title: The impact of aminopyrene trisulfonate (APTS) label in acceptor glycan substrates for profiling plant pectin β-galactosyltransferase activities.
abstract: Aminopyrene trisulfonate (APTS)-labelled disaccharides are demonstrated to serve as readily accessible acceptor substrates for galactosyltransferase activities present in Arabidopsis microsome preparations. The reductive amination procedure used to install the fluorophore results in loss of the ring structure of the reducing terminal sugar unit, such that a single intact sugar ring is present, attached via an alditol tether to the aminopyrene fluorophore. The configuration of the alditol portion of the labelled acceptor, as well as the position of alditol galactosylation, substantially influence the ability of compounds to serve as Arabidopsis galactosyltransferase acceptor substrates. The APTS label exhibits an unexpected reaction-promoting effect that is not evident for structurally similar sulfonated aromatic fluorophores ANDS and ANTS. When APTS-labelled β-(1 → 4)-Gal3 was employed as an acceptor substrate with Arabidopsis microsomes, glycan extension generated β-(1 → 4)-galactan chains running to beyond 60 galactose residues. These studies demonstrate the potential of even very short glycan-APTS probes for assessing plant galactosyltransferase activities and the suitability CE-LIF for CAZyme profiling.
authors.affiliation: Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
authors.forename: Stephan
authors.initials: S
authors.lastname: Goetz
authors.affiliation: Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
authors.forename: Martin
authors.initials: M
authors.lastname: Rejzek
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. Electronic address: rob.field@jic.ac.uk.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Field
mesh_terms: D000586:Amination; D017360:Arabidopsis / Q000166:cytology / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000378:metabolism; D004187:Disaccharides / Q000032:analysis* / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D005700:Galactosyltransferases / Q000378:metabolism*; D011721:Pyrenes / Q000737:chemistry*
chemical_list: D029681:Arabidopsis Proteins; D004187:Disaccharides; D005456:Fluorescent Dyes; D011721:Pyrenes; D005700:Galactosyltransferases
keywords: Acceptor substrate; Capillary electrophoresis; Fluorophore; Galactosyltransferase; Pectin