Purification of a calcium-activated neutral proteinase from human placenta

A calcium-activated neutral proteinase has been purified to homogeneity from human placenta. The purified enzyme is a dimer composed of Mr 73 000 and 30 000 subunits. Half-maximal activity is observed at 250 microM Ca2+. It requires reduced sulfhydryl groups and neutral pH for optimal activity. Leupeptin, antipain, E-64, sulfhydryl-blocking agents and endogenous proteinase inhibitor inhibit the purified enzyme. This paper is the first to describe the purification and characterization of a calcium-activated neutral proteinase from a human non-muscular parenchymatous organ.     

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.     

Calcium-induced localization of calcium-activated neutral proteinase on plasma membranes

The location of calcium-activated neutral proteinase (CANP) was determined in human erythrocytes by crosslinking CANP to co-localizing proteins using a photolabeling bifunctional reagent, 4,4'-dithiobisphenylazide (DTBPA). The crosslinked products were selectively isolated by immunoprecipitation with a polyclonal anti-CANP antibody and analyzed by SDS-polyacrylamide gel electrophoresis after cleavage of the crosslinkage. In the calcium-free incubation medium the main proteins crosslinked with CANP were cytosolic proteins such as hemoglobin. In the presence of calcium ions, on the other hand, membrane skeletal proteins such as spectrin, band 4.1, 4.2 and 6 proteins as well as band 3 were crosslinked with CANP. Addition of calcium ionophore further increased the amount of crosslinked membrane proteins. These results suggest that in the absence of calcium ions CANP exists diffusely in the cytoplasm and is crosslinked with cytoplasmic hemoglobin nonspecifically while in the presence of calcium ions CANP associated with membrane where it is crosslinked specifically with the lining proteins. Thus it is demonstrated biochemically that the localization of CANP is dynamic depending on the presence of calcium ions.     

Endogenous inhibitor of calcium activated neutral proteinase from human placenta: Purification and possible mechanism of proteinase regulation

An endogenous inhibitor of calcium activated neutral proteinase has been purified from human placenta. The procedure included chromatography on DEAE cellulose, Ultrogel AcA 22 and milli calcium activated neutral proteinase-sepharose in succession. Endogenous calcium activated neutral proteinase inhibitor was a tetramer with identical subunits of molecular weight 68 kDa. It was specific for milli calcium activated neutral proteinase (Calpain II) which is inhibited by the formation of an inactive enzyme-inhibitor complex and not by sequestering Ca₂₊ from the medium. Although micro calcium activated neutral proteinase (Calpain I) was not inhibited by endogenous calcium activated neutral proteinase inhibitor, it was protected from autolysis in the presence of the inhibitor. The placental endogenous calcium activated neutral proteinase inhibitor thus regulates Ca₂₊ activated proteolysis by ensuring micro calcium activated neutral proteinase activity, while inhibiting milli calcium activated neutral proteinase.     

Characterization of cDNA clones encoding a novel calcium-activated neutral proteinase from Schistosoma mansoni

To identify and characterize Schistosoma mansoni proteins that are recognized by infected hosts, we have used a pool of sera from infected humans to screen cDNA libraries constructed from poly(A)+ mRNA of adult S. mansoni. The deduced amino acid sequences of the three isolated clones showed a high degree of similarity to the large subunit of calcium-activated neutral proteinase (CANP) from humans and chicken. These overlapping clones, which include a nearly full-length clone with an open reading frame of 758 amino acid residues, together encode the entire large subunit of CANP. The deduced sequence of this S. mansoni protein can be divided into four domains (I-IV) that include the two domains characteristic of other large subunits of CANP: a thiol-protease domain (II) and a calcium-binding domain (IV) containing EF hand motifs. However, the schistosome protein is unique in having only three EF hand motifs in the calcium-binding domain and in having an additional EF hand motif that is shared between domains II and III. We have shown that these EF hand motifs are capable of binding 45Ca2+. Furthermore, the large subunit is S. mansoni contains an NH2-terminal sequence of 28 residues that is absent from the mammalian CANPs and has a high degree of similarity to the presumed receptor binding sequence of colicin Ia and Ib.     

Diabetic state-induced activation of calcium-activated neutral proteinase in mouse skeletal muscle

The effect of a diabetic state in the diabetic KK-CAy mouse on calcium activated neutral proteinase (CANP) of hind-limb skeletal muscles was investigated. In the diabetic state, there was an increased sensitivity to activation of CANP by calcium (Ca). In addition, there was an enhancement of maximal activity of the enzyme. The effect was induced by secondary modification of the diabetic state, but not genetical factors. Several lines of evidence suggest that the CANP is responsible for 92 K dalton protein in diabetic skeletal muscles. Among the evidence are the following: a) The 92 K band in the diabetic muscles was lower than in the prediabetic mouse and restored by the addition of 2 mM EDTA and 2 mM EGTA. b) The band was reduced by increasing the Ca content and neutral pH in the non-diabetic normal muscles. c) E-64-C, a CANP inhibitor, restored the 92 K component reduced by the diabetic state. Since the band in denervated muscles was not changed by the Ca chelating agents, the reduction of the band in the diabetic muscles is related with musculotrophic factors, not diabetic neuropathy. These results suggest that diabetic amyotrophy may be regarded as a phenomenon linked to an increase in intracellular Ca ions and an increase in CANP activity.     

