Molecular Cloning and Characterization of a Fasciola gigantica Nuclear Receptor Subfamily 1 (FgNR1)

Martviset, Pongsakorn; Chantree, Pathanin; Chaimon, Salisa; Torungkitmangmi, Nattaya; Prathaphan, Parisa; Ruangtong, Jittiporn; Sornchuer, Phornphan; Thongsepee, Nattaya; Sangpairoj, Kant; Adisakwattana, Poom

doi:10.3390/pathogens11121458

Cited by 8 publications

(12 citation statements)

References 74 publications

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“…Interestingly, the native FgRXRα-A can be localized concentratedly in the testes of the adult worm. It is correlated with our previous study of FgNR1, which was detected in the testes and tips of caeca 17 . As previously known, the NRs can interact with several sex hormones, such as testosterone and estrogen, and facilitate sexual development [54][55][56] .…”

Section: Discussionsupporting

confidence: 92%

“…The life cycle of F. gigantica , which involves the host’s liver, where many hormones and bile salts are accumulating, makes us interested in the NRs in this parasite. We suspected some possibilities that NRs would be the key molecules that regulate the parasite biology inside the host body 17 . After getting an infection with F. gigantica , the newly excysted juvenile (NEJ) is released from the infective metacercaria, penetrates the intestinal wall, and finally becomes an adult in the liver parenchyma and major bile ducts 2 .…”

Section: Introductionmentioning

confidence: 99%

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Molecular and biochemical characterizations of a Fasciola gigantica retinoid X receptor-α isoform A (FgRXRα-A)

Torungkitmangmi,

Chantree,

Chaimon

et al. 2024

Sci Rep

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Fascioliasis is a parasitic infection in animals and humans caused by the parasitic flatworm genus Fasciola, which has two major species, F. hepatica and F. gigantica. A major concern regarding this disease is drug resistance, which is increasingly reported worldwide. Hence, the discovery of a novel drug as well as drug targets is crucially required. Therefore, this study aims to characterize the novel drug target in the adult F. gigantica. In the beginning, we hypothesized that the parasite might interact with some host molecules when it lives inside the liver parenchyma or bile ducts, specifically hormones and hormone-like molecules, through the specific receptors, primarily nuclear receptors (NRs), which are recognized as a major drug target in various diseases. The retinoid X receptor (RXR) is a member of subfamily 2 NRs that plays multitudinous roles in organisms by forming homodimers or heterodimers with other NRs. We obtained the full-length amino acid sequences of F. gigantica retinoid X receptor-alpha (FgRXRα-A) from the transcriptome of F. gigantica that existed in the NCBI database. The FgRXRα-A were computationally predicted for the basic properties, multiple aligned, phylogeny analyzed, and generated of 2D and 3D models. Moreover, FgRXRα-A was molecular cloned and expressed as a recombinant protein (rFgRXRα-A), then used for immunization for specific polyclonal antibodies. The native FgRXRα-A was detected in the parasite extracts and tissues, and the function was investigated by in vitro binding assay. The results demonstrated the conservation of FgRXRα-A to the other RXRs, especially RXRs from the trematodes. Interestingly, the native FgRXRα-A could be detected in the testes of the parasite, where the sex hormones are accumulated. Moreover, the binding assay revealed the interaction of 9-cis retinoic acid and FgRXRα-A, suggesting the function of FgRXRα-A. Our findings suggested that FgRXRα-A will be involved with the sexual reproduction of the parasite by forming heterodimers with other NRs, and it could be the potential target for further drug development of fascioliasis.

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Section: Discussionsupporting

confidence: 92%

Section: Introductionmentioning

confidence: 99%

Molecular and biochemical characterizations of a Fasciola gigantica retinoid X receptor-α isoform A (FgRXRα-A)

Torungkitmangmi,

Chantree,

Chaimon

et al. 2024

Sci Rep

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“…Anti-rOvROPN1L antibodies were produced in two female BALB/c mice as previously described [ 36 , 37 ]. At 6 to 8 weeks of age, the mice were immunized at 2-week intervals via three intraperitoneal injections.…”

