THE SUBSTRATE-SPECIFICITY OF THE SNAIL LYMNAEA-STAGNALIS UDP-GLCNAC-GLCNAC-BETA-R BETA-4-N-ACETYLGLUCOSAMINYLTRANSFERASE REVEALS A NOVEL VARIANT PATHWAY OF COMPLEX-TYPE OLIGOSACCHARIDE SYNTHESIS

UDP-GlcNAc:GlcNAc beta-R beta 1-->4-N-acetylglucosaminyl-transferase ( beta 4-GlcNAcT) of the snail Lymnaea stagnalis is an enzyme with struc tural resemblance to mammalian beta 4-galactosyltransferases (beta 4-G alT), The enzyme, which is present in the prostate gland of the snail, was expressed in a recombinant form in insect cells using the baculov irus technology, This form was used to determine the enzyme's in vitro substrate specificity in order to assess its possible role in vivo, T he enzyme appeared to be a genuine GlcNAcT as no UDP-sugar other than UDP-GlcNAc could serve as an efficient donor substrate, Acceptor speci ficity studies showed that the enzyme is far more restricted in accept or usage than beta 4-GalT. Oligomers of beta 4-GlcNAc were relatively poor accepters, indicating that this enzyme is not involved in the syn thesis of chitin-like molecules, Both its polypeptide structure and ac ceptor specificity suggest that it neither is implicated in the synthe sis of the chitobiose core of N-linked glycans, Preferred substrates a re those that contain a beta-GlcNAc residue attached to the carbon-6 o f Gal or GalNAc residues, as found in vertebrate blood-group I-active and O-linked core 2- and 4-based oligosaccharides, respectively, By co ntrast, compounds in which GlcNAc is beta 6-linked to Man (as in N-lin ked glycans) are poor accepters, Analysis of the products formed in vi tro by H-1-NMR spectroscopy and acetolysis showed that the enzyme esta blishes a GlcNAc beta 1-->4GlcNAc linkage and shows branch specificity with a blood-group I-active trisaccharide substrate, Furthermore, the enzyme differs from P4-GalT in that it is not responsive to alpha-lac talbumin, It is proposed that the enzyme functions in a novel, variant pathway of complex-type oligosaccharide synthesis in the snail.