title: Heparan sulfate polymerization in Drosophila.
abstract: The formation of heparan sulfate (HS) chains is catalyzed by glycosyltransferases encoded by EXT (hereditary multiple exostosin gene) family members. Genetic screening for mutations affecting morphogen signaling pathways in Drosophila has identified three genes, tout-velu (ttv), sister of tout-velu (sotv), and brother of toutvelu (botv), which encode homologues of human EXT1, EXT2, and EXTL3, respectively. So far, in vitro glycosyltransferase activities have been demonstrated only for BOTV/DEXTL3, which harbors both N-acetylglucosaminyltransferase-I (GlcNAcT-I) and N-acetylglucosaminyltransferase-II (GlcNAcT-II) activities responsible for the chain initiation and elongation of HS, and no glucuronyltransferase-II (GlcAT-II) activity. Here we demonstrated that TTV/DEXT1 and SOTV/DEXT2 had GlcNAcT-II and GlcAT-II activities required for the biosynthesis of repeating disaccharide units of the HS backbone, and the coexpression of TTV with SOTV markedly augmented both glycosyltransferase activities when compared with the expression of TTV or SOTV alone. Moreover, the polymerization of HS was demonstrated on a linkage region analogue as an acceptor substrate by BOTV and an enzyme complex composed of TTV and SOTV (TTV-SOTV). In contrast to human, TTV-SOTV exhibited no GlcNAcT-I activity, indicating that BOTV/DEXT3, which is an EXT-Like gene and possesses GlcNAcT-I activity required for the initiation of HS, is indispensable for the biosynthesis of HS chains in Drosophila. Thus, all three EXT members in Drosophila, TTV, SOTV, and BOTV, are required for the biosynthesis of full-length HS in Drosophila.
authors.affiliation: Department of Biochemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Izumikawa
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Egusa
authors.forename: Fumiyasu
authors.initials: F
authors.lastname: Taniguchi
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Sugahara
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kitagawa
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D003470:Culture Media / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004187:Disaccharides / Q000737:chemistry; D029721:Drosophila Proteins / Q000235:genetics / Q000502:physiology; D004331:Drosophila melanogaster; D008040:Genetic Linkage; D016695:Glycosyltransferases / Q000737:chemistry; D006497:Heparan Sulfate / Q000737:chemistry*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D009844:Oligosaccharides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D017434:Protein Structure, Tertiary; D000099058:Exostosin 1
chemical_list: D003470:Culture Media; D018076:DNA, Complementary; D004187:Disaccharides; D029721:Drosophila Proteins; D016695:Glycosyltransferases; D006497:Heparan Sulfate; D008565:Membrane Proteins; D017351:N-Acetylglucosaminyltransferases; D009844:Oligosaccharides; D011108:Polymers; D000099058:Exostosin 1; C113073:TTV protein, Drosophila; C487132:Botv protein, Drosophila; C487133:Sotv protein, Drosophila; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I; C063313:alpha-1,6-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase