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Plant Physiol. 2018 May;177(1):24-37. doi: 10.1104/pp.18.00250. Epub 2018 Mar 19.

Multiplex Fluorescent, Activity-Based Protein Profiling Identifies Active α-Glycosidases and Other Hydrolases in Plants.

Author information

1
Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom.
2
Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shalimar- 190025, Jammu and Kashmir, India.
3
Chemische Biologie, Zentrum für Medizinische Biotechnologie, Fakultät für Biologie, Universität Duisburg-Essen, 45117 Essen, Germany.
4
Universidad Politécnica de Madrid, Escuela Técnica Superior de Ingenieria Agronómica, Alimentaria cv de Biosistemas, Ciudad Universitaria s/n, 28040 Madrid, Spain.
5
Gorlaeus Laboratories, Leiden Institute of Chemistry and Netherlands Center for Proteomics, 2333 CC Leiden, The Netherlands.
6
Instituto Botánico, Facultad de Farmacia, Universidad de Castilla-La Mancha, Campus Universitario s/n, Albacete 02071, Spain.
7
Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom renier.vanderhoorn@plants.ox.ac.uk.

Abstract

With nearly 140 α-glycosidases in 14 different families, plants are well equipped with enzymes that can break the α-glucosidic bonds in a large diversity of molecules. Here, we introduce activity-based protein profiling (ABPP) of α-glycosidases in plants using α-configured cyclophellitol aziridine probes carrying various fluorophores or biotin. In Arabidopsis (Arabidopsis thaliana), these probes label members of the GH31 family of glycosyl hydrolases, including endoplasmic reticulum-resident α-glucosidase-II Radial Swelling3/Priority for Sweet Life5 (RSW3/PSL5) and Golgi-resident α-mannosidase-II Hybrid Glycosylation1 (HGL1), both of which trim N-glycans on glycoproteins. We detected the active state of extracellular α-glycosidases such as α-xylosidase XYL1, which acts on xyloglucans in the cell wall to promote cell expansion, and α-glucosidase AGLU1, which acts in starch hydrolysis and can suppress fungal invasion. Labeling of α-glycosidases generates pH-dependent signals that can be suppressed by α-glycosidase inhibitors in a broad range of plant species. To demonstrate its use on a nonmodel plant species, we applied ABPP on saffron crocus (Crocus sativus), a cash crop for the production of saffron spice. Using a combination of biotinylated glycosidase probes, we identified and quantified 67 active glycosidases in saffron crocus stigma, of which 10 are differentially active. We also uncovered massive changes in hydrolase activities in the corms upon infection with Fusarium oxysporum using multiplex fluorescence labeling in combination with probes for serine hydrolases and cysteine proteases. These experiments demonstrate the ease with which active α-glycosidases and other hydrolases can be analyzed through ABPP in model and nonmodel plants.

PMID:
29555787
PMCID:
PMC5933116
DOI:
10.1104/pp.18.00250
[Indexed for MEDLINE]
Free PMC Article

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