Imitation Of Glutathione Peroxidase Using Human Selenium-containing Single Chain Fv

Oxidative stress is a cellular imbalance between the production and elimination of reactive oxygen species (ROS), such as superoxide radicals (O2.-), hydroxyl radicals(·OH)or hydrogen peroxide (H2O2). ROS are accumaulated when oxidative metabolism products increase or the antioxidative defenses lack in the organism. ROS have very strong oxidative ability. Excess production of ROS can damage proteins, DNA and lipid,which cause loss of cell function and, ultimately, death. Many studies suggest ROS play a key role in several human diseases, including heart diseases. Therefore, it is important to develop effective antioxidants for treatment of human dissease induced by oxidative stress.Aerobic living organisms have evolved relevant non-enzymatic and enzymatic antioxidant systems to eliminate excess ROS. Antioxidaive enzymes include superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX). GPX has been widely found in both animals and plants. So far, there are at least four isoforms of GPX in mammals. All of them are selenoenzymes. Selenium, in the form of selenocysteine (Sec), localizes in the active center of GPX. GPX catalyzes the reduction of hydroperoxides using GSH as the reducing substrate and selenocysteine (Sec) residues as catalytic group. GPX is substantially more efficient on a molar basis than the other enzymes. Therefore, GPX is more valuable in pharmaceutical applications.Therapeutic usage of native GPX is limited because of its limited availability, instability and high molecular weight. Many scientists have made a great deal of efforts to imitate the functions of GPX. The early GPX mimics have generally rather low activities because of lack of the GSH binding site. The key of imitation GPX is to generate a specific binding site for GSH. The technique for preparing monoclonal antibody should be an efficient method for preparing GPX mimics with substrate binding site.Conventional abzymes are produced on the basis of Pauling's transition state theory. However, native GPX catalyzes a double substrate (GSH and H2O2) reaction. The reaction mechanism of native GPX is very complicated and its transition state is not known until now. It is impossible to imitate GPX using transition state analog method. A new method, named hydrophobic modification method, for preparing abzyme with GPX activity is proposed from the viewpoint of structural chemistry by professor Luo's group. The main idea of this strategy is as follows: the polar groups of substrate GSH are modified by different hydrophobic groups and the modified substrates are used as haptens to immune mouse and obtain monoclonal antibodies. The antibodies possess not only hydrophobic cavity similar to that of the active site of native enzymes, but also specificity to substrate GSH. Finally, the catalytic group Sec is incorporated into the hydrophobic cavity by chemical mutation of the reactive serine residues.On the basis of this strategy, a series of mouse monoclonal abzymes, including Se-4A4, Se-3G5, Se-5C9 and Se-2F3 were obtained using S-substituted dinitrophenyl glutathione (GSH-S-DNP), GSH-S-DNPMe, GSSGMe and GSH-S-DNPBu as haptens, respectively. All of them display rather highly GPX activities. Among them, Se-2F3 against GSH-S-DNPBu exhibits the most highly catalytic efficiency. Nevertheless, intact antibody molecules are so large that it is difficult to enter into target tissues and cells. In order to produce pharmaceutical protein, single-chain Fv fragment (scFv) with GPX activity (Se-scFv-2F3) from Ab-2F3 was prepared using gene engineering method. However, mouse monoclonal antibodies or scFv may cause human immunoreaction by inducing human anti-mouse antibody. The generation of human abzyme with GPX activity is more important in pharmaceutical application. The technology of phage display antibody library is an efficient method for preparing human abzyme.In the present study, the main aim is to generate human catalytic scFv with GPX activity and study its antioxidant effect in cells.1. Synthesis of antigen GSH-S-DNPBuGSH-S-DNPBu was synthesized and used as target antigen based on a hydrophobic modification strategy and previous some studies from professor Luo's group. The modification of sulfhydryl group of GSH by dinitro chlorobenzene (DNCB) increased antigen