Faculty of Fisheries, Kagoshima University, Kagoshima, Japan.
Faculty of Fisheries, Kagoshima University, Kagoshima, Japan; The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima, Japan.
Biochimie. 2018 Jun;149:92-104. doi: 10.1016/j.biochi.2018.04.003. Epub 2018 Apr 7.
Sialidase catalyzes the removal of sialic acids from glycoconjugates. Different from Neu1 and Neu3 sialidases, Neu4 enzymatic properties such as substrate specificity and subcellular localization are not well-conserved among vertebrates. In fish only zebrafish and medaka neu4 genes have been cloned and their polypeptides have been characterized so far. Thus, characterization of Neu4 from other fish species is necessary to evaluate Neu4 physiological functions. Here, Nile tilapia was chosen for the characterization of Neu4 polypeptide considering that it is one of the major cultured fish all over the world and that its genomic sequences are now available. Coding DNA sequence of tilapia Neu4 was identified as 1,497 bp and its recombinant protein showed broad substrate specificity and optimal sialidase enzyme activity pH at 4.0. Neu4 activity was sustained even in neutral and alkali pH. Interestingly, immunofluorescence analysis revealed that major subcellular localization of tilapia Neu4 was nuclear, quite distinct from zebrafish (ER) and medaka Neu4 (lysosome). Bioinformatic analysis showed the existence of putative nuclear localization signal (NLS) in tilapia Neu4. In general, it is known that importin families bind to several proteins via NLS and transfer them into nucleus. Therefore, to determine the involvement of putative NLS in Neu4 nuclear localization, Neu4 mutant deleting NLS was constructed and expressed in cultured cells. As a result, NLS deletion significantly diminished the nuclear localization. Furthermore, treatment of importazole, interrupter of binding importin β and RanGTP, significantly suppressed Neu4 nuclear localization. In summary, tilapia Neu4 is a unique sialidase localized at nucleus and its transport system into nucleus is regulated by importin.
唾液酸酶催化糖缀合物中唾液酸的去除。与 Neu1 和 Neu3 唾液酸酶不同，Neu4 的酶学特性，如底物特异性和亚细胞定位，在脊椎动物中没有很好地保守。在鱼类中，只有斑马鱼和青鳉克隆了 neu4 基因，并且迄今为止已经对其多肽进行了表征。因此，有必要对其他鱼类的 Neu4 进行表征，以评估 Neu4 的生理功能。考虑到尼罗罗非鱼是世界上主要的养殖鱼类之一，并且其基因组序列现在已经可用，因此选择尼罗罗非鱼来表征 Neu4 多肽。鉴定出罗非鱼 Neu4 的编码 DNA 序列为 1497bp，其重组蛋白表现出广泛的底物特异性和最佳的唾液酸酶活性 pH 值为 4.0。Neu4 活性在中性和碱性 pH 值下仍能维持。有趣的是，免疫荧光分析显示，罗非鱼 Neu4 的主要亚细胞定位是核内，与斑马鱼（内质网）和青鳉 Neu4（溶酶体）明显不同。生物信息学分析表明罗非鱼 Neu4 存在潜在的核定位信号（NLS）。一般来说，已知 importin 家族通过 NLS 与几种蛋白质结合，并将它们转运到细胞核内。因此，为了确定潜在 NLS 在 Neu4 核定位中的作用，构建并在培养细胞中表达了缺失 NLS 的 Neu4 突变体。结果表明，NLS 缺失显著减少了 Neu4 的核定位。此外，用 importazole（一种抑制 importin β 结合和 RanGTP 的抑制剂）处理，显著抑制了 Neu4 的核定位。综上所述，罗非鱼 Neu4 是一种独特的定位于核内的唾液酸酶，其向核内的转运系统受 importin 调节。
