Study On Eukaryotic Expression And Characterization Of Selenoproteins With Antioxidant Activity

Reactive oxygen species (ROS) are reactive molecules that contain the oxygenatom. ROS coming from normal metabolism are important in order to the lifeactivities of the tissues and cells in vivo. ROS at low/moderate concentrations playphysiological roles in defending against infectious agents and regulating signaltransduction pathways and transcription factors. Mammalian cells possess elaboratedefense mechanisms to detoxify ROS and maintain the balance between productionand elimination of ROS, including glutaredoxin (Grx) and glutathione peroxidase(GPx, EC1.11.1.9) system.Selenium is an essential micronutrient for mammals, incorporation intoselenoproteins by Seleno-cysteine (Sec) and displays its physiology function informs of Sec containing enzyme. There are at least25distinct selenoenzymes in thehuman body. Moderate selenium deficiency has been linked to many conditions,such as increased cancer and infection risk, immune and inflammatory disorders,male infertility, decrease in immune and thyroid function, and several neurologicconditions, including Alzheimer’s and Parkinson’s disease. Selenium-dependent GPxis one of the members of the cellular antioxidative defense system and can catalyzethe reduction of a variety of hydroperoxides using glutathione as a reducing substrate.Sec, the21st amino acid, is the active site of GPx. Incorporation of Sec intoselenoproteins employs a unique mechanism that involves decoding of the UGAcodon. This process requires multiple features such as the selenocysteine insertionsequence (SECIS) element and several protein factors.In this research, we obtained recombinant human Grx1(hGrx1) in eukaryoticexpression systems and a novel GPx mimic, seleno-hGrx1, using hGrx1as a protein scaffold. Owing to that no catalytic tetrad, which was conserved in theselenium-dependent GPx family, was detected in seleno-hGrx1, it was difficult toobtain seleno-hGrx1with high GPx activity. Thus, we tried to obtain recombinanthuman GPx4(hGPx4) in the same eukaryotic expression systems and studied itscatalytic activity and catalytic mechanism.(1) Study on expression and characterization human Grx1from eukaryotic cellsGrx is a class of small proteins existing in bacteria, viruses, mammals. So far,two Grx isoforms were found in humans, including Grx1and Grx2. Grx playsimportant roles on prevention and treatment of many human diseases in vivo, such aslung disease, neurodegenerative diseases, atherosclerosis, and cataracts. In thisexperiment, we obtained recombinant hGrx1with antioxidant activity fromeukaryotic cells. HGPx1gene amplification primers were designed based on thecomplementary DNA sequence reported in NCBI and hGrx1gene was amplifiedfrom the complementary DNA library of HepG2cell line. Primers were designed toincorporate His-tag in the C-terminal of the protein expressed, which made itconvenient to purify the recombinant hGrx1. HGrx1gene was subcloned intoplasmid pSecTag2A, yielding pSecTag2A-hGrx1. The plasmid pSecTag2A were transfected into the eukaryotic cells HEK293T for over expressing recombinanthGrx1. Recombinant hGrx1with His-tag at its C-terminal was purified byimmobilized metal affinity chromatography (IMAC) and identified by Western blot.Recombinant hGrx1showed Grx activity of631±36U/μmol. Although Grx and GPxboth can use GSH as substrate, the reduction of H2O2by Grx is slower than that byGPx. However, Grx1could be an ideal protein scaffold for imitating GPx because ofthe natural binding site of GSH.(2) Study on expression and characterization seleno-hGrx1from eukaryoticcellsWe found that GPx activity of recombinant hGrx1was much lower than that ofnative GPx. As all known, Grx is an essential small molecular weight antioxidantenzymes in human, which utilizes GSH as substrate like GPx. It has been reported that a seleno-Grx was developed for imitating GPx using Grx from E. coli as aprotein scaffold, which showed significantly lower GPx activity than that of naturalbovine GPx. However, GPx activity of another seleno-Grx using mouse Grx asprotein scaffold was in the same order of magnitude as that of natural bovine GPx.These results indicated that species differences and the microenvironment of theactive center of homologous protein structure had significant influence on thecatalytic activity.Despite high GPx activity of mouse seleno-Grx, the human immune response tothis enzyme was an unavoidable problem. In this research, a novel GPx mimic wasdeveloped using hGrx1as a protein scaffold. Due to that Sec has a similar structureto Cys, replacement of Cys by Sec is predicted to cause little structural change.Cys-22was located in the GSH binding site based on computational analysis. Thus,the codon for Cys-22of hGrx1was replaced by Sec codon by site-directedmutagenesis. The mutated hGrx1gene was subcloned into plasmidpSelExpress1-leader, which could facilitate expression of selenoprotein. Therecombinant plasmid was transfected into HEK293T cells and then seleno-hGrx1was obtained, which showed GPx activity of108±27U/μmol.(3) Study on expression and characterization recombinant human GPx4fromeukaryotic cellsIn recent years, preparation of artificial mimics of GPx and the relatedinvestigation has attracted considerable attention. Although a variety of mimetichave been constructed, mass preparation of selenium-containing GPx in is still aneffective way to analyze its biological function. GPx4, also known as phospholipidhydroperoxide glutathione peroxidase, is a single polypeptide chain protein with amolecular mass of19.5kDa. In addition to reducing small peroxides like H2O2,GPx4is also capable of reducing membrane-bound hydroperoxides and lipids, suchas phospholipids and cholesterol hydroperoxides. HGPx4gene amplification primerswere designed based on the complementary DNA sequence reported in NCBI.HGPx4gene was amplified from the complementary DNA library of human hepatoma cell line and subcloned into plasmid pSelExpress1-leader. And then, weachieved the overexpression and purification of recombinant hGPx4in HEK293Tcells. GPx activity of recombinant hGPx4was measured to be1931U/μmol. Thecatalytic mechanism of recombinant hGPx4is similar to that of GPx1, which followsa ping–pong mechanism. Although the amount of recombinant hGPx4was lower, itsactivity was close to that of native GPx4. This research is of great importance forheterologous expression of mammalian selenium-dependent GPx4mutants ineukaryotic cell, and lays the foundation for clarifying the catalytic mechanism ofGPx4.