Partial Purification and Properties of a Cysteine Protease from Citrus Red Mite Panonychus citri

Several studies have reported that the citrus red mites Panonychus citri were an important allergen of citrus-cultivating farmers in Jeju Island. The aim of the present study was to purify and assess properties of a cysteine protease from the mites acting as a potentially pathogenic factor to citrus-cultivating farmers. A cysteine protease was purified using column chromatography of Mono Q anion exchanger and Superdex 200 HR gel filtration. It was estimated to be 46 kDa by gel filtration column chromatography and consisted of 2 polypeptides, at least. Cysteine protease inhibitors, such as trans poxy-succinyl-L-leucyl-amido (4-guanidino) butane (E-64) and iodoacetic acid (IAA) totally inhibited the enzyme activities, whereas serine or metalloprotease inhibitors did not affect the activities. In addition, the purified enzyme degraded human IgG, collagen, and fibronectin, but not egg albumin. From these results, the cysteine protease of the mites might be involved in the pathogenesis such as tissue destruction and penetration instead of nutrient digestion.     

Influence of 120 kDa Pyruvate:Ferredoxin Oxidoreductase on Pathogenicity of Trichomonas vaginalis

Trichomonas vaginalis is a flagellate protozoan parasite and commonly infected the lower genital tract in women and men. Iron is a known nutrient for growth of various pathogens, and also reported to be involved in establishment of trichomoniasis. However, the exact mechanism was not clarified. In this study, the author investigated whether the 120 kDa protein of T. vaginalis may be involved in pathogenicity of trichomonads. Antibodies against 120 kDa protein of T. vaginalis, which was identified as pyruvate:ferredoxin oxidoreductase (PFOR) by peptide analysis of MALDI-TOF-MS, were prepared in rabbits. Pretreatment of T. vaginalis with anti-120 kDa Ab decreased the proliferation and adherence to vaginal epithelial cells (MS74) of T. vaginalis. Subcutaneous tissue abscess in anti-120 kDa Ab-treated T. vaginalis-injected mice was smaller in size than that of untreated T. vaginalis-infected mice. Collectively, the 120 kDa protein expressed by iron may be involved in proliferation, adhesion to host cells, and abscess formation, thereby may influence on the pathogenicity of T. vaginalis.     

Use of inhibitors to identify essential cysteine proteinases of Trichomonas vaginalis

Designing cysteine proteinase inhibitors as antitrichomonal drugs requires knowledge of which cysteine proteinases are essential to the parasite. In an attempt to obtain such information, the effects of a number of cysteine proteinase inhibitors on trichomonad growth in vitro and proteinase activity were investigated. The broad specificity inhibitor trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane (known as E-64) had little effect on growth of Trichomonas vaginalis (27% inhibition at 280 μM, none at 28 μM) even though the addition of 2.8 μM E-64 to growth medium resulted in inhibition of all but two (apparent molecular masses: 35 k and 49 k) of the parasite's proteinases detected by gelatin SDS-PAGE. This shows that many of the parasite's cysteine proteinases are not essential for growth in axenic culture. In contrast, a peptidyl acyloxymethyl ketone, N-benzoyloxycarbonyl-Phe-Ala-CH₂OCO-(2,6,-(CF₃)₂)Ph, at 16 μM killed T. vaginalis and severely inhibited growth of Tritrichomonas foetus. Exposure of Trichomonas vaginalis to 16 μM of this compound for 1 h resulted in both the 35 kDa and 49 kDa proteinases being inhibited, whereas some other proteinases were unaffected. Similar distinctions between the inhibitor sensitivity of the parasite's cysteine proteinases were apparent when a biotinylated peptidyl diazomethyl ketone was used to detect active proteinases. These data suggest that the growth inhibitory effects of the peptidyl acyloxymethyl ketone are through inhibition of cysteine proteinases that are not affected when the parasites are grown in the presence of E-64. At least one of these enzymes, which include the 35 kDa and 49 kDa cysteine proteinases, must be essential and so a suitable target for chemotherapeutic attack.     

