Polysaccharide microarrays for high-throughput screening of transglycosylase activities in plant extracts.
Polysaccharide transglycosylases catalyze disproportionation of polysaccharide molecules by cleaving glycosidic linkages in polysaccharide chains and transferring their cleaved portions to hydroxyl groups at the non-reducing ends of other polysaccharide or oligosaccharide molecules. In plant cell walls, transglycosylases have a potential to catalyze both cross-linking of polysaccharide molecules and grafting of newly arriving polysaccharide molecules into the cell wall structure during cell growth. Here we describe a polysaccharide microarray in form of a glycochip permitting simultaneous high-throughput monitoring of multiple transglycosylase activities in plant extracts. The glycochip, containing donor polysaccharides printed onto nitrocellulose-coated glass slides, was incubated with crude plant extracts, along with a series of fluorophore-labelled acceptor oligosaccharides. After removing unused labelled oligosaccharides by washing, fluorescence retained on the glycochip as a result of transglycosylase reaction was detected with a standard microarray scanner. The glycochip assay was used to detect transglycosylase activities in crude extracts from nasturtium (Tropaeolum majus) and mouse-ear cress (Arabidopsis thaliana). A number of previously unknown saccharide donor-acceptor pairs active in transglycosylation reactions that lead to the formation of homo- and hetero-glycosidic conjugates, were detected. Our data provide experimental support for the existence of diverse transglycosylase activities in crude plant extracts.
Kosík
Ondrej
O
Institute of Chemistry, Centre for Glycomics, Slovak Academy of Sciences, Dúbravská cesta 9, 84538, Bratislava, Slovakia.
Auburn
Richard P
RP
Russell
Steven
S
Stratilová
Eva
E
Garajová
Sona
S
Hrmova
Maria
M
Farkas
Vladimír
V
D000327:Adsorption; D006031:Glycosylation; D006602:Hexosyltransferases / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D020128:Inhibitory Concentration 50; D046228:Microarray Analysis / Q000379:methods*; D010936:Plant Extracts / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D032388:Tropaeolum / Q000201:enzymology*
D010936:Plant Extracts; D011134:Polysaccharides; D006602:Hexosyltransferases