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The membrane attack complex/perforin (MACPF) superfamily consists of multifunctional proteins that form pores on the membrane surface of microorganisms to induce their death and have various immune-related functions. PFN2 is a perforin-like protein with an MACPF domain, and humans with deficient PFN2 levels have increased susceptibility to bacterial infection, which can lead to fatal consequences for some patients. Therefore, in this study, we confirmed the antimicrobial function of PFN2 in starry flounder (Platichthys stellatus). The molecular properties were confirmed based on the verified amino acid sequence of PsPFN2. In addition, the expression characteristics of tissue-specific and pathogen-specific PsPFN2 mRNA were also confirmed. The recombinant protein was produced using Escherichia coli , and the antimicrobial activity was then confirmed. The coding sequence of PFN2 (PsPFN2) in P. stellatus consists of 710 residues. The MACPF domain was conserved throughout evolution, as shown by multiple sequence alignment and phylogenetic analysis. PsPFN2 mRNA is abundantly distributed in immune-related organs such as the spleen and gills of healthy starry flounder, and significant expression changes were confirmed after artificial infection by bacteria or viruses. We cloned the MACPF domain region of PFN2 to produce a recombinant protein (rPFN2) and confirmed its antibacterial effect against a wide range of bacterial species and the parasite (Miamiensis avidus). • Perforin-2 (PFN2) from starry flounder (Platichthys stellatus) was identified. • PsPFN2 was highly expressed in the spleen, gills and PBLs of healthy starry flounder. • PsPFN2 mRNA expression levels were significantly regulated by S. parauberis and VHSV. • We confirmed the antimicrobial activities of rPsPFN2 against bacteria and M. avidus. [ABSTRACT FROM AUTHOR]

The membrane attack complex/perforin (MACPF) superfamily consists of multifunctional proteins that form pores on the membrane surface of microorganisms to induce their death and have various immune-related functions. PFN2 is a perforin-like protein with an MACPF domain, and humans with deficient PFN2 levels have increased susceptibility to bacterial infection, which can lead to fatal consequences for some patients. Therefore, in this study, we confirmed the antimicrobial function of PFN2 in starry flounder (Platichthys stellatus). The molecular properties were confirmed based on the verified amino acid sequence of PsPFN2. In addition, the expression characteristics of tissue-specific and pathogen-specific PsPFN2 mRNA were also confirmed. The recombinant protein was produced using Escherichia coli , and the antimicrobial activity was then confirmed. The coding sequence of PFN2 (PsPFN2) in P. stellatus consists of 710 residues. The MACPF domain was conserved throughout evolution, as shown by multiple sequence alignment and phylogenetic analysis. PsPFN2 mRNA is abundantly distributed in immune-related organs such as the spleen and gills of healthy starry flounder, and significant expression changes were confirmed after artificial infection by bacteria or viruses. We cloned the MACPF domain region of PFN2 to produce a recombinant protein (rPFN2) and confirmed its antibacterial effect against a wide range of bacterial species and the parasite (Miamiensis avidus). • Perforin-2 (PFN2) from starry flounder (Platichthys stellatus) was identified. • PsPFN2 was highly expressed in the spleen, gills and PBLs of healthy starry flounder. • PsPFN2 mRNA expression levels were significantly regulated by S. parauberis and VHSV. • We confirmed the antimicrobial activities of rPsPFN2 against bacteria and M. avidus. [ABSTRACT FROM AUTHOR]

Cathepsin Z (CTSZ) is a lysosomal cysteine protease that is known to be involved in the maintenance of homeostasis and the biological mechanisms of immune cells. In this study, we have confirmed the tissue specific expression of the cathepsin Z (PmCTSZ) gene in Pagrus major , and confirmed its biological function after producing recombinant protein using Escherichia coli (E. coli). Multiple sequence alignment analysis revealed that the active site of the cysteine proteases and three N -glycosylation sites of the deduced protein sequence were highly conserved among all of the organisms. Phylogenetic analysis revealed that PmCTSZ was included in the clusters of CTSZ and the cysteine proteases of other bony fish and is most closely related to Japanese flounder CTSZ. PmCTSZ was distributed in all of the tissues from healthy red sea bream that were used in the experiment and was most abundantly found in the spleen and gill. Analysis of mRNA expression after bacterial (Edwardsiella piscicida : E. piscicida and Streptococcus iniae : S. iniae) or viral (red seabream iridovirus: RSIV) challenge showed significant gene expression regulation in immune-related tissues, but they maintained relatively normal levels of expression. We produced recombinant PmCTSZ (rPmCTSZ) using an E. coli expression system and confirmed the biological function of extracellular rPmCTSZ in vitro. We found that bacterial proliferation was significantly inhibited by rPmCTSZ , and the leukocytes of red sea bream also induced apoptosis and viability reduction. • A cathepsin Z (CTSZ) was identified from red sea bream (Pagrus major). • PmCTSZ share high conserved cysteine proteases active site and three N-glycosylation sites with that of other species. • PmCTSZ mRNA was highly expressed in spleen and gill from healthy condition. • RPmCTSZ inhibited bacterial growth and induced leukocyte apoptosis. [ABSTRACT FROM AUTHOR]