Abstract
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.
Highlights
The complex-type of oligosaccharides are synthesized through elongation by glycosyltransferases after trimming of the precursor oligosaccharides transferred to proteins in the endoplasmic reticulum
The addition of a GlcNAc1– 6 at Man␣1– 6 residue of the core-PA structure (i.e. GnT-V product) increased the transfer rate about 150% (Gn3(6Ј,2Ј,2)core-PA against Gn2(2Ј,2)core-PA), while the addition of a GlcNAc1– 4 at the Man1– 4 residue of the core-PA structure (Gn3(2Ј,2,bis)core-PA, GnT-III products) prevented the transfer
A new 1,4-GlcNAc transferase was purified from the membrane fraction of bovine intestine
Summary
The complex-type of oligosaccharides are synthesized through elongation by glycosyltransferases after trimming of the precursor oligosaccharides transferred to proteins in the endoplasmic reticulum. N-Acetylglucosaminyltransferases (GnTs) take part in the formation of branches in the biosynthesis of complex-type sugar chains. Ʈ To whom correspondence should be addressed: Central Laboratories for Key Technology, KIRIN Brewery Co., Ltd., 1-13-5 Fukuura, Kanazawa-ku Yokohama 236, Japan. The complex-type sugar chains play important roles in many biological phenomena, such as control of glycoprotein hormone activity and cell-cell interactions. Highly branched complex-type sugar chains are essential for the effective expression of in vivo biological activity of human erythropoietin since the activity correlates to the ratio of tetra-antennary to biantennary oligosaccharides (2). Yoshimura et al (5) demonstrated that the sugar chains produced by the action of GnT-V are involved in invasion and cell attachment in the extravation stage of lung metastasis (5)
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