Abstract
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.
Highlights
As a lymphocyte subtype of white blood cells, B lymphocytes (B cells) are the principal components of the adaptive immune system and serve various immune functions, such as producing different antibodies and cytokines[1]
We have reported the identification of a B cell linker protein (BLNK) homolog named Lj-BLNK in the lamprey Lampetra japonica[26]
After induction with 0.25 mM isopropyl β -D-thiogalactopyranoside (IPTG), rLj-BLNK was expressed as a soluble His-tagged fusion protein in E. coli BL21
Summary
As a lymphocyte subtype of white blood cells, B lymphocytes (B cells) are the principal components of the adaptive immune system and serve various immune functions, such as producing different antibodies and cytokines[1]. The activation of BCR signaling leads to BLNK phosphorylation, which in turn recruits PLCγ , BTK, growth factor receptor-bound 2 (Grb2), Vav and Nck to the BCR complex[9] and initiates multiple signaling cascades involving kinases (p38mitogen-activated protein kinases (p38), c-Jun N-terminal kinases (JNKs) and extracellular-signal-regulated kinases (ERKs)), GTPases, and transcription factors (nuclear factor of activated T-cells (NFAT))[10,11,12] These reaction cascades lead to changes in cell metabolism, gene expression, and cytoskeletal organization, which can generate many distinct outcomes, including survival, tolerance (anergy), apoptosis, proliferation, and differentiation into antibody-producing cells or memory B cells[1]. We sought to demonstrate the role of Lj-BLNK in the VLRB-mediated adaptive immune response in lampreys
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