The substrate specificity of the snail Lymnaea stagnalis UDP-GlcNAc: GlcNAcβ-R β4-N-acetylglucosaminyltransferase reveals a novel variant pathway of complex-type oligosaccharide synthesis

Abstract

UDP-GlcNAc:GlcNAcβ-R β1→4-N-acetylglucosaminyltransferase (β4-GlcNAcT) of the snail Lymnaea stagnalis is an enzyme with structural resemblance to mammalian β4-galactosyltransferases (β4-GalT). The enzyme, which is present in the prostate gland of the snail, was expressed in a recombinant form in insect cells using the baculovirus technology. This form was used to determine the enzyme's ill vitro substrate specificity in order to assess its possible role in vivo. The enzyme appeared to be a genuine GlcNAcT as no UDP-sugar other than UDP-GlcNAc could serve as an efficient donor substrate. Acceptor specificity studies showed that the enzyme is far more restricted in acceptor usage than β4-GalT. Oligomers of β4-GlcNAc were relatively poor accepters, indicating that this enzyme is not involved in the synthesis of chitin-like molecules. Both ifs polypeptide structure and acceptor specificity suggest that it neither is implicated in the synthesis of the chitobiose core of N-linked glycans. Preferred substrates are those that contain a β-GlcNAc residue attached to the carbon-6 of Gal or GalNAc residues, as found in vertebrate blood-group I-active and O-linked core 2- and 4-based oligosaccharides, respectively. By contrast, compounds in which GlcNAc is β6-linked to Man (as in N-linked glycans) are poor acceptors. Analysis of the products formed in vitro by 1H-MMR spectroscopy and acetolysis showed that the enzyme establishes a GlcNAcβ1→4GlcNAc linkage and shows branch specificity with a blood-group I-active trisaccharide substrate. Furthermore, the enzyme differs from β4-GalT in that it is not responsive to α-lactalbumin. It is proposed that the enzyme functions in a novel, variant pathway of complex-type oligosaccharide synthesis in the snail.