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Functional characterization and gene expression profile of perforin-2 in starry flounder (Platichthys stellatus)
Kwang-Min Choi1, Dong-Hee Cho1, Min-Soo Joo1
1Department of Marine Biology & Aquaculture, College of Marine Science, Gyeongsang National University, 455, Tongyeong, 650-160, Republic of Korea.
Abstract:
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).
Insights
Starry flounder perforin-2 (PFN2) exhibits antimicrobial functions, defending against bacterial and parasitic infections. This study characterized PsPFN2
Area of Science:
- Immunology
- Marine Biology
- Biochemistry
Background:
- The membrane attack complex/perforin (MACPF) superfamily proteins are crucial for immune defense by forming pores on microbial membranes.
- Perforin-2 (PFN2), a member of the MACPF superfamily, plays a vital role in host defense, with deficiencies linked to increased susceptibility to bacterial infections in humans.
Purpose of the Study:
- To investigate and confirm the antimicrobial function of PFN2 in starry flounder (Platichthys stellatus).
- To characterize the molecular properties, tissue-specific, and pathogen-specific expression patterns of starry flounder PFN2 (PsPFN2).
Main Methods:
- Verified the amino acid sequence of PsPFN2 and performed multiple sequence alignment and phylogenetic analysis to confirm MACPF domain conservation.
- Analyzed tissue-specific and pathogen-specific expression of PsPFN2 mRNA in starry flounder.
- Produced recombinant PsPFN2 protein (rPFN2) using Escherichia coli and tested its antimicrobial activity against various pathogens.
Main Results:
- The PsPFN2 coding sequence comprises 710 residues, with a conserved MACPF domain.
- PsPFN2 mRNA was predominantly found in immune organs (spleen, gills) and showed altered expression upon bacterial or viral infection.
- Recombinant rPFN2 demonstrated significant antibacterial activity against multiple bacterial species and the parasite Miamiensis avidus.
Conclusions:
- Starry flounder PFN2 possesses conserved molecular properties and plays a significant role in the innate immune response against microbial infections.
- PsPFN2 is a key component of the immune system in starry flounder, offering protection against a broad spectrum of pathogens.
