Leishmania major Elongation Factor 1B Complex Has Trypanothione S-Transferase and Peroxidase Activity

Abstract

In the Trypanosomatidae, trypanothione has subsumed many of the roles of glutathione in defense against chemical and oxidant stress. Crithidia fasciculata lacks glutathione S-transferase, but contains an unusual trypanothione S-transferase activity that is associated with eukaryotic translation elongation factor 1B (eEF1B). Here we describe the cloning, expression, and reconstitution of the purified ␣, ␤, and ␥ subunits of eEF1B from Leishmania major. Individual subunits lacked trypanothione S-transferase activity. Only eEF1B, formed by reconstitution or co-expression of the three subunits, was able to conjugate a variety of electrophilic substrates to trypanothione or glutathionylspermidine, but not glutathione. In contrast to the C. fasciculata eEF1B, the L. major enzyme also displayed peroxidase activity against a variety of organic hydroperoxides. The enzyme showed no activity with hydrogen peroxide and greatest activity with linoleic acid hydroperoxide (1 unit mg ؊1 ). Kinetic studies suggest a ternary complex mechanism, with K m values of 140 M for trypanothione and 7.4 mM for cumene hydroperoxide and k cat ‫؍‬ 25 s ؊1 . Immunofluorescence studies indicate that the enzyme may be localized to the surface of the endoplasmic reticulum. These results suggest that, in addition to its role in protein synthesis, the Leishmania eEF1B may help protect the parasite from lipid peroxidation.Parasitic protozoa of the family trypanosomatidae cause disease and death throughout the tropical and subtropical world. Trypanosoma brucei infections are estimated to cause ϳ400,000 cases of sleeping sickness per year; Trypanosoma cruzi, the cause of Chagas' disease, chronically infects ϳ17 million people and Leishmania spp. are thought to cause 2 million cases of leishmaniasis per year. 1 Current chemotherapies for these diseases are, on the whole, ineffective and toxic (2, 3), whereas effective vaccines may never be developed. The development of effective treatments for these infections is therefore an urgent necessity.New anti-parasitic drugs can be developed from inhibitors of biochemical pathways that are essential for parasite survival but absent from the host. One such target is the thiol metabolism of trypanosomatids. Uniquely, this is dependent upon trypanothione (N 1 ,N 8 -bis(glutathionyl)spermidine or T[SH] 2 ) 2 (4), whereas their human hosts use glutathione ((␥-L-glutamyl-L-cysteinylglycine or GSH). Trypanothione is involved in protective processes, defending against oxidative stress through peroxidase systems (5), against reactive aldehydes through the glyoxalase system (6, 7), and against toxic xenobiotics through the trypanothione S-transferase (TST) (8). All these processes depend upon trypanothione being maintained in its dithiol form by trypanothione reductase. This NADPH-dependent flavoenzyme is essential to the trypanosomatids (9, 10) as, in these organisms, it is the only known route for the transfer of reducing equivalents from NADPH to low molecular mass thiols. In addition, because these parasites lack an...

Analysis of major intracellular proteins of Aspergillus fumigatus by MALDI mass spectrometry: Identification and characterisation of an elongation factor 1B protein with glutathione transferase activity

“…No glutathione peroxidase activity was detectable in ElfA preparations. Interestingly, Vickers et al [12] have shown that the intact eEF1B complex is necessary for peroxidase activity found in the eEF1Bc subunit of Leishmania major eEF1B. Microarray analysis of A. fumigatus gene expression has revealed that ElfA (termed elongation factor-1c) expression is up-regulated twofold within 1 h of a temperature shift (30-48°C) which suggests a role for ElfA in protecting against heat shock [33].…”

“…However, GST-like domains have also been detected in distinct proteins such as Ure2p, Mak16, and EF1Bc (a subunit of the EF1B complex) [11]. Furthermore, although GST activity has recently been ascribed to the elongation factor protein complex EF1B isolated from rice and trypanothione S-transferase activity detected in EF1B complex from Leishmania major, no detection of GST activity in native EF1Bc has been possible [12]. In an attempt to purify the GST proteins (GstA-C) from A. fumigatus, glutathione (GSH)-Sepharose affinity chromatography was employed.…”

Elongation Factor-2, a Th1 Stimulatory Protein of Leishmania donovani, Generates Strong IFN-γ and IL-12 Response in Cured Leishmania-Infected Patients/Hamsters and Protects Hamsters against Leishmania Challenge

“…This protein though, is involved in protein synthesis, has not been characterized in Leishmania species. The proteins of a similar family, EL-1a, EL-1B, and EL-4a, have been reported in various Leishmania species (14,28,29). Of these, EL-1a, cloned and characterized in L.donovani (29), has been attributed to have an essential role in survival and thus can serve as a good drug target (30).…”

Overexpression of MitochondrialLeishmania majorAscorbate Peroxidase Enhances Tolerance to Oxidative Stress-Induced Programmed Cell Death and Protein Damage

“…Considerable evidence has suggested that nonselenium GSH peroxidase-like enzymes in trypanosomatids metabolize fatty acid and phospholipid hydroperoxides (56), although the activity is very low compared to that of selenium-containing GSH peroxidase. Leishmania also possess trypanothione S transferase activity associated with the eukaryotic translation elongation factor 1B, which reacts preferentially with linoleic acid hydroperoxide but not with H 2 O 2 (54). Recent reports also demonstrate that overexpression of APX within Leishmania and T. cruzi is more resistant to exogenously added H 2 O 2 (18,57).…”