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.
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.
de Vries
T
T
Department of Chemistry and Biochemistry, San Francisco State University, California 94132, USA.
Srnka
C A
CA
Palcic
M M
MM
Swiedler
S J
SJ
van den Eijnden
D H
DH
Macher
B A
BA
D000818:Animals; D002240:Carbohydrate Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D005033:Ethylmaleimide / Q000494:pharmacology; D005647:Fucosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006154:Guanosine Diphosphate Fucose / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D006154:Guanosine Diphosphate Fucose; D005647:Fucosyltransferases; C019818:3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase; D005033:Ethylmaleimide