Purification of a proteinase and proteinase inhibitor from Tetrahymen a pyriformis

• 1.1. A cysteine proteinase and cysteine proteinase inhibitor have been purified from Tetrahymena.
• 2.2. The proteinase was purified by ammonium sulphate fractionation, gel filtration, ion exchange chromatography and affinity chromatography, and appeared homogeneous by gel filtration and electrophoresis (mol. wt approx 28,000). It hydrolysed BAPNA, degraded azocasein, and converted 80S ribosomes to subunits. Thiol reagents inhibited these activities.
• 3.3. The inhibitor was purified by heat treatment, ammonium sulphate fractionation and ion exchange chromatography, and appeared homogeneous by gel filtration and electrophoresis (mol. wt approx 12.500). The inhibitor was heat stable and it inhibited papain, as well as the Tetrahymena proteinase.

     

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.     

Partial characterization of a 29 kDa cysteine protease purified from Taenia solium metacestodes

A 29 kDa cysteine protease of Taenia solium metacestodes was purified by Mono Q anion-exchanger and Superose 6 HR gel filtration chromatography. The enzyme was effectively inhibited by cysteine protease inhibitors, such as iodoacetic acid (IAA) and trans-epoxy-succinyl-L-leucyl-amido (4-guanidino) butane (E-64) while inhibitors acting on serine- or metallo-proteases did not affect the enzyme activity. The purified enzyme degraded human immunoglobulin G (IgG), collagen and bovine serum albumin (BSA), but human IgG was more susceptible for proteolysis by the enzyme. To define the precise biological roles of the enzyme, more detailed biochemical and functional studies would be required.     

Arabidopsis cysteine proteinase inhibitor AtCYSb interacts with a Ca-dependent nuclease,AtCaN2

Plant cysteine proteinase inhibitors (cystatins) play important roles in plant defense mechanisms. Some proteins that interact with cystatins may defend against abiotic stresses. Here, we showed that AtCaN2, a Ca²⁺-dependent nuclease in Arabidopsis, is transcribed in senescent leaves and stems and interacts with an Arabidopsis cystatin (AtCYSb) in a yeast two-hybrid screen. The interaction between AtCYSb and AtCaN2 was confirmed by in vitro pull-down assay and bimolecular fluorescence complementation. Agarose gel electrophoresis showed that the nuclease activity of AtCaN2 against λDNA was inhibited by AtCYSb, which suggests that AtCYSb regulates nucleic acid degradation in cells.     

Purification of a 68-kDa cysteine proteinase from crude extract of Pneumocystis carinii

The present study intended to verify activities of cysteine proteinase of Pneumocystis carinii from rats and to purify the enzyme. In order to exclude the contamination of host-derived enzymes, concentrates of P. carinii was primarily treated with a mixture of proteinase inhibitors before lysis of P. carinii. A 68-kDa cysteine proteinase was finally purified from the crude extract of P. carinii by 4 sequential chromatographic methods. The enzyme showed an optimal activity at pH 5.5 in 0.1 M sodium acetate, and its activity was specifically inhibited by L-trans-epoxy-succinylleucylamido (4-guanidino) butane (E-64) and iodoacetic acid, suggesting that the enzyme is a cysteine proteinase. The 68-kDa proteinase weakly digested macromolecules such as collagen, hemoglobin and fibronectin. The present study demonstrated the activity of cysteine proteinase at the 68-kDa band of P. carinii, and purified and characterized the molecule.     

Purification and characterization of a lectin from the banana shrimp Fenneropenaeus merguiensis hemolymph

