Molecular characterization and expression analysis of ATP-gated P2X7 receptor involved in Japanese flounder (Paralichthys olivaceus) innate immune response

PLoS One. 2014 May 5;9(5):e96625. doi: 10.1371/journal.pone.0096625. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism*
  • Amino Acid Sequence
  • Animals
  • Cloning, Molecular
  • Cytokines / metabolism
  • DNA Primers
  • DNA, Complementary / metabolism
  • Edwardsiella tarda
  • Electrophysiology
  • Female
  • Fish Proteins / immunology
  • Fish Proteins / metabolism*
  • Flounder / immunology*
  • Flounder / metabolism*
  • Flounder / microbiology
  • Gene Expression Regulation
  • Immunity, Innate*
  • Kidney / metabolism
  • Male
  • Molecular Sequence Data
  • Phylogeny
  • Receptors, Purinergic P2X7 / immunology
  • Receptors, Purinergic P2X7 / metabolism*
  • Sequence Analysis, DNA
  • Sequence Homology, Amino Acid
  • Tissue Distribution
  • Vibrio

Substances

  • Cytokines
  • DNA Primers
  • DNA, Complementary
  • Fish Proteins
  • Receptors, Purinergic P2X7
  • Adenosine Triphosphate

Associated data

  • GENBANK/KC748421

Grants and funding

This project was sponsored by grants from the Natural Science Foundation of Tianjin (13JCYBJC38300; http://www.tstc.gov.cn/), the Science & Technology Development Fund of Tianjin Education Commission for Higher Education (20130607; http://www.tjmec.gov.cn/), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (http://fund.cscse.edu.cn/Login.aspx), FONDECYT Initiation grant from the Chilean government (11121302; http://www.fondecyt.cl/), the Research Initiation Fund provided by Tianjin Normal University (5RL120; http://www.tjnu.edu.cn/), the National Basic Research Program of China (2012CB114405; http://program.most.gov.cn/), and National Key Technology R&D Program (2011BAD13B07, 2012AA10A401 and 2012AA092205; http://program.most.gov.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.