cDNA Cloning and Expression of Bovine UDP-N-Acetylglucosamine:α1,3-d-Mannoside β1,4-N-Acetylglucosaminyltransferase IV

Abstract

UDP-N-acetylglucosamine:␣1,3-D-mannoside ␤1,4-Nacetylglucosaminyltransferase (GnT-IV) is one of the essential enzymes in the production of tri-and tetra-antennary Asn-linked sugar chains. Recently, we have successfully purified GnT-IV from bovine small intestine. Based on the partial amino acid sequence of the purified bovine GnT-IV enzyme, its cDNA has been cloned from bovine small intestine. The open reading frame is 1,605 base pairs long, and this sequence produced GnT-IV activity on transient expression in COS-7 cells. Although the deduced amino acid sequence does not have any significant homology with other known N-acetylglucosaminyltransferases (GnTs), the hydrophobicity profile showed a typical type II transmembrane protein structure, which is common to many glycosyltransferases. N-terminal amino acid sequencing of the purified GnT-IV revealed that 92 amino acids, including a transmembrane region, were truncated during purification. Of the three potential N-glycosylation sites Asn-458 was actually glycosylated in the purified enzyme, although this N-glycosylation site could be abolished without any reduction in GnT-IV activity. Serial deletions at both the N and C termini proved that the catalytic domain of GnT-IV is located in the central region of the enzyme. The GnT-IV mRNA level correlated with enzymatic activity in the various bovine tissues tested.UDP-N-acetylglucosamine:␣1,3-D-mannoside ␤1,4-N-acetylglucosaminyltransferase (GnT-IV) 1 (EC 2.4.1.145) is one of the essential enzymes in the synthesis of the multiantennary structure of Asn-linked sugar chains. It specifically generates the ␤1-4 linkage of GlcNAc to the ␣1-3 Man of the core structure of N-glycans (Man 3 GlcNAc 2 ). GnT-IV produces tetra-antennary structures in cooperation with GnT-V, which produces the ␤1-6 linkage of GlcNAc to the ␣1-6 Man of the core structure. This tetra-antennary structure is dominant in certain glycoproteins, such as erythropoietin (EPO) and ␣ 1 -acid glycoprotein, and very important for their bioactivity (1, 2). For instance, the in vivo biological activity of recombinant EPO has a positive correlation with the ratio of tetra-/biantennary sugar chains. Pharmacokinetics studies have shown that the bulky structure of the tetra-antennary branching prevents EPO from filtering out into the urine. The increase in the antenna number of ␣ 1 -acid glycoprotein observed under a chronic inflammatory state or liver damage suggested that some clinical situations and/or cellular disorders may also affect the branch formation of sugar chains (3). On the other hand, structural analyses of oligosaccharides in hepatoma cell lines and in choriocarcinoma have suggested that GnT-IV activity should be high in certain kinds of tumors (4 -7). Rat 3Y1 cells transformed with the adenovirus 12 gene also elevated the expression of the ␤1-4 GlcNAc on the ␣1-3 Man (8).To date, GnT-I, -II, -III, and -V have been purified and their cDNAs and/or genomic DNAs have been cloned (9 -23). Among the enzymes necessary to produce tetra-antennary sugar cha...

Cloning and characterization of the human UDP-N-acetylglucosamine: α-1,3-D-mannoside β-1,4-N-acetylglucosaminyltransferase IV-homologue (hGnT-IV-H) gene

“…GnT-IV is an essential enzyme for the synthesis of the multiantennary structure of asparagine (Asn)-linked oligosaccharides, which are mainly contained in erythropoietin and α1 acid glycoprotein (Gleeson and Schachter 1983;Minowa et al 1998). Together with GnT-V, this protein contributes to phenotypic changes in neoplastic transformed cells by inducing a structural change in cell surface carbohydrates, known as increased sialyation, and by 1-6-linked branching of complex-type Asn-linked oligosaccharides (Schachter, 1986).…”

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

“…Examples of the most highly branched N-linked complex-type glycan, which is a pentaantennary glycan with a bisecting 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)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Only GnT VI has not been purified, and its gene structure remains unknown.…”

Branched N-glycans and their implications for cell adhesion, signaling and clinical applications for cancer biomarkers and in therapeutics

“…The enzyme was identified for the first time in the hen oviduct by Schachter's group (10). A simple assay method was established by our group and the enzyme was successfully purified from bovine small intestine using affinity chromatography, in collaboration with our group in 1997 (43) and Minowa et al subsequently cloned its cDNA (44). The regulation of GnT-IV expression is particularly interesting because enzymatic activity increases during oncogenesis and differentiation.…”