Department of Fisheries Science, College of Fisheries and Ocean Sciences, Chonnam National University, 50, Daehak-ro, Yeosu, Jeonnam 59626, Korea.
Department of Biomedical and Electronic Engineering, College of Engineering, Chonnam National University, Yeosu, Jeonnam 59626, Korea.
Int J Mol Sci. 2018 Mar 13;19(3):842. doi: 10.3390/ijms19030842.
In this study, an 1888-bp carbonic anhydrase XII (CA XII) sequence was cloned from the brain of the pufferfish, . The cloned sequence contained a coding region of 1470-bp, which was predicted to translate into a protein of 490 amino acid residues. The predicted protein showed between 68-56% identity with the large yellow croaker (), tilapia (), and Asian arowana () CA XII proteins. It also exhibited 36% and 53% identity with human CA II and CA XII, respectively. The cloned sequence contained a 22 amino acid NH₂-terminal signal sequence and three Asn-Xaa-Ser/Thr sequons, among which one was potentially glycosylated. Four cysteine residues were also identified (Cys-21, Cys-201, Cys-355, and Cys-358), two of which (Cys-21 and Cys-201) could potentially form a disulfide bond. A 22-amino acid COOH-terminal cytoplasmic tail containing a potential site for phosphorylation by protein kinase A was also found. The cloned sequence might be a transmembrane protein, as predicted from in silico and phylogenetic analyses. The active site analysis of the predicted protein showed that its active site residues were highly conserved with tilapia CA XII protein. Homology modeling of the pufferfish CA XII was done using the crystal structure of the extracellular domain of human carbonic anhydrase XII at 1.55 Å resolution as a template. Semi-quantitative reverse transcription (RT)-PCR, quantitative PCR (q-PCR), and in situ hybridization confirmed that pufferfish is highly expressed in the brain.
在这项研究中，我们从河豚鱼的脑中克隆出了一段长 1888 个碱基的碳酸酐酶 XII（CA XII）序列。该克隆序列包含一个 1470 个碱基的编码区，预测可翻译成 490 个氨基酸残基的蛋白质。预测的蛋白质与大黄鱼（）、罗非鱼（）和亚洲龙鱼（）的 CA XII 蛋白具有 68-56%的同源性。它与人类 CA II 和 CA XII 的同源性分别为 36%和 53%。该克隆序列包含一个 22 个氨基酸的 NH₂-末端信号序列和三个 Asn-Xaa-Ser/Thr 序列，其中一个可能被糖基化。还鉴定了四个半胱氨酸残基（Cys-21、Cys-201、Cys-355 和 Cys-358），其中两个（Cys-21 和 Cys-201）可能形成二硫键。还发现了一个包含蛋白激酶 A 磷酸化潜在位点的 22 个氨基酸的 COOH-末端细胞质尾巴。根据计算机分析和系统发育分析预测，该克隆序列可能是一种跨膜蛋白。预测蛋白的活性位点分析表明，其活性位点残基与罗非鱼 CA XII 蛋白高度保守。使用分辨率为 1.55 Å 的人碳酸酐酶 XII 外显子结构晶体作为模板，对河豚鱼 CA XII 进行了同源建模。半定量 RT-PCR、定量 PCR（q-PCR）和原位杂交证实，河豚鱼 在脑中高度表达。
