title: Lysosomal localization of Japanese medaka (Oryzias latipes) Neu1 sialidase and its highly conserved enzymatic profiles with human.
abstract: 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.
authors.affiliation: Faculty of Fisheries, Kagoshima University, Kagoshima, Japan.
authors.forename: Sena
authors.initials: S
authors.lastname: Ryuzono
authors.affiliation: Faculty of Fisheries, Kagoshima University, Kagoshima, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Takase
authors.affiliation: Faculty of Fisheries, Kagoshima University, Kagoshima, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Oishi
authors.affiliation: Faculty of Fisheries, Kagoshima University, Kagoshima, Japan.
authors.forename: Asami
authors.initials: A
authors.lastname: Ikeda
authors.affiliation: Faculty of Fisheries, Kagoshima University, Kagoshima, Japan; The United Graduate School of Agricultural Science, Kagoshima University, Kagoshima, Japan.
authors.forename: Petros Kingstone
authors.initials: PK
authors.lastname: Chigwechokha
authors.affiliation: Faculty of Fisheries, Kagoshima University, Kagoshima, Japan; The United Graduate School of Agricultural Science, Kagoshima University, Kagoshima, Japan.
authors.forename: Aki
authors.initials: A
authors.lastname: Funahashi
authors.affiliation: Faculty of Fisheries, Kagoshima University, Kagoshima, Japan; The United Graduate School of Agricultural Science, Kagoshima University, Kagoshima, Japan.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Komatsu
authors.affiliation: Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, Sendai, Miyagi, Japan.
authors.forename: Taeko
authors.initials: T
authors.lastname: Miyagi
authors.affiliation: Faculty of Fisheries, Kagoshima University, Kagoshima, Japan; The United Graduate School of Agricultural Science, Kagoshima University, Kagoshima, Japan. Electronic address: shiozaki@fish.kagoshima-u.ac.jp.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Shiozaki
mesh_terms: D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D017124:Conserved Sequence; D029941:Fish Proteins / Q000235:genetics* / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D009439:Neuraminidase / Q000235:genetics* / Q000378:metabolism*; D009990:Oryzias / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D029941:Fish Proteins; C486385:NEU1 protein, human; D009439:Neuraminidase
keywords: Cathepsin A; Medaka; Model animal; Neu1; Sialidase