Expression Of Selenium-containing Glutathione S-transferase In Eukaryotic Cells And Study On Its Antioxidant Effect

Accumulating evidence shows that glutathione peroxidase (GPX), one of the mostimportant antioxidant selenoenzymes, plays an essential role in protecting cells and tissuesagainst oxidative damage by catalyzing the reduction of hydrogenperoxide by glutathione(GSH). Unfortunately, because of the limited availability and poor stability of GPX, it hasnot been used clinically to protect against oxidative stress. To overcome these problems it isnecessary to generate mimics of GPX.GPX has two notable zymologic characteristics: ability to recognize and combine withGSH, and unique catalytic Sec which is close to the functional group of GSH in thethree-dimensional structure. Several research groups have developed a number of GPXmimics by incorporating some structural features of the native enzyme.These researchesdemonstrated that most of these artificial GPX mimics have low GPX activity because theylack a critical substrate binding site. In order to improve the activity of GPX mimics, wemust find an ideal scaffold with a special GSH-binding site and a potential catalytic groupclose to the GSH-binding site. The crystal structure of human glutathione transferase Zeta1-1(hGSTZ1-1) indicates that it can bind GSH and its active site contains three highlyconserved residues (Ser14–Ser15–Cys16). Ser14, Ser15and Cys16are located close to thethiol of bound GSH and they can be easily modified to Sec. In previous studies, researchersmade chemical modification, which convert Ser to Sec, on the basis of hGSTZ1-1, andgenerated a new mimic---Se-hGSTZ1-1, with high GPX activity. However, althoughchemical mutation demonstrated the feasibility of engineering novel GPX activity in proteins,it is not specific, and site-directed substitution cannot be achieved, making it unfavorable forfurther development. In this study, we have used mechanism directed mutagenesis and theinclusion of a selenocysteine insertion sequence to engineer the expression in eukaryoticcells, of hGSTZ1-1with selenocysteine in the active site (seleno-hGSTZ1-1(Ser15Sec). Theresults suggest that seleno-hGSTZ1-1(Ser15Sec) has a high GPX activity. What's more, weexplored the antioxidant effect of Se-hGSTZ1-1in in oxidative stress challenged rat cardiacmyocytes. Specific as follows:1Expression and purification of seleno-hGSTZ1-1In this study, we use a vecor contained hGSTZ1-1, which was recombined by researcher in our lab and was suitable to express selenoprotein in eukaryotic cells, as ascaffold, in which15Ser was mutatd to Sec, by site-directed substitution, and6×his tag wasincorporated to detect expression and purication of recobinent protein, and then getrecombinant plasmid. HEK293T cells were transfected with these plasmids and werecultured in the media supplementing with sodium selenite. Western blotting showed that therecombinant protein was expressed in eukaryotic cells and prifide with IMAC successfully.2Study on the GPX activity and kinetics of seleno-hGSTZ1-1By the analysis of a three-dimensional structure of Seleno-hGSTZ1-1built by means ofhomology modeling, we found that there are two possible catalytic triads consisting of Sec15,Gln169and Trp18, or Sec15, Gln207and Trp18, resembling that of GPXs. The distancesbetween each of these atoms is too great to support the formation of stable hydrogen bonds.This potentially explains why the GPX activity of Seleno-hGSTZ1-1is lower than that ofsome natural GPX isoenzymes. Compared to Se-hGSTZ1-1, chemical modification ofhGSTZ1-1, which incorporated8.13moles selenium per mole of enzyme, and resultingSe-hGSTZ1-1had high (8602U/μmol) GPX activity, the selenium content ofseleno-hGSTZ1-1expressed in this research is1.06mol selenium per mol selenoenzyme,which is lower than that of Se-hGSTZ1-1generated by chemical modification and mayexplain the lower GPX activity of seleno-hGSTZ1-1(2266U/μmol). Another reason couldbe the quantity of catalytic triad in a protein molecule and the distances between thefunctional atoms of three amino acids. It is worthwhile to note that the mutation of Ser15Secby genetic engineering will alter both the primary structure and spatial confirmation of thehGSTZ1-1, optimizing the structure of catalytic triad for GPX activity.3Antioxidant effect of Se-hGSTZ1-1As one of the most important antioxidant enzymes, GPX protects cells and tissuesagainst oxidative damage, and plays an important role in protecting heart andcerebrovascular injuries induced by oxidative stress. In the present work, the antioxidanteffect of Se-hGSTZ1-1was investigated in rat cardiac myocytes. To explore the protection ofGPX in hydrogen peroxide (H2O2) challenged rat cardiac myocytes, malondialdehyde(MDA), lactate dehydrogenase (LDH), superoxide dismutase (SOD), the levels ofperoxisome proliferator-activated receptor gamma (PPAR-γ), nuclear factor-kappa B(NF-κB), and Bax were examined. The results demonstrated that exposure to H2O2for6and12hours induced the significant increases of MDA, LDH, and decrease of SOD in thecardiac myocytes, but incubation with Se-hGSTZ1-1at0.0005unit/ml or0.001unit/ml in two different ways(prophylactic treatment and therapeutic treatment), prevents oxidativestress induced by H2O2with enhances the expression of PPAR-γ and ameliorates the levelsof NF-κB and bax. All these results hints that Se-hGSTZ1-1has antioxidant activity in vitro,and might be a regulator of PPAR-γ, NF-κB, and bax in oxidative stress challenged ratcardiac myocytes.In conclusion, we expressed seleno-hGSTZ1-1(Ser15Sec) with GPX activity of2266U/μmol in eukaryotic cells. What's more, Se-hGSTZ1-1exhibits an antioxidant activity invitro, resemble as natural GPX.