title: Broad-range glycosidase activity profiling.
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.
authors: 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;
authors: Balakumaran
authors: B
authors: Chandrasekar
authors: ‖Gorlaeus Laboratories, Leiden Institute of Chemistry and Netherlands Center for Proteomics, Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors: Thomas
authors: T
authors: Colby
authors: §Plant Chemetics Laboratory, Max Planck Institute for Plant Breeding Research, Carl-von-Linne Weg 10, 50829 Cologne, Germany;
authors: Asif
authors: A
authors: Emran Khan Emon
authors: ‖Gorlaeus Laboratories, Leiden Institute of Chemistry and Netherlands Center for Proteomics, Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors: Jianbing
authors: J
authors: Jiang
authors: 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;
authors: Tram Ngoc
authors: TN
authors: Hong
authors: §Plant Chemetics Laboratory, Max Planck Institute for Plant Breeding Research, Carl-von-Linne Weg 10, 50829 Cologne, Germany;
authors: Joji Grace
authors: JG
authors: Villamor
authors: ‖Gorlaeus Laboratories, Leiden Institute of Chemistry and Netherlands Center for Proteomics, Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors: Anne
authors: A
authors: Harzen
authors: ‖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.
authors: Herman S
authors: HS
authors: Overkleeft
authors: 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;
authors: Renier A L
authors: RA
authors: van der Hoorn
mesh_terms: D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D001388:Aziridines / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D002473:Cell Wall / Q000201:enzymology; D003511:Cyclohexanols / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D015335:Molecular Probes / Q000737:chemistry / Q000378:metabolism*; D010802:Phylogeny; D040901:Proteomics / Q000379:methods*
chemical_list: D029681:Arabidopsis Proteins; D001388:Aziridines; D003511:Cyclohexanols; D015335:Molecular Probes; C063285:cyclophellitol; D006026:Glycoside Hydrolases