Design and Synthesis of Activity Probes for Glycosidases

Tsai, Charng Sheng; Li, Yaw-Kuen; Lo, Lee‐Chiang

doi:10.1021/ol0265315

Cited by 85 publications

(69 citation statements)

References 22 publications

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“…A previous method that made use of quinone methide chemistry produced cross-labeling when carried out in a mixture of proteins because, after initial reaction at the active site of a glycosidase, the glycoside that provided specificity was lost, and a reactive quinone methide was generated, which is known to leave the active site and to react with exposed nucleophilic residues anywhere on the protein or indeed on any protein in the mixture (46,47). A very recent attempt at proteomic analysis of exoglycosidases using a ligation strategy was more specific, but lacked the desired efficacy (15), with the tagged substrate reacting 10 4 times more slowly than the parent substrate.…”

Section: Discussionmentioning

confidence: 99%

Active-site Peptide “Fingerprinting” of Glycosidases in Complex Mixtures by Mass Spectrometry

Hekmat¹,

Kim²,

Williams³

et al. 2005

Journal of Biological Chemistry

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New proteomics methods are required for targeting and identification of subsets of a proteome in an activity-based fashion. Here, we report the first gel-free, mass spectrometry-based strategy for mechanism-based profiling of retaining ␤-endoglycosidases in complex proteomes. Using a biotinylated, cleavable 2-deoxy-2-fluoroxylobioside inactivator, we have isolated and identified the active-site peptides of target retaining ␤-1,4-glycanases in systems of increasing complexity: pure enzymes, artificial proteomes, and the secreted proteome of the aerobic mesophilic soil bacterium Cellulomonas fimi. The active-site peptide of a new C. fimi ␤-1,4-glycanase was identified in this manner, and the peptide sequence, which includes the catalytic nucleophile, is highly conserved among glycosidase family 10 members. The glycanase gene (GenBank TM accession number DQ146941) was cloned using inverse PCR techniques, and the protein was found to comprise a catalytic domain that shares ϳ70% sequence identity with those of xylanases from Streptomyces sp. and a family 2b carbohydrate-binding module. The new glycanase hydrolyzes natural and artificial xylo-configured substrates more efficiently than their cello-configured counterparts. It has a pH dependence very similar to that of known C. fimi retaining glycanases.With the completion of the genome sequences of many organisms, the field of proteomics faces several major tasks. One challenge is that of identification and assignment of structure/function to the tens of thousands of proteins encoded by prokaryotic and eukaryotic genomes. Another challenge is accurate quantitative analysis of changes in protein levels/activities that occur within a proteome as a response to biological perturbations that are due either to normal developmental and metabolic changes or to abnormalities associated with disease. Proteomic techniques such as comparative two-dimensional gel electrophoresis coupled with mass spectrometry, the isotope-coded affinity tagging approach (1), and variations of isotope-coded affinity tagging that identify sites of modification (e.g. glycosylation and phosphorylation) on proteins (2-4) fail to provide a direct assessment of protein function.Recently, several chemical strategies for activity-based protein profiling (ABPP) 4 in complex proteomes that target several enzyme groups, including oxidoreductases (5, 6), serine hydrolases (7, 8), cysteine proteases (9, 10), threonine proteases (11), metalloproteases (12), protein phosphatases (13), kinases (14), and exoglycosidases (15), have been employed. ABPP probes have two general features: 1) an active sitedirected (mechanism-based) inactivator or affinity label that reacts with a catalytic residue and forms a covalent adduct with the target enzyme(s) and 2) one or more reporter groups that enable rapid detection (e.g. a fluorophore) and/or affinity isolation (e.g. biotin) (16). As such, ABPP methods can provide direct information on post-translational forms of protein regulation (17). However, most of the ABPP research ...

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Section: Discussionmentioning

confidence: 99%

Active-site Peptide “Fingerprinting” of Glycosidases in Complex Mixtures by Mass Spectrometry

Hekmat¹,

Kim²,

Williams³

et al. 2005

Journal of Biological Chemistry

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“…The tag may incorporate a moiety, such as an alkyne or azide, for subsequent modification, such as by the Cu(I)-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC), to introduce a reporter (15,16). ABPP probes have been developed to monitor changes of specific enzymes associated with certain biological states, including serine hydrolases (17,18), cysteine proteases (19)(20)(21)(22), protein phosphatases (23)(24)(25), oxidoreductases (26), histone deacetylases (27), kinases (28,29), metalloproteases (30)(31)(32), and glycosidases (33)(34)(35)(36).…”

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confidence: 99%

Cell-permeable probe for identification and imaging of sialidases

Tsai

Yen

Lin

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Alkyne-hinged 3-fluorosialyl fluoride (DFSA) containing an alkyne group was shown to be a mechanism-based target-specific irreversible inhibitor of sialidases. The ester-protected analog DFSA (PDFSA) is a membrane-permeable precursor of DFSA designed to be used in living cells, and it was shown to form covalent adducts with virus, bacteria, and human sialidases. The fluorosialyl–enzyme adduct can be ligated with an azide-annexed biotin via click reaction and detected by the streptavidin-specific reporting signals. Liquid chromatography-mass spectrometry/mass spectrometry analysis on the tryptic peptide fragments indicates that the 3-fluorosialyl moiety modifies tyrosine residues of the sialidases. DFSA was used to demonstrate influenza infection and the diagnosis of the viral susceptibility to the anti-influenza drug oseltamivir acid, whereas PDFSA was used for in situ imaging of the changes of sialidase activity in live cells.

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“…[7] Of these, we considered the quinone methides-although the basis of the first glycosidase ABPs reported and able to label recombinant, purified enzymes-unsuitable leads due to their broad reactivity in complex samples. [8] Literature data on the two remaining classes appeared more promising. 2-Deoxy-2-fluoroglycosides were first reported in 1987 by Withers and co-workers.…”

Section: Resultsmentioning

confidence: 98%

Activity‐Based Profiling of Retaining β‐Glucosidases: A Comparative Study

Witte

Walvoort

et al. 2011

ChemBioChem

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Activity-based protein profiling (ABPP) is a versatile strategy to report on enzyme activity in vitro, in situ, and in vivo. The development and use of ABPP tools and techniques has met with considerable success in monitoring physiological processes involving esterases and proteases. Activity-based profiling of glycosidases, on the other hand, has proven more difficult, and to date no broad-spectrum glycosidase activity-based probes (ABPs) have been reported. In a comparative study, we investigated both 2-deoxy-2-fluoroglycosides and cyclitol epoxides for their utility as a starting point towards retaining β-glucosidase ABP. We also investigated the merits of direct labeling and two-step bio-orthogonal labeling in reporting on glucosidase activity under various conditions. Our results demonstrate that 1) in general cyclitol epoxides are the superior glucosidase ABPs, 2) that direct labeling is the more efficient approach but it hinges on the ability of the glucosidase to be accommodated in the active site of the reporter (BODIPY) entity, and 3) that two-step bio-orthogonal labeling can be achieved on isolated enzymes but translating this protocol to cell extracts requires more investigation.

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Design and Synthesis of Activity Probes for Glycosidases

Cited by 85 publications

References 22 publications

Active-site Peptide “Fingerprinting” of Glycosidases in Complex Mixtures by Mass Spectrometry

Active-site Peptide “Fingerprinting” of Glycosidases in Complex Mixtures by Mass Spectrometry

Cell-permeable probe for identification and imaging of sialidases

Activity‐Based Profiling of Retaining β‐Glucosidases: A Comparative Study

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