Identification of a major low‐molecular‐mass thiol of the trypanosomatid Crithidia fasciculata as ovothiol A

Abstract

An unidentified low-molecular-mass thiol, U23, previously detected as the 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin derivative in extracts of the trypanosome Crithidiu fusciculutu, was purified as the bimane derivative. Resonances attributable to U23 were discerned from those of the bimane label by comparison of the 'H-and IT-NMR spectra of monobromobimane and U23-bimane. The complete 'H-and I3C-NMR spectra of U23-bimane were assigned by means of 'H-'H correlation spectroscopy, 'H-''C correlation spectroscopy and 13C multiplicity determinations. The results indicated identity of U23 with 1-N-methyl-4-mercaptohistidine (ovothiol A), previously isolated from marine sources. This assignment was confirmed by NOE difference experiments, fast-atom-bombardment mass spectrometry of U23-bimane and ultraviolet/visible spectrophotometry of U23, which was isolated as the disulfide. The isolation of ovothiol A from a parasitic protozoan suggest that the 4-mercaptohistidines may have a wider distribution and function as Several antioxidant thi& than was hitherto realized. mechanisms which enable trypanosomes to withstand oxidative killing by phagocytic macrophages seem to be operative. The microbicidal effects of reactive oxygen species released by the macrophages can be evaded by interference with signal transduction processes which trigger the respiratory burst. Such a mechanism probably accounted for the observed reduction in the magnitude of the respiratory burst when neutrophils were incubated with the surface membrane acid phosphatase of Leishmania donovuni [l]. In addition trypanosomes, like other organisms exposed to an aerobic environment, depend on both enzymic and non-enzymic mechanisms for the removal of reactive oxygen species (reviewed in [2] and [3]). However, while they possess superoxide dismutase, trypanosomes are deficient in catalase and various peroxidases, including glutathione peroxidase, which, in mammalian cells, are responsible for the removal of hydrogen peroxide, the product of the superoxide-dismutase-catalysed reaction [4]. Hydrogen peroxide is the immediate precursor of the reactive hydroxyl radical, which can be regarded as the most damaging of the reactive oxygen intermediates [5]. A peroxidase activity dependent on the unique trypanosomal metabolite, trypanothione [2, 51, has been detected [6, 71. The enzyme, however, has not yet been characterized and its activity was estimated to be quite low when compared with glutathione peroxidase in mammalian tissues [3]. The lack of adequate enzymatic mechanisms for the detoxification of hydrogen peroxide is thought to render trypanosomes susceptible to oxidative stress and to form the basis for the anti-trypanosomal activity of several drugs [3].On the other hand, evidence was presented that trypanosomes are able to metabolize exogenously added peroxide [7, 81 and that this process is inhibited by sulfhydryl reagents. Low-molecular-mass thiols, and particularly trypanothione [2, 31, are, therefore, thought to play an important role in ...

Effects of buthionine sulfoximine nifurtimox and benznidazole upon trypanothione and metallothionein proteins in Trypanosoma cruzi.

“…It is probable that MTs and other thiolcontaining compounds, such as ovothiols (Steenkamp and Spies, 1994;Ariyanayagam and Fairlamb, 2001), may contribute to protection of the parasite against oxidative stress.…”

“…The most important thiol molecules are trypanothione (bis-glutathionyl spermidine, T(SH) 2 ), glutathione (GSH), ovothiols (Fairlamb and Cerami, 1985;Steenkamp and Spies, 1994;Repetto et al, 1996;Maya et al, 1997;Maya et al, 1999;Maya et al, 2001a;Ariyanayagam and Fairlamb, 2001;Steenkamp, 2002), and metallothionein-like proteins (MTs), as shown in this paper. All these compounds act as free radical scavengers by virtue of their thiol groups.…”

The effect of buthionine sulfoximine on the growth of Leishmania donovani in culture

“…fasciculata was cultured as described [8] and adapted by successive subculturing to growth in the presence of increasing concentrations of paraquat.…”

“…More recently it was found that Crithidia fasciculata [8] and Leishmania donovani [9] produce N Imethyl-4-mercaptohistidine or ovothiol A, a small aromatic thiol with exceptional antioxidant properties [10,11].…”

Thiohistidine Biosynthesis

“…[25,26] Trypanosoma cruzi, Trypanosoma brucei and Leishmania major are the human pathogens which cause tropical diseases such as sleeping disease, Chagas disease and leishmaniasis also produce ovothiol A. [27][28][29][30] Elucidation of the corresponding biosynthetic pathway may reveal novel strategies to treat these conditions, for which no efficient therapy is known. Furthermore, identification of the ovothiol A biosynthetic genes (see below) revealed that several plant pathogens such as Erwinia amylophora, the causative agent of fire blight and Phytophthora infestans which causes potato blight, are also ovothiol A producers.…”