A novel member of B-cell linker protein identified in lamprey, Lampetra japonica.

Abstract

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.