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Purification, properties and possible gene assignment of an α1,3-fucosyltransferase expressed in human liver

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Abstract

α1,3-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 α1,3-fucosyltransferase activity and had a specific activity of ∼ 5–6 µmol min−1 mg−1 and anM r ∼ 44 000 deduced from SDS-PAGE and HPLC analysis. The purified enzyme readily utilized Galβ1-4GlcNAc, NeuAcα2-3Galβ1-4GlcNAc and Fucα1-2Galβ1-4GlcNAc, with a preference for sialylated and fucosylated Type 2 acceptors. Fucα1-2Galβ1-4Glc and the Type 1 compound Galβ1-3GlcNAc were very poor acceptors and no incorporation was observed with NeuAcα2-6Galβ1-4GlcNAc. A polyclonal antibody raised against the liver preparation reacted with the homologous enzyme and also with the blood group Lewis gene-associated α1,3/1,4-fucosyltransferase purified from the human A431 epidermoid carcinoma cell line. No cross reactivity was found with α1,3-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 theFuc-TVI cDNA, suggests a provisional identification ofFuc-TVI as the major α1,3-fucosyltransferase gene expressed in human liver.

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Author notes

  1. Julia L. Clarke & Winifred M. Watkins

    Present address: Department of Haematology, Royal Postgraduate Medical School, Hammersmith Hospital, W12 0NN, London, UK

  2. Philip H. Johnson

    Present address: MRC Blood Group Unit, University College London, Wolfson House, 4, Stephenson Way, NW1 2HE, London, UK

Authors and Affiliations

  1. Division of Immunochemical Genetics, MRC Clinical Research Centre, Watford Road, HA1 3UJ, Harrow, Middlesex, UK

    Philip H. Johnson, Alastair S. R. Donald, Julia L. Clarke & Winifred M. Watkins

  2. Department of Haematology, Royal Postgraduate Medical School, Hammersmith Hospital, W12 0NN, London, UK

    Philip H. Johnson & Alastair S. R. Donald

Authors
  1. Philip H. Johnson
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  2. Alastair S. R. Donald
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  3. Julia L. Clarke
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  4. Winifred M. Watkins
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†Died June, 1991

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Johnson, P.H., Donald, A.S.R., Clarke, J.L. et al. Purification, properties and possible gene assignment of an α1,3-fucosyltransferase expressed in human liver. Glycoconjugate J 12, 879–893 (1995). https://doi.org/10.1007/BF00731250

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  • DOI: https://doi.org/10.1007/BF00731250

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