Substrate specificity and inhibition of UDP-GlcNAc:GlcNAcβ1-2Manα1-6R β1,6-N-acetylglucosaminyltransferase V using synthetic substrate analogues

UDP-GlcNAc:GlcNAc β1-2Manα1-6R (GlcNAc to Man) β1,6-N-acetylglucosaminyltransferase V (GlcNAc-T V) adds a GlcNAcβ1-6 branch to bi- and triantennary N-glycans. An increase in this activity has been associated with cellular transformation, metastasis and differentiation. We have used synthetic substrate analogues to study the substrate specificity and inhibition of the partially purified enzyme from hamster kidney and of extracts from hen oviduct membranes and acute myeloid leukaemia leukocytes. All compounds with the minimum structure GlcNAcβ1-2Manα1-6Glc/Manβ-R were good substrates for GlcNAc-T V. The presence of structural elements other than the minimum trisaccharide structure affected GlcNAc-T V activity without being an absolute requirement for activity. Substrates with a biantennary structure were preferred over linear fragments of biantennary structures. Kinetic analysis showed that the 3-hydroxyl of the Manα1-3 residue and the 4-hydroxyl of the Manβ- residue of the Manα1-6(Manα1-3)Manβ-R N-glycan core are not essential for catalysis but influence substrate binding. GlcNAcβ1-2(4,6-di-O-methyl-)Manα1-6Glcβ-pnp was found to be an inhibitor of GlcNAc-T V from hamster kidney, hen oviduct microsomes and acute and chronic myeloid leukaemia leukocytes.