RESUMO
Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to Zn2+ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis.
Assuntos
Animais , Sequência de Aminoácidos , Clonorchis sinensis/enzimologia , Análise por Conglomerados , Sequência Conservada , DNA Complementar/genética , Metabolismo Energético , Perfilação da Expressão Gênica , Mitocôndrias/metabolismo , Modelos Moleculares , Peso Molecular , Filogenia , Conformação Proteica , Homologia de Sequência de Aminoácidos , Ubiquitina-Proteína Ligases/químicaRESUMO
Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to Zn2+ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis.
Assuntos
Animais , Sequência de Aminoácidos , Clonorchis sinensis/enzimologia , Análise por Conglomerados , Sequência Conservada , DNA Complementar/genética , Metabolismo Energético , Perfilação da Expressão Gênica , Mitocôndrias/metabolismo , Modelos Moleculares , Peso Molecular , Filogenia , Conformação Proteica , Homologia de Sequência de Aminoácidos , Ubiquitina-Proteína Ligases/químicaRESUMO
Many immune down-regulatory molecules have been isolated from parasites, including cystatin (cystain protease inhibitor). In a previous study, we isolated and characterized Type I cystatin (CsStefin-1) of the liver fluke, Clonorchis sinensis. To investigate whether the CsStefin-1 might be a new host immune modulator, we induced intestinal inflammation in mice by dextran sodium sulfate (DSS) and treated them with recombinant CsStefin-1 (rCsStefin-1). The disease activity index (DAI) increased in DSS only-treated mice. In contrast, the DAI value was significantly reduced in rCsStefin-1-treated mice than DSS only-treated mice. In addition, the colon length of DSS only-treated mice was shorter than that of rCsStefin-1 treated mice. The secretion levels of IFN-gamma and TNF-alpha in the spleen and mesenteric lymph nodes (MLNs) were significantly increased by DSS treatment, but the level of TNF-alpha in MLNs was significantly decreased by rCsStefin-1 treatment. IL-10 production in both spleen and MLNs was significantly increased, and IL-10+F4/80+ macrophage cells were significantly increased in the spleen and MLNs of rCsStefin-1 treated mice after DSS treatment. In conclusion, rCsStefin-1 could reduce the intestinal inflammation occurring after DSS treatment, these effects might be related with recruitment of IL-10 secreting macrophages.
Assuntos
Animais , Feminino , Camundongos , Antígenos de Diferenciação/análise , Clonorchis sinensis/enzimologia , Colo/patologia , Cistatinas/metabolismo , Citocinas/metabolismo , Sulfato de Dextrana/toxicidade , Proteínas de Helminto/metabolismo , Fatores Imunológicos/metabolismo , Inflamação/induzido quimicamente , Interleucina-10/análise , Intestinos/efeitos dos fármacos , Linfonodos/imunologia , Macrófagos/química , Camundongos Endogâmicos C57BL , Índice de Gravidade de Doença , Baço/imunologiaRESUMO
During the past several decades, researches on parasite genetics have progressed from biochemical and serodiagnostic studies to protein chemistry, molecular biology, and functional gene studies. Nowadays, bioinformatics, genomics, and proteomics approaches are being applied by Korean parasitology researchers. As for Clonorchis sinensis, investigations have been carried out to identify its functional genes using forward and reverse genetic approaches and to characterize the biochemical and biological properties of its gene products. The authors review the proteins of cloned genes, which include antigenic proteins, physiologic and metabolic enzymes, and the gene expression profile of Clonorchis sinensis.
Assuntos
Animais , Humanos , Clonorquíase/parasitologia , Clonorchis sinensis/enzimologia , Regulação da Expressão Gênica , Proteínas de Helminto/genéticaRESUMO
We compared the DNA sequence difference of isolates of Clonorchis sinensis from one Korean (Kimhae) and two Chinese areas (Guangxi and Shenyang). The sequences of nuclear rDNA (18S, internal transcribed spacer 1 and 2: ITS1 and ITS2) and mitochondrial DNA (cytochrome c oxidase subunit 1: cox1) were compared. A very few intraspecific nucleotide substitution of the 18S, ITS1, ITS2 and cox1 was found among three isolates of C. sinensis and a few nucleotide insertion and deletion of ITS1 were detected. The 18S, ITS1, ITS2 and cox1 sequences were highly conserved among three isolates. These findings indicated that the Korean and two Chinese isolates are similar at the DNA sequence level.
Assuntos
Animais , Sequência de Bases , China , Clonorchis sinensis/enzimologia , Estudo Comparativo , DNA de Helmintos/química , DNA Mitocondrial/química , DNA Ribossômico/química , DNA Espaçador Ribossômico/química , Complexo IV da Cadeia de Transporte de Elétrons/genética , Marcadores Genéticos , Coreia (Geográfico) , Dados de Sequência Molecular , RNA Ribossômico 18S/genética , Alinhamento de Sequência , Especificidade da Espécie , Variação GenéticaRESUMO
Glutathione S-transferase (28GST) with molecular mass of 28 kDa is an antioxidant enzyme abundant in Clonorchis sinensis. In adult C. sinensis, 28GST was localized in tegumental syncytium, cytons, parenchyma, and sperm tails examined by immunoelectron microscopy. C. sinensis 28GST was earlier found to neutralize bioreactive compounds and to be rich in eggs. Accordingly, it is suggested that 28GST plays important roles in phase II defense system and physiological roles in worm fecundity of C. sinensis.