Purification of calcium-activated neutral proteinase (CANP) from purified myelin of bovine brain white matter

A calcium-activated neutral proteinase was purified from myelin of bovine brain white matter. Myelin purified in the presence of EDTA (2 mM) was homogenized in 50 mM Trisacetate buffer at pH 7.5, containing 4 mM EDTA, 1 mM NaN₃, 5 mM -mercaptoethanol and 0.1% Triton X-100 for two hours. After centrifugation at 87,000g for 1 hour, the supernatant was subjected to purification through successive column chromatography as follows: i) DEAE-cellulose, ii) Ultrogel (AC-34) filtration, iii) Phenyl-Sepharose, iv) a second DEAE-cellulose. The enzyme activity was assayed using azocasein as substrate. The myelin enzyme was purified 2072-fold and SDS-PAGE analysis of the purified enzyme revealed a major subunit of 72–76 K. The enzyme was inhibited by iodoacetate (1 mM), leupeptin (1 mM), E-64C (1.6 mM), EGTA (1 mM), antipain (2 mM) and endogenous inhibitor calpastatin (2 g). It required 0.8 mM Ca²⁺ for half-maximal activation and 5 mM Ca²⁺ for optimal activation. Mg²⁺ (5 mM) was ineffective while Zn²⁺ and Hg²⁺ were inhibitory. The pH optimum was ranged from 7.5–8.5. Treatment of myelin with Triton X-100 increased the enzyme activity by 10-fold suggesting it is membrane bound whereas the purufied enzyme was not activated by Triton X-100 treatment. The presence of CANP in myelin may mediate the turnover of myelin proteins and myelin breakdown in degenerative brain diseases.     

Cloning and molecular characterization of calpain, a calcium-activated neutral proteinase, from different strains of Schistosoma japonicum

cDNA coding for calpain of Schistosoma japonicum were cloned and sequenced, and serological basis of host responses to calpain were analyzed. cDNA of calpain from S. japonicum of two different isolates, Yamanashi strain (Sj-J) and Hunan strain (Sj-C), were 2, 468 bp and 2, 465 bp in length, including the same number (2, 274) of open reading frame. Nucleotide sequence and amino acid sequence between the two calpains are 99.1% and 98.8% identity, respectively. Sj-J and Sj-C calpains were considered to be translated as a preproenzyme, and a 746-amino acid mature enzyme contains eight motifs without a signal peptide at the N-terminal based on the deduced amino acid sequences. mRNA for calpain were detectable in different developmental stages, however, sera obtained from mice immunized with recombinant calpain showed enhanced binding to cercarial antigen. Human sera from S. japonicum-infected individuals recognized the large subunit of schistosomal calpain, and light-infected sera showed stronger reactivities to the recombinant calpain than moderate/high infection cases. When we tested synthetic peptides, there were four common human B cell epitopes in schistosomal calpain, all of which are shared with S. mansoni. Together with these results, calpain of S. japonicum seems to be not only a vaccine candidate, but also a target antigen for immunodiagnosis of human schistosomiasis.     

Specificity of neuropeptide degradation by two calcium-activated neutral proteases from human skeletal muscle

Two calcium-activated neutral proteases (CAPI & II) were purified from human skeletal muscle by anion exchange, gel filtration and affinity (antipain-Sepharose and Blue Ultrogel A4R) chromatography. The enzymes were homogenous as judged by polyacrylamide gel electrophoresis, and have similar properties with the exception of the Ca2+ concentration required for optimum activity (CAP I = 0.1 mM; CAP II = 1 mM). Both enzymes hydrolysed a wide variety of neuropeptides. In six cases, the products were separated and identified by hplc and amino acid analysis. Neurotensin was hydrolysed at Tyr3-Glu4; dynorphin1-13 at Arg8-Arg9; LH-RH at Gly6-Leu7; CCK-8 at Phe8-NH2, substance-P at Met10-NH2; somatostatin at Thr10-Phe11. Although differences in the rates of neuropeptide degradation were noted for the two CAP's the specificity was the same for these six peptides. It is suggested that conformational requirements may be more important than side chains adjacent to the cleavage site in directing the specificity of CAP.     

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.     

