lower-Ca2+-requiring form (derived microCANP) by limited autolysis in the presence of Ca2+. The NH2-terminal regions of the two subunits of mCANP were cleaved by autolysis, but the COOH-termini remained intact after autolysis. When native mCANP or derived microCANP was dissociated into subunits, the proteolytic activity of the large subunit was reduced to 2-5% of that of the native dimeric enzyme. The Ca2+-sensitivity of one hybrid CANP reconstituted from the large subunit of derived microCANP and the small subunit of native mCANP was similar to that of derived microCANP. However, the other hybrid molecule composed of the large subunit of native mCANP and the small subunit of derived microCANP required a high concentration of Ca2+ for activity, like native mCANP. These results indicate that the Ca2+-sensitivity of derived microCANP is determined by the structural change of the large subunit resulting from loss of its NH2-terminal region. The autolysis of the small subunit apparently has no effect on the reduction of the Ca2+-requirement.     

Comparison of low and high calcium requiring forms of the calcium-activated neutral protease (CANP) from rabbit skeletal muscle

Two distinct calcium-dependent neutral proteases (CANPs) with different sensitivities to calcium ions were purified concurrently by almost the same procedures from rabbit skeletal muscle and their enzymatic properties were compared (sensitivity to various divalent metal ions, the pH dependency and heat-stability of the activity, and the hydrolytic activity towards various substrates). They were further compared chemically in terms of the state of thiol groups, the amino acid compositions of subunits and the peptide fragments by digestion with S. aureus V8 protease. The low calcium requiring form of CANP (microCANP) was more sensitive to other divalent metal ions such as Sr2+ and Ba2+ than the high calcium requiring form of CANP (mCANP). The comparison of the pH dependency of these CANP activities showed that microCANP was active in a broader pH range than mCANP and the former was more heat-stable than the latter. Both CANPs had similar affinity to various substrates, but the hydrolytic velocity was several times higher with microCANP than with mCANP. Although they were inhibited by thiol protease inhibitors to the same extent, the states of thiol groups in them were quite different. The thiol group involved in the catalytic activity of the enzyme was exposed without adding Ca2+ in microCANP, whereas the group in mCANP became exposed only when sufficient Ca2+ was added. The large subunits of these two CANPs were different in their amino acid compositions and in the peptide fragment patterns produced by S. aureus V8 protease but the small subunits were indistinguishable from each other. These results led us to conclude that these two CANPs are quite different in nature and are not in a simple relationship, i.e., one of them is not derived from the other by autolysis or modification.     

Effect of metal ions on the structure and activity of calcium-activated neutral protease (CANP)

To clarify the mechanism of activation of calcium activated neutral protease (CANP, or mCANP: active at mM Ca2+), the structure of mCANP was examined by measuring CD spectra and by titration of SH groups in the presence of Mn2+. Mn2+ significantly increases the sensitivity of CANP to Ca2+ but CANP is not active with Mn2+ alone. The structural changes induced by Mn2+ were compared with those induced by Ca2+, and the structure of muCANP, which is active at microM Ca2+, was also examined for comparison. Mn2+ and Ca2+ induced the same structural changes of CANP. However, specific activation of the active site SH group by Ca2+ was not observed with Mn2+. Six moles of calcium bound to mCANP and the average dissociation constant of Ca2+ was 150 microM. The structure of muCANP was similar to that of mCANP in terms of the CD spectra. The titration of SH groups of muCANP indicated that the structure of muCANP was looser or SH groups were more exposed than in the case of mCANP. A model which can explain the activation of mCANP is proposed and the mechanism of activation is discussed based on the proposed model. The role of Ca2+ can be explained in terms of conformational change and activation of the active-site SH group of CANP.     

Degradation of Alzheimer's ß-Amyloid Protein by Human and Rat Brain Peptidases: Involvement of Insulin-Degrading Enzyme

We examined the degradation of Alzheimer's ß-amyloid protein (1–40) by soluble and synaptic membrane fractions from post mortem human and fresh rat brain using HPLC. Most of the activity at neutral pH was in the soluble fraction. The activity was thiol and metal dependent, with a similar inhibition profile to insulin-degrading enzyme. Immunoprecipitation of insulin-degrading enzyme from the human soluble fraction using a monoclonal antibody removed over 85% of the ß-amyloid protein degrading activity. Thus insulin-degrading enzyme is the main soluble ß-amyloid degrading enzyme at neutral pH in human brain. The highest ß-amyloid protein degrading activity in the soluble fractions occurred between pH 4–5, and this activity was inhibited by pepstatin, implicating an aspartyl protease. Synaptic membranes had much lower ß-amyloid protein degrading activity than the soluble fraction. EDTA (2mM) caused over 85% inhibition of the degrading activity but inhibitors of endopeptidases –24.11, –24.15, –24.16, angiotensin converting enzyme, aminopeptidases, and carboxypeptidases had little or no effect.     

