Properties of erythrocyte membrane binding and autolytic activation of calcium-activated neutral protease

The binding of a calcium-activated neutral protease (CANP) with high calcium sensitivity (muCANP) to erythrocyte membranes and its subsequent autolytic activation on the membranes were analyzed by an immunoblot technique. In the presence of calcium ions, muCANP bound to the erythrocyte membranes as a heterodimer of 79- and 28-kDa subunits and was converted quickly on the membranes to an active form with a 76-kDa large subunit. The active form was then released from the membranes to the soluble fraction. These sequential reactions, however, were not specific to inside-out vesicles, but occurred also, except for some Ca2+-independent binding, on right side-out vesicles. A rapid degradation of some membrane proteins was observed after binding of muCANP to the membranes. The binding of muCANP to erythrocyte membranes was inhibited by substrates and the endogenous CANP inhibitor, which is also a suicide substrate. These results strongly suggest that muCANP binds to membranes by recognition of membrane proteins as substrates and not at a special site for activation. Thus, a possible mechanism for muCANP activation on membranes is that muCANP first binds to substrates on membranes, is activated, and then degrades the substrates to deform the membrane structures.     

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.     

Degradation of neuropeptides by calcium-activated neutral protease

The substrate specificity of calcium-activated neutral protease (CANP) from monkey cardiac muscle was examined with various neuropeptides as substrates. The enzyme required mM order calcium ions for activation and had an enkephalinase activity, hydrolyzing Leu-enkephalin at the 1Tyr-2Gly and 3Gly-4Phe bonds. Furthermore, it showed the tendency to cleave especially the bonds around the paired basic amino acid residues in alpha- and beta-neoendorphins and dynorphin(1-13), while it could not hydrolyze substance P.     

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.     

Preferential localization of calcium-activated neutral protease in epithelial tissues

Immunohistochemical localization of calcium-activated neutral protease (CANP) in rabbit organs was determined using a monoclonal antibody against CANP. In most organs, epithelial tissues reacted intensely: these tissues include great alveolar and squamous alveolar cells in lung; interlobular artery, vein, and bile duct in liver; small vessels in skeletal muscle; glomeruli, juxtanglomerular cells, distal and collecting tubules in kidney; mucous epithelium in gallbladder; interstitial cells in testis; and cuboidal epithelial cells in brain choroid plexus. On the other hand, hepatocytes, epithelial cells which have ill defined basal lamina, were stained very faintly. These observations suggest that the physiological function of CANP is involved with transport systems in epithelial tissues through basal lamina.     

Phosphorylation of bovine cardiac calcium-activated neutral protease by protein kinase-C

Protein kinase C prepared from rat brain was used to phosphorylate a calcium-activated neutral protease, purified from bovine cardiac muscle. Attempts to phosphorylate the enzyme in the presence of calcium were unsuccessful, unless the protease inhibitor leupeptin was also present. Phosphorylation of the 74K subunit of the protease was completely inhibited in the absence of phosphatidylserine and diolein, indicating that phosphorylation of the enzyme was catalysed by the calcium and phospholipid-dependent protein kinase C.     

Hydrolysis of protamine by calcium-activated neutral protease (CANP)

To determine the substrate recognition mechanism in calcium-activated neutral protease (CANP), the hydrolytic velocities for some possible substrates were compared. In general, succinylated polypeptides were poorer substrates than unmodified ones, suggesting that CANP interacts with positively charged amino groups and/or repels negatively charged succinyl groups in substrates. Among the substrates examined, protamine was degraded quite rapidly in a restricted manner. This degradation of protamine was remarkably accelerated by the addition of salt, and, in the absence of salt, protamine was inhibitory as to the degradation of vimentin by CANP. Protamine was separated into components and the sites cleaved by CANP were determined. CANP cleaved the clupeine YII and Z components at two sites, both being arginyl-arginine bonds, and the amino acid sequences around these sites were almost identical between YII and Z. No other arginyl-arginine bond was cleaved at all. These results showed that CANP prefers basic amino acid side chains but its specificity is very restricted.     

Primary structure and evolution of calcium-activated neutral protease (CANP)

The amino acid sequences of two subunits (80K and 30K) of calcium-activated neutral protease (CANP) were examined to clarify the structure-function relationship of CANP. The 80K subunit is composed of four clear domains (I–IV from the N-terminus). Domain II is a cysteine proteinase domain homologous to cathepsins B, L, and H. Domain IV is a calcium binding domain with four consecutive EF-hand structures known as typical calcium-binding sites found in calmodulin. The 30K subunit also has a clear domain structure (two domains). The N-terminal domain, a Gly-rich hydrophobic domain, probably determines the location of CANP through association with cellular membrane. The C-terminal domain is a calmodulinlike calcium-binding domain highly homologous to IV in the 80K subunit. The protease activity ascribable to II is regulated by 2 moles of built-in calmodulins, though its precise regulation mechanism is unknown. These results are discussed together with the molecular evolution of CANP on the basis of the gene structures of the two subunits.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Binding to erythrocyte membrane is the physiological mechanism for activation of Ca2+-dependent neutral proteinase

In the presence of micromolar concentrations of Ca2+ the catalytic 80 kDa subunit of human erythrocyte procalpain binds to the cytosolic surface of the erythrocyte membrane. Binding is rapid, highly specific and is reversed by the removal of Ca2+. In the bound form the 80 kDa catalytic subunit undergoes a rapid conversion to calpain, the active 75 kDa Ca2+-requiring proteinase. The activated proteinase produces extensive degradation of membrane components, particularly of band 4.1 and 2.1 proteins. Binding to membranes may represent an obligatory physiological mechanism for the conversion of procalpain to calpain.     