Activation of intracellular calcium-activated neutral proteinase in erythrocytes and its inhibition by exogenously added inhibitors

Intracellular calcium-activated neutral proteinase (CANP) in rabbit erythrocytes was activated by an influx of Ca2+ into the cells. The catalytic large subunit changed from the original 79 kDa from to the 77 kDa and 76 kDa forms on activation just in the same manner as occurs in the autolytic activation of purified CANP in vitro. The activation required both extracellular Ca2+ and A23187, and was accompanied by the degradation of some membrane proteins and morphological changes in erythrocyte shape from discocytes to echinodisks, echinocytes, and spherocytes. Exogenously added Cbz-Leu-Leu-Leu-aldehyde inhibited the activation of intracellular CANP as well as the degradation of membrane proteins and the morphological changes indicating that the latter two processes are due to the action of CANP. Leupeptin and E64d were without effect on intracellular CANP.     

A novel proteinase from human pancreas

A cholesterol-binding protein was previously isolated from human pancreas [Sziegoleit (1982) Biochem. J. 207, 573-582] and shown to consist of a single polypeptide chain with an apparent Mr of 28 000 and an isoelectric point of pH 4.9. In further investigations, a proteolytic activity was observed to be present in preparations of this protein. The enzyme activity was not dissociable from the cholesterol-binding protein. It decreased in the presence of sodium dodecyl sulphate or urea parallel to degradation of the protein, indicating autodegradation in the presence of these denaturants. Glucagon digestion studies indicated the carbonyl bond of alanine to be a favoured site of the enzymic cleavage. The proteinase was inactive against chromogenic substrates relatively specific for elastase, trypsin and chymotrypsin, but was found to cleave benzyloxycarbonylalanine p-nitrophenyl ester efficiently. The enzyme was inactivated by phenylmethanesulphonyl fluoride and was thus classified as a serine proteinase. Autoradiographic studies demonstrated binding to serum alpha 1-antitrypsin and alpha 2-macroglobulin in a similar manner to that observed with other pancreatic endo-proteinases. The collective results indicate that the isolated protein, provisionally named 'cholesterol-binding pancreatic proteinase', is a novel proteinase of the human pancreas. Quantitative measurements indicate that it comprises 4-6% of total protein in pancreatic secretions.     

Third form of calcium-activated neutral proteinase from calf brain: purification, partial characterization and comparison of properties with other forms

A third form (CANP3) of calcium-activated neutral proteinase (CANP) has been purified, 3900-fold, to near homogeneity from calf brain cortex. The purification procedure is based on the one recently developed for the purification of CANP1 and CANP2. The molecular weight of CANP3, as judged on SDS-polyacrylamide gel electrophoresis was Mr 78,000. A protein with an apparent Mr 17,000 co-purified with the proteinase. At neutral pH (7.2), it was maximally active at 260 microM CaCl2. In the presence of CaCl2, CANP1 and CANP3 were autolyzed very rapidly, whereas the autolysis of CANP2 was slow and gradual. The autolyzed CANP1 and CANP3 responded differently to CaCl2; CANP1 lost activity completely, whereas CANP3 was fully active at 0.5 microM CaCl2. Despite the opposite behavior of these proteinases in the presence of Ca2+, no significant differences in the peptide maps of the three proteinases were observed. Neurofilaments, neurotubules and myelin basic protein (MBP) were degraded by each of the proteinases. Monoclonal antibodies raised against CANP2 reacted almost equally with CANP1 and CANP3. As with CANP1 and CANP2, leupeptin and sulfhydryl-modifying compounds, NEM and iodoacetic acid, inhibited the activity of CANP3.     

Autolytic activation of calcium-activated neutral protease

Degradation of vimentin by native low calcium ion-requiring protease (mu CANP) was compared to that by autodigested mu CANP. On activation with 5 mM barium ions, a lag time was observed for the case of native mu CANP. This provides direct evidence that native mu CANP is inactive as a protease and must be autolyzed to be activated. Most of the protease activity can be accounted for by autodigested mu CANP with a 76 K polypeptide but another species with 50 K polypeptide may also be active.     

A neutral proteinase from human gingival fibroblasts active against the c-terminal cross-linking region of type I collagen

An enzyme that was capable of releasing small fragments containing hydroxynorleucine from the c-terminal extra-helical region of the alpha 1 chain of reduced (tritiated) soluble type I collagen was found, along with collagenase, in medium that had been conditioned by the culture of human gingival fibroblasts (Gin-1). The enzyme was present in a latent form or forms and could be activated by treatment with either trypsin or p-hydroxymercuribenzoate. It was maximally active at neutral pH and inhibited by EDTA. It is suggested that this enzyme, acting within a region of the molecule which is of major importance in stabilizing fibrillar collagen through intermolecular cross-linking, could potentially play an important role in collagen turnover in vivo.