Partial characterization of an endogenous inhibitor of a calcium-dependent form of insulin protease

Insulin protease activity has resisted high-yield purification to homogeneity, due to its low amount in tissues, its instability, and its erratic recovery from several types of chromatography. This report outlines the preliminary characterization of a naturally-occurring insulin protease inhibitor that accounts for some of these problems in rat skeletal muscle. In these experiments, inhibitory activity was assayed by its effect upon hydrolysis of 125I-(A14)-insulin by the partially purified insulin protease activity of rat skeletal muscle cytosol. During Sephadex G-200 chromatography of cytosol at pH 7.5, inhibitory activity copurifies with insulin protease activity, and the incomplete resolution of the two activities contributes to the impression that insulin protease exists in distinct 180,000-dalton and 80,000-dalton forms. By contrast, during DEAE-Sephacel chromatography of cytosol at pH 7.5, inhibitory activity and insulin protease activity are resolved by eluting the resin with 50 mM NaCl and 200 mM NaCl, respectively. Post-DEAE-Sephacel inhibitor has an Mr(app) of 67,000 daltons or 80,000-120,000 daltons, as determined by high-performance liquid chromatography or Sephadex G-150 chromatography, respectively. Post-DEAE-Sephacel insulin protease activity exhibits a Km for insulin of 15 nM and resides in a 200,000-dalton neutral thiol protease which requires 50 micromolar calcium for its maximum insulin-degrading activity. The inhibitor reduces the enzyme's activity reversibly, nonprogressively, and non-competitively with respect to insulin, but it does not alter the enzyme's sensitivity to calcium ion. These observations suggest that calcium and an endogenous protease inhibitor may influence cellular degradation of insulin via previously unrecognized effects upon cytosolic insulin protease activity.     

Proteolysis of the calcium-dependent protease inhibitor by myocardial calcium-dependent protease

Bovine heart peak II calcium-dependent protease was capable of hydrolyzing its specific inhibitor protein at high molar ratios of protease to inhibitor. The proteolysis was inhibited by leupeptin and required millimolar calcium. Thus, it appeared to be attributable to the calcium-dependent protease and not to possible contaminating proteases in the purified preparations of inhibitor or calcium-dependent protease. Incubation of the purified inhibitor with the calcium-dependent protease produced a discrete pattern of inhibitor fragments on Western blots developed with an inhibitor-specific monoclonal antibody. Traces of similar or identical lower molecular weight immunoreactive material could be observed in Western blots of bovine heart extracts, and the immunoreactivity present as these lower molecular weight forms could be increased by incubation of the extracts with calcium ion. These results suggest that the inhibitor can be proteolyzed to low molecular weight forms which can be detected in cardiac tissue extracts, and that calcium-dependent protease(s) may be responsible for this phenomenon.     

Stimulation of rat platelet adenylate cyclase by an endogenous calcium-dependent protease-like activity

The stimulation of adenylate cyclase by various exogenous proteases has been described in several tissues. In this study, we describe a 2 to 7-fold increase of adenylate cyclase activity in a particulate preparation from rat platelets following prior exposure of the homogenate to calcium. Calmodulin alone was unable to increase the adenylate cyclase activity and trifluoperazine only partially inhibited the calcium-dependent activation. On the other hand, calcium had a slight stimulatory effect on the particulate preparation but this activation was greatly enhanced by the addition of supernatant. Only the combined addition of calcium, supernatant and calmodulin to washed particulate preparations reconstituted the activation seen in homogenates. The activation was significantly inhibited by leupeptin and thiol reagents. It is concluded that platelets contain a calcium-dependent protease-like activity that is able to increase adenylate cyclase activity in membrane fractions. This phenomenon may be involved in the regulation of adenylate cyclase activity in platelets.     

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.     

Rapid purification of calcium-activated protease by calcium-dependent hydrophobic-interaction chromatography

Both low Ca2+- and high Ca2+-requiring forms of Ca2+-activated protease (calpains I and II) were found to bind to phenyl-Sepharose in a calcium-dependent manner, suggesting that both enzymes expose a hydrophobic surface region in the presence of Ca2+. Inclusion of leupeptin in column buffers prevented the loss of activity during hydrophobic-interaction and substrate-affinity chromatography. Under these conditions calpain II (high calcium-requiring form) was rapidly purified from bovine brain and rabbit skeletal muscle using successive phenyl-Sepharose and casein-Sepharose columns.     

