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Updated: Sep 22, 2025
Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection
Published on: July 21, 2022
Molecular characterization, antiviral activity, and UV-B damage responses of Caspase-9 from Amphiprion clarkii
H M V Udayantha1, Anushka Vidurangi Samaraweera1, D S Liyanage1
1Department of Marine Life Sciences & Fish Vaccine Research Center, Jeju National University, Jeju, 63243, Republic of Korea.
Abstract:
Apoptosis plays a vital role in maintaining cellular homeostasis in multicellular organisms. Caspase-9 (casp-9) is one of the major initiator caspases that induces apoptosis by activating downstream intrinsic apoptosis pathway genes. Here, we isolated the cDNA sequence (1992 bp) of caspase-9 from Amphiprion clarkii (Accasp-9) that consists of a 1305 bp coding region and encodes a 434 aa protein. In silico analysis showed that Accasp-9 has a theoretical isoelectric point of 5.81 and a molecular weight of 48.45 kDa. Multiple sequence alignment revealed that the CARD domain is located at the N-terminus, whereas the large P-20 and small P-10 domains are located at the C-terminus. Moreover, a highly conserved pentapeptide active site (296QACGG301), as well as histidine and cysteine active sites, are also retained at the C-terminus. In phylogenetic analysis, Accasp-9 formed a clade with casp-9 from different species, distinct from other caspases. Accasp-9 was highly expressed in the gill and intestine compared with other tissues analyzed in healthy A. clarkii. Accasp-9 expression was significantly elevated in the blood after stimulation with Vibrio harveyi and polyinosinic:polycytidylic acid (poly I:C; 12-48 h), but not with lipopolysaccharide. The nucleoprotein expression of the viral hemorrhagic septicemia virus was significantly reduced in Accasp-9 overexpressed fathead minnow (FHM) cells compared with that in the control. In addition, other in vitro assays revealed that cell apoptosis was significantly elevated in poly I:C and UV-B-treated Accasp-9 transfected FHM cells. However, H248P or C298S mutated Accasp-9 significantly reduced apoptosis in UV-B irradiated cells. Collectively, our results show that Accasp-9 might play a defensive role against invading pathogens and UV-B radiation and H248 and C298 active residues are significantly involved in apoptosis in teleosts.
Insights
Amphiprion clarkii caspase-9 (Accasp-9) plays a key role in apoptosis and immune defense against pathogens and UV-B radiation. Specific active residues, H248 and C298, are crucial for Accasp-9-mediated apoptosis in teleosts.
Area of Science:
- Molecular Biology
- Immunology
- Cell Biology
Background:
- Apoptosis is essential for cellular homeostasis in multicellular organisms.
- Caspase-9 (casp-9) is a critical initiator caspase in the intrinsic apoptosis pathway.
- Understanding fish caspase-9 function is vital for aquatic immunology and disease resistance.
Purpose of the Study:
- To isolate and characterize the caspase-9 cDNA from Amphiprion clarkii (Accasp-9).
- To investigate the expression patterns and functional role of Accasp-9 in immune response and apoptosis.
- To identify key active residues involved in Accasp-9 function.
Main Methods:
- cDNA isolation and sequencing of Accasp-9.
- In silico analysis of protein properties (isoelectric point, molecular weight, domain structure).
- Phylogenetic analysis to determine evolutionary relationships.
- Gene expression analysis in different tissues and after immune stimulation (Vibrio harveyi, poly I:C, LPS).
- Functional assays using Accasp-9 overexpressed in fathead minnow (FHM) cells, including viral challenge and treatment with poly I:C and UV-B radiation.
- Site-directed mutagenesis of putative active residues (H248, C298) to assess their role in apoptosis.
Main Results:
- The Accasp-9 cDNA sequence (1992 bp) encodes a 434 amino acid protein with conserved caspase domains (CARD, P-20, P-10) and active sites (QACGG, H248, C298).
- Accasp-9 showed high expression in the gill and intestine and was significantly upregulated in blood following Vibrio harveyi and poly I:C stimulation.
- Overexpression of Accasp-9 in FHM cells reduced viral nucleoprotein expression and enhanced apoptosis upon poly I:C and UV-B treatment.
- Mutations in H248 or C298 significantly diminished Accasp-9-induced apoptosis in UV-B irradiated cells.
Conclusions:
- Accasp-9 plays a significant role in the innate immune response of Amphiprion clarkii against bacterial pathogens and UV-B radiation.
- The identified active residues H248 and C298 are critical for Accasp-9's pro-apoptotic function in teleosts.
- This study provides insights into the molecular mechanisms of apoptosis and immune defense in fish.
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