Expression And Proteomics Of Antioxidant Activity Of Methionine Sulfoxide Reductase A From Triaenodon Obesus

Portein redox is a chemical reaction that exists in living organisms generally and can be induced by physical factors,chemical factors and biological factors.Protein oxidation caused by Reactive Oxygen Species(ROS) is one of the important oxidation forms compare with others.The physiological level of ROS has a positive regulatory effect on cell proliferation and growth,but it will seriously damage DNA,protein,lipid and others biological macromolecules and resulting in loss of cell function and even irreversible damage when exceeds certain threshold value.Sulfur-containing amino acid,methionine(Met) is one of the most easily oxidized amino acids and forms two epimer include S-Methionine-Sulfoxide(S-Met-O) and R-Methionine-Sulfoxide(RMet-O)when it was oxidized.The former can be specifically repaired by methionine sulfoxide reductase A(MsrA).MsrA is expressed in all eukaryote,bacterial and some archaea,and is of great significance for maintaining protein biological homeostasis.Previous studies have found that sharks have higher ROS concentrations in vivo due to their predation habits and physiological structure.In order to ensure that protein are not damaged by excessive oxidation,we speculate that MsrA expressed in the shark is different from other species and has a superior reduction active.Therefore,we selected Triaenodon obesus(The genomic has not sequenced)MsrA(T-MsrA)as the research object to acquired cDNA sequences and explore function by molecular biology,proteomics and bioinformatics.The full-length cDNA sequence of T-MsrA was obtained from the mRNA of the liver of the Triaenodon obesus by reverse transcription-polymerase chain reaction(RT-PCR) and mass spectrometry and other methods.Analysis shows that the sequence is 1207 bp in total length,of which 684 bp is in the open reading frame(ORF) and contains 227 amino acids(including signal peptide).The experimentally expressed protein has a mass of 24.57 kDa(signal peptide is not expressed).The isoelectric point is predicted to be 8.46.At pH = 7.5,the protein secondary structure contains 30% alpha helix,16% beta sheet and 54% random coil.Substrate concentration is 1mM,pH = 7.5,T-MsrA enzyme at 37 ? The activity is 0.21 ?mol / min / mg,and the optimum temperature of the protease is 17 ° C and the optimum pH is 7.5.In vivo experiments found that overexpression of T-MsrA in E.coli(BL21-DE3) will cause significant differences in the expression of the proteome.The differentially expressed proteins include catalase,glutathione-S-transferase,allantoinase,etc.Among them,the expression of peroxidase is down-regulated,it is inferred that T-MsrA has reducing activity in the bacteria and inhibits the expression of endogenous peroxidase.The enrichment analysis of KEGG pathway for differential proteins found that it is related to multiple pathways,such as acetaldehyde acid dicarboxylic acid metabolism,arginine and proline metabolism,etc.It is inferred that T-MsrA overexpression activates the occurrence of one or more enrichment pathways and directly or indirectly inhibits the downregulation of bacterial peroxidase expression.Analysis of the methionine oxidation intensity of the E.coli protein group revealed that the methionine oxidation intensity of the transgenic experimental group was significantly lower than that of the stress group,proving that T-MsrA has reducing activity in bacteria.In vitro,using ?-crystallin A,cytochrome c(Cyt c)and cell protein as reactants co-incubated with T-MsrA(containing vitamin C as enzyme protein reducing agent)at 17? for 2h to analyze the intensity of methionine oxidized peptides after oxidized by UV.The results show that the oxidation intensity of the enzyme protein after repair is significantly reduced,and the oxidation intensity of vitamin C alone is also reduced,but not significant.Further analysis of the tryptic peptide modification of ?-crystallin A and Cyt c,it is inferred that T-MsrA has a certain reducing ability to the oxidation of protein tryptophan residues(Trp),so we synthesized peptides containing Trp but not Met,the results show that the Trp oxidation intensity of the peptide oxidation products after enzyme incubation was significantly lower than that of the oxidation group,confirming our inference.In addition,we found that besides the oxidative modification of protein methionine,UV can also cause other amino acids to undergo various modifications such as Cys beta elimination to form dehydroaniline(Dha),Trp's canine urinate and so on.It is worth noting that in the in vitro redox experiment of T-MsrA,we used vitamin C(Vc) as the reducing agent for the enzyme protein instead of the traditional MsrA reducing agent dithiothreitol(DTT),and obtained the expected results.In the discussion,we found that the activity of T-MsrA in repairing ?-crystallin A in vitro is higher than that of human MsrA in comparison with previous research data.It is speculated that this advantage comes from more Cys in the T-MsrA sequence.In addition,we discuss the use of different statistical algorithms to process omics data to increase the reliability of the data.In the peptide experiment,we also synthesized peptide sequences containing Met,and verified that T-MsrA has reducing activity in vitro from the peptide level.