Rong Xiao

Low concentrations of the recombinant toxin protein rLj-RGD3 suppress TNF-α-induced human renal carcinoma cell invasion

A hallmark of renal cell carcinoma (RCC) invasion is the degradation of the extracellular matrix (ECM) by the local production of gelatinase enzymes. Matrix metalloproteinase-9 (MMP-9)-induced cancer cell invasion is one of the pivotal steps in cancer metastasis. It has been reported that tumor necrosis factor-α (TNF-α), a regulator of MMP-9, can induce invasion in human renal carcinoma cells. Previous work in our laboratory has shown that rLj-RGD3, a recombinant RGD (Arg-Gly-Asp)-toxin protein from the buccal gland secretion of Lampetra japonica, possesses anti-tumor activity. In this study, we demonstrated that rLj-RGD3 suppressed TNF-α-induced MMP-9 secretion in 786-0 cells (human renal carcinoma cells). To investigate the regulatory effect of rLj-RGD3 on TNF-α-induced MMP-9 secretion, we pre-treated cells with rLj-RGD3. Interestingly, rLj-RGD3 had no significant effect on the constitutive secretion of MMPs. However, low concentrations of rLj-RGD3 decreased TNF-α-induced MMP-9 secretion. Functional studies revealed that rLj-RGD3 induced apoptosis and significantly inhibited the proliferation, migration, and invasion of 786-0 cells. Furthermore, the actin architecture in cells pre-treated with rLj-RGD3 was aggregated and disorganized. Our findings suggest that rLj-RGD3 may be used as a potential drug in renal cancer therapy.

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.

Data for the inhibition effects of recombinant lamprey CRBGP on the tube formation of HUVECs and new blood vessel generation in CAM models

In the present data article, lamprey cysteine-rich buccal gland protein (CRBGP) which belongs to cysteine-rich secretory proteins (CRISPs) family was recombinant and expressed in Rosetta blue cells. After identification, the recombinant protein was purified through affinity chromatograph. The inhibition effects of recombinant lamprey CRBGP (rL-CRBGP) on tube formation of human umbilical vein endothelial cells (HUVECs) and new blood vessel generation in chick chorioallantoic membrane (CAM) models were analyzed. This paper contains data related to research concurrently published in “Anti-angiogenic activities of CRBGP from buccal glands of lampreys (Lampetra japonica)” [1].

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

Cysteine‐rich buccal gland protein suppressed the proliferation, migration and invasion of hela cells through akt pathway

Cysteine‐rich buccal gland protein (CRBGP) as a member of cysteine‐rich secretory proteins (CRISPs) superfamily was isolated from the buccal glands of Lampetra japonica, the blood suckers in the marine. Previous studies showed CRBGP could suppress angiogenesis probably due to its ion channel blocking activity. Whether CRBGP could also affect the activity of tumor cells has not been reported yet. In this study, CRBGP suppressed the proliferation of Hela cells with an IC50 of 6.7 μM by inducing apoptosis. Both microscopic observation and Western blot indicated that CRBGP was able to induce the nuclei shrinking, downregulate the protein level of BCL2 and caspase 3 as well as upregulate the level of BAX in Hela cells, suggested that CRBGP might induce apoptosis of Hela cells in a mitochondrial‐dependent pathway. Furthermore, CRBGP could disturb F‐actin organization, which would finally cause the Hela cells to lose their shape and to lessen their abilities on adhesion, migration and invasion. Finally, CRBGP was shown to reduce the phosphorylation level of Akt, which indicated that CRBGP might inhibit the proliferation and metastasis of Hela cells through Akt pathway. CRBGP, as a voltage‐gated sodium channel blocker, also possesses the anti‐tumor abilities which provided information on the effects and action manner of the other CRISPs.