Isolation and Characterization of a Cysteine Protease of Freesia Corms

A protease, freesia protease (FP)-A, was purified to electrophoretic homogeneity from regular freesia (Freesia reflacta) corms in harvest time. The M _r of FP-A was estimated to be 24 k by SDS-PAGE. The optimum pH of the enzyme was 8.0 using a casein substrate. These enzymes were strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethane-sulfonylfluoride and EDTA. These results indicate that FP-A belongs to the cysteine proteases. The amino terminal sequence of FP-A was similar to that of papain, and the sequences was regarded to the conservative residues of cysteine protease. From the hydrolysis of peptidyl-pNAs, the specificity of FP-A was found to be broad. It was thought that FP-A was a new protease from freesia corms.     

The development of cysteine proteases in freesia corms during responses to chilling

Freesia protease (FP)-A has been found in regular freesia corms (Kaneda et al., Biosci. Biotechnol. Biochem. 61 (1997) 1554). New corms were generated from original corms that were kept for several months at 4°C. In this study, two proteases (FP-B and FP-C) have been found to new corms kept for 6 months at 4°C, and have increased during new corms enlargement.

 FPs were purified from the extracts of new corms, and the M_r of those were 24k (A), 25k (B), and 24.5k (C) by SDS-PAGE, respectively.

 The N-terminal sequences of FPs were identical to those of papain with respect to the conservative residues of cysteine protease. The sequence of FP-A was identical with those of FP-B within 20 residues of its N-terminal. It may be possible that FP-B was produced by some post-translational modifications from FP-A during the chilling. On the other hand, N-terminal sequence of FP-C was different from those of FP-A and FP-B. It was explained that FP-C was a new protease of freesia corm.     

Purification and Characterization of a Cysteine Protease from Corms of Freesia,Freesia reflacta

A protease (freesia protease B) has been purified to electrophoretic homogeneity from corms of freesia, Freesia reflacta by five steps of chromatography. Its M_r was estimated to be about 26,000 by SDS–PAGE. The optimum pH of the enzyme was 6.0–7.0 at 30°C using casein as a substrate. The enzyme was strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethanesulphonylfluoride and EDTA. These results indicate that freesia protease B is a cysteine protease. Nine sites of oxidized insulin B-chain were cleaved by freesia protease B in 24 h of hydrolysis. The four cleavage sites among them resembled those of papain. From the digestion of five peptidyl substrates the specificity of freesia protease B was found to be approximately broad, but the preferential cleavage sites were negatively charged residues at positions. Freesia protease B preferred also the large hydrophobic amino acid residues at the P₂ position, in a similar manner to papain. The amino terminal sequence of freesia protease B was identical with those of papain in regard to the conservative residues of cysteine protease.     

Changes in abscisic acid levels during dormancy release in freesia corms

A high level of free-abscisic acid (ABA) was detected when corms were still in deep dormancy. The level of free-ABA decreased as the corm dormancy disappeared and increased temporarily after complete release from dormancy. A gradual slight increase of bound-ABA was observed during dormancy release.Treatment of dormant corms with benzyladenine (BA) increased sprouting but the sprouts did not show normal growth. Ethylene treatment induced complete sprouting and subsequent normal growth. Changes in ABA levels and ethylene production are discussed in relation to dormancy release in freesia corms.     

Characterization of cysteine protease induced by oxidative stress in cells of Chlamydomonas sp. strain W80

