Guangzhou Key Laboratory of Subtropical Biodiversity and Biomonitoring, Institute of Modern Aquaculture Science and Engineering, School of Life Sciences, South China Normal University, Guangzhou 510631, China.
Guangdong Provincial Engineering Technology Research Center for Environmentally-Friendly Aquaculture, South China Normal University, Guangzhou 510631, China.
Int J Mol Sci. 2023 Apr 13;24(8):7179. doi: 10.3390/ijms24087179.
Tartrate-resistant acid phosphatase type 5 (TRAP5) is an enzyme that is highly expressed in activated macrophages and osteoclasts and plays important biological functions in mammalian immune defense systems. In the study, we investigated the functions of tartrate-resistant acid phosphatase type 5b from (OnTRAP5b). The OnTRAP5b gene has an open reading frame of 975 bp, which encodes a mature peptide consisting of 302 amino acids with a molecular weight of 33.448 kDa. The OnTRAP5b protein contains a metallophosphatase domain with metal binding and active sites. Phylogenetic analysis revealed that OnTRAP5b is clustered with TRAP5b of teleost fish and shares a high amino acid sequence similarity with other TRAP5b in teleost fish (61.73-98.15%). Tissues expression analysis showed that OnTRAP5b was most abundant in the liver and was also widely expressed in other tissues. Upon challenge with and in vivo and in vitro, the expression of OnTRAP5b was significantly up-regulated. Additionally, the purified recombinant OnTRAP5b ((r)OnTRAP5) protein exhibited optimal phosphatase activity at pH 5.0 and an ideal temperature of 50 °C. The , and of purified (r)OnTRAP5b were found to be 0.484 μmol × min × mg, 2.112 mM, and 0.27 s with respect to NPP as a substrate, respectively. Its phosphatase activity was differentially affected by metal ions (K, Na, Mg, Ca, Mn, Cu, Zn, and Fe) and inhibitors (sodium tartrate, sodium fluoride, and EDTA). Furthermore, (r)OnTRAP5b was found to promote the expression of inflammatory-related genes in head kidney macrophages and induce reactive oxygen expression and phagocytosis. Moreover, OnTRAP5b overexpression and knockdown had a significant effect on bacterial proliferation in vivo. When taken together, our findings suggest that OnTRAP5b plays a significant role in the immune response against bacterial infection in Nile tilapia.
抗酒石酸酸性磷酸酶 5 型（TRAP5）是一种在激活的巨噬细胞和破骨细胞中高度表达的酶，在哺乳动物免疫系统中发挥着重要的生物学功能。在本研究中，我们研究了 （OnTRAP5b）的抗酒石酸酸性磷酸酶 5b 的功能。OnTRAP5b 基因具有 975bp 的开放阅读框，编码一个由 302 个氨基酸组成的成熟肽，分子量为 33.448kDa。OnTRAP5b 蛋白含有一个金属磷酸酶结构域，具有金属结合和活性位点。系统进化分析表明，OnTRAP5b 与硬骨鱼类的 TRAP5b 聚类在一起，与硬骨鱼类的其他 TRAP5b 具有很高的氨基酸序列相似性（61.73-98.15%）。组织表达分析显示，OnTRAP5b 在肝脏中含量最丰富，在其他组织中也广泛表达。在体内和体外受到 和 刺激后，OnTRAP5b 的表达显著上调。此外，纯化的重组 OnTRAP5b（（r）OnTRAP5b）蛋白在 pH5.0 时表现出最佳的磷酸酶活性，最适温度为 50°C。纯化的（r）OnTRAP5b 的 Km、Vmax 和 kcat 值分别为 0.484μmol×min×mg、2.112mM 和 0.27s，以 NPP 为底物。其磷酸酶活性受到金属离子（K、Na、Mg、Ca、Mn、Cu、Zn 和 Fe）和抑制剂（酒石酸钠、氟化钠和 EDTA）的差异影响。此外，（r）OnTRAP5b 被发现可促进头肾巨噬细胞中炎症相关基因的表达，并诱导活性氧的表达和吞噬作用。此外，OnTRAP5b 的过表达和敲低对体内细菌增殖有显著影响。综上所述，本研究结果表明，OnTRAP5b 在尼罗罗非鱼抵抗细菌感染的免疫反应中发挥重要作用。
