Ovothiol and trypanothione as antioxidants in trypanosomatids

Abstract

The relative amounts of ovothiol A (N¹-methyl-4-mercaptohistidine) and trypanothione [N¹,N⁸-bis(glutathionyl)spermidine] have been determined in all life cycle stages of representative trypanosomatids (Leishmania spp, Crithidia fasciculata, Trypanosoma cruzi and T. brucei). Ovothiol A is present in all insect stages with intracellular concentrations of >1 mM for five species of Leishmania promastigotes and <0.25 mM for other trypanosomatids. In Leishmania promastigotes, ovothiol A can exceed trypanothione content particularly in late logarithmic and stationary phases of growth. In the other trypanosomatids, it represents less than 10% of the total thiol pool. Although amastigotes of L. major and L. donovani contain equivalent amounts of glutathione and trypanothione, ovothiol A is present in the former but absent in the latter. Ovothiol A is present in all developmental stages of T. cruzi but absent in bloodstream trypomastigotes of T. brucei. No ovothiol reductase activity could be detected in dialysed parasite extracts. Ovothiol disulphide is not a substrate for trypanothione reductase, although it can be reduced by the concerted action of trypanothione and trypanothione reductase. No ovothiol-dependent peroxidase activity was present in Leishmania extracts. Although ovothiol A can act as a non-enzymatic scavenger of hydrogen peroxide, it is less efficient than trypanothione. Second order rate constants were determined with trypanothione>glutathionylspermidine>ovothiol>glutathione. Given the presence of an active trypanothione peroxidase system in all these trypanosomatids, it is concluded that under physiological conditions, ovothiol is unlikely to play a major role in the metabolism of hydrogen peroxide in intact cells. Nonetheless, since ovothiol is absent in host macrophage, kidney and CHO cells, this metabolite may have other important functional roles in trypanosomatids that could be exploited as a chemotherapeutic target.

Introduction

Trypanosomatids possess a unique thiol-redox system based upon the low molecular mass thiol trypanothione [N¹,N⁸-bis(glutathionyl)spermidine] [1] and trypanothione reductase (TryR) [2]. Together with tryparedoxin (TryX) and tryparedoxin peroxidase (TryP) these proteins comprise the trypanothione peroxidase system [3], [4], [5], [6], [7], [8], [9]. The three-dimensional structure of all components of this pathway for peroxide metabolism have been reported recently [10], [11], [12]. It is important to note that trypanosomatids do not possess the selenium-dependent glutathione peroxidase/glutathione reductase (GR) system found in other cells [13]. Moreover, although host GR and parasite TryR belong to the same family of flavoprotein-disulphide oxidoreductases, the mutually exclusive specificity for their disulphide substrates [2] indicates that selective inhibition of TryR is possible. Various genetic strategies have been used to successfully validate TryR as a drug target in Leishmania spp [14], [15], [16] and in Trypanosoma brucei [17].

Although the trypanothione system appears to be the principal defence against oxidant damage in these parasites [13], [18], the discovery of ovothiol A (N¹-methyl-4-mercaptohistidine) in Crithidia fasciculata and L. donovani [19], [20], [21] prompted us to re-evaluate this conclusion. Ovothiols have three family members which differ in the number of methyl substituents on the α-amino group (n=0, 1 or 2 for ovothiol A, B and C, respectively) [22], [23]. Ovothiols are present in eggs of marine echinoderms and molluscs in millimolar concentrations and are thought to play an important role in defence against the intense respiratory burst that is triggered following fertilisation [24], [25], [26]. Two anti-oxidant properties of ovothiol may be involved. First, ovothiols can act as non-enzymatic scavengers of hydrogen peroxide (H₂O₂) forming water and ovothiol disulphide (OSSO). The latter is subsequently reduced non-enzymatically via thiol-disulphide exchange with glutathione (GSH), and the resulting glutathione disulphide (GSSG) being reduced by GR [26], [27]. Second, ovothiols possess free-radical scavenging properties [28], [29], which may protect against free radicals formed during cross-linking of the fertilisation envelope.

Given these characteristics of ovothiol, we have determined the prevalence of this thiol in the different life cycle stages of four representative trypanosomatids and examined the interaction of ovothiol with the trypanothione redox system in order to elucidate possible functions in these parasites.