title: Protection against peroxynitrite by pseudoperoxidase from Leishmania major.
abstract: Heme proteins share the ability to detoxify reactive nitrogen intermediates (NO and peroxynitrite). But, to date, no heme-containing enzymatic defense against toxic reactive nitrogen intermediates has been discovered in Leishmania species. We have cloned, expressed, and characterized a pseudoperoxidase from Leishmania major (LmPP) that is capable of detoxifying peroxynitrite (ONOO(-)). Optical, EPR, and resonance Raman spectral studies demonstrate that ONOO(-) can rapidly convert the six-coordinate ferric low-spin to a ferric high-spin form at neutral pH. Western blotting and immunofluorescence studies with anti-LmPP antibody show that the mature enzyme is located at the plasma membrane of amastigotes and is expressed eightfold higher in amastigotes compared to promastigotes. Moreover, to further investigate its exact physiological role in Leishmania, we have created LmPP-knockout mutants by gene replacement in L. major strains. IC(50) values for exogenously added H(2)O(2) or 3-morpholinosydnonimine (SIN1) show that deletion of LmPP in L. major renders the cell more susceptible to SIN1. The null mutant cells exhibit a marked decrease in virulence on infection with activated macrophages as well as inoculation into BALB/c mice. Collectively, these data provide strong evidence that LmPP plays an important role in the enzymatic defense against ONOO(-) within macrophages.
authors.affiliation: Division of Structural Biology and Bio-informatics, Indian Institute of Chemical Biology, Kolkata, India.
authors.forename: Moumita
authors.initials: M
authors.lastname: Bose
authors.forename: Rina
authors.initials: R
authors.lastname: Saha
authors.forename: Sumit
authors.initials: S
authors.lastname: Sen Santara
authors.forename: Supratim
authors.initials: S
authors.lastname: Mukherjee
authors.forename: Jayasree
authors.initials: J
authors.lastname: Roy
authors.forename: Subrata
authors.initials: S
authors.lastname: Adak
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D005260:Female; D006861:Hydrogen Peroxide / Q000633:toxicity; D018320:Leishmania major / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity; D016773:Leishmaniasis, Cutaneous / Q000469:parasitology; D008262:Macrophage Activation; D008264:Macrophages / Q000378:metabolism / Q000469:parasitology; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D008981:Molsidomine / Q000031:analogs & derivatives / Q000633:toxicity; D009195:Peroxidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D030421:Peroxynitrous Acid / Q000378:metabolism*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D015800:Protozoan Proteins; D030421:Peroxynitrous Acid; C002385:linsidomine; D006861:Hydrogen Peroxide; D008981:Molsidomine; D009195:Peroxidase; C580840:pseudoperoxidase, Leishmania major