Molecular cloning and characterization of Schistosoma japonicum aldose reductase

Antioxidant defense is an essential mechanism for schistosomes to cope with damage from host immune-generated reactive oxygen species. The evaluation of the effects of aldose reductase, an important enzyme that may be involved in this system, has long been neglected. In the present study, aldose reductase of Schistosoma japonicum (SjAR) was cloned and characterized. The activity of SjAR was assessed in vitro and was suppressed by the reported inhibitor, epalrestat. RT-PCR analysis revealed that SjAR was expressed at each of the development stages analyzed with increased levels in cercariae. The results also showed that SjAR was expressed at higher levels in adult male worms than in adult female worms. Indirect enzyme-linked immunosorbent assay and western blot analysis indicated that the purified recombinant SjAR (rSjAR) protein displayed a significant level of antigenicity. Immunolocalization analysis revealed that SjAR was mainly distributed in the gynecophoral canal of adult male worms. BALB/c mice immunized with rSjAR induced a 32.91 % worm reduction compared to the adjuvant group (P?<?0.01). Moreover, a 28.27 % reduction in egg development in the liver (P?>?0.05) and a 42.75 % reduction in egg development in the fecal samples (P?<?0.05) were also observed. These results suggested that SjAR may be a potential new drug target or vaccine candidate for schistosomes.     

Identification and characterization of glyceraldehyde 3-phosphate dehydrogenase from Fasciola gigantica

Fasciola gigantica is an important food-borne trematode responsible for the hepatobiliary disease, commonly known as fascioliasis. In F. gigantica, the glyceraldehyde 3-phosphate dehydrogenase (FgGAPDH) is a key enzyme of the glycolytic pathway and catalyzes the reversible oxidative phosphorylation of d-glyceraldehyde-3-phosphate (G-3-P) to 1,3-bisphosphoglycerate (1,3-BPG), with the simultaneous reduction of NAD⁺ to NADH. In the present study, we analyzed the sequence of FgGAPDH and investigated its structural, binding, and catalytic properties. Sequence alignment of FgGAPDH showed 100% identity with the sister fluke Fasciola hepatica GAPDH. The gapdh gene was cloned and expressed in Escherichia coli, and the recombinant protein was purified. The purified FgGAPDH exists as a homo-tetramer, composed of a ~ 37-kDa subunit under non-dissociating conditions at 300 mM salt concentration indicating that higher salt stabilizes the tetrameric state. The binding of the cofactor NAD⁺ caused a conformational rearrangement in the enzyme structure, leading to the stabilization of the enzyme. A homology model of FgGAPDH was constructed, the cofactor (NAD⁺) and substrate (G-3-P) were docked, and the binding sites were identified in a single chain. The inter-subunit cleft of GAPDH that has been exploited for structure-based drug design in certain protozoan parasites is closed in the case of FgGAPDH, similar to the human GAPDH. Thus, the conformation of FgGAPDH in this region is similar to the human enzyme. Therefore, GAPDH may not be a suitable target for drug discovery against fascioliasis. Still, the analysis of the structural and functional attributes of GAPDH will be significant in understanding the various roles of this enzyme in the parasite as well as provide new insights into the biochemistry of flukes.

     

Expression and characterization of glutathione peroxidase of the liver fluke,Fasciola gigantica

Parasitology Research - Glutathione peroxidase (GPx) is a key member of the family of antioxidant enzymes in trematode parasites including Fasciola spp. Because of its abundance and central role as...     

