Immunological functional studies of a putative lipocalin member LcApoM_AGPRP in large yellow croaker (Larimichthys crocea).

Abstract

Lipocalins play crucial roles in immune defense in vertebrates; however, their functional mechanisms in teleost fish remain poorly understood. In this study, the economically important marine fish large yellow croaker (Larimichthys crocea) was used as a model to investigate a novel lipocalin-like protein, LcApoM_AGPRP, and to elucidate its role in immune responses. A putative antibacterial protein, LcApoM_AGPRP, which lacks functional annotation in the genome, was identified in large yellow croaker. LcApoM_AGPRP was highly constitutively expressed in the liver, head kidney, ovary, gill and spermary of healthy large yellow croaker. It expression was significantly upregulated following LPS and Poly(I:C) stimulation, indicating a potential role in innate immune responses. Subcellular localization analysis demonstrated that LcApoM_AGPRP is predominantly localized in lysosomes, mainly distributed in the Golgi apparatus and mitochondria and partially distributed in the endoplasmic reticulum, implying its involvement in organelle-mediated immune defense. Functional assays showed that LcApoM_AGPRP possesses broad-spectrum antimicrobial activity. The recombinant protein exerts its antibacterial effects through multiple mechanisms, including disruption of bacterial membrane integrity, inhibition of bacterial motility, and interference with biofilm formation. Stability assays further confirmed that its antibacterial activity is maintained over a wide range of temperatures (25-100 °C) and salinities (0-40 ‰), highlighting its strong environmental adaptability. These properties suggest that LcApoM_AGPRP has considerable potential for application as an antibacterial agent, aquaculture feed additive, or food preservative. This study provides the first systematic identification and functional characterization of LcApoM_AGPRP, offering new insights into the innate immune system of teleost fish.