stability and the incorporation of aroma group enhanced antigen hydrophobicity. The carboxyl groups of GSH-S-DNP were esterifized with butanol to obtain GSH-s-DNPBu. The GSH-S-DNPBu maintains the basic structure of GSH and increases the hydrophobicity of antigen. The antibodies against GSH-S-DNPBu screened from phage display antibodies library may not only possess hydrophobic cavity, but amplify structural space in GSH binding site of activity center. Native GPX catalyzes a double substrate (GSH and ROOH) reaction. Amplified structural space may make ROOH easily access the active site of GPX, therefore increase GPX activity of abzyme. The structure and composition of the synthetical antigen was confirmed by 1H NMR.2. Affinity screening and selection of phage scFvs against GSH-S-DNPBu from phage display human scFv antibody libraryThe characteristic of phage display antibody library is that the genotype and phenotype of antibody are incorporated in the same phage. Antibody displays in surface of phage and the encoding gene of antibody is inserted in phage DNA. Specific phage clones binding to a certain antigen are enriched by the process of adsorption, elution and amplification. Therefore, the encoding genes of specific antibodies can be obtained from phage DNAs and used for further gene engineering operation. In our study, phage display human scFv antibody library was used to obtain the human scFv. ScFv is the smallest antibody fragment of retaining the ability to bind antigen. Therefore, scFv is easy to enter into the target tissue . In the present study, specific phage antibodies against GSH analog, GSH-S-DNPBu was screened from phage display human scFv antibody library. After three rounds of screening, the specificities of polyclonal populations of phage produced at each round of screening to GSH-S-DNPBu were determined by polyclonal phage-ELISA, whereas the specificities to GSH-S-DNPBu of monoclonal phages from 40 single infected bacterial clones, selected at random from bacterial clones of the third round of screening, were determined by monoclonal phage-ELISA. The results suggested that affinities to GSH-S-DNPBu of the polyclonal phage populations were increased stage by stage. Whereas, only three phage clones (A11, B3 and D5) from randomly selected 40 phage clones showed specific binding to GSH-S-DNPBu. The three phage clones were selected for the next experiment. The encoding genes of human scFvs were amplified by PCR method with phagemid DNA prepared from the selected positive clones by ELISA as templates. Analysis of PCR products by gel electrophoresis and sequencing showed that only phage clone B3 contained an amplified intact scFv gene of 750 bp, which was cloned into secretory expression vector pPElB and transfected in E. coli Rosetta to obtain soluble expressed human scFv (scFv-B3). The expressed scFv-B3 was purified by Ni2+-immobilized metal affinity chromatography (IMAC). The results of SDS-PAGE and western blot showed scFv-B3 was homogeneous and successfully expressed as soluble form.4. Preparation of selenium-containing human catalytic scFvThe selenium-containing human catalytic scFv (Se-scFv-B3) with GPX activity of 1288 U/μmol was prepared by chemically converting the active site serines of scFv-B3 into Secs.5. Antioxidative effects of selenium-containing human catalytic scFv (Se-scFv-B3)The antioxidative effect of Se-scFv-B3 was further investigated in primary neonatal rat cardiac myocytes using H2O2 injury system. The Se-scFv-B3 was demonstrated to protect cardiac myocytes against H2O2-induced damage by the partial increase of cell viability, the decline of malondialdehyde (MDA) production and lactate dehydrogenase (LDH) release, the inhibition of mitochondrial membrane potential (?ψm) collapse using MTT method, MDA detection reagent kit and LDH colorimetry kit and flow cytometry analysis. The decrease of intracellular ROS after treatment of rat cardiac myocytes by Se-scFv-B3 indicated that Se-scFv-B3 exerts an antioxidative mechanism similar to that of the natural GPX in cells. In the present study, human catalytic scFv with GPX activity (Se-scFv-B3) of 1288 U/μmol was successfully generated. Although the GPX activity of Se-scFv-B3 is lower than those of mouse monoclonal antibodies or scFv, as a human scFv, Se-scFv-B3 may have a high pharmaceutical value because of its lower antigenicity to human.