Characterization Of P2X7R Of Ayu, Plecoglossus Altivelis

P2X purinoceptor7(P2X7R), an ATP-gated ion channel, plays an important role during theinnate immune response in mammals. However, relatively little is known about the role of P2X7Rin the fish immune system. Using transcriptome sequencing and the rapid amplification of cDNAends (RACE) method., we cloned a cDNA sequence encoding ayu (Plecoglossus altivelis) P2X7R(aP2X7R) The full-length cDNA of aP2X7R consisted of2046nucleotides including a1725bpsingle open reading frame (ORF), a56bp5′-untranslated region (5′-UTR) and a265bp3′-UTR, apoly(A) tail. The predicted protein was composed of574amino acid residues with a P2X familysignature, two transmembrane domains, and a long C-terminal. aP2X7R transcripts were mainlydistributed in ayu immune tissues such as head kidney, spleen and liver. After challenge withListonella anguillarum, The expression of aP2X7R transcripts in head kidney, spleen, liver, heart,muscle, gill and intestine were all significantly increased at4hpi detected by RT-qPCR. The mostdramatic upregulation in aP2X7R mRNA expression was observed in the head kidneys (up to19.99-fold) at12hpi. Measured by RT-qPCR and Western blot, the aP2X7R mRNA and proteinwas also upregulated significantly in ayu macrophages upon bacterial challenge. An antibodyagainst the ectodomain of aP2X7R (aEPAb) and an antagonist (oATP) were used to block aP2X7R.aP2X7R siRNA was also used to knockdown the receptor expression in ayu macrophages. Celldeath induced by ATP was significantly inhibited in ayu macrophages after aEPAb, oATP, orsiRNA treatment. Moreover, aP2X7R ablation also resulted in suppression of phagocytic activityand ATP-induced bacterial killing in ayu macrophages. Our results indicated that aP2X7R wasupregulated after infection and mediated cell death, phagocytosis, and bacterial killing of ayumacrophages.