Identification and expression of Fasciola gigantica thioredoxin

In the present study, a cDNA encoding Trx from F. gigantica (FgTrx) was cloned by polymerase chain reaction (PCR). The sequence of FgTrx, analyzed by BLAST, SignalP, and ClustralW programs, showed 315 bp of an open reading frame (ORF), 12 bp 5’UTR, 78 bp 3’UTR, and the putative FgTrx peptide comprising of 104 amino acids, with a molecular weight of 11.68 kDa, with the active site containing five amino acids (tryptophan, cysteine, glycine, proline, cysteine) with a conserved dithiol motif from the two cysteines, and pI 5.86. The peptide had no signal sequence; hence, it was not a secreted protein. The recombinant FgTrx was expressed in Escherichia coli BL21 (DE3) and used for production for a polyclonal antibody in rabbits (anti-rFgTrx). The FgTrx protein expression, estimated by indirect ELISA using the rabbit anti-rFgTrx as probe, showed high levels in eggs, 2- and 4-week-old juveniles, and adult parasite. In a functional test, the rFgTrx exhibited specific activity that could be suppressed by an inhibitor (PX12). When tested by immunoblotting and immunohistochemistry, rabbit anti-rFgTrx reacted with natural FgTrx at a molecular weight of 11.68 kDa from eggs, metacercariae, NEJ, 2- and 4-week-old juveniles, and adult F. gigantica. The FgTrx protein was distributed at high levels in the tegument of 2- and 4-week-old juveniles, and the tegument, parenchyma, eggs, and reproductive organs of adult parasites. FgTrx may be one of the major factors acting against oxidative stresses that can damage the parasite; hence, it could be considered as a novel vaccine or drug target.     

Molecular confirmation that Fasciola gigantica can undertake aberrant migrations in human hosts

Two cases of aberrant migration by the liver fluke Fasciola gigantica in humans are reported. In both cases, subadult worms emerged through the skin. The identity of the worms was confirmed from their DNA sequences. This uncommon human pathogen might be more likely than F. hepatica to undertake aberrant migrations in humans.     

Acquired resistance of merino sheep against Fasciola gigantica

 Merino sheep acquired resistance against Fasciola gigantica, which is contrary to previous observations of infections with F. hepatica in that breed. The acquired resistance was manifest against young adult parasites. St. Croix sheep had previously been shown to have more resistance than European sheep against F. hepatica after primary infection; however, in F. gigantica infections in the present study there was no difference between the resistance levels of the breeds. Antigenic analysis of the host:parasite relationships could lead to identification of protective antigens suitable for use as vaccines. Received: 1 June 1996 / Accepted: 15 June 1996     

Functional analysis of novel aquaporins from Fasciola gigantica

Fascioliasis, caused by liver flukes of the genus Fasciola, is an important disease of ruminants. In order to identify a potential new drug target we have studied aquaporin (AQP) in Fasciola gigantica. AQPs facilitate the transport of water, glycerol and other small solutes across biological membranes. The structure, function, and pathology of AQPs have been extensively studied in mammals but data for AQPs from trematodes is still limited. In the present study, we have functionally characterized two closely related AQP isoforms, FgAQP-1 and FgAQP-2, from the trematode F. gigantica. Immunohistochemical analysis located the FgAQPs in the tegumental cells, their processes and the tegument itself. In addition, they were present in the epithelial linings of testes and ovary. Expression in Xenopus oocytes of these FgAQPs increased osmotic water permeability 3-4-fold but failed to increase glycerol and urea permeability. AQPs have two highly conserved NPA motifs that are important for the function of the channel pore. In FgAQP-1 and FgAQP-2 the first NPA motif is changed to TAA. Substitution of Thr with Asn in the TAA motif of FgAQP-1 increased its water permeability twofold but did not affect urea and glycerol impermeability while the substitution at the pore mouth of Cys204 by Tyr caused loss of water permeability. In addition, the FgAQPs did not increase methylamine and ammonia permeability after expression in yeast. In comparison to rat AQP-1 the described FgAQPs showed low water permeability and further in vivo analyses are necessary to determine their contribution to osmoregulation in Fasciola.     

Production and characterization of a monoclonal antibody against recombinant glutathione S-transferase (GST) of Fasciola gigantica

