TY - JOUR
T1 - The impact of aminopyrene trisulfonate (APTS) label in acceptor glycan substrates for profiling plant pectin β-galactosyltransferase activities
AU - Goetz, Stephan
AU - Rejzek, Martin
AU - Nepogodiev, Sergey A.
AU - Field, Robert A.
N1 - Funding Information:
These studies were supported by the UK BBSRC Institute Strategic Programme on Understanding and Exploiting Metabolism (MET) [ BB/J004561/1 ] and the John Innes Foundation . We thank Paul Dupree, University of Cambridge, for helpful discussions.
Publisher Copyright:
© 2016 The Authors
PY - 2016
Y1 - 2016
N2 - 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)-Gal3was 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.
AB - 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)-Gal3was 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.
KW - Acceptor substrate
KW - Capillary electrophoresis
KW - Fluorophore
KW - Galactosyltransferase
KW - Pectin
UR - http://www.scopus.com/inward/record.url?scp=84979498193&partnerID=8YFLogxK
U2 - 10.1016/j.carres.2016.07.017
DO - 10.1016/j.carres.2016.07.017
M3 - Article
C2 - 27479753
AN - SCOPUS:84979498193
VL - 433
SP - 97
EP - 105
JO - Carbohydrate Research
JF - Carbohydrate Research
SN - 0008-6215
ER -