Purification of native Sigma class glutathione transferase from Fasciola hepatica

Duncan, Joshua D.; Cutress, David; Morphew, Russell M.; Brophy, Peter M.

doi:10.1016/j.molbiopara.2018.04.006

Cited by 8 publications

(9 citation statements)

References 24 publications

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“…During infestation, adult F. hepatica can release GST (as one of important components of ESPs), which is not only involved in the immune responses of the host, but related to the resistance of F. hepatica to triclabendazole, and also reduced liver disease. Therefore, GST is often used as a candidate vaccine or drug target for research [ 14 , 16 , 35 ]. Previous research has reported that the main component of ESPs of this parasite may suppress immune responses of the host by modulating the function of antigen presentation cells (APCs) [ 30 ].…”

Section: Discussionmentioning

confidence: 99%

Molecular characterization of a novel GSTO2 ofFasciola hepaticaand its roles in modulating murine macrophages

et al. 2022

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Fascioliasis is an important zoonotic helminthic disease caused by Fasciola hepatica and poses a serious threat to global public health. To evade the immune response of its host (humans or animals), F. hepatica secretes various antioxidant enzymes such as glutathione transferase (GST) to facilitate its invasion, migration and parasitism in vivo. To investigate the biological functions of a novel omega-class GST (GSTO), the molecular features of GSTO2 of F. hepatica were analyzed by online software, and the biochemical properties in vitro of recombinant GSTO2 (rGSTO2) were dissected. Then, the regulatory roles of rGSTO2 protein in murine macrophages in vitro were further explored. The results revealed that the GSTO2 gene encodes 254 amino acids, which harbor the characteristic N-terminal domain (βαβαββα) and C-terminal domain (α-helical) of the cytoplasmic GST superfamily. GSTO2 was mainly expressed in F. hepatica vitelline follicles, intestinal tract, excretory pores and vitelline cells, with thioltransferase and dehydroascorbate reductase activities. Moreover, rGSTO2 protein could be taken up by murine macrophages and significantly inhibit the viability of macrophages. In addition, rGSTO2 protein could significantly promote apoptosis and modulate the expression of cytokines in macrophages. These findings suggested that F. hepatica GSTO2 plays an important role in modulating the physiological functions of macrophages, whereby this protein might be involved in immunomodulatory and anti-inflammatory roles during infection. This study provided new insights into the immune-evasion mechanism of F. hepatica and may contribute to the development of a potential anti-inflammatory agent.

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Section: Discussionmentioning

confidence: 99%

Molecular characterization of a novel GSTO2 ofFasciola hepaticaand its roles in modulating murine macrophages

et al. 2022

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“…All SDS-PAGE separations were conducted as described previously [ 63 ]. Successful rSmLEV1.3 purification was confirmed by SDS-PAGE separation, followed by Coomassie Blue staining [ 64 ] and anti-His 6 western blot analysis.…”

Section: Methodsmentioning

confidence: 99%

“…The anti-SmTSP-2 antibody was a kind gift from Professor Alex Loukas (James Cook University, Australia). For western blotting, 1.5 μg of parasite extract or 100 ng of each EV fraction was mixed with 2x SDS non-reducing sample buffer (0.143 M SDS + 0.125 M Tris (pH 6.8) + 20% v/v glycerol) and incubated for 5 min at 100°C, prior to separation by 1D-SDS-PAGE and transfer to polyvinylidene difluoride (PVDF) membrane as described previously [ 63 ]. Membrane blocking occurred for 2 h at room temperature in blocking buffer (PBS containing 0.2% fish skin gelatine 0.3% Tween 20).…”

Section: Methodsmentioning

confidence: 99%

“…Successful rSmLEV1.3 purification was confirmed by SDS-PAGE separation, followed by Coomassie Blue staining [64] and anti-His 6 western blot analysis. Predicted rSmLEV1.3 proteins bands were excised, and in-gel trypsin digestion followed by mass spectrometric analysis was conducted [63]. Subsequent sequence identification using Mascot (version2.2.1; Matrix Science) database search of the S. mansoni predicted proteins (genome version 7) confirmed the sole presence of rSmLEV1.3 (S2 Table ).…”

