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Polysaccharide microarrays for high-throughput screening of transglycosylase activities in plant extracts

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Abstract

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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Abbreviations

SR:

sulforhodamine

HEC:

hydroxyethyl cellulose

XET:

xyloglucan endotransglycosylase, EC 2.4.1.207

XTH:

xyloglucan endotransglycosylase/hydrolase

XG:

xyloglucan

XGOs:

xyloglucan-derived oligosaccharides

CEOs:

cellooligosaccharides

LAOs:

laminarioligosaccharides

XYLOs:

(1-4)-β-D-xylan derived oligosaccharides

MLGOs:

mixed-linkage (1-3;1-4)-β-d-glucan oligosaccharides

XLLG:

nonasaccharide Glc4Xyl3Gal2 derived from xyloglucan

TLC:

thin-layer chromatography

GPC:

gel-permeation chromatography

MALDI-TOF:

matrix-assisted laser-desorption/ionization time-of-flight

HPLC:

high-performance liquid chromatography

PMSF:

phenylmethylsulfonyl fluoride

DP:

degree of polymerization

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Acknowledgements

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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Authors and Affiliations

  1. Institute of Chemistry, Centre for Glycomics, Slovak Academy of Sciences, Dúbravská cesta 9, 84538, Bratislava, Slovakia

    Ondřej Kosík, Eva Stratilová, Soňa Garajová & Vladimír Farkaš

  2. Department of Genetics and Cambridge Systems Biology Centre, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR, UK

    Richard P. Auburn & Steven Russell

  3. The University of Adelaide, School of Agriculture, Food and Wine, Australian Centre for Plant Functional Genomics, Waite Campus, Glen Osmond, 5064, Australia

    Maria Hrmova

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  1. Ondřej Kosík
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  7. Vladimír Farkaš
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Correspondence to Vladimír Farkaš.

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Fig. S1

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)

Fig. S2

Effect of pH on the transglycosylation reactions with 10 μM XGO-SR catalyzed by a crude extract from germinated nasturtium seeds. The polysaccharide layouts are depicted in Fig. 1. (PDF 66.4 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

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