Qingwei Li

Molecular cloning, expression and antioxidant activity of a peroxiredoxin 2 homologue from Lampetra japonica

Peroxiredoxin (Prx) is a cellular antioxidant protein family that plays important roles in oxidative stress and immune cytotoxicity. In this study, we cloned a homologue of the Prx2 from the buccal gland of Lampetra japonica (L. japonica). L. japonica Prx2 (Lj-Prx2) contained two highly conserved motifs and shared more than 70% identity with the homologs from other vertebrate species. Phylogenetic analysis revealed that Lj-Prx2 is closely related to other available teleost Prx2. The real-time PCR results demonstrated that the Prx2 gene was widely expressed in adult lamprey. In addition, the expression of Prx2 gene was particularly up-regulated in red blood cells (RBCs) after the experimental animals were challenged with lipopolysaccharide (LPS) in vivo. Lj-Prx2 gene was subcloned into the pET23b vector and expressed in Escherichia coli BL21 (DE3). The recombinant L. japonica Prx2 (rLj-Prx2) was purified by using His Bind affinity chromatography. Polyclonal antibody to rLj-Prx2 was generated in New Zealand Rabbit. Western blot analysis showed that the Lj-Prx2 is present in the buccal gland secretion, suggesting the secretory feature of it. The function assays revealed that rLj-Prx2 has the capability to reduce the H2O2 when dithiothreitol (DTT) is used as a reducing equivalent and to protect DNA from oxidative damage. These findings suggested that Lj-Prx2 probably plays an essential role in antioxidant defense in RBCs to keep lamprey alive.

Identification and characterization of the lamprey high-mobility group box 1 gene.

High-mobility group box 1 (HMGB1), a highly conserved DNA-binding protein, plays an important role in maintaining nucleosome structures, transcription, and inflammation. We identified a homolog of HMGB1 in the Japanese lamprey (Lampetra japonica). The Lampetra japonica HMGB1 gene (Lj-HMGB1) has over 70% sequence identity with its homologs in jawed vertebrates. Despite the reasonably high sequence identity with other HMGB1 proteins, Lj-HMGB1 did not group together with these proteins in a phylogenetic analysis. We examined Lj-HMGB1 expression in lymphocyte-like cells, and the kidneys, heart, gills, and intestines of lampreys before and after the animals were challenged with lipopolysaccharide (LPS) and concanavalin A (ConA). Lj-HMGB1 was initially expressed at a higher level in the heart, but after treatment with LPS and ConA only the gills demonstrated a significant up-regulation of expression. The recombinant Lj-HMGB1 (rLj-HMGB1) protein bound double-stranded DNA and induced the proliferation of human adenocarcinoma cells to a similar extent as human HMGB1. We further revealed that Lj-HMGB1 was able to induce the production of tumor necrosis factor-α (TNF-α), a pro-inflammatory mediator, in activated human acute monocytic leukemia cells. These results suggest that lampreys use HMGB1 to activate their innate immunity for the purpose of pathogen defense.

Characterization, phylogenetic analysis and cDNA cloning of natterin-like gene from the blood of lamprey, Lampetra japonica

Lamprey as a living fossil of immunological origin and rich treasure of biological pharmaceutical development has caused attention of scholars. The cDNA library construction and EST sequencing of blood had been done previously in our lab, and bioinformatics analysis provided a gene fragment which is highly homologous with natterin family, named natterin-like. To elucidate the characterization and phylogeny of natterin-like genes in early evolution, we cloned the full-length cDNA of natterin-like gene from the blood of Lampetra japonica. The open reading frame of this sequence contained 942bp and encoded 313 amino acids, including a lectin-like domain and a pore-forming toxin-like domain. Using reverse transcription PCR, natterin-like mRNA was also detected in lamprey blood, kidney, heart, liver, medullary, gonad, but absent in lamprey intestine and gill. Our results suggested that in lampreys and most of other species, there might be only one natterin-like gene, which was fused by certain sequences during evolution and encoded proteins with more functions. It is similar between C terminal of natterin-like protein and Aerolysin in space structure and the lectin-like domain of natterin-like equivalent to glycoprotein binding motif of Aerolysin in function. We also propose that the defense mechanism against specific predators in historical evolution of lamprey. Our findings may provide insights into the function and characterization of natterin-like genes as well as other gene families in vertebrates and provide a foundation for identification and structural, functional, and evolutionary analyses of more natterin-like genes and other gene families.

