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2014 ; 13
(10
): 2787-800
Nephropedia Template TP
Chandrasekar B
; Colby T
; Emran Khan Emon A
; Jiang J
; Hong TN
; Villamor JG
; Harzen A
; Overkleeft HS
; van der Hoorn RA
Mol Cell Proteomics
2014[Oct]; 13
(10
): 2787-800
PMID25056938
show ga
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.
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