Glutathionylation of trypanosomal thiol redox proteins.
Trypanosomatids, the causative agents of several tropical diseases, lack glutathione reductase and thioredoxin reductase but have a trypanothione reductase instead. The main low molecular weight thiols are trypanothione (N(1),N(8)-bis-(glutathionyl)spermidine) and glutathionyl-spermidine, but the parasites also contain free glutathione. To elucidate whether trypanosomes employ S-thiolation for regulatory or protection purposes, six recombinant parasite thiol redox proteins were studied by ESI-MS and MALDI-TOF-MS for their ability to form mixed disulfides with glutathione or glutathionylspermidine. Trypanosoma brucei mono-Cys-glutaredoxin 1 is specifically thiolated at Cys(181). Thiolation of this residue induced formation of an intramolecular disulfide bridge with the putative active site Cys(104). This contrasts with mono-Cys-glutaredoxins from other sources that have been reported to be glutathionylated at the active site cysteine. Both disulfide forms of the T. brucei protein were reduced by tryparedoxin and trypanothione, whereas glutathione cleaved only the protein disulfide. In the glutathione peroxidase-type tryparedoxin peroxidase III of T. brucei, either Cys(47) or Cys(95) became glutathionylated but not both residues in the same protein molecule. T. brucei thioredoxin contains a third cysteine (Cys(68)) in addition to the redox active dithiol/disulfide. Treatment of the reduced protein with GSSG caused glutathionylation of Cys(68), which did not affect its capacity to catalyze reduction of insulin disulfide. Reduced T. brucei tryparedoxin possesses only the redox active Cys(32)-Cys(35) couple, which upon reaction with GSSG formed a disulfide. Also glyoxalase II and Trypanosoma cruzi trypanothione reductase were not sensitive to thiolation at physiological GSSG concentrations.
Melchers
Johannes
J
Biochemie-Zentrum, Universität, Heidelberg, Germany.
Dirdjaja
Natalie
N
Ruppert
Thomas
T
Krauth-Siegel
R Luise
RL
D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D005978:Glutathione / Q000031:analogs & derivatives* / Q000737:chemistry*; D007328:Insulin / Q000737:chemistry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013095:Spermidine / Q000031:analogs & derivatives* / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D014345:Trypanosoma
D004220:Disulfides; D007328:Insulin; D013438:Sulfhydryl Compounds; C012333:glutathionylspermidine; C044809:trypanothione; D005978:Glutathione; D003545:Cysteine; D013095:Spermidine