A novel sigma-like glutathione transferase of Taenia solium metacestode.
GSTs are a group of multifunctional enzymes, whose major functions involve catalysis of conjugation of glutathione thiolate anion with a multitude of bi-substrates or transportation of a range of hydrophobic ligands. Helminth GSTs are intimately involved in the scavenging of endogenously/exogenously-derived toxic compounds and xenobiotics. In this study, we identified a novel GST gene of Taenia solium metacestodes (TsMs), which is a causative agent of neurocysticercosis. The 804 bp-long cDNA encoded a 639 bp open reading frame (212 amino acid polypeptide), which exhibited the structural motif and domain organisation characteristic of GST. It formed a strong clade with trematode and insect sigmaGSTs. We designated this cDNA as TsM sigma-like GST (TsMsigmaGST). Native TsMsigmaGST identified through gel filtration combined with compatible immunoproteomics consisted of four isoforms at approximately 25 kDa with different pIs between 8.2 and 8.7. TsMsigmaGST showed an enzyme activity as a homodimer and was specifically expressed in the scolex cytosol. The recombinant TsMsigmaGST expressed in Escherichia coli showed sigma-like activity with 1-chloro-2,4-dinitrobenzene (CDNB). The Vmax and Km for CDNB and glutathione (GSH) were 1.08 and 0.78 micromol/min/mg, and 0.16 and 0.17 mM, respectively. Its optimal activity was observed at pH 8.0 and at 40 degrees C. The enzyme activity was potently inhibited by bromosulfophthalein, and to a lesser extent by rose bengal and triphenyltin chloride. Albendazole and praziquantel non-competitively inhibited both G- and H-sites of the enzyme. To our knowledge this is the first description of the sigma-class GST in cestode parasites. The enzyme might be involved in scavenging of intracellularly generated xenobiotics during homeostatic processes and anthelminthic metabolisms. Revelation of biochemical and biological properties of TsMsigmaGST might allow us to understand pathobiological events inherent to this long-standing parasitic disease, and thus to target therapeutic intervention.
Nguyen
Hung Anh
HA
Department of Molecular Parasitology, Sungkyunkwan University School of Medicine, and Center for Molecular Medicine, Samsung Biomedical Research Center, 300 Cheoncheon-dong, Jangan-gu, Suwon 440-746, Republic of Korea.
Bae
Young-An
YA
Lee
Eung-Goo
EG
Kim
Seon-Hee
SH
Diaz-Camacho
Sylvia Paz
SP
Nawa
Yukifumi
Y
Kang
Insug
I
Kong
Yoon
Y
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D016000:Cluster Analysis; D018076:DNA, Complementary / Q000235:genetics; D018509:DNA, Helminth / Q000737:chemistry / Q000235:genetics; D004137:Dinitrochlorobenzene / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015801:Helminth Proteins / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D041201:Taenia solium / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
D018076:DNA, Complementary; D018509:DNA, Helminth; D004137:Dinitrochlorobenzene; D015801:Helminth Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D005982:Glutathione Transferase