Yue Pang

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.

First evidence of protein G-binding protein in the most primitive vertebrate: Serum lectin from lamprey (Lampetra japonica)

The intelectins, a recently identified subgroup of extracellular animal lectins, are glycan-binding receptors that recognize glycan epitopes on foreign pathogens in host systems. Here, we have described NPGBP (novel protein G-binding protein), a novel serum lectin found in the lamprey, Lampetra japonica. RT-PCR yielded a 1005 bp cDNA sequence from the lamprey liver encoding a 334 amino acid secretory protein with homology to mammalian and aquatic organism intelectins. Gene expression analyses showed that the NPGBP gene was expressed in the blood, intestines, kidney, heart, gill, liver, adipose tissue and gonads. NPGBP was isolated by protein G-conjugated agarose immunoprecipitation, and SDS-PAGE analyses showed that NPGBP migrated as a specific band (∼35 and ∼124 kDa under reducing and non-reducing conditions, respectively). These results suggested that NPGBP forms monomers and tetramers. NPGBP gene expression was induced by in vivo bacterial stimulation, and NPGBP showed different agglutination activities against pathogenic Gram-positive bacteria, Gram-negative bacteria and fungi. The induction of NPGBP suggested that it plays an important role in defense against microorganisms in the internal circulation system of the lamprey. When incubated with an unrelated antibody, the specific binding between NPGBP and protein G was competitively inhibited, indicating that NPGBP and the Fc region of Ig bind to the same site on protein G. We thus assume that the tertiary structure of NPGBP is similar to that of the Fc region of Ig. Additionally, NPGBP can effectively promote endothelial cell mitosis. These findings suggest that NPGBP plays a role in the immune defense against microorganisms, and this study represents one of the few examples of the characterization and functional analysis of an aquatic organism intelectin.

Identification and characterization of the lamprey IRF gene

Interferon regulatory factors (IRFs) are named for their ability to bind to and regulate interferon genes when an organism becomes infected with a virus. Numerous studies have revealed the versatile and critical functions of IRFs. In this study, an IRF gene from Lampetra japonica was identified and analyzed using bioinformatic methods. The L. japonica IRF (Lj-IRF) shares high sequence homology with other vertebrate IRFs but low sequence homology with an ascidian IRF-like protein. We also used recombinant Lj-IRF protein (rLj-IRF) to immunize New Zealand rabbits to prepare specific anti-rLj-IRF polyclonal antibodies. Enzyme-linked immunosorbent assays (ELISAs) and Western blotting assays were performed to detect the valence and specificity of the antibody. FACS analysis revealed that the Lj-IRF protein was expressed in approximately 21.14% of leukocytes and 9.60% of supraneural body cells in L. japonica, with immunofluorescence staining indicating a cytoplasmic location. The immunohistochemistry results demonstrated that IRF is distributed in the epithelial cells of the heart, supraneural body, kidneys and gills but is not detectable in intestinals or oral gland tissues. However, the expression of IRF was upregulated in lamprey intestinal tissues upon stimulation with the rLj-HMGB1 protein. Lj-IRF gene expression levels were higher in the rLj-HMGB1-stimulated group than the control group, and the expression level of Lj-IRF was significantly increased in the intestines as determined by quantitative real-time PCR. These results provide a foundation for studying the origin and evolution of the innate immune system in lampreys.

A novel member of B-cell linker protein identified in lamprey, Lampetra japonica.

