Expression And Characterization Of GPx Mutants And Its Mimic

Reactive oxygen species (ROS) is a collective term that includes superoxide(O2·-), hydroxyl (·OH), hydrogen peroxide and some unstable intermediates of lipidperoxidation. There is a balance between the production and elimination of ROSunder normal physiological conditions. However, excessive generation of ROS willinduce a series of harmful effects, which are associated with a variety of diseases,such as cancer, atherosclerosis, Parkionson’s disease and diabetes. The harmfuleffects of ROS are balanced by the antioxidant action of antioxidant enzymes andnonenzymatic antioxidants. Central antioxidant enzymes include superoxidedismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx).GPx is an important antioxidant that can catalyze the reduction of hydrogenperoxide and lipid hydroperoxides to water and the corresponding alcohol, usingglutathione (GSH) as an essential cosubstrate. The pivotal role of GPx in protectionagainst oxidative stressors has been demonstrated. Sec encoded by UGA is thecatalytic group in the active site of GPx. To synthesize GPx, both prokaryotic andeukaryotic cells have their own mechanism of Sec insertion. Thus it is impossible todirectly prepare GPx with traditional recombinant DNA technology. Owing to thedifficulty in gene recombination technique and important biological roles of GPx,many research efforts have been made to mimic the function of GPx. However, thereare no reports of heterologous expression of natural hGPx.E.coli auxotrophic expression system is an effective method, which is based onthe following assumption: the aa-specific tRNA is aminoacylated with amino acidanalogue by aa-tRNA ligase when the amino acid is omitted. E.coli cysteineauxotrophic expression system employed in this dissertation just take the point thatthe E.coli strains of Cys auxotrophic cannot synthesize Cys itself, so it can make use of the Sec abundant in the culture to express target protein. Recently, a novelexpression system called the single protein production (SPP) system for theproduction of a single protein in E. coli has been developed. This system canproduce a protein of interest in the absence of other cellular proteins through theintroduction of MazF to host cells. At the same time, SPP dramatically reduces thecost of protein production without sacrificing protein yield by condensing the culture.Using this system, it is feasible to obtain high yield of recombinant selenoenzymefrom E. coli BL21(DE3)cys auxotrophs. In this study, we produced a series ofrecombinant hGPx1and hGPx2mutants with high activities, and the biologicalproperties of the mutants were also characterized. Furthermore, computersimulations of mutants were used to discuss the relationships between structure andfunction of protein. Based on the above results, hGSTZ1c-1c gene studied previouslywas modified, and the obtained seleno-hGSTZ1c-1c mutants showed some activitiestoward H2O2. Besides, the function of the catalytic center SSC was discussed.(1) Heterologous expression and characterization of human cellular GPx mutantsGPx1was the first seleno-containing enzyme to be identified, which is widelyexpressed and particularly abundant in erythrocytes, kidney and liver. The pivotalrole of GPx1in protection against oxidative stressors has been widely demonstrated.In this study, hGPx1gene was cloned from a cDNA library of the human hepatomacell line HepG2. The codon UGA encoding Sec49of hGPx1was first mutated toUGC encoding cysteine (Cys) and then biosynthetically converted to Sec duringexpression in an E. coli BL21(DE3)cys auxotrophic system. Seleno-hGPx1Secdisplayed a low GPx activity of522U/μmol. There were five Cys residues in onehGPx1molecule. The substitutions of the multi-Sec for Cys residues, especially atposition2,78,115,156, and202at the same time in the auxotrophic strain, caused aconsiderable change of the hGPx1structure and affected the catalytic activity.Considering the similarity between Cys and Ser, the five Cys residues were mutatedto Ser residues. The mutant seleno-hGPx1Sershowed a high activity of5278U/μmol,which was more than9-fold enhanced as compared with seleno-hGPx1Sec. The capability of catalytic activity of it toward H2O2was found to be much moreefficient than most of previous GPx mimics, and it even could be comparable tothose of natural GPx, such as bovine liver GPx (2084U/μmol), and rabbit liver GPx(5780U/μmol). Kinetic analysis of seleno-hGPx1Ser showed a typical ping-pongmechanism, which was similar to those of natural GPxs. This research was the firsttime to express hGPx1by E. coli BL21(DE3)cys auxotrophic system. It will be ofvalue in overcoming the problem of limited sources of natural GPx. And in future, itstill can help us to research and recognize natural hGPx1.