Molecular cloning and expression analysis of the ASC gene from mandarin fish and its regulation of NF-κB activation

Apoptosis-associated speck-like protein containing a CARD (ASC) is an adaptor protein that has a bipartite domain structure, an N-terminal PYRIN domain and a C-terminal caspase-recruitment domain (CARD). In this study, we cloned the mandarin fish ASC cDNA (mfASC), which consisted of 899 bp with a 115 bp 5′-UTR and a 181 bp 3′-UTR. The open reading frame encoded 201 amino acids. The mfASC shows 37% identity to an ASC orthologue from zebrafish. The mfASC has two protein-protein interaction domains, an N-terminal PYRIN domain and a C-terminal CARD domain. The mfASC gene structure was determined and had a length of 3954 bp with four exons separated by three introns. Northern blot analysis showed that mfASC mRNA is constitutively expressed in the head kidney, gill, hind kidney, spleen and intestine. In vitro studies, mfASC fused with green fluorescent protein appeared as a speck in the transfected 293T cells. When transiently overexpressed in 293T cells, mfASC inhibited NF-κB activity with or without tumor necrosis factor (TNFα) or lipopolysacharide (LPS) stimulation.