A novel caspase-associated recruitment domain (CARD) containing protein (CgCARDCP-1) involved in LPS recognition and NF-κB activation in oyster (Crassostrea gigas)

Fish Shellfish Immunol. 2018 Aug:79:120-129. doi: 10.1016/j.fsi.2018.05.018. Epub 2018 May 8.

Abstract

Caspase-associated recruitment domain (CARD) containing proteins play critical roles in molecular interaction and regulation of various signaling pathways, such as the activation of caspase and NF-κB singling pathway in the process of apoptosis or inflammation. In the present study, a novel CARD containing protein (designed CgCARDCP-1) was identified and characterized from oyster Crassostrea gigas. Molecular feature analysis revealed that, the open reading frame (ORF) of CgCARDCP-1 gene was 759 bp encoding a polypeptide of 253 amino acids with a conserved N-terminal CARD domain and two transcriptional coactivator p15 (PC4) domains in C-terminus. Homologous alignment showed that the amino acid sequence of CgCARDCP-1 shared 30%-46% identity with that of caspase-2. By RT-PCR detection, the mRNA transcripts of CgCARDCP-1 were found to be widely distributed in various tissues of oyster with the highest expression level in hemocytes and mantle. And CgCARDCP-1 protein was mostly distributed in the cytoplasm of oyster hemocytes as shown by immunohistochemistry. Moreover, the CgCARDCP-1 mRNA expression level in hemocytes was significantly up-regulated after lipopolysaccharide (LPS) and Vibrio splendidus stimulations. The recombinant CgCARDCP-1 displayed strong binding activity with LPS in vitro. In addition, after transfected into the HEK-293T cell with luciferase reporter system, CgCARDCP-1 could significantly promote the NF-κB activation (1.29-fold, p < 0.05) compared to that in the control group. These results collectively demonstrated that the CgCARDCP-1 might serve as a recognition molecule for LPS and a regulator of NF-κB activation in the immune response of oyster.

Keywords: CARD-containing proteins; Crassostrea gigas; LPS binding; NF-κB activation.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Caspases / chemistry
  • Caspases / genetics*
  • Caspases / immunology*
  • Crassostrea / enzymology
  • Crassostrea / genetics*
  • Crassostrea / immunology*
  • Gene Expression Profiling
  • Gene Expression Regulation / immunology*
  • Immunity, Innate / genetics*
  • Lipopolysaccharides / pharmacology
  • NF-kappa B / genetics
  • NF-kappa B / metabolism
  • Phylogeny
  • Sequence Alignment
  • Signal Transduction / immunology*

Substances

  • Lipopolysaccharides
  • NF-kappa B
  • Caspases