Molecular cloning and functional characterization of Caspase-8 in goldfish (Carassius auratus L.)

Abstract

Apoptosis, a form of programmed cell death, is essential for maintaining homeostasis within the internal environment. Caspase-8, an initiator caspase, plays a pivotal role in activating the caspase cascade during the apoptotic process. This study cloned and expressed Caspase-8 from goldfish, aimed to investigate the role of Caspase-8 in the immune response of fish to bacterial infections, specifically those caused by Aeromonas hydrophila. The Gf-Casp8 gene consists of 1425 base pairs, encoding a protein of 474 amino acids with a molecular weight of 54.55 kDa. Quantitative real-time PCR analysis revealed that Gf-Casp8 is highly expressed in the spleen and kidney, with lower expression levels in the muscle and heart. Moreover, Gf-Casp8 expression was significantly upregulated in kidney leukocytes following stimulation with A. hydrophila and LPS. Post-immunization, both mRNA and protein levels of Gf-Casp8 in the kidney were significantly increased. Subcellular localization analysis indicated that Gf-Casp8 is localized in both the cytoplasm and nucleus. In addition, flow cytometry analysis demonstrated that overexpression of Gf-Casp8 significantly enhances apoptosis in HEK293T cells. These results highlight the critical function of Gf-Casp8 in modulating apoptosis and antibacterial immune responses in goldfish.