Abstract
A new beta1,4-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 beta1-4-linked GlcNAc residue on the Manalpha1-6 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 beta1,6-N-acetylglucosaminylation of the Manalpha1-6 arm (GnT V product) is essential for its activity. This clearly distinguishes it from GnT IV, which is known to generate a beta1-4-linked GlcNAc residue only on the Manalpha1-3 arm. Based on these findings, we conclude that this enzyme is UDP-GlcNAc:GlcNAcbeta1-6(GlcNAcbeta1-2)Manalpha1-R [GlcNAc to Man]-beta1,4-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.
Highlights
To obtain insights into the functions of the N-linked glycans, it is essential to purify and to clone the enzymes responsible for their biosynthesis
GnT VI activity is defined as that catalyzing the transfer of GlcNAc to the Man␣1– 6 arm and forms GlcNAc1– 4Man␣1– 6 linkage
Substantial amounts of proteins were separated from GnT VI by Q-Sepharose FF and Ni2ϩchelating Sepharose FF chromatographies (Fig. 3, A and B)
Summary
GnTs, N-acetylglucosaminyltransferases; PA, 2-aminopyridine; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; DTT, dithiothreitol. GlcNAc residue, were found in hen ovomucoid (2, 3) and in the fish egg glycoprotein known as hyosophorin (4). GnTs I–V have been purified, and the corresponding genes have been cloned (5–19). GnT VI has not been purified, and its gene structure remains unknown. This study reports the purification of GnT VI from hen oviduct, which has been previously shown to have high activity (20). By successive column chromatographies using Q-Sepharose FF, Ni2ϩ-chelating Sepharose FF, and UDP-hexanolamine-agarose with a newly developed assay method (21) wherein pyridylaminated agalactotetraantennary oligosaccharide ([(2,4),(2,6)]-PA) (see Fig. 2) was used as an acceptor substrate and the reaction product was pyridylaminated agalactopentaantennary oligosaccharide ([(2,4),(2,4,6)]-PA) (see Fig. 2), this enzyme was purified 64,000-fold from a homogenate of hen oviduct. The purified enzyme was shown to have an absolute requirement of GlcNAc1–2(GlcNAc1– 6)Man␣1-R substrate sequence. The GnT VI enzyme is distinct from GnT IV, which generates a 1– 4linked GlcNAc residue only on the Man␣1–3 arm (13)
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