Synthesis and Testing of Mechanism‐Based Protein‐Profiling Probes for Retaining Endo‐glycosidases

Abstract

New functional proteomics methods are required for targeting and identification of subsets of a proteome in an activity‐based fashion. Glycosidases play critical roles in biology, yet a robust method for functional analysis of their activities and identities in biological proteomes is still lacking. An aryl 2‐deoxy‐2‐fluoro xylobioside inactivator was conjugated through cleavable and noncleavable linker arms to a biotin tag, thereby yielding two new active‐site‐directed reagents for activity‐based profiling of retaining β‐glycanases in complex proteomes. Crucially, these tagged reagents possess high specificity for their target enzymes with kinetic parameters similar to those of the untagged reagent. Western blotting showed that these reagents bind and covalently label active retaining β‐glycanases both in pure enzyme samples and in the secreted proteome of the soil bacterium Cellulomonas fimi. Such reagents therefore show great promise for future activity‐based targeting of glycanases.

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 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.…”

“…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. [2] Taking advantage of up to a 100-fold higher sensitivity of fluorescent ABPs [6] and the range of colours available, we report here a gel-based method for simultaneous in-gel visualisation of active retaining endo-b-1,4-glycanases with different specificities.…”

Chitosan-graft-polyethylenimine with improved properties as a potential gene vector

“…3,3 0 -Dithiodipropionic acid was used as the linker between chitosan and branched 800 Da PEI (Williams, Hekmat, & Withers, 2006). Chitosan is a weak base with a pK a value of about 6.2-7.0 (due to the D-glucosamine residue), thus shows a very poor aqueous solubility at neutral and alkaline pH values.…”