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.

Lampreys and hagfish belong to the class Gnathostomata, which contains extinct and modern jawless vertebrates. As a group of lower vertebrates, Gnathostomata not only share several primitive features, such as the innate immune response system of jawed vertebrates, but also exhibit adaptive immune reactions that involve antigen-specific immunological memory 20 . Although T-cell receptors (TCR) and BCRs are not present in jawless vertebrates, recent findings have revealed that they possess an alternative immune system that specifically recognizes and responds to external pathogens 21 . This alternative immune system uses genomic leucine-rich-repeat (LRR) cassettes for the combinatorial assembly of diverse antigen receptor genes encodinga vast number of variable lymphocyte receptors (VLRs) to resist pathogen invasion 22,23 . To date, three types of antigen receptors, VLRA, VLRB and VLRC, have been identified in lampreys 23 , and recent evidence has indicated that VLRA, VLRB and VLRC are expressed in different lymphocyte subsets that resemble the α β T, B and γ δ T cells of jawed vertebrates, respectively 23,24 . After infection by specific pathogens, VLRB lymphocyte-like cells exhibit increased expression of specific VLRB molecules, and the cells begin to secrete VLRB in a manner analogous to Ig secretion by B cells in jawed vertebrates 25 .
Although BLNK has been extensively studied in jawed vertebrates, little is known about BLNK immune function in jawless vertebrates. In a previous study, we have reported the identification of a BLNK homolog named Lj-BLNK in the lamprey Lampetra japonica 26 . In this study, the cloning and prokaryotic expression of a truncated Lj-BLNK cDNA fragment encoding the SH2 domain, the purification of recombinant Lj-BLNK fragments (rLj-BLNK), and the generation of polyclonal antibodies (PAbs) against rLj-BLNK are described. Using verified PAbs, the expression pattern of Lj-BLNK in immune-relevant tissues and the co-localization of Lj-BLNK and VLRB were investigated by western blotting, fluorescence activated cell sorting (FACS) analysis and immunofluorescence methods after LPS treatment. Here, we sought to demonstrate the role of Lj-BLNK in the VLRB-mediated adaptive immune response in lampreys.

Expression and purification of the lamprey rLj-BLNK protein.
A 336 bp cDNA fragmentof Lj-BLNK encoding the entire SH2 domain was amplified ( Supplementary Fig. S1) and cloned into a pMD19-T vector. A recombinant expression plasmid was reconstructedby inserting the cDNA fragment, which was flanked by XhoI and EcoRI restriction sites and generated by PCR, into the pET-28a (+ ) plasmid, as described in the Methods section. After induction with 0.25 mM IPTG, rLj-BLNK was expressed as a soluble His-tagged fusion protein in E. coli BL21. The purified rLj-BLNK migrated on a 15% SDS-PAGE gel as a single band with a molecular mass of approximately 14 kDa, which was consistent with the molecular mass predicted from the cDNA sequence (Fig. 1). By MALDI-TOF mass spectrometry analysis, the rLj-BLNK peptide mass fingerprints were identified with significant protein scores (p < 0.05) from Mascot server searches. A peptide with m/z of 1671.5 was determined to be K.GTVYNL.R, which is identical to a fragment of the rLj-BLNK protein sequence ( Supplementary Fig. S2). The verified rLj-BLNK was concentrated to 0.5 mg·ml −1 for the generation of a polyclonal antibody.

Titer and specificity analyses of polyclonal antibodies against the truncated rLj-BLNK protein.
Polyclonal antibodies against the truncated rLj-BLNK protein were purified from rabbit antiserum by CNBr-activated Sepharose 4B affinity chromatography. The ELISA results showed that the titer of rabbit anti-rLj-BLNK PAbs was higher than 1: 512,000 ( Fig. 2A). The specificity of the PAbs against Lj-BLNK was determined by western blotting analysis, and the antibody detected both rLj-BLNK and native Lj-BLNK in lymphocyte-like cells (Fig. 2B). Full-length Lj-BLNK was identified as a specific 83 kDa band, which is consistent with the theoretical value (Fig. 2B).

Expression of Lj-BLNK in VLRB + lymphocyte-like cells. To identify Lj-BLNK expression in VLRB +
lymphocyte-like cells, cells from non-treated and LPS-stimulated lampreys were separately incubated with anti-Lj-BLNK and anti-Lj-VLRB Abs and analyzed by FACS. The results showed that Lj-BLNK was expressed in 48% of lymphocyte-like cells (about 74% of VLRB + lymphocyte-like cells) from non-treated lampreys and in 59.9% of lymphocyte-like cells (about 90% of VLRB + lymphocyte-like cells) from LPS-stimulated lampreys (calculated from Fig. 3). The up-regulation of Lj-BLNK expression in VLRB + lymphocyte-like cells after LPS stimulation indicated that Lj-BLNK might be involved in the LPS-induced immune response of lamprey lymphocyte-like cells.

