Ryuzono Sena, Takase Ryo, Oishi Kazuki, Ikeda Asami, Chigwechokha Petros Kingstone, Funahashi Aki, Komatsu Masaharu, Miyagi Taeko, Shiozaki Kazuhiro
Faculty of Fisheries, Kagoshima University, Kagoshima, Japan.
Faculty of Fisheries, Kagoshima University, Kagoshima, Japan; The United Graduate School of Agricultural Science, Kagoshima University, Kagoshima, Japan.
Gene. 2016 Jan 10;575(2 Pt 2):513-523. doi: 10.1016/j.gene.2015.09.028. Epub 2015 Oct 8.
Desialylation in the lysosome is a crucial step for glycoprotein degradation. The abnormality of lysosomal desialylation by NEU1 sialidase is involved in diseases of mammals such as sialidosis and galactosialidosis. Mammalian Neu1 sialidase is also localized at plasma membrane where it regulates several signaling pathways through glycoprotein desialylation. In fish, on the other hand, the mechanism of desialylation in the lysosome and functions of Neu1 sialidase are still unclear. Here, to understand the significance of fish Neu1 sialidase, neu1 gene was cloned from medaka brain and the profiles of its polypeptides were analyzed. Open reading frame of medaka neu1 consisted 1,182 bp and the similarity of its deduced amino acids with human NEU1 was 57%. As this recombinant polypeptide did not show significant sialidase activity, medaka cathepsin A, known in mammals as protective protein activating Neu1, was cloned and then co-expressed with medaka Neu1 to examine whether medaka cathepsin A activates Neu1 activity. As a result, Neu1/cathepsin A showed a drastic increase of sialidase activity toward MU-NANA. Major substrate of medaka Neu1 was 3-sialyllactose and its optimal pH was 4.0. With immunofluorescence analysis, signal of overexpressed medaka Neu1 was found to coincide with Lysotracker signals (organelle marker of lysosome) and co-localized with medaka cathepsin A in fish hepatic Hepa-T1 cells. Furthermore, part of medaka Neu1 was also detected at plasma membrane. Medaka Neu1 possessed signal peptide sequence at N-terminal and incomplete lysosomal targeting sequence at C-terminus. Medaka neu1 gene was ubiquitously expressed in various medaka tissues, and its expression level was significantly higher than other sialidase genes such as neu3a, neu3b and neu4. The present study revealed the profiles of fish Neu1 sialidase and indicated its high conservation with human NEU1 for the first time, suggesting the presence of similar desialylation system in the medaka lysosome to human. Moreover, the present study showed the possibility of medaka as a model animal of human NEU1 sialidase.
溶酶体中的去唾液酸化是糖蛋白降解的关键步骤。NEU1唾液酸酶导致的溶酶体去唾液酸化异常与哺乳动物的疾病如唾液酸贮积症和半乳糖唾液酸贮积症有关。哺乳动物的Neu1唾液酸酶也定位于质膜，在那里它通过糖蛋白去唾液酸化调节多种信号通路。另一方面，在鱼类中，溶酶体中的去唾液酸化机制和Neu1唾液酸酶的功能仍不清楚。在此，为了解鱼类Neu1唾液酸酶的重要性，从青鳉脑克隆了neu1基因并分析了其多肽谱。青鳉neu1的开放阅读框由1182个碱基对组成，其推导的氨基酸与人NEU1的相似性为57%。由于这种重组多肽未显示出显著的唾液酸酶活性，因此克隆了在哺乳动物中作为激活Neu1的保护蛋白的青鳉组织蛋白酶A，然后将其与青鳉Neu1共表达，以研究青鳉组织蛋白酶A是否激活Neu1活性。结果，Neu1/组织蛋白酶A对MU-NANA的唾液酸酶活性急剧增加。青鳉Neu1的主要底物是3-唾液酸乳糖，其最适pH为4.0。通过免疫荧光分析，发现过表达的青鳉Neu1信号与溶酶体示踪信号（溶酶体的细胞器标记物）一致，并在鱼类肝脏Hepa-T1细胞中与青鳉组织蛋白酶A共定位。此外，在质膜上也检测到了部分青鳉Neu1。青鳉Neu1在N端具有信号肽序列，在C端具有不完整的溶酶体靶向序列。青鳉neu1基因在青鳉的各种组织中普遍表达，其表达水平显著高于其他唾液酸酶基因如neu3a、neu3b和neu4。本研究揭示了鱼类Neu1唾液酸酶的特征，并首次表明其与人NEU1具有高度保守性，这表明青鳉溶酶体中存在与人类相似的去唾液酸化系统。此外，本研究表明青鳉作为人类NEU1唾液酸酶模型动物的可能性。
