Generation Of Selenoprotein (Selenopeptide) And Its Antioxidant Ability

Free radical (FR) is a normal metabolism product in the body, and it plays animportant biological role such as bactericidal potency, virus inhibition inflammationelimination, etc. Reactive oxygen species (ROS) is the most important category of FR,including superoxide anions (O2s), hydroxyl radical (HO·), hydrogen peroxy radical(HOO·), singlet oxygen (1O2), etc. But, the accumulation of FR in the body will leadto excessive oxidation reactions under the incentive effect of smoking, alcoholism,radiation, inhalation of contaminated air, etc. And then, the injury of body could becaused. This phenomenon is known as oxidative stress. FR can irreversibly damage tounsaturated fatty acids, proteins, nucleic acids and any other important biologicalmacromolecules under the oxidative status. Furthermore, some diseases could occur,example for cancer, cardiovascular disease, hemorrhagic shock, ischemia reperfusioninjury, atherosclerosis, etc.There is an antioxidant defense system in the body. This system can be set tomaintain balances of FR. Antioxidants include two kinds of enzymes and non-enzymes, within the antioxidant enzymes i.e. antioxidase exert a major function.Natural antioxidant enzymes consists of superoxide dismutase (SOD), catalase (CAT),glutathione peroxidase (GPx), glutathione S-transferase (GST), glutathione reductase(GSR), etc. Since natural antioxidant enzyme possess a lot of defection, such as highcost for extract, a large molecular weight, poor stability, which limits theirpharmaceutical applications. So, the antioxidant drugs, which were from artificialantioxidant mimics prepared according to the natural antioxidant enzyme as a model,become a research hotspot in recent years. Among them, the studies about GPxenzyme mimic absorb more attention of scholars. Currently, GPx enzyme mimics include organic selenium compounds, metal complex, selenoprotein, supermolecularnanoenzyme, etc. Previous research has shown that most selenoprotein are enzymeswith antioxidant activity in the body, such as glutathione peroxidases (GPx),iodothyronine deiodinases (ID), thioredoxin reductases (TrxR), etc. Thus,selenoprotein become the development goal of antioxidant drug. In this study, threescenarios were used for the preparation of selenoproteins, which are computer-aidedmolecular design combined with chemical modification, single-protein production(SPP) system combined with chemical modification and SPP system combined withcysteine auxotrophic expression technique, respectively. Meanwhile, the biologicaleffects of selenoprotein prepared by the above-mentioned scenarios were investigatedin subcellular and cellular levels. Thereby optimize the preparation process ofselenoproteins and explore the mechanism of antioxidant selenoproteins.1. Preparation of selenoproteins with antioxidant activity by the method ofcomputer-aided molecular design combined with chemical modificationScreening technologies of selenoproteins with high antioxidant activity in thelaboratory involve the inducement method, the copy method, the introduction methodand phage antibody library display technique. However, these methods are complexand costly. The use of simulate screening via computer-aided molecular designmethod similar to drug screening can solve these problems, which instead of previousscreening method in laboratory. Researches on protein structure indicate that thefolded forms of the protein backbone are very limited. This inspired us to carry out alarge number of simulate screening to selenoproteins by means of the transformationabout the protein backbone according to the structure of the natural antioxidantenzymes, for instance substitutions of amino acid, insertions or delete of peptidefragments, and the fusion of domains. In this study, a novel human scFv was designedon the basis of the structure of human antibody and optimized via bioinformaticsmethods such as homologous sequence analysis, three-dimensional (3D) modelbuilding, binding-site analysis and docking. The new scFv was named WCD1, whichhas a higher binding capacity to GSH. The DNA sequence of the human scFv-WCD1was synthesized and cloned into the expression vector pET22b(+), then scFv-WCD1protein was expressed in soluble form in Escherichia coli (E. coli) BL21(DE3) andpurified by Ni2+-immobilized metal affinity chromatography (IMAC). The serineresidue of scFv in the active site was converted into selenocysteine (Sec) with the chemical modification method, thus, the human Se-scFv-WCD1with GPx activitywas obtained. And it exhibited a typical ping-pong kinetic mechanism. This showsthat the combination method of chemical modification and computer virtual screeningwas a clever strategy for preparation of antioxidant mimics, human Se-scFv. Thisscenario saves a lot of costs for multiple rounds screening used by phage antibodylibrary display technique.2. Preparation of selenoproteins with antioxidant activity by the method of SPPsystem combined with chemical modificationLarge-scale preparation strategy of protein through recombination technology ofgenetic engineering has been very mature.While this solves the problem about limitedresources of natural antioxidant enzymes, but a new trouble was also was brought.The new trouble was time-consuming, labor-intensive and high cost in the purificationprocess of protein. The SPP system can save purification cost through the productionof a single protein, because MazF of the SPP system can degrade almost all ACAsequence of mRNA in the intracellular. Therefore, ACA sequence in scFv-WCD1mRNA was knocked out used the codon degeneracy in a case without changing theamino acid sequence. The scFv-WCD1-lessACA can be only expressed in SPPsystem and no other background proteins in the cells could be expressed. Then, Secwas incorporated into the scFv-WCD1-lessACA by chemical modification andresulted in Se-scFv-WCD1-lessACA. The enzymatic characteristics ofSe-scFv-WCD1-lessACA were determined. Se-scFv-WCD1-lessACA andSe-scFv-WCD1have the same properties, such as GPx activity, the binding constantfor GSH, optimal pH, optimal temperature and enzyme kinetics. Moreover,Se-scFv-WCD1-lessACA was confirmed to have a strong antioxidant ability toprotect mitochondria against oxidative damage induced by Fe2+/VC(mitochondrialdamage model). This saves a lot of time and test material required for the purificationof proteins.3. Preparation of selenoproteins with antioxidant activity by the method of SPPsystem combined with cysteine auxotrophic expression techniqueVarious antioxidant enzymes can act cooperatively to scavenge the FR, in orderto the balance of oxidation-reduction system in body. Therefore, the research on thesynergism of multi-function antioxidant mimic is imperative. Recently, it wasdiscovered that the synergy between GPx and SOD plays a vital role in the antioxidant defense system. So, a65-mer peptide imitates (Se-CuZn-65P) of SOD andGPx was designed on the basis of native enzyme structural models under the aid ofthe computer, which contains a delicate dual-activity center based on the homologoussequence alignment in natural SOD3active center and the15-mer peptide with a GSHbinding site. The structure of Se-CuZn-65P contains that form the domains of theactive center of SOD and the catalytic triad of GPx upon the incorporation of Cu2+and Zn2+metals. And then, Se-CuZn-65P is expressed through the SPP systemcombined with cysteine auxotrophic expression technique. The results from Tricine-SDS-PAGE electrophoresis, RP-HPLC and MALDI-TOF MS show that the highpurity Se-CuZn-65P could be obtained successfully by this strategy. Enzymaticcharacterization also found that Se-CuZn-65P has higher SOD and GPx activity. It notonly efficiently effectuates fixed-point insertion of Sec, but also does not affect thespatial conformation and catalytic activity of the protein. The cell model of alcoholicliver disease (ALD) by way of ethanol induced L02cells was established forinvestigate the antioxidant activity of Se-CuZn-65P at the cellular level. The resultsdemonstrated that Se-CuZn-65P is not only almost no cytotoxicity, but also increaseincrease of cell viability, migration and decrease of ROS, malondialdehyde (MDA),lactate dehydrogenase (LDH), aspartate aminotransferase (AST). Se-CuZn-65P canalso inhibit apoptosis. Research on the mechanism of apoptosis revealed thatSe-CuZn-65P can maintain the integrity of the nucleus, and induce cell apoptosisthrough mitochondrial pathway. So Se-CuZn-65P has potential application forprevention of ALD.In summary, Strategies for preparation of selenoproteins can product a largenumber of antioxidant enzyme mimics with high purity and high activity in this study.The technical support was provided for preparation of selenoprotein and exploitationof novel selenium-containing antioxidant drug. It establishes the basis of clarificationabout catalytic mechanism and synergism of antioxidant enzymes.