The expression and localization patterns of Lj-BLNK and VLRB in immune-relevant lamprey tissues.
The expression patterns of VLRB and Lj-BLNK were examined by using western blotting of total protein extracted from livers, gills, kidneys, supraneural myeloid bodies and lymphocyte-like cells with or without LPS challenge. VLRB and Lj-BLNK were expressed at a much higher level in liver and the supraneural myeloid bodies than in other tissues (Fig. 4). In the LPS-stimulated group, the highest VLRB and Lj-BLNK expression levels were detected in lymphocyte-like cells and supraneural myeloid bodies, in which they were upregulated approximately 2.5-fold compared to corresponding controls (P < 0.05). The significant up-regulation of VLRB and Lj-BLNK in the same immune-relevant tissues such as the supraneural myeloid bodies and lymphocyte-like cells after challenge with LPS indicates that Lj-BLNK might play an important role in the LPS-induced immune response of the VLRB + subset of lymphocyte-like cells. To verify the cellular localization of VLRB and Lj-BLNK, the distribution of VLRB and Lj-BLNK in lamprey lymphocyte-like cells was further evaluated by confocal laser-scanning microscopy. VLRB and Lj-BLNK were distributed on the cell membrane or in the cytoplasm of the same cells (Fig. 5), thus demonstrating that they are co-expressed in the VLRB + subset of lymphocyte-like cells.

Discussion
In jawed vertebrates, B cell function depends on the ability of BCR to bind an antigen and to effectively induce an efficient biochemical cascade from the cell membrane to the nucleus. Thus, cell morphology is rearranged in the cytosol by cytoskeletal reorganization, and the transcription of new genes is activated in the nucleus to promote cellular differentiation and proliferation. It has been shown that at least three families of protein tyrosine kinases (PTK), including the Src-family of PTKs (including Fyn, Lyn, and Blk), Syk, and Btk, participate in these biochemical events 4 . BLNK was first identified as a new member of the PTK family by Fu et al. who have reported that BLNK interacts with PLCγ , Grb2, and Vav after BCR activation 27 . In jawless vertebrates, instead of the Ig fold-based BCR, lymphocyte-like cells express VLRBs containing LRRs that are similar to TLRs in structure but similar to Abs (secreted VLRB) and BCRs (membrane-anchored VLRB) in function 28 . Although the receptors and Abs in jawless vertebrates are well studied, the types of molecules participating in the cascade reaction from signal recognition to Ab production is still unclear. Interestingly, we have recently identified some PTK homologs (Btk, Syk, Fyn, BLNK, and Vav), PLCγ , and Grb2 in the lamprey L. japonica 26,[29][30][31] . The discovery and validation   of these BCR downstream signaling molecules in lampreys indicate that the intracellular signal transduction mechanisms of lamprey VLRB + lymphocyte-like cells and higher vertebrates B cells appear to be conserved, despite differences in the transmembrane signaling pathways (jawless vertebrates lack certain critical immune mediators, such as Igα and Igβ in the BCR complex).
LPS is a major component of the outer membrane of Gram-negative bacteria and has been used in mammals, amphioxus, fish and lamprey for immunological studies [32][33][34][35] . Consistently with these findings, we observed that the expression levels of VLRB and BLNK were upregulated approximately 2.5-fold in lymphocyte-like cells and supraneural myeloid bodies after stimulation with LPS (Fig. 4). It has been well documented that LPS is a potent activator of mature B cells in jawed vertebrates through Toll-like receptor 4 (TLR4) signaling 13 . However, of the 16 lamprey TLR genes and 4 adaptor molecules in the TLR signaling pathwaythat have been identified in the P. marinus genome by Kasamatsu et al. the critical molecules participating in the B cell response to LPS, including TLR4, TLR9, CD14, MD2, Type I IFN (IFNα /β ), TNF-α , IL-6 and IL-12p40, are absent in lampreys 36 . Their results have excluded the possibility that the TLR4-singnaling pathway is responsible for the activation of lymphocyte-like cells by LPS in lampreys, thus raising the possibility that a VLRB-dependent pathway is functionally related to the LPS-induced immune response of lamprey VLRB + lymphocyte-like cells.
The immunization of lamprey with bacteria, viruses, or mammalian cells induces VLRB lymphocytes to undergo lymphoblastoid transformation, proliferation, and differentiation into plasmacytes that secrete antigenreceptors as multivalent VLRB Abs 37 . During the Ab production process, some signaling molecules such as TCR-like, Syk and B cell adaptor protein are upregulated in lamprey VLRB + lymphocyte-likecells 24 . Consistently with these findings, we observed that Lj-BLNK was notably upregulated in VLRB + lymphocyte-like cells after stimulation with LPS (Fig. 3). At the same time, Lj-BLNK was localizedin the cytoplasm of VLRB + lymphocyte-like cells (Fig. 5). The similar expression pattern of VLRB and Lj-BLNK before and after LPS stimulation indicated that Lj-BLNK might play animportant role in the VLRB-mediated signal transduction process of lamprey VLRB + lymphocyte-like cells after LPS stimulation.
Monoclonal lamprey Abs (VLRBs) selectively bind various human glycans, including monosaccharides, disaccharides, trisaccharides, polysaccharides, and glycoproteins 38,39 . Although the mechanism of LPS activation in lamprey lymphocyte-like cells remains unknown, it is nonetheless possible that VLRB recognizes LPS with the help of adaptors and, in turn, activates VLRB + lymphocyte-like cells. Thus, further studies are still needed to shed light on the VLRB transmembrane signaling pathway and the recruitment of Lj-BLNK and Lj-BLNK-interacting molecules to understand the mechanism by which Lj-BLNK fulfills its function as an immune molecule in the lamprey adaptive immune system.

