Molecular cloning, structure and expressional profiles of two novel single-exon genes (PoCCR6A and PoCCR6B) in the Japanese flounder (Paralichthys olivaceus)

Highlights

• Two chemokine receptors (PoCCR6A and PoCCR6B) of the Japanese flounder were cloned and characterized.

• PoCCR6A and PoCCR6B were the first reported single-exonic CCR6 genes among various species.

• PoCCR6A and PoCCR6B were highly expressed in mucosal tissues of the Japanese flounder.

• PoCCR6A and PoCCR6B exhibited upregulated expression patterns during bacterial infection in four immune tissues.

Abstract

CCR6 is an important binding receptor of CCL20 and beta-defensins, and has multiple functions in the innate and acquired immune responses. In this study, we cloned the PoCCR6A and PoCCR6B genes of the Japanese flounder and studied the gene structure and expression patterns of these two genes in bacterial infection. The full-length PoCCR6A cDNA is 1415 bp and the open reading frame (ORF) is 1113 bp, encoding a 370-amino-acid peptide. The full-length PoCCR6B cDNA is 2193 bp and the ORF is 1029 bp, encoding a 363-amino-acid peptide. The structures of PoCCR6A and PoCCR6B indicate that they are single-exon genes. The predicted proteins encoded by PoCCR6A and PoCCR6B have the typical G-protein-coupled receptor (GPCR) family signature of seven transmembrane domains and several conserved structural features. A tissue distribution analysis showed that PoCCR6A is predominately expressed in the intestine, gill, and blood, and PoCCR6B in the gill, spleen, and liver. The expression patterns of the two chemokine receptors were analyzed during bacterial infection. In spleen and kidney, the expression of PoCCR6A was significantly upregulated at 24 h after infection, whereas the expression of PoCCR6B was steady at these time points. While in intestine, both of them were upregulated at 6 h–12 h after infection, and in gill the expression levels of them were upregulated at 24 h. The patterns of expression suggested that PoCCR6A and PoCCR6B play an important role in the immune response of the Japanese flounder, especially in the mucosal tissues.

Introduction

Chemokine receptors and their ligands have been identified as major factors initiating and controlling the migratory patterns of all immune cells, and are critical for the functions of the innate and adaptive immune responses [1], [2]. CCR6 is a nonpromiscuous chemokine receptor, with only one known chemokine ligand, CCL20. The CCL20–CCR6 pair is responsible for the chemoattraction of immature dendritic cells, effector/memory T cells, and B cells in the skin and mucosal surfaces under homeostatic and inflammatory conditions [3], [4]. Although CCL20 does not share the binding site of CCR6 with any other chemokines, human beta-defensin-1 (HBD-1) and −2 (HBD-2), small cationic antimicrobial peptides, also bind to and activate CCR6 [5]. Beta-defensins may promote the adaptive immune response by recruiting dendritic and T cells to the site of microbial invasion through their interactions with CCR6 [5]. In CCR6, the disulfide-bond-forming cysteines in the first (Cys118) and second (Cys197) extracellular loops are very important for its receptor activity, and the three-dimensional structures of the CCL20, HBD-1, and HBD-2 monomers are very similar [6], [7].

To date, 232 CCR6 gene sequences from different species have been submitted to the National Center for Biotechnology Information (NCBI) database (Nov. 2015), including those from mammals (160), birds (55), bony fishes (9), Crocodylia (2), amphibians (1), cartilaginous fishes (1), lizards (1), turtles (1), coelacanths (1), and viruses (1). The CCR6 gene is duplicated in the bony fishes and each gene has evolved independently and developed specialized functions [8]. Duplicated CCR6 sequences have been reported in Danio rerio and Epinephelus coioides [9]. In Epinephelus coioides, the expression patterns of CCR6A and CCR6B in healthy and Cryptocaryon irritans-infected fish indicate their important roles in the immune responses of bony fish [9].

The Japanese flounder (Paralichthys olivaceus) is an important marine flatfish, widely cultured in Asian countries. Compared with mammalian studies, the study of chemokines and their receptors in the Japanese flounder has focused on the gene and protein sequences, including those of several CCLs (Paol SCYA101–107) [10], [11], [12], [13], [14], CXCL13 [15], CXCL8 (IL-8) [16], IL-8 receptor (AB079600.1), CCR3 (AB081311.1), CCR9 (AB081312.1), and CCR6a (GU174266.1), whereas few functional analyses have been reported.

On human chromosome 6, RNASET2 (ribonuclease T2, location: NC_000006.12, 166929516–166956589) and FGFR1OP (FGFR1 oncogene partner, location: NC_000006.12, 166999317–167042418) are located near CCR6 (location: NC_000006.12, 167111807–167139141), and various tag single-nucleotide polymorphisms (SNPs) in the gene cluster are associated with autoimmune diseases (NCBI database). The SNPs (rs3093023 and rs3093024) are intron variants of CCR6 [17], [18], [19], [20]. The SNPs in fish CCR6 have not yet been determined.

In this study, we found that the previously identified Japanese flounder CCR6a gene (GU174266.1) was wrongly named, and that the sequence was highly similar to FGFR1OP. The real Japanese flounder CCR6A and CCR6B genes were cloned in this study. The gene structures of PoCCR6A and PoCCR6B were also analyzed. Their expression patterns in the tissues of healthy and Vibrio anguillarum-challenged fish were investigated to determine their functions in the immune response.