The Read-through Efficiency Of Selenocysteine And Expression Of Selenocysteine-containing GST In Escherichia Coli

Selenium is an essential element for mammals. Many of selenoenzymes are involved in redox reactions with selenocysteine acting as an essential component of the catalytic cycle, such as glutathione peroxidase, thioredoxin reductase, iodothyronine deiodinase and formate dehydrogenase. Interestingly, selenocysteine is encoded by the UGA codon and is incorporated into polypeptides co-translationally. Thus, selenocysteine is considered the twenty-first proteinogenic amino acid. Since the UGA codon retains its routine function in tissues expressing selenoproteins, there is a special regulatory mechanism for the incorporation of selenocysteine.In prokaryotes, the incorporation of selenocysteine requires four gene products: one tRNA and three proteins. In addition, recognition of UGA as a selenocysteins codon depends on secondary mRNA structure, i.e. stem loops called SECIS (Selenocysteine Inserting Sequence), which is located 11 nucleotides downstream of the UGA codon. The SECIS element from prokaryotes is containedwithin the open reading frame of mRNA, while eukaryotic SECIS is in the 5'- or 3'-untranslated region. Furthermore, the bacterial SECIS is species-specific, while the SECIS elements in eubacteria and eukaryotes are somewhat less species-restricted.Glutathione S-transferases (GST) constitute a family of multifunctional proteins able to detoxify endogenous and xenobiotic electrophiles by conjugation with glutathione (GSH). In the active site of GSTs, the catalytic residue is typically either Tyr or Ser, and is presumed to act by inducing and stabilizing a reactive thiolate at the conjugating sulfur residue of the GSH substrate. Glutathione peroxidase is a well-known selenoprotein, which functions as an antioxidant to protect from oxidative damage by catalyzing the reduction of a variety of hydroperoxides, using glutathione as the reducing substrate and a reactive selenocysteine residue in the active site. Structural determinations, detailed kinetic studies and modeling of enzyme-substrate complexes have desaibed how the selenocysteine (Sec) residue plays its critical role in the catalytic cycle by utilizing the specific redox properties of selenium.Both GST and GPX have glutathione-binding sites and thioredoxin- fold structures, but due to the use of different catalytic residues, i.e. Tyr (or Ser) on one hand and Sec on the other, they have completely different catalytic mechanisms. However, some overlap between their activities may exist in some aspects, beyond their shared GSH-dependency, since certain GSTs were reported to display GPX activity towards organic hydroperoxide. We previously reported the conversion of GST to a selenium-containing GST (GST-Sec) with GPX activity using chemical modification. However, the non-directed substitution hampered any further structure-function characterization of the seleno-GST thus produced. Use of peptide ligation mediated by Sec is also hampered due to size limitations of selenopeptide prepared by chemical synthesis (30, 31). Thus, we tried to achieve GST-Sec, using the selenoprotein synthesis machinery of E. coli. In addition, weconstructed a green fluorescent protein for quantitaviely detecting the read-through efficiency of the UGA codon, and examined the influence of co-expressing selA, selB and selC genes on read-through efficiency.1. Expression of selenocystein-containing GST in E. coli and its propertiesTo acquire the expression of GST-Sec in E. coli, codon UAU encoding Tyr7, the catalytic residue in the active site in Schistosoma japonicum GST, has been mutated to UGA, which encodes Sec. Because expression of a eukaryotic selenoprotein requires the introduction of a bacterial-type SECIS element immediately adjacent to the UGA codon inside the ORF, the minimal fdhF SECIS consisting of 17 nucleotides was introduced into the sjGST gene 11 nucleotides downstream of the UGA codon. Utilizing coexpression with the bacterial selA, selB and selC genes the yield of recombinant GST-Sec was about 2.9 mg per liter of bacterial culture, concomitant with formation of approximately 85% truncation product as the results of termination of translation at the selenocysteine-encoding UGA codon. The final yield of pure GST-Sec using the pPelB-approach was about 0.25 mg purified protein obtained from 1 liter of bacterial culture.Far-UV circular dichroism and intrinsic fluorescence measurements gave highly similar spectra of GST-S compared to the wild-type sjGST, suggesting that the overall fold was maintained. The mutations inferred as a result of the introduction of a SECIS element did not affect the glutathione binding capacity (Km = 53 ,mM for glutathione as compared to 63 ^M for the wild type enzyme) nor the GST activity (kcat = 14.3 s"1 vs. 16.6 s'1), provided that the catalytic Tyr residue was intact. When this residue was changed to selenocysteine, however, the glutathione binding capacity GST-Sec decreased 10-fold (Km = 570 ^M). It also failed to display any novel GPX activity towards three standard peroxide substrates (hydrogen peroxide, butyl hydroperoxide or cumene hydroperoxide). These results show that recombinant selenoproteins with internal selenocysteine residues may be heterologously produced in E. coli at sufficient amounts forpurification. We also conclude that introduction of a selenocysteine residue into the catalytic site of a glutathione S-transferase is not sufficient to induce GPX activity in spite of a maintained glutathione binding capacity.2. GFP as a reporter quantitatively detecting the read-through efficiency of selenocysteineSince GFP was described as reporter gene in 1994, it has been used extensively as a fusion tag, as a reporter, as well as in protein interaction experiments and in fluorescent energy resonance transfer assays. GFP is able to autocatalytically fluoresce without external agents other than oxygen.The Tr-GFP fusion was constructed by inserting a sel-gfp gene fragment into the multiple clone sites downstream of thioredoxin (Tr) in plasmid pET-32b. There were different combinations of UGA codons and SECIS elements in the DNA linker between thioredoxin and GFP. When the UGA codon was read through, the GFP located downstream would be translated. Then the fluorescence intensity of GFP can be used for quantitative detection of the UGA codon read-through efficiency. The results showed that the read-through efficiency of the UGA codon was increased by 1.32-fold in the presence of the SECIS element compared to that without a SECIS element, and the efficiency was futher increased up to 2.0-fold when GFP fusion was co-expressed with selA, selB and selC genes. These results suggest that GFP can be used as a rapid and convenient tool compared to a-galactosidase for measuring the read-through efficiency of the UGA codon in intact E. coli cells, and they support a potential use of GFP in measuring the Opal suppression in mammalian cell culture and tissue samples.3. The influence of selA, selB and selC genes on the read-through efficiency of selenocysteineTo assess the influence of selA jelB and selC genes on the read-through...