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.

唾液酸酶催化从糖缀合物中除去唾液酸。与Neu1和Neu3唾液酸酶不同，Neu4的酶学性质（例如底物特异性和亚细胞定位）在脊椎动物之间不是很好保守的。在鱼中只有斑马鱼和高加索neu4到目前为止，已经克隆了这些基因，并且已经鉴定了它们的多肽。因此，表征其他鱼类的Neu4对评估Neu4的生理功能是必要的。在这里，考虑到尼罗罗非鱼是世界上主要的养殖鱼类之一，并且现在已经有了其基因组序列，因此选择尼罗罗非鱼来表征Neu4多肽。罗非鱼Neu4的编码DNA序列鉴定为1,497 bp，其重组蛋白在4.0的条件下具有广泛的底物特异性和最佳的唾液酸酶活性pH。即使在中性和碱性pH值下，Neu4活性也得以维持。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的，与斑马鱼（ER）和medaka Neu4（溶酶体）完全不同。生物信息学分析表明，罗非鱼Neu4中存在假定的核定位信号（NLS）。通常，已知importin家族通过NLS与几种蛋白质结合，然后将它们转移到细胞核中。因此，为了确定推定的NLS参与Neu4核定位，构建了缺失Neu4突变体的NLS并在培养的细胞中表达。结果，NLS缺失显着减少了核定位。此外，治疗importazole，结合importinβ和RanGTP的中断者，可显着抑制Neu4核定位。总而言之，罗非鱼Neu4是一种独特的唾液酸酶，位于细胞核内，其向细胞核内的转运系统受到importin的调节。众所周知，importin家族通过NLS与几种蛋白质结合并转移到细胞核中。因此，为了确定推定的NLS参与Neu4核定位，构建了缺失Neu4突变体的NLS并在培养的细胞中表达。结果，NLS缺失显着减少了核定位。此外，治疗importazole，结合importinβ和RanGTP的中断者，可显着抑制Neu4核定位。总而言之，罗非鱼Neu4是一种独特的唾液酸酶，位于细胞核内，其向核内的转运系统受importin的调节。众所周知，importin家族通过NLS与几种蛋白质结合并转移到细胞核中。因此，为了确定推定的NLS参与Neu4核定位，构建了缺失Neu4突变体的NLS并在培养的细胞中表达。结果，NLS缺失显着减少了核定位。此外，治疗importazole，结合importinβ和RanGTP的中断者，可显着抑制Neu4核定位。总而言之，罗非鱼Neu4是一种独特的唾液酸酶，位于细胞核内，其向细胞核内的转运系统受到importin的调节。NLS删除大大减少了核定位。此外，治疗importazole，结合importinβ和RanGTP的中断者，可显着抑制Neu4核定位。总而言之，罗非鱼Neu4是一种独特的唾液酸酶，位于细胞核内，其向细胞核内的转运系统受到importin的调节。NLS删除大大减少了核定位。此外，治疗importazole，结合importinβ和RanGTP的中断者，可显着抑制Neu4核定位。总而言之，罗非鱼Neu4是一种独特的唾液酸酶，位于细胞核内，其向细胞核内的转运系统受到importin的调节。