Purification and characterization of a calcium-dependent protease from rat liver

A calcium-dependent protease, previously identified in rat liver and designated peak II [DeMartino, G. N. (1981) Arch. Biochem. Biophys. 211, 253-257], was purified and characterized. The calcium-dependent proteolytic activity was accounted for by an 80 000-dalton protein. Depending on the method of purification, we found that this protease could be associated with a 28 000-dalton subunit, which was devoid of protease activity. The catalytic characteristics of the two different forms of the protease were indistinguishable. Each was half-maximally activated by approximately 250 microM calcium.     

Structural characterization of a conserved, calcium-dependent periplasmic protease from Legionella pneumophila

The bacterial dinucleotide second messenger c-di-GMP has emerged as a central molecule in regulating bacterial behavior, including motility and biofilm formation. Proteins for the synthesis and degradation of c-di-GMP and effectors for its signal transmission are widely used in the bacterial domain. Previous work established the GGDEF-EAL domain-containing receptor LapD as a central switch in Pseudomonas fluorescens cell adhesion. LapD senses c-di-GMP inside the cytosol and relays this signal to the outside by the differential recruitment of the periplasmic protease LapG. Here we identify the core components of an orthologous system in Legionella pneumophila. Despite only moderate sequence conservation at the protein level, key features concerning the regulation of LapG are retained. The output domain of the LapD-like receptor from L. pneumophila, CdgS9, binds the LapG ortholog involving a strictly conserved surface tryptophan residue. While the endogenous substrate for L. pneumophila LapG is unknown, the enzyme processed the corresponding P. fluorescens substrate, indicating a common catalytic mechanism and substrate recognition. Crystal structures of L. pneumophila LapG provide the first atomic models of bacterial proteases of the DUF920 family and reveal a conserved calcium-binding site important for LapG function.     

Identification and characterization of inhibitory sequences in four repeating domains of the endogenous inhibitor for calcium-dependent protease

We reported previously the cDNA cloning of the endogenous inhibitor for calcium-dependent protease (CANP inhibitor, calpastatin) and the expression of its fragments in Escherichia coli. The CANP inhibitor has four internal repeating domains each spanning about 140 amino acid residues. The inhibitory activity arises from these domains which have a well-conserved sequence, TIPPXYR, in their central positions. The inhibitory activities of various fragments expressed in E. coli suggest the involvement of the regions around the well-conserved sequences. In this report, we describe further detailed investigation on the interaction site of the CANP inhibitor with CANP by truncating inhibitor fragments and by using chemically synthesized peptides. The results clearly indicate that the sequence around the well-conserved sequence, TIPPXYR, is an interaction site. A peptide as short as 23 amino acid residues retained inhibitory activity, but a 9-residue peptide corresponding to the conserved sequence, VTIPPKYRE had none. The inhibitory sequence is suggested as LGXKDREXTIPPXYRXLL. The analysis of the competition between an inhibitor peptide and an irreversible inhibitor, E-64 for the reaction with the active site suggests no involvement of the active site cysteine residue of CANP in the inhibitory interaction between CANP and the CANP inhibitor. The high specificity of the CANP inhibitor to CANP arises from its interaction with residues other than the active site cysteine residue, possibly the subsite for substrate-binding of CANP.     

Activation of a cyclic AMP-independent protein kinase by an endogenous ATP-requiring protease from lymphosarcoma cells

The activation of a cyclic AMP-independent protein kinase by an endogenous protease is described. The H4 phosphotransferase (Masaracchia, R. A., Kemp, B., and Walsh, D. A. (1977) J. Biol. Chem. 252, 7109-7117) from lymphosarcoma cells was isolated in a nonactive form. Activation required ATP and Mg2+ and was shown to be time-dependent. Although Mn2+ was capable of substituting for Mg2+ in the protein kinase reaction, no activation was observed when Mn2+ replaced Mg2+. The protein substrate histone H4 inhibited phosphotransferase activation at concentrations greater than 60 microM. The inhibition was complete in the presence of 100 microM H4. Comparable concentrations of bovine serum albumin did not inhibit the activation. The selective dependence on Mg2+ suggested separate activating and phosphotransferase activities. This was confirmed by heat denaturation in which the activation reaction was shown to be more sensitive to heat inactivation than was the phosphotransferase reaction. The activating enzyme was separated from the protein kinase by column chromatofocusing in the pH range 7-4. The pI of the activating enzyme was greater than 7.0. The pI values of the activated and nonactivated phosphotransferase were 4.8 and 5.3, respectively. The apparent molecular weight of the nonactivated phosphotransferase was 68,000; the activated enzyme was eluted from an S-200 Sephadex column with an apparent Mr = 52,000. Despite many similarities to a protease-activated Ca2+/phospholipid-dependent enzyme isolated from lymphocytes (Ogawa, Y., Takai, Y., Kawahara, Y., Kimura, S., and Nishizuka, Y. (1981) J. Immunol. 127, 1369-1374), the H4 phosphotransferase was not activated by Ca2+, phospholipids, or any combination thereof.     