A monoclonal antibody (MoAb) against a recombinant glutathione S-transferase (rGST) of F. gigantica was produced in BALB/c mice. Reactivity and specificity of this monoclonal antibody was assessed by ELISA and immunoblotting. Six stable clones, namely 3A3, 3B2, 3C6, 4A6, 4B1 and 4D6 were obtained, All these MoAb reacted with rGST and native GST at a molecular weight of 28 kDa and found to be IgG1, kappa-light chain isotypes. These MoAb cross-reacted with Schistosoma mansoni and Schistosoma japonicum antigens at molecular weights of 28 and 26 kDa, respectively, but no cross-reactions were detected with antigens of Eurytrema and Paramphistomum spp. The localization of GST in metacercaria, 7-week-old juvenile and adult F. gigantica was performed by immunofluorescence technique, using MoAb as well as polyclonal antibody (PoAb) to the native protein as probes. In general, all clones of MoAb gave similar results and the pattern was quite similar to staining by PoAb. The fluorescence was intense, which implied the presence of a high concentration of GST in the parenchymal tissue in all stages of the parasite. However, the parenchymal cells were not evenly stained which implied the existence of subpopulations of this cell type with regard to GST production and storage. In addition, in adult and juvenile stages a moderate fluorescence was present in the basal layer of the tegument, while light fluorescence was observed in the caecal epithelium, cells in the ovary, testis and vitelline gland of the adult. In the metacercaria stage, in addition to parenchymal tissue, the tegument and tegumental cells were stained relatively more intense with MoAb and PoAb than in other stages.     

Purification, characterization and kinetic properties of the multifunctional thioredoxin-glutathione reductase from Taenia crassiceps metacestode (cysticerci)

The multifunctional enzyme thioredoxin-glutathione reductase (TGR) was purified to homogeneity from the soluble fraction of Taenia crassiceps metacestode (cysticerci). Specific activities of 17.5 and 4.7 U mg(-1) were obtained with Plasmodium falciparum thioredoxin and GSSG, respectively, at pH 7.75. Under the same conditions, Km values of 17, 15, and 3 microM were respectively calculated for thioredoxin, GSSG and NADPH. The kcat/Km ratio of T. crassiceps TGR for both thioredoxin and GSSG falls in the range observed for typical thioredoxin reductases and glutathione reductases. Purified enzyme also showed glutaredoxin activity, with a specific activity of 19.2 U mg(-1) with hydroxyethyl disulfide as substrate. Both thioredoxin and GSSG disulfide reductase activities were fully inhibited by nanomolar concentrations of the gold compound auranofin, supporting the existence of an essential selenocysteine residue. Relative molecular mass of native enzyme was 136,000 +/- 3000, while the corresponding value per subunit, obtained under denaturing conditions, was 66,000 +/- 1000. These results suggest TGR exists as a dimeric protein. Isoelectric point of the enzyme was at pH 5.2. Moderate or high concentrations of GSSG, but neither thioredoxin nor NADPH, resulted in a markedly hysteretic kinetic, characterized by a lag time before the steady state velocity was reached. The magnitude of the lag time was dependent on GSSG and enzyme concentration. Preincubation of the enzyme with micromolar concentrations of GSH or DTT abolished the hysteresis, suggesting that a thiol-disulfide exchange mechanism is involved.     

Fasciola hepatica and Fasciola gigantica: comparative morphometric studies on the redial stage of both species

Experimental infections of Galba truncatula with Fasciola gigantica or F. hepatica were carried out under laboratory conditions (20°C) to determine the characteristics of rediae of both species via their morphometry and to find reliable measurements that might be efficiently used to discriminate between the rediae of both species of Fasciola. These results were compared to those of another snail: Radix natalensis, infected with either F. gigantica or F. hepatica under the same protocol. At day 28 post-exposure, abortive infections with F. hepatica were found in a group of R. natalensis. By contrast, live rediae were observed in the other three groups. The group of infected snails and the redial category significantly influenced the mean values of the seven measurements studied and those of three indices. Using the PSLD Fisher test, it was found that the index, distance from the anterior end of the body to the collar/length of the body, was an efficient means of distinguishing the rediae of F. hepatica from those of F. gigantica [second-appearing mother rediae (R1b) of the first generation, 0.14 instead of 0.22; daughter rediae (R2a) produced by the first mother rediae, 0.19 instead of 0.24]. Another index, distance from the anterior end of the body to the collar/diameter of the collar, could also be used to discriminate between rediae (R1b, 0.80 for F. hepatica instead of 1.09 for F. gigantica; R2a, 0.90 instead of 1.26, respectively). Compared to measurements recorded for the rediae of F. hepatica, rediae of F. gigantica can be characterized by the following measurements: the diameter of the pharyngeal lumen and the distance from the anterior end of the body to the collar for larvae developed in R. natalensis, and the length of the body and the distance from the posterior end of the body to lateral projections for those found in G. truncatula. The species of snail host and, consequently, its growth, as well as the species of Fasciola, had a significant influence on the morphometric characters of the redial stage.     

