Mass spectrometry characterization of trypanothione and novel peptides of medical importance isolated from Acanthamoeba polyphaga.

This paper presents unequivocal results about the presence of trypanothione and its precursor glutathionespermidine from the opportunistic human pathogen Acanthamoeba polyphaga. They were isolated by RP-HPLC as thiolbimane derivatives and characterized using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF/TOF). Additionally RP-HPLC demonstrated that thiol-bimane compounds corresponding to cysteine and glutathione were also present in A. polyphaga. Besides trypanothione, we want to report four new peptides in trophozoites, a tetrapeptide, a hexapeptide, a heptapeptide and a nonapeptide. Trypanothione and two of the thiol peptides, the hexapeptide and heptapeptide, are oxidized since the reduced forms increase in amount when the normal extract is treated by DTT or by electrolytic reduction that convert the oxidized forms to reduced ones. On the other hand, they disappear when the amoeba extract is treated with NEM or when the amoeba culture is treated with various inhibitors of NADPH-dependent disulfidereducing enzymes. Comparison of the thiol peptides, including trypanothione from A. polyphaga with extracts from human lymphocytes showed that they are not present in the latter. Therefore, some of the peptides here reported could be used as antigens for rapid detection of these parasites. In regard to the presence of the enzymes that synthesize and reduce trypanothione in A. polyphaga we suggest that they can be used as drug targets.

Identification of trypanothione from the human pathogen Entamoeba histolytica by mass spectrometry and chemical analysis.

In this paper, we present definitive data to show, from ESI (electrospray ionization) studies, that the thiol-bimane compound isolated and purified from Entamoeba histolytica trophozoites, corresponds unequivocally to the structure of trypanothione. Trypanothione disulphide was shown to have a molecular ion of m/z 722. It was further demonstrated by MALDI-TOF (matrix-assisted laser desorption ionization-time-of-flight) MS that this thiol compound also corresponds to the characteristic monoprotonated ion of trypanothione-(bimane)(2), which has a molecular ion of m/z 1103.95. The ion pattern of the thiol-bimane compound prepared from the commercial trypanothione standard is identical with the E. histolytica thiol-bimane compound. After HPLC separation, chemical amino acid analysis by dabsylation and dansylation of the thiol bimane compound from Entamoeba showed the presence of the following trypanothione components: glutamic acid, cysteic acid, glycine and spermidine. We can conclude from these highly reliable MS experiments and chemical analyses that E. histolytica contains the thiol compound trypanothione, which was previously thought to occur only in trypanosomatids.

Trypanothione reductase from the human parasite Entamoeba histolytica: a new drug target.

Although there is a general agreement that the protist Entamoeba histolytica lacks glutathione, it has been a matter of dispute as to whether this human parasite contains the glutathione derivative known as trypanothione. In the present study, we describe a gene for the TR (trypanothione reductase) obtained from E. histolytica by PCR amplification of its DNA. After Northern-blot analysis, the radiolabelled DNA probe from Trypanosoma cruzi hybridizes with the total RNA of Entamoeba, showing that the TR gene is expressed as mRNA. We have demonstrated the presence of the NADPH-dependent TR activity in vitro with partially purified extracts and showed also that the thiol-bimane compound isolated and purified from E. histolytica trophozoites, unequivocally corresponds, by matrix-assisted laser-desorption ionization-time-of-flight MS, to the characteristic monoprotonated ion of trypanothione-(bimane)(2) with m/z 1104.4 and the trypanothione-(bimane) with m/z 914.3. The PCR product consisted of 1476 bp (491 deduced amino acids), has sequences diagnostic for the reducing catalytic site (CVNVGC) as well as domains for binding NADPH, FAD I and FAD II that are present in all members of this group of disulphide-reducing enzymes, as well as those unique to TRs. The putative protein sequence is 86% identical with that of TR from T. cruzi and it is also clearly distinguishable from other related reductases by phylogenetic analysis. We can conclude, from these highly reliable experiments, that E. histolytica contains the TR enzyme and the thiol compound trypanothione that was previously supposed to occur only in trypanosomatids.