DataSheet_1_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.docx

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.

众多免疫性疾病可通过调节神经行为进行治疗，其中细胞外腺苷三磷酸（ATP）作为内源性危险相关分子模式信号分子的重要组成部分，在先天神经相关免疫反应中扮演着至关重要的角色。在炎症、损伤或细胞凋亡过程中，激活或应激细胞通过半通道（Pannexin, Panx）和连接蛋白（Connexin, Cx）释放ATP。除了参与ATP释放之外，Panxs和Cxs还具备关键的免疫功能。在本研究中，我们从斑马鱼（Lateolabrax maculatus）中鉴定并表征了pannexin1、三种connexin32异构体和connexin43，分别命名为LmPanx1、LmCx32.2、LmCx32.3、LmCx32.7和LmCx43。序列分析揭示了它们相似的拓扑结构：一个相对保守的C端区域和四个高度保守的跨膜（TM）结构域等。Panx1的每个细胞外（ECL）区域包含两个保守的半胱氨酸残基。与Panx1不同，Cx32和Cx43的每个ECL区域均包含三个保守的半胱氨酸残基，形成两个保守基序：ECL1中的CX6CX3C基序和ECL2中的CX4CX5C基序。此外，Panx1和Cx43与选定脊椎动物中的同源基因在基因组组织和同源性方面具有相似性。Cx32和Cx43在鱼类中位于同一基因座，但在两栖动物中分化为两个基因座。尽管表达水平存在差异，但所鉴定的基因在所有检查的组织中均呈组成性表达。所有基因在体内和体外受到病原相关分子模式（PAMP，如脂多糖和聚（I：C））刺激或细菌感染后均上调表达，但在刺激后6或12小时的大脑中表达下调。特别是，三个LmCx32异构体和LmCx43在头肾原淋巴细胞受到ATP刺激后上调表达；然而，在12小时后观察到LmCx32.3和LmCx43表达的下调。相反，ATP处理抑制了LmPanx1的表达。重要的是，我们展示了斑马鱼的Panx1、Cx43和Cx32定位于细胞膜上，并参与炎症诱导的ATP释放。综合来看，我们的研究结果证实了Panx1、Cx32和Cx43是重要的神经相关免疫反应基因，它们参与炎症诱导的ATP释放。