Characterization of specific and cross-reacting antigens of Fasciola gigantica by immunoblotting

Somatic antigens of F. gigantica, G. explanatum, S. spindale and hydatid cyst ingredients were analysed to identify the cross-reactive antigens among them using Western blot technique. When probed with F. gigantica infected cattle sera, the immunodominant 156 kDa and 28 kDa proteins of F. gigantica was found common amongst the antigens prepared from hydatid cysts ingredients like germinal layer, fertile and sterile, hydatid fluid, fertile and sterile, while another protein of 34 kDa was shared between F. gigantica and antigen prepared from protoscolices. In F. gigantica–buffalo system the proteins of 34 kDa and 28 kDa were found reactive with most of the antigens tested. Immunoaffinity chromatography using, F. gigantica infected rabbit immunoglobulins as legands isolated the immunodominant 34 kDa and 28 kDa proteins in dimer form and the same were found immunodominant in F. gigantica–cattle, F. gigantica–buffalo and F. gigantica–sheep system. No cross-reaction was noted with the sera of goats experimentally infected with Paramphistomum epiclitum. ELISA with the immunodominant proteins of 34 kDa and 28 kDa could be a feasible diagnostic tool for the early detection of bovine fasciolosis.     

Molecular cloning and expression of Cu/Zn-containing superoxide dismutase from Fasciola hepatica

The cytosolic superoxide dismutase (SOD) of Fasciola hepatica, a causative agent of fascioliasis, was purified and characterized. The enzyme consists of two identical subunits, each with an apparent molecular mass of 17.5 kDa. An analysis of the enzyme's primary structure and inhibition studies revealed that the enzyme is a copper/zinc-containing SOD (Cu/Zn-SOD). The enzyme activity was relatively stable in a broad pH range, from pH 7.0 to 10.0, and the enzyme showed maximum activity at pH 7.5. This enzyme also displayed strong antigenicity against sera of bovine and human subjects with fascioliasis. The SOD gene fragment was amplified by PCR with degenerate oligonucleotide primers derived from amino acid sequences conserved in the Cu/Zn-SODs of other organisms. An F. hepatica cDNA library was screened with the SOD gene fragment as a probe. As a result, a complete gene encoding the Cu/Zn-SOD was identified, and its nucleotide sequence was determined. The gene had an open reading frame of 438 bp and 146 deduced amino acids. Comparison of the deduced amino acid sequence of the enzyme with previously reported Cu/Zn-SOD amino acid sequences revealed considerably high homologies. The coding region of the F. hepatica Cu/Zn-SOD was cloned and expressed in Escherichia coli. Staining of native polyacrylamide gel for SOD activity of the expressed protein revealed SOD activity that was inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide. This means that the presence of the recombinant fusion protein is indicative of Cu/Zn-SOD. The expressed protein also reacted with sera of bovine and human subjects with fascioliasis, but it did not react with sera of uninfected bovine and human subjects.     

Molecular cloning and characterization of a new Japanese cedar pollen allergen homologous to plant isoflavone reductase family

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal pollinosis, and more than 10% of Japanese people suffer from this allergic disorder. However, only two major pollen allergens, Cry j 1 and Cry j 2, have been identified and exclusively characterized. OBJECTIVE: The aim of this study was to explore and identify important Japanese cedar pollen allergens other than Cry j 1 or Cry j 2. METHODS: C. japonica cDNA library was immunoscreened by rabbit antiserum raised against a partially purified cedar pollen allergen fraction. An isolated cDNA clone was inserted into a glutathione S-transferase (GST)-tagged Escherichia coli expression vector to obtain recombinant GST fusion protein. Non-fusion recombinant protein was purified by glutathione Sepharose affinity chromatography in conjunction with factor Xa cleavage of the GST moiety. IgE-binding ability of the recombinant protein was then evaluated by western blot analysis and enzyme-linked immunosorbent assay (ELISA). RESULTS: The cDNA encodes 306 amino acids with significant sequence similarity to those of plant isoflavone reductase-like proteins, which include a recently identified birch pollen allergen Bet v 5. Western blot analysis demonstrated that recombinant protein was recognized by cedar pollinosis patient IgE. In contrast to Bet v 5 being reported as a minor allergen, the recombinant protein exhibited 76% IgE binding frequency (19/25) against pollinosis patients. CONCLUSION: Here we identified the third member of Japanese cedar pollen allergen homologous to isoflavone reductase. Its high IgE-binding frequency implicates that the isoflavone reductase homologue might be an additional major pollen allergen in C. japonica.     

