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.     

Persisting antibody reaction in paragonimiasis after praziquantel treatment is elicited mainly by egg antigens

Antibody responses in serum and cerebrospinal fluid (CSF) samples from patients with active and chronic paragonimiasis and in sera from patients on whom follow-up studies were done after praziquantel treatment were analyzed using antigens of Paragonimus westermani prepared from eggs, metacercariae, juveniles of 4- and 7-week old, adult worms and recombinant protein of 28 kDa cruzipain-like cysteine protease (rPw28CCP). The patient sera/CSFs of active and chronic paragonimiasis revealed strong antibody reactions against the crude extracts of 4- and 7-week old juveniles as well as against those from egg and adult. rPw28CCP also showed specific reaction to the sera with active paragonimiasis. After the treatment, levels of specific antibodies in the sera gradually decreased to negative range in most patients. In some cases with persisting high antibody levels, however, the reactions at 27 kDa egg protein were sustained throughout the observation period of 34 months. The reactions at 35 and 32 kDa in adult extract and rPw28CCP disappeared rapidly after the treatment. Persistent antibody reactions even after successful treatment are provoked by continuous antigenic challenge from eggs which were not resolved by treatment.     

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.     

IgG antibody responses in early experimental sparganosis and IgG subclass responses in human sparganosis

Antigenic components in the crude extracts of Spirometra mansoni plerocercoid were analyzed in early experimental infections and in IgG subclass observed in clinical sparganosis. By IgG immunoblot, sera obtained serially from experimental mice, fed 5 spargana each, were reacted with the crude extracts. Protein bands at 36-26 kDa and 103 kDa showed positive reactions since two weeks after infection. In a differential immunoblot, in which a monospecific antibody against sparganum chymase at 36 kDa was pre-treated, the reactions at 36-26 kDa disappeared, indicating that the sparganum chymase and its degradation products invoked IgG antibody reactions. When 69 patients sera of human sparganosis were examined for their IgG subclass responses, IgG4 levels showed the highest reaction which was followed by IgG1. The IgG4 antibody also reacted mainly with 36-31 kDa protease. These results indicate that 36 kDa chymase of S. mansoni plerocercoid is the main antigenic component inducing IgG antibody response in early stage of experimental sparganosis and for specific IgG subclass reactions in human sparganosis.     

Diagnostic Efficacy of a Recombinant Cysteine Protease of Spirometra erinacei Larvae for Serodiagnosis of Sparganosis

The mature domain of a cysteine protease of Spirometra erinacei plerocercoid larva (i.e., sparganum) was expressed in Escherichia coli, and its value as an antigen for the serodiagnosis of sparganosis was investigated. The recombinant protein (rSepCp-1) has the molecular weight of 23.4 kDa, and strongly reacted with the sparganum positive human or mice sera but not with negative sera by immunoblotting. ELISA with rSepCp-1 protein or sparganum crude antigen (SeC) was evaluated for the serodiagnosis of sparganosis using patient''s sera. The sensitivity and specificity of ELISA using rSepCp-1 protein were 95.0% (19/20) and 99.1% (111/112), respectively. In contrast, the sensitivity and specificity of ELISA with SeC were 100% (20/20) and 96.4% (108/112), respectively. Moreover, in experimentally infected mice, the sensitivity and specificity of both ELISA assays were 100% for the detection of anti-sparganum IgG. It is suggested that the rSepCp-1 protein-based ELISA could provide a highly sensitive and specific assay for the diagnosis of sparganosis.     

Identification and characterization of a mitochondrial manganese superoxide dismutase of Spirometra erinacei

A gene encoding the manganese superoxide dismutase (Mn-SOD) of Spirometra erinacei was identified, and the biochemical properties of the recombinant enzyme were partially characterized. The S. erinacei Mn-SOD gene consisted of 669 bp, which encoded 222 amino acids. A sequence analysis of the gene showed that it had typical molecular structures, including characteristic metal-binding residues and motifs that were conserved in Mn-SODs. An analysis of the N-terminal presequence of S. erinacei Mn-SOD revealed that it had physiochemical characteristics commonly found in mitochondria-targeting sequences and predicted that the enzyme is located in the mitochondria. A biochemical analysis also revealed that the enzyme is a typical Mn-SOD. The enzyme was consistently expressed in both S. erinacei plerocercoid larvae and adult worms. Our results collectively suggested that S. erinacei Mn-SOD is a typical mitochondrial Mn-SOD and may play an important role in parasite physiology, detoxifying excess superoxide radicals generated in the mitochondria.     

