title: Fluorescent mannosides serve as acceptor substrates for glycosyltransferase and sugar-1-phosphate transferase activities in Euglena gracilis membranes.
abstract: Synthetic hexynyl α-D-mannopyranoside and its α-1,6-linked disaccharide counterpart were fluorescently labelled through CuAAC click chemistry with 3-azido-7-hydroxycoumarin. The resulting triazolyl-coumarin adducts, which were amenable to analysis by TLC, HPLC and mass spectrometry, proved to be acceptor substrates for α-1,6-ManT activities in mycobacterial membranes, as well as α- and β-GalT activities in trypanosomal membranes, benchmarking the potential of the fluorescent acceptor approach against earlier radiochemical assays. Following on to explore the glycobiology of the benign protozoan alga Euglena gracilis, α-1,3- and α-1,2-ManT activities were detected in membrane preparations, along with GlcT, Glc-P-T and GlcNAc-P-T activities. These studies serve to demonstrate the potential of readily accessible fluorescent glycans as substrates for exploring carbohydrate active enzymes.
authors.affiliation: Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
authors.forename: Irina M
authors.initials: IM
authors.lastname: Ivanova
authors.affiliation: Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Nepogodiev
authors.affiliation: Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Saalbach
authors.affiliation: Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
authors.forename: Ellis C
authors.initials: EC
authors.lastname: O'Neill
authors.affiliation: Biomedical and Life Sciences, Lancaster University, Furness Building, Lancaster LA1 4YG, UK; College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Urbaniak
authors.affiliation: College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.
authors.forename: Michael A J
authors.initials: MA
authors.lastname: Ferguson
authors.affiliation: School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors.forename: Sudagar S
authors.initials: SS
authors.lastname: Gurcha
authors.affiliation: School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors.forename: Gurdyal S
authors.initials: GS
authors.lastname: Besra
authors.affiliation: Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK. Electronic address: rob.field@jic.ac.uk.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Field
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D005056:Euglena gracilis / Q000201:enzymology*; D005453:Fluorescence; D016695:Glycosyltransferases / Q000378:metabolism*; D007700:Kinetics; D008362:Mannosides / Q000737:chemistry / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D008362:Mannosides; D016695:Glycosyltransferases
keywords: Enzyme assays; Euglena gracilis; Fluorescent glycans; Glycosyltransferases; N-acetylglucosamine-1-phosphate transferase