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.
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xyloglucan endotransglycosylase, EC 184.108.40.206
(1-4)-β-D-xylan derived oligosaccharides
mixed-linkage (1-3;1-4)-β-d-glucan oligosaccharides
nonasaccharide Glc4Xyl3Gal2 derived from xyloglucan
matrix-assisted laser-desorption/ionization time-of-flight
high-performance liquid chromatography
degree of polymerization
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This work was supported by a grant from the European Commission 6th Framework Programme, contract number MRTN-CT-2004-512265, acronym WallNet and grant no. 2/0011/09 from the Scientific Grant Agency VEGA, Slovakia. A UK BBSRC grant awarded to S. R. supported R. A. Technical assistance provided by Mrs. L. Fischerová and Mr. T. Lipka is greatly appreciated.
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Effect of 100 μM concentrations of non-labelled oligosaccharides (XGOs, CEOs, LAOs) on the microarray reaction between XG and XGOs-SR catalyzed by crude extract from germinated nasturtium seeds. Incubation proceeded at 30°C for 4 h. Control incubation was performed in the absence of nonlabelled oligosaccharides. The inhibition was apparent with only XGOs. The polysaccharide layouts are depicted in Fig. 1. (PDF 186 kb)
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Kosík, O., Auburn, R.P., Russell, S. et al. Polysaccharide microarrays for high-throughput screening of transglycosylase activities in plant extracts. Glycoconj J 27, 79–87 (2010). https://doi.org/10.1007/s10719-009-9271-8
- Plant cell wall