Selenium metabolism in Trypanosoma: characterization of selenoproteomes and identification of a Kinetoplastida-specific selenoprotein

Abstract

Proteins containing the 21st amino acid selenocysteine (Sec) are present in the three domains of life. However, within lower eukaryotes, particularly parasitic protists, the dependence on the trace element selenium is variable as many organisms lost the ability to utilize Sec. Herein, we analyzed the genomes of Trypanosoma and Leishmania for the presence of genes coding for Sec-containing proteins. The selenoproteomes of these flagellated protozoa have three selenoproteins, including distant homologs of mammalian SelK and SelT, and a novel multidomain selenoprotein designated SelTryp. In SelK and SelTryp, Sec is near the C-terminus, and in all three selenoproteins, it is within predicted redox motifs. SelTryp has neither Sec- nor cysteine-containing homologs in the human host and appears to be a Kinetoplastida-specific protein. The use of selenium for protein synthesis was verified by metabolically labeling Trypanosoma cells with 75Se. In addition, genes coding for components of the Sec insertion machinery were identified in the Kinetoplastida genomes. Finally, we found that Trypanosoma brucei brucei cells were highly sensitive to auranofin, a compound that specifically targets selenoproteins. Overall, these data establish that Trypanosoma, Leishmania and likely other Kinetoplastida utilize and depend on the trace element selenium, and this dependence is due to occurrence of selenium in at least three selenoproteins.

Figure 1

Structures and nucleotide sequence alignment of SelK SECIS elements. Functionally important nucleotides in the apical loop and the Quartet (SECIS core) are shown in bold (in the structure) or in red (in the alignment). Conserved nucleotides are highlighted.

Figure 2

Amino acid sequence alignment of SelK sequences. Sec (indicated by U) is shown in red, and the corresponding Cys in blue. The position of these residues in the sequences is indicated by a red star above the sequences. Conserved residues are highlighted. ORFs were predicted in the following sequences: 4 265 315.c000313905.Contig1 (T.congolense), Tb927_10_v4 (T.brucei brucei), 1 585 712.c000312726.Contig1 (T.gambiense), Contig8734 (T.vivax), Tcruzi.chrunknown.4757 (T.cruzi), LmjF36_01_20040630_V4.0 (L.major), LI0706f02.p1k (L.infantum), brazil1129f08.q1k (L.braziliensis).

Figure 3

Structures and nucleotide sequence alignment of SelT SECIS elements. Functionally important nucleotides in the apical loop and the Quartet (SECIS core) are shown in bold (in the structure) or in red (in the alignment). Conserved nucleotides are highlighted. Separate alignments for Trypanosoma and Leishmania are shown due to lack of homology between SECIS elements from these two groups of organisms.

Figure 4

Amino acid sequence alignment of SelT sequences. Conserved residues are highlighted. Sec (indicated by U) is shown in red, and the corresponding Cys in blue. The following sequences were used to construct the alignment: tviv326d03.p1k_1 and tviv326d03.p1k_2 (T.vivax), gamb21f07.q1k_2 (T.gambiense), Tb927.5.860 and Tb927.5.870 (T.brucei brucei), congo270d08.q1k_5 and congo270d08.q1k_6 (T.congolense), Tc00.1047053505163.60 and Tc00.1047053505163.70 (T.cruzi), LmjF35.1110 (L.major), LI0881h10.q1k (L.infantum), brazil1006d02.p1k and brazil74d01.q1k (L.braziliensis).

Figure 4

Amino acid sequence alignment of SelT sequences. Conserved residues are highlighted. Sec (indicated by U) is shown in red, and the corresponding Cys in blue. The following sequences were used to construct the alignment: tviv326d03.p1k_1 and tviv326d03.p1k_2 (T.vivax), gamb21f07.q1k_2 (T.gambiense), Tb927.5.860 and Tb927.5.870 (T.brucei brucei), congo270d08.q1k_5 and congo270d08.q1k_6 (T.congolense), Tc00.1047053505163.60 and Tc00.1047053505163.70 (T.cruzi), LmjF35.1110 (L.major), LI0881h10.q1k (L.infantum), brazil1006d02.p1k and brazil74d01.q1k (L.braziliensis).

Figure 5

Structures and nucleotide sequence alignments of SelTryp SECIS elements. Functionally important nucleotides in the apical loop and the Quartet (SECIS core) are shown in bold (in the structure) or in red (in the alignment). Conserved nucleotides are highlighted. Separate alignments for Trypanosoma and Leishmania are shown due to lack of homology between SECIS elements from these two groups of organisms.

Figure 6

Amino acid sequence alignment of SelTryp sequences. Conserved residues are highlighted. Sec (indicated by U) is shown in red. Sequences used in the alignment were as follows: Tc00.1047053507485.100 (T.cruzi), tviv195d03.q1k_2 (T.vivax), Tb927.4.3410 (T.brucei brucei), gamb564d12.p1k_15 (T.gambiense), congo936h09.q1k_1 (T.congolense), LmjF34.0950 (L.major), LinJ34.0860 (L.infantum). Location of rhodanese domains, metallo-β-lactamase domain and the CxxU motif is indicated above the sequences. Active site cysteines in rhodanese domains are highlighted in blue and additional conserved cysteines in SelTryps in green. Conserved histidines that may be involved in metal coordination are shown in pink.

Figure 6

Amino acid sequence alignment of SelTryp sequences. Conserved residues are highlighted. Sec (indicated by U) is shown in red. Sequences used in the alignment were as follows: Tc00.1047053507485.100 (T.cruzi), tviv195d03.q1k_2 (T.vivax), Tb927.4.3410 (T.brucei brucei), gamb564d12.p1k_15 (T.gambiense), congo936h09.q1k_1 (T.congolense), LmjF34.0950 (L.major), LinJ34.0860 (L.infantum). Location of rhodanese domains, metallo-β-lactamase domain and the CxxU motif is indicated above the sequences. Active site cysteines in rhodanese domains are highlighted in blue and additional conserved cysteines in SelTryps in green. Conserved histidines that may be involved in metal coordination are shown in pink.

Figure 7

Selenoprotein expression in T.cruzi. T.cruzi epimastigote cells were radiolabeled with ⁷⁵Se and collected after 40 h of DMEM culture. Proteins were electrophoresed under reducing conditions on a 10% SDS–PAGE gel, transferred onto a PVDF membrane, analyzed by PhosphorImager (A and B) exposure time 30 min and 72 h, respectively and stained with Coomassie blue (C). Predicted molecular masses of T.cruzi selenoproteins were 9.5 kDa (SelK), 28.8 kDa (SelT) and 89 kDa (SelTryp). Lane 1: supernatant of epimastigote cells after sonication; lane 2: resuspended pellet after epimastigote sonication; lane 3 total epimastigote extract; lane 4: molecular weight markers (Sigmawide). An arrow on (B) shows a specific selenoprotein band, probably corresponding to SelK.

Figure 8

Effect of auranofin on parasite growth. (a) Bloodstream and (b) procyclic T.brucei brucei (strain 449) cell cultures were incubated with different concentrations (shown on a logarithmic scale) of the gold-compound auranofin. Indicated are the percentages of viable cells after 18 h of incubation compared to a culture in the absence of auranofin. All experiments were done in duplicate. The error bars indicate the standard deviation.

Figure 9

Phylogenetic tree of Sec tRNAs. Phylogenetic tree of Sec tRNAs was constructed using ClustalX program for alignments of tRNA sequences and calculating the distances between them. TreeView was used for tree visualization.