The B-cell linker protein (BLNK) is an adaptor molecule and plays an important role in signal transduction of B-cell receptor (BCR) and pre-B-cell antigen receptor [1,2]. The BLNK contains a conserved C-terminal Src homology 2 (SH2) domain, a proline-rich region and an N-terminal acidic region [2]. As an adaptor protein, BLNK can bind with some signaling molecules such as Bruton’s tyrosine kinase (BTK), growth factor receptor-bound protein 2 (Grb2), and spleen tyrosine kinases (Syk) through its SH2 domain [3]. When BCR signaling pathway was activated, phosphorylation of BLNK could recruit phospholipase Cg (PLCg), BTK, Grb2, guanine nucleotide exchange factor Vav (Vav) and noncatalytic region of tyrosine kinase adaptor protein (Nck), and regulate downstream signaling pathways [4]. Agnathans, represented by lamprey and hagfish, are the oldest vertebrates currently proved to possess the adaptive immune defenses [5]. Though T-cell receptor and BCR do not exist in jawless vertebrates, recent findings in lamprey have revealed that it possesses an alternative immune system that could specifically recognize and respond to external pathogens [6]. The handling of lamprey (Lampetra japonica) and all experimental procedures were approved by the Animal Welfare and Research Ethics Committee of the Institute of Dalian Medical University (Permit Number: SYXK2004-0029). Adult lampreys were collected from Tongjiang section of the Heilongjiang River (Tongjiang City, China) in December 2012. Adult lampreys (200–220 g in weight) were divided into two groups (20 animals per group): one group animals were immunized with 0.1 mg of LPS (Escherichia coli 0111:B4) (Sigma-Aldrich, St Louis, USA) in 0.1 ml PBS and the control animals were injected with 0.1 ml PBS only. The animals were immunized at 8-day intervals by four intraperitoneal injections. Based on the analysis expressed sequence tags (EST) of the cDNA library constructed with lamprey lymphocyte-like cells by our lab previously, a BLNK ortholog was found using Basic Local Alignment Search Tool (BLAST) in the National Center for Biotechnology Information (http://www. ncbi.nlm.nih.gov/). Total RNA was isolated from lamprey lymphocyte-like cells [7] using RNAiso (TaKaRa, Dalian, China) reagent following the manufacturer instructions, and dissolved in DEPC-treated water and stored at 2808C. First strand 30 and 50 RACE-cDNAs were synthesized from 3 mg of total RNA by Reverse transcriptase M-MLV at 308C for 10 min, 428C for 60 min, 708C for 15 min with the 30-CDS primer and 50-CDS primer and Random 9 mers primer following the manufacturer instructions (TaKaRa). The 30and 50-end sequences of Lj-BLNK were obtained by polymerase chain reaction (PCR) with outer primer, inner primer (TaKaRa) and specific primers (Supplementary Table S1). Taq DNA polymerase (TaKaRa) was used for amplification with the following cycling conditions: 948C for 5 min, followed by 40 amplification cycles at 948C for 30 s, 658C for 30 s, 728C for 2 min and a final extension step at 728C for 10 min. Products were analyzed by electrophoresis in a 2% agarose gel stained with ethidium bromide. The target band of PCR product was isolated and purified, subcloned into a pMD19-T vector using a DNA Ligation kit (TaKaRa) and then subject to DNA sequencing (TaKaRa). Total RNAs were separately extracted from different lamprey tissues including lymphocyte-like cells, gill, heart, liver, intestine, and kidney using RNAiso reagent (TaKaRa), and the total RNAs were treated with DNase I (TaKaRa), and then subject to reverse transcription using PrimeScript RT reagent kit (Perfect Real Time) (TaKaRa). Real-time quantitative PCR experiments were performed with a TaKaRa TP800 Real Time PCR System (TaKaRa) using 2 ml cDNA with 16.8 ml SYBR green PCR mastermix (TaKaRa) and 0.6 ml of each specific primer (Supplementary Table S1). The efficiency of the primers was analyzed in 10-fold serial dilutions of cDNA by calculating the slope of the regression line of the cycle thresholds (Cts) versus the relative concentration of cDNA. The GAPDH of L. japonica was used as an Acta Biochim Biophys Sin 2014, xx: 1–5 |a The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmu027.

A novel protein derived from lamprey supraneural body tissue with efficient cytocidal actions against tumor cells

BackgroundIn previous research, we found that cell secretion from the adult lamprey supraneural body tissues possesses cytocidal activity against tumor cells, but the protein with cytocidal activity was unidentified.MethodsA novel lamprey immune protein (LIP) as defense molecule was first purified and identified in jawless vertebrates (cyclostomes) using hydroxyapatite column and Q Sepharose Fast Flow column. After LIP stimulation, morphological changes of tumor cells were analysed and measured whether in vivo or in vitro.ResultsLIP induces remarkable morphological changes in tumor cells, including cell blebbing, cytoskeletal alterations, mitochondrial fragmentation and endoplasmic reticulum vacuolation, and most of the cytoplasmic and organelle proteins are released following treatment with LIP. LIP evokes an elevation of intracellular calcium and inflammatory molecule levels. Our analysis of the cytotoxic mechanism suggests that LIP can upregulate the expression of caspase 1, RIPK1, RIP3 to trigger pyroptosis and necroptosis. To examine the effect of LIP in vivo, tumor xenograft experiments were performed, and the results indicated that LIP inhibits tumor growth without damage to mice. In addition, the cytotoxic action of LIP depended on the phosphatidylserine (PS) content of the cell membrane.ConclusionsThese observations suggest that LIP plays a crucial role in tumor cell survival and growth. The findings will also help to elucidate the mechanisms of host defense in lamprey.

Cell secretion from the adult lamprey supraneural body tissues possesses cytocidal activity against tumor cells.

The supraneural body was identified in the adult lamprey, and its secretions induced the death of a variety of tumor cells but had no effect on normal cells. The cell secretions from different lamprey tissues were separated, and these secretions killed human tumor cells to varying degrees. The cell secretions induced remarkable cell morphological alterations such as cell blebbing, and the plasma membrane was destroyed by the secretions. In addition, the secretions induced morphological alterations of the mitochondria, cytoskeletal structure, and endoplasmic reticulum, eventually leading to cell death. These observations suggest the presence of a novel protein in the lamprey and the possibility of new applications for the protein in the medical field.