A lectin from the hemolymph of the banana shrimp Fenneropenaeus merguiensis was purified by affinity chromatography on a fetuin-agarose column following by gel filtration on a Superose-12 column. The native molecular mass of purified F. merguiensis lectin (FmL) determined by gel filtration was 316.2 kDa and its carbohydrate content was estimated to be 4.4%. By SDS-PAGE analysis, purified FmL consisted of 32.3 kDa and 30.9 kDa subunits. These data suggest that this lectin is an oligomer. Two-dimensional electrophoresis showed that it had a pI value of 6.0 and was mainly composed of glycine, serine, histidine, glutamic acids and glutamine, with relatively lower amounts of methionine and tyrosine. Purified FmL expressed higher agglutination activity against rabbit and rat erythrocytes than with those from human, and its activity was Ca²⁺-dependent. The hemagglutinating activity of FmL was stable up to 55 °C and at pH 7.5–8. N-acetylated sugars, such as ManNAc, GlcNAc, GalNAc, and NeuNAc were strong inhibitors of the FmL induced hemagglutinating activity with NeuNAc being most effective. Porcine stomach mucin and fetuin were the most potent inhibitors of FmL. Purified FmL caused selective agglutination of Vibrio harveyi, and Vibrio parahemolyticus both pathogens of this Penaeus species and to a lesser extent Vibrio vulnificus but had no effect on the non-pathogenic strains; Vibrio cholerae, Salmonella typhi and Escherichia coli. Its bacterial agglutination was also completely inhibited by NeuNAc, mucin, fetuin and also anti-FmL antibody. This observation indicates that FmL may contribute to the defense response of this species of penaeid shrimps to potentially pathogenic bacteria.     

Biological and biochemical modulation of Trichomonas vaginalis KT9 isolate after shifting of culture medium from TPS-1 into TYM

To evaluate the biological and biochemical characteristics of Trichomonas vaginalis KT9 isolate, the growth and size of trichomonads, pathogenicity in mouse, protein profiles and proteinase activity were examined after shifting the medium from TPS-1 into TYM. Generation time of trichomonads in TYM medium was 4.5 hr in comparison to TPS-1 with 7.1 hr. Size of trichomonads cultured in TPS-1 medium (8.5 ± 0.9 × 6.0 ± 0.9 µm) was significantly smaller than those in TYM medium (10.9 ± 1.4 × 8.2 ± 0.9 µm). Trichomonads cultured in TYM medium produced subcutaneous abscess in 9 out of 10 mice, whereas those in TPS-1 medium produced abscesses in 2 out of 10 mice. In SDS-PAGE, trichomonad lysates from both media showed ten common bands. However, trichomonads in TYM medium showed additional bands of 136 kDa, 116 kDa and 40 kDa in comparison to those in TPS-1 with 100 kDa. By immunoblot with T. vaginalis-immunized rabbit sera, T. vaginalis cultivated in both TYM and TPS-1 media showed 5 common bands, and unique bands of 116 kDa, 105 kDa, and 86 kDa were observed in trichomonads in TYM while a 140 kDa band in those in TPS-1. In gelatin SDS-PAGE, trichomonads in TYM degraded gelatin stronger than those in TPS-1. Also protease activity of trichomonads in TYM was significantly higher than that of trichomonads in TPS-1 using Bz-Pro-Phe-Arg-Nan as a substrate. According to the results, it is assumed that the shift from TPS-1 into TYM medium for cultivation of T. vaginalis might modulate the biological and biochemical properties of T. vaginalis in vitro.     

Purification and characterization of a cysteine proteinase from silkworm eggs

Eggs of the silkworm, Bombyx mori, contain a high level of a proteinase which is most active in acidic pH region. The proteinase was purified from an extract of eggs by a six-step procedure which included conventional chromatographic fractionations. The molecular mass of the proteinase was estimated to be 350 kDa by gel filtration and 47 kDa by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels, suggesting an octameric structure. The amino acid composition was found to resemble that of mammalian lysosomal cysteine proteinases, in particular cathepsin L. The NH2-terminal 10-residue sequence is Val-Gln-Phe-Phe-Asp-Leu-Val-Lys-Glu-Glu-. The enzyme appears to be a member of the class of cysteine proteinases since it was strongly inhibited by sulfhydryl-reactive compounds and N-[N-(1,3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine (E-64). The enzyme hydrolyzed various protein substrates, such as hemoglobin, vitellogenin, vitellin, and lipophorin, with maximal activity around pH 3-3.5. The specificity of the cleavage sites in the oxidized B chain of insulin was rather well defined and there was high affinity for hydrophobic residues at the P2 and P3 positions. The cysteine proteinase is thought to be involved in protein degradation during embryonic development of silkworm eggs.     

