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Protein Sci. Jan 2008; 17(1): 176–182.
PMCID: PMC2144598

Selenoproteinless animals: Selenophosphate synthetase SPS1 functions in a pathway unrelated to selenocysteine biosynthesis


Proteins containing the 21st amino acid, selenocysteine (Sec), have been described in all three domains of life, but the composition of selenoproteomes in organisms varies significantly. Here, we report that aquatic arthropods possess many selenoproteins also detected in other animals and unicellular eukaryotes, and that most of these proteins were either lost or replaced with cysteine-containing homologs in insects. As a result of this selective selenoproteome reduction, fruit flies and mosquitoes have three known selenoproteins, and the honeybee, Apis mellifera, a single detected candidate selenoprotein. Moreover, we identified the red flour beetle, Tribolium castaneum, and the silkworm, Bombyx mori, as the first animals that lack any Sec-containing proteins. These insects also lost the Sec biosynthesis and insertion machinery, but selenophosphate synthetase 1 (SPS1), an enzyme previously implicated in Sec biosynthesis, is present in all insects, including T. castaneum and B. mori. These data indicate that SPS1 functions in a pathway unrelated to selenoprotein synthesis. Since SPS1 evolved from a protein that utilizes selenium for Sec biosynthesis, an attractive possibility is that SPS1 may define a new pathway of selenium utilization in animals.

Keywords: selenophosphate synthetase, selenocysteine, selenoproteome, Sec-insertion machinery

Selenocysteine (Sec), the 21st naturally occurring protein amino acid, is inserted into nascent polypeptides at UGA codons. The use of Sec instead of cysteine (Cys) often results in a higher enzyme activity, providing a competitive advantage to the organisms that utilize it (Kim and Gladyshev 2005). In eukaryotes, the number of proteins containing Sec (i.e., selenoproteins) varies from zero in higher plants to >30 in fish (Novoselov et al. 2002; Castellano et al. 2005; A.V. Lobanov and V.N. Gladyshev, unpubl.). Insertion of Sec requires the presence of a complex machinery that includes Sec tRNA, elongation factor EFsec, SECIS-binding protein 2 (SBP2), selenophosphate synthetase 2 (SPS2), phosphoseryl-tRNA kinase, SECp43, and Sec synthase (SecS), as well as a cis-acting stem–loop structure, the Sec insertion sequence (SECIS) element in the 3′-UTR (Hatfield et al. 2006).

Recently, we have shown that organisms with large selenoproteomes often associate with aquatic environments (Lobanov et al. 2007). As the Sec pathway is ancient, the first Sec-utilizing organisms likely lived in water. Mammals, insects, and certain other groups of organisms made successful and independent transitions from aquatic to terrestrial environments. In this process, some selenoproteomes were significantly reduced. Small terrestrial animals, such as fruit flies, have small selenoproteomes (Castellano et al. 2001; Martin-Romero et al. 2001), and Caenorhabditis elegans has only a single selenoprotein (Taskov et al. 2005). In higher plants and fungi, selenoproteins were completely lost.

An additional protein implicated in the pathway of Sec biosynthesis is selenophosphate synthetase 1 (SPS1) (Low et al. 1995). Previous research has shown that SPS1 has an important function in fruit flies and participates in oxidative stress defense (Morey et al. 2003a,b). Moreover, SPS1 knockout is lethal in flies, and a mutation in the SPS1 gene leads to larval lethality and increased apoptosis (Alsina et al. 1998). It is thought that selenophosphate synthetases, including eukaryotic SPS1, SPS2, and prokaryotic SelD, generate selenophosphate, an activated biological form of selenium. Many organisms utilizing Sec, such as mammals and fish, possess both SPS1 and SPS2, the latter itself a selenoprotein. It was reported (Kim et al. 1997) that replacement of Sec in mouse SPS2 with Cys reduced enzyme activity but did not completely inactivate the enzyme. SPS1 was also suggested to be responsible for Sec recycling (Tamura et al. 2004), and has been reported to interact with SecS in vitro and in vivo and coimmunoprecipitate with SecS and SecP43 from nuclear and cytosolic fractions (Small-Howard et al. 2006).

However, Drosophila SPS2 was found to synthesize selenophosphate from selenide in in vitro assays, whereas SPS1 did not support this reaction (Persson et al. 1997; Xu et al. 2006). Moreover, recent studies showed that knockdown of SPS1 in mammalian cells had no effect on selenoprotein biosynthesis, whereas knockdown of SPS2 severely impaired selenoprotein expression (Xu et al. 2007). In addition, mammalian SPS2 was active in synthesis of selenophosphate, whereas SPS1 was not active (Xu et al. 2006).