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.     

Cloning of the novel putative apoptosis-related gene of Spirometra erinacei (Order Pseudophyllidae)

We postulated that apolysis was processed in accordance with apoptotic changes occurring in a cestode, Spirometra erinacei (Pseudophyllidea). We cloned the novel putative apoptosis-associated gene from S. erinacei via screening of a S. erinacei cDNA library with a ced-3 gene (activator of apoptosis) probe from Caenorhabditis elegans. We identified a 261-bp cDNA sequence, which encodes for an 86-amino acid protein. The cloned gene expression was observed in the neck and gravid proglottids via Northern blotting, using cloned cDNA inserts as probes, but the clone was not expressed in any of other tissues. We suggest that this gene may be involved in the apolysis of S. erinacei during normal tissue development and differentiation in cestode parasites.     

A 54 kDa cysteine protease purified from the crude extract of Neodiplostomum seoulense adult worms

As a preliminary study for the explanation of pathobiology of Neodiplostomum seoulense infection, a 54 kDa protease was purified from the crude extract of adult worms by sequential chromatographic methods. The crude extract was subjected to DEAE-Sepharose Fast Flow column, and protein was eluted using 25 mM Tris-HCl (pH 7.4) containing 0.05, 0.1, 0.2 and 0.4 M NaCl in stepwise elution. The 0.2 M NaCl fraction was further purified by Q-Sepharose chromatography and protein was eluted using 20 mM sodium acetate (pH 6.4) containing 0.05, 0.1, 0.2 and 0.3 M NaCl, respectively. The 0.1M NaCl fraction showed a single protein band on SDS-PAGE carried out on a 7.5-15% gradient gel. The proteolytic activities of the purified enzyme were specifically inhibited by L-trans-epoxy-succinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid. The enzyme, cysteine protease, showed the maximum proteolytic activity at pH 6.0 in 0.1 M buffer, and degraded extracellular matrix proteins such as collagen and fibronectin with different activities. It is suggested that the cysteine protease may play a role in the nutrient uptake of N. seoulense from the host intestine.     

A 27 kDa cysteine protease secreted by newly excysted Paragonimus westermani metacercariae induces superoxide anion production and degranulation of human eosinophils

Eosinophil degranulation plays a crucial role in tissue inflammatory reactions associated with helminth parasitic infections and allergic diseases. Paragonimus westermani, a lung fluke causing human paragonimiasis, secretes a large amount of cysteine proteases, which are involved in nutrient uptake, tissue invasion, and modulation of hos's immune responses. There is, however, limited information about the response of eosinophils to direct stimulation by cysteine proteases (CP) secreted by P. westermani. In the present study, we tested whether degranulation and superoxide production from human eosinophils can be induced by stimulation of the 2 CP (27 kDa and 28 kDa) purified from excretory-secretory products (ESP) of P. westermani newly excysted metacercariae (PwNEM). A large quantity of eosinophil-derived neurotoxin (EDN) was detected in the culture supernatant when human eosinophils isolated from the peripheral blood were incubated with the purified 27 kDa CP. Furthermore, the 27 kDa CP induced superoxide anion production by eosinophils in time- and dose-dependent manners. In contrast, the purified 28 kDa CP did not induce superoxide production and degranulation. These findings suggest that the 27 kDa CP secreted by PwNEM induces superoxide production and degranulation of human eosinophils, which may be involved in eosinophil-mediated tissue inflammatory responses during the larval migration in human paragonimiasis.     