Molecular cloning and characterization of cynomolgus monkey Fas

The Fas-FasL system plays a crucial role in the maintenance of homeostasis in the immune system. To characterize the Fas/FasL system in macaque monkeys that are commonly used as experimental primates, we cloned and sequenced Fas cDNA derived from the cynomolgus monkey. The predicted amino acid sequence consists of 331 amino acids with a calculated molecular weight of 35,800. The extracellular cysteine-rich motif of cynomolgus Fas is highly homologous to that of humans (96%), whereas the intracellular death domain has a relatively low similarity to that of humans (86%). An agonistic Fas antibody (CH11) or cynomolgus FasL induced apoptosis in human Fas-transfected K562 cells in the presence of CHX but not in the cynomolgus Fas transfectant. CH11 and FasL failed to trigger apoptosis in the transfectant expressing human-cynomolgus chimera Fas consisting mostly of human-derived extracellular region and cynomolgus-derived intracellular portion. On the other hand, the transfectant expressing cynomolgus-human chimera Fas with human-derived intracellular region underwent apoptosis upon exposure to FasL. In addition, the virus-transformed, Fas-positive cynomolgus monkey cell line was highly sensitive to FasL. These findings suggest that the lack of apoptotic activity in the cynomolgus Fas transfectant in the human cell line might be related to the species-specific structure of Fas, especially of the death domain.     

Molecular characterization and cloning of sheep mitochondrial DNA

Summary Mitochondrial DNA from the liver of a single Rasa Aragonesa sheep has been isolated and characterized. The size of the genome, determined by restriction enzyme analysis, was found to be 16.58 kbp. The cleavage sites for the restriction endonucleases BamHI, HindIII, EcoRI, BglII, PvuII, BstEII and PstI were mapped, and the gene organization deduced through heterologous hybridization using different cloned fragments of the rat mitochondrial genome. Fragments representative of the entire sheep genome were cloned in plasmid vectors pGEM3Z and pUN121.     

Molecular cloning and characterization of mouse CD97

The EGF-TM7 family (CD97 and EMR1) is a group of class II seven-span transmembrane receptors predominantly expressed by cells of the immune system. Recently, we have identified CD55, a regulatory molecule of the complement cascade, as a cellular ligand of human CD97 (hCD97). In this study, the molecular properties of mouse CD97 (mCD97) are described. Like hCD97, mCD97 has an extended extracellular region with several epidermal growth factor-like (EGF) domains. Due to alternative RNA splicing, isoforms with three and four EGF domains exist, designated mCD97(EGF1,2,4) and mCD97(EGF1,2, 3,4) respectively. All EGF domains, except for the N-terminal one, possess a calcium-binding site. In a third isoform mCD97(EGF1,2,X,3, 4), a sequence of 45 amino acids was found between the second and third EGF domain that does not correspond to any known protein module. Using newly generated mCD97 mAb, we show that analogous to the blood expression pattern of hCD97, mCD97 can be found on lymphoid and myeloid cells. Adhesion of mouse erythrocytes and splenocytes to COS cells expressing mCD97(EGF1,2,4) or mCD97(EGF1,2, 3,4) could be blocked by mouse CD55 (mCD55) antibody, identifying mCD55 as a cellular ligand for mCD97. Consistent with the necessity of directly linked EGF domains for the integrity of the CD55-binding site on hCD97, no adhesion was detected to the largest mouse isoform mCD97(EGF1,2,X,3,4). Remarkably, we found that the interaction between CD97 and CD55 is phylogenetically restricted, as indicated by the selective adhesion of primate erythrocytes to hCD97 transfectants, and of mouse and rat erythrocytes to mCD97 transfectants respectively.