A Proteinase from Germinating Barley : I. Purification and Some Physical Properties of a 30 kD Cysteine Endoproteinase from Green Malt

A proteinase was purified from germinated barley (green malt from Hordeum vulgare L. cv Morex) by acidic extraction, ammonium sulfate fractionation and successive chromatographies on CM-cellulose, hemoglobin sepharose, Sephadex G-75 and organomercurial agarose columns. The overall purification and final recovery were 290-fold and 7.5%, respectively. The purified enzyme was homogeneous on analytical gel electrophoresis, yielding a single protein associated with protease activity. An apparent molecular weight of about 20 kilodaltons was estimated for the native enzyme from gel filtration. SDS-gel electrophoresis revealed a single polypeptide of about 30 kilodaltons. The optimum pH for the hydrolysis of hemoglobin was around 3.8. The enzyme was strongly inhibited by leupeptin but was insensitive to phenylmethylsulfonyl fluoride, indicating that it was a cysteine proteinase. It hydrolyzed several large proteins from various origins. The ability of the enzyme to digest barley storage proteins in vitro was examined using SDS-gel electrophoresis. The hydrolysis patterns obtained showed that the enzyme rapidly hydrolyzed the large hordein polypeptides into relatively small fragments. The results of this study suggest that this 30 kilodalton enzyme is one of the predominant cysteine proteinases secreted into the starchy endosperm during barley germination and that it plays a major role in the mobilization of storage proteins.     

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.     

Purification and characterization of inhibitor against the cysteine proteinase of Rana catesbeiana

1. Inhibitors of cysteine proteinase were found in tadpole tail of metamorphosing bullfrog. 2. One of the inhibitors was purified by affinity chromatography with CM-papain agarose, gel filtration with Superose 12 and ion exchange chromatography with Mono S. 3. The molecular weight of the inhibitor was 130,000-140,000 and the isoelectric point was pH 9.6. 4. The inhibitor had inhibitory effects on ficin, papain and tadpole tail cysteine proteinase. 5. The inhibitor is possibly involved in the regulation of muscle degradation in tail regression of metamorphosing tadpole.     

Trichomonas vaginalis: The adhesins AP51 and AP65 bind heme and hemoglobin

Trichomonas vaginalis is the cause of human trichomoniasis, the most common non-viral sexually transmitted disease worldwide. Although acquisition of iron by binding to host hemoglobin through distinct receptor(s) has been described, no specific heme- or hemoglobin-binding site has been reported in this parasite.To determine the presence of hemoglobin-binding protein(s), membrane proteins were subjected to hemoglobin-affinity chromatography. Eluted proteins were analysed by SDS-PAGE. Two protein bands of 48 and 63 kDa were detected. Competition assay with an excess amount of hemoglobin or hemin in hemoglobin-affinity chromatography could block the 63- and 48-kDa bands, respectively.Further analysis by mass spectrometry indicated that the 48- and 63-kDa proteins had identity with two T. vaginalis adhesins: AP51 and AP65, respectively. This study confirms the existence of multifunctional proteins in T. vaginalis, and suggested that AP51 and AP65, besides serving as adhesion molecules, could also act as heme- and hemoglobin-binding proteins.     

Involvement of PI3K/AKT and MAPK Pathways for TNF-α Production in SiHa Cervical Mucosal Epithelial Cells Infected with Trichomonas vaginalis

Trichomonas vaginalis; induces proinflammation in cervicovaginal mucosal epithelium. To investigate the signaling pathways in TNF-α production in cervical mucosal epithelium after T. vaginalis infection, the phosphorylation of PI3K/AKT and MAPK pathways were evaluated in T. vaginalis-infected SiHa cells in the presence and absence of specific inhibitors. T. vaginalis increased TNF-α production in SiHa cells, in a parasite burden-dependent and incubation time-dependent manner. In T. vaginalis-infected SiHa cells, AKT, ERK1/2, p38 MAPK, and JNK were phosphorylated from 1 hr after infection; however, the phosphorylation patterns were different from each other. After pretreatment with inhibitors of the PI3K/AKT and MAPK pathways, TNF-α production was significantly decreased compared to the control; however, TNF-α reduction patterns were different depending on the type of PI3K/MAPK inhibitors. TNF-α production was reduced in a dose-dependent manner by treatment with wortmannin and PD98059, whereas it was increased by SP600125. These data suggested that PI3K/AKT and MAPK signaling pathways are important in regulation of TNF-α production in cervical mucosal epithelial SiHa cells. However, activation patterns of each pathway were different from the types of PI3K/MAPK pathways.     

