Ryuzono Sena, Takase Ryo, Kamada Yuko, Ikenaga Takanori, Chigwechokha Petros Kingstone, Komatsu Masaharu, Shiozaki Kazuhiro
Faculty of Fisheries, Kagoshima University, Kagoshima, Japan.
Faculty of Science, Kagoshima University, Kagoshima, Japan.
Biochimie. 2017 Apr;135:63-71. doi: 10.1016/j.biochi.2017.01.008. Epub 2017 Jan 20.
Sialidase catalyzes the removal of sialic acids from glycoconjugates. Recently, medaka sialidase Neu1 has been cloned and its enzymatic properties were investigated. Although enzymatic properties of this sialidase, such as optimal pH and substrate specificity, exhibits high similarity with human NEU1, Neu1 physiological functions in fish are still unclear. Here, to understand Neu1 significance in medaka embryogenesis, sialidase translation knockdown was carried out with one-cell stage fertilized egg using morpholino oligo injection. Neu1 exhibited desialylation of α2-3 sialic acid linkage in vitro and lysosomal localization in medaka caudal fin primary cells. Chloroquine treatment, inhibitor of lysosomal enzymes, caused an accumulation of α2-3 sialo-glycoproteins in the primary cells. During the embryogenesis neu1 mRNA level was elevated until 3.5 day post fertilization (dpf) while an initial decrease of α2-3 sialo-glycoprotein was observed around the same developmental stage. Neu1 knockdown by morpholino oligo induced some abnormal phenotypes such as delay of yolk sac absorption and small embryos. Sialidase-knockdown embryos also showed increase of heart rate in 5.5 and 6.5 dpf. Furthermore, about 37% decrease of hatching rate was observed in Neu1-MO treated embryos compared with control MO. Embryos showing severe phenotypes stopped embryogenesis at the late stage of development. Alteration of embryonic sialo-glycoproteins induced by morpholino injection was examined by lectin blotting to clarify the mechanism of abnormal development. As a result, degradation of several α2-3 sialo-glycoproteins was suppressed in Neu1-MO embryo, possibly induced by the interruption of lysosomal desialylation toward yolk glycoprotein. Our results suggest that medaka Neu1 could be crucial for embryonic development through the degradation of yolk sac nutrition.
唾液酸酶催化从糖缀合物中去除唾液酸。最近，青鳉唾液酸酶Neu1已被克隆，并对其酶学性质进行了研究。尽管这种唾液酸酶的酶学性质，如最适pH和底物特异性，与人类NEU1表现出高度相似性，但Neu1在鱼类中的生理功能仍不清楚。在此，为了了解Neu1在青鳉胚胎发育中的意义，使用吗啉代寡核苷酸注射在单细胞期受精卵中进行了唾液酸酶翻译敲低。Neu1在体外表现出对α2-3唾液酸连接的去唾液酸化作用，并且在青鳉尾鳍原代细胞中定位于溶酶体。溶酶体酶抑制剂氯喹处理导致原代细胞中α2-3唾液酸糖蛋白的积累。在胚胎发育过程中，neu1 mRNA水平在受精后3.5天（dpf）之前升高，而在相同发育阶段观察到α2-3唾液酸糖蛋白最初减少。通过吗啉代寡核苷酸敲低Neu1诱导了一些异常表型，如卵黄囊吸收延迟和胚胎较小。唾液酸酶敲低的胚胎在5.5和6.5 dpf时也显示心率增加。此外，与对照吗啉代相比，在Neu1-MO处理的胚胎中观察到孵化率降低约37%。表现出严重表型的胚胎在发育后期停止胚胎发育。通过凝集素印迹法检测吗啉代注射诱导的胚胎唾液酸糖蛋白的变化，以阐明异常发育的机制。结果，在Neu1-MO胚胎中，几种α2-3唾液酸糖蛋白的降解受到抑制，这可能是由于溶酶体对卵黄糖蛋白的去唾液酸化作用中断所致。我们的结果表明，青鳉Neu1可能通过卵黄囊营养的降解对胚胎发育至关重要。
