Cloning and expression analysis of a novel high-mobility group box 2 homologue from Lampetra japonica

Abstract

High-mobility group box 2 (HMGB2) is a nonhistone architectural protein that plays important roles in many biological processes. In this study, we cloned a homologue of the HMGB2 from the lymphocyte-like cells of Lampetra japonica (L. japonica). Sequence analysis reveals that L. japonica HMGB2 contains two highly conserved motifs and shares more than 70 % identity with the homologues from other vertebrate species. Subsequently, Lj-HMGB2 was subcloned into the pET-28a(+) and pIRES2 AcGFP1-Nuc vector and expressed in Rosetta blue (DE3) and Hela cell lines, respectively. The recombinant L. japonica HMGB2 (rLj-HMGB2) with apparent molecular mass of 22 kDa was further purified by His-Bind affinity chromatography. Real-time quantitative PCR indicates that the expression level of Lj-HMGB2 was particularly up-regulated in intestines after challenged with lipopolysaccharide, while up-regulated in lymphocyte-like cells and heart after challenged with concanavalin A in vivo. In addition, rLj-HMGB2 could induce the generation of proinflammatory mediators in the activated human acute monocytic leukemia cell line (THP1), which suggested that Lj-HMGB2 may participate in the immune response of the lampreys.

Fig. 1

ORF and structural schematic of Lj-HMGB2. Nucleotide and deduced amino acid sequences of lamprey HMGB2 ORF (a). Nucleotide (upper line) and amino acid (lower line) sequences are numbered from the initiation of methionine. Stop codon is marked with an asterisk. Peptides corresponding to the single underlined sequence (His₂₇–Lys₄₃) are lysine-rich nuclear localization sequences (NLSs). Three cysteine (Cys₂₃, Cys₄₅ and Cys₁₀₄) residues appear in bold. Structural schematic of Lj-HMGB2 domains (b)

Fig. 2

The expression level of Lj-HMGB2 in different tissues from adult lampreys after LPS or ConA challenge. Real-time quantitative RT-PCR was done with RNA samples from gills, intestines, lymphocyte-like cells, heart and kidneys of L. japonica stimulated with PBS, LPS and ConA. The lamprey GAPDH was used as an internal control to calibrate the cDNA template for all the samples. The significant differences (P < 0.05) in HMGB2 expression between the challenged groups and the blank group were indicated with asterisks (**P < 0.01)

Fig. 3

Lj-HMGB2 gene amplification, rLj-HMGB1 purification and Western blotting. a Amplification of Lj-HMGB2 gene fragment by PCR. b Expression and purification of recombinant six-His-tagged Lj-HMGB2 proteins. Lane M low molecular weight protein marker, lane 1 crude lysate of E. coli before induction, lane 2 lysate of E. coli transfected by Pet28a(+)-Lj-HMGB2 expression vector, lane 3 purified recombinant Lj-HMGB2 protein. c Western blotting analysis of Lj-HMGB2 by rabbit anti-Lj-HMGB2 polyclonal antibodies. Lane 1 L. japonica lymphocyte-like lysate, lane 2 human HMGB1 protein, lane 3 recombinant Lj-HMGB2 protein

Fig. 4

Lamprey HMGB2 induced TNF-α production in THP-1 monocytic cells. THP-1 cells (1 × 10⁹ cell/l) were incubated with rLj-HMGB2, rLj-HMGB1 (50 ng/ml), LPS (50 ng/ml), rLj-HBP1 (50 ng/ml) or human HMGB1 (50 ng/ml) for various lengths of time. Human HMGB1 and LPS were used as a positive control, and rLj-HBP1 was used as a negative control. The concentration of released TNF-α in the culture supernatants was measured by ELISA

Fig. 5

GFP gene transfection results in Hela cells (10 × 10). GFP expression mediated by pIRES2-AcGFP1-Nuc plasmid (b), pIRES2-AcGFP1-Nuc-LjHMGB2 plasmid (d), Hela cells with no plasmid transfected (used as negative control) (f), the corresponding images under bright field (a, c, e)

Fig. 6

Lj-HMGB2 mRNA expression in transfected Hela cells. PCR showing levels of Lj-HMGB2 and GAPDH mRNA expression in transfected Hela cells and in untransfected Hela cells