PURIFICATION AND CHARACTERIZATION OF GDP-L-FUCOSE-N-ACETYL-BETA-D-GLUCOSAMINIDE-ALPHA-1-]6FUCOSYLTRANSFERASE FROM CULTURED HUMAN SKIN FIBROBLASTS - REQUIREMENT OF A SPECIFIC BIANTENNARY OLIGOSACCHARIDE AS SUBSTRATE

GDP-L-fucose-N-acetyl-beta-D-glucosaminide alpha-1 --> 6-fucosyltransferase which catalyzes the transfer of fucose from GDP-L-fucose to the asparagine-linked N-acetyl-beta-D-glucosamine of N-linked glycoproteins has been purified 37,000-fold from cultured human skin fibroblasts. The K(m) values for the substrate asialoagalactotransferrin glycopeptide, and GDP-L-fucose were 66 and 4.2-mu-M, respectively. The V(max) was 1.4-mu-mol/mg/min. The key step in enzyme purification was affinity chromatography using the immobilized substrate asialoagalactotransferrin glycopeptide-CH-Sepharose. The affinity-purified enzyme had a minimum substrate requirement for a biantennary oligosaccharide with GlcNAc in terminal position, having a K(m) value of 55-mu-M. It was heretofore unexpected that the oligosaccharide would serve as substrate, since the site of enzyme activity is GlcNAc-1-linked to Asn. Although the presence of amino acids on this oligosaccharide enhanced the activity 3-fold, it is proposed that this may be the result of an alpha/beta anomeric mixture (2:1) of oligosaccharide used in these studies with only the beta-anomer active as substrate. The implication is that the amino acid is required only to retain the beta-anomeric position of the substrate. Removal of GlcNAc or addition of Gal to either the oligosaccharide or glycopeptide destroyed the ability to serve as substrates. In addition, di-N-acetylchitobiose, tri-N-acetylchitotriose and GlcNAc-beta-1 --> Asn were nonpermissible substrates. This rigid substrate requirement is unique among fucosyl-transferases thus far reported, since the natural substrates for the other enzymes may be substituted by one of several disaccharides.