Fluorescent mannosides serve as acceptor substrates for glycosyltransferase and sugar-1-phosphate transferase activities in Euglena gracilis membranes

TY - JOUR

T1 - Fluorescent mannosides serve as acceptor substrates for glycosyltransferase and sugar-1-phosphate transferase activities in Euglena gracilis membranes

AU - Ivanova, Irina M.

AU - Nepogodiev, Sergey A.

AU - Saalbach, Gerhard

AU - O'Neill, Ellis C.

AU - Urbaniak, Michael D.

AU - Ferguson, Michael A. J.

AU - Gurcha, Sudagar S.

AU - Besra, Gurdyal S.

AU - Field, Robert A.

N1 - 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 Martin Rejzek for help with HPLC analyses and Sakonwan Kuhaudomlarp for assistance with protein sequence analysis.

PY - 2017/1/13

Y1 - 2017/1/13

N2 - 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.

AB - 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.

KW - Euglena gracilis

KW - Glycosyltransferases

KW - Fluorescent glycans

KW - N-acetylglucosamine-1-phosphate

KW - transferase

KW - Enzyme assays

U2 - 10.1016/j.carres.2016.11.017

DO - 10.1016/j.carres.2016.11.017

M3 - Article

C2 - 27960097

SN - 0008-6215

VL - 438

SP - 26

EP - 38

JO - Carbohydrate Research

JF - Carbohydrate Research

ER -