Unlike known Chlamydomonas species, Chlamydomonas sp. strain W80, which was isolated from seawater, shows tolerance to salt and cadmium. In this study, we purified and characterized cysteine protease from Chlamydomonas sp. strain W80 cells and also investigated their response to oxidative stress. The protease was purified 2760-fold with a yield of 2.6% by five steps of successive chromatography. This protease had a pH optimum of 8.0 and was specific only for tert -butoxycarbonyl (Boc)-Leu-Arg-Arg-4-methylcoumaryl-7-amide (MCA) (Boc-LRR-MCA) and Boc-Val-Leu-Lys-MCA as substrates among eight fluorogenic peptides tested. The K _m value was estimated to be 44.4 μ M for Boc-LRR-MCA. The molecular weight of the protease was determined to be approximately 102 kDa by Superdex 200 gel filtration and 60 kDa by SDS–PAGE, suggesting that this enzyme is a dimer. This enzyme was inhibited by the cysteine protease inhibitors leupeptin and N-ethylmaleimide but neither inhibited by phenylmethylsulfonyl fluoride or ethylenediaminetetraacetic acid nor activated by metal cations. These findings indicate that this enzyme is likely a cysteine protease. When strain W80 was grown under oxidative stress in the presence of methyl viologen and cadmium chloride, cysteine protease activity was about 30–90% higher than normal, whereas no changes were observed in carbon enrichment or senescence. It is likely that this protease is upregulated in response to oxidative stress and plays a role in the maintenance of cell metabolism under oxidative stress conditions.     

Cysteine protease is a major exotoxin of pathogenic luminous Vibrio harveyi in the tiger prawn, Penaeus monodon

The role of an extracellular cysteine protease, produced by pathogenic luminous Vibrio harveyi strain 820514 originally isolated from diseased tiger prawn (Penaeus monodon), in the disease process in the prawns was studied. The protease was lethal to P. monodon with an LD50 value of 0.3 microgram protein g-1 prawn. The lethal toxicity of the extracellular products (ECP) of the bacterium was neutralized by pre-incubation of the ECP with rabbit antiserum to the cysteine protease. Pre-incubation of ECP with CuCl2 (an inhibitor of cysteine protease) also inhibited toxicity. This suggests that cysteine protease is the major toxin produced by the bacterium. The present protease is the first toxic cysteine protease to be found in Vibrio species.     

Partial characterization of a 17 kDa protein of Clonorchis sinensis

A 17 kDa protein from Clonorchis sinensis adults was purified by a procedure including Sephacryl S-200 HR gel filtration and Q-Sepharose anion exchange chromatography. The protein was proved to be a cysteine protease as it showed hydrolytic activity toward Cbz-Phe-Arg-AMC in the presence of dithiothreitol and was inhibited by specific inhibitors such as iodoacetic acid or trans epoxy-succinly-L-leucyl-amido(4-guanidino) butane. The polyclonal antibody raised against the protein reacted to 17 kDa proteins of trematodes such as Paragonimus westermani, Fasciola hepatica, Opisthorchis viverrini, Gymnophalloides seoi, and Metagonimus yokogawai. The antibody recognized the 17 kDa and 16 kDa cysteine proteases purified from C. sinensis, P. westermani, and G. seoi as well. These results suggest that the 17 kDa protein may be a cysteine protease commonly present in trematodes.     

A cysteine protease from maize isolated in a complex with cystatin

We recently purified a latent but SDS-activated protease complex (40, 15- or 13-kDa proteins) from maize [Yamada et al. (1998) Plant Cell Physiol. 39: 106]. Here, we revealed that the complex was composed of a cysteine protease (40 kDa) and a cystatin, cysteine protease inhibitor (15- or 13-kDa). This is the first report on the isolation of a complex consisting of a cystatin and a target cysteine protease from plants. Cloning of the cysteine protease revealed that it had low homology (25-30%) to other maize cysteine proteases cloned to date but was highly homologous to other plant cysteine proteases such as rice oryzain alpha (84%) and the homologs (50-80%). The cysteine protease expressed in Escherichia coli showed the same substrate and inhibitor specificities as the protease of the complex, demonstrating that the isolated cDNA clone exactly encodes the protease of the complex. The protease expressed in E. coli itself was active but not latent, probably because it was not bound to cystatin. It is most likely that in vitro activation of the protease complex by SDS is caused by the release of bound cystatin. The mRNA of protease was expressed in various tissues except for seeds.     

Evidence for the proenkephalin processing enzyme prohormone thiol protease (PTP) as a multicatalytic cysteine protease complex: activation by glutathione localized to secretory vesicles.

