The first non-mammalian CXCR5 in a teleost fish: molecular cloning and expression analysis in grass carp (Ctenopharyngodon idella)

Abstract

Background: Chemokines, a group of small and structurally related proteins, mediate chemotaxis of various cell types via chemokine receptors. In mammals, seven different CXC chemokine receptors denoted as CXCR1 to CXCR7 have been reported. However, the chemokine receptor CXCR5 has not been reported in other vertebrates.

Results: In the present study, the genomic sequence of CXCR5 was isolated from the grass carp Ctenopharyngodon idella. The cDNA sequence of grass carp CXCR5 (gcCXCR5) consists of 1518 bp with a 43 bp 5' untranslated region (UTR) and a 332 bp 3' UTR, with an open reading frame of 1143 bp encoding 381 amino acids which are predicted to have seven transmembrane helices. The characteristic residues (DRYLAIVHA) and conserved cysteine residues are located in the extracellular regions and in the third to seventh transmembrane domains. The deduced amino acid sequence shows 37.6-66.6% identities with CXCR5 of mammals, avian and other fish species. The grass carp gene consists of two exons, with one intervening intron, spaced over 2081 bp of genomic sequence. Phylogenetic analysis clearly demonstrated that the gcCXCR5 is clustered with those in other teleost fish and then in chicken and mammals. Real-time PCR analysis showed that gcCXCR5 was expressed in all tested organs/tissues and its expression level was the highest in trunk kidney, followed by in the spleen. The expression of gcCXCR5 was significantly modulated by immunostimulants such as peptidoglycan (PGN), lipopolysaccharide (LPS), polyinosinic-polycytidylic acid sodium salt (Poly I:C) and phytohaemagglutinin (PHA).

Conclusion: The cDNA and genomic sequences of CXCR5 have been successfully characterized in a teleost fish, the grass carp. The CXCR5 has in general a constitutive expression in organs/tissues examined, whereas its expression was significantly up-regulated in immune organs and down-regulated in brain, indicating its potential role in immune response and central nervous system.

Figure 1

The alignment of the amino acid sequences of gcCXCR5 (GenBank accession no. FJ825363) with CXCR5 sequences of other vertebrates by using CLUSTAL W program. Symbol (*) represents identical residues, (:) conservative substitution, (.) similar residues and (-) missing residues. The seven transmembrane domains are underlined, cysteines in the transmembrane domains are indicated by gray shadow and the conserved four cysteines presumably forming disulfide bonds are boxed. Broken underline indicates sequence DRYLAIVHA and uninterrupted underline NXS/T indicates N-glycosylation site. The sequences of CXCR5 used for analysis are derived from the GenBank, with their accession numbers listed in Table 2.

Figure 2

Phylogenetic relationship of CXCR members. A neighbor-joining phylogenetic tree of chemokine receptor sequences was constructed based on analysis of protein sequences by the computer program CLUSTER W. The sequences of the CXCR used for analysis are derived from the GenBank and SwissProt, and their accession numbers are listed in Table 2. The bootstrap confidence values shown at the nodes of the tree are based on 1000 bootstrap replications. Homo sapiens, Human; Mus musculus, Mouse; Gallus gallus, Chicken; Danio rerio, Zebrafish; Cyprinus carpio, Common carp; Ctenopharyngodon idella, Grass carp; Oncorhynchus mykiss, Rainbow trout; Petromyzon marinus, Sea lamprey; Tetraodon nigroviridis, Tetraodon; Takifugu rubripes, Fugu; Gasterosteus aculeatus, Stickleback.

Figure 3

Schematic diagram of exon-intron arrangement of CXCR5 genes from human (Gene ID 643), mouse (Gene ID 12145), cow (Gene ID 497021), fugu (ENSTRUG00000011814) and grass carp (GenBank accession no. FJ825364). Boxes represent exons and lines adjacent to exons represent introns. Open reading frames and untranslated regions are shown as black boxes and white boxes, respectively. The number of nucleotides in each exon and intron is, respectively, shown above and below the corresponding element.

Figure 4

Expression of gcCXCR5 in different organs/tissues revealed by real-time PCR. Total RNA was obtained from heart (H), trunk kidney (TK), brain (BR), liver (L), head kidney (HK), intestine (I), spleen (SP), blood (BL), gill (G) and muscle (M), respectively, then were reverse-transcribed to cDNA. Gene expression was measured by means of real-time quantitative PCR and is shown relative to the gene expression of β-actin.

Figure 5

Expression pattern of grass carp CXCR5 gene induced by peptidoglycan (PGN), lipopolysaccharide (LPS), polyinosinic-polycytidylic acid sodium salt (Poly I:C) and phytohaemagglutinin (PHA) in vivo. Total RNA was obtained from brain (BR), spleen (SP), gill (G), trunk kidney (TK), liver (L), intestine (I), head kidney (HK), thymus (T) and blood (BL), respectively. The transcriptional changes are shown in ratio in comparison with control fish, as determined by quantitative real-time PCR. Each column and bar represents the mean ± SE of three individual fish.