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.