Molecular cloning of the cDNA for the large subunit of the high-Ca2+-requiring form of human Ca2+-activated neutral protease

A nearly full-length cDNA clone for the large subunit of high-Ca2+-requiring Ca2+-activated neutral protease (mCANP) from human tissues has been isolated. The deduced protein, determined for the first time as an mCANP, has essentially the same structural features as those revealed previously for the large subunits of the low-Ca2+-requiring form (muCANP) [Aoki, K., Imajoh, S., Ohno, S., Emori, Y., Koike, M., Kosaki, G., & Suzuki, K. (1986) FEBS Lett. 205, 313-317] and chicken CANP [Ohno, S., Emori, Y., Imajoh, S., Kawasaki, H., Kisaragi, M., & Suzuki, K. (1984) Nature (London) 312, 566-570]. Namely, the protein, comprising 700 amino acid residues, is characterized by four domains, containing a cysteine protease like domain and a Ca2+-binding domain. The overall amino acid sequence similarities of the mCANP large subunit with those of human muCANP and chicken CANP are 62% and 66%, respectively. These values are slightly lower than that observed between muCANP and chicken CANP (70%). Local sequence similarities vary with the domain, 73-78% in the cysteine protease like domain and 48-65% in the Ca2+-binding domain. These results suggest that CANPs with different Ca2+ sensitivities share a common evolutionary origin and that their regulatory mechanisms are similar except for the Ca2+ concentrations required for activation.     

Regional and Subcellular Distribution of ATP-Citrate Lyase and Other Enzymes of Acetyl-CoA Metabolism in Rat Brain

The activities of ATP-citrate lyase in frog, guinea pig, mouse, rat, and human brain vary from 18 to 30 μmol/h/g of tissue, being several times higher than choline acetyltransferase activity. Activities of pyruvate dehydrogenase and acetyl coenzyme A synthetase in rat brain are 206 and 18.4 μmol/h/g of tissue, respectively. Over 70% of the activities of both choline acetyltransferase and ATP-citrate lyase in secondary fractions are found in synaptosomes. Their preferential localization in synaptosomes and synaptoplasm is supported by RSA values above 2. Acetyl CoA synthetase activity is located mainly in whole brain mitochondria (RSA, 2.33) and its activity in synaptoplasm is low (RSA, 0.25). The activities of pyruvate dehydrogenase, citrate synthase, and carnitine acetyltransferase are present mainly in fractions C and B_p. No pyruvate dehydrogenase activity is found in synaptoplasm. Striatum, cerebral cortex, and cerebellum contain similar activities of pyruvate dehydrogenase, citrate synthase, carnitine acetyltransferase, fatty acid synthetase, and acetyl-CoA hydrolase. Activities of acetyl CoA synthetase, choline acetyltransferase and ATP-citrate lyase in cerebellum are about 10 and 4 times lower, respectively, than in other parts of the brain. These data indicate preferential localization of ATP-citrate lyase in cholinergic nerve endings, and indicate that this enzyme is not a rate limiting step in the synthesis of the acetyl moiety of ACh in brain.     

Purification of a calcium-activated neutral proteinase from bovine brain

A calcium-activated neutral proteinase (CANP) resolved into three components has been partially purified from bovine brain. The method of isolation has resulted in 22,000, 7,100, and 8,000-fold purification for CANP I, II and III respectively. All three fractions require Ca2+ for activation. The characterization of the purified CANP I has shown that it is activated by 250 microM Ca2+ and the enzyme loses its activity when incubated in the presence of Ca2+ without substrate. Mg2+ is ineffective. The enzyme degrades neurofilament triplet proteins, tubulin and casein efficiently. The myelin basic protein is hydrolyzed after longer incubation. Bovine serum albumin and histones are unaffected. The enzyme is active at pH 5.5 to 9.0 with optimum between pH 7.5 and 8.5. It has a Km of 1.8 X 10(-7) M for the 69,000 dalton neurofilament protein. The enzyme is inhibited by sulphydryl blocking reagents and also by EGTA, leupeptin and E-64c. The SDS-PAGE analysis of the enzyme fractions has shown a major band at 66-68,000 daltons and two minor bands at 60,000 and 48-50,000 daltons for CANP I; a major band at 48-50,000 daltons and a minor band at 30-32,000 daltons for CANP II and a predominant doublet at 30-32,000 daltons with a minor band at 48-50,000 daltons for CANP III. The degradation of neurofilament proteins suggests that the CANP(s) may be involved in the turnover of these proteins.     

Identification of Ca2+-Activated Neutral Protease in the Peripheral Nerve and Its Effects on Neurofilament Degeneration

Rat sciatic nerve segments were incubated in five different media. Disappearance of neurofilament (NF) triplet proteins (200K, 160K, and 68K MW) occurred in medium containing Ca²⁺ and was inhibited by the addition of E-64-c or leupeptin. Therefore, the presence in the peripheral nerve of an enzyme whose properties are similar to those of Ca²⁺-activated neutral protease (CANP) is suggested. The extraction of crude CANP from rat sciatic nerve was performed. CANP activity was completely recovered (0.129 ± 0.008 U/g) in the precipitate salted out by the addition of 0 to 50% saturated ammonium sulfate to the soluble fraction of the peripheral nerve (crude CANP). Properties of the crude CANP were examined using NF as a substrate and were found to be similar to those of the CANP extracted from skeletal muscle. Identification of the crude CANP with the CANP extracted from rat skeletal muscle was performed using the immunoreplica method. Bands corresponding to 73K were detected in both CANPs.