Acceptor specificity of different length constructs of human recombinant alpha 1,3/4-fucosyltransferases. Replacement of the stem region and the transmembrane domain of fucosyltransferase V by protein A results in an enzyme with GDP-fucose hydrolyzing activity

J Biol Chem. 1995 Apr 14;270(15):8712-22. doi: 10.1074/jbc.270.15.8712.

Abstract

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 1H NMR spectroscopy. Thin layer chromatography immunostaining revealed that FucT-IV preferred the distal GlcNAc residue in nLc6Cer, whereas Fuc-TV preferred the proximal Gl-cNAc residue. Incubation of Fuc-TIV or Fuc-TV with VI3NeuAcnLc6Cer resulted in products with the sialyl-LewisX epitope as well as the VIM-2 structure. To identify polar groups on acceptors that function in enzyme binding, deoxygenated substrate analogs were tested as acceptors. 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Carbohydrate Sequence
  • Cell Line
  • Cell Membrane / enzymology
  • Ethylmaleimide / pharmacology
  • Fucosyltransferases / antagonists & inhibitors
  • Fucosyltransferases / chemistry
  • Fucosyltransferases / metabolism*
  • Guanosine Diphosphate Fucose / metabolism*
  • Humans
  • Hydrolysis
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Molecular Sequence Data
  • Recombinant Proteins / antagonists & inhibitors
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Staphylococcal Protein A / chemistry
  • Staphylococcal Protein A / metabolism*
  • Substrate Specificity

Substances

  • Recombinant Proteins
  • Staphylococcal Protein A
  • Guanosine Diphosphate Fucose
  • Fucosyltransferases
  • 3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase
  • Ethylmaleimide