A novel BTK-like protein involved in immune response in Lethenteron japonicum.

Brutons tyrosine kinase (BTK) plays an important role in the development, differentiation and signal transduction of B-lymphocyte. Lymphocyte-like cells and unique variable lymphocyte receptors have been found in the Japanese lamprey Lethenteron japonicum, an agnatha known to be lacking T or B lymphocyte, and the oldest agantha in existence. In this study a BTK-like gene from L. japonicum was identified and characterized. It contained a 1992-bp open reading frame (ORF), a 157-bp 5-untranslated region (UTR) and a 567-bp 3-UTR. Phylogenetic analysis indicated it had high sequence homology with BTKs from other vertebrates. Real-time PCR analysis showed that the transcript of the L. japonicum BTK-like gene was distributed in various immune associated tissues, and the level of which was upregulated in leukocytes after the animal was stimulated with LPS. This observation was further confirmed by FACS and western blot analyses. These results indicated that the BTK-like gene identified in L. japonicum may play an important role in the immune response of lamprey.

Cloning of Arctic lamprey Lethenteron camtschaticum cd9 with roles in the immune response

In this study, the cd9 gene, a member of the tetraspanin superfamily and involved in various cellular processes, was cloned from Lethenteron camtschaticum. Both real-time PCR and immunohistochemical assays showed broad distribution of cd9 in various L. camtschaticum tissues. In addition, expression levels of Cd9 mRNA were up-regulated in the liver and heart after stimulation by lipopolysaccharide. Flow cytometric analyses demonstrated that cd9 was detected on the leukocytes and that the expression level was higher on granulocytes than on lymphocytes, which implied that cd9 was mainly involved in innate immunity.

A novel CD29-like protein expressed in Japanese lamprey (Lethenteron japonicum) and involved in immune response

CD29 is the integrin beta1 subunit. Integrin family members are membrane receptors involved in cell adhesion and recognition in a variety of processes including embryogenesis, tissue repair and immune response. In this study, a novel CD29-like gene (LCD29) was identified and characterized in Japanese lamprey (Lethenteron japonicum), an agnathan that occupies a critical phylogenic position between cephalochordates and gnathostomes. After a partial cDNA sequence of LCD29 was found from the leucocyte cDNA library, the full-length cDNA was obtained by means of 3 and 5 RACE, respectively. LCD29 encodes 780 amino acids and shares high sequence homology with other vertebrates. Both real-time PCR and immunohistochemical assays have demonstrated the wide distribution of the LCD29 in lamprey tissues, and FACS analysis has shown that the expression level of this protein is higher in granulocytes than in lymphocytes. Furthermore, after the lampreys were stimulated with lipopolysaccharide, the levels of LCD29 mRNA were obviously up-regulated in the leucocytes, intestine, heart and gill tissues. Most importantly, an interaction between LCD29 and LCD9 in the intestine has been found with co-immunoprecipitation assays for the first time. The results obtained in the present study will help us to understand the function of LCD29 in immune response in jawless vertebrates.

Effects of the recombinant toxin protein rLj-RGD3 in multidrug-resistant human breast carcinoma cells.

We have previously reported that Lj-RGD3, a novel RGD-toxin protein, was isolated from the buccal gland of Lampetra japonica. The recombinant protein rLj-RGD3 has anti-invasive and anti-adhesive activity in tumor cells (HeLa cells) and endothelial cells (ECV304 cells) in vitro, and inhibits αvβ3, αvβ5, and β1 integrin-mediated adhesion. In this study, we investigated the bioactivity of rLj-RGD3 in the drug-resistant MCF-7/Adr breast carcinoma cell line and drug-sensitive parental line MCF-7, and found that rLj-RGD3 inhibited the growth of both cell lines. Biological function studies revealed that rLj-RGD3 could induce the apoptosis in MCF-7/Adr, which was more prevalent than that in the drug-sensitive parental line MCF-7. In addition, rLj-RGD3 inhibited the adhesion of MCF-7/Adr cells to fibronectin. Furthermore, rLj-RGD3 prevented invasion of MCF-7/Adr cells through an artificial matrigel basement membrane. In summary, rLj-RGD3 may be used as a potential drug in multidrug-resistant breast cancer therapy.

