Purification, properties and possible gene assignment of an alpha 1,3-fucosyltransferase expressed in human liver.
alpha 1,3-Fucosyltransferase solubilized from human liver has been purified 40,000-fold to apparent homogeneity by a multistage process involving cation exchange chromatography on CM-Sephadex, hydrophobic interaction chromatography on Phenyl Sepharose, affinity chromatography on GDP-hexanolamine Sepharose and HPLC gel exclusion chromatography. The final step gave a major protein peak that co-chromatographed with alpha 1,3-fucosyltransferase activity and had a specific activity of approximately 5-6 mumol min-1 mg-1 and an M(r) approximately 44,000 deduced from SDS-PAGE and HPLC analysis. The purified enzyme readily utilized Gal beta 1-4GlcNAc, NeuAc alpha 2-3Gal beta 1-4GlcNAc and Fuc alpha 1-2Gal beta 1-4GlcNAc, with a preference for sialylated and fucosylated Type 2 acceptors. Fuc alpha 1-2Gal beta 1-4Glc and the Type 1 compound Gal beta 1-3GlcNAc were very poor acceptors and no incorporation was observed with NeuAc alpha 2-6Gal beta 1-4GlcNAc. A polyclonal antibody raised against the liver preparation reacted with the homologous enzyme and also with the blood group Lewis gene-associated alpha 1,3/1,4-fucosyltransferase purified from the human A431 epidermoid carcinoma cell line. No cross reactivity was found with alpha 1,3-fucosyltransferase(s) isolated from myeloid cells. Examination by Northern blot analysis of mRNA from normal liver and from the HepG2 cell line, together with a comparison of the specificity pattern of the purified enzyme with that reported for the enzyme expressed in mammalian cells transfected with the Fuc-TVI cDNA, suggests a provisional identification of Fuc-TVI as the major alpha 1,3-fucosyltransferase gene expressed in human liver.
Johnson
P H
PH
Division of Immunochemical Genetics, MRC Clinical Research Centre, Harrow, Middlesex, UK.
Donald
A S
AS
Clarke
J L
JL
Watkins
W M
WM
D000645:Ammonium Sulfate; D002240:Carbohydrate Sequence; D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange; D005647:Fucosyltransferases / Q000235:genetics* / Q000302:isolation & purification*; D005815:Genetic Code; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D012995:Solubility; D013379:Substrate Specificity
D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase; D000645:Ammonium Sulfate