National and Provincial Joint Laboratory of Exploration and Utilization of Marine Aquatic Genetic Resources, National Engineering Research Center of Marine Facilities Aquaculture, Zhejiang Ocean University, Zhoushan, 316022, PR China.
Marine Fishery Institute of Zhejiang Province, Key Lab of Mariculture and Enhancement of Zhejiang Province, Zhoushan, 316100, China.
Dev Comp Immunol. 2022 Oct;135:104463. doi: 10.1016/j.dci.2022.104463. Epub 2022 Jun 8.
As an important member in SR-As, member 5 (SCARA5) can swallow apoptotic cells and foreign bodies, and participate multiple signaling pathways to inhibit tumor occurrence, development growth and metastasis. To explore its immune function, SCARA5 was identified from the yellow drum (Nibea albiflora) according to its transcriptome data, and its full-length cDNA was 6968 bp (named as NaSCARA5, GenBank accession no: MW070211) encoding 497 amino acids with a calculated molecular weight of 55.12 kDa, which had the typical motifs of SR family, such as transmembrane helix region, coil region, Pfam collagens region and SR region. BLASTp and the phylogenetic relationship analysis illustrated that the sequences shared high similarity with known SCARA5 of teleosts. Quantitative real time RT-PCR analysis showed that NaSCARA5 was expressed in intestine, stomach, liver, kidney, gill, heart and spleen, with the highest in the spleen (24.42-fold compared with that in heart). After being infected with Polyinosinic:polycytidylic acid (PolyI:C), Vibrio alginolyticus and Vibrio parahaemolyticus, NaSCARA5 mRNA were up-regulated with time dependent mode in spleen, which suggested that NaSCARA5 might play an important role in the immune process of fish. The extracellular domain of NaSCARA5 was successfully expressed in BL21 (DE3), and yielded the target protein of the expected size with many active sites for their conferring protein-protein interaction functions. After being purified by Ni-NAT Superflow resin and renatured, it was found to bind all the tested bacteria (V.parahaemolyticus,V.alginolyticus and Vibrio harveyi). The eukaryotic expression vector of the NaSCARA5-EGFP fusion protein was constructed and transferred into epithelioma papulosum cyprini (EPC) cells, and it was mainly expressed on the cell membrane indicating that NaSCARA5 was a typical transmembrane protein. The aforementioned results indicated that NaSCARA5 played a significant role in the defense against pathogenic bacteria infection as PRRs, which may provide some further understandings of the regulatory mechanisms in the fish innate immune system for SR family.
作为 SR-As 的重要成员，成员 5（SCARA5）可以吞噬凋亡细胞和异物，并参与多种信号通路抑制肿瘤的发生、发展和转移。为了探索其免疫功能，根据黄颡鱼（Nibea albiflora）的转录组数据鉴定出 SCARA5，并获得其全长 cDNA，长度为 6968bp（命名为 NaSCARA5，GenBank 登录号：MW070211），编码 497 个氨基酸，预测分子量为 55.12kDa，具有 SR 家族的典型特征，如跨膜螺旋区、卷曲区、Pfam 胶原蛋白区和 SR 区。BLASTp 和系统发育关系分析表明，该序列与已知硬骨鱼类的 SCARA5 具有高度相似性。定量实时 RT-PCR 分析表明，NaSCARA5 在肠道、胃、肝、肾、鳃、心和脾中均有表达，其中脾中表达量最高（与心相比，表达量为 24.42 倍）。在用 Polyinosinic:polycytidylic acid (PolyI:C)、溶藻弧菌和副溶血弧菌感染后，NaSCARA5mRNA 随时间呈依赖性上调，表明 NaSCARA5 可能在鱼类免疫过程中发挥重要作用。NaSCARA5 的细胞外结构域在 BL21（DE3）中成功表达，产生了具有许多活性位点的预期大小的靶蛋白，这些活性位点赋予了它们蛋白-蛋白相互作用的功能。经 Ni-NAT Superflow 树脂纯化和复性后，发现该蛋白能与所有测试的细菌（副溶血弧菌、溶藻弧菌和哈维弧菌）结合。构建了 NaSCARA5-EGFP 融合蛋白的真核表达载体，并转染到鲤鱼上皮瘤细胞（EPC）中，发现它主要表达在细胞膜上，表明 NaSCARA5 是一种典型的跨膜蛋白。上述结果表明，NaSCARA5 作为 PRRs 在防御病原菌感染中发挥了重要作用，这可能为进一步了解鱼类先天免疫系统中 SR 家族的调控机制提供一些依据。