(2) Expression of recombinant seleno-hGPx1mutants with high activities and studyon their structures and activitiesTo further study the relationship between structure and properties of hGPx1,Cys-2, Cys-78, Cys-115, Cys-156and Cys-202in hGPx1were mutated to Ser in turnbecause untargeted substitution of Sec in place of Cys resulted in the decline ofrecombinant selenoenzyme activity. In this study, a series of hGPx1mutants wereproduced for the first time in an E. coli BL21(DE3)cys auxotrophic strain using theSPP system. The results of this work showed that the catalytic activities of themutants increased progressively with decreasing number of noncatalytic Sec residues.Seleno-hGPx1-C2/78/115/156/202S with all Cys residues changed to Ser showed thehighest activity (21,268U/lmol), which was more than9-fold higher than bovineliver GPx. This increase could be explained by structural analysis of hGPx1mutantsbased on homology modeling and binding site analysis. Although the structure ofthis intermediate has not been solved to date, the distance between the Se atom ofSec in the catalytic center and the S atom of GSH is believed to be a key factor forits formation. These results lead to the hypothesis that the conversion of noncatalyticSec residues to Ser may optimize the structure of seleno-GPx in this expressionsystem and consequently increase the catalytic efficiency. In addition, to comparewith the method of auxotroph expression, this combined expression system not onlyincreases the protein yield and reduce the production cost, but also greatly improvesthe activity of the protein. (3) Expression of recombinant seleno-hGPx2mutant and study on its structure andactivityGastrointestinal glutathione peroxidase (GPx2) is regarded as the first line ofdefense against oxidative stress caused by ingested pro-oxidants or gut microbes. Inorder to produce bioactive recombinant hGPx2, the gene of hGPx2was designedwith the conversion of the codons for four Cys residues to the codons for Serresidues and the codon for Sec-40was changed to the codon for Cys. Thisrecombinant seleno-hGPx2mutant was obtained using a SPP system in a Cysauxotrophic strain. The activity of this mutant was in the same order of magnitude asthat of hGPx4, but about one order of magnitude lower than that of hGPx1andhGPx3. The results obtained from the kinetic analysis demonstrated that it followeda typical ping-pong mechanism similar to native GPx. As there was no report on theactivity of purified GPx2, this research was valuable in recognizing native GPx2. Inaddition, a three-dimensional structure of seleno-hGPx2mutant was constructed,which could facilitate further analysis of the role and the catalytic mechanism ofnative GPx2.(4) Catalytic activity of seleno-hGSTZ1c-1c based on site-directed mutagenesisHuman glutathione transferase zeta1c-1c (hGSTZ1c-1c) was considered to bean ideal protein scaffold to imitate GPx owing to the natural binding site of GSH. Inorder to obtain GPx mimics, four Cys residues (Cys-137, Cys-154, Cys-165andCys-205) were mutated to Ser at first to avoid introducing more Sec residues. Thenthe three (14,15,17) residues near GSH were respectively mutated to Cys, andbiosynthetically converted to Sec in Cys auxotrophic expression system. Theseleno-hGSTZ1c-1c mutants15C,14C/15C and17C showed some activities towardH2O2. Subsequent study of nonseleno-containing mutants suggested that Ser-14andSer-15may play crucial roles in catalysis, while Ser-14may play a role in binding ofGSH and Ser-15probably had a synergistic effect in catalysis. All of these findingswill be helpful for further study in this area.