If SPS1 is involved exclusively in Sec biosynthesis, the mutation of this gene may have a phenotype similar to that resulting from disruption of the overall Sec pathway. However, while SPS1 is essential in fruit flies, disruption of the eEFsec gene had no effect on viability, and in fact, life spans of mutant and wild-type flies were similar (Hirosawa-Takamori et al. 2004). Thus, despite numerous studies, the role of SPS1 in selenoprotein biosynthesis remains unresolved.

In this work, we analyzed the selenoproteomes of aquatic and terrestrial arthropods and found that they were selectively reduced in insects. In the course of this study, we identified the first animals that do not have Sec-containing proteins and have also lost the Sec biosynthesis and insertion machinery. The presence of SPS1 in these organisms indicates that this protein is involved in a pathway unrelated to selenoprotein biosynthesis.

Results and Discussion

Reduced selenoproteomes in insects

Several computational approaches have been developed in recent years for the identification of selenoprotein genes (Kryukov et al. 1999; Lescure et al. 1999; Castellano et al. 2001). The genome of D. melanogaster was one of the first analyzed, and found to contain three selenoprotein genes (Castellano et al. 2001; Martin-Romero et al. 2001). Comparative analysis of Drosophila pseudoobscura and D. melanogaster genomes identified the same three selenoproteins (i.e., SPS2, BthD, and G-rich) (Lobanov et al. 2007). In the current work, we examined the selenoprotein content of other species of insects for which completely sequenced genomes are available as well as that of other arthropods.

Interestingly, aquatic arthropods had many more selenoproteins than their terrestrial counterparts even though none of them had their genomes fully sequenced (Fig. 1). Selenoproteins detected in aquatic arthropods were previously identified in vertebrates and green algae. However, in insects, many of these proteins were present in the form of Cys-containing homologs. As a result, all selenoproteomes of terrestrial arthropods, including Anopheles gambiae, Apis mellifera, D. pseudoobscura, D. melanogaster, B. mori, and Tribolium castaneum, were small and had 0–3 selenoproteins.

Figure 1.
Occurrence of selenoproteins in insects and other arthropods. Eighteen selenoprotein families detected in any arthropod sequences or in a reference organism, cnidarian Nematostella vectensis, are shown at the top of the figure. Organism names shown in ...

In addition to having Cys versions of selenoproteins, many insects lost several selenoproteins altogether, including SelH, SelU, and SelM. The widespread occurrence of these proteins in eukaryotes (Table 1), including arthropods, and their lack in all or a subset of insects, suggests loss of these proteins in insects rather than their gain in other organisms. Similarly, the widespread occurrence of other aquatic arthropod selenoproteins in eukaryotes and the presence of their Cys-containing homologs in insects suggest replacement of Sec with Cys in insect proteins (Fig. 1). Thus, there was a gradual reduction in the number of selenoproteins in insects. For example, Locusta migratoria has at least five selenoproteins, fruit flies and mosquitoes three, and the honeybee only one detected candidate selenoprotein.

Table 1.
Selenoproteomes of representative eukaryotic organisms

Animals that lack selenoproteins

Interestingly, our selenoprotein searches revealed no selenoproteins in B. mori and T. castaneum. T. castaneum, the red flour beetle, is a model organism widely used in population ecology and genetics as well as in molecular and developmental genetics (Brown et al. 2002). B. mori, “silkworm of the mulberry tree,” is a popular model for insect genetics, second only to the fruit fly (Xia et al. 2004). The lack of selenoproteins in these organisms was surprising since all previously examined animals had selenoproteins. Since it was possible that our search strategy did not identify some selenoproteins, we analyzed genomic sequences for the presence of different components of Sec biosynthesis and incorporation machinery.

The use of tRNAscan-SE, a standard program for identification of tRNA genes, did not detect Sec tRNA in B. mori and T. castaneum, whereas a similar search found such structures in several other examined insects. We also carried out an alternative search that utilized BLASTN against insect tRNA sequences and an independent tRNA search program, Aragorn (Laslett and Canback 2004), but Sec tRNA again could not be found. We further analyzed for occurrence of SBP2, SECp43, PSTK, and SecS in arthropods (Table 2). These proteins could be detected in all tested insects with the exception of B. mori and T. castaneum. (In addition, the honeybee had some components of the Sec machinery but several components were not detected. Whether this organism is capable of synthesizing Sec-containing proteins is not clear.) The lack of components of Sec biosynthesis and insertion machinery in B. mori and T. castaneum is indicative of the loss of this machinery during evolution. Thus, results of the searches for these components are consistent with the lack of selenoproteins in these organisms. As a whole, these data provide evidence that B. mori and T. castaneum are the first known animals that do not synthesize Sec-containing proteins.

