Translationally controlled tumor protein of Brugia malayi functions as an antioxidant protein

Abstract

Translationally controlled tumor protein (TCTP) is one of the most abundantly expressed proteins in the filarial parasites as well as in the other organisms. Several functions have been suggested for TCTP family of proteins ranging from calcium binding to histamine release function. However, its physiological function is still a mystery. Previous studies showed that the expression of TCTP is increased several-fold during oxidative stress. In the present work, we report the putative antioxidant function of Brugia malayi TCTP (BmTCTP). When tested in vitro, rBmTCTP could be reduced by a variety of reducing agents including thioredoxin. Such reduced form of rBmTCTP was able to protect DNA from oxidative damage, suggesting that BmTCTP may have an antioxidant function in the parasite. Sequence analysis of filarial TCTPs revealed that there are three cysteine amino acids located in the central portion of the protein. Subsequent targeted residue modification studies showed that these cysteine residues in rBmTCTP are critical for its antioxidant function. To determine the significance of this finding, rBmTCTP was overexpressed in vivo in Escherichia coli and subjected to oxidative stress. These studies showed that rBmTCTP significantly protected cells form oxidative damage. Taken together, these findings suggest that BmTCTP might be functioning as a non-classical antioxidant protein in the filarial parasites.

Fig. 1

Multiple sequence alignment of the cysteine amino acids of various TCTPs (human, mouse, rabbit, B. malayi, W. bancrofti, and O. volvulus) was determined by a Clustal W alignment. The analysis showed that cysteine amino acids of mammalian TCTPs and parasite TCTPs are highly conserved

Fig. 2

DNA nicking assay (a–e). Approximately 200 ng of variously treated DNA was resolved on a 1% agarose gel followed by staining with Etbr, and bands were visualized using a UV transilluminator. a ϕX174 DNA was incubated in the presence of rBmTCTP that was reduced by DTT (reduced BmT) or rWbTCTP that was reduced by DTT (reduced WbT). Results show that the reduced form of BmT and WbT substantially decreased the formation of nicked DNA (NF), leaving majority of the DNA in the supercoiled form (SF) similar to that of the untreated ϕX174 DNA (C₁). However, incubation of ϕX174 DNA in MFO buffer alone (C₂) or in the presence of BSA or a control recombinant protein, S. mansoni G box binding factor (SmG), resulted in significant nicking of the DNA. b Dose-dependent inhibition of DNA nicking by reduced BmT. Addition of N-ethylenemaleimide modified TCTP (N-BmT) instead of reduced BmT almost completely reversed its DNA protecting ability. Increasing the concentration of reduced BmT in the reaction mixture substantially decreased the nicked form of DNA. However, addition of N-BmT had no protective effect. c Time kinetics studies showed that the DNA protecting effect of reduced rBmTCTP is time-dependent and is present up to 4 h after initiating the incubation. d TCTP reduced by Trx system also efficiently protects DNA from oxidative damage compared to unreduced rBmTCTP Lane 1 Untreated ϕX174 DNA, Lane 2 ϕX174 DNA incubated with unreduced rBmTCTP, Lane 3 ϕX174 DNA incubated with rBmTCTP reduced with Trx. e Addition of DTT-reduced rBmTCTP conferred protection from oxidative damage, and this effect was reversed when N-BmT was used instead of reduced BmT. f Five micrograms of E. coli Trx was resolved on 15% SDS-PAGE, transferred to nitrocellulose membrane, probed with biotinylated rBmTCTP, and developed by ECL. Arrow shows that rBmTCTP interacts with Trx (lane 1). Similarly, 5 μg of rBmTCTP was resolved on 12% SDS-PAGE, transferred to nitrocellulose membrane, incubated with Trx, and probed with a horse radish peroxidase labeled-rabbit anti-Trx antibody and color-developed using an ECL kit (lane 3). Arrow shows the binding of Trx with rBmTCTP. Lanes 2 and 4 were negative controls using BSA. Data presented are representative of one of three similar experiments

Fig. 3

a Disulfide reduction of rBmTCTP by Trx system. rBmTCTP was added to a reaction mixture containing 3 μM of TR and 3 μM of Trx in TE buffer containing 400 μM NADPH. Reference controls included reaction mixtures without the addition of Trx or without the addition of TR. Samples were incubated at room temperature, and oxidation of NADPH was monitored spectrophotometrically at 340 nm. Readings were taken every 30 s from 0.5 to 10 min, and the reference readings were subtracted from the test reaction to determine NADPH consumption. Results are representative of three similar experiments. b Overexpression of rBmTCTP conferred resistance to H₂O₂ damage in E. coli cells. Cells transfected with rBmTCTP or blank vectors were serially diluted from 1:10 to 1:100 and plated on to LB agar plates containing 1.2 mM H₂O₂. After incubation for 12 h at 37°C, growth of bacterial cells was evaluated. Results show that cell transfected with the blank vector failed to grow under these conditions, whereas cells transfected with rBmTCTP showed growth even up to a dilution of 1:50 in the presence of H₂O₂. Data represent results from one of two similar experiments