Differentiation of the tegument and associated structures in Diphyllobothrium dendriticum Nitsch (1824) (Cestoda: Pseudophyllidea). An electron microscopical study

Grammeltvedt Anne-François 1973. Differentiation of the tegument and associated structures in Diphyllobothrium dendriticum Nitsch 1824 (Cestoda : Pseudophyllidea). An electron microscopical study. International Journal for Parasitology3: 321–327. The differentiation of the tegument and associated structures of the coracidium, procercoid, plerocercoid and adult is described. The embryophore is composed of four zones and is covered by a fibrous layer resembling a glycocalyx. The oncospheral plasma membrane is extensively folded. A typical cestode tegument, with a distal and perinuclear cytoplasm, is probably already existing in the coracidium. The formation of the microvilli starts after about three days in the copepod host. In young procercoids ribosomes and Golgi complexes were observed in the distal cytoplasm. These organelles disappear at later stages. The infective procercoid has a typical tegument. The microvilli are shaped like a thorn compressed from the sides. They have an electron dense tip and a less dense base in which microfilaments are seen. Bodies, called disc-shaped and lamellated bodies, are described. The microvilli of the plerocercoid are characterized by a great variation in shape. The villi are bounded by two unit membranes. The lamellated bodies are especially well developed. The adult microvilli are uniform in shape. The lamellated bodies are few in young adults and disappear in mature worms.     

Component proteins and protease activities in excretory-secretory product of sparganum.

Spirometra mansoni plerocercoid (sparganum) was incubated in saline at 4 degrees C or 37 degrees C up to 100 hours. Protein contents in the excretory-secretory product (ESP) were rather constant (mean 7.7 mg of protein/gram of sparganum) in the preparations. Reducing SDS-PAGE of ESP showed similar protein subunit compositions with those in crude extract. Antigenic 36 and 31 kDa proteins were major bands in ESP. ESP exhibited specific activities of protease (2.9-5.3 units/mg) at pH 6.0 and pH 7.5. Presence of protease activity in ESP may be a supporting evidence that hitherto known cysteine protease of sparganum is possibly secreted.     

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.     

Excretory bladder: the source of cysteine proteases in Paragonimus westermani metacercariae

The cysteine proteases of Paragonimus westermani metacercariae are involved in metacercarial excystment, host immune modulation, and possibly in tissue penetration. In order to clarify the origin of the enzymes, 28 and 27 kDa cysteine proteases in metacercarial excretory-secretory products were purified through the FPLC system using Mono Q column chromatography. The polyclonal antibodies to the enzymes were produced in BALB/c mice. Immunolocalization studies revealed that both cysteine proteases were distributed at the linings of excretory bladder and excretory concretions of the metacercariae. It was suggested that the excretory epithelium of P. westermani undertake the secretory function of metacercarial cysteine proteases, in addition to its role as a route for eliminating waste products.     

A new method for concentration of proteins in the calcareous corpuscles separated from the spargana of Spirometra erinacei

Calcareous corpuscles are a characteristic structure found in larval and adult stage cestodes. These corpuscles are known to contain several protein components and to possess protein-binding activity. However, the proteins bound to calcareous corpuscles in situ have not been studied. The present study was undertaken to identify the proteins on calcareous corpuscles. Calcareous corpuscles were purified from the plerocercoids (= spargana) of Spirometra erinacei, and serially dissolved using 0.1 M sulfamic acid solution. Collected supernatants were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. The results showed that only the fraction remaining after the 19th dissolved fraction contained proteins. A total of 20 protein molecules were detected in gel, with major bands at 56, 53, 46, 40, 35, 29, 28, 24.5, 21, 19, 16, 13, 10 and 8 kDa. In particular, the proteins corresponding to the 21 and 16 kDa bands were most abundant. Our results demonstrated for the first time the protein contents of the calcareous corpuscles of spargana. Further studies on the functions of these proteins are required.     

