ACCEPTOR SPECIFICITY OF DIFFERENT LENGTH CONSTRUCTS OF HUMAN RECOMBINANT ALPHA-1,3/4-FUCOSYL-TRANSFERASES - REPLACEMENT OF THE STEM REGION AND THE TRANSMEMBRANE DOMAIN OF FUCOSYL-TRANSFERASE-V BY PROTEIN-A RESULTS IN AN ENZYME WITH GDP-FUCOSE HYDROLYZING ACTIVITY

The acceptor specificity of recombinant full-length, membrane-bound fucosyltransferases, expressed in COS-7 cells, and soluble, protein-A chimeric forms of alpha 1,3-fucosyltransferase (Fuc-T) III, Fuc-TIV, and Fuc-TV was analyzed toward a broad panel of oligosaccharide, glycolipid, and glycoprotein substrates. Our results on the full-length enzymes confirm and extend previous studies. However, chimeric Fuc-Ts showed increased activity toward glycoproteins, whereas chimeric Fuc-TIII and Fuc-TV had a decreased activity with glycosphingolipids, compared to the full-length enzymes. Unexpectedly, chimeric Fuc-TV exhibited a GDP-fucose hydrolyzing activity. In substrates with multiple acceptor sites, the preferred site of fucosylation was identified. Fuc-TIII and Fuc-TV catalyzed fucose transfer exclusively to OH-3 of glucose in lacto-N-neotetraose and lacto-N-tetraose, respectively, as was demonstrated by H-1 MMR spectroscopy. Thin layer chromatography immunostaining revealed that FucT-IV preferred the distal GlcNAc residue in nLc(6)Cer, whereas Fuc-TV preferred the proximal GlcNAc residue. Incubation of Fuc-TIV or Fuc-TV with VI(3)NeuAcnLc(6)Cer resulted in products with the sialyl-Lewis(X) epitope as well as the VIM-2 structure. To identify polar groups on accepters that function in enzyme binding, deoxygenated substrate analogs were tested as accepters. All three Fuc-Ts had an absolute requirement for a hydroxyl at C-6 of galactose in addition to the accepting hydroxyl at C-3 or C-4 of GlcNAc.