Department of Marine Life Sciences & Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province, 63243, Republic of Korea; Marine Science Institute, Jeju National University, Jeju Self-Governing Province, 63333, Republic of Korea.
Department of Applied Biosciences, Kyungpook National University, Daegu, 41566, Republic of Korea.
Fish Shellfish Immunol. 2021 Apr;111:152-159. doi: 10.1016/j.fsi.2021.01.016. Epub 2021 Feb 6.
The tetraspanin superfamily proteins are transmembrane proteins identified in a diverse range of eukaryotic organisms. Tetraspanins are involved in a variety of essential biological functions, including cell differentiation, adhesion, migration, signal transduction, intracellular trafficking, and immune responses. For an infection to occur, viruses must interact with various cell surface components, including receptors and signaling molecules. Tetraspanin CD63 is involved in the organization of the cell membrane and trafficking of cellular transmembrane proteins that interact with many viruses. In this study, the cd63 gene was characterized by studying its expression and function in a zebrafish model. The functional domains and structural features of Cd63, such as the Cys-Cys-Gly (CCG) motif in the large extracellular loop and cysteine residues, are conserved in zebrafish. We confirmed that cd63 was expressed in immune system organs, such as the axial vein and pronephric duct, during the embryonic development of zebrafish. To better understand the role of cd63 in the zebrafish immune system, we established cd63-deficient zebrafish lines using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. A 19 bp insertion mutation was generated in single guide RNA (sgRNA) target sequence of exon 3 of the cd63 gene, to create a pre-mature stop codon. We then analyzed the expression of cd63-related genes cxcr4a and cxcr4b in wild type (WT) and cd63-deficient zebrafish. We believe our study provides an important model that could be used to investigate the roles of cd63 in viral infection in vivo.
四跨膜蛋白超家族蛋白是在各种真核生物中鉴定出的跨膜蛋白。四跨膜蛋白参与多种重要的生物学功能，包括细胞分化、黏附、迁移、信号转导、细胞内运输和免疫反应。为了发生感染，病毒必须与各种细胞表面成分相互作用，包括受体和信号分子。四跨膜蛋白 CD63 参与细胞膜的组织和与许多病毒相互作用的细胞跨膜蛋白的运输。在这项研究中，通过在斑马鱼模型中研究其表达和功能来表征 cd63 基因。Cd63 的功能域和结构特征，如大细胞外环中的 Cys-Cys-Gly (CCG) 基序和半胱氨酸残基，在斑马鱼中是保守的。我们证实 cd63 在斑马鱼胚胎发育过程中在免疫系统器官（如轴向静脉和前肾管）中表达。为了更好地了解 cd63 在斑马鱼免疫系统中的作用，我们使用成簇规律间隔短回文重复序列/CRISPR 相关蛋白 9 (CRISPR/Cas9) 系统建立了 cd63 缺陷型斑马鱼系。在 cd63 基因外显子 3 的单指导 RNA (sgRNA) 靶序列中产生了 19 个碱基的插入突变，从而产生了一个提前终止密码子。然后，我们分析了野生型 (WT) 和 cd63 缺陷型斑马鱼中 cd63 相关基因 cxcr4a 和 cxcr4b 的表达。我们相信我们的研究提供了一个重要的模型，可以用于研究 cd63 在体内病毒感染中的作用。
