Yinglun Han

Lamprey Variable Lymphocyte Receptors Mediate Complement-Dependent Cytotoxicity

An alternative adaptive-immune system is present in the most basal vertebrates—lampreys and hagfish—the only surviving jawless vertebrates. These eel-like fish use leucine-rich repeat–based receptors for Ag recognition instead of the Ig-based receptors used in jawed vertebrates. We report that in Japanese lamprey (Lampetra japonica), variable lymphocyte receptor (VLR)B interacts with C1q and C3 proteins to mediate complement-dependent cytotoxicity for bacteria and tumor cells. The immune-based lysis involves deposition of VLRB and C1q-like protein complex on the surface of target cells, activation of C3, and ultimate disruption of cell wall integrity. The demonstration of functional interaction between VLRB and complement components in lamprey provides evidence for the emergence of cooperative innate and adaptive-immune responses at a pivotal point in vertebrate evolution, before or in parallel with the evolution of Ig-based Abs and the classical complement-activation pathway.

Identification and characterisation of the immune response properties of Lampetra japonica BLNK.

B cell linker protein (BLNK) is a central linker protein involved in B cell signal transduction in jawed vertebrates. In a previous study, we have reported the identification of a BLNK homolog named Lj-BLNK in lampreys. In this study, a 336 bp cDNA fragment encoding the Lj-BLNK Src homology 2 (SH2) domain was cloned into the vector pET-28a(+) and overexpressed in Escherichia coli BL21. The recombinant fragment of Lj-BLNK (rLj-BLNK) was purifiedby His-Bind affinity chromatography, and polyclonal antibodies against rLj-BLNK were raised in male New Zealand rabbits. Fluorescenceactivated cell sorting (FACS) analysisrevealed that Lj-BLNK was expressed in approximately 48% of the lymphocyte-like cells of control lampreys, and a significant increase in Lj-BLNK expression was observed in lampreys stimulated with lipopolysaccharide (LPS). Western blotting analysis showed that variable lymphocyte receptor B (VLRB) and Lj-BLNKwere distributed in the same immune-relevant tissues, and the levels of both were upregulated in supraneural myeloid bodies and lymphocyte-like cells after LPS stimulation. Immunofluorescence demonstrated that Lj-BLNK was localized in VLRB+ lymphocyte-like cells. These results indicate that the Lj-BLNK protein identified in lampreys might play an important role in the VLRB-mediated adaptive immune response.

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.

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.

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.

A Novel Vav3 Homolog Identified in Lamprey, Lampetra japonica, with Roles in Lipopolysaccharide-Mediated Immune Response

Vav guanine nucleotide exchange factor 3 (Vav3), a Rho family GTPase, regulates multiple cell signaling pathways including those of T- and B-cell receptors in vertebrates through mediating the activities of the Rho family members. Whether the lamprey possesses Vav3 homolog and what role it plays in immune response remain unknown. Gene cloning, recombinant expression, antibody production and expression pattern analyses were performed to characterize the lamprey Vav3 in the current study. The lamprey Vav3 is closer to jawed vertebrates’ Vav3 molecules (about 53% identities in general) than to Vav2 molecules of jawless and jawed vertebrates (about 51% identities in general) in sequence similarity. Conserved motif analysis showed that the most distinguished parts between Vav3 and Vav2 proteins are their two Src-homology 3 domains. The relative expression levels of lamprey vav3 mRNA and protein were significantly up-regulated in lamprey lymphocytes and supraneural myeloid bodies after mixed-antigens stimulation, respectively. In addition, lamprey Vav3 were up-regulated drastically in lymphocytes and supraneural myeloid bodies after lipopolysaccharide (LPS) rather than phytohemagglutinin (PHA) stimulation. Lamprey Vav3 distributed in the cytoplasm of variable lymphocyte receptor B positive (VLRB+) lymphocytes, and the number of plasmacytes (VLRB and lamprey Vav3 double positive) in blood lymphocytes also increased after LPS stimulation. Our results proved that lamprey Vav3 was involved in the LPS-mediated immune reaction of lamprey and provided a clue for the further study of the precise role lamprey Vav3 played in the signaling pathway of lamprey VLRB+ lymphocytes.