Production and properties of an alkaline protease by Aureobasidium pullulans

A strain of the yeast-like fungus Aureobasidium pullulans was grown on whey to produce an extracellular protease. The protease was totally inhibited by the serine inhibitor, phenyl methyl sulphonyl fluoride (PMSF), and partially inhibited by the chelating agent EDTA. The enzyme showed maximal activity in the alkaline range with an optimum pH of 9·5–10·5. The optimum temperature for protease activity was 41C. As well as being active against the non-specific proteolytic substrate Azocoll, the protease readily degraded purified α-casein. A molecular weight of 27000 ± 350 was determined for the protease using gel filtration chromatography.     

Production and purification of a calcium-dependent protease from Bacillus cereus BG1

The production and purification of a calcium-dependent protease by Bacillus cereus BG1 were studied. The production of the protease was found to depend specifically on the calcium concentration in the culture medium. This suggests that this metal ion is essential for the induction of protease production and/or stabilisation of the enzyme after synthesis. The calcium requirement is highly specific since other metal ions (such as Mg²⁺ and Ba²⁺, which both activate the enzyme) are not able to induce protease production. The most appropriate medium for growth and protease production comprises (g L^–1) starch 5, CaCl₂ 2, yeast extract 2, K₂HPO₄ 0.2 and KH₂PO₄ 0.2. The protease of BG1 strain was purified to homogeneity by ultrafiltration, heat treatment, gel filtration on Sephacryl S-200, ion exchange chromatography on DEAE-cellulose and, finally, a second gel filtration on Sephacryl S-200, with a 39-fold increase in specific activity and 23% recovery. The molecular weight was estimated to be 34 kDa on SDS-PAGE. The optimum temperature and pH of the purified enzyme were determined to be 60°C and 8.0, respectively, in 100 mM Tris-HCl buffer + 2 mM CaCl₂.     

LapG, required for modulating biofilm formation by Pseudomonas fluorescens Pf0-1, is a calcium-dependent protease

Biofilm formation by Pseudomonas fluorescens Pf0-1 requires the cell surface adhesin LapA. We previously reported that LapG, a periplasmic cysteine protease of P. fluorescens, cleaves the N terminus of LapA, thus releasing this adhesin from the cell surface and resulting in loss of the ability to make a biofilm. The activity of LapG is regulated by the inner membrane-localized cyclic-di-GMP receptor LapD via direct protein-protein interactions. Here we present chelation and metal add-back studies demonstrating that calcium availability regulates biofilm formation by P. fluorescens Pf0-1. The determination that LapG is a calcium-dependent protease, based on in vivo and in vitro studies, explains the basis of this calcium-dependent regulation. Based on the crystal structure of LapG of Legionella pneumophila in the accompanying report by Chatterjee and colleagues (D. Chatterjee et al., J. Bacteriol. 194:4415-4425, 2012), we show that the calcium-binding residues of LapG, D134 and E136, which are near the critical C135 active-site residue, are required for LapG activity of P. fluorescens in vivo and in vitro. Furthermore, we show that mutations in D134 and E136 result in LapG proteins no longer able to interact with LapD, indicating that calcium binding results in LapG adopting a conformation competent for interaction with the protein that regulates its activity. Finally, we show that citrate, an environmentally relevant calcium chelator, can impact LapG activity and thus biofilm formation, suggesting that a physiologically relevant chelator of calcium can impact biofilm formation by this organism.     

Nicking of single chain Clostridium botulinum type A neurotoxin by an endogenous protease

Botulinum neurotoxin (NT) serotype A isolated from cells from young cultures (approximately 8 h) of Clostridium botulinum type A is a approximately 150 kDa single chain protein. Supernatant from older cultures (96 h) yields approximately 150 kDa dichain NT composed approximately 50 and approximately 100 kDa subunits, that remain associated by disulfide and noncovalent bonds. This had led to the assumption that an endogenous protease cleaves a peptide bond at 1/3rd the distance from the N- or C-terminals of the single chain protein. An endogenous protease that causes such a cleavage (nicking) has now been purified greater than 1,000-fold from C. botulinum type A (Hall strain) culture; this culture also produces the single chain NT and eventually yields the dichain NT. The purified protease nicked the pure preparation of single chain type A NT, in vitro at pH 5.6, into a dichain form that was indistinguishable from the dichain NT normally isolated from 96 h cultures. The protease appears specific for nicking serotype A NT because it did not nick single chain serotype B and E NT nor did it enhance toxicity of serotype A, B and E NT.     

