Purification and characterization of UDP-N-acetylglucosamine: alpha1,3-D-mannoside beta1,4-N-acetylglucosaminyltransferase (N-acetylglucosaminyltransferase-IV) from bovine small intestine.
A new beta1,4-N-acetylglucosaminyltransferase (GnT) which involves in branch formation of Asn-linked complex-type sugar chains has been purified 224,000-fold from bovine small intestine. This enzyme requires divalent cations, such as Mn2+, and catalyzes the transfer of GlcNAc from UDP-GlcNAc to biantennary oligosaccharide and produces triantennary oligosaccharide with the beta1-4-linked GlcNAc residue on the Manalpha1-3 arm. The purified enzyme shows a single band of Mr 58,000 and behaves as a monomer. The substrate specificity demonstrated that the beta1-2-linked GlcNAc residue on the Manalpha1-3 arm (GnT-I product) is essential for the enzyme activity. beta1-4-Galactosylaion to this essential beta1-2-linked GlcNAc residue or N-acetylglucosaminylation to the beta-linked Man residue (bisecting GlcNAc, GnT-III product) blocks the enzyme action, while beta1-6-N-acetylglucosaminylation to the Manalpha1-6 arm (GnT-V product) increases the transfer. Based on these findings, we conclude that the purified enzyme is UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,4-N-acetylglucosaminyltransferase IV (GnT-IV), that has been a missing link on biosynthesis of complex-type sugar chains.
Oguri
S
S
Central Laboratories for Key Technology, KIRIN Brewery Co., Ltd., 1-13-5 Fukuura, Kanazawa-ku, Yokohama 236, Japan.
Minowa
M T
MT
Ihara
Y
Y
Taniguchi
N
N
Ikenaga
H
H
Takeuchi
M
M
D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007421:Intestine, Small / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D017351:N-Acetylglucosaminyltransferases; C038111:alpha-1,3-mannosylglycoprotein beta-1,4-N-acetylglucosaminyltransferase