Gene Cloning, Expression And Biological Activity Of A Peroxiredoxin2Homologue From Lampetra Japonica

Peroxiredoxin (Prx), as a class of antioxidants, is highly conserved in evolution. Prxs are present in all living organisms and possesss various biological functions, including antioxidant activity, immune cytotoxicity and mediation of cell proliferation, differentiation and apoptosis. Lampreys are considered to be the most scientifically accessible model of the remaining jawless vertebrates. Researches in the evolution and development of immunological structures in lamprey may contribute to better understand how vertebrates could have evolved. So far researches have focused mainly on Prxs in fish and mammalian, little is known about the existence of Prx in lamprey. Here, we report the molecular cloning and characterization of a Prx2homologue from L. japonica by analyzing the expressed sequence tags (ESTs) of the buccal gland cDNA library. Our researches may contribute to understand the relationship of Prx2between invertebrates and vertebrates. Furthermore, adult lampreys attach to the host fishes with a sucking mouth and feed on the blood and body fluids. Some compounds in the buccal gland secretion, such as anaesthetic, anticoagulant and vasodilator, are indicated to facilitate parasitic lampreys to prey upon hosts. In the present study, we obtained Prx2gene from L. japonica and this is the first report about Prx gene in lamprey. The expression pattern of Lj-Prx2gene was examined. The antioxidant activity of the recombinant protein was identified.In this paper, a single EST homologous to Prx2was found among the extensive EST sequences from the cDNA library of L. japonica buccal gland. The ORF of Prx2is594bp in length that ends at the termination codon TGA. It encodes a protein of197amino acids with a predicted molecular mass of21.7kDa. The L. japonica Prx2(Lj-Prx2) possesses the main feature of the typical2-Cys Prx given that it contains two highly conserved cysteine residues which are surrounded by two motifs FFYPLDFTFVCPTEI and GEVCPA, respectively. There is no signal peptide in Lj-Prx2. Lj-Prx2shows high sequence similarities (>70%) with Prxl and Prx2in other species. To further analyze the relationship between Lj-Prx2and other vertebrate Prxl and Prx2, a phylogenetic tree was constructed. Although the deduced amino acid sequence of Lj-Prx2shares77%identity with channel catfish NKEF-A, it does not group with the Prx1subfamily. As seen from the sequence comparison and phylogenetic tree, our study demonstrated that Lj-Prx2should be classified into the Prx2subfamily and clustering of Prx2is identical with eukaryotic classification..To determine the expression of Lj-Prx2gene in various tissues, we performed real time quantitative PCR assay with GAPDH as an internal control. Our result demonstrated that Lj-Prx2gene was ubiquitously expressed in a variety of tissues including buccal glands, hearts, livers, kidneys, gills, intestines, RBCs and white blood cells. The strongest expression was detected in RBCs and weakest in white blood cells. Obviously, the level of Lj-Prx2gene expression in RBCs remained at a high level in LPS-stimulated group, indicating that Lj-Prx2can act as a very effective antioxidant to remove oxidative stress in RBCs to protect lamprey from damage by ROS and therefore ensure its own survival.Then the Lj-Prx2gene cloned into pET23b was expressed as a histidine tag fusion protein in Escherichia coli BL21(DE3) with the treatment of IPTG. And we prepared polyclonal antibody against rLj-Prx2. Immunohistochemistry of buccal gland showed that Prx2mainly existed in epithelial cell and dense connective tissue cells. Then we performed western blotting analyses and observed that the polyclonal antibody could recognize both rLj-Prx2and natural Prx2protein in the buccal gland secretion, indicating that the Prx2from buccal gland of L. japonica is secretory. Expression of Prx2in buccal gland secretion may be related to the parasitic habit of lamprey. Due to the fact that during a long-term parasitical state, lamprey is likely to endure ROS generated by metabolic processes and immune defenses from host, we predict that the lamprey buccal gland can secrete an antioxidant to suppress the oxidative assault from host and make lamprey alive.The peroxidase activity of rLj-Prx2was measured by the decrease of H2O2in the reaction mixture with or without DTT. As expected, when DTT was present in the reaction mixture, the rate of H2O2degradation was gradually increased as the concentration of recombinant protein increased. It was higher than the control group without rLj-Prx2. In comparison, the rLj-Prx2had almost no effect on the degradation of H2O2without DTT These results show that the peroxidase activity of rLj-Prx2is thiol-dependent. To evaluate the ability of rLj-Prx2in protecting DNA from oxidative damage, a thiol-dependent MFO system was chosen. In the MFO system, the supercoiled plasmid DNA was converted into nicked form. The rLj-Prx2was found to prevent supercoiled DNA from degrading in a dose-dependent manner, suggesting that rLj-Prx2can act as an antioxidant protein.In conclusion, we have cloned a homologue of the typical2-Cys Prx2from the buccal gland of L. japonica. Lj-Prx2, as a secretory protein, has the ability to remove H2O2and protect DNA from oxidative injury. The potential effects of Prx2on antioxidant defense and counteracting oxidative damage from the host may contribute to better understand how Prx2 could have evolved. Lj-Prx2may play an important role in metabolism and immune defense.