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----6fucosyltransferase 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 Km values for the substrate asialoagalactotransferrin glycopeptide, and GDP-L-fucose were 66 and 4.2 microM, respectively. The Vmax was 1.4 mumols/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 Km value of 55 microM. 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 fucosyltransferases thus far reported, since the natural substrates for the other enzymes may be substituted by one of several disaccharides.
Department of Pediatrics, School of Medicine, University of Pennsylvania, Philadelphia.
J A
JA
Voynow
R S
RS
Kaiser
T F
TF
Scanlin
M C
MC
Glick
D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D005347:Fibroblasts / Q000201:enzymology; D005647:Fucosyltransferases / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000138:chemical synthesis / Q000378:metabolism*; D012867:Skin / Q000201:enzymology*; D013379:Substrate Specificity
D009844:Oligosaccharides; D005647:Fucosyltransferases; C020559:Glycoprotein 6-alpha-L-fucosyltransferase