Generation Of A 35-Mer Peptide With Superoxide Dismutase And Glutathione Peroxidase Activities By Genetic Engineering

Reactive oxygen species (ROS) are products of the normal metabolic activities of aerobic living organism and are produced in response to various stimuli. Under normal conditions, there is a balance between the production of ROS and their destruction. In certain pathogenic states the production of ROS is enhanced and the excess ROS damage various biomacromolecules including RNA, DNA, protein, sugars and lipids, and therefore results in ROS-mediated diseases. ROS-related diseases include reperfusion injury, inflammatory process, age-related diseases, neuronal apoptosis, cancer and cataract. In order to scavenge ROS, the living organism has several lines of defense system, including enzymatic and non-enzymatic action. The enzymatic antioxidant system consists of glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD). The non-enzymatic antioxidant system includes vitamine E, ascorbate, glutathione (GSH) and uric acid. Due to their pivotal role in scavenging ROS, the enzymatic antioxidant system could act as promising antioxidant drug. However, GPx has some drawbacks such as solution instability, limited cellular accessibility, immunogenicity, short half-lives, costs of production, and proteolytic digestion. And elevation of some kinds of SOD could cause disorder in organisms when other antioxidant enzymes such as GPx and CAT keep constant. Those factors limit the pharmacological use of the naturally occurring enzymes. Scientists have made a great deal of efforts to study their catalytic mechanisms as well as their relationships between them, and prepared many mimics that have the antioxidant ability. But each one of these mimixcs imitated a single enzyme. Actually, the antioxidant enzymes must cooperate with each other to effectively scavenge reactive oxygen species (ROS) because anyone of them can not scavenge all forms of ROS alone. For example, SOD can scavenge 02-·, but the product, H2O2, may still distroy the biomolecules. And GPx can reduced H2O2 to H2O by oxiditing GSH to GSSG, but it can do nothing to 02-·. In addition, the destroyed biomolecules by ROS are still the berden of cells, and it is very important to repair or scavenge them for cells. GST can repair or scavenge the destroyed biomolecules and regenerate S-thiolated protein destroyed by oxidants. In organism, there is a mini system, which is composed from SOD, GPx, GST and glutathione reductase (GR), and plays roles in antioxidation, detoxification and repair of injury. SOD catalyzes dismutation of 02-·to H2O2, GPx reduces H2O2 to H2O and GST repairs the macromolecules oxidized by ROS and regenerate S-thiolated protein.The available information from structural biology indicates that most proteins arise by limited modifications of preexisting protein scaffolds acquiring novel functional properties by recombination of preexisting modules such as amino acid substitution, insertion or deletion of peptide segments, or fusion of different structural domains. This principle can be exploited in the redesign of existing enzymes for novel efficiently antioxidant functions. Based on this principle and the mimics prepared recently of SOD and GPx, we designed a bifunctional enzyme with activity of GPx and SOD by the methods of genetic engineering. And we further construct a SOD, Gpx and GST trifunctional enzyme model by genetic fusion technology.This project purposes that a 35-mer polypeptide was linked by 17-mer SOD and 15-mer GPx with a 3 amino acid linker. After comparing of homology sequence of nature enzymes, two and three-dimensional structure prediction, and molecular dynamics simulation, we insert the DNA sequence encoding the new 35-mer peptide into the pGEX-2T vector at the C-terminus of the gene encoding Schistosoma japonicum GST (SjGST). The fusion protein is expressed in cysteine auxotrophic expression system with copper ion. Then, we cleavages the fusion protein to obtain the 35-mer peptide by using of the thrombin. Thus, as the SOD activity center, copper is coordinated by histidine residues in N-terminus of 35-mer peptide. Selenocysteine, the catalytic residue of GPX was incorporated instead of the cysteine of the 35-mer peptide C-terminus in cysteine auxotrophic expression system. This new peptide enzyme will possess low-molecular, lack of immunogenic response, longer half-life in the blood, improved cellular permeability and clear structure-functional relationship. We anticipate that the antioxidant enzyme mimics with synergism may hold promise for the treatment of human diseases and possession of potential applications in medicine as potent antioxidants. Design and generation of multifunctional mimics will become main current for development of artificial enzyme field.