Purification and Characterization of UDP-GlcNAc: GlcNAcβ1–6(GlcNAcβ1–2)Manα1-R [GlcNAc to Man]-β1, 4-N-acetylglucosaminyltransferase VI from Hen Oviduct

• Published in: The Journal of biological chemistry Vol. 275; no. 42; p. 32598
• Main Authors: Tomohiko Taguchi, Tomoya Ogawa, Sadako Inoue, Yasuo Inoue, Yoshihiro Sakamoto, Hiroaki Korekane, Naoyuki Taniguchi
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 20.10.2000
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M004673200

A new β1,4- N -acetylglucosaminyltransferase (GnT) responsible for the formation of branched N -linked complex-type sugar chains has been purified 64,000-fold in 16% yield from a homogenate of hen oviduct by column chromatography procedures using Q-Sepharose FF, Ni 2+ -chelating Sepharose FF, and UDP-hexanolamine-agarose. This enzyme catalyzes the transfer of GlcNAc from UDP-GlcNAc to tetraantennary oligosaccharide and produces pentaantennary oligosaccharide with the β1–4-linked GlcNAc residue on the Manα1–6 arm. It requires a divalent cation such as Mn 2+ and has an apparent molecular weight of 72,000 under nonreducing conditions. The enzyme does not act on biantennary oligosaccharide (GnT I and II product), and β1,6- N -acetylglucosaminylation of the Manα1–6 arm (GnT V product) is essential for its activity. This clearly distinguishes it from GnT IV, which is known to generate a β1–4-linked GlcNAc residue only on the Manα1–3 arm. Based on these findings, we conclude that this enzyme is UDP-GlcNAc:GlcNAcβ1–6(GlcNAcβ1–2)Manα1-R [GlcNAc to Man]-β1,4- N -acetylglucosaminyltransferase VI. This is the only known enzyme that has not been previously purified among GnTs responsible for antenna formation on the cores of N -linked complex-type sugar chains.