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

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...

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Bibliographic Details
Published in:Glycoconjugate journal 1992-10, Vol.9 (5), p.241-249
Main Authors: JOHNSON, P. H, WATKINS, W. M
Format: Article
Language:English
Subjects:
alpha-L-Fucosidase
Analytical, structural and metabolic biochemistry
Biological and medical sciences
Chromatography, Affinity
Chromatography, Gel
Chromatography, Ion Exchange
Electrophoresis, Polyacrylamide Gel
Enzymes and enzyme inhibitors
Fucose - metabolism
Fucosyltransferases - biosynthesis
Fucosyltransferases - genetics
Fucosyltransferases - isolation & purification
Fundamental and applied biological sciences. Psychology
Humans
Lactose - analysis
Lewis Blood-Group System - genetics
Milk, Human - enzymology
Molecular Weight
Oligosaccharides - chemistry
Oligosaccharides - metabolism
Transferases
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Summary: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.
ISSN:0282-0080
1573-4986
DOI:10.1007/BF00731136