LrHMGB1 Shares Structural Similarities with Human HMGB1, and Its Expression Is Induced in Bacterial Infection, Antiviral Vaccination, and Pathogen-Associated Molecular Patterns Stimulation.

Abstract

The high-mobility group box 1 (HMGB1) protein is a highly conserved nonhistone chromosomal protein ubiquitously present in almost all cell types. In the nucleus, it facilitates DNA repair and replication, V(D)J recombination, stabilization of nucleosome, and in the cytoplasm, it regulates autophagy and apoptosis. In addition to these intracellular functions, HMGB1 also facilitates activation of innate immune responses and plays key roles in host defense. To investigate its role in fish, we cloned and characterized HMGB1 in Labeo rohita (LrHMGB1), the most important freshwater fish species in the Indian subcontinent. The full-length cDNA sequence of LrHMGB1 consisted of 787 bp having an open reading frame of 618 bp encoding 205 amino acids (aa) polypeptide, with an estimated molecular mass of 23.61 kDa and isoelectric point (pI) of 5.96. Motif search of LrHMGB1 revealed two homologous DNA-binding domains, the A-box and B-box comprising 8-78 aa and 94-162 aa, respectively, and a negatively charged acidic C-terminal tail (179-204) that consisted of 26 consecutive aspartic and glutamic acid residues. The amino acids sequence of LrHMGB1 protein and the secondary structure having helix (H) and coils (C) in tandem in the A- and B-box region and only coils in the acidic tail region shared significant similarity with mouse and human HMGB1. In addition to the three prominent motifs (A-box, B-box, and the acidic tail), the site of acetylation (lys27-29), phosphorylation (serine38,41,45,52), methylation (lys43), and oxidation (cysteine44,105) in LrHMGB1 was also conserved across the fish species, mouse, and human. LrHMGB1 was expressed during embryogenesis and was widely expressed in various organs/tissues having highest expression in blood. In response to bacterial infection, antiviral vaccination, and pathogen-associated molecular patterns stimulations, LrHMGB1 gene expression was significantly (p < 0.05) induced in various organs and tissues. These results together suggest an evolutionary conserved structure and function of HMGB1 from fish to human.