Pufferfish insulin-like growth factor binding protein 5 regulates host antimicrobial immune responses by modulating cytokines expression

Abstract

Insulin-like growth factor binding protein 5 (IGFBP5) plays crucial roles in growth and development, but its immunological functions in teleosts remain poorly understood. This study characterized the molecular features and immune function of IGFBP5 (ToIGFBP5) in pufferfish Takifugu obscurus. Bioinformatic analysis revealed the ToIGFBP5 coding sequence spans 798 bp, encoding a 265-amino acid protein featuring a signal peptide, an IGF-binding domain, and a thyroglobulin type-1 domain, with predicted tertiary structure comprising seven α-helices and seven β-strands. Basal tissue expression profiling via RT-qPCR detected ToIGFBP5 transcripts ubiquitously, with the highest abundance in liver, followed by head kidney, intestine, and gills. Following Vibrio challenge, ToIGFBP5 exhibited rapid, significant upregulation in head kidney and liver tissues. To functionally dissect a predicted heparin-binding motif (HBM), recombinant plasmids expressing wild-type or HBM-deleted protein were constructed. Overexpression of pToIGFBP5-WT prior to bacterial challenge significantly amplified pro-inflammatory cytokines (IL-1β, IL-8, and TNF-α) expression in head kidney and liver at 12 hpi and significantly enhanced bacterial clearance compared to controls. Crucially, these immunomodulatory and antibacterial effects were markedly attenuated in fish overexpressing pToIGFBP5-ΔHBM. These results indicate that ToIGFBP5 acts as an immune mediator in T. obscurus, modulating innate immunity against pathogenic Vibrio species primarily through its HBM-dependent mechanism.