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Mol Cell Proteomics. 2014 Oct;13(10):2787-800. doi: 10.1074/mcp.O114.041616. Epub 2014 Jul 23.

Broad-range glycosidase activity profiling.

Author information

1
From the ‡Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, United Kingdom; §Plant Chemetics Laboratory, Max Planck Institute for Plant Breeding Research, Carl-von-Linne Weg 10, 50829 Cologne, Germany;
2
‖Gorlaeus Laboratories, Leiden Institute of Chemistry and Netherlands Center for Proteomics, Einsteinweg 55, 2333 CC Leiden, The Netherlands.
3
§Plant Chemetics Laboratory, Max Planck Institute for Plant Breeding Research, Carl-von-Linne Weg 10, 50829 Cologne, Germany;
4
‖Gorlaeus Laboratories, Leiden Institute of Chemistry and Netherlands Center for Proteomics, Einsteinweg 55, 2333 CC Leiden, The Netherlands renier.vanderhoorn@plants.ox.ac.uk h.s.overkleeft@chem.leidenuniv.nl.
5
From the ‡Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, United Kingdom; From the ‡Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, United Kingdom;

Abstract

Plants produce hundreds of glycosidases. Despite their importance in cell wall (re)modeling, protein and lipid modification, and metabolite conversion, very little is known of this large class of glycolytic enzymes, partly because of their post-translational regulation and their elusive substrates. Here, we applied activity-based glycosidase profiling using cell-permeable small molecular probes that react covalently with the active site nucleophile of retaining glycosidases in an activity-dependent manner. Using mass spectrometry we detected the active state of dozens of myrosinases, glucosidases, xylosidases, and galactosidases representing seven different retaining glycosidase families. The method is simple and applicable for different organs and different plant species, in living cells and in subproteomes. We display the active state of previously uncharacterized glycosidases, one of which was encoded by a previously declared pseudogene. Interestingly, glycosidase activity profiling also revealed the active state of a diverse range of putative xylosidases, galactosidases, glucanases, and heparanase in the cell wall of Nicotiana benthamiana. Our data illustrate that this powerful approach displays a new and important layer of functional proteomic information on the active state of glycosidases.

PMID:
25056938
PMCID:
PMC4189003
DOI:
10.1074/mcp.O114.041616
[Indexed for MEDLINE]
Free PMC Article

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