Section: Anti-smlev13 Polyclonal Antibody Production and Western Blot Analysismentioning

confidence: 99%

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Schistosoma mansoni Larval Extracellular Vesicle protein 1 (SmLEV1) is an immunogenic antigen found in EVs released from pre-acetabular glands of invading cercariae

et al. 2021

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Extracellular Vesicles (EVs) are an integral component of cellular/organismal communication and have been found in the excreted/secreted (ES) products of both protozoan and metazoan parasites. Within the blood fluke schistosomes, EVs have been isolated from egg, schistosomula, and adult lifecycle stages. However, the role(s) that EVs have in shaping aspects of parasite biology and/or manipulating host interactions is poorly defined. Herein, we characterise the most abundant EV-enriched protein in Schistosoma mansoni tissue-migrating schistosomula (Schistosoma mansoni Larval Extracellular Vesicle protein 1 (SmLEV1)). Comparative sequence analysis demonstrates that lev1 orthologs are found in all published Schistosoma genomes, yet homologs are not found outside of the Schistosomatidae. Lifecycle expression analyses collectively reveal that smlev1 transcription peaks in cercariae, is male biased in adults, and is processed by alternative splicing in intra-mammalian lifecycle stages. Immunohistochemistry of cercariae using a polyclonal anti-recombinant SmLEV1 antiserum localises this protein to the pre-acetabular gland, with some disperse localisation to the surface of the parasite. S. mansoni—infected Ugandan fishermen exhibit a strong IgG1 response against SmLEV1 (dropping significantly after praziquantel treatment), with 11% of the cohort exhibiting an IgE response and minimal levels of detectable antigen-specific IgG4. Furthermore, mice vaccinated with rSmLEV1 show a slightly reduced parasite burden upon challenge infection and significantly reduced granuloma volumes, compared with control animals. Collectively, these results describe SmLEV1 as a Schistosomatidae-specific, EV-enriched immunogen. Further investigations are now necessary to uncover the full extent of SmLEV1’s role in shaping schistosome EV function and definitive host relationships.

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“…Immediately prior to performing tandem mass spectrometry the dried peptides were re-suspended in 20 μL 0.1% v/v formic acid. For 2D SDS PAGE protein spots, the method of Duncan et al (2018) was utilized for LC-MSMS. For whole A. perfoliata Orbitrap Fusion™ Tribrid™ mass spectrometer (Thermo Scientific™), with EASY-Spray™ Source, coupled to an UltiMate™ 3000 RSLCnano System (Thermo Scientific™) as per methods of Rooney et al (2022).…”

Section: Trypsin Digestion and Mass Spectrometrymentioning

confidence: 99%

A dominance of Mu class glutathione transferases within the equine tapeworm Anoplocephala perfoliata

Northcote,

Wititkornkul,

Cutress

et al. 2024

Parasitology

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The most common equine tapeworm, Anoplocephala perfoliata, has often been neglected amongst molecular investigations and has been faced with limited treatment options. However, the recent release of a transcriptome dataset has now provided opportunities for in-depth analysis of A. perfoliata protein expression. Here, global, and sub-proteomic approaches were utilized to provide a comprehensive characterization of the A. perfoliata soluble glutathione transferases (GST) (ApGST). Utilizing both bioinformatics and gel-based proteomics, GeLC and 2D-SDS PAGE, the A. perfoliata ‘GST-ome’ was observed to be dominated with Mu class GST representatives. In addition, both Sigma and Omega class GSTs were identified, albeit to a lesser extent and absent from affinity chromatography approaches. Moreover, 51 ApGSTs were localized across somatic (47 GSTs), extracellular vesicles (EVs) (Whole: 1 GST, Surface: 2 GSTs) and EV depleted excretory secretory product (ESP) (9 GSTs) proteomes. In related helminths, GSTs have shown promise as novel anthelmintic or vaccine targets for improved helminth control. Thus, provides potential targets for understanding A. perfoliata novel infection mechanisms, host–parasite relationships and anthelmintic treatments.

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Purification of native Sigma class glutathione transferase from Fasciola hepatica

Cited by 8 publications

References 24 publications

Molecular characterization of a novel GSTO2 ofFasciola hepaticaand its roles in modulating murine macrophages

Molecular characterization of a novel GSTO2 ofFasciola hepaticaand its roles in modulating murine macrophages

Schistosoma mansoni Larval Extracellular Vesicle protein 1 (SmLEV1) is an immunogenic antigen found in EVs released from pre-acetabular glands of invading cercariae

A dominance of Mu class glutathione transferases within the equine tapeworm Anoplocephala perfoliata

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