The Read-through Expression Of Se-containing Single Chain Antibody With A Catalytic Triad And Research On Its Biological Effects

As a trace element, Se plays an important role in mammals. Studieshave shown that many diseases were related to selenium deficiency, suchas hypothyroidism, decreased reproductive capacity, cardiovasculardisease and some immune system diseases. In vivo, Se mainly constitutedthe domain of selenoprotein, which plays a biological function. As activecenter of many selenium-containing enzyme, Se involved in the body’s resistance to oxidative damage, as well as participate in immuneresponses. Most of the selenium-containing enzyme carrying Se is theform of selenocysteine （Sec）. Interestingly, selenocysteine is encoded bythe UGA codon and is incorporated into polypeptides co-translationally.Thus, selenocysteine is considered the twenty-first proteinogenic aminoacids. Most of the known selenoprotein are enzymes involved in theoxidation-reduction reaction. In addition to yeast, selenoprotein is presentin the vast majority of in vivo. Common selenium protease including GPx,TrxR and ID, etc. In these mammals selenoprotein, Rotruek firstlydemonstrated that GPx was a selenium-containing enzyme in1973, andSe constituted the active ingredient of the enzyme. So, the research ofselenoproteins was beginning. Currently, GPx is mammalianselenium-containing enzyme which was studied the most in-depth. Thestudies show that the GPx can clear the hydrogen peroxide （H₂O₂） andorganic hydroperoxide efficiently, and it played the important role on the resist oxidative damage.However, natural GPx has some shortcomings such as unstable andpreparation difficulties. So a lot of studies on GPx mimics were carriedout and had been successful. Some GPx mimics were obtained, such assome of the synthesis of small molecule mimics and small molecularweight peptide enzyme.The selenium-containing single chain antibody Se-scFv2F3, a GPxantibody mimics, was obtained by chemical mutation Ser into Sec baseon the hydrophobic cavity theory by our team, and its GPx activity was3394U/μmol. Se-scFv2F3can penetrate the tissue and itsimmunogenicity was low, because its low molecular weight. In addition,Se-scFv2F3can be highly expressed in a prokaryotic system. Especially,the structure of Se-scFv2F3, including to its mutation, can be easilyanalysis using magnetic resonance imaging and X-ray diffraction.Therefore, it is more suitable for use as a drug. Though, there were a variety of methods about preparation of theselenoprotein such as the original peptide synthesis and chemicalmutagenesis, etc. But these methods present the disadvantages of highcost, low yield, and protein denaturation of chemical mutagenesis,especially the lack of express directional property. So the selenoproteinsprepared method use of biological engineering becoming the focus ofattention. In order to find a simple method which can avoid denaturationof selenoproteins, some researchers obtain successfully the selenoproteinby Cys auxotrophic expression system in E. coli. Luo also expressedContaining selenium Se-scFv-2F3successfully by the Cys auxotrophicexpression system, which present higher GPx activity （2359U/μmol）. ButCys auxotrophic expression system needs to mutate the Cys in catalyticsites to other amino acids for keeping the structure of selenoprotein. If theamino acid mutation will bring about the change of selenoproteinstructure, it is too much trouble in the research and application in future. Therefore, the E. coli Sec insertion mechanisms directly was tried inorder to express selenoprotein. Via this system, selenium-containingglutathione-S-transferase （GST） was expressed successfully by our team.Read-through expression of scFv-2F3also held a preliminary attempt,obtained the Se-scFv-2F3. But the expressing quantity is very low, andnot for further dynamic analysis. These two examples illustrate feasibilityof the application in expressing of Se-scFv-2F3.The structure topology studies show that Se is an essential elementof the catalytic reaction of GPx. And Se exerts its biological function byform of Sec. Sec can restore the peroxide in vivo, thereby it can protectcell membranes and other tissues from oxidative damage. Sec must crossthe stop codon UGA, in order to parameter into selenoproteins expression.This means Sec incorporation may be involved to a special regulationmechanism, so that the UGA still maintain its normal function. Inprokaryotes, the Sec insertion mechanism involves one cis-acting element and four trans-acting factors. The cis-acting element is an Sec insertionsequence （SECIS）, which is a special Stem-loop structure and ispositioned at the3’ end of UGA codon. The The four trans-acting factorsare:①SelA: Sec synthase for catalytic Ser;②SelB: specifictranslation factor;③SelC: Ser-tRNAsec to identified UGA;④SelD:Selenophosphate synthetase. The completed of Sec insertion process needthe four trans-acting factors and the SECIS, they help Sec translation bystop codon UGA and expression of selenoproteins. In addition, it isnecessary for the expression of Sec:①Minimum SECIS element, whichis a composition consisting of17nucleotides of the stem-loop structure;②A particular mRNA codon UGA is3’ end of the downstream11nucleotides at the stem-loop structure. So, SECIS element, located at thedownstream of the Sec codon （UGA）, is necessary for expression ofselenoprotein in E. coli. However, the spatial conformation of the targetprotein may be affected, and thus the enzyme activity will be low. The key of obtaining high active GPx mimic Se-scFv-2F3is to findthe position of catalytic site Sec, this position is recognition site of theGPx substrates GSH. In order to find the