Identification and characterization of the lamprey high-mobility group box 1 gene

Abstract

High-mobility group box 1 (HMGB1), a highly conserved DNA-binding protein, plays an important role in maintaining nucleosome structures, transcription, and inflammation. We identified a homolog of HMGB1 in the Japanese lamprey (Lampetra japonica). The Lampetra japonica HMGB1 gene (Lj-HMGB1) has over 70% sequence identity with its homologs in jawed vertebrates. Despite the reasonably high sequence identity with other HMGB1 proteins, Lj-HMGB1 did not group together with these proteins in a phylogenetic analysis. We examined Lj-HMGB1 expression in lymphocyte-like cells, and the kidneys, heart, gills, and intestines of lampreys before and after the animals were challenged with lipopolysaccharide (LPS) and concanavalin A (ConA). Lj-HMGB1 was initially expressed at a higher level in the heart, but after treatment with LPS and ConA only the gills demonstrated a significant up-regulation of expression. The recombinant Lj-HMGB1 (rLj-HMGB1) protein bound double-stranded DNA and induced the proliferation of human adenocarcinoma cells to a similar extent as human HMGB1. We further revealed that Lj-HMGB1 was able to induce the production of tumor necrosis factor-α (TNF-α), a pro-inflammatory mediator, in activated human acute monocytic leukemia cells. These results suggest that lampreys use HMGB1 to activate their innate immunity for the purpose of pathogen defense.

Figure 1. Schematic of the Lj-HMGB1 structural domains.

Figure 2. Sequence alignment of Lj-HMGB1 with HMGB1/2/3 of other species using ClustalX.

The accession numbers of the amino acid sequences extracted from the EXPASY database are as follows: human HMGB1 (P09429); mouse HMGB1 (P63158); chick HMGB1 (Q9PUK9); frog HMGB1 (Q7SZ42); human HMGB2 (P26583); mouse HMGB2 (P30681); chick HMGB2 (P26584); frog HMGB2 (Q32NS7); human HMGB3 (O15347); mouse HMGB3 (O54879); chick HMGB3 (P40618); frog HMGB3 (Q1XCD9); Lj-HMGB1 (Lampetra japonica, HQ615991); Lf-HMGB1 (Lampetra fluviatilis, Q91070); amphioxus HMG1/2 (Q6PUE4); and sea urchin HMG1 (P40644). Identical (asterisk) and similar (colon) residues are indicated. Dashes represent gaps inserted into the alignment.

Figure 3. Phylogenetic relationship of lamprey HMGB1 with other HMGB members.

A phylogenetic tree was constructed based on the amino acid sequences of HMGB from Fig. 2. The number at each node indicates the percentage of bootstrapping after 1000 replications. The bar (0.05) indicates genetic distance.

Figure 4. Lj-HMGB1 mRNA expression is significantly upregulated in gill tissue after treatment with LPS or ConA.

The lamprey HMGB1 mRNA levels were determined using real-time quantitative RT-PCR in various lamprey tissues. Total RNA was extracted from the gills, intestines, heart, lymphocyte-like cells and kidneys of lampreys after stimulation with LPS or ConA. Lamprey GAPDH served as an internal control to calibrate the cDNA template for all of the samples, and PBS served as a negative treatment control. The significant differences (p<0.05) in HMGB1 expression between the challenged groups and the control group are indicated with asterisks.

Figure 5. Expression and purification of recombinant Lj-HMGB1.

A. SDS-PAGE. Total protein was separated by 12% SDS-PAGE under reducing conditions and stained with Coomassie Brilliant Blue R-250. Lane M, low molecular weight protein maker; Lane 1, crude lysate pre-induction; Lane 2, crude lysate post-induction; Lane 3, purified rLj-HMGB1. B. Western blot showing the specificity of the anti-Lj-HMGB1 antibody. Purified rLj-HMGB1 and proteins from crude homogenates of various lamprey tissues were probed with a rabbit anti-Lj-HMGB1 antibody. Lane 1, purified rLj-HMGB1; Lane 2, purified rLj-HBP1 (His-tagged negative control); Lanes 3–6, lymphocyte-like cells (lane 3), crude homogenate of the intestines (lane 4), kidneys (lane 5), gills (lane 6) and heart (lane 7) from L. japonica.

Figure 6. EMSA of lamprey HMGB1 binding to double-stranded polynucleotides.

Double-stranded lamprey GAPDH DNA (100 ng, 101 bp and 12 bp in length) was incubated with purified Lj-HMGB1 protein at various concentrations, and aliquots were taken for electrophoresis on a 2% agarose gel (A) and a 20% native PAGE gel (B). Lane 1, Tris buffer; Lane 2, 10 ng of Lj-HMGB1; Lane 3, 20 ng of Lj-HMGB1; Lane 4, 30 ng of Lj-HMGB1; Lane 5, 30 ng of BSA; Lane 6, 10 ng of human HMGB1. (C). DNA hydrolysis in the presence of rLj-HMGB1. pEGFP-N1 DNA (4730 bp, 100 ng) was hydrolyzed by DNase I in the presence of rLj-HMGB1. Tris buffer (lane 2), BSA (lane 4), rLj-HMGB1 (lane 6) or human HMGB1 (lane 8) were incubated with pEGFP-N1 DNA at a quantitative ratio of 1∶10 in 20 mM Tris-HCl buffer containing 2 mM MgCl₂ (pH 8.0) at room temperature for 10 min. DNase I (0.05 units, TaKaRa) was then added to each sample to hydrolyze pEGFP-N1 DNA at 37 °C for 10 min. Tris buffer (lane 1), BSA (lane 3), rLj-HMGB1 (lane 5) or human HMGB1 (lane 7) incubated with pEGFP-N1 DNA without DNase I served as the controls. The aliquots were analyzed on 1% agarose gels.

Figure 7. Lamprey HMGB1 induced TNF-α production in THP-1 monocytic cells.

THP-1 cells (1×10⁹ cell/L) were incubated with rLj-HBP1 (50 ng/ml), rLj-HMGB1 (50 ng/ml), LPS (50 ng/ml), or human HMGB1 (50 ng/ml) for various lengths of time. Human HMGB1 was 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.

Figure 8. Lamprey HMGB1 induced proliferation of MCF-7 cells.

MCF-7 cells were treated with different concentrations of rLj-HMGB1, Hu-HMGB1, or rLj-HBP1 for 24 h, and MTT assays were used to examine proliferation. Cells incubated with rLj-HBP1 were used as a negative control. “*" indicates a significant increase at p<0.05, “**" indicates a significant increase at p<0.01.

Figure 9. Model of the functions of lamprey HMGB1.

Lamprey HMGB1 has various roles, such as binding DNA, which may maintain DNA structure, and inducing cell proliferation, which may promote the evolutional stability of the parasitic lamprey. After LPS/ConA stimulation, the Lj-HMGB1 gene was up-regulated in gills, which in turn promoted the release of proinflammatory mediators, such as TNF-α, to alert the lampreys to underlying danger and prevent the invasion of bacteria or viruses or other exogenous injuries.