title: Molecular characterization of Clonorchis sinensis secretory myoglobin: delineating its role in anti-oxidative survival.
abstract: BACKGROUND
  Clonorchiasis is a globally important, neglected food-borne disease caused by Clonorchis sinensis (C. sinensis), and it is highly related to cholangiocarcinoma and hepatocellular carcinoma. Increased molecular evidence has strongly suggested that the adult worm of C. sinensis continuously releases excretory-secretory proteins (ESPs), which play important roles in the parasite-host interactions, to establish successful infection and ensure its own survival. Myoglobin, a hemoprotein, is present in high concentrations in trematodes and ESPs. To further understand the biological function of CsMb and its putative roles in the interactions of C. sinensis with its host, we explored the molecular characterization of CsMb in this paper.
  
  
  METHODS
  We expressed CsMb and its mutants in E. coli BL21 and identified its molecular characteristics using bioinformatics analysis and experimental approaches. Reverse transcription PCR analysis was used to measure myoglobin transcripts of C. sinensis with different culture conditions. The peroxidase activity of CsMb was confirmed by spectrophotometry. We co-cultured RAW264.7 cells with recombinant CsMb (rCsMb), and we then measured the production of hydrogen peroxide (H2O2) and nitric oxide (NO) in addition to the mRNA levels of inducible nitric oxide synthase (iNOS), Cu-Zn superoxide dismutase (SOD1) and Mn superoxide dismutase (SOD2) in activated RAW264.7 cells.
  
  
  RESULTS
  In the in vitro culture of adult worms, the transcripts of CsMb increased with the increase of oxygen content. Oxidative stress conditions induced by H2O2 increased the levels of CsMb transcripts in a dose-dependent manner. Furthermore, CsMb catalyzed oxidation reactions in the presence of H2O2, and amino acid 34 of CsMb played an essential role in its reaction with H2O2. In addition, CsMb significantly reduced H2O2 and NO levels in LPS-activated macrophages, and CsMb downregulated iNOS and SOD expression in activated macrophages.
  
  
  CONCLUSIONS
  The present study is the first to investigate the peroxidase activity of CsMb. This investigation suggested that C. sinensis may decrease the redox activation of macrophages by CsMb expression to evade host immune responses. These studies contribute to a better understanding of the role of CsMb in the molecular mechanisms involved in ROS detoxification by C. sinensis.
authors.forename: Mengyu
authors.initials: M
authors.lastname: Ren
authors.forename: Lei
authors.initials: L
authors.lastname: He
authors.forename: Yan
authors.initials: Y
authors.lastname: Huang
authors.forename: Qiang
authors.initials: Q
authors.lastname: Mao
authors.forename: Shan
authors.initials: S
authors.lastname: Li
authors.forename: Honglin
authors.initials: H
authors.lastname: Qu
authors.forename: Meng
authors.initials: M
authors.lastname: Bian
authors.forename: Pei
authors.initials: P
authors.lastname: Liang
authors.forename: Xueqing
authors.initials: X
authors.lastname: Chen
authors.forename: Jinsi
authors.initials: J
authors.lastname: Ling
authors.forename: Tingjing
authors.initials: T
authors.lastname: Chen
authors.forename: Chi
authors.initials: C
authors.lastname: Liang
authors.forename: Xiaoyun
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, Guangdong 510080, China. xuerong2@mail.sysu.edu.cn.
authors.forename: Xuerong
authors.initials: X
authors.lastname: Li
authors.forename: Xinbing
authors.initials: X
authors.lastname: Yu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D002460:Cell Line; D003003:Clonorchiasis / Q000469:parasitology; D003004:Clonorchis sinensis / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D005786:Gene Expression Regulation; D015801:Helminth Proteins / Q000235:genetics / Q000378:metabolism*; D006861:Hydrogen Peroxide; D008264:Macrophages / Q000469:parasitology; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009211:Myoglobin / Q000235:genetics / Q000378:metabolism*; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D011487:Protein Conformation; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism
chemical_list: D000975:Antioxidants; D015801:Helminth Proteins; D009211:Myoglobin; D009569:Nitric Oxide; D006861:Hydrogen Peroxide; D052247:Nitric Oxide Synthase Type II; D013482:Superoxide Dismutase