Fluorescently labeled substrates for monitoring α1,3-fucosyltransferase IX activity.

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  • Additional Information
    • Source:
      Publisher: Wiley-VCH Country of Publication: Germany NLM ID: 9513783 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1521-3765 (Electronic) Linking ISSN: 09476539 NLM ISO Abbreviation: Chemistry Subsets: MEDLINE
    • Publication Information:
      Original Publication: Weinheim, Germany : Wiley-VCH
    • Subject Terms:
      Fluorescent Dyes/*chemistry ; Fucosyltransferases/*metabolism; Chromatography, Thin Layer ; Fucosyltransferases/genetics ; Glycosylation ; Helicobacter pylori/enzymology ; Humans ; Mass Spectrometry ; Recombinant Proteins/genetics ; Recombinant Proteins/metabolism ; Substrate Specificity
    • Abstract:
      Fucosylation is often the final process in glycan biosynthesis. The resulting glycans are involved in a variety of biological processes, such as cell adhesion, inflammation, or tumor metastasis. Fucosyltransferases catalyze the transfer of fucose residues from the activated donor molecule GDP-β-L-fucose to various acceptor molecules. However, detailed information about the reaction processes is still lacking for most fucosyltransferases. In this work we have monitored α1,3-fucosyltransferase activity. For both donor and acceptor substrates, the introduction of a fluorescent ATTO dye was the last step in the synthesis. The subsequent conversion of these substrates into fluorescently labeled products by α1,3-fucosyltransferases was examined by high-performance thin-layer chromatography coupled with mass spectrometry as well as dual-color fluorescence cross-correlation spectroscopy, which revealed that both fluorescently labeled donor GDP-β-L-fucose-ATTO 550 and acceptor N-acetyllactosamine-ATTO 647N were accepted by recombinant human fucosyltransferase IX and Helicobacter pylori α1,3-fucosyltransferase, respectively. Analysis by fluorescence cross-correlation spectroscopy allowed a quick and versatile estimation of the progress of the enzymatic reaction and therefore this method can be used as an alternative method for investigating fucosyltransferase reactions.
      (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
    • Contributed Indexing:
      Keywords: carbohydrates; chromatographic methods; enzymes; fluorescent probes; glycosylation
    • Accession Number:
      0 (Fluorescent Dyes)
      0 (Recombinant Proteins)
      EC 2.4.1.- (FUT9 protein, human)
      EC 2.4.1.- (Fucosyltransferases)
    • Publication Date:
      Date Created: 20131122 Date Completed: 20140722 Latest Revision: 20210830
    • Publication Date:
      20240829
    • Accession Number:
      10.1002/chem.201302601
    • Accession Number:
      24258785