Anionic sites on the membrane intercalated particles of human erythrocyte ghost membranes. Freeze-etch localization

Freeze-fracture and freeze-etching techniques disclose exclusive association of a ferritin derivative (with high isoelectric point, used as a marker for anionic sites) with the regions at the outer and inner surface of the membrane of human erythrocyte ghosts which correspond to the membraneintercalated particles. At the outer surface the sites include sialoglycoprotein. Exclusive association of anionic sites and membrane particles, and comparison of the number of sialic acid residues and intercalated particles implies clustering of acidic groups over discrete sites at the surface. Association of the label with the outer and inner surface regions which correspond to the membrane intercalated particles, provides further support for the concept of protein-containing structures which are intercalated and traverse the hydrophobic matrix of membrane regions with bilayer organization.     

Proteolytic activation of calcium-activated, phospholipid-dependent protein kinase by calcium-dependent neutral protease

A Ca2+-dependent protease I), which hydrolyzes casein at Ca2+ concentrations lower than the 10(-5) M range, is purified roughly 4000-fold from the soluble fraction of rat brain. This protease is able to activate Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) by limited proteolysis analogously to the previously known Ca2+-dependent analogously to the previously known Ca2+-dependent protease (Ca2+ protease II) which is active at the millimolar range of Ca2+ (Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616). The protein kinase fragment thus produced shows a molecular weight of about 5.1 X 10(4), and is significantly smaller than native protein kinase C (Mr = 7.7 X 10(4). Although protein kinase C may be normally activated in a reversible manner by the simultaneous presence of phospholipid and diacylglycerol at Ca2+ concentrations less than 10(-6) M, this enzyme fragment is fully active without any lipid fractions and independent of Ca2+. The limited proteolysis of protein kinase C is markedly enhanced in the velocity by the addition of phospholipid and diacylglycerol, which are both required for the reversible activation of the enzyme. However, casein hydrolysis by this protease is not affected by phospholipid and diacylglycerol. Available evidence suggests that, at lower concentrations of this divalent cation, Ca2+ protease I reacts preferentially with the active form of protein kinase C which is associated with membrane, and converts it to the permanently active form. In contrast, the inactive form of protein kinase C, which is free of membrane phospholipid, does not appear to be very susceptible to the proteolytic attack. It remains unknown, however, whether this mechanism of irreversible activation of protein kinase C does operate in physiological processes. It is noted that Ca2+ protease II, which is active at higher concentrations of Ca2+, proteolytically activates protein kinase C irrespective of the presence and absence of phospholipid and diacylglycerol.     

Purification and characterization of a calcium-activated neutral protease from monkey cardiac muscle

A calcium-activated neutral protease (CANP) was purified from monkey cardiac muscle by a method involving column chromatography on DEAE-cellulose, Sepharose CL-6B, DEAE-Sephacel, organomercurial-Sepharose 4B, and Sephadex G-150 in succession. This protease required both millimolar concentration of Ca2+ and the SH-group for activation, and it was maximally active around pH 8.0. It was strongly inhibited by thiol protease inhibitors such as iodoacetic acid, antipain, leupeptin, and epoxysuccinic acid derivatives. The molecular weight of this protease was estimated to be 110,000 by gel filtration. Upon nondenaturing electrophoresis the purified protease gave two bands, both of which were active at millimolar concentration of Ca2+, indicating the existence of two forms of the protease. The less acidic band (form I CANP) contained two components with molecular weights of 74,000 and 28,000 and the more acidic one (form II CANP) contained components with molecular weights of 74,000 and 26,000. The protease was synergistically activated by Mn2+ and Ca2+ at a concentration where Mn2+ or Ca2+ alone was not effective. In the presence of millimolar level of Ca2+, limited autolysis reduced the Ca2+-requirement of this protease. The proteolysis of myofibrils by this protease resulted in the production of a component with a molecular weight of 30,000 as well as various other higher and lower molecular weight peptide fragments.     

Characterization of neutral glycosphingolipids from porcine erythrocyte membranes

1. Six neutral GSL fractions were purified from porcine erythrocyte membranes. 2. They were identified to be LacCer (14% of total neutral GSLs), 2-hydroxy acid-rich and -poor Gb3Cer (3 and 7%, respectively) and Gb4Cer (71%) by means of NMR spectrometry. 3. Monohexosylceramides (5%) were composed of GlcCer and GalCer with near amount. 4. All these GSL classes contained a high concentration (more than 20% of total acids in each class) of 2-hydroxy fatty acids. 5. GalCer and GlcCer contained considerable amounts of C16- and C18-acids, and of C18-phytosphingosine, whereas C24-acids and C18-sphingosine were predominant in the other GSLs. 6. A minor GSL fraction (less than 1% of total neutral GSLs) which migrated more slowly than Gb5Cer on a thin layer plate and composed of several GSL components contained L-fucose.