Section: Methodsmentioning

confidence: 99%

Production and Immunological Characterization of scFv Specific to Epitope of Opisthorchis viverrini Rhophilin-Associated Tail Protein 1-like (OvROPN1L)

et al. 2023

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(1) Background: Opisthorchis viverrini is a significant health problem in the Mekong subregion of Southeast Asia, causing aggressive cholangiocarcinoma. Current diagnostic procedures do not cover early diagnosis and low infection. Hence, an effective diagnostic tool is still required. Immunodiagnosis seems promising, but attempts to generate monoclonal antibodies have not yet been successful. This study aims to develop a single-chain variable antibody fragment (scFv) against Rhophilin-associated tail protein 1-like (ROPN1L), the sperm-specific antigen of adult O. viverrini, which has not been reported elsewhere. (2) Methods: The target epitope for phage screening was L3-Q13 of OvROPN1L, which showed the highest antigenicity to human opisthorchiasis analyzed in a previous study. This peptide was commercially synthesized and used for phage library screening. The isolated phage was produced in a bacterial expression system and tested for specificity in vitro and in silico. (3) Results: One of fourteen phages, named scFv anti-OvROPN1L-CL19, significantly bound to rOvROPN1L compared with non-infected hamster fecal extracts. This phage clone was successfully produced and purified using Ni-NTA chromatography. Indirect ELISA demonstrated that scFv anti-OvROPN1L-CL19 has a high reactivity with O. viverrini-infected hamster fecal extracts (12 wpi, n = 6) in comparison with non-infected hamster fecal extracts (0 wpi, n = 6), while the polyclonal rOvROPN1L antibodies did not show such a difference. Molecular modeling and docking confirmed our in vitro findings. (4) Conclusion: scFv anti-OvROPN1L-CL19 could be used as an effective material for developing O. viverrini-immunodiagnostic procedures in the future.

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“…FgCyst fragments were then subcloned into a pQE-30 expression vector (Qiagen, Hilden, Germany) that contained an N-terminus histidine tag, then transformed into M15 E. coli . For rFgCyst production, the positive transformant of M15/pQE-30 containing FgCyst was cultured until the OD 600 reached 0.4–0.6 before being induced with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 37 °C for 2 h. Bacterial cells were harvested and rFgCyst was purified under native conditions using high-performance Ni Sepharose ® (Cytiva, Uppsala, Sweden) [ 42 ].…”

Section: Methodsmentioning

confidence: 99%

Type I Cystatin Derived from Fasciola gigantica Suppresses Macrophage-Mediated Inflammatory Responses

et al. 2023

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There is an inverse relationship between the high incidence of helminth infection and the low incidence of inflammatory disease. Hence, it may be that helminth molecules have anti-inflammatory effects. Helminth cystatins are being extensively studied for anti-inflammatory potential. Therefore, in this study, the recombinant type I cystatin (stefin-1) of Fasciola gigantica (rFgCyst) was verified to have LPS-activated anti-inflammatory potential, including in human THP-1-derived macrophages and RAW 264.7 murine macrophages. The results from the MTT assay suggest that rFgCyst did not alter cell viability; moreover, it exerted anti-inflammatory activity by decreasing the production of proinflammatory cytokines and mediators, including IL-1β, IL-6, IL-8, TNF-α, iNOS, and COX-2 at the gene transcription and protein expression levels, as determined by qRT-PCR and Western blot analysis, respectively. Further, the secretion levels of IL-1β, IL-6, and TNF-α determined by ELISA and the NO production level determined by the Griess test were decreased. Furthermore, in Western blot analysis, the anti-inflammatory effects involved the downregulation of pIKKα/β, pIκBα, and pNF-κB in the NF-κB signaling pathway, hence reducing the translocation from the cytosol into the nucleus of pNF-κB, which subsequently turned on the gene of proinflammatory molecules. Therefore, cystatin type 1 of F. gigantica is a potential candidate for inflammatory disease treatment.

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Molecular Cloning and Characterization of a Fasciola gigantica Nuclear Receptor Subfamily 1 (FgNR1)

Cited by 8 publications

References 74 publications

Molecular and biochemical characterizations of a Fasciola gigantica retinoid X receptor-α isoform A (FgRXRα-A)

Molecular and biochemical characterizations of a Fasciola gigantica retinoid X receptor-α isoform A (FgRXRα-A)

Production and Immunological Characterization of scFv Specific to Epitope of Opisthorchis viverrini Rhophilin-Associated Tail Protein 1-like (OvROPN1L)

Type I Cystatin Derived from Fasciola gigantica Suppresses Macrophage-Mediated Inflammatory Responses

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