Study On Imitation Of Glutathione Peroxidase Using Small Molecular Human Antibody

Reactive oxygen species (ROS), including superoxide anion (O2–?), hydroxyl radical (·OH), hydrogen peroxide (H2O2), hydroperoxide (ROOH), nitric oxide (NO) and singlet oxygen (1O2) are normal products of aerobic metabolism. Excess production of ROS can cause damage to cellular membranes and key biological molecules, and is a serious causative factor in many diseases. Mammalian cells possess elaborate defense mechanisms to detoxify ROS and maintain the balance between the production and elimination of ROS, including enzymatic and non-enzymatic action. Glutathione peroxidase (GPX, EC. 1. 11. 1. 9) is one of the important members of the mammalian antioxidant enzyme system. Up to now,at least six members of GPX family have been identified in humans, they are classic or cytosolic GPX (cGPX, GPX1), gastrointestial GPX (giGPX, GPX2), plasma GPX (pGPX, GPX3), phospholipid hydroperoxide GPX (PHGPX, GPX4), epididymis-specific GPX (GPX5), olfactory epithelium or embryonic tissues GPX (GPX6). Although they have remarkable differences on locations, structures of subunit, primary sequences and characters of enzymology, the major function of these peroxidases is considered to be the removal of hydrogen peroxide and lipid hydroperoxides, maintenance of cellular redox state, and protection of lipid membranes and other cellular components from oxidative damage.The most types of GPX are selenoenzymes that use GSH as one substrate and selenocysteine (Sec) as catalytic group to catalyze the redox reactions. The researches indicate that GPX is substantially more efficient on a molar basis than the other antioxidative enzymes, and a potential drug for prevention and therapy of many diseases. Owe to the disadvantages of limited availability, poor stability and high molecular weight of native GPX, its therapeutic usage is limited. Artificial imitation of GPX not only can provide more potential antioxidative drugs, but also be propitious to the research of catalytic mechanism. So far, the imitations of GPX which mainly include small molecular chemical enzyme, selenium-containing abzyme, bioimprinting enzyme and semi- synthesize enzyme, have a good development. Native GPX catalyzes a double substrates (GSH and H2O2) reaction, and the mechanism is very complicated and its transition state is not known, so it is difficult to imitate GPX using transition state analog method. Based on these facts, a new method, named hydrophobic modification method, for preparing abzyme with GPX activity is proposed from the viewpoint of structural chemistry by professor Luo. The main idea of this method is as follows: the polar groups of substrate GSH are modified by different hydrophobic groups and then the substrate analogs are used as haptens to prepare the monoclonal antibodies. The antibodies prepared by this method not only can bind specially to substrate GSH, but also possess hydrophobic cavity similar to that of the active site of native enzymes. After incorporating the catalytic group Sec into the hydrophobic cavity by chemical mutation, the abzyme with GPX activity is acquired. On the basis of this strategy, a series of mouse monoclonal abzymes with high GPX activity were obtained, and a smaller molecular selenium-containing single chain Fv fragment 2F3 (Se-scFv-2F3) was prepared further. All of them displayed rather highly GPX activities, but they may cause human immunoreaction because of their mouse origin. In order to prepare the human catalytic antibody with GPX activity, the hydrophobicly modified GSH was used as antigen, and the phage display antibody library technology was applied to acquire the human scFv-B3. It successfully imitates GPX after introducing Sec by chemical mutation, but has much lower activity than the mouse abzyme.In order to acquire more and better small molecular human abzymes, for Se-scFv-B3, site-directed mutation based on the theoretical analysis of structure was carried on to improve its catalytic activity. The binding site was studied using theoretical and experimental methods. On the other hand, the phage display antibody library technology was used to obtain more human anti-GSH scFvs, and structural analysis was also performed to study the binding site and the catalytic group. In addition, the methods of genetic engineering was tried to acquire selenium-containing scFv with auxotrophic selenium-containing protein expression system. Heavy chain variable region (VH) fragment of the antibody was also used to try to imitate GPX, for acquiring smaller human abzyme.1. Improving GPX activity of human single-chain Fv abzyme Se-scFv-B3 by site-directed mutation based on the structural analysisThree-dimensional structure of a protein is the foundation of the function, the study of dimensional structure has very important significance, and the theoretical anticipation for the protein is a simple method to understand its structural information. In order to study the substrate binding sites and the catalytic group of Se-scFv-B3, and provide theoretical foundation for the further research, we do cooperate with Institute of Theoretical Chemistry. A three-dimensional structure of scFv-B3 was constructed by means of homology modeling and binding site analysis was carried out. Computer-aided docking and energy minimization calculations of the antibody-GSH complex were also performed. These studies show that scFv-B3 contains two hydrophobic pockets, and that one or both could be presumed to be the GSH binding site(s). However, no Ser was found in either pocket, the serines surrounding the pocket were deemed to be too far from the sulfhydryl of GSH, possibly demonstrating why the Se-scFv-B3 has lower catalytic activity. After a more detailed analysis of the binding sites, Ala180 in Site 1 and Ala44 in Site 2 were chosen to be mutated to serines to acquire the two mutated protein respectively. After chemical modification, the