Purification and properties of a fibrinogenase from the venom of Naja nigricollis

A fibrinogenolytic proteinase from the venom of Naja nigricollis was purified by chromatography on Bio-Rex 70 and Phenyl-Sepharose. The purified enzyme, designated proteinase F1, was homogeneous by the criterion of SDS-polyacrylamide gel electrophoresis, and consisted of a single chain with a molecular weight of 58 000. Purified proteinase F1 had approximately 15-fold more proteinase activity than the crude venom, based on its ability to inactive α₂-macroglobulin. The enzyme acted on only the Aα-chain of fibrinogen and left the Bβ- and γ-chains intact. The pH optimum for this fibrinogenolytic activity was in the range of pH 8 to 10. In addition to its activity on fibrinogen, proteinase F1 was active on α₂-macroglobulin and fibronectin, but did not degrade casein, hemoglobin or bovine serum albumin. The enzyme was not inhibited by inhibitors of serine proteinases, cysteine proteinases or acid proteinases, but only by the metalloproteinase inhibitor, EDTA. The inhibition by EDTA could be prevented by Zn²⁺, but not by Ca²⁺ or Mg²⁺.     

Identification of a novel 4 kDa immunoglobulin-A-binding peptide obtained by the limited proteolysis of jacalin

Jacalin, an IgA-binding lectin from jackfruit (Artocarpus heterophyllus) seeds, was isolated by the passage of PBS extracts of seeds over an affinity matrix containing IgA-Sepharose-4B. It was further purified by HPLC. When analyzed by SDS-PAGE under both reducing and nonreducing conditions, the native jacalin was dissociated into two subunits of 12 and 15.4 kDa. Both the subunits could bind IgA. Peptide mapping performed with radioiodinated jacalin indicated that both the subunits were susceptible to proteolysis by Staphyloccous aureus V8 proteinase. One degradation product was a small peptide of 4 kDa. This small proteolytic fragment also bound IgA. The amino-termini of the two major IgA binding subunits, 12 and 15.4 kDa, were identical. The 4 kDa IgA-binding proteolytic fragment of jacalin had a different amino-terminal sequence, suggesting that the region of jacalin which binds IgA does not remain close to the amino-terminus of the peptide.     

Inactivation of the serine proteinase operon (proMCD) of Staphylococcus warneri M: Serine proteinase and cysteine proteases are involved in the autolysis

Unlike other members of coagulase negative staphylococci (CNS), strain warneri has proMCD operon, a homologue of sspABC proteinase operon of S. aureus. The proM and proC encode serine glutamyl endopeptidase and cysteine protease respectively, whereas proD directs homologue of SspC, putative cytoplasmic inhibitor which protects the host bacterium from premature activation of SspB. We determined whole nucleotide sequence of proMCD operon of S. warneri M, succeeded in expression of these genes, and investigated their functions by gene inactivation and complementation experiments. In gelatin zymography of the culture supernatant, a 20-kDa band corresponding to PROC cysteine protease was detected. By Western blotting, PROD was also confirmed in the cytoplasmic protein fraction. PROC and PROD showed significant similarity to SspB and SspC of S. aureus (73% and 58%, respectively). Inactivation mutants of proMCD, proCD and proD genes were established, separately. In the proMCD mutant, degradation/processing of extracellular proteins was drastically reduced, suggesting that PROM was responsible for the cleavage of extracellular proteins. By the proD mutation, the growth profile was not affected, and secretion of PROC was retained. Extracellular protein profiles of the proCD and proD mutants were not so different each other, but autolysin profiles were slightly dissimilar, around 39–48 kDa and 20 kDa bands in zymogram. Experiments in buffer systems showed that autolysis was significantly diminished in proMCD mutant, and was promoted by addition of purified PROM. The proC gene was cloned into a multicopy plasmid, and introduced into the proMCD mutant. Compared with the wild type, autolysis of the proC-complemented strain was definitely enhanced by addition of purified PROM. These results suggested that PROM and PROC affected the coccal autolysis, through processing of the autolysin.