Indentification And Characterization Of Novel Aldo-keto Reductase BmAKR1 And BmAKR2 From Babesia Microti

Babesia microti is a kind of apicomplexan parasites in mammalian red blood cells casuing human babesiosis and is tranmitted by ticks.The clinical presentation also includes hemolysis,headache,fatigue,fever,shaking and anemia.Elderly people,low immunity and immunodeficiency usually suffer with severe infections or death.Current treatment for human babesiosis consists of two drug combinations,atovaquone+azithromycin or quinine+clindamycin,however,therapeutic regimen are associated with significant side effects and drug failures.Alde-keto reductases?AKRs?are a large family of enzymes that occur in nearly all phyla.These enzymes have broad substrate specificity and reduce carbonyl substrates such as:aldehyde,ketone sugar aldehydes,keto-steroids,quinones and lipid peroxidation by-products.AKRs were deemed important drugs target involved in various physiological functions,such as detoxification,antioxidant response and drug metabolism.In order to identify important drug targets,the transciptome sequences of B.microti at erythrocyte stage were analyzed,and we found two important expressed sequence tags of aldo-keto reductase.We obtained the sequence of full-length cDNA of two aldo-keto reductase by RACE-PCR.We name the two aldo-keto reductases as BmAKR1 and BmAKR2,respectively.The full-length BmAKR1 cDNA contained an open reading frame of 2,145 bp in length,which encoded a 714-amino acid polypeptide,isoelectric 6.26.There are 8 introns in this gene in genome compared with R1 genome sequence.The full-length BmAKR2 cDNA contained an open reading frame of 2490 bp in length,which encoded a829-amino acid polypeptide,isoelectric 5.24.There are 4 introns in this gene in genome compared with R1 genome sequence.The coding sequence of BmAKR1 without the putative signal peptide and BmAKR2 functional domain were subcloned into prokaryotic PGEX-4T-1 expression vectors to generate a recombinant BmAKR.Recombinant BmAKR1?rBmAKR1?and recombinant BmAKR-HX?rBmAKR2-HX?with N-terminal GST-tag had a molecular mass of 109 kDa and 66KDa,which coincided with the predicted size of the recombinant BmAKR-GST.rBmAKR was purified from E.coli,and used in the production of antibodies.The result of Western Blot show,using anti-rBmAKR antiboday,respectively,a specific protein band with a molecular weight of approximately 83.5 kDa and 96kDa was detected in iRBCs but not in the un-infected cells.The result of Indirect immunofluorescence?IFA?showed that both BmAKR1 and BmAKR2 were located in the nuclei of B.microti merozoites in mouse RBCs.Recombinant BmAKR was used to detect anti-BmAKR antibody level in serum of mice days post infecting by Enzyme linked immunosorbent assay?ELISA?.The results showed that the two kinds of BmAKR were not the antigen of the host immune response to B.microti.The results of real time PCR?RT-PCR?showed BmAKR1 mRNA relative expression levels were highest at 8 DPI,however BmAKR2 mRNA relative expression levels were highest at 5 DPI.The result of antioxidant test show DCF fluorescence intensity in iRBCs of the 150?M H₂O₂ group was significantly higher than that of the untreated group,which was indicative of augmented ROS production.Furthermore,BmAKR1 expression was 3 fold of the untreated groups,suggesting that BmAKR1 may be involved in implication for anti-oxidation.In order to explore the role of BmAKR1in drug metalism relationship,BmAKR1 mRNA relative expression levels were analyzed after treatment with the four drugs-robenidine,atovaquone,artemisinin,quinine.BmAKR1 relative expression levels in iRBCs were significantly upregulated when exposed to robenidine and atovaquone.Furthermore,the robenidine-induced expression of BmAKR1 was dose-dependent.However,the atovaquone-induced expression of the BmAKR1 showed no discernible differences between the high-dose regimen and the low-dose regimen.The relative expression levels of BmAKR1 showed no significant difference after artemisinin or quinine treatment,respectively.These findings indicate that BmAKR1 may be involved in drugs response.Generally,in present study,we cloned and indentified of two novel aldo-keto reductase from B.microti—BmAKR1 and BmAKR2,both locate in nucleus,butthey have different transcription character.The result of antioxidant test show BmAKR1 has anti-oxidation function.Meanwhile,BmAKR1 is involved in robenidine and atovaquone response.The result show BmAKR1 is an important functional moleculea to explore furtherly.