Identification and characterisation of a functional peroxidoxin from Leishmania major.
Leishmania spp. encounter damaging oxygen metabolites from endogenous metabolic processes as well as from exogenous sources, such as inside the gut of the sandfly vector and within host macrophages. The recently described peroxidoxin protein family form part of a novel pathway for metabolising hydrogen peroxide that, in trypanosomatids, links peroxide reduction to NADPH oxidation via trypanothione. Here we report the cloning and characterisation of the Leishmania major peroxidoxin gene, tryparedoxin peroxidase (TryP). TryP is a multi-copy gene arranged in a complex tandem array located on the size polymorphic homologues of chromosome 15. Northern analysis showed that TryP expresses a single 1.6 kb mRNA throughout promastigote development. TryP encodes a 22-kDa protein with two conserved cysteine-containing domains that defines it as a 2-Cys peroxidoxin. Purified recombinant TryP protein catabolised hydrogen peroxide in the presence of the tryparedoxin homologue from Crithidia fasciculata (Cf-TryX), trypanothione, trypanothione reductase and NADPH. The demonstration that L. major utilises a three-protein peroxidase system confirms that this is a mechanism of protection against oxidative damage in this parasite.
Levick
M P
MP
Department of Medicine, University of Cambridge, Addenbrooke's Hospital, UK.
Tetaud
E
E
Fairlamb
A H
AH
Blackwell
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D018076:DNA, Complementary; D020224:Expressed Sequence Tags; D017125:Genes, Protozoan; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D018320:Leishmania major / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010545:Peroxides / Q000378:metabolism; D015800:Protozoan Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013095:Spermidine / Q000031:analogs & derivatives / Q000378:metabolism; D013379:Substrate Specificity
D018076:DNA, Complementary; D010545:Peroxides; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D009249:NADP; C044809:trypanothione; D006861:Hydrogen Peroxide; D010544:Peroxidases; C110959:tryparedoxin peroxidase; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase; D005978:Glutathione; D013095:Spermidine