The cysteine protease known as "prohormone thiol protease" (PTP) has been identified as a major proenkephalin processing enzyme in secretory vesicles of adrenal medulla (known as chromaffin granules). This study provides the first demonstration that PTP exists as a multicatalytic cysteine protease complex that can be activated by endogenous glutathione present in chromaffin granules. The high molecular mass nature of PTP, of approximately 185 kDa, was demonstrated by elution of a single peak of 35S-enkephalin precursor cleaving activity by Sephacryl S200 gel filtration chromatography and by a single band of 35S-enkephalin precursor cleaving activity detected on radiozymogram gels under native buffer conditions. Importantly, when 0.1% SDS was included in radiozymogram gels, PTP activity was resolved into three bands of proteolytic activity with apparent molecular masses of 88, 81, and 61 kDa. These activities were all cysteine proteases, since they were inhibited by the cysteine protease inhibitor E-64c but not by pepstatin A or EDTA that inhibit aspartyl protease and metalloprotease, respectively. Purification of native PTP by preparative gel electrophoresis indicated that PTP was composed of four polypeptides of 66, 60, 33, and 29 kDa detected on SDS-PAGE gels. These four protein subunits accounted for the three catalytic activities of PTP, as demonstrated on 35S-enkephalin precursor radiozymogram gels. Results also indicated that the electrophoretic mobilities of the four subunits differed under reducing compared to nonreducing conditions. The multicatalytic activities of the PTP complex all require reducing conditions for activity, which can be provided by endogenous reduced glutathione in chromaffin granules. These novel findings provide the first evidence for a role of a multicatalytic cysteine protease complex, PTP, in chromaffin granules that may be involved in the proteolytic processing of proenkephalin and perhaps other precursors into active neuropeptides.     

Protease inhibitors divert amyloid precursor protein to the secretory pathway

Addition of cysteine protease inhibitors to cells expressing amyloid precursor protein (APP) resulted in a >2-fold increase in appearance of the secreted extracellular domain of APP in the media. This was accounted for by increased flux of APP into the secretory pathway since protease inhibitors also caused a twofold increase in newly translated, incompletely glycosylated APP detected by pulse-labeling. These results show that a portion of newly translated APP molecules are normally rapidly degraded by cysteine protease(s) but can enter the secretory pathway when degradation is inhibited. Newly translated APP molecules are thus still competent for posttranslational processing in distal cellular compartments. Their degradation thus may not result from misfolding but merely susceptibility to an endoplasmic reticulum localized cysteine protease.     

Partial purification and characterization of a cysteine protease inhibitor from the plerocercoid of Spirometra erinacei

Helminthic cysteine proteases are well known to play critical roles in tissue invasion, nutrient uptake, and immune evasion of the parasites. In the same manner, the sparganum, the plerocercoid of Spirometra mansoni, is also known to secrete a large amount of cysteine proteases. However, cysteine protease inhibitors regulating the proteolytic activities of the cysteine protease are poorly illustrated. In this regard, we partially purified an endogenous cysteine protease inhibitor from spargana and characterized its biochemical properties. The cysteine protease inhibitor was purified by sequential chromatographies using Resource Q anion exchanger and Superdex 200 HR gel filtration from crude extracts of spargana. The molecular weight of the purified protein was estimated to be about 11 kD on SDS-PAGE. It was able to inhibit papain and 27 kDa cysteine protease of spargana with the ratio of 25.7% and 49.1%, respectively, while did not inhibit chymotrypsin. This finding suggests that the cysteine protease inhibitor of spargana may be involved in regulation of endogenous cysteine proteases of the parasite, rather than interact with cysteine proteases from their hosts.     

Possible involvement of radical intermediates in the inhibition of cysteine proteases by allenyl esters and amides

In order to investigate crystallographically the mechanism of inhibition of cysteine protease by alpha-methyl-gamma,gamma-diphenylallenecarboxylic acid ethyl ester 3, a cysteine protease inhibitor having in vivo stability, we synthesized N-(alpha-methyl-gamma,gamma-diphenylallenecarbonyl)-L-phenylalanine ethyl ester 4. Reaction of 4 with thiophenol, the SH group of which has similar pK(a) value to that of cysteine protease, produced oxygen-mediated radical adducts 6 and 7 in ambient air but did not proceed under oxygen-free conditions. Catalytic activities of two thiol enzymes including cathepsin B were also lowered in the absence of oxygen. These results suggest that cysteine protease can act through an oxygen-dependent radical mechanism.     

