Purification of the Lewis blood-group gene associated α-3/4-fucosyltransferase from human milk: an enzyme transferring fucose primarily to Type 1 and lactose-based oligosaccharide chains

Abstract

A soluble Lewis blood-group gene associated α-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 α-3-L-fucosyltransferase activity directed towardsN-acetylglucosamine in Type 2 (Galβ1-4GlcNAc-R) acceptors from an α-3/4-fucosyltransferase fraction acting on both Type 1 (Galβ1-3GlcNAc-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 toN-acetylglucosamine in both Type 1 and Type 2 acceptors and to theO-3 position of glucose in lactose-based oligosaccharides. The enzyme preparation at this stage resembled previously described α-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 α-3-fucosyltransferase activity acting solely on Type 2 acceptors and left a residual α-3/4-fucosyltransferase that retained strong α-4 activity with the Type 1 acceptor, lacto-N-biose 1, and α-3 activity with 2′-fucosyllactose, but had relatively little α-3 activity withN-acetyllactosamine and virtually no capacity to transfer fucose to glycoproteins withN-linked oligosaccharide chains having unsubstituted terminal Type 2 structures.