Molecular Characterization and Expression Analysis of ATP-Gated P2X7 Receptor Involved in Japanese Flounder (Paralichthys olivaceus) Innate Immune Response

Abstract

ATP-gated P2X7 receptor (P2RX7) channel is a key component for purinergic signaling and plays important roles in the innate immune response in mammals. However, the expression, molecular properties and immune significances of P2RX7 in lower vertebrates are still very limited. Here we identified and characterized a novel bony fish P2RX7 homologue cDNA, termed poP2RX7, in Japanese flounder (Paralichthys olivaceus). PoP2RX7 protein shares about 60–88% sequence similarity and 45–78% sequence identity with known vertebrate P2RX7 proteins. Phylogenetic analysis placed poP2RX7 and other P2RX7 proteins within their own cluster apart from other P2RX members. While the functional poP2RX7 channel shares structural features in common with known P2RX7 homologs, electrophysiological studies revealed that BzATP, the more potent agonist for known mammalian and fish P2RX7s, shows similar potency to ATP in poP2RX7 activation. poP2RX7 mRNA constitutively expressed in all examined tissues from unstimulated healthy Japanese flounder with dominant expression in hepatopancreas and the lowest expression in head kidney, trunk kidney, spleen and gill. poP2RX7 mRNA expression, however, was significantly induced in Japanese flounder head kidney primary cells by Poly(I:C) and bacterial endotoxin LPS stimulations. In vivo experiments further revealed that poP2RX7 gene expression was substantially up-regulated by immune challenge with infectious bacteria Edwardsiella tarda and Vibrio anguillarum. Moreover, activation of poP2RX7 results in an increased gene expression of multifunctional cytokines IL-1β and IL-6 in the head kidney primary cells. Collectively, we identified and characterized a novel fish P2RX7 homolog which is engaged in Japanese flounder innate immune response probably through modulation of pro-inflammatory cytokines expression.

Characterization of UDP-Activated Purinergic Receptor P2Y6 Involved in Japanese Flounder Paralichthys olivaceus Innate Immunity

“…In particular, dominant expression of Japanese flounder P2Y 6 R mRNA transcripts was found in the liver while the least expression was in the spleen ( Figure 3 ). We have shown previously the transcriptional presence of P2X2 , P2X4 , P2X7 , P2Y 2 , and P2Y 12 receptors in various Japanese flounder tissues [ 12 , 18 , 19 , 20 ]. The diversity of purinergic receptor subtypes present in Japanese flounder tissues and cells, addresses an essential role of purinergic receptors in fish through triggering different downstream signaling events.…”

Divalent metal modulation of Japanese flounder (Paralichthys olivaceus) purinergic P2X7 receptor

“…We previously reported that this role is conserved in the Japanese flounder, i.e. activation of poP2X7R can induce an increased gene expression of the proinflammatory cytokines IL-1b and IL-6 in the Japanese flounder head kidney primary cells [15]. Moreover, we have demonstrated the release of ATP by connexin43 and pannexin1 channels in the Japanese flounder immune cells under inflammatory conditions, further supporting the role of extracellular ATP and its associated membrane receptors in fish innate immune response [25,26].…”

“…Oocytes were manually defolliculated and incubated 30 min with type III collagenase as previously described . The pIRES‐EGFP/poP2XR7 plasmid was generated as previously described . Oocytes were injected intranuclearly with 4 ng Japanese flounder or rat P2X7R cDNA.…”

“… have characterized the pharmacological properties of P2X7R from the gilthead seabream ( Sparus aurata L.) by electrophysiological recordings. Recently, we have identified a functional P2X7R homolog from the teleost fish Japanese flounder ( Paralichthys olivaceus ) ortholog, termed poP2X7R, and studied its engagement in the Japanese flounder innate immune response . However, the effects of divalent cations on P2X7Rs in teleost are still lacking.…”

Identification and expression analysis of nascent polypeptide-associated complex alpha gene in response to immune challenges in Japanese flounder Paralichthys olivaceus

“…Bacterium Edwardsiella tarda was cultured overnight from a single colony with marine LuriaeBertani medium as described in our previous study [19]. The bacterial cultures were then pelleted, washed twice and re-suspended in sterilized physiological saline.…”