Preparation, Identfication, and Activity Assay of Lamprey (Lampetra japonica) Natural Intelectins

Intelectins play an important role in innate immune response. In a previous study, lamprey inteletins purified by galactose-Sepharose were inactive and insoluble. Herein, we provided a simple and effective method to purify natural intelectins from the serum of lamprey (Lethenteron japonicum) using proteinG agarose. SDS-PAGE, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and mass spectrometry (MS) were used to analyze the purified proteins. The purified proteins were identified to be lamprey serum lectin and intelectinB. The activity analysis results indicated that the proteins had certain extent agglutination activity. The effective method will be useful to study their immune functions and molecular mechanisms.

Lamprey serum can kill HeLa and NB4 tumor cells.

Nature consists primarily of living materials, many of which are pathogenic microorganisms capable of killing and converting other organisms into copies of themselves [1,2]. For self-defense, eukaryotes have gradually evolved immune systems, including the innate and adaptive immune systems [3–7]. Innate immunity has been found in plants, fungi, and metazoans; while jawed vertebrates have an evolved adaptive immune system in addition to the innate immunity defense [8]. Agnathans, represented by lamprey [9] and hagfish, are the oldest vertebrates that possess the adaptive immune defenses, although whether TCR and BCR exist in jawless vertebrates remains unknown. This adaptive immune system undergoes germline genomic rearrangements of insertion of diverse LRRs to generate various lymphatic receptors (VLRs). Three types of VLRs, VLRA, VLRB, and VLRC, have been identified in lampreys. VLRA and VLRB are expressed in lymphocytes that resemble T cells and B cells of jawed vertebrates, respectively. After being infected by a specific pathogen, B-cell-like lymphocytes increased the expression of VLRB, and VLRB was secreted in a manner analogous to the secretion of immunoglobulins by B cells [10,11]. In this study, we report that lamprey serum can kill HeLa and NB4 cells in vitro. The phenomenon of cell killing in lamprey is different from the traditional cytotoxic effect in jawed vertebrates, and the results might be helpful for the research on the early diagnosis and therapy of neoplastic diseases of human beings. Adult lampreys were collected from the Tongjiang Section of the Heilongjiang River (Tongjiang, China) and housed in sand-lined aquariums at 208C. HeLa and NB4 cells were supplied by College of Life Science, Liaoning Normal University (Dalian, China). HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (SigmaAldrich, St Louis, USA) and NB4 cells were maintained in RPMI 1640 medium (Sigma-Aldrich). Both media were supplemented with 10% fetus bovine serum (Sigma-Aldrich), 100 U/ml penicillin (Sigma-Aldrich), and 100 mg/ml streptomycin (Sigma-Aldrich). Cells were cultured in an incubator humidified with 5% CO2 and 95% air at 378C. Normal adult lamprey (200–220 g in weight) was tailsevered, and blood was drawn into a 10-ml plastic centrifuge tube and allowed to clot at 48C overnight. Serum was separated by centrifugation (1680 g) for 10 min at 48C and was stored in a 1.5-ml centrifuge tube at 2208C. Before use, 5 ml of lamprey serum was dialyzed (molecular weight cut-off, 10 kDa) against phosphate buffered saline (PBS, three changes, a total of 2 l) for 24 h and adjusted to a final protein concentration of 20 mg/ml. Cell death analyses were performed using propidiumiodide (PI; Sigma-Aldrich) staining with subsequent fluorescence-activated cell sorting (FACS) analysis. HeLa and NB4 cells (5 10) were incubated with 20 mg/ml lamprey serum dialyzate for 15 min at room temperature, and PBS was used as a negative control. After that, the cell cultures were centrifuged at 150 g for 5 min, and the cells were collected, washed, and resuspended in 1 ml of cold PBS. The cells were further incubated with 100 ml of PI for 15 min at room temperature, and then 400 ml of 1 annexinbinding buffer was added for flow cytometry (FACSAria II; BD Corporation, New York, USA) analysis. The results showed that incubation of lamprey serum with HeLa and NB4 cells for 15 min caused outer-membrane disruption and led to the formation of blebs (Fig. 1A). At this time, the membrane quality was assessed by monitoring the membrane integrity. There was a higher percentage of cell death for HeLa (98.3%) and NB4 (96.2%) cells incubated with lamprey serum than for HeLa (18.8%) and NB4 (17.6%) cells incubated with PBS (Fig. 1B). Most cell death occurred within a 15-min incubation in lamprey serum (Fig. 2, P , 0.01). For morphological studies on cell death, HeLa and NB4 cell suspensions (5 10 cells/ml in PBS) were centrifuged at 150 g for 5 min, and then cells were collected and resuspended in 200 ml of 20 mg/ml lamprey serum dialyzate (PBS as negative control) at room temperature for 15 min. After that, the cells were fixed with 2.5% glutaraldehyde (Sigma-Aldrich) in PBS for 2 h at room temperature and Acta Biochim Biophys Sin 2014, 46: 623–626 | 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/gmu039. Advance Access Publication 21 May 2014

