Prokaryotic Expression, Structure And Characterization Of Recombinant Human GPx4and Its Mimic

Free radicals can be defined as molecules or molecular fragments containing oneor more unpaired electrons in atomic or molecular orbitals. Reactive oxygen species(ROS) are intermediate products of normal metabolism and they can be either harmfulor beneficial to living systems. ROS at low/moderate concentrations playphysiological roles in defending against infectious agents and regulating signaltransduction pathways and transcription factors. The harmful effects of ROS occur athigh concentrations and the excess ROS can cause damage to lipids, proteins andnucleic acids in cells, causing the condition termed oxidative stress. Oxidative stresshas been implicated in a number of human diseases as well as in the ageing process.The balance between beneficial and harmful effects of ROS is essential for thesurvival of organisms and achieved by the antioxidant action of non-enzymatic andenzymatic antioxidants.Selenium is well established as an essential trace mineral of fundamentalimportance to human health and this element is mainly incorporated into thepolypeptide chain as part of selenocysteine (Sec). At least25proteins are identified sofar which have different tissue specificity and maintain normal physiological functionof many body tissues. Glutathione peroxidase (GPx) is a member of selenoenzymesand acts as as a free radical scavenger using GSH as reducing substrate. The catalyticcenter of GPx is known as catalytic triad consisting of a Sec, a Trp, and a Gln residue.Sec is also known as the21stamino acid and encoded by UGA, which usuallyfunctions as a stop codon. The incorporation of Sec into protein requires a specialmechanism. The selenocysteine insertion sequence (SECIS) and multi trans-actingfactors are necessary to achieve the incorporation. And the fact that the mechanismdiffers from prokaryotes to eukaryotes makes it difficult to directly heterologouslyexpress recombinant mammalian selenoproteins in E. coli. Due to the disadvantages of limited availability, poor stability and high molecular weight of native GPx, itstherapeutic usage is limited. Researchers’ study focused on artificial imitation of GPx.In this research, selenocysteine-containing hGSTZ1c-1c and its various mutantswere prepared by using cysteine auxotrophic strain to obtain GPx mimics with highactivity. And the role of different residues in the SSC motif on binding GSH and theeffect of introducing Sec into hGSTZ1c-1c were explored by site-directedmutagenesis and computational analysis. Furthermore, we produced recombinanthuman GPx4mutants using cysteine auxotrophic strain and discussed the impact ofnon-catalytic Sec on the structure of the protein. A strategy was proposed forenhancing the catalytic activity of recombinant enzyme and a new GPx4mutant withhigh activity was obtained.(1) Characterization of catalytic activity and structure of selenocysteine-containinghGSTZ1c-1c based on site-directed mutagenesis and computational analysisGlutathione peroxidase (GPx, EC1.11.1.9) is an important class of antioxidantenzymes that protects cells from oxidative damage. As radical scavengers, GPxscatalyze the GSH-dependent reaction and thereby reduce different types of peroxidesto their respective alcohols. Because of the natural binding site of GSH, humanglutathione transferase zeta1c-1c (hGSTZ1c-1c) has been considered to be an idealprotein scaffold for imitating glutathione peroxidase. The SSC motif(Ser14-Ser15-Cys16) highly conserved in most of GSTs was regarded as the activecenter in catalysis. It has been reported that the Sec-14and Sec-15ofselenium-containing hGSTZ1-1(Seleno-hGSTZ1-1) produced by chemicallyconverting Ser to Sec residue contributes significantly to the GPx activity. But it wasdifficult to prepare selenoenzyme in large amount due to the restrictions of chemicalmethod. Instead, Cys auxotrophic expression system was used to produce GPxmimics. The aim of this study is to investigate the changes of catalytic activity fromhGSTZ1c-1c to Seleno-hGSTZ1c-1c produced by using Cys auxotrophic expressionsystem. Furthermore, the role of different residues in the SSC motif on binding GSHand the effect of introducing Sec into hGSTZ1c-1c were explored by site-directed mutagenesis and computational analysis. In this report, several residues near GSHwere mutated to selenocysteine (Sec) or cysteine (Cys) residues and the impacts of thesubstitutions on different activities were discussed. Mutations of Ser-14or/and Ser-15to Cys or Sec residues resulted in dramatic loss of catalytic activity of hGSTZ1c-1cwith chlorofluoroacetic acid (CFA) as substrate, indicating the importance of thehydroxyl groups in Ser-14and Ser-15. And