location, the homologous modelof scFv-2F3was built and the molecular dynamics simulation wasperformed, catalytic site of scFv-2F3imitate GPx is52Ser. Furtheranalysis also found that if the catalytic site Ser52mutation into Sec52,can and the surrounding Phe29and Gln72form similar natural GPxcatalytic triplet （Sec52Gln72and Trp29） structure, so as to stabilize itscatalytic structure and improve the activity. The role of catalytic triad hasbeen verified by E. coli auxotroph expression system to express theSe-scFv-2F3. The GPx activity increased significantly after theintroduction of catalytic triplet （4235U/μmol）. This study introduced GPxcatalytic triad and combined with E. coli Sec insertion mechanism,read-through expressed containing selenium single antibody（Se-scFv-WUQ） with a catalytic triad. The coding sequence of Se-scFv-WUQ was redesigned on the basisof previous research about Sec insertion mechanism and computer theorypredicts. Base on this, the single-chain antibody scFv-2F3was choosed inthe Ser52mutation into Sec52, and to the introduction of heterologousselenoproteins minimal ECIS element in E. coli expression directly. Inorder to ensure the conformation of a minimum SECIS stem-loopstructure, codon GGT of glycine57will be mutated to GGG andassurance a base pairing in the downstream of minimal SECIS stem-loopstructure. Then, the three-dimensional structure of the scFv-2F3after theintroduction of SECIS element was predicted by computer, the resultsshowed that the introduction of SECIS is in protein surface, and more tothe outside relative to the catalytic triplet position.There was only littlechange about the structure of the target protein. Catalytic triad in theenzyme molecule can play an important function of stabling catalyticintermediates and increasing enzyme activity. Thus, the read-through expression vector of selenium-containing scFv-2F3with GPx catalytictriad was built. While the catalytic triad structure was aslo beenintroducted into the read-through expression of the sequence. It not onlycan realize the positioning expression of Sec and catalytic triad, but alsostudy the contribution of the catalytic amino acids in the catalytic triad.This can further reveal the catalytic mechanism of Se-scFv-2F3.It is necessary for expression of Se-scFv-WUQ successfully toco-expression with four trans-acting factors. It also had been proved thefour trans-acting factors played the important role in the expression ofselenoprotein in previous studies. Therefore, we co-transformated theexpression vector and the vector containing the SelA, SelB, SelC genes,the engineering strain of the high expression of selenoprotein wasobtained finally by means of selD in E. coli.The low efficiency of Sec insertion will result in the inefficient of translation initiation, generally, the expression efficiency of Sec inselenoprotein was only1³%of the normal amino acids and often lead tothe low expression of selenoprotein. In order to maximize solubleexpression quantity and easy purification, the expression vector with ahistidine tag and signal peptide of pPelB was used.Se-scFv-WUQ with the catalytic triad was expressed successfully.Because the His6-Tag at C-terminus, and Se-scFv-WUQ was purified viaNi2+metal ion chelation chromatography and proved by Western blotting,the results showed that the Se-scFv-WUQ was expressed successfully.The studies above mentioned established the theoretical and experimentalbasis for the design and efficient expression of simulation enzyme.The enzymatic activity of the Se-scFv-WUQ was assayed in order toevaluate the nature of the enzymatic activity, the results showed a certainGPx activity （872U/μmol）. Moreover, the introduction of catalytic triadof GPx （Sec52, Gln72and Trp29） enhanced GPx activity of Se-scFv-WUQ and verified our theoretical prediction.In order to study its medicinal prospects, the biological effects ofSe-scFv-WUQ were studied. Because mitochondrial is the main place ofROS generation. Once the dynamic balance state of ROS is destructed,mitochondrial damage will occur, and there will be a disease signal. Themore badly in damage, the more seriously in certain diseases. Thus,mitochondria can be as the oxidative damage model to evaluate biologicaleffect of the Se-scFv-WUQ. Damage model of myocardial mitochondrialinduced by Fe2+/Vc free radical was used to research Se-scFv-WUQeffect on the oxidative damage of mitochondria. The results showed thatSe-scFv-WUQ has a certain protective effect on oxidative damage ofmyocardial mitochondrial.Oxidative damage model of human normal liver cell L02inducedH₂O₂also was set up, the protection of damage induced H₂O₂was observed. The results showed that Se-scFv-WUQ have certain protectioneffect on oxidative damage of L02cells, can reduce intracellular MDAgeneration, improve the survival rate, prevent cellular LDH leakage, etc.Se-scFv-WUQ also can maintain good permeability of cell membrane toresistance free radical damage, in order to maintain intracellular GSHlevels in the normal level. Therefore, the research of Se-scFv-WUQprovides a theoretical basis for further study to GPx mimics.Small-molecule GPx mimics also were researched based on interests.The small-molecule GPx mimic6–CySeCD was studied in protectingNIH3T3fibroblasts in mice induced H₂O₂and HaCaT humanimmortalized cutin cells induced by UVB. Results showed that6-CySeCD can obviously protect the oxidative damage of NIH3T3andHaCaT cells.6-CySeCD Can improve the cell survival rate, reduce theMDA content and inhibit ROS generation in the cell.6-CySeCD also have certain effect on HaCaT cell apoptosis inhibition and maintenanceof the cell cycle. As a result,6-CySeCD is expected to become a newkind of antioxidant drugs.