title: Specific enzyme complex of beta-1,4-galactosyltransferase-II and glucuronyltransferase-P facilitates biosynthesis of N-linked human natural killer-1 (HNK-1) carbohydrate.
abstract: Human natural killer-1 (HNK-1) carbohydrate is highly expressed in the nervous system and is involved in synaptic plasticity and dendritic spine maturation. This unique carbohydrate, consisting of a sulfated trisaccharide (HSO(3)-3GlcAβ1-3Galβ1-4GlcNAc-), is biosynthesized by the successive actions of β-1,4-galactosyltransferase (β4GalT), glucuronyltransferase (GlcAT-P and GlcAT-S), and sulfotransferase (HNK-1ST). A previous study showed that mice lacking β4GalT-II, one of seven β4GalTs, exhibited a dramatic loss of HNK-1 expression in the brain, although β4GalT-I-deficient mice did not. Here, we investigated the underlying molecular mechanism of the regulation of HNK-1 expression. First, focusing on a major HNK-1 carrier, neural cell adhesion molecule, we found that reduced expression of an N-linked HNK-1 carbohydrate caused by a deficiency of β4GalT-II is not likely due to a general loss of the β1,4-galactose residue as an acceptor for GlcAT-P. Instead, we demonstrated by co-immunoprecipitation and endoplasmic reticulum-retention analyses using Neuro2a (N2a) cells that β4GalT-II physically and specifically associates with GlcAT-P. In addition, we revealed by pulldown assay that Golgi luminal domains of β4GalT-II and GlcAT-P are sufficient for the complex to form. With an in vitro assay system, we produced the evidence that the kinetic efficiency k(cat)/K(m) of GlcAT-P in the presence of β4GalT-II was increased about 2.5-fold compared with that in the absence of β4GalT-II. Finally, we showed that co-expression of β4GalT-II and GlcAT-P increased HNK-1 expression on various glycoproteins in N2a cells, including neural cell adhesion molecule. These results indicate that the specific enzyme complex of β4GalT-II with GlcAT-P plays an important role in the biosynthesis of HNK-1 carbohydrate.
authors.affiliation: Department of Biological Chemistry, Human Health Sciences, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Kouno
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Kizuka
authors.forename: Naoki
authors.initials: N
authors.lastname: Nakagawa
authors.forename: Toru
authors.initials: T
authors.lastname: Yoshihara
authors.forename: Masahide
authors.initials: M
authors.lastname: Asano
authors.forename: Shogo
authors.initials: S
authors.lastname: Oka
mesh_terms: D000818:Animals; D018959:CD57 Antigens / Q000096:biosynthesis*; D002460:Cell Line; D005700:Galactosyltransferases / Q000172:deficiency / Q000378:metabolism*; D005786:Gene Expression Regulation; D014453:Glucuronosyltransferase / Q000172:deficiency / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D053858:Metabolic Networks and Pathways; D051379:Mice; D018345:Mice, Knockout
chemical_list: D018959:CD57 Antigens; D005700:Galactosyltransferases; C419407:beta-1,4-galactosyltransferase II; D014453:Glucuronosyltransferase