Sumi Kanij Rukshana, Kim Soo Cheol, Natarajan Sathishkumar, Choi Kap Seong, Choi Myeong Rak, Kim Hoy-Taek, Park Jong-In, Nou Ill-Sup, Gilmour Kathleen M, Kho Kang Hee
Department of Fisheries Science, College of Fisheries and Ocean Sciences, Chonnam National University, 50, Daehak-ro, Yeosu, Jeonnam 59626, Republic of Korea.
Department of Biomedical and Electronic Engineering, College of Engineering, Chonnam National University, Yeosu 59626, Republic of Korea.
Gene. 2018 Jan 15;640:57-65. doi: 10.1016/j.gene.2017.10.008. Epub 2017 Oct 10.
Carbonic anhydrase VI (CA VI) has been characterized as a secretory isozyme in mammals. Our present study confirmed the occurrence of CA VI in pufferfish (Takifugu rubripes). In this study, genomic sequence information for the CA VI of pufferfish was used for molecular cloning. We cloned a 1821 bp cDNA sequence, which consisted of a complete coding sequence of 1623bp and a deduced amino acid sequence of 540 amino acids from the open reading frame. A BLAST search indicated that this protein exhibits 53%, 79%, and 67% identity with human, tilapia, and gar CA VI, respectively. It also shows 63%-77% identity with other fish CA VI-like sequences (zebrafish, Asian arowana, salmon, and large yellow croaker). Moreover, alignment of two or more sequences revealed that the protein sequence of pufferfish CA VI has 34%-37% identity with mammalian and fish CA II sequences. An NH-terminal signal peptide of 18 amino acids in length was predicted in the pufferfish CA VI sequence. Three potential N-linked glycosylation sites and two cysteine residues (Cys-28 and Cys-209) that are likely to form one disulfide bond were present in pufferfish CA VI. In silico and phylogenetic analyses revealed that pufferfish CA VI is an extracellular secretory protein. Active site analysis indicated that this protein is a low-activity CA isozymes due to a characteristic Val/Ile substitution at position 207. Homology modeling of puffer CA VI was performed using the crystal structure of human carbonic anhydrase XIV as a template structure, based on high similarity. Reverse transcription-polymerase chain reaction (PCR), quantitative PCR and in situ hybridization results revealed that, the pufferfish CA VI is highly expressed in liver tissue.
碳酸酐酶VI（CA VI）在哺乳动物中被表征为一种分泌型同工酶。我们目前的研究证实了河豚（红鳍东方鲀）中存在CA VI。在本研究中，河豚CA VI的基因组序列信息被用于分子克隆。我们克隆了一个1821 bp的cDNA序列，其由1623 bp的完整编码序列和来自开放阅读框的540个氨基酸的推导氨基酸序列组成。BLAST搜索表明，该蛋白与人、罗非鱼和雀鳝的CA VI分别具有53%、79%和67%的同一性。它与其他鱼类CA VI样序列（斑马鱼、亚洲龙鱼、鲑鱼和大黄鱼）也显示出63%-77%的同一性。此外，两个或更多序列的比对显示河豚CA VI的蛋白序列与哺乳动物和鱼类CA II序列具有34%-37%的同一性。在河豚CA VI序列中预测到一个长度为18个氨基酸的N端信号肽。河豚CA VI中存在三个潜在的N-糖基化位点和两个可能形成一个二硫键的半胱氨酸残基（Cys-28和Cys-209）。电子分析和系统发育分析表明，河豚CA VI是一种细胞外分泌蛋白。活性位点分析表明，由于在207位存在特征性的Val/Ile取代，该蛋白是一种低活性的CA同工酶。基于高度相似性，以人碳酸酐酶XIV的晶体结构为模板结构对河豚CA VI进行了同源建模。逆转录-聚合酶链反应（PCR）、定量PCR和原位杂交结果显示，河豚CA VI在肝脏组织中高度表达。
