The Effect Of Amino Acid Mutations In The Peroxide Oxidoreductase Prx1 Of Babesia Voles On Its Function

Babesia is a kind of apical complex protozoa living in mammalian red blood cells.Pathogens are mainly transmitted through tick bites,blood transfusion,organ transplantation and placenta vertical transmission.The Babesia microti is a human-to-animal Babesia parasite that causes red blood cell lysis,jaundice,hemolytic anemia,palliative fever and renal insufficiency after infection,with a mortality rate of 10%.Babesiosis is considered to be a emerging disease threatening global health,but there is a lack of effective and convenient therapeutic drugs.Therefore,it is necessary to study drugs and their target molecules.Babesia live in oxygen-rich red blood cells and produce a large amount of reactive oxygen species(ROS),which can destroy biological macromolecules.To counteract this effect,the parasite has a series of antioxidant molecules,such as thioredoxin,peroxidoreductase,glutathione and superoxide dismutase.In order to study the potential of antioxidant molecule as a drug target,the full-length gene sequence of peroxidoreductase Prx1 was cloned and recombined in our laboratory.The purified recombinant protein was obtained and its basic function was verified.In this study,the key active sites of the molecule will be mutated and the relationship between its structure and function will be studied.1.The q-PCR method was used to detect the prx1 expression level of Babesia in different days after infection.It was found that the expression level of prx1 increased slowly in the 2-7 days of infection,and then decreased rapidly after reaching the peak on the 8th day.This indicates that the molecule is closely related to the infection process.2.Based on the bioinformatics analysis,the key role of the 50 th cysteine and the 170 th cysteine of prx1 was determined.The primer containing the mutation site was designed to be successfully constructed by the laboratory pET-30a(+)-The BmPrx1 plasmid was used as a template,and PCR was carried out to obtain a mutant plasmid in which the 50 th cysteine,the 170 th cysteine,and the two sites were simultaneously mutated.The three mutant plasmids were transformed into BL21 competent cells for Prokaryotic expression.A higherconcentration of the protein was obtained by purifying the protein using the his tag.In reducing SDS-PAGE,wild-type,C50 S,C170S,C50S-C170 S proteins are all monomeric,with a size of 23 KDa.In non-reducing SDS-PAGE,wild type,170 S is a dimeric form,C50 S and C50SC170 S is in monomeric form,so the 50 th cysteine is critical for the formation of its Prx1 dimer.3.The effect of amino acid mutation on the antioxidant activity of pBluescript plasmid was studied.Fe3+in Chinese MFO system could result in the absence of pBluescript plasmid.Wild-type Prx1 could protect pBluescript plasmid from oxidative damage in MFO system.The ability to protect pBluescript plasmid from oxidative damage was lost after mutation of 50 th cysteine,while 170 th cysteine was lost after mutation.It has no effect on this function.Among the ability of Prx1 to catalyze hydrogen peroxide determined by ferric thiocyanate method,the mutation of 170 cysteine leads to the decrease of reaction rate,the mutation of 170 cysteine and the simultaneous mutation of 50 th and 170 th cysteine lead to the loss of the ability of Prx1 to catalyze hydrogen peroxide,and50 th cysteine is crucial to the ability of Prx1 to catalyze hydrogen peroxide.Prx1 has peroxidase activity,and the activity of cysteine at position 50 and 170 is almost lost after mutation.Therefore,cysteine at both sites is essential for its peroxidase activity.4.Prx1 protein can bind to DNA and protect DNA from degradation by nuclease.Molecular chaperone activity assay:Prx1 protein can inhibit the aggregation of MDH in a concentration-dependent manner.After cysteine 50 and cysteine 170,they could still inhibit the aggregation of MDH.It is possible that the mutation site is not the active site of the molecular chaperone function.In conclusion,These results indicated that peroxide reductase Prx1 of Babesia was closely related to the antioxidant function of the parasite,and had the function of molecular chaperone.Structurally,the 50 th and 170 th cysteines are the key sites of enzyme activity,but they have nothing to do with chaperone function.Cysteine 50 is essential for the formation of Prx1 dimer.These findings are important for elucidating the biological function of the molecule and discovering drug targets.