Evidence that serpin architecture intrinsically supports papain-like cysteine protease inhibition: engineering alpha(1)-antitrypsin to inhibit cathepsin proteases

The closely related serpins squamous cell carcinoma antigen-1 and -2 (SCCA-1 and -2, respectively) are capable of inhibiting cysteine proteases of the papain superfamily. To ascertain whether the ability to inhibit cysteine proteases is an intrinsic property of serpins in general, the reactive center loop (RCL) of the archetypal serine protease inhibitor alpha(1)-antitrypsin was replaced with that of SCCA-1. It was found that this simple substitution could convert alpha(1)-antitrypsin into a cysteine protease inhibitor, albeit an inefficient one. The RCL of SCCA-1 is three residues longer than that of alpha(1)-antitrypsin, and therefore, the effect of loop length on the cysteine protease inhibitory activity was investigated. Mutants in which the RCL was shortened by one, two, or three residues were effective inhibitors with second-order rate constants of 10(5)-10(7) M(-)(1) s(-)(1). In addition to loop length, the identity of the cysteine protease was of considerable importance, since the chimeric molecules inhibited cathepsins L, V, and K efficiently, but not papain or cathepsin B. By testing complexes between an RCL-mimicking peptide and the mutants, it was found that the formation of a stable serpin-cysteine protease complex and the inhibition of a cysteine protease were both critically dependent on RCL insertion. The results strongly indicate that the serpin body is intrinsically capable of supporting cysteine protease inhibition, and that the complex with a papain-like cysteine protease would be expected to be analogous to that seen with serine proteases.     

Presence of anti-cystatin C-positive dendritic cells or macrophages and localization of cysteine proteases in the apical bud of the enamel organ in the rat incisor.

Cystatin C, a cysteine protease inhibitor, was examined in the apical buds of rat incisors by immunohistochemistry, because in transition and maturation zones most of the dendritic cells in the papillary layer are anti-cystatin C-positive. Anti-cystatin C-labeled cells were sparse and localized to the proliferation and differentiation zones, constituting the apical bud of 5-week-old rat incisors. These cells were considered macrophages or dendritic cells, based on their reactivity with OX6 and ED1, as well as their ultrastructure. Basement membrane at the periphery of apical bud was also labeled by anti-cystatin C antibody. The apical buds included a few apoptotic fragments and weak reactivity with antibody to cathepsin L, a cysteine protease. Reactivity to anti-cystatin C and anti-cathepsin L antibodies was also detected in the apical bud of newborn rat incisors. These results suggest that the cystatin C-positive macrophages or dendritic cells are involved in normal incisor formation. They may be related to the clearance of apoptotic cells or protection from putative cysteine protease activity.     

cDNA cloning of a novel cysteine protease of Plasmodium falciparum

The cDNA for a novel Plasmodium cysteine protease (falcipain-2) has been isolated from a Plasmodium falciparum cDNA library. A 602 bp fragment was amplified from P. falciparum by PCR using degenerate oligonucleotide primers. The primers were designed based upon the amino acids flanking the active site cysteine and asparagine residues that are conserved in the eukaryotic cysteine proteases. This fragment was used to screen a P. falciparum cDNA library and isolated a 2.1 kb clone that encoded a novel cysteine protease. The sequence of the 2.1 kb clone predicted a 56 kDa protein containing a typical signal sequence, a prosequence and a 24.7 kDa mature protease with 37% identity to falcipain-1, a hemoglobin-degrading cysteine protease of P. falciparum. Northern blot analysis detected a 2.1 kb message in trophozoites. Taken together, we have isolated a novel cysteine protease of P. falciparum, which may play an important role at the late stages of the erythrocytic cycle of the parasite.     