Degradation products of insulin generated by hepatocytes and by insulin protease

The enzymatic mechanisms for insulin breakdown by hepatocytes have not been established, nor have the degradation products been identified. Several lines of evidence have suggested that the enzyme insulin protease is involved in insulin degradation by hepatocytes. To identify the products of insulin generated by insulin protease and to compare them with those produced by hepatocytes, we have incubated insulin specifically iodinated at either the B-16 or the B-26 tyrosines with insulin protease and with isolated hepatocytes, separated the products on high performance liquid chromatography (HPLC), and identified the B-chain cleavages. Insulin-sized products were obtained by Sephadex G-50 filtration. These insulin-sized products were injected on reverse-phase HPLC, and the peaks of radioactivity were identified. The product patterns generated by the enzyme and by hepatocytes were essentially identical with both isomers. The products were also sulfitolized to prepare the S-sulfonate derivatives of the B-chain and B-chain peptides. Again, the patterns on HPLC generated by the enzyme and by hepatocytes with both isomers were identical. Each of the original product peaks was also sulfitolized and injected separately on HPLC to relate B-chain peptides with product peaks. Again, the peptide compositions of the product peaks for both enzyme and hepatocytes were essentially identical. To identify the cleavage sites in the B-chain of insulin produced by insulin protease, the peptides from the degradation of [125I]iodo(B-26)insulin were purified and submitted to automated Edman degradation to identify the cycle in which radioactivity appeared. Seven peptides with cleavages on the amino side of the B26 residue were identified, and the cleavage sites were determined. Cleavages were found between B-9 and B-10 (Ser-His), B-10 and B-11 (His-Leu), B-14 and B-15 (Ala-Leu), B-13 and B-14 (Glu-Ala), B-16 and B-17 (Tyr-Leu), B-24 and B-25 (Phe-Phe), and B-25 and B-26 (Phe-Tyr). Peptides were also isolated from [125I]iodoinsulin incubated with isolated hepatocytes, and the cleavage sites in several of these were determined. These agreed exactly with the cleavage sites identified generated by the enzyme. The major peptides generated by the degradation of [125I]iodo(B-16)insulin were also isolated and sequenced, again showing identical cleavage sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Carboxy terminally truncated forms of ribophorin I are degraded in pre- Golgi compartments by a calcium-dependent process

Two COOH terminally truncated variants of ribophorin I (RI), a type I transmembrane glycoprotein of 583 amino acids that is segregated to the rough portions of the ER and is associated with the protein-translocating apparatus of this organelle, were expressed in permanent HeLa cell transformants. Both variants, one membrane anchored but lacking part of the cytoplasmic domain (RL467) and the other consisting of the luminal 332 NH2-terminal amino acids (RI332), were retained intracellularly but, in contrast to the endogenous long lived, full length ribophorin I (t 1/2 = 25 h), were rapidly degraded (t 1/2 less than 50 min) by a nonlysosomal mechanism. The absence of a measurable lag phase in the degradation of both truncated ribophorins indicates that their turnover begins in the ER itself. The degradation of RI467 was monophasic (t 1/2 = 50 min) but the rate of degradation of RI332 molecules increased about threefold approximately 50 min after their synthesis. Several pieces of evidence suggest that the increase in degradative rate is the consequence of the transport of RI332 molecules that are not degraded during the first phase to a second degradative compartment. Thus, when added immediately after labeling, ionophores that inhibit vesicular flow out of the ER, such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) and monensin, suppressed the second phase of degradation of RI332. On the other hand, when CCCP was added after the second phase of degradation of RI332 was initiated, the degradation was unaffected. Moreover, in cells treated with brefeldin A the degradation of RI332 became monophasic, and took place with a half-life intermediate between those of the two normal phases. These results point to the existence of two subcellular compartments where abnormal ER proteins can be degraded. One is the ER itself and the second is a non-lysosomal pre-Golgi compartment to which ER proteins are transported by vesicular flow. A survey of the effects of a variety of other ionophores and protease inhibitors on the turnover of RI332 revealed that metalloproteases are involved in both phases of the turnover and that the maintenance of a high Ca2+ concentration is necessary for the degradation of the luminally truncated ribophorin.     

