Cloning and characterization of apoptosis-associated speck-like protein containing a CARD domain (ASC) gene from Japanese flounder Paralichthys olivaceus.

Abstract

Apoptosis-associated speck-like protein containing a CARD domain (ASC) is a critical adaptor molecule in multiple inflammasome protein complexes that mediate inflammation and host defense. However, few studies have been performed in lower vertebrates such as in teleost. Here we identified and characterized a novel ASC gene (namely PoASC) from Japanese flounder Paralichthys olivaceus. The complete cDNA sequence of PoASC contains a 22bp 5'-untranslated sequence, a 612bp open reading frame, and a 438bp 3'-untranslated sequence. The deduced PoASC protein is comprised of 203 amino acids with a conserved N-terminal PYD domain and a C-terminal CARD domain and shows 35-62% sequence identity with other vertebrate ASC proteins. PoASC mRNA transcripts was detected in various Japanese flounder tissues and is dominantly expressed in hepatopancreas. Oligomeric speck-like structures were observed when PoASC was exogenously expressed in Japanese flounder FG-9307cells. Immune challenge experiments revealed that PoASC gene expression was significantly induced in the Japanese flounder head kidney macrophages and peripheral blood leukocytes by the canonical TLR ligands LPS, Poly(I:C) and zymosan stimulations. In addition, the induction of PoASC was also observed in Edwardsiella tarda challenged head kidney and gill tissues. Furthermore, we for the first time showed that extracellular ATP, an important signaling molecule in triggering innate immune response and activation of NLR inflammasome, significantly up-regulates PoASC expression in the Japanese flounder head kidney macrophages in a dose-dependent manner. Together, these findings addressed the involvement of PoASC in TLR and extracellular ATP-mediated innate immune signaling in the Japanese flounders.