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

Abstract

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 ...

Specificity Fingerprinting of Retaining β‐1,4‐Glycanases in theCellulomonas fimiSecretome Using Two Fluorescent Mechanism‐Based Probes

“…[13,14] These molecules label the catalytic nucleophile A C H T U N G T R E N N U N G because the fluoroglycosyl-enzyme intermediate (Scheme 1 B) forms faster than it breaks down, thus it accumulates and can be studied by proteomic methods. [4,5,15] In contrast, 2-deoxy-2-fluoro glycosides cannot label inverting glycosidases because they employ a single-displacement catalytic mechanism with no intermediate or enzymic nucleophile. [12] Results and Discussion…”

“…We first demonstrated the specificity-based labelling of retaining endo-b-1,4-glycanases by the two ABPs using a mixture of three enzymes: two GH family 10 mixed-specificity endo-xylanases/cellulases (Cex [17][18][19] and Cfx catalytic domain, Cfx cd ) [5,20] from C. fimi and a GH family 11 endo-xylanase (Bcx) [17] from Bacillus circulans. GH family 11 glycanases are exclusively xylan-specific while GH family 10 enzymes can also cleave cellulose, though usually with at least 100-fold lower k cat /K m .…”

“…We had previously shown through kinetic analysis that the potential negative impact of the tag on the reactivity of the probes with their target endo-b-glycanases is minimised by appending the tag to the sugar hydroxyl that is A C H T U N G T R E N N U N G normally linked to the rest of the polysaccharide A C H T U N G T R E N N U N G substrate. [4,5] The fact that the two ABPs incorporate two fluorophores with nonoverlapping excitation and emission spectra allows for multiplexing of the probes. [16] Testing ABPs in enzyme…”

“…[1][2][3] The specificity of the ABP depends critically on the mode of action and design of the A C H T U N G T R E N N U N G labelling component incorporated. Recently, we developed a gel-free, mass spectrometry-based ABPP (MS-ABPP) methodology for the active-site peptide fingerprinting of retaining endo-b-glycosidases [4,5] which led to the discovery of a previously unrecognised glycanase in the extracellular proteome of the soil bacterium Cellulomonas fimi. While biotinylated ABPs could be used in gel-based studies to visualise labelled enzymes through Western blotting, [4] the approach is cumbersome and does not allow for the visualisation of different activities simultaneously.…”

A mechanism-based inactivator of glycoside hydrolases involving formation of a transient non-classical carbocation

“…Growing awareness of the importance of GHs to fundamental biological processes, and of their utility in biotechnology, is stimulating interest in both identifying and modulating the activities of these enzymes. For application in biological samples in particular there has been increasing attention paid to developing techniques that capitalize on mechanism-based enzyme inactivators and their adaptation to generate activity-based probes [1][2][3][4][5] .…”