Autoproteolysis of the small subunit of calcium-dependent protease II activates and regulates protease activity

Calcium-dependent protease II (CDP-II) from bovine heart is a heterodimer with subunit molecular weights of 80,000 and 26,000. Previous studies have demonstrated that the protease requires 350 microM Ca2+ for half-maximal activity and that the large subunit contains both the catalytic and Ca2+ binding functions of the enzyme. The function of the small subunit has been unclear. We have examined the effect of Ca2+ on structural and catalytic properties of CDP-II in the presence and absence of substrate proteins. When incubated with Ca2+ in the absence of substrate, CDP-II undergoes a series of autoproteolytic cleavages that sequentially reduce the small subunit's molecular weight from 26,000 to 24,000 to 22,000 to 17,000. During this time there is no detectable change in the 80-kDa subunit, which remains associated with the autolyzed small subunit. The rate of autoproteolysis is dependent on temperature and on the concentration of Ca2+ (half-maximal rate at approximately 600 microM Ca2+). The first cleavage appears to be unimolecular because its rate is unaffected by CDP-II concentration or by the presence of exogenous protein substrates. Subsequent cleavages result in the formation of the 80-kDa/17-kDa heterodimer and appear to occur by bimolecular reactions; rates of these reactions were slowed by decreasing CDP-II concentrations and by the presence of protein substrates. Autoproteolysis of the small subunit has two distinct functional consequences, each of which is associated with different forms of the autolyzed protease. Our results indicate that the 80-kDa/26-kDa form of CDP-II represents an inactive proenzyme and that the initial Ca2+-dependent cleavage of the 26-kDa subunit results in activation of the protease. The activated enzyme hydrolyzes protein substrates with a Ca2+ concentration requirement of 350 microM for half-maximal rates. The further autoproteolysis, which results in the formation of the 80-kDa/17-kDa heterodimer, serves to reduce the Ca2+ concentration requirement for protease activity by 25-fold. Thus, these results provide evidence for specific roles of the small subunit in the regulation of CDP-II activity.     

Calcium-dependent and phosphorylation-stimulated proteolysis of lipocortin I by an endogenous A431 cell membrane protease

Purified placental lipocortin I but not lipocortin II was proteolyzed during A431 cell membrane-catalyzed phosphorylation reactions. Proteolysis was Ca2+-dependent but was not prevented in the presence of a variety of inhibitors of Ca2+-dependent proteases, suggesting that the Ca2+ effect is a property of lipocortin I itself. Proteolysis was inhibited by Triton X-100 or dithiothreitol and was temperature-dependent, occurring at 30 degrees C but not at 0 degrees C. Tyrosine phosphorylation and proteolysis are distinct events as both phosphorylated and nonphosphorylated lipocortins could be cleaved by the membrane protease, but prephosphorylation enhanced the rate of proteolysis 2-fold during the initial reaction and by 60 min almost half of the phosphorylated lipocortin was proteolyzed. Cleavage of the 38-kDa phosphotyrosyl lipocortin I generated a truncated 37-kDa form of lipocortin which retained the phosphate label, indicating that proteolysis occurred at a site N-terminal to the site of tyrosine phosphorylation, possibly at tryptophan 12. Ando, Y., Imamura, S., Hong, Y.-M., Owada, M.K., Kakunaga, T., and Kannagi, R. [1989) J. Biol. Chem. 264, 6948-6955) have recently reported that in vitro cleavage at sites in the N-terminal tail region of lipocortin I by exogenously added proteases dramatically enhanced the Ca2+ sensitivity of phospholipid binding by lipocortin. The demonstrated ability of an endogenous membrane protease to catalyze a similar and specific cleavage in a Ca2+-dependent manner indicates that this event may occur in the cell where it would have important effects on the functional properties of lipocortin I.     

Stimulation by phspholipid of calcium-dependent phosphorylation of endogenous proteins from mammalian tissues

The occurrence of endogenous substrate proteins for calcium-dependent protein kinase, augmented by either phospholipid or calmodulin, was examined in extracts of several tissues. Pancreas, vas deferens, adrenal and liver were found to contain substrate proteins for phospholipid-sensitive protein kinase. Phosphorylation of pancreatic substrate protein for phospholipid-senstivie protein kinase was rapid and highly sensitive to Ca²⁺, being detectable within 15 s a following exposure to Ca²⁺ and phosphatidylserine and at concentrations of Ca²⁺ as low as 0.5 μM. These findings suggest that the phospholipid-sensitive protien kinase system may serve to mediate some effects of Ca²⁺ in a variety of mammalian cell types.