Characterization of Pannexin1, Connexin32, and Connexin43 in Spotted Sea Bass (Lateolabrax maculatus): They Are Important Neuro-Related Immune Response Genes Involved in Inflammation-Induced ATP Release

Abstract

Many immunological diseases can be treated by regulating neurobehavior, in which extracellular ATP is a vital member of endogenous danger-associated molecular pattern signaling molecule that plays a crucial part in innate neuro-related immunity. It is actively released through pannexin (Panx) and connexin (Cx) hemichannels from activated or stressed cells during inflammation, injury, or apoptosis. In addition to participating in ATP release, Panxs and Cxs also have crucial immune functions. In this study, pannexin1, three connexin32 isoforms and connexin43 were identified and characterized in spotted sea bass (Lateolabrax maculatus), which were named LmPanx1, LmCx32.2, LmCx32.3, LmCx32.7, and LmCx43. Their similar topological structures were discovered by sequence analysis: a relatively unconserved C-terminal region and four highly conserved transmembrane (TM) domains, and so on. Each extracellular (ECL) region of Panx1 has two conserved cysteine residues. Unlike Panx1, each ECL region of Cx32 and Cx43 contains three conserved cysteine residues, forming two conserved motifs: CX₆CX₃C motif in ECL1 and CX₄CX₅C motif in ECL2. Furthermore, Panx1 and Cx43 share similar genomic organization and synteny with their counterparts in selected vertebrates. Cx32 and CX43 were located in the same locus in fish, but diverged into two loci from amphibian. Moreover, despite varying expression levels, the identified genes were constitutively expressed in all examined tissues. All genes were upregulated by PAMP [lipopolysaccharide and poly(I:C)] stimulation or bacterial infection in vivo and in vitro, but they were downregulated in the brain at 6 or 12 h after stimulation. Especially, the three LmCx32 isoforms and LmCx43 were upregulated by ATP stimulation in primary head kidney leukocytes; however, downregulation of LmCx32.3 and LmCx43 expression were noted at 12 h. Conversely, ATP treatment inhibited the expression of LmPanx1. Importantly, we showed that the spotted sea bass Panx1, Cx43, and Cx32 were localized on the cellular membrane and involved in inflammation-induced ATP release. Taken together, our results demonstrated that Panx1, Cx32, and Cx43 are important neuro-related immune response genes involved in inflammation-induced ATP release.

Keywords: ATP release; Lateolabrax maculatus; connexin32; connexin43; innate immunity; pannexin1.

Figure 1

Multiple sequence alignment analysis of Panx1 (A), Cx32 (B), and Cx43 (C). The N-terminal region, four TM domains (TM1-4), the intracellular loop (ICL1), the extracellular loops (ECL1-2), and the C-terminal region of LmPanx1, LmCx32, and LmCx43, are marked above the alignment. Symbol (▲) indicates the conserved cysteine residues. The K or R residue in position 75 and the classic innexin-specific P-X-X-X-W motif are boxed in green and red, respectively. In addition, the CNX domain, connexin-ccc domain, and PDZ domain are boxed in yellow, purple, and red, respectively. The LmPanx1, LmCx32, and LmCx43 are shown in bold. The accession numbers of sequences are shown in Figure 2. Hs, Homo sapiens; Mm, Mus musculus; Gg, Gallus gallus; Xt, Xenopus tropicalis; Dr, Danio rerio; Po, Paralichthys olivaceus; Ms, Morone saxatilis; Lc, Larimichthys crocea; Lm, Lateolabrax maculatus; Ss, Salmo salar; Cc, Cyprinus carpio; Om, Oncorhynchus mykiss.

Figure 2

Phylogenetic tree analysis of Panx1 (A), Cx32 (B), and Cx43 (C). Phylogenetic tree was constructed using the NJ method and run for 10,000 replications. The LmPanx1, LmCx32, and LmCx43 are shown in bold.

Figure 3

Genomic organization of Panx1 (A), Cx32 (B), and Cx43 (C). Blank and solid boxes indicate UTR and coding exon, respectively. The size (bp) of exons and introns is indicated. Note that the size of exons and introns is disproportionate.

Figure 4

Gene synteny of Panx1 (A) and Cx32 and Cx43 (B). The LmPanx1, LmCx32, and LmCx43 genome sequence data were obtained from the spotted sea bass genome database (https://www.ncbi.nlm.nih.gov/genome/43909). Synteny information for other vertebrates was retrieved from the Ensembl database (http://www.ensembl.org/index.html). Arrows indicate transcription orientations.

Figure 5

Expression analysis of LmPanx1, LmCx32, and LmCx43 in tissues by qPCR. The expression level of each gene was normalized to that of EF1α. Data are presented as mean ± SEM (N = 4).

Figure 6

Expression of LmPanx1 (A), LmCx32.2 (B), LmCx32.3 (C), LmCx32.7 (D), and LmCx43 (E) after LPS, poly(I:C), or Edwardsiella tarda challenge. Spotted sea bass was i.p. injected with LPS (5 mg/kg body weight), poly(I:C) (5 mg/kg body weight), E. tarda (5 × 10⁴ CFU/fish), or PBS (control) The relative expression levels of target genes were normalized to that of EF1α. Data are shown as mean +SEM (N = 4). *p < 0.05, **p < 0.01 are considered significant difference.

Figure 7

Expression of LmPanx1, LmCx32, and LmCx43 in primary head kidney leukocytes after stimulation with LPS, poly(I:C) (A) or ATP (B). Primary head kidney leukocytes were isolated from the spotted sea bass head kidney and stimulated with LPS (100 µg/mL), poly(I:C) (50 µg/mL), 100 µM ATP, 1 mM ATP, or PBS (control). The relative expression levels of target genes were normalized to that of EF1α. The data are shown as mean +SEM (N = 4). *p < 0.05, **p < 0.01 are considered significant difference.

Figure 8

Subcellular localization of LmPanx1, LmCx32, and LmCx43 in HEK293 T cells (A) and LPS-induced extracellular ATP release in primary head kidney leukocytes (B) or HEK293 T cells (C). (A) HEK293 T cells were transfected with pEGFP-N1-LmPanx1, pEGFP-N1-LmCx32.2, pEGFP-N1-LmCx32.3, pEGFP-N1-LmCx32.7, or pEGFP-N1-LmCx43 plasmids. At 24 h posttransfection, the cells were stained with DAPI and observed under a confocal microscope. (B, C) The primary head kidney leukocytes were stimulated with LPS (100 µg/mL) or PBS (control). HEK293 T cells were transfected with pcDNA3.1-LmPanx1, pcDNA3.1-LmCx32.2, pcDNA3.1-LmCx32.3, pcDNA3.1-LmCx32.7, or pcDNA3.1-LmCx43. After 24 h, the cells were stimulated with LPS (100 µg/mL). The supernatant was collected at 15 and 30 min after stimulation and the ATP levels were subsequently measured. The mock transfected and empty plasmid transfected cells served as controls. Data are shown as mean +SEM (N = 4). *p < 0.05, **p < 0.01 are considered significant difference.