Isolation of the protease component of maize cysteine protease-cystatin complex: release of cystatin is not crucial for the activation of the cysteine protease

The maize cysteine protease complex, which required SDS for its activation in vitro, is a 179 kDa trimeric complex (P-I)3 of a cysteine protease (P) [EC 3.4.22] and a cysteine protease inhibitor (I), cystatin [Yamada et al. (1998) Plant Cell Physiol. 39: 106, Yamada et al. (2000) Plant Cell Physiol. 41: 185]. Here, we show the mechanism of the SDS-dependent activation of the trimeric (P-I) complex and stabilization of the activated protease by its specific substrates. The cystatin-free cysteine protease isolated by preparative SDS-PAGE was still specifically activated by SDS, and its profile of SDS-dependency was exactly the same as that of the trimeric (P-I) complex. It is, therefore, evident that an SDS-dependent conformational change of the protease itself, rather than the release of cystatin from the complex, is crucial for the activation. Pre-treatment analysis with SDS revealed that SDS was required for the initiation of the activation of the trimeric (P-I) complex. Furthermore, we found that once the protease was activated, if there was no substrate, it was rapidly inactivated under optimum conditions of proteolysis, and showed that such inactivation was not due to autolysis of the protease. In contrast, addition of specific substrates prevented the inactivation, and thus we presumed that the activity of the cysteine protease is regulated by both activation by conformational change and rapid inactivation after consumption of substrates.     

Investigation of sequential behavior of carboxyl protease and cysteine protease activities in virus-infected Sf-9 insect cell culture by inhibition assay

Proteases produced during the culture of Spodoptera frugiperda Sf-9 cells infected with Autographa californica nuclear polyhedrosis virus (AcNPV) were assayed with various protease inhibitors. This inhibitory analysis revealed that: (1) carboxyl and cysteine proteases were predominantly produced by the insect cells infected with recombinant AcNPV, the gene of which encoded a variant of green fluorescent protein in a portion of the polyhedrin gene of the baculovirus, and (2) the protease activity was almost completely blocked by pepstatin A (carboxyl protease inhibitor) and E64 (cysteine protease inhibitor) in an additive manner in the presence of EDTA. Utilizing the additive property of the inhibitors, the inhibition-based protease assay discriminated between the two protease activities and elucidated the sequential behavior of the carboxyl and cysteine proteases produced in the virus-infected Sf-9 cell culture. The carboxyl protease(s) existed in the virus-infected cells all the time and their level in the medium continuously increased. Uninfected cells also contained a carboxyl protease activity, the level of which was similar to that of the virus-infected cells. At a certain time after virus infection, the cysteine protease activity was largely increased in the virus-infected cells and a significant amount of the protease(s) was released into the medium, due to the cell membranes losing their integrity. The behavior of intracellular and extracellular cysteine protease activities coincided with that of a recombinant protein whose expression was under the control of the viral polyhedrin promoter. Similar examinations with wt-AcNPV-infected and uninfected insect cells showed that the inhibition-based protease assay was useful for analyzing the carboxyl protease and cysteine protease activities emerging in the insect cell (Sf-9)/baculovirus expression system.     

The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants

Programmed cell death (PCD) is a process by which cells in many organisms die. The basic morphological and biochemical features of PCD are conserved between the animal and plant kingdoms. Cysteine proteases have emerged as key enzymes in the regulation of animal PCD. Here, we show that in soybean cells, PCD-activating oxidative stress induced a set of cysteine proteases. The activation of one or more of the cysteine proteases was instrumental in the PCD of soybean cells. Inhibition of the cysteine proteases by ectopic expression of cystatin, an endogenous cysteine protease inhibitor gene, inhibited induced cysteine protease activity and blocked PCD triggered either by an avirulent strain of Pseudomonas syringae pv glycinea or directly by oxidative stress. Similar expression of serine protease inhibitors was ineffective. A glutathione S-transferase-cystatin fusion protein was used to purify and characterize the induced proteases. Taken together, our results suggest that plant PCD can be regulated by activity poised between the cysteine proteases and the cysteine protease inhibitors. We also propose a new role for proteinase inhibitor genes as modulators of PCD in plants.