Trypanothione S-transferase activity in a trypanosomatid ribosomal elongation factor 1B

Abstract

Trypanothione is a thiol unique to the Kinetoplastida and has been shown to be a vital component of their antioxidant defenses. However, little is known as to the role of trypanothione in xenobiotic metabolism. A trypanothione S-transferase activity was detected in extracts of Leishmania major, L. infantum, L. tarentolae, Trypanosoma brucei, and Crithidia fasciculata, but not Trypanosoma cruzi. No glutathione S-transferase activity was detected in any of these parasites. Trypanothione S-transferase was purified from C. fasciculata and shown to be a hexadecameric complex of three subunits with a relative molecular weight of 650,000. This enzyme complex was specific for the thiols trypanothione and glutathionylspermidine and only used 1-chloro-2,4-dinitrobenzene from a range of glutathione S-transferase substrates. Peptide sequencing revealed that the three components were the alpha, beta, and gamma subunits of ribosomal eukaryotic elongation factor 1B (eEF1B). Partial dissociation of the complex suggested that the S-transferase activity was associated with the gamma subunit. Moreover, Cibacron blue was found to be a tight binding inhibitor and reactive blue 4 an irreversible time-dependent inhibitor that covalently modified only the gamma subunit. The rate of inactivation by reactive blue 4 was increased more than 600-fold in the presence of trypanothione, and Cibacron blue protected the enzyme from inactivation by 1-chloro-2,4-dinitrobenzene, confirming that these dyes interact with the active site region. Two eEF1Bgamma genes were cloned from C. fasciculata, but recombinant C. fasciculata eEF1Bgamma had no S-transferase activity, suggesting that eEF1Bgamma is unstable in the absence of the other subunits.

Figure 1

Structures of glutathione (γ-L-glutamyl-L-cysteinylglycine) and trypanothione (N¹,N⁸-bis(glutathionyl)spermidine)

Figure 2. SDS-PAGE of C. fasciculata TST purification fractions

Samples (10 μg protein in each lane) from the purification were separated in a 10% SDS-PAGE gel. Lane 1, clarified cell extract lane 2, 2-10% redissolved PEG 6000 precipitate lane 3, Q-sepharose eluant lane 4, Phenyl sepharose eluant lane 5, Superdex 200 eluant. Lanes 6 and 7 were additional samples of the Superdex 200 pool with 1mM DTT being added to the sample in lane 6 and the sample in lane 7 containing no reducing agent.

Figure 3. Analytical size-exclusion chromatography of purified C. fasciculata TST. (A)

Elution profiles of TST activity and protein from a Superdex 200 HR analytical size exclusion column. The absorbance at 280 nm is shown with a solid line, the fraction numbers are shown above this trace and are corrected for the delay volume between the fraction collector and the UV flowcell. The TST activity of fractions was measured using the microtitre plate assay and shown in filled circles joined with a dotted line. (B) Determination of the relative molecular mass of this species, with the sample (○) shown relative to a set of analytical standards (●). (C) SDS-PAGE analysis of 10 μl-samples taken from the 0.5 ml fractions collected during this separation. The four visible bands, subsequently identified as the eEF1Bα, β and γ subunits of the eEF1B complex and an aminoacyl-tRNA synthetase are labelled at the right-hand side of the gel.

Figure 4. Cross-linking of the TST complex

(A) 10% Laemmli SDS-PAGE analysis of the products of the reaction between increasing concentrations of BS³ and TST (lane 1 - no BS³ lane 2 - 75 μM lane 3 - 200 μM and lane 4 – 1 mM. The negative control used the monomeric proteins BSA (66 kDa) and carbonic anhydrase (29 kDa), which were treated with 1 mM BS³ in the presence (lane 5) or absence (lane 6) of TST. The positive control in lane 7 shows the products from treatment of the tetrameric protein aldolase with 75 μM BS³, with the masses of the products formed indicated on the right-hand side of the gel. (B) The same samples were analysed in a 6% Weber SDS-PAGE gel. The masses on the left of the gel are from a cross-linked phosphorylase b marker and again the masses of the aldolase products are indicated on the right-hand side of the gel. Samples as described in panel A.

Figure 5. Separation of the subunits of the TST complex using thiocyanate

Samples of TST were incubated in the presence and absence of 1.5 M NaSCN and then applied to a size-exclusion column. (A) Elution profiles of these samples, the untreated control is shown in red and the NaSCN-treated sample in blue. The elution positions and masses of protein standards are indicated with arrows. (B) TST activity profiles of the fractions collected from this separation, the untreated control is indicated with red triangles and the NaSCN-treated sample with blue squares. (C and D) SDS-PAGE analysis of these column fractions, with Panel C corresponding to the untreated control and Panel D to the NaSCN-treated sample.

Figure 6. Inhibition of TST by Cibacron blue

Inhibition of TST by Cibacron blue in the presence (○) and absence (●) of 0.5% (w/v) BSA in the assay buffer.

Figure 7. Time-dependent inhibition of TST by reactive blue 4

(A) Time-dependent inactivation of TST by reactive blue 4. TST was incubated with the inhibitors in 20 mM (Na⁺) phosphate pH 7.5 at 25°C and the residual activity determined at intervals, as described in the methods. The incubations contained either 100 μM Cibacron blue () or reactive blue 4 at 2 μM (○), 10 μM (●), 20 μM (□), 50 μM (■), 75 μM (Δ) and 100 μM (▲). (B) Observed rates of TST inactivation by reactive blue 4 as a function of the concentration of inhibitor.

Figure 8. Covalent modification of eEF1Bγ by reactive blue 4

Reactive blue 4-inactivated TST was analysed by MALDI-TOF mass spectrometry. The two samples were prepared as described in the text, with the TST sample shown in panel A being an untreated negative control and the TST sample shown in panel B being inactivated by incubation with 50 μM reactive blue 4 for 1 h.

Figure 9. Interactions between TST substrates and triazine dyes

Incubations were performed in 20 mM (Na⁺) phosphate pH 7.5 at 25°C and the residual activity determined at intervals, as described in the methods. (A) Time-dependent inactivation of TST by CDNB. The incubations contained no CDNB (○), 500 μM CDNB (●), 500 μM CDNB and 500 μM T[SH]₂ (■) and 500 μM CDNB and 100 μM Cibacron blue (▲). (B) Effects of thiols on the inactivation of TST by reactive blue 4. The incubations contained no reactive blue 4 (○), 50 μM reactive blue 4 (●), 50 μM reactive blue 4 and 1 mM GSH (□), 50 μM reactive blue 4 and 500 μM T[SH]₂ (■) and 50 μM reactive blue 4 and 500 μM DTT (Δ).

Figure 10. Alignment of the sequences of trypanosomatid eEF1Bγ proteins

The predicted amino-acid sequences of the C. fasciculata eEF1Bγ genes isolated by RT-PCR (RT-PCR C.f.) and intergenic PCR (Full-length C.f.) are shown aligned with the sequences of the Homo sapiens (P26641), T. brucei (temporary name - TRYPtp_ends-17b11.p1k_188), L. infantum (Q9BHZ6) and T. cruzi (P34827) proteins. Gaps are indicated with dashes and the alignment shaded according to similarity, the colours indicate black background, white text - over 80% identity dark grey background, white text - over 60% identity and light grey background, black text – over 40% identity.