title: N-Acetylglucosaminyltransferase IX acts on the GlcNAc beta 1,2-Man alpha 1-Ser/Thr moiety, forming a 2,6-branched structure in brain O-mannosyl glycan.
abstract: Mammals contain O-linked mannose residues with 2-mono- and 2,6-di-substitutions by GlcNAc in brain glycoproteins. It has been demonstrated that the transfer of GlcNAc to the 2-OH position of the mannose residue is catalyzed by the enzyme, protein O-mannose beta1,2-N-acetylglucosaminyltransferase (POMGnT1), but the enzymatic basis of the transfer to the 6-OH position is unknown. We recently reported on a brain-specific beta1,6-N-acetylglucosaminyltransferase, GnT-IX, that catalyzes the transfer of GlcNAc to the 6-OH position of the mannose residue of GlcNAcbeta1,2-Manalpha on both the alpha1,3- and alpha1,6-linked mannose arms in the core structure of N-glycan (Inamori, K., Endo, T., Ide, Y., Fujii, S., Gu, J., Honke, K., and Taniguchi, N. (2003) J. Biol. Chem. 278, 43102-43109). Here we examined the issue of whether GnT-IX is able to act on the same sequence of the GlcNAcbeta1,2-Manalpha in O-mannosyl glycan. Using three synthetic Ser-linked mannose-containing saccharides, Manalpha1-Ser, GlcNAcbeta1,2-Manalpha1-Ser, and Galbeta1,4-GlcNAcbeta1,2-Manalpha1-Ser as acceptor substrates, the findings show that (14)C-labeled GlcNAc was incorporated only into GlcNAcbeta1,2-Manalpha1-Ser after separation by thin layer chromatography. To simplify the assay, high performance liquid chromatography was employed, using a fluorescence-labeled acceptor substrate GlcNAcbeta1,2-Manalpha1-Ser-pyridylaminoethylsuccinamyl (PAES). Consistent with the above data, GnT-IX generated a new product which was identified as GlcNAcbeta1,2-(GlcNAcbeta1,6-)Manalpha1-Ser-PAES by mass spectrometry and (1)H NMR. Furthermore, incorporation of an additional GlcNAc residue into a synthetic mannosyl peptide Ac-Ala-Ala-Pro-Thr(Man)-Pro-Val-Ala-Ala-Pro-NH(2) by GnT-IX was only observed in the presence of POMGnT1. Collectively, these results strongly suggest that GnT-IX may be a novel beta1,6-N-acetylglucosaminyltransferase that is responsible for the formation of the 2,6-branched structure in the brain O-mannosyl glycan.
authors: Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors: Kei-ichiro
authors: K
authors: Inamori
authors: Takeshi
authors: T
authors: Endo
authors: Jianguo
authors: J
authors: Gu
authors: Ichiro
authors: I
authors: Matsuo
authors: Yukishige
authors: Y
authors: Ito
authors: Shigeru
authors: S
authors: Fujii
authors: Hiroko
authors: H
authors: Iwasaki
authors: Hisashi
authors: H
authors: Narimatsu
authors: Eiji
authors: E
authors: Miyoshi
authors: Koichi
authors: K
authors: Honke
authors: Naoyuki
authors: N
authors: Taniguchi
mesh_terms: D001921:Brain / Q000201:enzymology; D001923:Brain Chemistry; D006801:Humans; D008358:Mannose / Q000031:analogs & derivatives / Q000737:chemistry*; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D009419:Nerve Tissue Proteins; D011134:Polysaccharides; C479741:MGAT5B protein, human; D017351:N-Acetylglucosaminyltransferases; D008358:Mannose