subsequent study by molecular modelingsuggested that Ser-15was probably involved in catalysis, while Ser-14may play acrucial role in binding and orientation of GSH and possibly had a synergistic effectwith Ser-15in catalysis. On the contrary, the result of converting Cys-16to Serindicated its trivial role in catalysis. The investigations of theselenocysteine-containing hGSTZ1c-1c (Seleno-hGSTZ1c-1c) and the mutant S17Cimplied that the substitutions of multi-Sec for Cys residues could lead to subtlechange in the structure of the protein molecule and concomitant change in catalyticactivity as a direct result. This finding provides evidence that the protein scaffoldcontaining fewer cysteines should be chosen for imitating GPx using Cys auxotrophicexpression system to avoid unexpected structural changes.(2) Prokaryotic expression of recombinant Se-GPx4and study on its structure andactivityCytosolic GPx4is a single polypeptide chain protein with a molecular mass of19.5kDa, which does not undergo major post-translational modification. cysteineauxotrophic strain has been proved to efficiently achieve the introduction of Sec intoproteins. In this study, recombinant GPx4was produced by using this E. coli strain.Cys-10and Cys-16were mutated to Ser residues for they may cause the recombinantprotein aggregation. Although recombinant GPx4was obtained with high purity andstability and showed higher GPx activity (22U/μmol) than Ebselen (0.99U/μmol),but two orders of magnitude lower than native GPx4. There were five non-catalyticSec residues besides Sec-46in recombinant GPx4which were unspecificallyintroduced by cysteine auxotrophic strain. And the five Sec were seleninic acidsbecause they could not be reduced by GSH. The substitution of multi-Sec for Cys in recombinant GPx4may lead to structural change for the structural differencesbetween Sec and Cys. And this difference caused the active hydroxyl group of Sec-46orientating toward the inside of the protein in comparison to the putative native GPx4and thereby directly interfered with binding of GSH to the recombinant enzyme. Inthe catalytic reaction, the rapid reactions of the selenenic acid with GSH and of theresulting selenenyl sulfide (E-SeSG) with a second GSH to produce the selenol appearto be very important, because these reactions ensure that the selenium moiety in theenzyme is not irreversibly inactivated. Lower reduction efficiency might be the mainreason for the loss of the activity of recombinant GPx4. In this study, it is the firsttime to prepare recombinant human GPx4in prokaryotic expression system. Althoughthe activity of recombinant GPx4is not as high as native GPx4, but the resultsrepresent a solid step forward on the path toward heterologous expression ofrecombinant GPx in E. coli.(3) A modification of recombinant GPx4mutantThe substitution of multi-Sec for Cys in recombinant GPx4may lead to structuralchange and thereby cause the loss of the normal GPx activity. So we need an effectiveway to improve the activity by modifying recombinant GPx4. Ser and Cys exhibitvery similar physical and chemical properties and the substitution of Ser for Cys inrecombinant GPx4wouldn’t lead to severe structural change. In this study, Cys-148,Cys-107, Cys-37, Cys-75and Cys-2in GPx4were mutated to Ser in turn and theresponding Se-GPx4mutants were prepared by using the “combined” expressionsystem. The results indicated that the catalytic activity of recombinant GPx4wasenhanced following the number of non-catalytic Sec decreased. The mutant showedthe highest catalytic activity (646U/μmol) when all the Sec residues in recombinantSe-GPx4mutant were replaced by Ser, which was15fold higer than non-mutatedrecombinant GPx4. A three-dimensional structure of this recombinant Se-GPx4mutant was constructed and the catalytic triad was therefore detected. Unlikenon-mutant recombinant GPx4, the active hydroxyl group of E(mutant)-SeOH isorientated toward the same direction compared with the putative native GPx4. And the increase of the catalytic activity of recombinant Se-GPx4mutant may attribute tothis. In addition, a new way to prepare recombinant Se-GPx4was established by usingcysteine auxotrophic strain combined with SPP system. The “combined” expressionsystem has been proved to express selenoprotein with higher efficiency of solubleprotein production and lower cost of protein production than cysteine auxotrophicexpression system. These results indicated the feasibility of substituting Ser for Cys toenhance the catalytic activity of recombinant Se-GPx4produced in prokaryotic host.This research is of great significance for the heterologous expression of recombinantSe-GPx, and lay the foundation for investigating the clear structure and catalyticmechanism of GPx.