Cloning, Expression And Characterization Of The Aldehyde Dehydrogenase 9 And Glutathione Peroxidase 4 From Lamprey

Lampreys are one of the oldest class jawless vertebrates, gained attention with its unique evolutionary status and unique immune system, become the new model when studying the origin and evolution of vertebrate innate immune system and antioxidant system. In this paper, we report for the first time the molecular cloning and characterization of aldehyde dehydrogenase 9(ALDH9) and glutathione peroxidase 4(GPx4) from Japanese lamprey(Lampetra japonica) leukocytes and we analyze the genes by using gene cloning, recombination and bioinformatics methods, the researches laying the groundwork for better understanding protein properties and activity.Aldehyde dehydrogenases(ALDHs), which oxidize aldehyde to corresponding acids, play a major role in the detoxification of various endogenous and exogenous aldehydes. To date, ALDH has been studied in mammals and microorganisms, but it has not been studied in fish and amphibians. In this study, we report for the first time the molecular cloning and prokaryotic expression of aldehyde dehydrogenase 9(LjALDH9) from Lampetra japonica. The open reading frame of LjALDH9 was 1566 bp, encoding 521 amino acids with a predicted molecular mass of 55.68 kDa. LjALDH9 protein had a signal peptide(1-29) and Aldedh domain with the active site Cys315. The predicted molecular mass of LjALDH9 without the signal peptide was 52.92 kDa and the theoretical isoelectric point was 5.57. The recombinant protein was existing in the form of inclusion bodies, it was successfully assayed enzyme activity through denaturing, refolding and purification, the result showed that the most suitable reaction conditions were pH 7.0, 16-23°C and Mn2+ as the activator. Real-time quantitative PCR revealed that LjALDH9 was highly expressed in the buccal gland, it provides theoretical proof that LjALDH9 plays an important role in the parasitic life phase of lamprey.The antioxidant enzyme, glutathione peroxidase(GPx), is capable of reducing complex lipid hydroperoxides, hydrogen peroxide and organic hydroperoxides which can maintain normal ROX level to avoid the oxidative damage of DNA, protein and lipid. In this study, we report for the first time the molecular cloning of glutathione peroxidase 4(LjGPx4) genomic DNA sequences from Lampetra japonica. The open reading frame of LjGPx4 was 477 bp, encoding 158 amino acids with a predicted molecular mass of 18.06 kDa and the theoretical isoelectric point was 7.75. The active site of the enzyme contains a selenocysteine encoded by a TGA termination codon at position 33. The complete genomic sequence was 6541 bp and revealed a six exon-five intron structure. Furthermore, upon in vivo stimulation of lampreys with lipopolysaccharide(LPS), LjGPx4 mRNA was upregulated in leukocytes, showed its involvement in the antibacterial immune response of lamprey, provide evidence for the evolutionary existence of this antioxidant enzyme playing a vital role in innate immune responses in jawless vertebrates.