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  • A genomic DNA sequence was retrieved

    2020-01-13

    A genomic DNA sequence was retrieved (Scaffold_30) from the fugu genomic database ( v3.0), which encodes the homolog of CXCL8 of many other vertebrates and specific primers CXCL8F1 and CXCL8R1 () were designed for 3′-RACE and 5′-RACE PCR, respectively. First-strand thymus cDNA was used as template. A cDNA sequence that exhibits distinguishing features of CXCL8 was isolated (GenBank Accession no. ). This sequence showed significant identity to IL8 of flounder (67%), trout (57%), chickens (41%) and human (a). Four cysteine residues (Cys-35, 37, 61 and 78) that are essential for the formation of the tertiary structure of the CXC chemokine are conserved in fugu CXCL8. The two cysteine residues that are characteristic features of CXCL8 were separated by an arginine residue. The 3′-UTR contained six repeats of an ATTTA sequence; these repeats are known to shorten the half-life of several cytokines and growth factors, and are also thought to cause rapid mRNA degradation . The ELR motif that is thought to mediate the neutrophil chemotaxis in mammals is absent in fugu CXCL8. Although trout has a similar motif (DLR) at this position , fugu CXCL8 contains EQH. In human CXCL8, a mutation from ELR to DLR retained neutrophil attracting activity at lower potency . Strieter et al. reported that CXCL8s that lack ELR motif, PF4, IP-10 and MIG are potent inhibitors of both CXC (ELR) chemokine and basic fibroblast growth factor-induced angiogenesis in human . Taken together, we infer that both ELR CXC and non-ELR CXC might play important roles in the inflammatory responses of the immune system. Two fugu genomic DNA sequences (Scaffold_36 and Scaffold_184), which are homologous to the flounder IL8R were also retrieved from the fugu genomic database ( v4.0). For cDNA cloning, specific primers for 5\'-RACE (CXCR1RA, CXCR1RB for type 1, and CXCR2R1, CXCR2R2 for type 2, ) and 3\'-RACE (CXCR1FA, CXCR1FB for type 1, and CXCR2F1, CXCR2F2 for type 2, ) were designed. The first-strand cDNA from phytohemagglutinin (PHA)-stimulated PBL was amplified by thermal cycling: 35 cycles, each Fungicidin at 95°C for 15s, 55°C for 30s and 68°C for 2min, and a final elongation for 10min at 72°C in a total volume of 20μl of reaction mixture (1×reaction buffer, 200μM dNTP, 5 units of Advantage2 DNA polymerase (Clontech) and 200nM of each specific primer). After 35 cycles, nested PCR was performed using a touchdown protocol: 15 cycles at 95°C for 15s, 68°C for 30s (lowered by 1°C every cycle), and 68°C for 1min, followed by another 20 cycles at 95°C for 15s, 55°C for 30s, and 68°C for 1min, with a final extension at 70°C for 10min using an NUP-A primer and gene specific primers. The PCR products were subcloned into the pGEM-T easy vector (Promega) and sequenced. Two CXCRs (CXCR1 and CXCR2) were isolated, which display similar structural features of mammalian IL8RA and IL8RB. CXCR1 has two transcripts; short (1495bp, GenBank accession no. ) and long transcript (1654bp, GenBank accession no. ). Though both transcripts have the same coding region, the longer transcript has an additional 161bp in its 5\'-UTR region. The presence of two transcripts could be the result of an alternative splicing. However, we cannot rule out the possibility that the different 5\'-UTR would play some roles in the translation of the protein. The CXCR2 cDNA encompasses 1633 nucleotides, including an open reading frame of 1077 nucleotides that encodes 359 amino acid residues (GenBank accession no. ). Both CXCRs of fugu showed high homology to other vertebrates (28–56%) and the homology between CXCR1 and CXCR2 was 44% (b). Analysis of deduced amino acid sequences by TMHMM v.2.0 revealed that both receptors have seven transmembrane domains, which are characteristic of the G-protein-coupled receptor family (b). Two cysteine residues, one in the N-terminal domain and the other in the third extracellular domain are also conserved in fugu (b). A DRY motif, which may be important in regulating signaling , is found in fugu CXCRs. Like mammalian CXCR, the C-terminal intracellular segment of fugu CXCRs is predicted to be enriched in serine and threonine residues. These essential features that are conserved in fugu suggest that these chemokine receptors may have a functional role in immune inflammatory-response in fish.