N-Acetylglucosaminyltransferase IX Acts on the GlcNAcβ1,2-Manα1-Ser/Thr Moiety, Forming a 2,6-Branched Structure in Brain O-Mannosyl Glycan*

By K. Inamori, T. Endo, J. Gu, I. Matsuo, Yukishige Ito, S. Fujii, H. Iwasaki, H. Narimatsu, E. Miyoshi, K. Honke and 1 more. Journal of Biological Chemistry, 2004

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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 β1,2-N-acetylglucosaminyltransferase (POMGnT1), but the enzymatic basis of the transfer to the 6-OH position is unknown. We recently reported on a brain-specific β1,6-N-acetylglucosaminyltransferase, GnT-IX, that catalyzes the transfer of GlcNAc to the 6-OH position of the mannose residue of GlcNAcβ1,2-Manα on both the α1,3- and α1,6-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 GlcNAcβ1,2-Manα in O-mannosyl glycan. Using three synthetic Ser-linked mannose-containing saccharides, Manα1-Ser, GlcNAcβ1,2-Manα1-Ser, and Galβ1,4-GlcNAcβ1,2-Manα1-Ser as acceptor substrates, the findings show that 14C-labeled GlcNAc was incorporated only into GlcNAcβ1,2-Manα1-Ser after separation by thin layer chromatography. To simplify the assay, high performance liquid chromatography was employed, using a fluorescence-labeled acceptor substrate GlcNAcβ1,2-Manα1-Ser-pyridylaminoethylsuccinamyl (PAES). Consistent with the above data, GnT-IX generated a new product which was identified as GlcNAcβ1,2-(GlcNAcβ1,6-)Manα1-Ser-PAES by mass spectrometry and 1H NMR. Furthermore, incorporation of an additional GlcNAc residue into a synthetic mannosyl peptide Ac-Ala-Ala-Pro-Thr(Man)-Pro-Val-Ala-Ala-Pro-NH2 by GnT-IX was only observed in the presence of POMGnT1. Collectively, these results strongly suggest that GnT-IX may be a novel β1,6-N-acetylglucosaminyltransferase that is responsible for the formation of the 2,6-branched structure in the brain O-mannosyl glycan.

Subfields: Immunology, Molecular Biology, Organic Chemistry

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