Purification of the Lewis blood-group gene associated alpha-3/4-fucosyltransferase from human milk: an enzyme transferring fucose primarily to type 1 and lactose-based oligosaccharide chains.
A soluble Lewis blood-group gene associated alpha-3/4-L-fucosyltransferase has been purified from human milk by a series of steps involving hydrophobic chromatography on Phenyl Sepharose 4B, ion exchange chromatography on CM-Sephadex C-50, affinity chromatography on GDP-hexanolamine Sepharose 4B and gel filtration on Sephacryl S-200. The first step separated alpha-3-L-fucosyltransferase activity directed towards N-acetylglucosamine in Type 2 (Gal beta 1-4GlcNAc-R) acceptors from an alpha-3/4-fucosyltransferase fraction acting on both Type 1 (Gal beta 1-3GlcNAc-R) and Type 2 acceptors. Further purification of this latter fraction on CM-Sephadex and GDP-hexanolamine Sepharose gave a single peak of fucosyltransferase activity that catalysed the addition of fucose to N-acetylglucosamine in both Type 1 and Type 2 acceptors and to the O-3 position of glucose in lactose-based oligosaccharides. The enzyme preparation at this stage resembled previously described alpha-3/4-fucosyltransferase preparations purified from human milk. However, gel filtration of this preparation on Sephacryl S-200 or Sephadex G-150 separated further amounts of alpha-3-fucosyltransferase activity acting solely on Type 2 acceptors and left a residual alpha-3/4-fucosyltransferase that retained strong alpha-4 activity with the Type 1 acceptor, lacto-N-biose 1, and alpha-3 activity with 2'-fucosyllactose, but had relatively little alpha-3 activity with N-acetyllactosamine and virtually no capacity to transfer fucose to glycoproteins with N-linked oligosaccharide chains having unsubstituted terminal Type 2 structures.
Johnson
P H
PH
Division of Immunochemical Genetics, MRC Clinical Research Centre, Harrow, Middlesex, UK.
Watkins
W M
WM
D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005643:Fucose / Q000378:metabolism*; D005647:Fucosyltransferases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D007785:Lactose / Q000032:analysis*; D007983:Lewis Blood Group Antigens / Q000235:genetics*; D008895:Milk, Human / Q000201:enzymology*; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism; D005644:alpha-L-Fucosidase
D007983:Lewis Blood Group Antigens; D009844:Oligosaccharides; D005643:Fucose; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase; C019818:3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase; D005644:alpha-L-Fucosidase; D007785:Lactose