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. Molecular & Cellular Proteomics

Activity‐based protein profiling of hydrolytic enzymes induced by gibberellic acid in isolated aleurone layers of malting barley

“…Signals were also detected for JOGDA1, which targets cathepsin‐B‐like proteases, and JOPD1, which targets vacuolar processing enzymes . Multiple strong signals were displayed upon labeling with JJB70, which targets retaining β‐glycosidases and with FP‐Rh, which targets serine hydrolases . For MVB072, which targets the catalytic beta subunit of the proteasome, clear signals were observed (Fig.…”

Plant carbohydrate‐active enzymes in bamboo Neosinocalamus affinis: identification, classification and function in lignocellulose biosynthesis in herbivore defence

“…The CAZy database contains 161 GH families (Xu et al ), which are enzymes that hydrolyse the glycosidic bond of carbohydrates (Chandrasekar et al ). In plants, numerous genes have been identified to be GH family members (Chandrasekar et al ), thereby indicating a functional redundancy or differential response. In the N. affinis transcriptome, 222 unigenes were clustered into 36 GH families, of which the top 10 families were GH1, GH3, GH5, GH9, GH13, GH16, GH17, GH19, GH28 and GH152 (Fig.…”

Beta galactosidases in Arabidopsis and tomato–a mini review

“…ABPP involves chemical probes that label the active site residue of proteins in an activity-dependent manner. We have recently introduced cyclophellitol aziridine-based probes to monitor broad range of active glycosidases in plants [59]. To monitor the in vivo BGAL activity, Arabidopsis plants have also been treated with the artificial substrate X-Gal [60].…”