Meng Gou

rLj-RGD3 induces apoptosis via the mitochondrial-dependent pathway and inhibits adhesion, migration and invasion of human HeyA8 cells via FAK pathway

Ovarian carcinoma is a tumor derived from ovary, which brings relatively higher mortality rate among the fatal gynecological cancers. Recently, lots of studies have concentrated on the anti-tumor effects of Arg-Gly-Asp (RGD) motif containing peptides due to their integrin binding properties. In order to meet the criterion of genetic engineering drugs, a recombinant RGD toxin protein (rLj-RGD3) without a His-tag was cloned from the buccal glands of Lampetra japonica in the present study. After endotoxin removal, the His-tag removed rLj-RGD3 was shown to inhibit the proliferation of HeyA8 cells. According to the confocal microscope, flow cytometry and western blot analysis, rLj-RGD3 could trigger HeyA8 cells apoptosis by changing mitochondrial membrane potential, arrangement of F-actin, protein level of BCL2, BAX, caspase 3, and cleaved caspase 3, concentration of cytoplasmic calcium, as well as phosphorylation level of ERK/JNK/p38. Furthermore, rLj-RGD3 was also able to suppress the adhesion, migration, and invasion processes of HeyA8 cells by disturbing the organization of F-actin and reducing the level of p-FAK. In addition, rLj-RGD3 could inhibit the adhesion of HeyA8 cells to extracellular matrix proteins by competitively binding to integrins, indicated that rLj-RGD3 might act as an anti-tumor drug to treat ovarian carcinoma patients in the future.

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.

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.

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].

Identification and characterization of aldehyde dehydrogenase 9 from Lampetra japonica and its protective role against cytotoxicity

Aldehyde dehydrogenases (ALDHs), which oxidize aldehyde to corresponding acids, play a major role in the detoxification of various endogenous and exogenous aldehydes. In this study, we cloned and characterized ALDH9 (designated LjALDH9) from Arctic lamprey Lampetra japonica. The open reading frame of LjALDH9 was 1566 bp, encoding 521 amino acids with a predicted molecular mass of 55.68 kDa. LjALDH9 protein had a signal peptide and Aldedh domain with the active site Cys315. In addition, LjALDH9 shares high sequence homology with ALDH9 of jawed vertebrates. Real-time quantitative PCR revealed that LjALDH9 was highly expressed in the buccal gland. A reactive LjALDH9 protein was obtained by prokaryotic expression, two-step-denaturing and refolding and affinity purification. During enzyme activity analysis of recombinant LjALDH9, we found that the most suitable reaction conditions were pH7.0, 16-23 °C and Mn(2+) as the activator. Our study provides theoretical proof that LjALDH9 plays an important role in the parasitic life phase of lamprey.

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.

Target-directed isolation and identification of a serum lectin from lamprey (Lampetra japonica) by chromatographys and MALDI-TOF/TOF

A 105-kDa polymer lectin was purified from lamprey (Lampetra japonica) serum by chromatography methods including cation ion-exchange chromatography with a SP-Sepharose™ XL column and size exclusion chromatography with a Superdex 200 column. The target fractions were collected according to the direction of hemagglutinating activity. The results revealed that the active fractions could adsorb on SP-Sepharose column and showed a 280 nm UV absorbance peak corresponding to molecular weights of 105 kDa in the following size exclusion chromatography. The target fractions with hemagglutinating activity were further checked by Native- PAGE and SDS-PAGE. Two single bands at around 105 kDa and 35 kDa were displayed by two electrophoresis methods respectively, indicating that the protein exists as a trimer in solution. After Native-PAGE and SDS-PAGE, two bands were excised from the gels respectively and further identified by MALDI-TOF/TOF as serum lectin (gi: 13094239). The lectin was able to agglutinate rabbit red blood cells (RRBCs) and sheep red blood cells (SRBCs) in vitro. The lectin isolated from lamprey serum in the current study might be helpful for deeply understanding the innate immune molecules dependent immune defence in jawless vertebrates which have been proved recently that they possess a lymphocyte-based system of anticipatory immunity with variable lymphocyte receptors as mediators.

Molecular cloning, expression pattern and phylogenetic analysis of Guanine nucleotide exchange factor Vav2 in lamprey, Lampetra japonica

The Guanine nucleotide exchange factor Vav2 (Vav2) is a member of the Vav family that serves as an important regulators for the Rho family of Ras-related GTPases. In the current study, an ortholog (Lj-Vav2) of Vav2 was identified in the lamprey (Lampetra japonica). To elucidate the phylogenetic relationship of Vav2, the metazoan genome databases were analyzed to mine the ortholog of Vav. It was found that Vav2 genes were only existed in vertebrates and Lj-Vav2 was the original one found in agnathans. The evolutionary dynamics of conserved motifs of Vav2 were explored using combined amino acid sequence as markers, and it is revealed that the Calponin homology (CH) domain, Dbl-homologous (DH) domain, Pleckstrin homology (PH) domain, Cysteine-rich (C1) domains, Src homology 3 (SH3) domains and Src homology 2 (SH2) domain were conserved throughout the Vav2 gene family in vertebrates during gene evolution. Relative quantitative real-time PCR analysis showed that the Lj- Vav2 was distributed in the heart, kidney, supraneural myeloid body, liver, gill and lymphocyte-like cells. The Lj- Vav2 was found to be expressed in these tissues, and the level of which was upregulated in lymphocyte-like cells after the animal was stimulated with LPS. These results indicated that the Lj-Vav2 might be involved in the immune response of lymphocyte-like cells in lamprey. Meanwhile, our findings provided a foundation for further investigation of the function of Lj-Vav2 in the primary vertebrate.