Study On Molecular Modification Of Human Extracellular Superoxide Dismutase

There are three kinds of superoxide dismutase: intracellularsuperoxide dismutase(CuZnSOD), extracellular superoxide dismutase(EC-SOD) and Mn superoxide dismutase(Mn-SOD). EC-SOD is firstisolated from mammal by Stefan L. Marklund. It is the mainSOD-isoenzyme in extracellular matrix. EC-SOD is a tetramericsecretory glycoprotein with high affinity for heparin sulfateproteoglycans. Each subunit has one coper and one zinc atom. Theprimiry structure of EC-SOD contains three parts. The N-terminalregion supports the quaternary structure and contains the consensusfor N-linked glycosylation at Asn89. The C-terminal region mediatesthe binding of EC-SOD to components in extracellular matrix. Thecentral region of human EC-SOD has the activity site and isapproximately 50% identical to the final two-thirds of Cu/Zn-SODand contains all the ligands essential for the coordination of the activesite Cu(II) and Zn(II) ions. The protein is too large for structuredetermination by NMR. Indeed difficulties in crystallization ofEC-SODcaused probably by glycosylation lead to the failure indetermination of EC-SOD's three-dimensional structure by X-raycrystallography.With a wealth of information provided in this field over the lastfew years, the significance of EC-SOD in biology and pathologybegins to be understood. EC-SOD preserves nitric oxide bioactivityand protect against extracellular oxidative damage throughscavenging superoxide,. We can detected EC-SOD activity in plasma,lymph and synovial fluid. It prevents and cure cardiovascular andpulmonary diseases, osteoarthritis and inflammation. In clinictreatment it can be used in operation to cure ischemia-reperfusioninjury, traumatic brain injury and acute respiratory distress syndrome.And it also can protect cells from death or apoptosis which areassociated with aging and some diseases such as Huntingdon's diseaseand Parkinson's disease.Homologous genes recombination is an important mechanism ofgene evolution,and an important method of getting new enzymes inmolecular biology. We reported here the production of a new protein,which is a modified human EC-SOD at the activation site usinghomologous genes recombination technology.Because of their high lecleof homology at the activation sitebetween EC-SOD and CuZnSOD, we replace the activation site ofEC-SOD with that of CuZnSOD's. The mutant protein compromisesthe N-terminal and C-terminal domains of hEC-SOD with activationsite of hCuZnSOD. We constructed the fusion protein using stick-feetmutagenesis technique.During the past 15 years, the methylotrophic yeast Pichia pastorishas been developed into a highly successful system for the productionof a variety of heterologous proteins. All expression vectors have beendesigned as Escherichia coli/P. pastoris shuttle vectors, containing anorigin of replication for plasmid maintenance in E. coli. We selectedvector pPIC9 which undergoes secretery expression of our gene usingthe α-factor secretion signal. The constructed gene was cloned intothe vector, which was introduced into pichia pastoris by yeasttransformation kit. The results indicated that the constructed gene wasexpressed in pichia pastoris successfully.The sample containing fusion protein was applied to ananion-exchange chromatographic column, DEAE-Sepharose, and thefusion protein was eluted in a pulse of 0.5 M NaCl in the buffer. ThenThe sample containing fusion protein was applied to Sephacryl-S200.The fusion protein eluted was indicaded to be designed protein bySDS-PAGE.We also measured the activity of the SOD. The activity is higherthan human EC-SOD, and the protein binds to heparin-sepharose withapproximately the same affinity as hEC-SOD.