A novel member of lymphocyte-specific protein tyrosine kinase protein identified in lamprey, Lampetra japonica

Lymphocyte-specific protein tyrosine kinase p56 (Lck) is a member of the Src family of non-receptor protein tyrosine kinase. Lck plays an important role in mediating T-cell receptor (TCR) signal transduction and the development, differentiation, proliferation, and activation of T-cells [1]. Lck contains N-terminus of myristylation sequence, a unique aminoterminus region, followed by Src homology 3 (SH3) and SH2 domains and a C-terminus of tyrosine kinase catalytic domain [2]. Lck can associate with the inner face of the plasma membrane through its myristoyl glycine and palmitoyl cysteines in the amino-terminus [3].Following the myristylation sequence, there is the unique region, a short region of 80 amino acids. This unique region is involved in the interaction of Lck with specific cellular proteins [4,5]. Downstream of this unique region are SH3 and SH2 domains which are involved in protein–protein interactions [6]. The tyrosine kinase domain is the catalytic domain of Lck catalyzing the transfer of the gamma-phosphate from ATP to tyrosine residues in proteins. The catalytic domain of human Lck contains a site of autophosphorylation (Tyr-394), which plays an important role in regulating the protein kinase activity [7]. Agnathans, represented by lamprey and hagfish, are the oldest vertebrates currently identified possessing the adaptive immune defenses [8]. A recent study of jawless vertebrate has provided a clue for the origin of adaptive immune defense. Though TCR and B-cell receptor system do not exist in jawless vertebrates, lamprey has been confirmed to possess an alternative immune system that could specifically recognize and respond to external pathogens [9]. 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. Adult lampreys were purchased from Tongjiang section of the Heilongjiang River (Tongjiang, China) in December. Adult lampreys (200–220 g in weight) were divided into two groups (20 animals per group); one group of animals was immunized with 0.1 mg of lipopolysaccharide (LPS) (Sigma-Aldrich, St Louis, USA) in 0.1 ml phosphate-buffered saline (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 expressed sequence tag analysis of the cDNA library which was constructed with lamprey lymphocyte-like cells in our laboratory, a Lck homolog was found using Basic Local Alignment Search Tool (BLAST) in the National Center for Biotechnology Information (NCBI; http://www. ncbi.nlm.nih.gov/). Total RNA was isolated from lamprey lymphocyte-like cells [10] using RNAiso (TaKaRa Biotechnology, Dalian, China) reagent following the manufacturer instructions, and dissolved in Diethyl pyrocarbonate-treated water and stored at 2808C. First strand 30and 50-RACEcDNAs were synthesized from 5 mg of total RNA by Reverse Transcriptase M-MLV at 308C for 10 min, 428C for 30 min, 708C for 15 min, 958C for 5 min, 48C for 60 min with the 30-coding sequence primer and 50-coding sequence primer and Random 9-mers primer following the manufacturer instructions (TaKaRa Biotechnology). The 30and 50-end sequences of Lj-Lck were obtained by polymerase chain reaction (PCR) with outer primer, inner primer (TaKaRa Biotechnology), and specific primers (Supplementary Table S1). LA Taq DNA polymerase (TaKaRa Biotechnology) was used for amplification with the following cycling conditions: 948C for 3 min, followed by 40 amplification cycles at 948C for 30 s, 558C 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 Acta Biochim Biophys Sin 2014, 46: 820–825 |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/gmu066. Advance Access Publication 25 July 2014