Molecular characterization and functional analysis of Granzyme B in large yellow croaker (Larimichthys crocea).

Abstract

This study successfully cloned and functionally characterized the granzyme B gene (LcGzmB) in large yellow croaker (Larimichthys crocea, Lc), elucidating its dual regulatory roles in inflammatory modulation and antimicrobial defense. Comprehensive bioinformatic analyses revealed that LcGzmB harbors a canonical Tryp-SPc serine protease domain, shares the closest phylogenetic relationship with teleost GzmB orthologs, and exhibits high structural conservation with human GzmB. Tissue distribution analysis showed constitutive LcGzmB expression predominantly in the spleen under physiological conditions, while bacterial challenge with Pseudomonas plecoglossicida induced significant transcriptional upregulation in multiple immune organs (head kidney, spleen, gill, skin, and liver), with peak expression at 12 h post-infection. Integrated metabolomic and mechanistic studies demonstrated that recombinant LcGzmB (rLcGzmB) profoundly modulates lipid metabolic networks, particularly through suppressing the secretion of linoleic acid derivatives (e.g., 9 S-HODE, an endogenous ligand and activator of PPARγ). Functionally, LcGzmB orchestrates pro-inflammatory responses through a dual mechanism: inhibition of PPARγ-mediated anti-inflammatory signaling and potentiation of NF-κB activation. This immunomodulatory cascade culminates in increased expression of key pro-inflammatory cytokines (LcIL-1β, LcIL-6, LcIL-8, and LcTNF-α1), thereby augmenting host immune defenses. Collectively, these findings provide the first comprehensive evidence in teleost fish of GzmB-mediated immunometabolic regulation, offering novel evolutionary insights into granzyme-dependent immunity across vertebrates.