Cloning and expression of trypanothione reductase from a New World Leishmania species.
Trypanothione disulfide (T[S]2), an unusual form of glutathione found in parasitic protozoa, plays a crucial role in the regulation of the intracellular thiol redox balance and in the defense against oxidative stress. Trypanothione reductase (TR) is central to the thiol metabolism in all trypanosomatids, including the human pathogens Trypanosoma cruzi, Trypanosoma brucei and Leishmania. Here we report the cloning, sequencing and expression of the TR encoding gene from L. (L.) amazonensis. Multiple protein sequence alignment of all known trypanosomatid TRs highlights the high degree of conservation and illustrates the phylogenetic relationships. A 3D homology model for L. amazonensis TR was constructed based on the previously reported Crithidia fasciculata structure. The purified recombinant TR shows enzyme activity and in vivo expression of the native enzyme could be detected in infective promastigotes, both by Western blotting and by immunofluorescence.
Castro-Pinto
Denise Barçante
DB
Laboratory of Trypanosomatid Biochemistry, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, RJ, Brazil.
Genestra
Marcelo
M
Menezes
Gustavo B
GB
Waghabi
Mariana
M
Gonçalves
Antonio
A
De Nigris Del Cistia
Catarina
C
Sant'Anna
Carlos Mauricio R
CM
Leon
Leonor L
LL
Mendonça-Lima
Leila
L
D000595:Amino Acid Sequence; D000818:Animals; D000909:Antibodies, Helminth / Q000032:analysis; D003001:Cloning, Molecular; D015870:Gene Expression; D007891:Leishmania / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D007896:Leishmaniasis / Q000469:parasitology / Q000662:veterinary; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D000909:Antibodies, Helminth; D015800:Protozoan Proteins; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase