title: Molecular cloning and biochemical characterization of medaka (Oryzias latipes) lysosomal neu4 sialidase.
abstract: Glycoconjugates are known to be involved in many physiological events in vertebrates. Sialidase is one of the glycosidases, which removes sialic acid from glycoconjugates. In mammals, the properties and physiological functions of sialidases have been investigated, while there is little understanding of fish sialidase. Here, to investigate the significance of fish neu4 sialidase, neu4 gene was cloned from medaka brain mRNA and identified. Sialidase-specific motifs (GPG, YRVP and Asp-Box) were well conserved in the medaka neu4 polypeptide. Optimal pH of medaka neu4 sialidase was 4.6, but its activity was sustained even at neutral and weak alkaline pH. The neu4 considerably cleaved sialic acid from 4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid and sialyllactose, but not from ganglioside and fetuin, which are good substrates for human NEU4. neu4 activity was mostly detected in mitochondria/lysosome fraction after biochemical fractionation, and indirect immunofluorescence assays revealed neu4 localization in lysosome in neu4 overexpressed cells. Next, developmental change in medaka neu4 and other sialidase mRNA levels were estimated by real-time PCR. Each sialidases showed different expression patterns in embryonic development: neu4 was up-regulated at late developmental stage in embryo, and neu3a mRNA level was quite high in 0.5 dpf. On the other hand, neu3b expression was drastically increased after hatching, suggesting that each sialidase may play a different role in embryonic development.
authors.affiliation: Faculty of Fisheries, Kagoshima University, 4-50-20 Shimo-arata, Kagoshima, 890-0056, Japan, shiozaki@fish.kagoshima-u.ac.jp.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Shiozaki
authors.forename: Sena
authors.initials: S
authors.lastname: Ryuzono
authors.forename: Naoto
authors.initials: N
authors.lastname: Matsushita
authors.forename: Asami
authors.initials: A
authors.lastname: Ikeda
authors.forename: Kazuki
authors.initials: K
authors.lastname: Takeshita
authors.forename: Petros Kingstone
authors.initials: PK
authors.lastname: Chigwechokha
authors.forename: Masaharu
authors.initials: M
authors.lastname: Komatsu
authors.forename: Taeko
authors.initials: T
authors.lastname: Miyagi
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000818:Animals; D001921:Brain / Q000378:metabolism*; D005591:Chemical Fractionation; D003001:Cloning, Molecular; D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D019084:Fluorescent Antibody Technique, Indirect / Q000662:veterinary; D018507:Gene Expression Regulation, Developmental / Q000235:genetics / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000201:enzymology*; D009439:Neuraminidase / Q000235:genetics* / Q000378:metabolism; D009990:Oryzias / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D012333:RNA, Messenger; D009439:Neuraminidase
references.citation: Biochem J. 2007 Dec 1;408(2):211-9
references.pmid: 17708748
references.citation: Biochem J. 2007 Dec 15;408(3):395-406
references.pmid: 17708749
references.citation: Nat Methods. 2011 Sep 29;8(10):785-6
references.pmid: 21959131
references.citation: J Biol Chem. 2006 Mar 24;281(12):7756-64
references.pmid: 16428383
references.citation: FEBS Lett. 2004 May 21;566(1-3):178-82
references.pmid: 15147891
references.citation: J Biol Chem. 2007 Apr 27;282(17):12484-91
references.pmid: 17327233
references.citation: Oncogene. 2007 Apr 12;26(17):2483-90
references.pmid: 17334392
references.citation: Toxicol Sci. 2007 Feb;95(2):356-68
references.pmid: 17093204
references.citation: Glycobiology. 2005 Feb;15(2):153-64
references.pmid: 15385431
references.citation: FEMS Microbiol Lett. 2013 Apr;341(1):18-26
references.pmid: 23320941
references.citation: Oncogene. 2009 Mar 5;28(9):1218-29
references.pmid: 19151752
references.citation: Biochem Biophys Res Commun. 2001 Jan 26;280(3):726-32
references.pmid: 11162581
references.citation: Glycobiology. 2012 Jul;22(7):880-96
references.pmid: 22377912
references.citation: Int J Cancer. 2001 Jun 15;92(6):797-804
references.pmid: 11351298
references.citation: PLoS One. 2013 Jun 12;8(6):e65457
references.pmid: 23776483
references.citation: Biochem Biophys Res Commun. 1996 Apr 25;221(3):826-30
references.pmid: 8630046
references.citation: Cancer Sci. 2009 Apr;100(4):588-94
references.pmid: 19215228
references.citation: Biochimie. 2013 Feb;95(2):280-9
references.pmid: 23032629
references.citation: Proteomics. 2004 Jun;4(6):1633-49
references.pmid: 15174133
references.citation: Curr Opin Chem Biol. 2008 Oct;12(5):565-72
references.pmid: 18625334
references.citation: J Mol Biol. 1999 Dec 17;294(5):1351-62
references.pmid: 10600390
references.citation: PLoS One. 2012;7(8):e44193
references.pmid: 22952925
references.citation: Biochem J. 2005 Aug 15;390(Pt 1):85-93
references.pmid: 15847605
references.citation: J Biol Chem. 2009 Aug 7;284(32):21157-64
references.pmid: 19506080
references.citation: J Biol Chem. 2004 Aug 27;279(35):37021-9
references.pmid: 15213228
references.citation: Mech Dev. 2004 Jul;121(7-8):605-18
references.pmid: 15210170
references.citation: Biochem Biophys Res Commun. 2005 Jul 29;333(2):367-73
references.pmid: 15979459
references.citation: Am J Pathol. 2008 Oct;173(4):1042-56
references.pmid: 18772331
references.citation: Glycobiology. 2010 Feb;20(2):148-57
references.pmid: 19797320
references.citation: J Biol Chem. 2011 Jun 17;286(24):21052-61
references.pmid: 21521691
references.citation: FEBS Lett. 2007 Feb 6;581(3):406-12
references.pmid: 17234188