Identification and characterisation of the anti-oxidative stress properties of the lamprey prohibitin 2 gene.

The highly conserved protein prohibitin 2 (PHB2) has been implicated as a cell-surface receptor in the regulation of proliferation, apoptosis, transcription, and mitochondrial protein folding. In the present study, we identified a Lampetra morii homologue of PHB2, Lm-PHB2, showing greater than 61.8% sequence identity with its homologues. Phylogenetic analysis indicated that the position of Lm-PHB2 is consistent with lamprey phylogeny. Expression of the Lm-PHB2 protein was nearly equivalent in the heart, liver, kidneys, intestines, and muscles of normal lampreys. However, the Lm-PHB2 protein was down-regulated in the myocardia of lampreys challenged for 5 days with adriamycin (Adr), followed by a significant up-regulation 10 days after treatment. Inxa0vitro, recombinant Lm-PHB2 (rLm-PHB2) protein could significantly enhance the H2O2-induced oxidative stress tolerance in Chang liver (CHL) cells. Further mechanism studies indicated that the nucleus-to-mitochondria translocation of Lm-PHB2 was closely involved in the oxidative stress protection. Our results suggests that the strategies to modulate Lm-PHB2 levels may constitute a novel therapeutic approach for myocardial injury and liver inflammatory diseases, conditions in which oxidative stress plays a critical role in tissue injury and inflammation.

Identification and characterisation of ROS modulator 1 in Lampetra japonica.

Reactive oxygen species (ROS) are a heterogeneous group of highly reactive molecules that oxidise targets in biological systems. ROS are also considered important immune regulators. In this study, we identified a homologue of reactive oxygen species modulator 1 (Romo1) in the Japanese lamprey (Lampetra japonica). The L japonica Romo1 (Lj-Romo1) gene shares high sequence homology with the Romo1 genes of jawed vertebrates. Real-time quantitative PCR demonstrated the wide distribution of Lj-Romo1 in lamprey tissues. Furthermore, after the lampreys were stimulated with lipopolysaccharide (LPS), the level of Lj-Romo1 mRNA was markedly up-regulated in the liver, gill, kidney, and intestine tissues. Lj-Romo1 was localised to the mitochondria and has the capacity to increase the ROS level in cells. The results obtained in the present study will help us to understand the roles of Romo1 in ROS production and innate immune responses in jawless vertebrates.

Identification and characterization of a novel IκB-ɛ-like gene from Lamprey (Lampetra japonica) with a role in immune response

Nuclear factor of kappa B (NF-κB) is a stimuli-activated transcription factor, regulates the expression of a diverse array of genes. Inhibitor of kappa B-epsilon (IκB-ε) is an inhibitor of NF-κB, which retains NF-κB in an inactive state in the cytoplasm. Lampreys (Lampetra japonica) belong to the lowest class of vertebrates with little information about its IκBs. We have identified a cDNA sequence IκB-ε-like in the lamprey and the deduced amino acid sequence of IκB-ε-like. It contains a conserved DSGxxS motif and six consecutive ankyrin repeats, which are necessary for signal-induced degradation of the molecule. Phylogenetic analysis indicated it had high sequence homology with IκB-εs from other vertebrates. FACS analysis showed that IκB-ε-like located in cytoplasm of leukocytes. The degradation of IκB-ε-like could be observed in leukocytes of L. japonica stimulated with lipopolysaccharide. These results indicate that IκB-ε proteins are conserved across vertebrates and the NF-κB-like signaling pathway may exist in the oldest agnatha.