activity of the anterior one up to about 2.16 times that of the Se-scFv-B3, but the latter one did not offer a significant improvement. This indicates that Site 1 is the predominant binding site for GSH, which was consistent with the foregoing conjecture and the results of energy calculation. By this study, the GPX activity of Se-scFv-B3 was improved and the Site1 preliminarily confirmed to be the substrate binding site based on both theoretical and experimental research. All of these observations will be very useful for further work in this area.2. Preparation of the selenium-containing scFv-D8 with high GPX activity and study of its active siteIn view of the amount limitation of the selection of phage scFvs during screening scFv-B3, the other round of screening was done using GSH-S-DNP as antigen. A new scFv-2D8 with high affinity to GSH analog was acquired and determined to have different sequence from scFv-B3. The gene of scFv-2D8 was cloned into the vector of pPELB, and then induced to express as soluble form in E. coli Rosetta. The yield of purified protein showed high GPX activity of 1684U/μmol after being converted into selenium-containing scFv by chemically modification, which is 400U/μmol higher than that of Se-scFv-B3(1288U/μmol).In order to investigate the catalytic group of this abzyme, a three-dimensional structure of scFv-2D8 is built by means of homology modeling, and the binding site analysis and computer-aided docking are performed on the one hand. Based on the structural information deduced from the theoretically modeled complex, two regions are chosen as candidate binding sites and Ser207 in Site 1 and (or) Ser65 in Site 2 are possible active residue after being converted into Sec. Thereinto, Sec65 is more likely to be the catalytic based on theoretical research. On the other hand, site-directed mutation is carried on, and two scFv mutants S207A (Ser207→Ala207) and S65A (Ser65→Ala65) are expressed and purified. After chemically converting the active Sers into Secs, the GPX activity of the latter one declined remarkably, and that of the anterior one declined at a certain extent. All of these indicated that Sec65 play an important role in the catalytic reaction.3. Discussion on the biosynthesis of seleno-scFv-2D8 in auxotrophic expression systemPreparation of selenium-containing scFv by chemical modification has some disadvantages such as higher cost, lower yield. Moreover, it is incapable of specifically mutating Ser into Sec on the target location, and in some cases, other hydroxyl groups in the protein are inevitably converted into selenols, which will hamper the further structure studies. For solving these problems, a method, which could bioincorporate Sec into the binding site of the scFv by using a cysteine auxotrophic expression system, was attempted to prepare seleno-scFv-2D8. The Cys of the protein expressed in this system will be substituted by Sec when selenocysteine is added to the culture medium to replace the cysteine. Determination of the catalytic group was the precondition of preparing selenium-containing abzyme using this method, and in the foregoing investigation, Sec65 was presumed to be the most possible catalytic group in the abzyme Se-scFv-2D8, and Sec 207 might play important role in it too. So, the Ser65 or Ser207 in the wild-type plasmid containing scFv-2D8 gene is mutated to cysteine, and the two mutated plasmids as well as the wild-type one is expressed respectively in the cysteine auxotrophic expression system. The three scFvs are all successfully expressed and purified from the system, but both the two seleno-mutant did not show high GPX activities compared with seleno-scFv-2D8 (wild-type). In view of the fact that there are four cysteines forming two disulfide bonds in scFv-D8, and the disulfide bond is important in the stability of the protein structure, so the substitution of Sec may result in the structural changes. Molecular simulation studies for the seleno-scFv-2D8 (4Cys→Sec) are performed, and the information from the structural analysis can help us to explain the results above. Although in the seleno-scFv-D8 (4Cys→Sec), the four selenocysteines also formed two diselenide bridges, the bond length of the diselenide bridge is longer than the disulfide bond, thus may result in the change of the structure and the location of the binding site. The results of the binding-site analysis for seleno-scFv-2D8 (4Sec) showed that only one possible binding pocket was found in seleno-scFv-2D8 (4Sec), which is different from that of Se-scFv-2D8, and Ser65 or Ser207 is not the proper candidate of catalytic group anymore. In this study, three seleno-scFv-2D8s are prepared successfully by biological conversion in cysteine auxotrophic expression system, although the GPX activities of them are not famous. And we can deduce a conclusion that when preparing a seleno-mimic of GPX in cysteine auxotrophic expression system, the characters of the protein must be fully considered, and the mimic protein containing multi-cysteines especially disulfide bond except catalytic group may be inappositely expressed in this system.4. Imitation of GPX using selenium-containing single domain antibodyIt is well known that the three hypervariable regions of variable region of heavy chain (VH) and the three ones in light chain compose of the antibody's antigen-binding site together, and in which variable region of heavy chain may be more important. The VH gene of scFv-2D8 is amplified by PCR method and cloned into the vector pPELB. The single domain antibody 2D8-VH is expressed in E.coli Rosetta as soluble form, and purified by IMAC. After introducing Sec by chemical modification, the selenium-containing single domain antibody shows a higher GPX activity. The results indicate that we first successfully imitate GPX using human single domain antibody which has smaller molecular weight, it will lay a foundation for preparing much smaller GPX mimics with higher activity.