2. Academic Validation
  3. The self-activation and LPS binding activity of executioner caspase-1 in oyster Crassostrea gigas

The self-activation and LPS binding activity of executioner caspase-1 in oyster Crassostrea gigas

  • Dev Comp Immunol. 2017 Dec:77:330-339. doi: 10.1016/j.dci.2017.09.002.
Guangxia Lu 1,  Zichao Yu 2,  Mengmeng Lu 2,  Dongyang Liu 2,  Feifei Wang 2,  Yichen Wu 2,  Yu Liu 2,  Chao Liu 2,  Lingling Wang 1,  Linsheng Song 3
Affiliations

Affiliations

  • 1 Liaoning Key Laboratory of Marine Animal Immunology and Disease Control, Dalian Ocean University, Dalian 116023, China; Laboratory of Marine Fisheries Science and Food Production Process, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China.
  • 2 Liaoning Key Laboratory of Marine Animal Immunology and Disease Control, Dalian Ocean University, Dalian 116023, China.
  • 3 Liaoning Key Laboratory of Marine Animal Immunology and Disease Control, Dalian Ocean University, Dalian 116023, China; Laboratory of Marine Fisheries Science and Food Production Process, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China. Electronic address: [email protected].
PMID: 28888538 DOI: 10.1016/j.dci.2017.09.002
Abstract

Executioner caspases play important roles in apoptotic pathway and immune defense, which is considered to coordinate the execution phase of Apoptosis by cleaving multiple structural and repair proteins. However, the knowledge about the activation mechanism and function of executioner caspases in mollusks, especially marine bivalves is limited. In the present study, the full-length cDNA sequence of Caspase-1 was cloned from oyster Crassostrea gigas, which encoded a predicted protein containing a small subunit (p10) and large subunit (p20) with a conserved Caspase active site QACRG similar to that of human executioner Caspase-3/7. SDS-polyacrylamide gel electrophoresis and western blot results demonstrated that the CgCaspase-1 zymogen could be cleaved into p20p10, p20 and p10 in prokaryotic expression systems, and the C-terminus of CgCaspase-1 was also cleaved into p20 and p10. Both of the recombinant CgCaspase-1 (rCgCaspase-1) and the C-terminus of CgCaspase-1 (rCgCaspase-1-C) exhibited similar Caspase activity towards proteolytic substrate Ac-DMQD-pNA and Ac-DEVD-pNA. However, the recombinant N-terminus of CgCaspase-1 (rCgCaspase-1-N) did not display any Caspase activity. Moreover, the inhibitor of both Caspase-3/7 and pan-caspase could significantly inhibit the proteolytic activity of rCgCaspase-1. The strong binding activities towards lipopolysaccharide (LPS) of both rCgCaspase-1 and rCgCaspase-1-C were revealed by ELISA techniques and western blotting. A high level of CgCaspase-1 mRNA transcripts was detected in the gills and hemocytes by quantitative Real-Time PCR, and the CgCaspase-1 protein was mainly located in the cytoplasm of oyster hemocytes by immunofluorescence assay. These results collectively suggested that CgCaspase-1 was a homolog of executioner Caspase-3/7, which could be self-activated through proteolytic cleavage in prokaryotic expression systems, and performed Caspase and LPS binding activities in the innate immune response of oyster.

Keywords

Caspase-1; Crassostrea gigas; LPS recognition receptor; Large subunit and small subunit; Self-activation.

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