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.

Characterization of the LECT2 gene and its protective effects against microbial infection via large lymphocytes in Lampetra japonica

ABSTRACT Leukocyte cell‐derived chemotaxin 2 (LECT2) is a multifunctional protein of the innate immune system that defends against bacterial infections and chemotactic activity. However, its precise function in lamprey remains unclear. In this study, a novel LECT2 gene was first cloned from Lampetra japonica. The full‐length cDNA sequence of L‐LECT2 consists of a 606‐bp ORF encoding a protein of 201 amino acid residues. L‐LECT2 has greater than 50% sequence identity with its homologs in jawed vertebrates. FACS and immunohistochemistry assays were used to determine that the L‐LECT2 protein was primarily distributed in the intestines and supraneural body tissues of lamprey, also marginally detectable in leukocytes. However, the expression of L‐LECT2 was differentially upregulated in the intestines and heart after treatment with LPS. The recombinant L‐LECT2 resulted in significant promoting migration of the leukocytes in vitro. Our data demonstrate that L‐LECT2 treatment could enhance phagocytosis in lamprey large lymphocytes. Thus, our results suggest that LECT2 can modulate the host defense in lamprey and mediate antibacterial protection against E.coli through large lymphocytes. HighlightsA novel LECT2 gene was cloned from Lampetra japonica.The expression of L‐LECT2 was differentially upregulated in the intestines and heart after treatment with LPS.L‐LECT2 treatment resulted in significant promoting migration of the leukocytes in vitro.L‐LECT2 treatment could enhance phagocytosis and bacterial killing in lamprey large lymphocytes.

Bacterial community composition in the gut content of Lampetra japonica revealed by 16S rRNA gene pyrosequencing

The composition of the bacterial communities in the hindgut contents of Lampetrs japonica was surveyed by Illumina MiSeq of the 16S rRNA gene. An average of 32385 optimized reads was obtained from three samples. The rarefaction curve based on the operational taxonomic units tended to approach the asymptote. The rank abundance curve representing the species richness and evenness was calculated. The composition of microbe in six classification levels was also analyzed. Top 20 members in genera level were displayed as the classification tree. The abundance of microorganisms in different individuals was displayed as the pie charts at the branch nodes in the classification tree. The differences of top 50 genera in abundance between individuals of lamprey are displayed as a heatmap. The pairwise comparison of bacterial taxa abundance revealed that there are no significant differences of gut microbiota between three individuals of lamprey at a given rarefied depth. Also, the gut microbiota derived from L. japonica displays little similarity with other aquatic organism of Vertebrata after UPGMA analysis. The metabolic function of the bacterial communities was predicted through KEGG analysis. This study represents the first analysis of the bacterial community composition in the gut content of L. japonica. The investigation of the gut microbiota associated with L. japonica will broaden our understanding of this unique organism.

The archaic roles of the lamprey NF-κB (lj-NF-κB) in innate immune responses.

Highlightslj‐NF‐&kgr;B gene was identified from lamprey after screening of a leukocytes cDNA library for the first time.We found lj‐NF‐&kgr;B could bind DNA and regulates genes expression.We found the ANKs of lj‐NF‐&kgr;B were the inhibition signal for the RHD of lj‐NF‐&kgr;B.Our research provides a new paradigm of the vertebrate NF‐&kgr;B family origin. ABSTRACT The nuclear factor‐kappa B (NF‐&kgr;B) is a pleiotropic transcription factor regulating the expression of genes involved in various biological processes including the immune response and inflammation. Lamprey is regarded as a key species to provide meaningful clues for understanding the evolution of immune system; nevertheless, no information about lamprey NF‐&kgr;B is reported. Thus, we have characterized a NF‐&kgr;B homolog in lamprey (lj‐NF‐&kgr;B) for the deeper understanding of the role it played in lamprey immune system. The sequence and 3D structure analyses demonstrate that lj‐NF‐&kgr;B contained a Rel homology domain (RHD) and seven ankyrin repeats domains (ANKs), which would exhibit functional similarities to NF‐&kgr;B superfamily proteins. This hypothesis was further proved by experiments. We found that the RHD of lj‐NF‐&kgr;B could interact with a mammalian &kgr;B response element, translocate to the nucleus to modulate gene (IL‐6, IL‐1&bgr; and TNF‐&agr;) expression, and the nuclear localization signals (NLS) was essential for the nuclear translocation. Furthermore, the ANKs of lj‐NF‐&kgr;B are the inhibition signal for the RHD of lj‐NF‐&kgr;B. The present results allow us to surmise that the lj‐NF‐&kgr;B should play a key role in immune response of lamprey, and the function of NF‐&kgr;B has been maintained during evolution.