Novel Activity‐Based Probes for Broad‐Spectrum Profiling of Retaining β‐Exoglucosidases In Situ and In Vivo

Kallemeijn, Wouter W.; Li, Kah‐Yee; Witte, Martin D.; Marques, André R. A.; Aten, Jan; Scheij, Saskia; Jiang, Jianbing; Willems, Lianne I.; Voorn-Brouwer, Tineke; Roomen, Cindy P. A. A. van; Ottenhoff, Roelof; Boot, Rolf G.; Elst, Hans van den; Walvoort, Marthe T. C.; Florea, Bogdan I.; Codée, Jeroen D. C.; Marel, Gijsbert A. van der; Aerts, J.M.F.G.; Overkleeft, Herman S.

doi:10.1002/ange.201207771

Cited by 45 publications

(80 citation statements)

References 28 publications

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“…The synthesis of KY371 and JJB111 has been described previously (33). Aliquots of these compounds are available upon request.…”

Section: Methodsmentioning

confidence: 99%

“…JJB70 carries a bodipy as its reporter tag and is slightly different from the previously described ABP3 (33) in that it has the triazole linker in reverse orientation (Fig. 1A).…”

Section: Labeling Of Leaf Proteomes With Cyclophellitol-aziridinementioning

confidence: 97%

“…JJB111 (previously called ABP4) and KY371 carry biotin and alkyne minitags, respectively (Fig. 1A) (33). The electrophilic acyl-aziridine group in the probes reacts with the nucleophilic active site glutamate/aspartate residue of retaining glycosidases, resulting in a reversible covalent ester bond (Fig.…”

Section: Labeling Of Leaf Proteomes With Cyclophellitol-aziridinementioning

confidence: 99%

“…KY371 carries an alkyne minitag. B, Mechanism of labeling retaining glycosidases (Kallemeijn et al, 2012). The nucleophilic oxygen of one of the two catalytic glutamic acids attacks the electrophilic carbon next to nitrogen in the aziridine ring to form a covalent, reversible ester bond.…”

Section: Labeling Of Leaf Proteomes With Cyclophellitol-aziridinementioning

confidence: 99%

“…Cyclophellitol-aziridine-based probes were previously used in animal proteomes to target retaining glucosidases (33). Here we established and applied glycosidase profiling in plants.…”

mentioning

confidence: 99%

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Broad-range Glycosidase Activity Profiling

Chandrasekar

Colby²,

Emon

et al. 2014

Molecular & Cellular Proteomics

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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

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“…The synthesis of KY371 and JJB111 has been described previously (33). Aliquots of these compounds are available upon request.…”

Section: Methodsmentioning

confidence: 99%

“…JJB70 carries a bodipy as its reporter tag and is slightly different from the previously described ABP3 (33) in that it has the triazole linker in reverse orientation (Fig. 1A).…”

Section: Labeling Of Leaf Proteomes With Cyclophellitol-aziridinementioning

confidence: 97%

Section: Labeling Of Leaf Proteomes With Cyclophellitol-aziridinementioning

confidence: 99%

Section: Labeling Of Leaf Proteomes With Cyclophellitol-aziridinementioning

confidence: 99%

“…Cyclophellitol-aziridine-based probes were previously used in animal proteomes to target retaining glucosidases (33). Here we established and applied glycosidase profiling in plants.…”

mentioning

confidence: 99%

See 3 more Smart Citations

Broad-range Glycosidase Activity Profiling

Chandrasekar

Colby²,

Emon

et al. 2014

Molecular & Cellular Proteomics

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α‐Boryl Isocyanides Enable Facile Preparation of Bioactive Boropeptides

et al. 2013

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Organelle‐Specific Activity‐Based Protein Profiling in Living Cells

et al. 2014

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A multimodal activity-based probe for targeting acidic organelles was developed to measure subcellular native enzymatic activity in cells by fluorescence microscopy and mass spectrometry. A cathepsin-reactive warhead conjugated to a weakly basic amine and a clickable alkyne, for subsequent appendage of a fluorophore or biotin reporter tag, accumulated in lysosomes as observed by structured illumination microscopy (SIM) in J774 mouse macrophage cells. Analysis of in vivo labeled J774 cells by mass spectrometry showed that the probe was very selective for cathepsins B and Z, two lysosomal cysteine proteases. Analysis of starvation-induced autophagy, a catabolic pathway involving lysosomes, showed a large increase in the number of tagged proteins and an increase in cathepsin activity. The organelle-targeting of activity-based probes holds great promise for the characterization of enzyme activities in the myriad diseases linked to specific subcellular locations, particularly the lysosome.

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Novel Activity‐Based Probes for Broad‐Spectrum Profiling of Retaining β‐Exoglucosidases In Situ and In Vivo

Cited by 45 publications

References 28 publications

Broad-range Glycosidase Activity Profiling

Broad-range Glycosidase Activity Profiling

α‐Boryl Isocyanides Enable Facile Preparation of Bioactive Boropeptides

Organelle‐Specific Activity‐Based Protein Profiling in Living Cells

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