Table 2.
Occurrence of SPS1 and components of Sec insertion machinery in insectsa

Interestingly, a recent study reported searches for selenoprotein genes in the B. mori genome, and claimed the identification of five selenoprotein genes containing in-frame UGA codons, including glutathione-S-transferase and four novel selenoproteins (Ping et al. 2006). However, examination of the structures reported in that study revealed that the SECIS-like structures identified in the 3′-UTRs of all five transcripts do not fit the canonical SECIS model, and could only be found with very relaxed settings of SECISearch. This mode can only be implemented for the identification of unusual SECIS elements and has a high false positive rate. In addition, all candidate B. mori selenoproteins have weak homology in the region flanking putative Sec residues, whereas in true selenoproteins this region tends to be highly conserved. In any event, the lack of any identifiable components of Sec biosynthesis and insertion machinery is a strong argument against the use of Sec in these predicted proteins.

SPS1 functions in a pathway unrelated to selenoprotein biosynthesis

Although B. mori and T. castaneum lacked both selenoprotein genes and Sec machinery, we detected SPS1 genes in these organisms (Table 2). The introduction of this paper contains an overview of the previous studies that examined the roles of selenophosphate synthetases in Sec biosynthesis. Critical analysis of these data indicates that the role of SPS2 in this pathway is well established, whereas the role of SPS1 is controversial. Our comparative genomics data clearly show that SPS1 is preserved even if no Sec-containing proteins are present in animals. Thus, this protein must function in a pathway unrelated to Sec biosynthesis. Such a Sec-independent pathway is essential in fruit flies (e.g., patufet mutation, a P-element insertion in the SPS1 gene that is lethal in homozygous flies at larval and pupal stages and also leads to a marked disruption in the size and morphology of the imaginal disks). It would be interesting to determine what the nature of this pathway is and whether selenium has a role in it. SPS1 may be involved in redox homeostasis, as previous data showed that it protects fruit flies from ROS damage (Morey et al. 2003b) and its antioxidant activity is dependent on p53 in mammalian cells (Chung et al. 2006).


Arthropods were found to possess selenoproteins that also could be found in other animals and unicellular eukaryotes. However, whereas many Sec-containing proteins occur in aquatic arthropods, insects, which are terrestrial arthropods, have few such proteins. We show that there was a selective selenoproteome reduction in insects through the loss of some selenoprotein genes and conversion of other Sec-containing proteins into Cys-containing homologs. Our analysis revealed the first animals completely lacking both Sec biosynthesis and selenoprotein genes (e.g., the silkworm, B. mori, and the red flour beetle, T. castaneum). These animals, however, encode the sps1 gene. Thus, SPS1 functions in a pathway unrelated to Sec biosynthesis. Our finding addresses the controversy regarding the function of SPS1 and suggests that future studies might search for a new pathway that utilizes this protein. Since SPS1 evolved from an ancestral SPS/SelD, a protein involved in the Sec pathway, an attractive possibility is that SPS1 may define a new pathway of selenium utilization in animals, including mammals.

Materials and Methods

EST, whole genome shotgun (WGS), and nonredundant (NR) data sets were obtained from NCBI. BLAST programs, also from NCBI, were used for homology searches. Human, fish, Drosophila, and Plasmodium sequences corresponding to known components of the Sec biosynthesis and insertion machinery were used as query sequences. B. mori sequences were obtained from the Silkworm Genome Database (http://www.ab.a.u-tokyo.ac.jp/genome/) and T. castaneum from Baylor College of Medicine (http://www.hgsc.bcm.tmc.edu/projects/tribolium/).

Analyses of selenoproteomes were carried out as described elsewhere (Kryukov et al. 2003). First, homologous sequences with UGA corresponding to Sec in known selenoproteins were identified using TBLASTN, and their 3′-UTRs were examined for the presence of SECIS elements. In addition, sequences satisfying primary sequence consensus were identified using SECISearch (Kryukov et al. 2003). Then, secondary structure criteria and additional filters were applied. SECIS candidates were further analyzed with BLASTN to identify structures conserved between different species, and TBLASTX was used to analyze their upstream regions for selenoprotein ORFs.

Aragorn (Laslett and Canback 2004), tRNAscan-SE (Schattner et al. 2005) and BLASTN programs were used to identify Sec tRNAs in analyzed genomes. Identified selenoproteins were placed in the phylogenetic scheme using taxonomy data and the Tree of Life web project (www.tolweb.org).


This study is supported by NIH GM061603 to V.N.G., and by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, and Center for Cancer Research to D.L.H.


Reprint requests to: Vadim N. Gladyshev, Department of Biochemistry, University of Nebraska, Lincoln, NE 68588, USA; e-mail: ude.lnu@1vehsydalgv; fax: (402) 472-7842.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.073261508.


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