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Cloning and characterization of the trout perforin
Sofia Athanasopoulou1, Dimitra Marioli, Angeliki Mikrou
1Department of Biology, School of Medicine, University of Patras, Rion 26 500 Patras, Greece.
Abstract:
The pore-forming protein, perforin is one of the effectors of cell-mediated killing. A perforin cDNA clone was isolated from rainbow trout (Oncorhynchus mykiss) after screening of a spleen cDNA library. The full-length cDNA is 2070 bp in size, encoding for a polypeptide of 589 amino acids. The predicted amino acid sequence of the trout perforin is 64, 58 and 40% identical to those of Japanese flounder, zebrafish and human perforins, respectively. Although its membrane attack complex/perforin (MACPF) domain is conserved, trout perforin shows low homology to human and trout terminal complement components (C6, C7, C8 and C9), ranging from 19 to 26% identity. Expression analysis reveals that the trout perforin gene is expressed in the blood, brain, heart, kidney, intestine and spleen. Phylogenetic analysis of proteins which belong to the MACPF superfamily clusters the trout perforin in the same group with other known perforins.
Insights
Researchers identified and characterized the rainbow trout perforin gene, a key protein in cell-mediated killing. This finding advances our understanding of immune responses in fish.
Area of Science:
- Immunology
- Molecular Biology
- Fish Science
Background:
- Perforin is a crucial pore-forming protein effector in cell-mediated cytotoxicity.
- Understanding perforin's role is vital for fish immunology and disease resistance.
Purpose of the Study:
- To isolate and characterize the perforin gene from rainbow trout (Oncorhynchus mykiss).
- To analyze the sequence, homology, and expression patterns of trout perforin.
- To determine the phylogenetic relationship of trout perforin within the MACPF superfamily.
Main Methods:
- Screening of a rainbow trout spleen cDNA library to isolate the perforin gene.
- Full-length cDNA sequencing and amino acid sequence prediction.
- Homology analysis with known perforin and complement proteins.
- Gene expression analysis across various trout tissues.
- Phylogenetic analysis of MACPF superfamily proteins.
Main Results:
- A full-length cDNA of 2070 bp encoding a 589-amino acid polypeptide was identified.
- Trout perforin shares 64%, 58%, and 40% amino acid identity with Japanese flounder, zebrafish, and human perforins, respectively.
- The membrane attack complex/perforin (MACPF) domain is conserved, but homology to complement components (C6-C9) is low (19-26%).
- The trout perforin gene is expressed in blood, brain, heart, kidney, intestine, and spleen.
- Phylogenetic analysis places trout perforin within the perforin cluster of the MACPF superfamily.
Conclusions:
- The identified rainbow trout perforin is a functional homolog of perforin found in other vertebrates.
- Its conserved MACPF domain suggests a similar pore-forming function in cytotoxic pathways.
- Differential expression across tissues indicates a systemic role in immune surveillance and response.
- Phylogenetic clustering confirms its evolutionary relationship with other perforins.