Purification and characterization of a 33 kDa serine protease from Acanthamoeba lugdunensis KA/E2 isolated from a Korean keratitis patient

In order to evaluate the possible roles of secretory proteases in the pathogenesis of amoebic keratitis, we purified and characterized a serine protease secreted by Acanthamoeba lugdunensis KA/E2, isolated from a Korean keratitis patient. The ammonium sulfate-precipitated culture supernatant of the isolate was purified by sequential chromatography on CM-Sepharose, Sephacryl S-200, and mono Q-anion exchange column. The purified 33 kDa protease had a pH optimum of 8.5 and a temperature optimum of 55 degrees C. Phenylmethylsulfonylfluoride and 4-(2- Aminoethyl)-benzenesulfonyl-fluoride, both serine protease specific inhibitors, inhibited almost completely the activity of the 33 kDa protease whereas other classes of inhibitors did not affect its activity. The 33 kDa enzyme degraded various extracellular matrix proteins and serum proteins. Our results strongly suggest that the 33 kDa serine protease secreted from this keratopathogenic Acanthamoeba play important roles in the pathogenesis of amoebic keratitis, such as in corneal tissue invasion, immune evasion and nutrient uptake.     

Litomosoides sigmodontis cystatin acts as an immunomodulator during experimental filariasis.

During chronic filariasis, parasite-specific cellular responsiveness is profoundly down-regulated. Cystatins, a group of cysteine protease inhibitors, have been implicated in this suppressive activity. In an attempt to investigate the effects of cystatins in vivo, we isolated and expressed a 14 kDa protein of the rodent filaria Litomosoides sigmodontis with substantial homologies to cystatins from human pathogenic filariae. Cystatin was detected in antigen preparations of several developmental stages of L. sigmodontis, as well as in the supernatants of in vitro cultured adult worms. On closer examination, L. sigmodontis cystatin (Ls-Cystatin) migrated as two separate bands at 14 and 15 kDa. When cystatin was introduced into the peritoneal cavity of C57BL/6 mice via micro-osmotic pumps, the production of nitric oxide was profoundly reduced upon microfilarial challenge and, at the same time, synthesis of TNF-alpha mRNA became up-regulated. Furthermore, antigen-specific proliferative response of spleen cells to circulating L. sigmodontis microfilariae was significantly diminished in the presence of cystatin, whereas the antibody production was not suppressed. In vaccination trials, using the L. sigmodontis/BALB/c mouse model of filariasis, L. sigmodontis cystatin did not generate protective effects in terms of adult worm recovery, however, lower numbers of patent infections, i.e. less infections with microfilaraemia were observed in vaccinated animals. These results suggested that cystatin acts as an immunomodulatory molecule during the course of a filarial infection, and its neutralisation might contribute to generate protective immune responses.     

Leukocyte accumulation in sparganosis: further characterization of an eosinophil chemotactic factor of the plerocercoid of Spirometra erinacei

An eosinophil chemotactic (ECF) was partially purified from plerocercoids of Spirometra erinacei by a combination of anion-exchange chromatography on DE52 and gel filtration on Sephacryl S-200. The molecular weight of ECF was estimated to be 25,000-45,000 by high-pressure liquid chromatography. The ECF was bound with concanavalin A-Sepharose. The ECF was sensitive to periodate oxidation and to heating (56 degrees C, 30 min). On isoelectric focusing, eosinophil chemotactic activity was clearly revealed at pI 4.1. These results suggest that ECF of S. erinacei plerocercoid is an acidic glycoprotein. An intradermal injection of ECF eosinophil attractions in the normal guinea pig skin.     

Comparison of carbohydrate moieties of sparganum proteins of the snake,mouse and those of adult worm

The carbohydrate moieties of larval sparganum proteins in two different species, the snakes, Elaphe rufodorsata, the Balb/c mouse and those of the adult worm, Spirometra erinacei, were compared using five different lectins including GNA, SNA, MAA, PNA and DSA. The GNA positive 53 kDa molecule, which is excretory-secretory protease in the sparganum from the snake showed a stage specific and developmental regulation. We also suggested that sparganum glycosylation may be involved in immune evasion and differentiation into an adult worm.