Animals and stimulation by LPS. Adult lampreys (L. japonica) from the Tongjiang section of the
Heilongjiang River (Tongjiang City, Heilongjiang Province, China) were purchased 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 LPS (Escherichia coli 0111:B4, Sigma-Aldrich, St. Louis, MO) in 0.1 ml PBS, andthe other group of animals (control group) was injected with 0.1 ml PBS only. The animals were immunized at 8-day intervals by four intraperitoneal injections and sacrificed with an intraperitoneal injection of ketamine (70 mg kg −1 ) to harvest the tissues for analysis 29,40 . Ketamine (Alfasan International B.V, Woerden, The Netherlands) was prepared as 100 mg ml −1 in normal saline for use when necessary. This work was approved by the Animal Welfare and Research Ethics Committee of the Institute of Dalian Medical University (Permit Number: SYXK2004-0029), and the methods were performed in accordance with the approved guidelines. Polyclonal antibodies against rLj-BLNK were purified with CNBr-activated Sepharose 4B per the manufacturer's instructions (GE Healthcare, New York, NY, USA). The antibody concentration was adjusted to 1.35 mg ml −1 , and the antibody was stored at −20 °C in PBS. The titer of the antibodies was determined with an enzyme-linked immunosorbentassay (ELISA). The specificity of the polyclonalantibodies was confirmed by western blotting using rLj-BLNK and lysates from lymphocyte-like cells.

Fluorescence-activated cell sorting (FACS) analysis. Lymphocyte-like cells isolated from control and
LPS-stimulated animals were plated intubes and fixed for 20 mins in a 4% paraformaldehyde solution in PBS at room temperature. Then, the cells were permeabilized with 0.1% Triton X-100 at room temperature for 10 mins. After being washed three times, the cells were blocked with normal donkey serum for 1 h and incubated with rabbit anti-rLj-BLNK polyclonal and mouse anti-VLRB monoclonal antibodies (200-fold) in PBS overnight at 4 °C, then washed three times with PBS. The cells were incubated with FITC-conjugated donkey anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA, 500-fold) for 30 mins at 37 °C in the dark followed by three washes with PBS. The cells were suspended with PBS and analyzed on a FACSAria II flow cytometer (BD Biosciences, San Jose, CA, USA). Cells incubated with FITC-conjugated goat anti-rabbit IgG and PE-conjugated goat anti-mouse IgG were used as isotype controls. Data analysis was performed using FlowJo software (Tree Star).
Western blotting. Total proteins were separately isolated from the immune-relevanttissues (livers, gills, kidneys, supraneural myeloid bodies and lymphocyte-like cells) of control and LPS-stimulated animalsusing tissue lysis buffer (Beyotime, Shanghai, China). The protein concentrations were determined using a BCA Protein Assay kit (Beyotime, Shanghai, China). The total protein samples from five lamprey tissues were separated by 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% BSA (Beyotime, Shanghai, China) and separatelyincubated with rabbit anti-rLj-BLNK (200-fold) or mouse anti-VLRB antibodies (200-fold) overnight at 4 °C; this was followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or HRP-conjugated goat anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, USA, 500-fold) 41 . The membranes were developed using an enhanced chemiluminescent substrate (Beyotime, Shanghai, China). Densitometric analysis was performed using Gel-Pro Analyzer software (Exon-Intron, Inc. Loganville, PA, USA). The optical density data in triplicate from three independent experiments were normalized to lamprey β -actin detected with a rabbit polyclonal antibody against human β -actin (Sigma-Aldrich, St. Louis, MO, USA, 5000-fold) and calibrated to the levels in control samples.
Scientific RepoRts | 6:25308 | DOI: 10.1038/srep25308 Immunofluorescence. Lamprey lymphocyte-like cells were fixed and permeabilized as described in the fluorescence activated cell sorting (FACS) analysis section. After being washed three times, cells were blocked with normal goat serum (Beyotime, Shanghai, China) for 1 h and incubated with rabbit anti-Lj-BLNK or mouse anti-VLRB antibodies (200-fold) in PBS overnight at 4 °C, then washed three times with PBS. The cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG and Alexa Fluor 555-conjugated goat anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, USA, 400-fold). After two additional PBS washes, the lymphocyte-like cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO, USA, 200-fold). All of the labeled cells were examined with a confocal laser scanning microscope (LSM710, Zeiss, Oberkochen, Germany) and analyzed using Zeiss ZEN LE software.
Statistical analysis. All of the detection experiments were performed in triplicate, and the data were expressed as the mean ± SEM. The differences between the two groups were analyzed using Student's t-test in the SPSS statistical software package. Differences were considered statistically significant at P < 0.05.