Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1996 Dec 24;93(26):15086-91.
doi: 10.1073/pnas.93.26.15086.

Identification of a novel selD homolog from eukaryotes, bacteria, and archaea: is there an autoregulatory mechanism in selenocysteine metabolism?

M J Guimarães  1 , D Peterson, A Vicari, B G Cocks, N G Copeland, D J Gilbert, N A Jenkins, D A Ferrick, R A Kastelein, J F Bazan, A Zlotnik
Affiliations

Identification of a novel selD homolog from eukaryotes, bacteria, and archaea: is there an autoregulatory mechanism in selenocysteine metabolism?

M J Guimarães et al. Proc Natl Acad Sci U S A. .

Abstract

Escherichia coli selenophosphate synthetase (SPS, the selD gene product) catalyzes the production of monoselenophosphate, the selenium donor compound required for synthesis of selenocysteine (Sec) and seleno-tRNAs. We report the molecular cloning of human and mouse homologs of the selD gene, designated Sps2, which contains an in-frame TGA codon at a site corresponding to the enzyme's putative active site. These sequences allow the identification of selD gene homologs in the genomes of the bacterium Haemophilus influenzae and the archaeon Methanococcus jannaschii, which had been previously misinterpreted due to their in-frame TGA codon. Sps2 mRNA levels are elevated in organs previously implicated in the synthesis of selenoproteins and in active sites of blood cell development. In addition, we show that Sps2 mRNA is up-regulated upon activation of T lymphocytes and have mapped the Sps2 gene to mouse chromosome 7. Using the mouse gene isolated from the hematopoietic cell line FDCPmixA4, we devised a construct for protein expression that results in the insertion of a FLAG tag sequence at the N terminus of the SPS2 protein. This strategy allowed us to document the readthrough of the in-frame TGA codon and the incorporation of 75Se into SPS2. These results suggest the existence of an autoregulatory mechanism involving the incorporation of Sec into SPS2 that might be relevant to blood cell biology. This mechanism is likely to have been present in ancient life forms and conserved in a variety of living organisms from all domains of life.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Identification of a novel selD homolog. (a) Schematic representation of the nucleotide sequences of mouse and human Sps2 (MoSPS2 and HuSPS2, respectively) shows the relative positions of the in-frame TGA codon as well as other TGA codons likely used as termination signals (see also Fig. 4). Thick lines denote the effective open reading frame (ORF). Human SPS1 (HuSPS1) and the E. coli SPS (EcSPS) sequences feature different codons opposite the Sps2 TGA. (b) Protein sequence alignment of the available SPS homologs. Reverse-lettered residues are identical among the six sequences; boxed amino acids are chemically conserved; dots are gaps in the alignment. Large boxes highlight the Walker A- and B-like motifs characteristic of nucleotide-binding folds; a Gly-rich active site pattern (where ⋄ is Sec—U in one-letter amino acid code (4)—Cys or Thr); Gly residues are marked by (○), and an invariant Asp (•) starts a predicted turn. Dashed lines represent predicted hydrophobic β-strands (19). (c) Dendrogram showing the divergence of SPS forms. Boxes around the SPS2, M. jannaschii, and H. influenzae molecules denote their special character as selenoenzymes. SPS1 and EcSPS have likely mutated this codon to Thr and Cys, respectively.
Figure 2
Figure 2
Northern blot and species distribution analyses of Sps2. (A) Sps2 is preferentially expressed in tissues where known selenoproteins are produced and in sites of blood cell development. YS, yolk sac; AGM, AGM region (see text); FL, fetal liver; H, head of the embryo; Plac, placenta; BM, bone marrow; S.Muscle, skeletal muscle; A.Fat, perivisceral abdominal fat; FDCP, hematopoietic FDCPmixA4 cell line; STO, fibroblast cell line STO (15); N2a, neuronal cell line N2a (15); 28S, ribosomal RNA. (B) Sps2 is a T-cell activation gene. Lanes: 1, unstimulated CD4+ CD8− T cells; 2, CD4+ CD8− T cells stimulated with anti-CD3; 3, unstimulated macrophages; 4, macrophages stimulated with lipopolysaccharide; 5, serum-deprived fibroblasts; 6, serum-stimulated fibroblasts. (C) SPS is highly conserved among a variety of animal organisms as shown by probing a Zooblot with the 5′ untranslated region (UTR) of mouse Sps2.
Figure 3
Figure 3
Sps2 maps in the distal region of mouse chromosome 7. Sps2 was placed on mouse chromosome 7 by interspecific backcross analysis. The segregation patterns of Sps2 and flanking genes in 146 backcross animals that were typed for all loci are shown at the top of the figure. Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J × M. spretus)F1 parent. The shaded boxes represent the presence of a C57BL/6J × M. spretus)F1 parent. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome 7 linkage map showing the location of Sps2 in relation to linked genes is shown at the bottom. Recombination distances between loci in centimorgans are shown to the left of the chromosome. The positions of loci cited in this study can be obtained from GDB (Genome Data Base, The Johns Hopkins University, Baltimore).
Figure 4
Figure 4
Readthrough of the in-frame TGA codon of mouse Sps2 in COS-7 cells and analysis of the contribution of the 3′ UTR to the degree of efficiency of translation. The inverted triangle represents the positioning of the FLAG sequence at the N terminus of the Sps2 cDNA constructs. Anti-FLAG-immunopurified lysates were analyzed by Western blotting using anti-FLAG M2 antibody. Mammalian cells were transfected with the pME18X expression vector without insert as control (VECTOR).
Figure 5
Figure 5
Simplified diagram to represent the three available scenarios in Sec metabolism: (a) Mus musculus and Homo sapiens (Eukaryotes). (b) H. influenzae (Bacteria) and M. jannaschii (Archaea). (c) E. coli (Bacteria). The wider arrow in SPS2 denotes the likely difference in activity, yet to be demonstrated (1), between this enzyme and SPS1.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Stadtman T C. Annu Rev Biochem. 1996;65:83–100. - PubMed
    1. Stadtman T C. J Biol Chem. 1991;266:16257–16260. - PubMed
    1. Burk R F, Hill K E. Annu Rev Nutr. 1993;13:65–81. - PubMed
    1. Bock A, Forchhammer K, Heider J, Leinfelder W, Sawers G, Veprek B, Zinoni F. Mol Microbiol. 1991;5:515–520. - PubMed
    1. Bock A, Forchhammer K, Heider J, Baron C. Trends Biochem Sci. 1991;16:463–467. - PubMed

Publication types

MeSH terms

Associated data